EVOLUTION OF ANTICANCER PROPERTIES FROM HERBAL PLANTS

(SUMMER TRAINING PROJECT REPORT) CARRIED OUT AT

TRIMS LABORATORIES-VISAKHAPATNAM.

Submitted By:
ASIF NIHAZ . SHAIK M.SC Biotechnology Batch: 2010-2011 Enroll No. : A7104107146 S.V.R.M COLLEGE NAGARAM , GNT DISTRICT

ACKNOWLEDGEMENT

I offer my thanks and gratitude to TRIMS LABORATORIES for giving me an opportunity to learn Anti cancer properties of various Herbal plants at their lab. I take this opportunity with immense pleasure to extend my sense of gratitude to my project guide Mr. sessei (operations-head) at TRIMS LABS for his most relevant support and encouragement throughout the training without which the successful completion of the target project would not have been possible. I would like to express my special gratitude to Ms. vissu for her kind guidance and assistance throughout my training period. I am also thankful to Mr. John for his support during the project and deeply indebted to management, all the employees who were very much cooperative during my project and I really appreciate the organizational culture of TRIMS LABS. Last but not the least I would like to thank all the researchers, scholars and scientists who made their research work available on net as going through it gave me great insight and knowledge about the subject in hand. Place: Visakhapatnam Date: September15, 2010 With Sincere Thanks ASIF NIHAZ SHAIK

INDEX
1. 2. 3. 4. 5. 6. 7. ABSTRACT INTRODUCTION REVIEW OF LITERATURE AIMS AND OBJECTIVES METHODS AND METHODOLOGY RESULTS AND DISCUSSION REFERENCES

INTRODUCTION
Cancer is a class of diseases in which a group of cells display uncontrolled growth, invasion that intrudes upon and destroys adjacent tissues, and sometimes metastasis, or spreading to other locations in the body via lymph or blood. These three malignant properties of cancers differentiate them from benign tumors, which do not invade or metastasize.

Researchers divide the causes of cancer into two groups: those with an environmental cause and those with a hereditary genetic cause. Cancer is primarily an environmental disease, though genetics influence the risk of some cancers.Common environmental factors leading to cancer include: tobacco, diet and obesity, infections, radiation, lack of physical activity, and environmental pollutants. These environmental factors cause or enhance abnormalities in the genetic material of cells.Cell reproduction is an extremely complex process that is normally tightly regulated by several classes of genes, including oncogenes and tumor suppressor genes. Hereditary or acquired abnormalities in these regulatory genes can lead to the development of cancer. A small percentage of cancers, approximately five to ten percent, are entirely hereditary.

The presence of cancer can be suspected on the basis of symptoms, or findings on radiology. Definitive diagnosis of cancer, however, requires the microscopic examination of a biopsy specimen. Most cancers can be treated. Possible treatments include chemotherapy, radiotherapy and surgery. The prognosis is influenced by the type of cancer and the extent of disease. While cancer can affect people of all ages, and a few types of cancer are more common in children, the overall risk of developing cancer increases with age. In 2007 cancer caused about 13% of all human deaths worldwide (7.9 million). Rates are rising as more people live to an old age and lifestyles change in the developing world.

Types of cancer
The three most common cancers in men in the United States are:

* Prostate cancer * Lung cancer * Colon cancer

In women in the United States, the three most common cancers are:

* Breast cancer * Colon cancer * Lung cancer

Some cancers are more common in certain parts of the world. For example, in Japan, there are many cases of stomach cancer, but in the United States, this type of cancer is pretty rare. Differences in diet may play a role.

Some other types of cancers include:

* Brain cancer * Cervical cancer * Hodgkin's lymphoma * Kidney cancer * Leukemia * Liver cancer * Non-Hodgkin's lymphoma * Ovarian cancer * Skin cancer * Testicular cancer * Thyroid cancer * Uterine cancer

Symptoms of Cancer

A broad spectrum of non-specific cancer symptoms may include:

Persistent Fatigue: Fatigue is one of the most commonly experienced cancer symptoms. It is usually more common when the cancer is advanced, but still occurs in the early stages of some cancers. Anemia is commonly the culprit -- a condition that is associated with many types of cancer, especially types affecting the bowel. Fatigue is a symptom of both malignant and non-malignant conditions and should be evaluated by a physician.

Unintentional Weight Loss: While it may be a welcome surprise to lose weight without trying, it can be a red flag for many illnesses, including cancer. Losing 10 pounds or more unintentionally definitely warrants a visit to the doctor. This type of weight loss can occur with or without loss of appetite. Remember, weight loss can be a symptom of cancer, but is also a symptom of many other illnesses, too.

Pain Typically, pain is not an early symptom of cancer, except in some cancer types like those that spread to the bone. Pain generally occurs when cancer spreads and begins to affect other organs and nerves.

Lower pack pain is cancer symptom that is associated with ovarian cancer and colon cancer. Shoulder pain can also be a symptom of lung cancer. Pain in the form of headaches can be associated with brain tumors (malignant and benign).

Stomach pains can be related to types of cancer, like stomach cancer, pancreatic cancer, and many others. Stomach pain can be a very vague symptom because so many illnesses can cause stomach pain.

Fever: A fever is a very non-specific symptom of many mild to severe conditions, including cancer. In relation to cancer, a fever that is persistent or one that comes and goes frequently can signal stress on the immune system. Fevers are commonly associated with types of cancer that affects the blood, like leukemia and lymphoma, but are also common in people whose cancer has spread.

Bowel Changes: If you experience constipation, diarrhea, blood in the stools, gas, thinner stools, or just a general overall change in bowel habits, see your doctor. These symptoms are most commonly associated with colon cancer, but are also related to other cancer types.

Chronic Cough: A persistent, new cough or a cough that won't go away or becomes worse needs to be evaluated by a doctor. Blood and/or mucus may accompany the cough and can be caused many conditions. In relation to cancer, a chronic cough with blood or mucus can be symptom of lung cancer.

Keep in mind that these are very general, vague symptoms of cancer. If you have one or two of these symptoms, it is not a red flag for cancer but more an indication to your doctor to run certain medical tests. The symptoms listed above are experienced by most people with cancer at various stages of their disease, but are also linked to many other non-cancerous conditions.

Current Reseach on Prostate Cancer

* Prevention
A number of ongoing studies around the world are looking at the effects of a variety of vitamin and mineral supplements on prostate health. Selenium, vitamin E, vitamin D, soy compounds, lycopene, and multivitamin tablets are all being studied for any possible benefit (or harm) that they may offer in terms of prevention.

* Diagnosis
Recent advances in medical imaging have brought about better methods for detecting cancer that has spread outside of the prostate.

Enhanced MRI", for example, is a fairly new technique that is being studied. It involves a special technique that causes the patients lymph nodes to light up on the MRI if cancer exists in them. This technique is being studied to determine if it is significantly better than standard MRI.

*Standard Treatments
Significant improvement in the way that radiation is administered to prostate cancer patients has happened in recent years and more advances are coming. With the aid of sophisticated computers that help to plan the treatments, radiation oncologists can now much more accurately pinpoint the radiation and minimize surrounding tissue damage. More and more research is being done to make radiation even more precise.

* Immune Therapy and Vaccines

Prostate cancer vaccines have been a source of significant excitement for a number of years now. The hope is that, after injecting a small amount of a substance into the body, the body will mount a large immune response to attack both the substance injected as well as prostate cancer cells. This is one of the hottest areas in current prostate cancer research.

The National Cancer Institute offers more information about cancer vaccines.

*Drugs to Slow Cancer Growth

Many drugs are being developed to slow or stop the growth of cancers. Some drugs act to block the cancerous cells from dividing, others inhibit their movement, and others block their ability to make their own blood vessels. All of these drugs may eventually have application to prostate cancer. Overall, prostate cancer research is an exciting field with many breakthroughs in research occurring every year from prevention to treatment.

TYPES OF CELL LINE

WHAT IS A CELL LINE?

A cell line is a product of immortal cells that are used for biological research. Cells used for cell lines are immortal, that happens if a cell is cancerous. The cells can perpetuate division indefinitely which is unlike regular cells which can only divide approximately 50 times. These cells are 'useful' for experimentation in labs as they are always available to researchers as a product and do not require what is known as 'harvesting' (the acquiring of tissue from a host) every time cells are needed in the lab. Different types of cell lines MCF-7 CELL LINEMeaning- Michigan Cancer Foundation-7 Organism-Human Origin tissue-Mammary gland Morphology-Invasive breast ductal carcinoma MCF-7 is a breast cancer cell line isolated in 1970 from a 69 year old Caucasian woman.MCF-7 is the acronym of Michigan cancer foundation-7, referring to the institute in detroit where the cell line was established in 1973 by Herbert Soule and co-workers. The Michigan cancer foundation is now known as the Barbara Ann Karmanos cancer institute.

Prior to MCF-7, it was not possible for cancer researchers to obtain a mammary cell line that was capable of living longer than a few months.

Characteristics Primary tumor-Invasive breast ductal carcinoma Origin of cells-pleural effusion Presence of estrogen receptors Proliferative response of estrogens Presence of progesterone receptors Absence of ERBB2 gene amplification Tumorigenicity in mice is present, but only with estrogen supplementation Phenotype-luminal epithelial This cell line retained several characteristics of differentiated mammary epithelium including the ability to process estradiol via cytoplasmic estrogen receptors and the capability of forming domes. Tumor necrosis factor alpha (TNF alpha) inhibits the growth of MCF-7 breast cancer cells. Treatment with anti estrogens can modulate the secretion of insulin like growth factor binding proteins. PIK3CA helical mutations were identified in MCF-7 but with low AKT activation.

CULTURE OF MCF-7 CELL LINESMedium-RPM1 1640 or L-15 media can used.MEM cocktail or DMEM can also be used. Eagles MEM-Supplemented with 10% FBS,1% Penicillin/Streptomycin. Can also add nonessential amino acids (0.1Mm), insulin (10micro grams/ml) and sodium pyruvate (1Mm). Add 10nM estrogen to media for a 3-4X increase in cell numbers.

Culture conditionsTemp-37 c Concentrated CO2 in atmosphere-5%

Hep2 cell line

Line established from human laryngeal carcinoma in 56 year old Caucasian male. Extensively used in viral studies. HEp-2 (human laryngeal carcinoma), WI-38 (human embryonic lung fibroblasts), and MRC-5 (human embryonic lung fibroblasts) cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD) and cultured in Eagle's modified essential medium (EMEM; Life Technologies GIBCO BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum, 2 mM glutamine, and penicillin/streptomycin (Life Technologies GIBCO BRL) at 37 C in 5% CO2 Designation: Hep-2 Organism: Homo sapiens (human) Tissue: Larynx Morphology: epithelial Celltype: epidermoid carcinoma Growth Properties: monolayer Description: The cells are positive for keratin by immunoperoxidase staining. Culture Medium: Minimum essential medium Eagle supplemented with 2 mM Lglutamine and Earle's BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids 1.0 mM sodium pyruvate and 10% fetal bovine serum Sub culturing: Remove medium, add fresh 0.025% trypsin/0.02% EDTA solution, rinse and remove trypsin. Allow flask to sit at room temperature (or at 37C) until cells detach. Add fresh medium, aspirate and dispense into new flasks. Split Ratio: A ratio of 1:4 to 1:10 is recommended Fluid Renewal:2 to 3 times weekly Freeze Medium:CM-1 (CLS Cell Lines Service)Sterility: Tests for mycoplasma, bacteria and fungi were negative Biosafety Level:1 Karyotype: hyper triploid, with abnormalities including dicentrics, breaks, pulverizations and minutes HeLa Markers:yesIsoenzymes:G6PD, A Reverse Transcriptase: negative Products: Keratin

Maintaining cells in culture

Cells are grown and maintained at an appropriate temperature and gas mixture (typically, 37 C, 5% CO2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed. Aside from temperature and gas mixture, the most commonly varied factor in culture systems is the growth medium. Recipes for growth media can vary in pH, glucose concentration, growth factors, and the presence of other nutrients. The growth factors used to supplement media are often derived from animal blood, such as calf serum. One complication of these blood-derived ingredients is the potential for contamination of the culture with viruses or prions, particularly in biotechnology medical applications. Current practice is to minimize or eliminate the use of these ingredients wherever possible and use chemically defined media, but this cannot always be accomplished. Alternative strategies involve sourcing the animal blood from countries with minimum BSE/TSE risk such as Australia and New Zealand, and using purified nutrient concentrates derived from serum in place of whole animal serum for cell culture. Plating density (number of cells per volume of culture medium) plays a critical role for some cell types. For example, a lower plating density makes granulosa cells exhibit estrogen production, while a higher plating density makes them appear as progesterone producing theca leutin cells. Cells can be grown in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so that they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or micro carrier, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is organotypic culture which involves growing cells in a three-dimensional environment as opposed to two-dimensional culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue, but is technically challenging to maintain because of many factors (e.g. diffusion).

METHODS AND METHODOLOGY

PROCEDURE-

Thawing Cells
1. Place frozen cells in 37C water bath for approximately 2 minutes or until cells are thawed. 2. Place cells into MEM media (which should be at room temperature) + 10% FBS for ten minutes. 3. Do a Trypan blue viability count on the cells. ____ x 50,000 = ____________________cells/ml x _____ml = ___________________________cells ________________________Percent viable 4. Centrifuge the cells at 1200 RPM for seven minutes at room temperature. 5. Resuspend the cells to a final concentration of 105 cells/ml.

Culturing the Cells

1. 2. 3. 4.

Place ~2.5 x 106 cells per culture flask (25 ml of 1 x 105 resuspended cells). Close the flask lid, but not too tightly. Place the flask with cells in the incubator at 37C with 5% CO2. Check the flasks daily for changes in media color and/or monolayer.

Changing the Cell Culture Media

1. Always leave one flask alone and feed the following day for fear of contamination problems. 2. 3. Cultures should be fed every two/three days once the media starts to change color. Take the culture flasks out of the incubator and place in the laminar flow hood.

4. Remove 50% to 75% of current media from the flask. Replace the amount taken with room temperature MEM media (10%FBS).

5.

Place the flask back into the incubator and record the information on the data sheet.

Sub culturing Cells

1. Remove present culture media.

2. Add 10 ml of 0.025% - 0.25% trypsin, and let the cells sit for 10 minutes at room temperature. It may be necessary to bang the culture flasks on the hood counter to remove any sticky cells from the flask surface. 3. Immediately after the ten minutes have passed add room temp MEM media (10%FBS) to inactivate the trypsin. 4. ml. Perform a trypan blue viability count and adjust the cell concentration to 1 x 105 cells per

____ x 50,000 = ____________________cells/ml x _____ml = ___________________________cells ________________________Percent viable 5. 6. Add 2.5 x 106 cells per culture flask (25 ml of 1 x 105 resuspended cells). Close the flask lid, but leave slightly lose.

7. Place culture flasks back into the incubator and check daily for media changes/monolayer formation. 8. Record information on data sheet.

RESULTS AND DISSCUSSION

CYTOTOXICITY

Cytotoxicity is the quality of being toxic to cells. Examples of toxic agents are a chemical substance, an immune cell or some types of venom. Measuring cytotoxicityCytotoxicity assays can be used to measure cytotoxicity. They include MTT assay SRB(Sulforhodamine B)assay

MTT assay Colorimetric assay for measuring activity of enzymes that reduce MTT or close dyes (MTS, XTT, WSTs) to formazan dyes, giving a purple color. Used to assess the viability and the proliferation of cells. Also used to determine cytotoxicity of potential medicinal agents and toxic materials, since those agents would stimulate or inhibit cell viability and growth.

MTT- [3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide] A yellow color tetrazol Reduced to purple formazan in living cells Solubilization solution (usually dimethyl sulfoxide) is added to dissolve the insoluble purple formazan product into a colored solution. Then absorbance of colored solution can be quantified by measuring at a certain wave length (usually between 500 and 600 nm) by a spectrophotometer.

SignificanceReduction of MTT takes place when reductase enzymes are active, and therefore conversion is often used as a measure of viable (living) cells. Changes in metabolic activity can give large

changes in MTT or MTS results while the number of viable cells is constant. When the amount of purple formazan produced by cells treated with an agent is compared with the amount of formazan produced by untreated control cells, the effectiveness of the agent in causing death, or changing metabolism of cells, can be deduced through the production of a dose-response curve. Advantage- Its rapidly and precision and the lack of any radio isotope. PROTOCOL1) Plate 500-10,000 cells in 200ul media per well in a 96 well plate. Leave 8 wells empty for blank controls. 2) Incubate (37C, 5% CO2) overnight to allow the cells to attach to the wells. 3) Add 2ul of drug of interest dissolved in DMSO to each well. Place on a shaking table, 150rpm for 5 minutes, to thoroughly mix the samples into the media. 5) Incubate (37C, 5% CO2) for 1-5 days to allow the drug to take effect. 6) Make 2ml or more of MTT solution per 96 well plate at 5mg/ml in PBS. Do not make a stock as MTT in solution is not stable long-term. 7) Add 20ul MTT solution to each well. Place on a shaking table, 150rpm for 5 minutes, to thoroughly mix the MTT into the media. 8) Incubate (37C, 5% CO2) for 1-5 hours to allow the MTT to be metabolized. 9) Dump off the media. (dry plate on paper towels to remove residue if necessary. 10) Resuspend formazan (MTT metabolic product) in 200ul DMSO. Place on a shaking table, 150rpm for 5 minutes, to thoroughly mix the formazan into the solvent. 11) Read optical density at 560nm and subtract background at 670nm. Optical density should be directly correlated with cell quantity. MTT: DMSO: Thiazolyl Blue Tetrazolium Bromide Dimethyl sulfoxide