Melanoma One Of The Fast Growing Type Cancer Biology Essay

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Melanoma is a one of the fast growing type of cancer. In spite of intensive research and large number of chemotherapy trials, the prospect of melanoma cancer is still poor. In our efforts to find new epidermal growth factor receptor inhibitor (EGFRI), Tarceva analogue (Lrina Tarceva) was investigated for its ability to inhibit the activity of Tyrosine kinase of epidermal growth factor receptor in melanoma B16 cells. In order to assess its application in future, the cytotoxic effect of the Tarceva were determinate by colorimetric MTT assays using Murine melanoma B16 cells. Results indicated that Tarceva shown an inhibition of cell viability at various dose concentration. Tarceva exhibited good cytotoxic effect at 10k ng/ml in B16 cell line. Therefore, this study suggests that Tarceva is candidate anti-melanogenic agent and it might be effective as a novel treatment regimen for melanoma cancer.

INTRODUCTION

The word "cancer" is a generic term used to describe more than 100 tumour disorder. Cancer is a universal disease but each of individual cancer differs from one country to another country. It is very important to understand reason of cancer than the past because it was predicted in world health report that by 2010 it will be leading cause of death. Cancer is leading cause of death in the western world, whereas cancer is second leading cause of death in United States of America but after 1999 it became number one leading killer in the people who has age more than 85.

British surgeon, Sir Rickman John Godlee was the first man who has done first surgery to remove brain tumour in 1884 and then surgery had become well known in Russia in 1887. In 1895, roentgen discovered x-ray which was to become very important in cure of cancer. After three years, Pierre and Marie Curie made outstanding discovery of radium. Both x-ray and radium can be dangerous. The first treatment of x-rays and radium were affected, the first death from exposure to radiation occurs too. There has been further development in twentieth century towards cure the cancer. The chemotherapy era began during world war-2. In World War- 2, Leon Jacobsen was the first man to show the effect of nitrogen mustard on lymphomas. After few years, Sidney Farber started diagnosis of cancer with folic acid antagonist. In 1950, the drug methotrexate shows strong effect in cure of carcinoma and it was the first strong impact of chemotherapy in cancer. By 1970, the chemotherapy was use as a combination to cure acute lymphocytic leukaemia in children which lead to maximizing their life

Cancer is an abnormal growth of cells caused by multiple changes in gene expression and inhibits the cell proliferation which leads to cell death. In normal cell biological mechanism properly control the cycle of cells throughout its life when cancer happens to anybody, it throws the control away from basic biological mechanism. Genes which are recognised as "the proto - oncogenes" or "suppressor genes" control the growth of cell. When gene mutation or translocation takes place within a chromosome, possibilities arises for a proto - oncogene to lose its power to govern cell replication and also become oncogene. These generic changes happen due to different factors which also build the final path in the biological mechanism of cancer. Cancer cell requires more energy due to the uncontrolled requirement for metabolism. So they stay together and cannot be separated. It cannot be worked as mature cells. These disorder form in different tissue in the body and lead to different types of illnesses. There are many kind of cancer like leukaemia, breast cancer, lymph sarcoma, Hodgkin's disease, testicular teratomas, retinoblastoma, ovarian carcinoma, prostatic carcinoma, head and neck cancer, carcinoma pancreas, bronchogenic carcinoma, etc.

The four prevalent types of cancer are carcinomas, sarcomas, leukaemias, and lymphomas. Highly percent of population are suffering from carcinomas, which form in skin or epithelium cells of internal organs, gland and body cavities. In sarcomas, cells are shifting in connective tissues like cartilage, bone and muscle. The main site of sarcomas is legs and arms. Cancer like leukaemias and lymphomas do not generate solid tumours. Leukaemias mainly found in bone marrow which drives to the over production of immature leukocytes. Lymphomas mainly found in lymphatic system.

Tumour is described as an uncontrolled growth of tissue. There are two types of tumours as below,

Malignant tumour:

Malignant tumour is that kind of tumour which can move from one tissue to another tissue via lymph or blood. They are not localised. They do not compress surrounding tissue.

Benign tumours :-

Benign tumour is that kind of tumour which cannot move from one tissue to another tissue. They elaborate by occupying space and destruct the tissue. They grow slowly. It constricts surrounding tissue. Benign tumour may decrease the flow of blood because it forms near a blood vessel. Benign tumours are localised.

There are many cause of cancers such as smoking, dietary factor, obesity, physical activity, occupation, genetic susceptibility, infectious agent, reproductive factor, socioeconomic status, environmental pollution, ultraviolet light, radiation, electric and magnetic field.

Treatment of cancer

The art of cancer treatment is in finding the right balance between tumour care and injury to normal cell. It is important to choose a most reliable treatment for particular cancer. There are many components which are taken into account for the treatment like cancer is malignant or benign, age of patient, toxicity of drug. Treatment is mainly applied with a view to have a successful cure but in case if it did not come out as desire than the secondary goal of the treatment will be giving the patient more life. Now days knowledge about cancer is elaborate, so the constantly changing in treatment of cancer.

Cancer can be preventing by three different ways.

Primary prevention: - In this stage administration of chemo preventive agent or the removal of environmental carcinogens which lead to reduction of the impact of carcinogen. Now day's life style adjustment is the more effective primary prevention method. For reducing the cancer incident there are many well known factors like lowering exposure to carcinogens, lowering dietary alteration, lowering body weight, doing more physical exercise, avoiding infection or with the help of medical treatment.

Secondary prevention: - secondary prevention includes screening and early identification method like mammogram, prostate specific antigen test, colonoscopy, which can detect abnormal changes before they transfer into cancerous cells. In this way cancer can be prevented before it goes bigger. There are many example of secondary prevention where the improvement of precancerous lesion trying to reverse carcinogenesis.

Tertiary prevention: - Final prevention includes taking care of settled disease and also prevention of risk associated with the disease. Final prevention is also known as cancer controller which includes choice of factors for patient care like quality of life, adjuvant therapies and surgical intervention.

Cancer process start from the initiated tumour cell than it goes to mild, moderate, sever dysplasia, invasive carcinoma and finally form the metastatic disease. So, it is important to find out that which treatment is reliable to stop these changes as early as possible. Early detection of cancer is the best method to stop the cancer in initial stage. Symptoms like pain, an unusual lump, bleeding which increase the doubt of colon cancer. Other symptoms like changes in the colour of skin which give the sign of melanoma. For women, two effective methods for early detection of breast cancer are mammography and ultrasound examination. Digital mammography made up of electronic detector system that shows the data on computer. Mammography is the first effective test which decrease the death by 30% .ovarian cancer is detected at an advanced stage but the diagnosis can provide better result in early stage. The mass spectroscopy with high sensitivity used in detection of ovarian cancer in early stage.

Tumour has been detected by the some classical markers like carcinoembryonic antigen, squamous cell carcinoma antigen and neuro specific enolase. They are useful for detecting only the few not the all. During the last few years the use of prostate specific antigen has increased to detect the prostate cancer in addition to digital rectal examination. The result of this method is very low and gives birth to unnecessary prostate biopsies. The research and advances in the utilisation of proteomics to analyse blood, plasma, saliva and related body fluids are of great importance for biomarkers in studies of cancer. These tools can be used in high risk patient with nasopharyngeal carcinoma (NPC). NPC is a malignant disease and the main reason for this disease is epstcin-brr virus infection. It has been speculated that a group of biomarker can be assembled to undertake NPC incidence, tumour progression and initial identification while the intervention of therapeutic go on.

Surgery, radiation and chemotherapy are widely used in diagnosis of cancer.

Surgery: - Surgery is the important character in diagnosis, staging and curing the advanced tumour. In surgery, the part of the body which is affected by the cancer is separated from the body so, that the cancer cannot be spread. When cancer is in the beginning stage, in this stage it stays at only one place and it is the best stage for doing the surgery which will give the better cure. Main requirement for the surgery is backbone from the other special things which includes anaesthesiology, antibiotic therapy, blood transfusion services, pathology and critical nursing care. Solid tumour like dukes A or T1 colon tumour, early prostate, breast, oral cavity cancer and malignant melanomas can be affected by surgery in early stage they do not need high technological aspect.

Extended radical mastectomy is the broad surgery used to cure the breast cancer. The main aim of radical mastectomy is stop the spreading of cancer by removing all of the lymphatic drainage pathways in the breast region but radical mastectomy does not lead to greater quality of life and more expensive than "lesser" surgery. The "lesser" surgery is the best simple mastectomy which is used to treat best cancer. Only breast tissue and superimposed skin are removed by this treatment. This treatment is often applied as a combination of radiotherapy. Other type of treatment is local excision or tylectomy which includes taking away of cancerous tumour as well as margin of healthy tissue around it.

Radiotherapy: - radiation forms the highly reactive radicals in the intracellular material which can destruct the DNA by breaking DNA bonds leading to cell death. radiotherapy used in the treatment of many cancer such as head and neck cancer, cancer of cervix, prostate and early Hodgkin disease and many of brain tumour of young people. There are two types of technique like teletherapy and brachytherapy which are used to treat the cancer. In teletherapy, the patient is treated from distance with the help of cobalt machine or by accelerator, where as in brachytherapy, patient is treated by temporarily putting radioactive sources in their body tissue. Brachytherapy also provided via different instruments for ex. For delivering low dose rate caesium is used and for delivering high dose rate iridium or cobalt is used.

There are many types of radiotherapy which are used in the treatment of cancer.

External beam radiation therapy: - in this therapy x-ray beam applied at tumour. This therapy covers a wide area which includes three different kind of tumour volume such as 1) gross tumour volume which covers regions familiar to cancer cell. 2) Clinical target volume which includes area where the possibility of cancer cell is very high plus the first volume type. 3) Planning tumour volume which covers a gap left by the clinical target volume.

Internal radiation therapy, where as covers very small areas in comparison to external beam radiation therapy but this radiation therapy needs more dose of radiation.

Intensity modulated radiation therapy (IMRT): - two decades ago Anders Brahme and Alan Cormack invented the CT- scanner in accordance of π-meson therapy and they also suggested the use of non uniform fields. Their idea was relied on the decision of using mathematical tricks and irradiation scheme, which help to deliver dose to target and also help in reducing the delivery of dose to the normal tissue around the target. In IMRT, there are two types of delivery system like robotic pencil beam and rotary fan beam with intensity modulation.

(3) Chemotherapy: - in chemotherapy diagnosis of cancer with the help of drug which kill the cancer cell and inhibit the cell growth. Most chemotherapic agent has toxic effect on normal cells such as bone marrow and mucous membrane cells which have fast turnover rate. So it is important to choose anticancer drugs which have less toxic effect on normal cell and the drugs that have a greater controlling effect on the cancer cells. Development in chemotherapy extremely based on the natural evolution of the cancer and for schematic anticancer agent had to be orient for each tumour type. Classic chemotherapy agents interfere with the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein, or with the appropriate functioning of the preformed molecule which lead to cell death.

The four ways of chemotherapy used in the treatment of cancer.

Primary induction treatment mainly used for advanced stages of disease or for cancer which do not have other effective treatment approaches. The main aim of this therapy is palliate tumour related symptoms, better quality of life and maximizing tumour progression. Many of cancer like Hodgkin's and non Hodgkin's lymphoma, germ cell cancer and choriocarcinoma are cured by this therapy.

Neoadjuvant chemotherapy use chemotherapy for treating patient who are suffering from localized cancer. For treating those other therapies are available like surgery but it is not completely effective. Now days, Neoadjuvant therapy is applied in the cure of anal cancer, bladder cancer, breast cancer, oesophageal cancer and non small cell lung cancer.

Adjuvant treatment to other methods of treatment such as surgery and/or radiation therapy and make them more effective.

This method is direct instillation; the medicine is directly injected in the cancer affected part of the body.

Combination chemotherapy: - cancer has been unable to treat by single dose because this dose is clinically tolerable. So combination of two doses is used and it provides the maximum cell kill within the range of toxicity tolerated by the host for each drug as long as dosing is not compromised. These combination therapies were developed based on mechanism of action of chemotherapic agent.

For example: - anti malarial drugs like chloroquin use as combination of anticancer drug like 5-flurouracil. Recently this combination has been shown a good effect on human colon cancer. Chloroquin used as anticancer agent as well as chemo sensitizer. Chloroquin inhibit autohagy in HT-29 cells and enhance the effect of 5-flurouracil. Autohagy is a catabolic process which protects the cell from starvation. Cancer cell used this mechanism for protection against anticancer agent, so chloroquin inhibit this autohagy and enhance the anticancer effect of 5-flurouracil.

(4) Targeted therapy: - recently, some novel targeted drugs have been shown an inhibitory effect on the epidermal growth factor receptor or the vascular endothelial growth factor in various types of cancer. Some molecules have the power to target specific mutation and other factors that are comparatively unique to cancerous cell. So, this therapy allows more toxic effect on cancerous cell and less effect on normal cell. Targeted therapy divided into two groups which are used in treatment of cancer. a) Small molecules have ability to target specific mutation. b) Monoclonal antibody act versus specific antigen on proteins.

Drug used in cancer treatment

Anticancer agents are drugs that are used in treatment of cancer. These drugs have cytotoxic effect on cancer cell. Antineoplastic agents are divided into different types of classes such as alkaylating agent, antimetabolites, antibiotics, hormones, vinca alkaloids, topoisomerase inhibitor, taxan, and miscellaneous agent.

Alkaylating agent: - alkaylating agent forms highly reactive carbonium ion which substitute the alkyl group in cellular macromolecules. This alkyl group interact with cellular DNA it leading to abnormal base pair or cross link. This result in death of cells by preventing replication of DNA. In this process, nucleic acid like guanine is more capable for alkylation, but adenine and cytosine are also involved. Some cancer like lymphomas, Hodgkin's disease, breast cancer, myeloma, bladder cancer and leukaemias are treated by alkaylating agent.

Drugs including alkaylating agents: - buslfane, mechlorethamine, cyclophosphamide, chlorambucil, malphalan, carmustin, lomustin, cis- platin, dacarbazine and thiotepa.

Antimetabolites: - these compounds prevent the synthesis of DNA and RNA by inhibiting the production of Purine and pyrimidines. Purine and pyrimidines are essential metabolites. Antimetabolites block the enzyme like dihydrofolate rductase enzyme which is involved in the production of metabolite compound and it lead to inhibition of nucleotide synthesis.

Antimetabolites drugs: - methotrexate, cytarabine, 5-flurouracil, 6-mercaptopurine, 6-thioguanine, azathioprine, gemcitabine.

Antibiotics: - this compound obtained from microorganism. They act as both antimicrobial as well as anti toxic. Antibiotic have many different mechanism by which they obtain their cytotoxic effect. Most of antibiotic compound intercalate between DNA strands and inhibit the function of template. Some antibiotic interact with DNA and form the complex with DNA. The result in an inhibition of protein synthesis.

Antibiotics drugs: - dactinomaycin, doxorubicin, mitomycin, mithramycin, mitoxantrone, daunorubicin and bleomycin.

Hormone: - they altered the growth of tumour and the tumour is dependent on the hormones. Hormones do not have any cytotoxic effect. For ex. Endocrine therapy is beneficial to patient suffering from advanced prostate cancer by promoting hormones refractory. It also reduces pain and having the main aim of maximizing life of patient.

Hormonal drugs:-dexamethason, prednisone, tamoxifen, flutamide, finasteride and fluoxymesterone.

Vinca alkaloids: - these drugs interfere with microtubule protein like tubulin and inhibit the formation of microtubule by preventing microtubule assembly, cause disruption of mitotic spindle lead to cell death.

Vinca alkaloids drug: - vincristine and vinblastin.

Topoisomerase inhibitor: - it is a derivative of podophayllotoxin. It blocks the topoisomerase enzyme and causes collapse in the structure of the DNA.

Topoisomerase inhibitor drugs: - etoposide and teniposide.

Taxanes: - mechanism action of Taxanes is opposite to that of vinca alkaloids. In this mechanism depolymerisation of microtubules is prevented and increases the stability of microtubule, and induces apoptosis.

Taxanes drugs: - paclitaxel (taxol) and docetaxel.

Miscellaneous agent: - these agents are differs than other major classes of Antineoplastic agent by their mechanism of action.

Miscellaneous drugs: - procarbazine, hydroxyurea, mitoten and L-asparaginase.

Limitation of drug treatment

In treatment of cancer, one of the major problems is drug resistance at the time of initial treatment that described as an innate drug resistance or following a beginning period of response to the anticancer drug that described as acquired drug resistance. Drug resistance has been observed for most types of anticancer drugs.

Drug resistance occur through a many of various mechanism. Some of mechanisms are described bellow: -

Reduced intracellular drug concentration.

The main reason for this mechanism is reduced cellular uptake of drug or increased efflux. Examples for this mechanism are vinca alkaloids, dectinomycine, anthracycline and epipodophyllotoxin.

Enhanced drug inactivation.

Drugs like alkaylating agent, cytarabine and doxorubicin can develop the resistance through this mechanism. For alkaylating agent, increase activity of glutathione transferase, which lead to rising in production of glutathione. This glutathione interfere with alkaylating agent and prevent the cross link with the DNA.

Reduce drug activation.

Drugs such as cytarabine, fluorouracil, 6-mercaptopurine and methotrexate are the best examples for this mechanism.

Modified production of receptor or enzyme.

Methotrexate is the best example that develops resistance through this mechanism. In this mechanism, amplification of DHFR gene occur which lead to increases the activity of DHFR enzyme so, high amount of methotrexate concentration are required for similar effect.

Reduced affinity of receptor or enzyme for the drug.

Antimetabolites, tyrosine kinase inhibitor, doxorubicin and etoposide develop the resistance due to this mechanism. This mechanism involves reduction in drug-enzyme binding affinity due to mutation of enzyme by amino acid.

Increased repair of drug induced DNA damage.

Alkaylating agent, cis-platin and mitomycin are the example for this mechanism. In this case some enzyme repairs the DNA damage by excising the damaged DNA base pair and resynthesizing the missing segment of DNA.

Reduction in activation of mechanism critical enzyme.

This mechanism involves reduction in enzyme activity for drugs. For example, reductions of activity of topoisomerase for drug like podophyllotoxin.

Multidrug resistance.

Multidrug resistance occurred in tumour cells that can become resistance to group of drugs that are dissimilar in structure and can act through unrelated cytotoxic mechanism, or both. This resistance involve over expression of p-gp which is responsible for removing drug from the cell. It also results of mechanism like increase of the glutathione detoxification pathway, or modification in topoisomerase enzyme system.

Adverse effect is the main factor limiting the usefulness of anticancer agent. Cancer such as granulocytopenia, thrombocytopenia and aplastic anaemia are the main cause of serious toxicity like bone marrow depression. Nausea, vomiting, mucosal injury and diarrhoea can observe in the gastrointestinal toxicity. In comparison of leukopenia and thrombocytopenia, it is not dangerous to life. Other adverse effect like alopecia involves hair loss occurs with most forms of chemotherapy. Drugs like doxorubicin can cause total hair loss within 4 to 6 week. Alopecia occurs due to damage to the cells in hair follicles. Some serious dose limiting side effects like ototoxicity, nephrotoxicity, and neurotoxicity occur with the use of some chemotherapic agent like cisplatin. In this case cisplatin induced ototoxicity is mainly result of reactive oxygen species.

Some drug-drug interactions in oncology lead to the side effect, for example interaction between cisplatin and topotecan. In this case cisplatin reduce the renal and total plasma clearance of topotecan and methotrexate which lead to renal damage. Renal clearance of methotrexate also reduced by some non-anticancer drugs like penicillin, probenicide, sulphonamides and NSAIDs which lead to methotrexate induced toxicity.

Testing of new drugs

New develop methods of testing anticancer drugs contains continuous evaluation and controversies. Well characterised and diverse testing system can prove beneficial for the drug discovery. Drug testing occurs in different areas like 1) invitro studies 2) drug supply and manufacturing 3) drug formulation 4) invivo studies in animal model. It is important to develop informative, less expensive and less time consuming drug testing method.

Invitro toxicological study

Invitro toxicology described as an application of cell culture techniques in toxicological studies. This test allow the examination of direct effect of anticancer agent and it also not get influence by variation in absorption, metabolism, and excretion, which are normally found in invivo test. Invitro test include examination of drug ability to bind to receptors, inhibit specific enzyme and stabilize or destabilize multiprotin complex. This test can be developed in intact cell line or cell free system. Stabilized cell lines can be characterized in detail for cytogenetic isozyme and drug sensitivity profile.

Invivo drug testing

Even though the development of invitro laboratory model is progressing there assessment is quite limited only to the invivo animal model system. For investing mammalian biology and disease, mouse and rat model is more useful than the higher mammals. The performance of any particular animal model is dependent on validity, selectivity, predictability and reproducibility of the model. Different kind of animal model are developed for human tumour in oncology. These models are divided into three big groups such as 1) spontaneous model which consist of animals produced naturally 2) engineered model which involves transgenic animal 3) transplanted tumour models which can be implemented for developing anticancer drug

Clinical trial of anticancer drug

Manufacturer submits the safety and effectiveness of drug data to the FDA which is taken from animal studies. If the drug achieves the desirable effect and if it is approved then the new investigated drug goes for clinical trial in human. Clinical trial in human occurs in four different phases. Before new drug comes in market, it must pass from phase 1 to phase III testing. Phase IV testing starts after finishing phase III and phase IV stops after marketing begins. Drug takes 5 to 9 years for passing through all 4 phases. Lots of healthy volunteers and patients with disease are included in testing for getting reliable projection of results. This trial should consist of different ages and sex of the high risk population and requires established treatment as control for comparison.

Cytotoxic effect of Tarceva (lrina Tarceva) on B16 melanoma cells

250px-Erlotinib_Structural_Formulae.png srassay.tif

Figure-1 Tarceva structure Figure-2 Tarceva Analogue (lrina Tarceva) (made by Dr. John Spencer, Uni. of Greenwich)

Tarceva is a cancer drug and it is described as an epidermal growth factor receptor inhibitor and it is also called tyrosine kinase inhibitor. Tarceva used to treat patient with advanced Non-Small-Cell lung cancer (NSCLC) and pancreatic cancer for effective treatment and it can provide prolong survival and better quality of life. It is also used in treatment of some other cancer like ovarian cancer and cancer of the head and neck. Tarceva is given orally at least one hour before a meal or two hours afterwards.

Mechanism of Action: - Tarceva is developed to inhibit cell growth by acting on a protein epidermal growth factor receptor (EGFR) that is present on the surface of cell. Tarceva competing with the ATP for binding of the intracellular tyrosine kinase and it inhibit the activity of tyrosine kinase which leads to inhibition of autophosphorylation. It gives the result in inhibition of protein synthesis and it prevents the cell growth.

It was approved in 2004 as a mono therapy for treatment of NSCLC after failure of at least one chemotherapy regimen. In 2005 greater improvement in overall survival was shown by Tarceva for first time when it was combined with gemcitabine in phase III trial. Tarceva was used as a first line treatment for pancreatic cancer.

The approved dose of erlotinib is 150 mg/d but some studies suggest that erlotinib have similar response rate and progression free survival by 25 mg/d. Accumulating evidence has confirmed the hypothesis that gefitinib and Erlotinib have similar response rate and disease control rates in pre-treated patient with NSCLC.

Adverse Effect: - Erlotinib has been show adverse effect on pulmonary. In East Asian patients, near fatal interstitial pneumonitis with acute lung injury due to Erlotinib was found. Some adverse effect like diarrhoea and cutaneous rash associated with the Erlotinib. It is also responsible for some other side effects like loss of appetite, fatigue, loss of fertility, and a sore mouth.

Resistant of Tarceva: - Continues Tarceva is the best therapy for treating the patient but it is proved inaccurate against drug resistant tumour. In this case, resistance of drug is related with the presence of secondary mutation in epidermal growth factor receptor (EGFR) that substitute methionine for threonine at position 790 in kinase domain of the protein. MET amplification has also been recorded in 20% of Tyrosine Kinase Inhibitor resistant lung cancer. The mitogen activated protein kinase (MAPK) / extra cellular signal regulated kinase (ERK) and P13K signal pathway are usually exhibited by tumours and these tumours are made by EGFR. This variable signalling pathway remains active in Tarceva resistant tumours, despite continuous administration of Tarceva.

Tarceva combined with other agent: -

Carboplatin: - The combination of Tarceva and carboplatin has shown good effect in patient with platinum sensitive disease like epithelial ovarian cancer.

Sorafenib: - Currently, some studies suggest that combination of Tarceva and sorafenib has increase the killing efficiency in patient with stage III B/IV NSCLS, despite a possible drug reaction and has an acceptable safety profile.

Cell Culture: -

In cell culture model, cells are grown with the help of nutrient solution that are required for the cell growth. Cell culture is carried out in tissue culture dishes of different size. For identification of cancer protective substance, cell culture model is a best experiment system. There are many types of culturing systems like dish culture, 3 D culture, Co culture, and high throughput screening.

Substrate: - cells are growing on different charged substrates like Polystyrene or acid washed glass with net negative charged and plastic with net positive charge. In this case cells are not directly attached to the synthetic substrate but they are attached via cell secrete matrix product which gives ligands for the interacting receptor like integrins.

Medium: - some common media like water, salt and media supplements such as tryptose or peptone are used and they must be sterilised by autoclave. Labile media like trypsin and serum is sterilised through a filtration. Cytokines, vitamins, calcium and planer polar compound are used as a growth factor.

Primary Culture: - In Primary Culture, Tissue passed through different stages like rinsing, dissection, and mechanical or enzymatic disaggregation in trypsin and collagenase, and it becomes small cluster of cells. This cluster of cells transferred to a fresh dish and then the cell population allowed for further growth in correct medium.

Subculture: - Monolayer of cells transferred from primary culture vessel to another clean vessel and diluted by dissociating the cells of monolayer in trypsin that lead to single cell suspension. Subculture increases the population of cell, which are used for more experiment and characterization. In subculture, monolayer is rinsing with PBS, and then cold trypsin is added after removing the rinse. It incubates for 5 to 15 min.

Cryopreservation: - Cells can be preserving by freezing to maintain and protect from contamination, and incubator failure. Some preservative like glycerol and dimethyl sulfoxide (DMSO) are used for the preservation.

Contamination: - The main problem of cell culture is microbial contamination. This contamination is decrease by the use of laminar air flow cabinet and antibiotic like penicillin. Other risk like cross contamination occurs due to handling continuous cell lines or use of other cell lines in laboratory. So, cross contamination can be avoided by taking some precautions.

Aim: -

Now days, there is no preclinical evaluation available for the Tarceva drugs in melanoma cancer. So, the aim of this study is to identify the influence of Tarceva analogue (Lrina Tarceva) on B16 melanoma cell proliferation in invitro. This study used to identify possible additive, adverse effect and resistance of Tarceva.

Method and Material: -

Materials:

RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), B16 Melanoma cell line, phosphate-buffered saline (PBS), antibiotics (penicillin G, streptomycin) and L-glutamine, Dextran, polyethyleneimine(PEI), cisplatin ,Tarceva analogue, Trypsin-ethylenediamine tetra acetic acid(EDTA) solution, 70% ethanol, haemocytometer, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell culture chamber, Gilson micro pipette, multichannel pipette, incubator, microtitre reader, 96-well plates, cell culture chamber.

Methods used:

Culture of B16 cells

B16 melanoma cell with fetal bovine serum are used to prepare Cell culture. Antibiotic like penicillin streptomycin were added. Culture is incubated at 370c with 5% of co2. Then the cell culture chamber are taken out from incubator and the chamber is rinsed with new media and after that clean the cell surface two times with phosphate buffer saline (PBS), which will help to balance the isotonicity of the cell and also become helpful to rinse the container containing cells. 1ml of Trypsin ethylenediaminetetraacetic acid (EDTA) should be added to the cell chamber. Cells are disaggregated by photolytic enzymes Trypsin EDTA. Trypsin EDTA digest the protein which help in holding the cell to the chamber wall and again incubate for 4-5minute into the incubator, the cell gets separated from wall of the chamber float on to the solution, that makes solution turbid. The turbid cell culture is transferred from cell chamber to trough. Again clean cell chamber with the help of cell media to remove remaining cell from the chamber and the solution is added to the trough. Take out the cell solution with the help of pipette from trough and put a one drop on one chamber of the haemocytometer and it observed under the microscope. Find out number of cells. The cells should be 5Ã-105/ml .If the cell concentration isn't in range and if it is different than it can be adjusted by diluting the media with the help of additional media. 100microlitter culture media is transferred by sucker to 96 well plates. Prepare two more plates in the same way. Incubate the plate at 37oC for 24hours with 5% of CO2.

Putting a drugs on the cells

Cells were cultured in 96-well plate at a density of 5x104 cells/well. To allow attachment cells should be left as it is for 24 hours than cells were treated with 5mg/ml stock solution of Tarceva analogue, cisplatin, dextrin and polyethyleneimine ( all these compounds are diluted using DMSO and they should be sterilized by membrane filter with the help of injection). From 5mg/ml stock solution makes 1:50 and 1:100 dilutions for 100k and 50k concentration in fisher tube, the solution should be transferred in to trough and multi channel pipette used to do an alternative serial dilution for multiple of one and multiple of five respectively. After the preparation, cells were incubated at various concentrations of drugs for 72 hours at 37oC in 5% CO2 atmosphere.

Assaying cell viability

Cell growth determination is done by calculating viable cells after staining it with a vital dye. In the mitochondria of living cells, yellow colour substance MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) is transferred to purple formazan. The main cause of this transformation is the activeness of mitochondria reductase enzymes and this transformation is directly attached to number of living cells. The optical density of coloured can be calculated at a particular wavelength. The absorption is comparative to the solvent employed.

In this experiment, viable cells were determined with the help of MTT assay. MTT reagents were added to the 96-well plates and incubate for 4 hour, while this incubation is going on yellow colour MTT is transferred in to purple formazan. Detach the culture medium and well plate which were on tissue paper to get proper culture removal. For getting equal distribution of purple colour, dissolving formazan crystals and measure optical density 10microlitter DMSO were added to each 96 well plates. Absorbance was measured at 540nn with the help of colorimetric microtitre plate reader to automatically fix the optical density in each well. Results were shown as the mean of wells viability and standard deviation.

Result: -

TABLE-1 DATA OF DEXTRAN

Drug concentration(ng/ml)

0

5

10

50

100

500

1k

5k

10k

50k

100k

Blank

OD 540

1.614

1.500

1.536

1.596

1.654

1.563

1.572

1.578

1.506

1.436

1.366

0.044

1.516

1.491

1.412

1.531

1.531

1.444

1.550

1.356

1.397

1.413

1.368

0.081

1.384

1.518

1.406

1.442

1.491

1.431

1.457

1.390

1.584

1.409

1.381

0.077

1.618

1.348

1.261

1.378

1.407

1.397

1.384

1.323

1.520

1.380

1.416

0.063

1.668

1.254

1.395

1.387

1.517

1.331

1.403

1.453

1.342

1.413

1.427

0.052

1.605

1.432

1.353

1.331

1.404

1.385

1.331

1.387

1.418

1.377

1.545

0.067

1.447

1.164

1.408

1.646

1.468

1.353

1.471

1.402

1.585

1.375

1.467

0.065

1.475

1.398

1.454

1.563

1.551

1.442

1.500

1.439

1.539

1.500

1.476

0.057

Average

1.541

1.388

1.403

1.484

1.503

1.418

1.459

1.416

1.486

1.413

1.431

0.063

Average-blank

1.478

1.325

1.340

1.421

1.440

1.355

1.395

1.353

1.423

1.350

1.368

(%) growth

100

89.7

90.7

96.2

97.4

91.7

94.4

91.5

96.3

91.3

92.5

Standard deviation

0.10

0.13

0.08

0.12

0.08

0.07

0.08

0.08

0.09

0.04

0.06

Standard Error of mean

6.8

8.6

5.3

7.8

5.5

4.8

5.6

5.2

6.1

2.8

4.2

TABLE-2 DATA OF POLYETHYELENIMINE (PEI)

Drug concentration(ng/ml)

0

5

10

50

100

500

1k

5k

10k

50k

100k

Blank

OD 540

0.248

1.023

0.978

1.089

0.841

1.507

1.013

1.279

1.089

0.196

0.198

0.058

1.097

0.907

0.967

1.003

0.933

1.676

0.863

1.037

1.016

0.197

0.205

0.061

1.054

0.925

0.926

1.002

0.8

1.256

1.045

1.169

0.933

0.21

0.217

0.058

1.008

0.82

0.895

0.982

0.855

1.424

0.915

1.024

0.977

0.195

0.227

0.055

1.117

0.92

0.821

0.961

0.906

1.393

0.981

1.063

1.007

0.209

0.21

0.066

1.032

1.003

1.019

1.09

0.964

1.524

1.221

1.034

0.911

0.198

0.204

0.089

1.034

1.074

1.154

1.116

0.862

1.369

1.106

1.23

1.052

0.187

0.194

0.061

1.257

0.977

1.015

1.176

1.043

1.708

1.283

1.273

1.08

0.18

0.225

0.072

Average

0.981

0.956

0.972

1.052

0.901

1.482

1.053

1.139

1.008

0.197

0.210

0.065

Average-blank

0.916

0.891

0.907

0.987

0.836

1.417

0.988

1.074

0.943

0.132

0.145

(%) growth

100

97.3

99.0

107.8

91.2

154.7

107.9

117.2

103.0

14.4

15.8

Standard deviation

0.31

0.08

0.10

0.08

0.08

0.15

0.14

0.11

0.07

0.01

0.01

Standard Error of mean

33.45

8.68

10.77

8.29

8.52

16.83

15.76

12.18

7.12

1.10

1.32

TABLE-3 DATA OF CISPLATIN

Drug concentration(ng/ml)

0

5

10

50

100

500

1k

5k

10k

50k

100k

Blank

OD 540

1.293

1.42

0.932

1.303

1.527

1.223

1.069

1.098

0.77

0.091

0.081

0.047

1.066

1.257

1.073

1.123

1.342

1.44

1.249

0.979

0.737

0.155

0.136

0.082

1.061

1.175

1.047

1.237

1.195

1.139

1.257

1.109

0.634

0.191

0.116

0.079

1.485

1.112

1.399

1.448

1.248

0.978

1.351

1.283

0.698

0.125

0.094

0.068

1.1

0.891

1.051

1.182

0.996

0.993

1.281

1.14

0.84

0.112

0.092

0.055

1.383

1.12

0.936

1.14

1.044

1.203

1.289

1.185

0.795

0.137

0.103

0.073

1.101

1.061

0.94

1.126

1.105

1.05

1.468

1.251

0.896

0.183

0.092

0.064

1.247

1.127

1.122

1.427

1.12

1.011

1.213

1.033

1.475

0.108

0.087

0.074

Average

1.217

1.145

1.063

1.248

1.197

1.130

1.272

1.135

0.856

0.138

0.100

0.068

Average-blank

1.149

1.078

0.995

1.181

1.129

1.062

1.204

1.067

0.788

0.070

0.032

(%) growth

100

93.8

86.6

102.7

98.3

92.4

104.8

92.8

68.6

6.1

2.8

Standard deviation

0.16

0.15

0.15

0.13

0.17

0.16

0.11

0.10

0.26

0.04

0.02

Standard Error of mean

14.0

13.3

13.4

11.5

15.1

13.7

9.9

9.0

22.9

3.1

1.6

TABLE-4 DATA OF TARCEVA

Drug concentration(ng/ml)

0

5

10

50

100

500

1k

5k

10k

50k

100k

Blank

OD 540

1.178

1.185

1.1

1.054

1.096

1.022

1.121

1.195

0.947

0.105

0.083

0.048

1.143

1.022

1.001

1.067

1.001

1.011

1.136

1.161

1.225

0.129

0.131

0.095

1.125

1.177

0.998

1.049

1.094

1.048

1.108

1.225

1.094

0.098

0.107

0.091

1.13

1.077

1.047

1.05

1.026

0.969

1.092

1.212

1.187

0.09

0.092

0.073

1.085

1.091

0.946

1.053

0.985

1.067

1.217

1.104

1.073

0.086

0.108

0.056

1.148

0.984

1.009

1.114

1.161

1.153

1.181

1.311

1.158

0.125

0.105

0.061

1.107

1.153

1.081

1.022

0.961

1.097

1.12

1.207

1.172

0.089

0.095

0.066

1.123

1.217

1.226

1.214

1.035

1.32

1.406

1.286

1.279

0.087

0.11

0.06

Average

1.130

1.113

1.051

1.078

1.045

1.086

1.173

1.213

1.142

0.101

0.104

0.069

Average-blank

1.061

1.045

0.982

1.009

0.976

1.017

1.104

1.144

1.073

0.032

0.035

(%) growth

100

98.4

92.6

95.1

92.0

95.9

104.0

107.8

101.1

3.1

3.3

Standard deviation

0.03

0.08

0.09

0.06

0.07

0.11

0.10

0.07

0.10

0.02

0.01

Standard Error of mean

2.6

7.8

8.1

5.7

6.3

10.4

9.7

6.2

9.7

1.6

1.4

In the present study, B16 cells were used as the cell model for investigating the efficacy of Tarceva on melanogenesis. To show the cytotoxic effect of drugs on B16 melanoma cells, an MTT assay was done first.

Figure-3 Effect of Dextran and PEI on B16 cell viability in melanoma. Melanoma cells (5x104) were cultured for 72 h with different doses of Dextran and PEI. IC50 was determined by an MTT assay. Data shown are the means ± SD of Dextran and PEI.

Figure-3 shown the cell viability of B16 melanoma treated in polyethyleneimine and Dextran. This graph compares the positive and negative control of drug. This provides the information that after treatment for 72 hours, Dextran did not affect cell viability. So, there was no effect of environment on cells and it gave negative control. In contrast the polyethyleneimine shows the cytotoxic effect on B16 melanoma cells. So, it gave positive control. Murine B16 cells are resistant to the lethal effect of PEI up to 10k ng/ml concentration but after 10k ng/ml the figure show the sudden reduction in viability of B16 melanoma cells by PEI. This graph also show the amount of PEI required to inhibit 50percent of a cells population. The data suggests that for B16 melanoma cells, IC50 value was 25k ng/ml of PEI after 72 hours incubation with the drug.

Figure-4 MTT assay was performed after incubation of Murine B16 melanoma cells treated with different concentration of cisplatin for 72 h at 370C in 5% CO2. OD was measured at 540 nm with colorimetric microtitre plate reader. Values are the mean ± SEM of cisplatin.

Murine B16 melanoma cells viability curves after 72 h incubation with cisplatin was presented on fig.4. The rise in concentration of cisplatin causes decrease in viability of B16 melanoma cells. We can see rise in the cytotoxic effect of cisplatin after 1k ng/ml whereas the graph also shows dramatic increased in cytotoxic effect after 10k ng/ml. The data also shows the IC50 value of cisplatin was 22k ng/ml after 72 hours incubation with the drug.

Figure-5 Effect of Tarceva on the growth of B16 melanoma. Cells were cultivated as a monolayer for 24 h before incubation with Tarceva. IC50 were determined by MTT assay. The data represented means ± SD of the Tarceva. OD was measured at 540 nm with colorimetric microtitre plate reader.

To determine cytotoxic effects of Tarceva, we treated Murine B16 melanoma cells with Tarceva at various concentration (5, 10, 50, 100, 500, 1k, 5k, 10k, 50k, 100k ng/ml) In figure 5 Murine B16 melanoma cells decreased significantly in the Tarceva range of 10k-100k ng/ml. The cytotoxic effect remains steady between ranges of 50k to 100k ng/ml of cisplatin drug concentration. The graph shows the IC50 values was 30k ng/ml of Tarceva.

The data show that growth inhibition of PEI, cisplatin and Tarceva were 84%, 97%, and 98% respectively. So, Tarceva and cisplatin had the similar effect to inhibit the cell growth. Tarceva inhibit the B16 Cells in small constriction range (10k-50k ng/ml) as compared to cisplatin (1k-50k ng/ml) and PEI (5k-50k ng/ml). So, Tarceva was more accurate in comparison of PEI and cisplatin in small range. We can say that on based of IC50 that cisplatin need less concentration (22k ng/ml) in comparison of Tarceva (30k ng/ml) and PEI (25k ng/ml).

Figure-6 effect of molarity of Dextran on B16 melanoma cells.

The figure 6 provided the information that after treatment for 72 hours, the molecular weight of Dextran did not affect cell viability at various concentrations. So, it confirmed that there was no environmental effect on cells viability. It was used as a negative control.

Figure-7 Effect of molarity of PEI on B16 cell viability. Data are mean ± SD of PEI.

This graph show that the effect of molarity of PEI on B16 cells. It is clear that the cell growth of B16 decreased when the concentration of PEI increased.

Figure-8 Effect of Molarity of cisplatin on B16 cell viability. Data are mean ± SD of cisplatin.

The graph shows the effect of molarity on B16 cells. It also represents that viability of B16 melanoma cells decreased with the high molar concentration of cisplatin.

Figure-9 Effect of molarity of Tarceva on B16 cell viability.

In this case, the number of mole of drug Tarceva increased the viability of cell reduced significantly. B16 melanoma cells reduced rapidly in the range of molarity of Tarceva of 25x10-3 - 25x10-2 M. From all result it is clear that Tarceva is a potent drug on melanoma B16 cells.

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