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Cancer is a collective term used for a group of diseases that are characterized by the loss of control of the growth, division, and spread of a group of cells, leading to a primary tumor that invades and destroys adjacent tissues. It may also spread to other regions of the body through a process known as metastasis, which is the cause of 90% of cancer death.
The terms cancer, malignant neoplasm (new growth) and malignant tumor are synonymous; they are distinguished from benign tumors by the properties ofdedifferentiation,
invasiveness and the ability to met stasis (spread to other party of the body). (Both benign and malignant tumors manifest uncontrolled proliferation.)
Carcinoma is a malignant neoplasm of epithelial origin. It is a tumor that arises in the tissues that line the body`s organs like the nose, the colon,prostrate, urinary bladder, and the urethra. Most of all cancer cases are carcinomas. The common symptoms include is dysphagia and weight loss.Growth of the tumor, be related to swallowing, or be related to metastases into the surrounding esophageal lymph nodes may results in pain.
These are the tumors originate in fat,muscle,bone or connecting tissue. The tumor may arise in the femur (thigh bone) or the pelvis. It may also develop in the skull or the flat bones of the trunk. Clinical symptoms are few. The most common is pain and occasionally swelling at the site of the tumor. Fever may also be present .The tumor spreads easily, often to the lungs and other bones.
Leukemia's are cancers of the blood or blood-forming organs. When leukemia develops, the body produces a large number of abnormal blood cells. In most types of leukemia, the abnormal cells are white blood cells. The leukemia cells usually look different from normal blood cells, and they do not function properly. Leukemia can either be acute or chronic. In acute leukemia the abnormal blood cells are blasts that remain very immature and cannot carry out their normal functions. The number of blasts increases rapidly, thus creating a greater and earlier impact on the victim. In chronic leukemia, some blast cells are present which are comparatively more mature, and thus can carry out some of their normal functions.
Lymphomas affect the lymphatic system, a network of vessels and nodes that acts as the body`s filter. The lymphatic system distributes nutrients to blood and tissue, and prevents bacteria and other foreign "invaders" from entering the bloodstream. There are over 20 types of lymphoma. Hodgkin`s disease is one type of lymphoma. All other lymphomas are grouped together and are called non-Hodgkin`s lymphoma. Non-Hodgkin`s lymphoma may occur in a single lymph node, a group of lymph nodes, or in another organ. This type of cancer can spread to almost any part of the body, including the liver, bone marrow, and spleen.
Cell cycle inhibition
The cell cycle refers to the process of cell division. In normal tissues this process is tightly controlled by the activity of a number of key cellular proteins. A loss of cell cycle control may lead to an inappropriate proliferation of cells and ultimately leads to tumor formation. The cell cycle consists of:
G1 = growth and preparation of the chromosomes for replication
S = synthesis of DNA (and centrosomes)
G2 = preparation for chromosome duplication
M = mitosis
Chemotherapy is the treatment of cancer with drugs (anticancer drugs) that can destroy cancer cells. In current usage, the term "chemotherapy" usually refers to cytotoxic drugs which affect rapidly dividing cells in general.
Chemotherapy refers to the use of medication and drugs for treatment of cancer. Most chemotherapeutic drugs are cytotoxic and they work by killing cells. They do this by preventing the formation of new DNA or by blocking some other essential function in the cell. There is a limit on how much of these drugs could be safely used. Since all the chemotherapy drugs interfere with growth of the cells, they all have some form of undesired side effects.
ASSAY FOR PROLIFERATION STUDIES
Tryphan blue assay
Trypan Blue is a blue acid dye that has two azo chromophores group. Trypan blue will not enter into the cell wall of plant cells grown in culture. Trypan Blue is an essential dye, use in estimating the number of viable cells present in a population. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye and then visually examined to determine whether cells take up or exclude dye. In the protocol presented here, a viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm. An advantage of the trypan blue exclusion test is that is a simple and rapid method able to provide approximate results.
MATERIALS REQUIRED IN MEM:
Monolayer culture bottle of Hep 2 cells
5ml, 10ml serological pipette
Minimal essential media (MEM) with 10%, 2% foetal calf serum
TPVG (Trypsin, PBS, versene, glucose)
Discarding jar, inverted microscope, dessicator
Gloves, spirit, cotton, label pad, marker pen
MATERIALS REQUIRED IN CYTOTOXICITY ASSAY:
Monolayer culture in log phase
Drug extract (different concentrations)
MEM without FCS
5ml sterile storage vial
Tissue paper, spirit, cotton, marker pen and gloves
Micropipette and tips
MINIMAL ESSENTIAL MEDIA PREPARATION:
Media is defined as a complex source of nutritional supplementation vital for the growth proliferation and maintenance of cells in vitro.
The MEM vial is dissolved in the pre sterilized Millipore distilled water and mixed well, closed and sterilized at 15lbs 121°c for 15mins. Allow ingredients in the quantity, depending on the concentration of foetal calf serum (2% or 10%) mix well by shaking. Take care avoid spills pass CO2 using sterile pipette, Shake the bottle, check Ph and adjust to 7.2 to 7.4. The MEM bottles are kept for 2 days at 37°c and checked for sterility, PH drop and floating particles they are then transferred to the refrigerator.
PREPARATION OF INGREDIENT:
Penicillin and Streptomycin: (Concentration 100IU of Penicillin and 100 Âµg 0f Streptomycin)
Dissolve both antibiotics in sterile Millipore distilled water, so as to give a final concentration 100 IU of penicillin and 100Âµg of streptomycin/ml. Mix well and distribute in 1ml aliquots. Store at -20° C Check sterility.
Fungizone (amphotericin B): (conc: 20Âµg/ml)
Dissolve in sterile Millipore distilled water so as to give a final concentration of 20Âµg/ml and distribute in 1ml aliquots in vials. Store at -20°c. Check sterility.
Weigh 3g of l-glutamine accurately and dissolve in 100ml sterile Millipore distilled water and mix well. Filter through Millipore membrane filter 0.22Âµ and distribute in 5ml aliquots in vials. Store at -20°c. Check sterility.
4. 7.5% sodium-bi-carbonate
Weigh requisite quantity of sodium-bi-carbonate (to give 7.5% solution) accurately and dissolve in 100ml of sterile Millipore distilled water. Filter through what man filter paper No.4, distribute into bottles and at 121°c, 15lbs, 15mins. Cool and store at +4°c.
5. Foetal calf serum
Bring FCS at room temperature. Inactivated at 56°c in water bath forÂ½ hour and cool at room temperature. If floating particles are seen filter through Seitz filter. Distribute in 100ml,50ml, 20ml quantities in sterile bottles. Store at -20°c.
6. Trypsin, PBS,versene, glucose solution: (TPVG)
2% Trypsin: 100ml
Weigh 2g of trypsin accurately; dissolve in 100 ml sterile Millipore distilled water with magnetic stirrer for Â½ hour. Filter through membrane filter. Store at -20°c.
Weigh 200mg of EDTA accurately. Dissolve in 100 ml of sterile Millipore distilled water. Autoclave at 15lbs/15mins.
10% Glucose -100ml
Weigh 1g of glucose accurately. Dissolve in 100 ml of sterile Millipore distilled water and filter through whatman filter paper and autoclave at 15lbs/15mins.
PBS - 840ml
Penicillin & streptomycin -5ml
Mix all ingredients and adjust the pH to 7.4 with 0.1 N HCl or 0.1 N NaOH. Distribute in 100 ml aliquots. Store at -20°c.
MAINTENANCE OF CELL LINE:
Hep-2 cell line obtained from National centre for cell science,Pune.
Maintenance of cells involves the following operations:
Dispersion and Sub culturing (seeding) of cells.
Preservation of cells in repository.
Revival of cells from repository.
SUBCULTURING AND MAINTENANCE OF CELL LINE:
1. Bring the medium and TPVG to room temperature for thawing.
2. Observe the tissue culture bottles for growth, cell degeneration, pH and turbidity by seeing in inverted microscope.
3. If the cells become 80% confluent it goes for sub culturing process
4. Wipe the mouth of the bottle with cotton soaked in spirit to remove the adhering particles.
5. Discard the growth medium in a discarding jar keep distance between the jar and the flask.
6. Then add 4 - 5 ml of MEM without FCS and gently rinsed with tilting. The dead cells and excess FCS are washed out and then discard the medium.
7. TPVG was added over the cells. And incubate at 37° C for 5 minutes for disaggregation. The cells become individual and it's present as suspension.
8. Add 5ml of 10% MEM with FCS by using serological pipette.
9. Gently give passaging by using serological pipette. If any clumbs is present then repeat the process.
10. After passaging split the cells into 1:2, 1:3 ratio for cytotoxicity studies for plating method
"Seeding of cells":
After homogenize take one ml of suspension and pour in to 24 well plates. In each well add 1ml of the suspension and kept in a desiccators in 5% CO2 atmosphere. After 2 days incubation observe the cells in inverted microscope. If the cells became 80% confluent.
In order to study the antitumor activity of a new drug, it is important to determine the cytotoxicity concentration of the drug. Cytotoxicity tests define the upper limit of the extract concentration, which is non-toxic to the cell line. The concentration nontoxic to the cells is chosen for antiviral assay.
After the addition of the drug, cell death and cell viability was estimated. The result is confirmed by additional metabolic intervention experiment such as MTT assay.
PERFORMANCE OF DRUG CYTOTOXICITY ASSAY
Cytotoxicity is the toxicity or damage caused to the cells on addition of drug. After the addition of the drug, cell viability is estimated by staining techniques, where by cells are treated with Trypan blue. Trypan blue is excluded by live cells, but stains dead cells blue. The results are confirmed by additional metabolic intervention experiments such as MTT assays.
Monolayer cultures in log phase.
Drug extract (different concentrations)
MEM without FCS
0.45 ï filter
5mL sterile storage vial
Tissue paper, Marker pen, Spirit, Cotton and Gloves
1 mL, 2 mL pipettes, Micropipette and tips
Discarding jar with 1 % Hypochlorite solutio
Stock drug concentration
100 mg of drug is dissolved in 10 mL of serum free MEM giving a concentration of 10mg / 1 mL. The stock is prepared fresh and filtered through 0.45 ï filter before each assay. Working concentrations of drug ranging from 10mg/ml to 0.3125mg/mlare prepared as follow
Preparation of working stock of 1 mg /mL
To 4.5 ml MEM add 0.5 mL of stock to give a working concentration of 1mg/mL
Drug concentration can be prepared from the working stock in MEM without FCS. Prepare required volume of test sample for each concentration.
1.48hr monolayer culture of Hep2 cells at a concentration of one lakh /ml /well (10 cells / ml / well) seeded in 24 well titer plate.
2. The plates were microscopically examined for confluent monolayer, turbidity and toxicity if the cells become confluent.
3. The growth medium (MEM) was removed using micropipette. Care was taken so that the tip of the pipette did not touch the cell sheet.
4. The monolayer of cells was washed twice with MEM without FCS to remove the dead cells and excess FCS.
5. To the washed cell sheet, add 1ml of medium (without FCS) containing defined concentration of the drug in respective wells.
6. Each dilution of the drug ranges from 1:1 to 1:64 and they were added to the respective wells of the 24 well titer plates.
7. To the cell control wells add 1ml MEM (w/o) FCS.
8. The plates were incubated at 37°c in 5% CO2 environment and observed for cytotoxicity using inverted microscope.
MTT assay is called as (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide.MTT assay was first proposed by Mossman in 1982.
After incubation, remove the medium from the wells carefully for MTT assay.
In each well wash with MEM (w/o) FCS for 2 - 3 times. And add 200Âµl of MTT conc of (5mg/ml).
And incubate for 6-7hrs in 5% CO2 incubator for Cytotoxicity.
After incubation add 1ml of DMSO in each well and mix by pipette and leave for 45sec
If any viable cells present formazan crystals after adding solublizing reagent (DMSO) it shows the purple color formation.
The suspension is transferred in to the cuvette of spectrophotometer and an O.D values is read at 595nm by taking DMSO as a blank.
Cell viability (%) = Mean OD/Control OD x 100
Sample : CD - 8
TOXICITY-3 NORMAL Hep 2 CELLLINE
Sample : CD-6
TOXICITY-3 Normal Hep 2 Cell line