1.1 History of Cancer
Human beings have had cancers throughout the recorded history. The earliest evidence of cancer is found among the fossilized bone tumours, human mummies in ancient Egypt, and ancient manuscripts. Bone remains of mummies have revealed growths suggestive of the bone cancer, osteosarcoma. Bony skull destruction as seen in cancer of the head and neck has been found, too. Our oldest description of cancer (although the word cancer was not used) was discovered in Egypt and dates back to about 1600 B.C. It is called the Edwin Smith Papyrus, and is a copy of part of an ancient Egyptian textbook on trauma surgery. It describes 8 cases of tumours or ulcers of the breast that were treated by cauterization, with a tool called the fire drill (Kardinal and Yarbro, 1979). A century is only a small segment in the timeline measuring the history of science through the ages, but for cancer research, the last 100 years overshadow all of the years that came before. Cancer is a generic term for a group of more than 100 diseases. The origin of word 'cancer' is derived from the Greek word crab, most likely applied to the disease because the finger-like spreading projections observed in a carcinoma brought to mind the shape of a crab (Diamondopolous, 1996). Hippocrates used the terms carcinos and carcinoma to describe non-ulcer forming and ulcer-forming tumours. The Roman physician, Celsus (28-50 B.C.), later translated the Greek term into cancer, the Latin word for crab. However, the word cancer has no scientific meaning in the nomenclature of diseases. Galen (130-200 A.D.), another Roman physician, used the word oncos (Greek for swelling) to describe tumours. Although the crab analogy of Hippocrates and Celsus is still used to describe malignant tumours, Galen's term is now used as a part of the name for cancer specialists - oncologists.
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Humoral theory: The earliest theory of cancer was based on the humoral theory of disease articulated by Hippocrates (460-370 BC). Hippocrates believed that the body had 4 humors (body fluids) -- blood, phlegm, yellow bile, and black bile. When the humors were balanced, a person was healthy. Too much or too little of any of them caused disease. An excess of black bile in various body sites was thought to cause cancer. This theory of cancer was passed on by the Romans and was embraced by the influential doctor Galen. Galen's humoral theory (130-200AD) of the causation of cancer which lasted for thousands of years and it postulated that cancer was caused due to solidification of excess of black bile in certain parts of the body.
Lymph theory: The theory that replaced the humoral theory of cancer was the lymph theory. Stahl and Hoffman theorized that cancer was composed of fermenting and degenerating lymph varying in density, acidity, and alkalinity. The eminent surgeon John Hunter (1723-1792) agreed that tumours grow from lymph constantly thrown out by the blood. The work of Le Dran (1685-1770) was an important landmark in the evolution of oncology which stated that cancer starts as a local disease and it later spreads through the lymphatic vessels to lymph nodes and eventually into the general circulation (Galluci, 1985).
Parasite theory: In the 17th and 18th centuries, some researchers believed that cancer was contagious. In fact, the first cancer hospital in France was forced to move from the city in 1779 because of the fear of the spread of cancer throughout the city (Plimmer, 1903).
Blastema theory: In 1838, German pathologist Johannes Muller demonstrated that cancer is made up of cells and not lymph, but he believed that cancer cells did not arise from normal cells. Muller proposed that cancer cells arose from budding elements (blastema) between normal tissues. His student, Rudolph Virchow (1821-1902), the famous German pathologist, later determined that all cells, including cancer cells, are derived from other cells (Raven, 1990).
Chronic irritation theory: Rudolph Virchow proposed that chronic irritation was the cause of cancer, but he falsely believed that cancers "spread like a liquid." A German surgeon, Karl Thiersch, showed that cancers metastasize through the spread of malignant cells and not through some unidentified fluid (Raven, 1990).
Trauma theory: Despite advances in the understanding of cancer, from the late 1800s until the 1920s, trauma was thought by some to cause cancer. This belief was maintained despite the failure of injury to cause cancer in experimental animals.
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It was after the 1920s that various chemotherapeutic drugs, carcinogens and various advanced means of diagnostic techniques have been developed. Chemotherapy and Radiation are extensively used in the treatment of cancer from the past few decades. Though not all cancers are treated but at least some are. Hence, it is evident that the efforts put on the research across the past centuries have revolutionized the understanding of cancer for us today.
1.2 Central Nervous system:
The central nervous system (CNS) consists of spinal cord and brain. The spinal cord is present in vertebral canal and is made up of white matter outside and grey matter inside. The spinal cord conducts sensory information from the nerves all over the body to the brain as well as conducts motor information via impulses from the brain to various effectors in body such as skeletal, cardiac, smooth muscles and glands. Spinal cord also, plays a role as a minor centre of various reflexes in the body (Brooker, 1997).
The brain is present inside the cranial cavity and is made up of white matter outside and grey matte inside. The brain receives sensory impulses from spinal cord and its own 12 cranial nerves, which it processes to initiate appropriate motor responses in co-ordinated manner. Brain and spinal cord is protected by three protective layers of connective tissue, they are - duramater forms outer, arachnoid forms middle and pia mater forms inner layer. Cerebrospinal fluid (CSF) is present throughout the central nervous system (Brooker, 1997).
Brain consists of three main parts - Forebrain (Prosencephalon), Midbrain (Mesencephalon) and Hindbrain (Rhombencephalon). The forebrain consists of cerebral hemispheres (telencephalon) and Diencephalon. The left cerebral hemisphere deals with right side of the body and vice-versa. Each cerebral hemisphere consists of frontal, parietal, occipital and temporal lobes. The diencephalon is made up of thalamus, lateral geniculate nucleus (LGN), hypothalamus and posterior lobe of pituitary. The thalamus acts as a relay station for all the information transmitted from the lower parts of CNS to the cerebral cortex while LGN receives and processes impulses from the optic nerve prior to cerebral cortex. The hypothalamus consists of autonomic nervous system and thus controlling normal homeostasis of body and plays inevitable role in release of hormones in blood. The posterior pituitary secretes two hormones in blood released from the hypothalamus (Brodal, 2005).
The Midbrain is relatively small in human brain and together with medulla oblongata and pons, forms brainstem. Hindbrain consists of 3 structures - Medulla oblongata, pons and cerebellum (Figure 1). The medulla oblongata contains respiratory, vasomotor and cardiac centres and is a junction for crossover of motor tracts from spinal cord and brain. Pons is a bridge-like structure relays impulses from parts of cerebral cortex to the cerebellum. Pons also plays role in respiratory reflexes (Brooker, 1997). The cerebellum is made up of two convoluted hemispheres and it plays major role in the co-ordinated motility of a person. It is a center for learning skills and thus contributes to the process of cognition (Brodal, 2005).
Despite increase in the knowledge about cancer, and improvement in treatment strategies and diagnostic techniques, it still remains the leading cause of death worldwide (World Health Organisation (WHO), 2008) and accounted for 7.9 million deaths in 2007. In the UK, cancer is responsible for 126,000 deaths per year (NHS, 2008). Moreover, cancer mortality is expected to continue rising, with an estimated 12 million deaths worldwide in 2030 (WHO, 2008). It is estimated that 1,437,180 Americans will be diagnosed with cancer and 565,650 of them will die of cancer of all sites in 2008 (American Cancer Society (ACS), 2008).
Gliomas are a heterogenous group of neoplasm that comprise the majority of tumours originating in the central nervous system (CNS). Brain tumours are masses or growth of abnormal cells in the brain and typically are categorized as either primary or secondary (Burton & Prados, 2000). Primary brain tumours comprise about 2% of all newly diagnosed cancers every year in the UK (Greenlee et al., 2000). In the USA, about 7000 cases of malignant brain tumours were newly diagnosed in 1987 (Kimmel et al., 1987). In American's Cancer Society surveillance in 2007, it was found that almost 20,500 new cases of malignant brain tumours were diagnosed, whilst in the UK the average annual incidence of cerebral glioma in the age range 15-64 years was 5.9 per 100,000 per year (Grant et al., 1996). In other European countries such as Switzerland, glioblastomas has an incidence with 3.55 new cases per 100,000 population per year (Kleihues & Ohgaki, 2007).
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2.1 Brain tumours
Brain tumours are masses or growth of abnormal cells in the brain and typically are categorized as either primary or secondary. Those which originate in the brain are called as primary tumours while secondary tumours are formed when cancer cells from other parts of the body, such as the lung or breast, spread to the brain (Aminoff, 2004).
Primary brain tumours originate in the brain and can be non-cancerous (benign) or cancerous (malignant) and are less common as compared to secondary brain tumours. However, unlike cancers elsewhere in the body, primary malignant brain tumours rarely spread from the brain (Aminoff, 2004). Many primary brain tumours are benign, which means that they remain in the part of the brain in which they started and do not spread into and destroy other areas of the brain and other parts of the body. If a benign tumour can be removed successfully it does not cause any further problems. Successful removal of benign tumour also depends upon the location of the tumour. Sometimes it is difficult to remove benign tumours as they might damage the surrounding sensitive brain cells (Figure 2). Malignant brain tumours grow more rapidly destroying surrounding brain cells. A brain tumor, primary or secondary can cause a variety of signs and symptoms because it can directly press on or invade brain tissue. The signs and symptoms include headaches that gradually become more frequent and more severe, new onset or change in pattern of headaches, difficulty with balance, personality or behavior changes, hearing problems, vision problems, such as blurred vision, double vision or loss of peripheral vision, confusion in everyday matters, hormonal (endocrine) disorders, unexplained nausea or vomiting, gradual loss of sensation or movement in an arm or a leg, speech difficulties etc.
Gliomas are the type of brain tumours that arise from glial cells in the brain. These heterogenous groups of neoplasms comprise the majority of tumours originating in the central nervous system (CNS) (Burton & Prados, 2000). Glial cells, which are the most common cells in the brain have various roles in supporting and protecting nerve cells like supplying energy and nutrients and also help to maintain the blood-brain barrier. Glial cells are of various types each with different functions. Astrocytes (Figure 3) transport nutrients and supports neurons in place, oligodendrocytes provides insulation (myelin) to neurons, microglia digests dead neurons and pathogens and Ependymal cells line the ventricles and secrete cerebrospinal fluid (Louis et al., 2001).
Figure 3: Diagram showing an astrocyte - a type of glial cell.
Depending on the cells of origin, there are three main types of glial tumours i.e. astrocytoma, oligodendroglioma and ependymoma (Louis et al., 2001). Gliomas vary in their aggressiveness, or malignancy. When the specific tumour diagnosis is made by the pathologist, the tumour is also "graded." This number grade is based on how normal - or abnormal - the tumour cells appear when examined under a microscope by the American brain tumour association.
Primary brain tumours originate in the brain and can either be non-cancerous (benign) or cancerous (malignant). Secondary brain tumours result from cancer that began elsewhere and has spread to the brain. Depending on the cells of origin, there are three main types of glial tumours i.e. astrocytoma, oligodendroglioma and ependymoma (Louis et al., 2001). In adults, the most frequently encountered of these are high-grade or malignant neoplasms of astrocytic and oligodendrocytic lineage (Burton & Prados, 2000). However, unlike cancers elsewhere in the body, primary malignant brain tumours rarely spread from the brain (Aminoff, 2004). Malignant gliomas are the most common primary CNS tumours (Zhu et al., 2005) and inspite of intensive clinical investigation and many therapeutic approaches, the treatment for primary brain tumours remain inadequate (Kim & Glantz, 2006). The WHO system further graded these, on the basis of histological degrees of malignancy with Grade I being the least malignant to Grade IV being the most malignant (Louis et al., 2001). The degree of malignancy is based on the presence or absence of increased cellularity, nuclear atypia, mitosis, endothelial proliferation and necrosis. Grade I and Grade II tumours are considered to be low-grade gliomas while Grade III and Grade IV tumours are high-grade gliomas. Low-grade tumours are usually circumscribed and grow slowly over a period of time while high-grade tumours are comparatively aggressive having poor prognosis. Some of the low-grade gliomas undergo malignant transformation to high-grade neoplasms with age, lifestyle and time. Glioblastoma multiforme [GBM] and anaplastic astrocytoma [AA] are the most malignant and aggressive high-grade glioma (WHO Grade IV and III respectively), having a combined incidence of 5-8/100,000 population (Avgeropoulos and Batchelor, 1999). In 2007, the WHO Classification of Tumours of the Nervous System revised the classification and added three new tumours (Table 2): angiocentric glioma (AG), pilomyxoid astrocytoma (PMA), and pituicytoma to the section on glioma (Louis et al., 2007; Brat et al., 2007).
Table 2: WHO Classification of Glioma (Taken from Louis et al., 2007).
Mixed and other common types of glioma
Astrocytes are star-shaped neuroglial cells that provide structural support for neurons and maintain electrolyte and neurotransmitter homeostasis in the brain. Astrocytoma is the tumour that develops from astrocytes in the CNS. The low grade astrocytomas i.e. grade I and II are less common than the high grade astrocytomas. Astrocytomas can be classified into two classes - those with narrow zones of infiltration which includes pilocytic astrocytoma, subependymal giant cell astrocytoma and pleomorphic xanthoastrocytoma. While the second class is those with diffuse zones of infiltration and it includes low-grade anaplastic astrocytomas and glioblastoma. Pilocytic astrocytomas correspond to WHO grade I and is common in children and youths. It contains compact bipolar cells exhibiting biphasic pattern alongwith Rosenthal fibres and microcysts and granular bodies (Kleihues and Cavenee, 1999). Giant cell astrocytoma is a form of glioblastoma with dominant multinucleated giant cells occurring commonly due to high TP53 mutations and is a rare type of brain tumour. Pleomorphic xanthoastrocytomas, common in children and youth, corresponds histologically to WHO grade II (Kleihues and Cavenee, 1999) and it is found to occur in superficial region of cerebrum with involvement of meninges. They consist of fibrillary and giant, multinucleated neoplastic astrocytes.
Diffuse astrocytoma is referred to as low grade (WHO grade II) astrocytomas of adults (Kleihues et al., 1993). Diffuse astrocytomas are capable of arising at any site of CNS due to their characteristic high cellular differentiation, slow growth and diffuse infiltrative nature. Anaplastic astrocytoma corresponds to WHO grade III and expresses increased cellularity and mitotic activity in addition to diffuse astrocytoma. It occurs similarly, like diffuse astrocytoma, due to TP53 mutation, RB alterations (25%), p19ARF deletion (15%) and CDK4 amplification (10%) (Kleihues and Cavenee, 1999). Mostly, it progresses to glioblastoma in about 2 years due to its increased ability of proliferation (Watanabe et al., 1997).
2.3.2 Oligodendroglioma (ODs):
Oligodendrogliomas (ODs), the glial brain tumours originating from oligodendroglial cells, are divided into WHO grade II and WHO anaplastic grade III tumours. The WHO grade II ODs are well differentiated cells that grow slowly while the latter grow faster. The WHO grading of ODs plays vital role in prognosis as well as predicting patient survival due to the indolent natured course of ODs (Dehghani et al., 1998; Wharton et al., 1998; Hagel et al., 1999). ODs cells are diffuse, well-differentiated that infiltrate to neighbouring brain structures and are found to be typically located in the cerebral hemispheres (Kleihues and Cavenee, 1999). OD show microvascular proliferation due to increased cytological atypia and mitotic activity (Kleihues and Cavenee, 1999).
The genetic alterations leading to ODs cells include Loss of Heterozygosity (LOH) on the long arm of chromosome 19 in majority cases (von Deimling et al., 1992; Reifenberger et al., 1994; Bello et al., 1995; Kraus et al., 1995) and LOH on the short arm of chromosome 1 in some cases (Reifenberger et al., 1994). Anaplastic OD include some features of WHO grade II tumours and areas of tumour necrosis (Kleihues and Cavenee, 1999). They are graded according to their likely rate of growth, from grade one (slowest growing) to grade four (fastest growing). Grade three and four gliomas are considered high-grade gliomas. Grade three gliomas include anaplastic astrocytoma, anaplastic ependymoma, anaplastic oligodendroglioma and anaplastic oligoastrocytoma. Grade four gliomas are usually glioblastomas. In adults, the most frequently encountered of these are high-grade or malignant neoplasms of astrocytic and oligodendrocytic lineage (Burton & Prados, 2000). Malignant gliomas are the most common primary CNS tumours (Zhu et al., 2005) and despite of intensive clinical investigation and many therapeutic approaches, the treatment for primary brain tumours remain inadequate (Kim & Glantz, 2006).
Glioblastoma is the most common and aggressive form of glial tumor arising from astrocytes. It is composed of a heterogeneous mixture of poorly differentiated neoplastic astrocytes. Usually, glioblastoma are found to affect adults in the subcortical white matter of cerebral hemispheres and less commonly brainstem and spinal cord. Glioblastoma develops and progresses very rapidly thus, increasing intracranial pressure (Kleihues and Cavenee, 1999). These gliomas exhibit ultimate degree of malignancy due to presence of both important processes, proliferation and neoangiogenesis and the ability to migrate to other regions of brain through the extra-cellular spaces (Lefranc, 2007).
Glioblastoma can be classified into two types- primary and secondary. Primary glioblastoma accounts for about 60% of adults and is common in men than females (Ohgaki and Kleihues, 2007) and develop de novo i.e. without any prior clinical or histopathological evidence of a less malignant precursor lesion (Kleihues and Cavenee, 1999). In more than 50% of cases, the history of disease is very short, about three months. Secondary glioblastoma accounts for 40% of adults aged between 45 to 50 years and is found to be common in females than males (Ohgaki and Kleihues, 2007). Glioblastoma may develop from diffuse astrocytomas and anaplastic astrocytomas (Kleihues and Cavenee, 1999).
Glioblastoma exhibits variable histopathology as some lesions show increased cellular and nuclear polymorphism while some are monotonous (Kleihues and Cavenee, 1999). The centre of glioblastoma consists of necrotic tumor cells while the periphery consists of viable tumor cells. Also, the tumor cells are arranged characteristically in a line in the cortex and around neurons (Burger et al., 1989).
Glioblastoma results from accumulation of greatest number of genetic changes among all other brain tumours. Some of these genetic changes include TP53 mutations, loss of heterozygosity on chromosomes 10q, 1p, 19q, 17q and EGFR amplification, p16INK4a deletion and PTEN mutations (von Deimling et al., 1993; Lang et al., 1994; Kraus et al., 2000; Kraus et al., 2001; Hilton et al., 2004; Ohgaki et al., 2004; Homma et al., 2006; Ohgaki and Kleihues 2007).
Diagnosis of high-grade glioma is provisionally made through a computed tomography (CT) scan or Magnetic Resonance Imaging (MRI). With the help of CT scan and MRI, the tumour can be detected as well as information on size, location, morphology and spread of tumour can be obtained. The diagnosis is then confirmed and the tumour classified histologically, either at the time of surgical resection or by a single-event biopsy if surgery is not possible. There is a growing understanding of the molecular genetics of gliomas, which is allowing a more accurate classification of glioma and may give an indication of prognosis and likely response to treatment. For example, Jean Yves and his team identified several types of deletions of chromosome 1 which were recorded using array CGH analysis. It was concluded that the complete loss of the short arm of chromosome 1 combined with complete loss of the long arm of chromosome 19 signifies a good prognosis. Partial loss of the short arm of chromosome 1, on the other hand, characterizes more aggressive tumours (Idbaih et al., 2005).
Usually, diagnosis is also carried out by conducting neurological tests (nerve tests) which thus, help to assess the effect of tumor on the nervous system. Diagnosis of large glioma mass and its grade can be done accurately by MRI-guided stereotactic brain biopsy. This diagnosis can further assist to guide subsequent therapy. However, accuracy of biopsy will be adversely affected on excess further usage of MR. (McGirt et al., 2003). Glioblastoma multiforme is diagnosed by fine needle aspiration biopsy (Schultz et al., 2005).
Histological diagnosis of gliosarcoma is confirmed on presence of tissue with gliomatous or mesenchymal differentiation as well as reticulin formation (Frankel et al., 1992; Paulus et al., 1994; Biernat et al., 1995; Boerman et al., 1996; Reis et al., 2000). Oligodendroglioma is diagnosed histologically, on presence of mild to moderate glial neoplasms with no or low mitotic activity (Kleihues and Cavenee, 1999). Also, further research in diagnosis of gliomas is been done by designing immunotherapeutic strategies based on pharmacogenomic findings (Yamanaka et al., 2008). This contributes to new strategies of treatment based on immunotherapy. Standard therapy for patients with this disease will be reviewed, together with more novel approaches such as targeted molecular therapies, angiogenesis inhibitors, immunotherapies, gene therapies and intratumoral therapies. Surgery and radiation remain the primary modalities of therapy for malignant brain tumours. Chemotherapy is another option for the treatment of glioma but the role of chemotherapy in malignant gliomas has been inconclusive (Kim & Glantz, 2006). A recent trial by the European Organisation for Research and Treatment at the Cancer Institute of Canada showed that combining radiation therapy with temozolomide (TZM) for newly diagnosed glioblastoma patients significantly improves survival benefit over radiation alone. TZM is a novel methylating agent currently under investigation for treatment of recurrent high grade gliomas (Tentori et al., 2003).
Glioblastoma is among the most common brain tumours leading to mortality, since glioblastoma is difficult to treat. On diagnosis of glioma, the standard form of treatment consists of maximal surgical resection of the tumour, radiotherapy and concomitant and adjuvant chemotherapy. This treatment is standard because of major challenges like tumour heterogeneity, location of tumour as it can be beyond the reach of surgical intervention and rapid, aggressive tumour relapse. Success of therapy depends on grade of tumour, extent of surgical tumour removal, location and age. The recurrent gliomas can be treated by surgery in appropriate patients as low grade tumours are less likely to resurface while; older patients are treated with less aggressive therapy like radiation etc. Surgery cannot be used to treat glioma developed in brain stem since biopsy and not removal of tumour is possible and brain stem is too delicate area to be operated (Barnet, 2004)
High grade tumours are treated with surgery followed by radiotherapy as it helps to control the growth, spread and symptoms of tumour. Long course of radiotherapy is not suitable for patients who are not fit or take enough self-care and hence may be offered a short course of palliative radiotherapy or conformal radiotherapy in which radiation beam is shaped to fit the tumor (Barnet, 2004). Radiation is effective in glioma treatment as the electrons and free radicals damage the DNA helix in tumor cells to produce single or double strand breaks and contribute to compete for oxygen as malignant brain tumours are found to have low oxygen tensions (Vinken et al., 2000). Radiotherapy in addition to surgery can increase the survival of patient from 3- 4 months to 7 - 12 months. However, the response may vary. Brachytherapy which involves insertion of temporary or permanent radioactive sources within tumor mass is of rare or limited use for treating gliomas. Thus, with interstitial brachytherapy, large dose of radiation is delivered to large tumor mass while simultaneously reducing radiation exposure to surrounding normal tissues (Barnet, 2004).
2.4 Glial cell lines:
In this study, two commercial glial cell lines have been used i.e. 1321N1 and U87MG. 1321N1 is a human brain astrocytoma grade II cell line showing glial morphology (Figure 4). The growth medium used for this cell line is Dulbecco's Modified Eagle Medium (DMEM) mixed with 10% Foetal bovine serum and 2mM L-glutamine.
Figure 4: A typical view of 70% confluent 1321N1 cells in a T75 flask.
U87MG is a human glioblastoma astrocytoma cell line of epithelial-like morphology of a 44 years old Caucasian female (Figure 5). It is a higher grade (grade IV) cell line as compared to 1321N1. The growth medium used for this cell line is Eagle's Minimum Essential Medium (EMEM) mixed with 10% Foetal bovine serum, 2mM L-glutamine, 1% Non Essential Amino Acids and 1mM Sodium Pyruvate.
Figure 5: A typical view of 60% confluent U87MG cells in a T75 flask.
HYPOTHESIS AND AIMS OF RESEARCH.
Novel compounds, such as 1-(2-Hydroxyphenyl)-2-(2-nitrophenyl)propan-1-one, 1-(2-Hydroxyphenyl)-2-(2-nitrophenyl)ethanone, Methyl 4-(1-(2-hydroxyphenyl)-1-oxopropan-2-yl)-3-nitrobenzoate, 1-(5-Bromo-2-hydroxyphenyl)-2-(2-nitrophenyl)propan-1-one and 1-(2-Hydroxyphenyl)-2-(2,4-dinitrophenyl)ethanone as well as numerous 2-arylindoles and related structures, may prevent or reduce cell proliferation leading to greater cytotoxic effect on glial cells compared to conventional anti cancer compounds. The main aim of this study is to investigate the effects of novel compounds by comparing their effects with conventional anti- cancer drugs on glioma cell lines in vitro.
i) To study and observe the effects of numerous novel compounds on glial cell lines in an in vitro chemo sensitivity system using different doses and incubation times.
ii) To investigate the effect of commercial anti-cancer drugs like Cisplatin, Taxol, Temoxifin, Etoposide, Gemcitabine, Carmustine etc alone or in combination on glial cell lines in an in vitro chemo sensitivity system using different doses and incubation times.
iii) To develop tissue culture techniques using U87, 1321N1, GOS3, U373, IN1265, IN859 cell lines.
iv) To synthesize a number of analogues of the most promising compounds employed in this study.
v) To elucidate the mechanisms of action of the active compounds, measuring apoptosis, gene expression and cytosolic free calcium levels.
vi) To analyse the data obtained statistically and present these as the PhD thesis.