In the ancient world there were Elisha stories and Elijah stories. The related Torah portion deals with issues of ritual purity – eruptions on the skin.
4:42 This is probably the source of the text for the marriage at Canaan from the NT. The parable of loaves and fishes. The intent here is magical. Consider the Hasidic tale of travel to escape a pogrom and there is one cart jammed full of people and people along the road. There is always room if we are willing to love one another a bit more. Here, with a sense of sharing, the bread could suffice. Consider the birkat hamazone – the blessing after the meal – which assumes there is enough food in the world for everyone. "When you have eaten and are satisfied you shall bless the lord your God for the good land he has given you." Remember that "Haftarah" is the departure reading – read at the end of the meal and when you leave for the synagogue. These are communal prayers as contrasted to individual prayers. Note also that the Reform movement changed the departure blessing by making it shorter by deleting several paragraphs in the nature of encores.
5:1 A valiant Aramean soldier who had a skin disease. The name Naaman is very close in sound to the Hebrew word that means faithful. Note the trope marks for vocalization developed by the Masoretes. http://en.wikipedia.org/wiki/Masoretes An Israelite girl suggest that he see a prophet in Samaria who will cure his skin.
5:4 Naaman gets permission from his king to go to Israel and takes a considerable sum of money with him to see the Israeli king. The king of Israel, upon being called upon to cure Naaman, tears his clothes and accused the Arameans of picking a quarrel with him. Which may be correct. Or the girls message became terribly garbled. There are elements of comedy here.
5:8 Elisha sends a message to the king and asks that Naaman be sent to him.
5:9 Elisha suggest that Naaman bathe in the Jordon but this annoys and upsets Naaman who expects a magical cure with a show. He compares the Jordan to the greater rivers of Damascus. "So he walked off in a fury." Recall again the more elaborate procedures for cure of skin dieseases set forth in the Torah portion. The reference to the number seven may refer to the observed movable objects in the sky – sun, moon and five planets. There are other cycles in the ancient world but the Israelites' used seven – which was adopted by the Greeks and then the Romans.
5:13 Naaman follows the instruction and his flesh becomes purified. This is a very simple solution compared to that the Israelites had to follow to be purified after the tenth plague which was very complex. Sometimes complexity is an indication of importance. It suggests that the people had to be tested to evaluate their will to be free.
5:16 Elisha refuses any gift for the cure. Naaman hopes that he will be forgiven when he goes home and is forced to enter the Temple of Rimmon and bow down. LL: Compare this to the feelings of the Marranos and Conversos in Spain (http://www.jewishvirtuallibrary.org/jsource/Judaism/Marranos.html) who were forced to convert during the inquisition. This Haftarah must have given them some solace. Issues of idolatry are treated with nuance here. It is interesting to consider the various vignettes in this account – the scene before the king and before the tent of Elisha. What defines a folk tale is that there is no definitive form for the story. Note that it is the waters of the Jordan that works and that Naaman takes away with him some of the earth of the soil of Israel. This points to Israel as a sacred place – particularly to a community in exile. There is also the notion here that a skin disease can be reflective of stress or other disturbance within an individual – which is erased by belief. Consider the Christian belief in taking the waters at Lourdes.
PG: The first real Zionists were Protestants who returned to the Old Testament and found there a path to redemption via the Jews returning to the land. This is still a powerful force today with evangelical Christians. Zionism among Jews did not become a powerful force until the 20th C.
Today is Shabat Sahor – which precedes Purim. Shabat remembered. This portion is read in honor of Shabat Purim. Next week we will read the red heifer from the Book of Numbers and then from the Book of Exodus – introducing the new moon. This is done because of the festival ""Maftir" means a reading of a portion of the Torah in honor of the person reading the Haftarah.
p. 1334 26::17 Amalek is the desert tribe that attacked the Israelites after they had crossed the Red Sea. "Amalek" has become a metaphor for anti-Semitism. Heyman is thought to be a descendant of this tribe. To blot out the memory and "Do not forget." Is a conundrum. The composition of this paragraph takes place well after the events described. Also, there are no archeological remains of the Amaleks – is it a literary construct? Hillel and Shamai are two noted Rabbinic commentators but the names mean praise and contempt. Are they actually literary constructs as well? We know nothing about the literary traditions of the time. Compare to a Greek play – which also often appears to be founded on actual events. See p. 447 in Exodus – the story of Amalek at 17:1. "Is the Eternal present among us or not?" And the next line is Amalek. When you question God's presence you admit the enemy to your presence is the traditional rabbinic view. But is the enemy exterior or is it within us? This contradiction seems to plumb our inner psychological state. But without an antagonist do we fall into complacency? Is an antagonist necessary for growth? In the beginning of their journey the Israelites need the assistance of God. By the time they get to the promised land they defeat their enemies on their own.
This raises the question as to what we mean by "faithfulness" In Jewish thought it was the starting point. In Christianity per Paul and Martin Luther it is the acceptance of Jesus. How is Moses holding up his arms? Is it in compassion or victory? See Gustave Dore's painting.
The analogous Torah portion deals with the incineration of Aaron's sons for making an improper sacrifice at the altar.
6:1 The Ark had been carried off from its shrine site in Shiloh by the Philistines and David has successfully recaptured it. Now there is a parade. Note that David does not appear in the Torah – only in the prophetic books. Also, there is no reference to the Ark in the Torah segment.
6:6 The oxen stumbles. Uzzah touches the ark and is struck down by God. Or at least this is the chroniclers explanation of this otherwise inexplicable event. This may also be a difficult translation. Could Uzzah have been crushed under the wheels of the cart? Or did he fall off a cliff in his urgency to save the cart. Any action which is not a matter of negligence is considered "an act of God." It could also be argued that Uzzah sacrificed himself to save the Ark. But God got "furious" which suggests a direct action. How is this different from the actions of Aaron's sons – who were also punished for sacrilige?
6: 9 David is terrified to take the Ark. It could be dangerous. He wonders if the Ark should go to Jerusalem or be returned to Shiloh. But the family he leaves the Ark with is blessed. How were they blessed? Perhaps a good crop or the birth of sheep. AF: How do the people know God's feelings – that he is angry or is happy/blessing? PG: This is more reflective of the views of the author that is creating the text – if it were to be fiction. For non-fiction it is more difficult to ascertain internal states. Here, the author himself is divinely inspired to know the mind of God. LL: Compare the last scene in Raiders of The Lost Ark – where only the righteous were safe from the wrath of God.
6:12 David takes custody of the Ark and skips/dances before it. PG: This story was likely told in a variety of ways but the redactor has selected this version. Or combined two or more versions.
6:16 Saul's daughter, David's wife, sees him skipping/dancing and comes to despise him for his un-kingly behavior. Note that Michal is the only woman in scripture who is identified as loving her husband. Until know.
6:17 David passes out "goodie bags" to the people in celebration.
6:20 Michal takes David to task for his behavior. Here it says she had no children – suggested as punishment – but elsewhere it is suggested she had five children perhaps by a previous spouse.
7:1 David is in a cedar palace and the Ark is in a tent. What does this comparison mean? David must build an elaborate palace for the Ark – which is eventually achieved by Solomon in the construction of the first Temple.
7:4 God wants a cedar temple for the Ark and tells Nathan that in a dream. But God also suggests that he really doesn't need it – that He is everywhere.
7:8 "I have been with you wherever you have gone…" Nathan learns that David will be able to establish a dynasty.


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In my observational studies, I focus on the energetic, brilliant objects known as blazars. These objects are part of a larger class called Active Galactic Nuclei, or "AGN," consisting of actively accreting super-massive black holes in the cores of very old and distant galalxies. Blazars emit every kind of light observable, from radio waves through gamma rays. The amount of light emitted from blazars changes over time in a non-periodic fashion on timescales ranging from minutes to decades. This variability makes blazars interesting objects to observe over the course of a single night, several days, or even months or years. The observed characteristics of the variability in different kinds of light allow astronomers to test physical and computational models predicting blazar behavior.
Florida Gulf Coast University Faculty websites at Scholar.


Google Analytics is a web-analytic tool offered by Google which can be used to track visitors on any webpage. You can easily track your website traffic by setting up Google Analytics on Blogger. In this step-by-step guide, I will teach you how to add Google Analytics to Blogger blog.
Before starting the detailed guide on Google Analytics, let us first discuss some benefits of using Google Analytics on your blog.
Why should you Use Google Analytics on your Blog?
How to add Google Analytics to Blogger?
How to test whether Google Analytics is working or not?
Helps you determine the conversion rate.
Google Analytics is also helpful in monitoring your Google Ads account.
It gives you a complete overview of your audience.
These are some of the main benefits of using Google Analytics. Now let us move to our main topic i.e. How to add Google Analytics to Blogger?
First, you have to sign up for Google Analytics using your Google account. Click on the below link to sign up for Google Analytics.
Select Reporting Time Zone: Here you need to enter the timezone according to which your analytic data will be shown.
After filling the above details, just click on Get Tracking ID.
Following Step 1, you can easily get your Google Analytics Tracking ID. Now its time to add your tracking ID to Blogger.
Go to Settings >> Others in Dashboard.
Scroll the page & enter your tracking ID in Analytics Web Property ID under the Google Analytics section.
Click on the Save Settings on the top of the page.
Copy the Google Analytics tag from your Dashboard.
Visit Theme >> Edit HTML.
Just after the <head> paste the Google Analytics code.
That's It. You have successfully setup Google Analytics on your Blogger blog. In the above 2 steps, we have discussed a complete guide on how to setup google analytics on blogger.
If you want to check whether Google Analytics in working or not, just send some test traffic to your site and take an eye on real-time visitors on your Analytics Dashboard. If Google Analytics is working properly, you will see a change in your real-time statistics.
Get Google Analytics Tracking ID.
You can also test your Google Analytics account by sending some test traffic.
I hope you love this complete step-by-step guide on adding Google Analytics to Blogger. If you have more questions/suggestions. Feel free to share with me on the comment section.
How to add the Custom theme in Blogger?
Some great tips here, have you thought about doing a wordpress version?
I will keep this post handy. Great information.
Great post, a complete step-by-step guide. Would be very informative and very helpful for new bloggers too.
Thanks for the amazing post. Even I was planning to set up Google Analytics for my blog. This would certainly help me to set it up easily.
This article is really helpful as I'm using blogger. I will set up my Google Analitics asap. Thanks.
You have given full tutorial on google analytics and made it easy for anyone to set up without much technical knowledge.
Thanks, Indu for your comment.
This is very helpful…… Much needed for setting up my newly built Blogspot blogs…… Looking forward for more input from you…..
Your step by step directions with pictures were so easy to understand. I wish I used Blogger now! Please do this for Word Press next.
You can easily set up Google Analytics on WordPress by using a plugin called MonsterInsights. Thanks for your comment.
Nice one Harish! I will try to set up my Google Analytics! Great Great Great post form you!
GA helps with the correct stats if any site. Thanks for this simple DIY guide. I love it!
Hi Sayanti, I think after reading this post. You can easily setup GA at your own. Thanks for your comment.
I will save this. Great one for sharing this. google analytics topic Thank you.
Thanks Chai for your comment.
I've actually been thinking about making the transition to blogger from WordPress. Wp has given me multiple headaches and blogger looks fairly more simple.
I don't recommend you to move from WordPress.com to Blogger. It is so because WordPress comes with a lot of features that Blogger don't have. If you're thinking of making a change I suggest WordPress.org with Bluehost.


I've been thinking about this for about two years now, but it's finally made it to the head of my "things to do" list: I want to build a short test track using the techniques I plan to use for the new layout: code 55 flex track and turnouts made using the Fast Tracks soldering jigs.
There are several reasons for this: first, I want to refresh my flex-track skills. Second, I want to learn how to use the jigs to make turnouts. Third, I want a fairly complex interlocking where I can try out electronics for detecting trains and controlling signals and interlocking those with turnouts, as well as interfacing all of that to DCC and JMRI running on a computer. And finally, I need to test some trains and see if they have any issues with this type of track.
So the first order of business was to figure out what I want the interlocking to look like. I started by sketching out an interlocking with a couple of tracks and some sidings, which was a nice, generic, interlocking, but not really representative of what I want to model. I'm modeling high-density urban commuter passenger lines in Tōkyō, and those are double-track with few sidings.
So that turned my thoughts to the junction between the Chūō Line and Sōbu Line at Ochanomizu Station, and the set of interlockings just to the west of there, between Ochanomizu and Suidōbashi stations. I've done a lot of research on that area, and know the layout of the track and associated signals fairly well. It has a mix of 3, 4 and 5-lamp signal heads, so I can test most and maybe all of the signal types I'd use. Plus it's a very complex environment, which makes for a good test.
Today's post is about my latest (and more successful) attempt to learn to use XTrackCAD for layout design (see diagram above). I've made a few half-hearted attempts in the past, but was always turned off by the amount of up-front work needed to learn the dang thing. It's not at all obvious, at least not to me. This time I started knowing it was going to be a pain, but with the commitment to see that through.
Much of what I learned was basic, but some of it was very specific to what I'm doing, which is a flex track layout in Japanese N scale. If you weren't already aware, Japanese N is 1:150 scale rather than the usual 1:160 used in American/European N, and, oddly, for Japanese Shinkansen models, but I'm modeling normal trains for the most part. And I'm also planning to hand-lay at least some turnouts using the Fast Tracks jigs, although that turned out to be a lot simpler to design in XTrackCAD than I'd expected.
All of my Japanese-themed layouts to date have used sectional track, either Kato Unitrack or Tomix Finetrack. I haven't built a layout using flex track in more than twenty years. And that layout was a relatively simple one, modeling an American freight shortline, with low-speed trains and no "mainline" trackage. That let me cut a few corners.
But now that I'm thinking of building a new layout using flex track, and particularly one with mainline track for both moderate-speed commuter and high-speed Shinkansen, it's time for me to confront two of the more complicated aspects of trackwork that I've so far been able to avoid: transition curves and superelevation (for more information on these, see my Easements page). And it turns out, neither is really complicated after all.


text:
Navigating Patient Assistance Programs
December 2016, Vol 7, No 11 - Patient Support Services
Washington, DC—In the past 6 years, more than $4 billion in charitable assistance has been dedicated to patients in need. In 2015 alone, the Patient Access Network (PAN) Foundation provided $950 million in cancer care support. The only people with deep enough pockets to support this type of assistance are drug manufacturers, adding to an already complicated regulatory environment for charitable assistance programs, said Daniel J. Klein, President and Chief Executive Officer, PAN Foundation, at the Sixth Annual Conference of the Association for Value-Based Cancer Care.
Although many manufacturers offer their own patient assistance programs, they differ from charitable programs. Whereas manufacturer programs typically only cover their drugs, foundations provide assistance with other expenses in addition to drugs.
Medicare Beneficiaries
The PAN Foundation also assists Medicare beneficiaries—something drug manufacturer programs cannot do. Approximately 85% to 90% of the people the PAN Foundation assists are Medicare beneficiaries, many of whom are under 500% of the federal poverty level.
"Why is this important? Everybody here knows how much the cost of oncology treatment has increased over the past decade. It's gone from about $5000 a month to $10,000 a month in terms of treatment cost…we're particularly concerned about that within Medicare," said Mr Klein.
In 2016, Medicare drug plans had deductibles totaling less than $360, with an initial cost-sharing of $825. People who are caught in the coverage gap pay approximately $1875 out of pocket, according to Mr Klein.
"A little over $3000 out of pocket just to get into the catastrophic period that picks up pretty much all of the costs, except for 5%," he said. Mr Klein explained that that 5% can be a significant barrier for patients.
"When you're down at 200% of the federal poverty level, which is above the level that you can get low-income subsidy in Medicare, even a drug costing $7500 a year is going to push you into being underinsured. Just that single drug."
Patient assistance programs lower the financial barriers to cancer care, which, in turn, improves access, adherence, and patient outcomes, Mr Klein said.
"People who are cutting their dosage of medication in half or skipping it tend not to get the maximum benefit from treatment. There's lots of research on the impact of lowering the financial barriers to care," Mr Klein said. "We provide the safety net, the only safety net, available for Medicare patients. Under the various rules and regulations, there's really no other place for Medicare patients to go to who are struggling with out of pocket costs," he added.
Access to Costly Drugs
Lack of access to drugs is something that Pamela Gavin, Chief Operating Officer, National Organization for Rare Disorders (NORD), Danbury, CT, knows all too well.
"Prior to the establishment of NORD, there were only 10 therapies on the market for rare diseases developed in the 10 years prior to the movement that led to the Orphan Drug Act," she said.
"If we look at the Orphan Drug Act, and the therapies approved under that law prior to 1983, and you look at the next 30 years, and how that's progressed, it's been probably one of the most successful pieces of healthcare legislation, when it comes to innovation that we've experienced in this country," Ms Gavin added.
According to Ms Gavin, in the past 2 years alone, there have been nearly 90 drugs approved under the orphan drug designation.
Ms Gavin and Mr Klein argue that the emergence of innovative drugs, however, does not fix the root of the problem.
"In the environment that we're operating in right now, the demand greatly outweighs the capacity for us even collectively—if we look at Dan's organization, NORD, and many others that are out there—the demand greatly outweighs the need. It's not a fix. It's not a solution long-term," said Ms Gavin.
August 2019, Vol 10, No 4 | Payers' Perspectives In Oncology: ASCO 2019 Highlights
Autologous Mesothelin-Targeted T-Cells Induce Responses in Pleural Solid Tumors
Chicago, IL—A mesothelin-targeted chimeric antigen receptor (CAR) T-cell immunotherapy demonstrated encouraging antitumor activity in combination with pembrolizumab in patients with mesothelin-associated malignant pleural solid tumors, primarily mesothelioma, who had disease progression after platinum-containing chemotherapy, according to results presented at ASCO 2019. [ Read More ]
August 2017, Vol 8, No 4 | Payers' Perspectives In Oncology: ASCO 2017 Highlights
Olaparib First PARP Inhibitor to Show Advantage in BRCA-Positive Breast Cancer
Chicago, IL—The PARP inhibitor olaparib (Lynparza) significantly improved progression-free survival (PFS) compared with standard chemotherapy in women with HER2-negative metastatic breast cancer with a germline BRCA mutation. Disease progression was delayed by approximately 3 months with olaparib in the multinational, randomized, open-label, phase 3 OlympiAD clinical trial, reported Mark E. Robson, MD, Clinic Director, Clinical Genetics Service, Memorial Sloan Kettering Cancer Center, New York City, at the 2017 ASCO annual meeting. [ Read More ]

redpajama_set_name: RedPajamaCommonCrawl


When I was a kid I loved to play with cars. They were much more fun than dolls that just sat there and didn't do anything. I passed on my love of boy toys to my daughters as well. My house has always had fire trucks and cars along with the dolls. One of my kids favorite cars are the Shake 'n Go cars by Fisher Price.
They love these cars! And I love them because my kids can play with them without my help! They shake the car, set it down, and away it goes. The longer you shake the car, the farther it goes. While you shake the car, you can hear the engine vroom! My twins get a great kick out of hearing the engine roar.
They make the cars in different colors and themes. Isabella's favorite is this Toy Story car.
You can purchase a Fisher Price Shake 'n Go Toy at various retailers.
You can visit Fisher Price here to see a complete list of their shake 'n go toys.
One lucky Twingle Mommy reader will win a Shake n' Go toy of their own.
1. You must be a follower through Google Friend Connect.
2. Leave a comment telling me who would love this toy in your house.
My 2 1/2 year old son Jack would LOVE this! He is sooo in to cars right now!!
cool post allison. my boys would love to play with this!
Hi I'm a new follower from MFM.
New follower from the Good Friends Just Click hop. I'd love to win this for my son!
New follower from the "Good Friends Just Click" blog hop. Come visit me at The Blessed Couponer and follow back :) Thanks!
love your blog its really nice.
My daughter would so love this toy.
My two year old little peapod would adore a toy like this. You are too sweet to post a giveaway like this. Good luck to everyone!
New follower from GFC. My Nephew would love this toy.
My husband would love this toy the most!
I'm your newest follower from the Happy Hump Day Blog Hop. If you get a minute please drop by and follow back.
My two year old son would looooove this!!
Great giveaway! I'm a new follower from the hop!
I also have a giveaway at: http://stylendecordeals.blogspot.com/2011/03/cinnamon-burst-cheerios-review-and.html Doesn't represent my blog very well though!
I'm your newest follower from the Bee Friendly blog hop. Look forward to reading your posts.
I have 2 boys and they would both love this car!
I would like to win this for my 2 year old son.
My son would totally love this toy!
My boy, while he's still young would LOVE this!
My little girl would LOVE this!!
My 12 month old son would LOVE this!
My 3 1/2 year old son Matthew and my 19 month old daughter LOVE Toy Story and they would adore this car!
my 18m old son would love this!!!
I am now following you from the Explore Plan Learn blog hop. Would love a follow back. Also would love to be entered to win if it's available to Canadians.
Im following via GFC .
My nephew Gabe would love this! Hes always into cars and things that are colorful.
Oops sorry both on one comment..
My daughter would love this..
would both LOVE this car.
*May put away for Ryan's Birthday in a couple months.
My son Matthew will love this car!
My son will love this--and if not him, his father definitely will.
My 22 month old daughter would LOVE this!
My 3 year old nephew would love this.


text:
Scream Factory's Ginger Snaps - Blu-ray Release Details and Cover Art
[ Read the Ginger Snaps (2000) Movie Review ]
[ Movie Page: Ginger Snaps (2000) ]
As good werewolf movies go, Ginger Snaps (2000) is definitely in the top werewolf movies list. No wonder Scream Factory will be releasing the Ginger Snaps Blu-ray. Special features and extras for the Blu-ray has not been released yet but are expected this upcoming May 2014. John Fawcett's (Spartacus: War of the Damned, Lost Girl, Spartacus: Gods of the Arena) Ginger Snaps was originally released in 2000 and stars Emily Perkins (Ginger Snaps franchise), Katharine Isabelle (Ginger Snaps franchise, American Mary) and Kris Lemche (The Frankenstein Theory, Final Destination 3).
From the Scream Factory Facebook Page:
We end January with yet another key art reveal for you. Check out the new key art for our upcoming Collector's Edition of the Canadian sisters-in-cahoots werewolf favorite GINGER SNAPS (DVD & Blu-ray combo out in July)!
Some of you may have seen this before as artist Joel Robinson (The Vincent Price Collection) adapted this from an existing piece of artwork he created prior to Scream Factory which we thought was cool to begin with and liked it enough to represent this fun cult film.
Ginger Snaps (2000)
Directed by John Fawcett
Movie Overview | Review | Trailer
Official Synopsis: Is becoming a woman analogous, in some deep psychological way, to becoming a werewolf? Ginger is 16, edgy, tough, and, with her younger sister, into staging and photographing scenes of death. They've made a pact about dying together. In early October, on the night she has her first period, which is also the night of a full moon, a werewolf bites Ginger. Within a few days, some serious changes happen to her body and her temperament. Her sister Brigitte, 15, tries to find a cure with the help of Sam, a local doper. As Brigitte races against the clock, Halloween and another full moon approach, Ginger gets scarier, and it isn't just local dogs that begin to die.
Cast: Katharine Isabelle, Emily Perkins, Jesse Moss, Kris Lemche, Steven Taylor, Joey Paul Gowdy, Lucy Lawless, John Bourgeois, Christopher Redman, Pak-Kwong…
Genres: Horror, Thriller, Drama, Fantasy, Romance
Source: official Facebook page
Ginger Snaps (2000) - Trailer
Ginger Snaps Back: The Beginning (2004) - Trailer
Ginger Snaps 2: Unleashed (2004) - Trailer


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Anti-Stealth Chinese Radar gets installed at Mianwali, Pakistan
File Photo Chinese Anti-Stealth JY-27A Radar installed at Mianwali
Chinese Anti-Stealth JY-27A Radar has been installed at Mianwali airbase.
According to report, satellite imagery on August 29 has revealed that a JY-27A radar developed by China Electronics Technology Group Corporation was installed at the Mianwali airbase.
The report said that the "counter-very-low-observable" radar may have arrived in Pakistan between June 5 and August 29.
The Chinese JY-27A long-range air surveillance and guidance radar is not only capable of detecting advanced stealth aircraft but also guiding missiles to destroy them, a senior Chinese radar designer had told Global Times a few months ago.
The radar could be deployed on vehicles, on land and warships, the Global Times reported quoting the Chinese military officials.
Pakistan, Turkey, Azerbaijan agree to 'resolve global issues in line with int'l law': Qureshi
Corona claims 32 more lives in last 24 Hrs
Blackout: After major breakdown, power being gradually restored in cities across Pakistan


text:
Trichomonas vaginalis induces apoptosis via ROS and ER stress response through ER–mitochondria crosstalk in SiHa cells
Fei Fei Gao1,2 na1,
Juan-Hua Quan3 na1,
Min A. Lee4 na1,
Wei Ye5,
Jae-Min Yuk1,2,
Guang-Ho Cha2,
In-Wook Choi2 &
Young-Ha Lee1,2
Trichomonas vaginalis causes lesions on the cervicovaginal mucosa in women; however, its pathogenesis remains unclear. We have investigated the involvement of the endoplasmic reticulum (ER) in the induction of apoptosis by T. vaginalis and its molecular mechanisms in human cervical cancer SiHa cells.
Apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), ER stress response and Bcl-2 family protein expression were evaluated using immunocytochemistry, flow cytometry, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide dye staining and western blotting.
Trichomonas vaginalis induced mitochondrial ROS production, apoptosis, the ER stress response and mitochondrial dysfunction, such as MMP depolarization and an imbalance in Bcl-2 family proteins, in SiHa cells in a parasite burden- and infection time-dependent manner. Pretreatment with N-acetyl cysteine (ROS scavenger) or 4-phenylbutyric acid (4-PBA; ER stress inhibitor) significantly alleviated apoptosis, mitochondrial ROS production, mitochondrial dysfunction and ER stress response in a dose-dependent manner. In addition, T. vaginalis induced the phosphorylation of apoptosis signal regulating kinase 1 (ASK1) and c-Jun N-terminal kinases (JNK) in SiHa cells, whereas 4-PBA or SP600125 (JNK inhibitor) pretreatment significantly attenuated ASK1/JNK phosphorylation, mitochondrial dysfunction, apoptosis and ER stress response in SiHa cells, in a dose-dependent manner. Furthermore, T. vaginalis excretory/secretory products also induced mitochondrial ROS production, apoptosis and the ER stress response in SiHa cells, in a time-dependent manner.
Trichomonas vaginalis induces apoptosis through mitochondrial ROS and ER stress responses, and also promotes ER stress-mediated mitochondrial apoptosis via the IRE1/ASK1/JNK/Bcl-2 family protein pathways in SiHa cells. These data suggest that T. vaginalis-induced apoptosis is affected by ROS and ER stress response via ER–mitochondria crosstalk.
Trichomonas vaginalis is a flagellated protozoan parasite that infects the female reproductive tract and the male urethra [1]. According to the 2019 report of the World Health Organization, the global prevalence estimates of trichomoniasis were 5.3% in women and 0.6% in men, and the total estimated new cases were 156.0 million (103.4–231.2 million) worldwide in 2016 [2, 3]. This parasite causes vaginitis and cervicitis in women and asymptomatic urethritis and prostatitis in men. In addition, the host inflammatory response against the parasite is predicted to result in multiple adverse health effects, such as a high incidence of premature births [4] and increased risk of cervical cancer [5]. However, the pathogenesis for T. vaginalis infection is poorly understood.
Apoptosis, a cellular event induced by the activation of a series of enzymes known as caspases, occurs when a cell is damaged beyond repair, infected with a pathogen or stressed due to DNA damage or toxic chemicals [6]. Mitochondria are important in the regulation and transmission of apoptotic signals, which are regulated by maintaining a balance among the levels of the Bcl-2-family proteins [7]. Several studies have presented various mechanisms underlying the induction of apoptosis by T. vaginalis infection using in vitro models [8,9,10,11,12,13,14]. Trichomonas vaginalis-induced apoptosis of cells has been described via the production of reactive oxygen species (ROS) [8, 9], secretion of cysteine proteases from T. vaginalis [10,11,12] and mitochondrial dysfunction, including an altered mitochondrial membrane potential (MMP) and an imbalance in Bcl-2 family protein expression [13, 14]. However, the role of the endoplasmic reticulum (ER) in T. vaginalis-induced apoptosis has not been elucidated.
ROS function as intermediates in cellular processes, such as inflammatory responses, cell-cycle progression, apoptosis, aging and cancer, and are produced in various organelles and via different enzyme systems, including mitochondria, ER, peroxisomes and NADPH oxidase [15]. The mitochondrion is a major source of ROS [16]. Previous reports have shown that ROS induce apoptosis in T. vaginalis-infected human neutrophils and cervical cancer cells through caspase-3 activation and nuclear factor kappa B (NF-κB) inactivation, respectively [8, 9]. ROS also activate NLRP3 inflammasome formation in human prostate epithelial RWPE-1 cells [17]. However, the interaction between ROS and ER stress in T. vaginalis-infected cells has not been studied.
The ER plays a role in protein folding and assembly, lipid biosynthesis, vesicular traffic and cellular calcium storage [18]. Its function can be disturbed by various factors, including the expression of misfolded or unfolded proteins, changes in calcium homeostasis, redox status and energy storage. These dysfunctions cause proteotoxicity in the ER, collectively termed ER stress, which leads to activation of the unfolded protein response (UPR) [18]. ER stress induces dissociation of the chaperone glucose-regulated protein 78 (GRP78) from the three ER transmembrane protein stress sensors, named inositol-requiring enzyme (IRE), protein kinase RNA (PKR)-like ER kinase (PERK) and activating transcription factor 6 (ATF6). IRE1α is an ER transmembrane protein that activates the UPR to maintain ER and cellular function. PERK is a trans-ER membrane serine/threonine kinase which is activated by misfolded proteins in the ER lumen. Phospho (p)-PERK activates eukaryotic initiation factor-2α (eIF2α), which suppresses protein synthesis. ATF6 is a transmembrane glycoprotein, and is cleaved after the accumulation of misfolded proteins in the ER. Activation of these three protein sensors induces the UPR, which increases the expression of CCAAT/enhancer-binding protein–homologous protein (CHOP). CHOP is a proapoptotic transcription factor that plays an important role in ER stress-induced apoptosis. Caspase-12 is a prodeath protease located on the outer surface of the ER membrane that is responsible for ER stress-induced apoptosis [18, 19].
Uncontrolled severe oxidative stress triggers a series of proapoptotic signaling pathways, including the ER stress response and mitochondrial dysfunction, ultimately resulting in cell apoptosis [20]. Many researchers have reported the induction of ER stress responses in cells infected by bacteria [21, 22]. ER stress has also been investigated in cells infected by protozoan parasites, including Plasmodium, Toxoplasma, Cryptosporidium and Leishmania [23]. However, ER stress induction in cells infected with T. vaginalis has not yet been explored. Therefore, in the present study, our aim was to study the involvement of ER stress response in apoptosis induction, as well as its potential molecular mechanisms, in T. vaginalis-infected human cervical cancer SiHa cells. Thus, we evaluated the cytotoxicity, apoptosis, ROS production, MMP, ER stress response and Bcl-2 family protein levels in T. vaginalis-infected SiHa cells with or without specific inhibitors using lactate dehydrogenase (LDH) assays, immunocytochemistry, flow cytometry, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) staining and western blotting.
Reagents and antibodies
The CytoTox 96® Non-Radioactive Cytotoxicity Assay was obtained from Promega (Madison, WI, USA). Life Technologies CellROX® oxidative stress reagents (Thermo Fisher Scientific, Waltham, MA, USA) were purchased from Alfagene Ltd. (Carcavelos, Portugal). The MitoSOX™ Red Mitochondrial Superoxide indicator was purchased from Thermo Fisher Scientific. The JC-1 MitoMP detection kit was obtained from Dojindo Laboratories (Kumamoto, Japan). The FITC Annexin V Apoptosis Detection Kit was purchased from BD Biosciences (San Diego, CA, USA). The ER stress inhibitor 4-phenylbutyric acid (4-PBA), the ROS scavenger N-acetyl-l-cysteine (NAC) and the C-Jun N-terminal kinase (JNK) inhibitor SP600125 were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
ER Stress Antibody Sampler Kit, anti-ATF6, anti-p-PERK, anti-phospho eukaryotic translation initiation factor 2α (ani-p-eIF2α), anti-poly (ADP-ribose) polymerase (anti-PARP), anti-cleaved caspase-3, anti-caspase-3, the pro-Apoptosis Bcl2 Family Member Antibody Sampler Kit, the pro-Survival Bcl2 Family Member Antibody Sampler Kit, anti-phospho-apoptosis signal-regulating kinase 1 (anti-p-ASK1, Ser967), anti-phospho-ASK1 (Thr845), anti-ASK1, anti-phospho-JNK (anti-p-JNK, Thr183/Tyr185), anti-JNK (56G8) and anti-β-actin antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-CHOP and anti-phospho-IRE1α (anti-p-IRE1α) antibodies were obtained from Abcam (Cambridge, MA, USA). The secondary antibodies anti-rabbit horseradish peroxidase (HRP) and anti-mouse HRP were obtained from Jackson Immuno Research Laboratories (West Grove, PA, USA). Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody–Alexa Fluor 568 was obtained from Thermo Fisher Scientific.
Culture of SiHa cells
Cells of the human cervical cancer cell line SiHa (ATCC® HTB-35™) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbeco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA) and antibiotic–antimycotic (Gibco BRL) in a 5% CO2 atmosphere at 37 ˚C.
Trichomonas vaginalis cultures
The T. vaginalis T016 strain was cultured according to previously reported methods [8, 12]. Briefly, T. vaginalis T016 isolate was cultured in screw-capped glass tubes containing Diamond's trypticase yeast-extract maltose (TYM) medium (NAPCO, Winchester, VA, USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich, St Louis, MO, USA) in 5% CO2 at 37 ˚C for 24 h. The cultured parasites were monitored for motility, and their viability was determined before each experiment by staining with trypan blue (> 99%).
Preparation of T. vaginalis excretory/secretory products
Trichomonas vaginalis excretory/secretory products (ESP) was prepared as described previously [12]. To prepare the T. vaginalis ESP, freshly purified trophozoites (1 × 107 cells/mL) were incubated with TYM medium at 37 °C for 1 h in 5% CO2. After centrifugation for 30 min at 10,000 g, the ESP-containing supernatant was filtered through a 0.2-μm pore. The T. vaginalis ESP concentrations were measured by the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin (BSA) as the standard. The samples were kept at − 70 °C until use.
SiHa cells were seeded on 96-well plates [for the LDH and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt [MTS] assays), on 12-well coverslips for immunofluorescence and ROS detection and in 100-mm culture dishes (for western blotting), at various densities and grown to confluence at 37 °C in 5% CO2.
Based on our previous studies [9, 12], live T. vaginalis trophozoites were incubated in SiHa cells in mixed-medium (DMEM:TYM, 2:1) at a multiplicity of infection (MOI) of 2 or 5 for 2 and 6 h, at 37 °C in 5% CO2. Following the predetermined incubation time, cytotoxicity, apoptosis, ROS production, MMP, induction of ER stress and its mechanism of action and Bcl-2 family-related protein expressions were evaluated in T. vaginalis-infected SiHa cells by the MTS assay, LDH assay, flow cytometry, immunocytochemistry and western blotting.
To determine the role of ROS in T. vaginalis-infected cells, cells were pretreated with the ROS scavenger NAC and then evaluated for apoptotic features, MMP and ER stress response. To assess the involvement of ER stress in apoptosis induction in T. vaginalis-infected SiHa cells, cells were pretreated with ER stress inhibitor 4-PBA, NAC or the JNK inhibitor SP600125, and then the apoptotic features, ROS production, MMP, Bcl-2 family proteins and ASK1/JNK pathways were evaluated. We also checked the induction of mitochondrial ROS, apoptosis and ER stress response in SiHa cells after treatment with T. vaginalis ESPs. Untreated SiHa cells were used as controls. Each experiment was performed at least three times in triplicate.
LDH assay
The LDH assay was performed for cytotoxicity quantification with the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's protocol. Briefly, 1 × 104 cells were seeded in 96-well plates and infected with T. vaginalis at various MOI (1, 2, 5, 10) for the indicated times (0, 0.5, 2, 6, 12 and 24 h) in an incubator (5% CO2, 90% relative humidity, 37 °C). Then, 50 µl of the culture supernatant from all infected and control wells was transferred into a new 96-well plate, and 50 µl of CytoTox 96® reagent was added to each sample aliquot. The plate was covered with foil to protect the samples from light and incubated for 30 min at room temperature. After adding 50 µl of stop solution to each well, the absorbance of the solution was measured immediately at 490 nm using a microplate reader (TECAN, Crailsheim, Germany). LDH release levels in the media were quantified and compared to maximum LDH release values according to the kit instructions.
MTS assay
The MTS assay was performed to detect cytotoxicity with the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit according to the manufacturer's instructions. Briefly, 1 × 104 SiHa cells were seeded in 96-well plates and stimulated with various concentrations of NAC (0, 0.2, 1, 5 mM), 4-PBA (0, 0.2, 1, 2 mM) and SP600125 (0, 0.3, 3, 30 µM) for the indicated times (0, 2, 6, 12, 24 h) in an incubator (5% CO2, 37 °C). Then, 20 µl of CellTiter 96® AQueous One Solution Reagent was pipetted into each well of the 96-well assay plate containing the samples in 100 µl of culture medium, following which the samples were incubated at 37 °C for 1–4 h in a humidified, 5% CO2 atmosphere and the absorbance recorded at 490 nm using a 96-well plate reader.
Measurement of ROS generation by confocal microscopy
SiHa cells (1 × 104 cells/well; 12-well plates) seeded on a coverslip were cultured in DMEM supplemented with 10% FBS and the culture medium replaced when the cells reached 80% confluence. To evaluate the generation of ROS, SiHa cells were first infected with live T. vaginalis (MOI 2 and 5) or treated with 100 µg/ml T. vaginalis ESP for 2 and 6 h, then incubated with 5 µM MitoSOX reagent or CellROX reagent and finally incubated for 10 min at 37 °C, 5% CO2 in the dark. The stained cells were imaged using a laser confocal microscope (model TCS SP8; Leica Microsystems GmbH, Wetzlar, Germany). All experiments were performed on triplicate samples, and fluorescence intensity was calculated using ImageJ software; the graph was plotted using SigmaPlot version 12.5 (Systat Software Inc., San Jose, CA, USA).
ROS-based flow cytometric assay
SiHa cells were cultured in 12-well plates and then treated with 100 µg/ml T. vaginalis ESP for 2 and 6 h or infected with T. vaginalis at MOI 2 and 5 for 0, 2 and 6 h; the cells were pretreated or not with the indicated concentrations of NAC (0, 0.2, 1, 5 mM). The cells were then washed with phosphate-buffered saline (PBS), removed from the well with 2.5% trypsin–EDTA and re-suspended in PBS with 5 µM MitoSOX reagent or 5 µM CellROX reagent and incubated at 37 ℃, protected from light, for 30 min. After gentle washing (3 times), the cells were re-suspended in FACS buffer (1% BSA in PBS) and immediately subjected to the acquisition of 10,000 events by a FACScan instrument (BD Biosciences). The results were expressed as a histogram emitting the corresponding fluorescence and as a bar graph representing the mean fluorescence intensity of all the groups.
MMP assay
The MMP was measured using the JC-1 MitoMP Detection Kit (Dojindo, Kumamoto, Japan). Briefly, SiHa cells were seeded onto coverslips in 12-well plates at a density of 1 × 104 cells/well and infected with T. vaginalis at various conditions with or without specific inhibitors. The cells were then incubated with 4 μM JC-1 fluorescence dye at 37 °C for 30 min in dark and rinsed three times with Hanks' Balanced Salt Solution (HBSS). The stained cells were mounted onto microscope slides in VECTASHIELD HardSet Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA), and images were collected using a laser confocal microscope (model TCS SP8; Leica Microsystems GmbH). The intensities of green (excitation/emission wavelength: 485/538 nm) and red (excitation/emission wavelength: 485/590 nm) fluorescence were analyzed for ≥ 6 microscopic fields in each sample.
SiHa cells were seeded onto coverslips in 12-well plates at a density of 1 × 104 cells/well and incubated for 24 h. The cells were pretreated with or without specific inhibitors for 2 h and then infected with T. vaginalis at MOI 5 for 6 h. The cells were washed with HBSS and fixed with freshly prepared 4% paraformaldehyde for 1 h at room temperature, then washed three times in PBS containing 0.3% Triton X-100 (PBS-T) for 5 min. The cells were then blocked with 1% BSA in 0.3% PBS-T for 30 min at room temperature, incubated with CHOP primary antibody for 2 h at room temperature, washed to remove the excess primary antibody and then incubated with the appropriate fluorescently labeled secondary antibody (anti-mouse Alexa Fluor 647) for 2 h at room temperature. After mounting with VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories, Inc.) to stain the nucleus, fluorescence images were acquired using a confocal microscope (Leica Microsystems GmbH).
Western blotting analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis were performed to determine the expression of numerous proteins. The 1 × 107 SiHa cells were cultured with complete medium (DMEM supplemented with 10% FBS and 1 × antibiotic–antimycotic) in 100-mm dishes and then subjected to serum deprivation for 4 h to remove any stimulation from serum factors. The SiHa cells were then treated with 100 µg/ml T. vaginalis ESP for 0, 2 and 6 h or infected by T. vaginalis at various MOIs (1, 2, 5 and 10) for the indicated times (0, 0.5, 2, 6, 12 and 24 h). SiHa cells were pretreated with various concentrations of NAC (0, 0.2, 1, 5 mM), 4-PBA (0, 0.2, 1, 2 mM) and SP600125 (0, 0.3, 3, 30 µM) for 2 h, and subsequently infected with T. vaginalis at MOI 5 for 6 h. After washing with PBS, proteins were extracted using the PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Gyeonggi, Korea). The extract was incubated with the complete protease inhibitor cocktail (Roche, Basel, Switzerland) for 30 min on ice followed by boiling for 10 min, and then centrifuged at 14,000 g for 15 min at 4 °C. The supernatant was collected, and equal amounts of protein from different samples were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were immersed for blocking at 5% skim milk in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) containing 0.1% Tween-20 (TBST) for 1 h at room temperature. After one washing in TBST, the membranes were incubated overnight at 4 °C with the primary antibodies diluted in TBST supplemented with 5% BSA (1:1000). Following three consecutive washes with TBST, the membranes were incubated for 90 min with HRP-conjugated anti-mouse or anti-rabbit secondary antibody diluted 1:2000 with 5% skim milk, as described above. The washed membranes were soaked with Immobilon Western Chemiluminescent HRP Substrate (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), and chemiluminescence was detected with a Fusion Solo System (Vilber Lourmat, Collegien, France). Band intensity was quantified using ImageJ software (NIH, Bethesda, MD, USA). These experiments were repeated at least three times.
Apoptosis detection by FITC Annexin V/propidium iodide
We used the BD Biosciences FITC Annexin V Apoptosis Detection Kit to measure T. vaginalis-induced SiHa cell apoptosis. Briefly, the SiHa cells were plated into a 6-well plate and infected with T. vaginalis at MOI 2 and 5 for the indicated times (0, 2 and 6 h), following which both floating and adherent cells were collected and washed twice with cold PBS. The cells were then resuspended in 1 × Binding Buffer at a concentration of 1 × 106 cells/ml, and 100 µl of the solution (1 × 105 cells) was transferred to a new FACS tube and 5 µl of FITC Annexin V and 5 µl of propidium iodide (PI) were added. The cells in the FACS tube were then gently vortexed and incubated for 15 min at room temperature in the dark. After 400 µl of 1 × Binding Buffer was added to each FACS tube, the cells were analyzed using a FACSCanto II flow cytometer (BD Biosciences) within 1 h.
All assays were performed in triplicate, and at least three independent experiments were conducted per test series. The results are presented as the mean ± standard deviation (SD). Statistical analysis of the data was performed using unpaired, two-tailed Student's t-tests. A P value < 0.05 indicated statistical significance.
Trichomonas vaginalis-induced cytotoxicity and mitochondrial ROS production in SiHa cells in a parasite burden- and infection time-dependent manner
To determine the adequate MOI and infection time for induction of apoptosis and ER stress response in live T. vaginalis-infected SiHa cells, we performed the LDH assay and western blotting under various conditions. LDH-dependent cytotoxicity was significantly increased by 30 min after infection and increased with increasing MOIs in SiHa cells between 2 and 6 h after infection (see Additional file 1: Figure S1a). The levels of apoptosis- and ER stress-related proteins also increased by 30 min after infection, and their protein bands were apparent at 2 and 6 h after infection. However, the band densities of some protein bands were reduced from 12 h after infection onwards. Among the various MOIs of live T. vaginalis parasites tested, the expressions of apoptosis- and ER stress-related proteins changed in a dose-dependent manner at MOI 2 and 5 (see Additional file 1: Figure S1b).
Based on the results shown in Additional file 1 and from our previous studies [9, 12], we selected MOI 2 and 5 as an adequate parasite burden, and 2 and 6 h as an adequate T. vaginalis infection period to induce apoptosis and the ER stress response. The cytotoxicity of SiHa cells infected with T. vaginalis increased with the MOI and in a time-dependent manner (Fig. 1a). As oxidative stress is an important factor in the cytotoxicity associated with apoptosis [15, 16], we checked the cellular and mitochondrial ROS levels in T. vaginalis-infected SiHa cells. Cellular ROS levels were significantly increased in SiHa cells (MOI 2) from 2 h after infection and had increased further at 6 h post-infection. Cellular ROS levels were significantly higher in cells infected with T. vaginalis at MOI 5 than in cells infected with T. vaginalis at MOI 2 (Fig. 1b, c). Similarly, mitochondrial ROS production in the SiHa cells significantly increased from 2 h after infection, and increased in direct proportion to parasite burden and infection time (Fig. 1d, e). These findings suggested that T. vaginalis induces cytotoxicity and mitochondrial ROS production in SiHa cells in a parasite burden- and infection time-dependent manner.
Trichomonas vaginalis-induced cytotoxicity and mitochondrial ROS production in SiHa cells. SiHa cells were infected with live T. vaginalis trophozoites at MOI 2 and 5 for 0, 2 or 6 h. a The LDH level in the medium, which is related to cell death, was measured by LDH assay at the indicated conditions. The data represent the mean value ± standard deviaiton (SD) of at least three independent experiments. Asterisks indicate significant difference (***P < 0.001) compared with untreated control cells under the same conditions. b, c Cellular ROS production was measured with CellROX reagent, a fluorogenic probe for measuring oxidative stress in live cells by confocal microscopy (b) and flow cytomery (c). d, e Mitochondrial ROS production was determined by confocal microscopy (d) and flow cytometry with MitoSOX, a mitochondrial ROS dye (e). Plots (c, e) depict the CellROX or MitoSOX-positive cells as determined by fluorescence analysis of flow cytometry results. Data shown are representative of three independent experiments with similar results. Asterisks indicate significant difference (***P < 0.001) in mean fluorescence compared with untreated control cells under the same conditions Scale bars: 10 µm. Abbreviations: CTL, Untreated control SiHa cells; Tv, SiHa cells infected with live T. vaginalis
Trichomonas vaginalis-induced mitochondrial apoptosis through ROS in SiHa cells
To determine whether the observed cytotoxicity is associated with apoptosis, we conducted western blotting and flow cytometry to detect apoptotic signals in the T. vaginalis-infected SiHa cells. The protein levels of cleaved PARP and caspase 3, both apoptosis indicator proteins [6, 24], were increased with increasing MOI (parasite burden) at 6 h after infection; they were also apparently increased from 2 h after infection in T. vaginalis-infected SiHa cells and increased further in a infection time-dependent manner (Fig. 2a). In addition to western blotting, flow cytometric analysis confirmed that both early and late apoptosis were significantly increased in T. vaginalis-infected cells in an infection time-dependent manner (Fig. 2b). Nucleus fragmentation and cellular shrinkage, a characteristic morphological change of apoptosis [6], were found in T. vaginalis-infected cells at 2 and 6 h after infection by DAPI staining; however, uninfected cells (0 h) grew well with a distinctly complete nucleus (Fig. 1b, d). These results suggested that T. vaginalis induces apoptosis in SiHa cells in a MOI- and infection time-dependent manner.
Trichomonas vaginalis induced mitochondrial apoptosis in SiHa cells. SiHa cells were infected with live T. vaginalis trophozoites at MOI 2 and 5 for 0, 2 or 6 h. a PARP and caspase-3 proteins were assessed using western blotting analysis, and anti-β-actin was used as a loading control. Lanes: Tv only, T. vaginalis parasite protein without SiHa cells; M, protein marker; CTL, untreated control SiHa cells; Tv, SiHa cells infected with live T. vaginalis. b Ratio of apoptosis was measured and analyzed by Annexin V-FITC/PI staining and flow cytometry. Cells that are considered to be viable are both FITC Annexin V and propidium iodide (PI) negative; cells that are necrotic cells are FITC Annexin V negative and PI positive; cells that are in early apoptosis are FITC Annexin V positive and PI negative; and cells that are in late apoptosis or already dead are are both FITC Annexin V and PI positive. Data are presented as the means ± SD, and asterisks indicate significant difference (***P < 0.001) compared to the control group. c SiHa cells were pretreated with various concentrations of an ROS scavenger (NAC) for 2 h, and subsequently infected with T. vaginalis at MOI 5 for 6 h. PARP and cleaved caspase 3 protein levels were assessed by western blotting. d JC-1 staining was observed by confocal fluorescence microscopy. In JC-1-stained cells, red fluorescence is visible in cells with high mitochondrial membrane potential, while green fluorescence of JC-1 monomer is present in cells with low mitochondrial potential. e SiHa cells were pretreated with various concentration of NAC for 2 h and subsequently infected with T. vaginalis at MOI 5 for 6 h. JC-1 staining was observed using confocal fluorescence microscopy. Scale bars: 10 µm. Data shown are representative of three independent experiments with similar results
Based on the results shown in Fig. 1d, e, we evaluated whether ROS are involved in the induction of apoptosis in T. vaginalis-infected SiHa cells. We therefore pretreated cells with the ROS scavenger NAC and then evaluated the expression of apoptosis-related proteins and MMP. We first determined the cytotoxic effects of NAC on cell viability using the MTS assay. Treatment of the SiHa cells with 0.2–1 mM NAC for 6 h elicited no significant differences in cell viability compared with cells in the medium only-treated group; however, cells treated with 5 mM NAC for 6 h showed slightly reduced viability (see Additional file 2: Figure S2a). Both cellular and mitochondrial ROS production were significantly suppressed in the T. vaginalis-infected cells following NAC pretreatment in a concentration-dependent manner (see Additional file 3: Figure S3a, b).
As shown in Fig. 2c, in the NAC-pretreated SiHa cells, T. vaginalis-induced cleavages of PARP and caspase-3 were significantly suppressed in a dose-dependent manner. The permeabilization of the mitochondrial outer membrane is a crucial step in the progression of mitochondrial apoptosis [6, 7]; thus, we also investigated whether the T. vaginalis-induced ROS-dependent apoptosis in SiHa cells was related to the mitochondrial apoptotic pathways. After T. vaginalis infection, JC-1 staining, an indicator of MMP [25], revealed a notable decrease in MMP levels, as indicated by the reduction in red fluorescence signal and an increase in green JC-1 fluorescence signal, in a parasite burden- and infection time-dependent manner compared with the signals in untreated control cells (Fig. 2d). However, these changes in JC-1 dye fluorescence were suppressed by NAC pretreatment in a dose-dependent manner (Fig. 2e). Collectively, these results suggested that T. vaginalis induces mitochondrial apoptosis through ROS and that ROS act upstream of PARP and caspase-3 during apoptosis induction.
Trichomonas vaginalis-induced ER stress response and ER stress-mediated apoptosis in SiHa cells
Endoplasmic reticulum stress is known to contribute to apoptosis [19, 20]. To check whether T. vaginalis causes ER stress in SiHa cells, we first measured the activities of three major ER stress sensor proteins in T. vaginalis-infected SiHa cells. As shown in Fig. 3a and Additional file 1: Figure S1b, the expression levels of phosphorylated IRE1α and PERK as well as of cleaved ATF6 were significantly increased in a parasite burden- and infection time-dependent manner. The downstream cascades of PERK, including eIF2α, ATF4 and CHOP, were also activated by T. vaginalis infection. Caspase-12 was consistently activated, whereas the protein level of procaspase-12 was decreased. In addition, Ero1-Lα, a luminal glycoprotein that plays a role in the formation of disulfide bonds of secreted proteins and membrane proteins [18], was also upregulated in response to T. vaginalis infection.
Trichomonas vaginalis-induced ER stress response and ER stress-dependent apoptosis in SiHa cells. a SiHa cells were infected with T. vaginalis under the indicated conditions, and ER stress-related proteins were evaluated by western blotting analysis. b, c SiHa cells were pretreated with various concentrations of an ER stress inhibitor (4-PBA) for 2 h and subsequently infected with T. vaginalis at MOI 5 for 6 h. The ER stress-related (b) and apoptosis-related (c) protein levels were assessed using western blotting analysis. Anti-β-actin was used as an internal control. The data shown are representative of three independent experiments with similar results
Next, to confirm whether T. vaginalis-induced apoptosis is mediated by ER stress in SiHa cells, the cells were pretreated with the ER stress inhibitor 4-phenylbutyric acid (4-PBA). 4-PBA is a low-molecular-weight fatty acid that prevents misfolded protein aggregation by interacting with hydrophobic regions in unfolded proteins [26]. There was no difference in cell viability between the SiHa cells treated with 2 mM 4-PBA for 6 h and cell in the untreated control group (see Additional file 2: Figure S2b). The expressions of ER stress-related proteins were reversed in the 4-PBA-pretreated T. vaginalis-infected SiHa cells in a dose-dependent manner as compared with those in the untreated T. vaginalis-infected cells (Fig. 3b). Surprisingly, 4-PBA pretreatment attenuated the levels of cleaved PARP and caspase-3 in T. vaginalis-infected SiHa cells in a dose-dependent manner (Fig. 3c). These results demonstrated that T. vaginalis induces ER stress response as well as ER stress-mediated apoptosis in SiHa cells.
Trichomonas vaginalis-induced ROS-dependent ER stress responses in SiHa cells
Endoplasmic reticula and mitochondria are closely related, both structurally and functionally [20]. Thus, to evaluate the effects of mitochondrial ROS in the induction of ER stress response in T. vaginalis-infected SiHa cells, we pretreated the SiHa cells with NAC and investigated the expression of ER stress-related proteins. Pretreatment with NAC significantly reduced the expression of Ero1-Lα, p-IRE1α, CHOP, p-PERK, p-elF2α, ATF4, and cleaved ATF6 in the T. vaginalis-infected SiHa cells in a dose-dependent manner, whereas the expression level of procaspase-12 was increased (Fig. 4a). Confocal microscopy imaging also confirmed that NAC pretreatment reduced T. vaginalis-induced upregulation of CHOP [also known as DNA damage-inducible gene 153 (GADD153)] in a dose-dependent manner (Fig. 4b). These results indicated that T. vaginalis induces ER stress response through mitochondrial ROS in SiHa cells, which leads to apoptosis.
Trichomonas vaginalis-induced ROS-dependent ER stress response in SiHa cells. SiHa cells were pretreated with various concentrations of NAC for 2 h and subsequently infected with T. vaginalis at MOI 5 for 6 h. a The ER stress-related protein levels were assessed using western blotting analysis. Anti-β-actin was used as a loading control. b Representative images obtained by confocal fluorescence microscopy stained with CHOP antibody. The data shown are representative of three independent experiments with similar results. Scale bars: 10 µm
Trichomonas vaginalis-induced ER stress-mediated mitochondrial dysfunction in SiHa cells
We also investigated whether ER stress affects mitochondrial functions in T. vaginalis-infected SiHa cells. Members of the Bcl-2 family proteins are major regulators of mitochondrial integrity and mitochondria-dependent caspase activation, and MMP is the key indicator of mitochondrial apoptosis [6, 7, 25]. Therefore, we investigated mitochondrial functions, including alternation of MMP and Bcl-2 family members, in T. vaginalis-infected SiHa cells. JC-1 dye staining revealed that T. vaginalis induced MMP depolarization in the SiHa cells; however, these changes in fluorescence were suppressed by 4-PBA pretreatment (Fig. 5a).
Trichomonas vaginalis-induced ER stress-dependent mitochondrial dysfunction in SiHa cells. a SiHa cells were pretreated with various concentrations of 4-PBA for 2 h and subsequently infected with T. vaginalis at MOI 5 for 6 h. SiHa cells were stained with JC-1, and fluorescence was detected under a confocal microscope. The figure shows representative confocal images of JC-1 aggregate (red) and monomer (green) fluorescence, respectively. b, c SiHa cells were infected with T. vaginalis at MOI 2 and 5 for 6 h. Pro-apoptosis Bcl-2 (b) and pro-survival Bcl-2 (c) family members were evaluated by western blotting. d, e SiHa cells were pretreated with various concentrations of 4-PBA for 2 h and subsequently infected with T. vaginalis at MOI 5 for 6 h. Pro-apoptosis Bcl2 (d) and pro-survival Bcl-2 (e) family members were evaluated by western blotting. Anti-β-actin was used as a loading control. The data shown are representative of three independent experiments with similar results. Scale bars: 10 µm
The expressions of p-Bad (Ser112), Bax, Bak, Bik and Puma were significantly increased in the T. vaginalis-infected SiHa cells (Fig. 5b), whereas the Bcl-xL and Mcl-1 levels were decreased in a parasite burden-dependent manner (Fig. 5c). The expression levels of p-Bad (Ser 112), Bax, Bak Bik, and Puma were significantly decreased in the 4-PBA-pretreated T. vaginalis-infected SiHa cells in a dose-dependent manner (Fig. 5d), whereas the levels of Bcl-xL and Mcl-1 proteins were increased (Fig. 5e).
These data indicated that MMP depolarization and Bcl-2 family protein imbalance were recovered in T. vaginalis-infected SiHa cells after treatment with 4-PBA. Thus, our results clearly indicated that T. vaginalis induces mitochondrial dysfunction via ER stress in SiHa cells, leading to apoptosis.
Trichomonas vaginalis-induced ER stress-mediated mitochondrial apoptosis via the IRE1/ASK1/JNK/Bcl-2 family protein pathways in SiHa cells, dose-dependently
Previous studies have reported that the IRE1/ASK1/JNK signaling cascade is one of the main downstream targets for the regulation of ER stress-induced cell apoptosis [19, 20]. Therefore, we investigated whether T. vaginalis activates ER stress-dependent mitochondrial apoptosis through the ASK1/JNK pathway in SiHa cells. We found a substantial increase in the phosphorylation levels of both ASK1 (Ser83, Ser967 and Thr845) and JNK in the T. vaginalis-infected SiHa cells in a parasite burden- and infection time-dependent manner (Fig. 6a). First, we investigated whether ASK1/JNK activation was related to ER stress in the T. vaginalis-infected SiHa cells. Western blotting analysis revealed that 4-PBA pretreatment attenuated the T. vaginalis-induced elevation of ASK1 and JNK phosphorylation levels in a dose-dependent manner (Fig. 6b). These data indicated that the T. vaginalis-induced ER stress activates the ASK1/JNK pathway in SiHa cells.
Trichomonas vaginalis-induced ES stress-dependent mitochondrial apoptosis via ASK1/JNK pathways in SiHa cells. a SiHa cells were infected with T. vaginalis at the indicated MOI for 6 h or at MOI 5 for the indicated time. Cells were lysed, and the ASK1/JNK phosphorylation levels were assessed by western blot analysis. b SiHa cells were pretreated with various concentrations of 4-PBA for 2 h and subsequently infected with T. vaginalis at MOI 5 for 6 h. ASK1/JNK phosphorylation levels were assessed by western blot analysis. c–e SiHa cells were pretreated with various concentrations of a JNK inhibitor (SP600125) for 2 h and subsequently infected with T. vaginalis at MOI 5 for 6 h. The protein levels of p-JNK, JNK, Bak, Bax, PARP and cleaved caspase 3 (c) and p-Bcl-2 (Ser70), Bcl-2, Bcl-xL and Mcl-1 (d) were assessed using western blotting analysis. Anti-β-actin was used as a loading control. JC-1 staining was observed by confocal imaging (e). All the results presented are representative of three independent experiments with similar results. Scale bars: 10 µm
Next, we investigated whether the ASK1/JNK/Bcl-2 family protein pathway is associated with mitochondrial apoptosis in T. vaginalis-infected SiHa cells. SiHa cells were preincubated with the JNK inhibitor SP600125, and then MMP, Bcl-2 family proteins and apoptosis-related proteins were investigated. The SiHa cells treated with 30 μM SP600125 for 6 h did not significantly differ from cells in the untreated control group in terms of cell viability (see Additional file 2: Fig. S2c). Western blotting analysis revealed that Bak, Bax, cleaved PARP and caspase-3 levels were decreased in a dose-dependent manner in the SP600125-pretreated T. vaginalis-infected SiHa cells (Fig. 6c). In contrast, Bcl-xL and Mcl-1 levels were increased by pretreatment with SP600125 in the T. vaginalis-infected SiHa cells in a dose-dependent manner (Fig. 6d). In addition, SP600125 pretreatment substantially increased MMP levels in the T. vaginalis-infected SiHa cells, as demonstrated by increased red and reduced green JC-1 fluorescence, in a dose-dependent manner (Fig. 6e). These observations provided evidence that T. vaginalis induces ER stress-mediated mitochondrial apoptosis via the IRE1/ASK1/JNK/Bcl-2 family protein signaling pathways in SiHa cells.
Trichomonas vaginalis excretory/secretory product (ESP) induced mitochondria ROS, apoptosis and ER stress response in SiHa cells
Trichomonas vaginalis is an extracellular protozoan parasite; it adheres to host cells and hydrolyzes organic components of host cells for survival, which is its mode of infection and pathogenesis [27,28,29]. There have been several reports of the ESP of T. vaginalis having cytotoxic effects on host cells [12, 14, 30]. Thus, we evaluated whether T. vaginalis ESP can induce the mitochondrial ROS production, ER stress response and apoptosis in SiHa cells. Cellular ROS levels were significantly increased in SiHa cells by T. vaginalis ESP from 2 h after treatment and increased further at 6 h after treatment (Fig. 7a, b). Similarly, mitochondrial ROS levels were also significantly increased after treatment with T. vaginalis ESP in an incubation time dependently (Fig. 7c, d).
Trichomonas vaginalis ESP induced apoptosis and ER stress response and generated mitochondrial ROS production in SiHa cells. SiHa cells were treated with 100 μg/ml T. vaginalis ESP for 0, 2 or 6 h. a, b Cellular ROS production was measured by confocal microscopy (a) and flow cytometry (b) with the CellROX oxidative stress reagent, a fluorogenic probe for measuring oxidative stress in live cells. Plots depict the CellROX-positive cells as determined by fluorescence analysis of flow cytometry. Asterisks indicate significant difference (***P < 0.001) in mean fluorescence compared with the untreated control cells under the same conditions. c, d Mitochondrial ROS production was determined by confocal microscopy (c) and flow cytometry (d) with MitoSOX, a mitochondrial ROS dye. Plots depict the MitoSOX-positive cells as determined by fluorescence analysis of flow cytometry. Data shown are representative of three independent experiments with similar results. Asterisks indicate significant difference (***P < 0.001) in mean fluorescence compared with the untreated control cells under the same conditions. e Apoptosis- and ER stress-related protein expression levels were evaluated by western blot. The data shown are representative of three independent experiments with similar results. Scale bars: 10 µm. Abbreviations: CTL, Untreated control SiHa cells; ESP, SiHa cells treated with T. vaginalis ESP
Next, we evaluated the expressions of apoptosis- and ER stress-related proteins in SiHa cells after treatment with T. vaginalis ESP. The protein levels of cleaved PARP and caspase 3 significantly increased in cells from 2 h after T. vaginalis ESP treatment, and further increased at 6 h post-treatment (Fig. 7e). Similarly, the protein levels of p-IRE1α, CHOP, p-PERK and cleaved ATF6 were significantly increased in cells after treatment with T. vaginalis ESP, incubation in a time-dependent manner (Fig. 7e). The results suggest that ESP from live T. vaginalis induce mitochondrial ROS production, apoptosis and ER stress response in the SiHa cells in an incubation time dependent manner.
Trichomonas vaginalis primarily causes lesions in the vagina and cervix in women [1]. SiHa is a human cervical cancer cell line that is used as an in vitro model for cervical cancer and cervicovaginal infection, such as papilloma virus [31], Candida [32] and Trichomonas infection [9, 12]. Thus, we used the SiHa cell line as a host cell to investigate the pathogenesis of human trichomoniasis. In this study, we investigated the involvement of ER stress in apoptosis induction in T. vaginalis-infected SiHa cells and its molecular mechanisms. Trichomonas vaginalis induced mitochondrial apoptosis through ROS in SiHa cells and also induced ER stress response directly and in an ROS-dependent manner in SiHa cells. In addition, Trichomonas vaginalis induced ER stress-mediated apoptosis in SiHa cells through the PERK/CHOP and IRE1/ASK1/JNK/Bcl-2 family protein pathways. This study is the first to demonstrate ER stress involvement in apoptosis induction in T. vaginalis-infected mammalian cells via the ROS–ER stress–mitochondrial apoptosis pathways.
We and other researchers have previously reported that T. vaginalis induces apoptosis in various cells [8,9,10,11,12,13,14] and that ER stress is involved in apoptosis induction via various agents, including some pathogens [21,22,23]. In this study, we also confirmed the induction of mitochondrial apoptosis in T. vaginalis-infected SiHa cells by western blotting and flow cytometry. We then checked whether T. vaginalis infection induces ER stress responses in SiHa cells. Perturbation of the physiological status of the ER triggers an ER-related stress response or UPR, which is distinguished by the action of three signaling proteins: IRE1α, PERK and ATF6 [18, 19]. We found that T. vaginalis infection increased the protein levels of p-IRE1α, p-PERK, ATF4, cleaved ATF6 and CHOP and decreased the levels of procaspase-4 and procaspase-12 in SiHa cells. These results demonstrate that T. vaginalis has the capacity to induce an ER stress response in SiHa cells. However, pretreatment with the ER stress inhibitor 4-PBA was found to significantly attenuate the levels of ER stress-related proteins in T. vaginalis-infected cells. Furthermore, 4-PBA pretreatment attenuated the cleavages of PARP and caspase-3 proteins in T. vaginalis-infected SiHa cells in a dose dependent manner. These findings indicate that ER stress and apoptosis interact with each other and that ER stress plays a pivotal role in apoptosis induction in T. vaginalis-infected SiHa cells, similar to that observed in Toxoplasma gondii-infected neural cells [33] and Leishmania infantum-infected macrophages [34]. ER stress-mediated apoptosis may be caused by activation of the ER-specific cysteine protease caspase-12 or transcription factor CHOP [18, 19].
Having established the involvement of ER stress in apoptosis induction in T. vaginalis-infected SiHa cells, we subsequently examined the role of ROS in promoting ER stress. Several studies have shown that ROS generation is one of the most critical factors for apoptosis induction in various mammalian cells infected with pathogens [8, 9, 21]. ROS-dependent caspase-3 activation plays an important role in the induction of apoptosis induction of human neutrophils induced by T. vaginalis [8]. It has been shown that the ER communicates with mitochondria via calcium and ROS signaling [19, 20]. In the present study, T. vaginalis induced mitochondrial ROS and ER stress responses in SiHa cells, and pretreatment with NAC or 4-PBA significantly suppressed mitochondrial ROS production, along with the expression levels of apoptosis- and ER stress-related proteins, in T. vaginalis-infected cells. These observations provide evidence of apoptotic crosstalk signaling between the ER and mitochondria via ROS, thereby indicating that T. vaginalis induces both ROS-dependent mitochondrial apoptosis and ROS-dependent ER stress-mediated apoptosis in SiHa cells. Similar findings were also reported in previous studies, in which Mycobacterium avium was shown to induce apoptosis via an ROS-dependent ER stress response in macrophages [21], and silibinin, a biologically active compound of milk thistle, was found to induce ER stress-mediated mitochondrial ROS-dependent apoptosis in prostate cell lines [35].
ER and mitochondria have been identified as key players in the sensing and perception of cellular stress [20]. Based on the aforementioned observations, we proceeded to investigate whether ER stress mediates mitochondrial apoptotic pathways in T. vaginalis-infected SiHa cells. Mitochondria and the ER play important roles in the regulation and transmission of apoptotic signals, with the regulatory mechanisms determined by a balance among Bcl-2-family proteins [20, 36]. Under severe and sustained ER stress conditions, the intrinsic apoptotic pathway can be activated by the IRE1-mediated activation of tumor necrosis factor receptor-associated factor 2 (TRAF2), which in turn stimulates the ASK1/JNK kinase cascade [19, 37]. In the present study, we found that T. vaginalis induced ASK1 and JNK phosphorylation in SiHa cells, whereas pretreatment with either 4-PBA or SP600125 contributed to a reduction in the levels of p-IRE1α, cleaved caspase-3, cleaved PARP, p-ASK1 and p-JNK proteins in T. vaginalis-infected SiHa cells. In addition, pretreatment with 4-PBA or SP600125 was found to enhance MMP, Bcl-xl, and Mcl-1 protein levels but reduce Bax and Bak protein levels in the infected cells. These results indicate that T. vaginalis induces apoptosis through ER stress-mediated mitochondrial dysfunction in SiHa cells via the IRE1/ASK1/JNK/Bcl-2 family protein pathways, and finally led to apoptosis. These findings can be explained in part by the fact that Bax and Bak are localized to both the mitochondria and ER and that their overexpression promotes apoptotic calcium mobilization during ER-mediated mitochondrial cell death [38]. A similar apoptotic mechanism has been proposed for endothelial cells treated with silica nanoparticles, in which apoptosis was induced via ER stress-associated IRE1/JNK signaling to the mitochondria mediated by Bcl-2 family members [39].
We also considered the cytopathogenic factors in T. vaginalis-infected cells. Trichomonas vaginalis is an extracellular protozoan parasite that produces a variety of proteases, such as cysteine proteases and metalloproteinases, which are important regulators of pathogenesis, invasion and survival [10,11,12, 27,28,29]. In previous studies, we and other researchers have reported that the ESP of live T. vaginalis, which comprises proteases, play critical roles in the induction of apoptosis in T. vaginalis-infected cells [9, 12, 14]. In the present study, we confirmed the induction of mitochondrial ROS production, apoptosis and ER stress responses in SiHa cells after treatment with T. vaginalis ESP, the results of which were consistent with those observed in live T. vaginalis-infected cells. We assume that these effects can be attributed to the interaction between T. vaginalis ESP and the surface proteins of host cells, which subsequently induce a range of pathogenetic events, such as ROS production, ER stress response and cell death [12, 14, 30]. However, the findings of a few studies have indicated that in human trichomoniasis, the death of host cells can also proceed via necrosis rather than the induction of apoptosis [28], and the difference between these results could be attributed to differences in the cell lines used, parasite strains, adherence ability and/or culture conditions.
Our results showed that live T. vaginalis induced apoptosis through mitochondrial ROS and ER stress responses and promoted the ER stress response both directly and in an ROS-dependent manner in human cervical cancer SiHa cells, dependent on the parasite burden and infection time. In addition, T. vaginalis induced ER stress-mediated mitochondrial dysfunction and finally led to apoptosis of SiHa cells through the IRE1/ASK1/JNK/Bcl-2 family protein pathways. Trichomonas vaginalis ESP also promoted mitochondrial ROS production, apoptosis and the ER stress response in SiHa cells in an incubation time-dependent manner. Collectively, our results suggest that T. vaginalis infection induces apoptosis as well as the ER stress response via ROS–ER stress-mitochondrial apoptosis pathways by interacting with the mitochondria and ER in human cervical cancer SiHa cells, and that ER stress is one of the major factors contributing to the apoptosis of T. vaginalis-infected cells. These pathogenesis mechanisms may be associated with the ESP released from live T. vaginalis (Fig. 8). Our findings will potentially contribute to enhancing our current understanding of the molecular mechanisms underlying the pathogenesis of human trichomoniasis.
Schematic model of apoptosis by T. vaginalis in SiHa cells. Trichomonas vaginalis induces apoptosis through mitochondrial ROS and ER stress response, and also induces ROS-dependent ER stress response and ER stress-mediated mitochondrial dysfunction through the IRE1/ASK1/JNK/Bcl-2 family protein pathways, finally leading to apoptosis of the SiHa cells. In addition, T. vaginalis ESP also exerted mitochondrial ROS production, apoptosis and ER stress response in SiHa cells. Collectively, T. vaginalis infection induces apoptosis via ROS–ER stress-mitochondrial apoptosis pathways by interaction with mitochondria and ER in human cervical cancer SiHa cells, and this pathogenesis may be involved in the ESP released from live T. vaginalis
All datasets generated during the present study are included in the published article including the Additional files.
ASK1:
Apoptosis signal regulating kinase 1
ATF:
Activating transcription factor
CHOP:
CCAAT/enhancer-binding protein–homologous protein
ESP:
Excretory/secretory products
ER:
IRE1:
Inositol-requiring enzyme 1
JC-1:
5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide
JNK:
C-Jun N-terminal kinases
Lactate dehydrogenase
MMP:
MOI:
Multiplicities of infection
MTS:
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt
NAC :
PARP:
Poly (ADP‐ribose) polymerase
4-PBA:
4-Phenylbutyric acid
PERK:
Protein kinase RNA-like ER kinase
ROS:
TRAF2:
Tumor necrosis factor receptor-associated factor 2
UPR:
Unfolded protein response
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This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (grant NRF-2019R1A2C1088346) at Chungnam National University, and the National Natural Science Foundation of China (grant 81971389), the Science Foundation of Guangdong Medical University (grant GDMUZ201801) and the Natural Science Foundation of Guangdong Province (grant no. 2019A1515011888).
Fei Fei Gao, Juan-Hua Quan and Min A. Lee contributed equally to this work
Brain Korea 21 FOUR Project for Medical Science, Chungnam National University College of Medicine, Daejeon, 35015, Korea
Fei Fei Gao, Jae-Min Yuk & Young-Ha Lee
Department of Medical Science and Department of Infection Biology, Chungnam National University College of Medicine, 6 Munhwa-dong, Jung-gu, Daejeon, 35015, Korea
Fei Fei Gao, Jae-Min Yuk, Guang-Ho Cha, In-Wook Choi & Young-Ha Lee
Department of Gastroenterology, The Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China
Juan-Hua Quan
Department of Obstetrics and Gynecology, Chungnam National University, DeaJeon, 35015, Korea
Min A. Lee
Department of Obstetrics and Gynecology, The Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China
Wei Ye
Fei Fei Gao
Jae-Min Yuk
Guang-Ho Cha
In-Wook Choi
Young-Ha Lee
FFG, JHQ, MAL and YHL designed and conceived the experiments. FFG, JHQ, MAL, WY, JMK, GHC, IWC and YHL carried out the experiments. FFG, JHQ, MAL, WY, JMK, GHC, IWC and YHL performed the data analysis. FFG, JHQ, MAL and YHL drafted and revised the manuscript. All authors read and approved the final manuscript.
Correspondence to Young-Ha Lee.
Additional file 1: Figure S1
. Trichomonas vaginalis-induced cell cytotoxicity and ER stress in SiHa cells. SiHa cells were infected with T. vaginalis at various MOIs (1, 2, 5 and 10) for the indicated times (0, 0.5, 2, 6, 12 and 24). a The percentages of LDH-dependent cytotoxicity in the medium was measured by LDH assay. The data represent the mean value ± standard deviation (SD) of at least three independent experiments. Asterisks indicate significant difference (**P < 0.01, ***P < 0.001) compared with untreated control cells under the same conditions. b The levels of apoptosis- and ER stress-related protein were measured by western blot, and anti-β-actin was used as a loading control. c Equal volume without quantification at each lane after collecting the T. vaginalis-infected SiHa cells. The expected protein loss which might be caused by apoptosis was not observed until 24 h after infection. The β-actin levels in the whole cell lysate from samples loaded with equal volume did not show any significant difference from samples loaded with equal protein amount (Fig. S1c vs. Fig. S1b)
. Effects of various concentrations of NAC (ROS scavenger), 4-PBA (ER stress inhibitor) and SP600125 (JNK1/2 inhibitor) on the viability of human cervical cancer SiHa cells. SiHa cells treated with 0.2, 1 and 5 mM of NAC (a), 0.2, 1 and 2 mM of 4-PBA (b) and 0.3, 3 and 30 µM of SP600125 (c), in 5% CO2 at 37°C for 0, 2, 6, 12 and 24 h. Cell viability was checked by the MTS assay. The data represent the mean ± SD of at least three independent experiments
. Trichomonas vaginalis-induced ROS production was suppressed with NAC pretreatment in SiHa cells. SiHa cells were pretreated with different concentrations of NAC and then infected with T. vaginalis at MOI 5 for 6 h. a Cellular ROS production was detected by CellROX. Percentages of CellROX-positive cells, as determined by densitometric analysis of fluorescence images. b Mitochondrial ROS production was detected by MitoSOX reagent. Percentages of MitoSOX positive cells, as determined by densitometric analysis of fluorescence images. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P < 0.001) compared with the untreated control cells. The data shown are representative of three independent experiments with similar results. Scale bars: 10 µm.
Gao, F.F., Quan, JH., Lee, M.A. et al. Trichomonas vaginalis induces apoptosis via ROS and ER stress response through ER–mitochondria crosstalk in SiHa cells. Parasites Vectors 14, 603 (2021). https://doi.org/10.1186/s13071-021-05098-2
Human cervical cancer SiHa cells
Endoplasmic reticulum stress
Protozoa and protozoan diseases
Spatial inequality, infectious diseases and disease control


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