How to go about testing (in-vitro & in-vivo) the anti-cancer or anti-proliferative activity of a natural plant extract compound against bone/osseous cancer. And which bone cancer cell lines could be used for the same in In-vitro analysis. I don't have much knowledge in the field of Cancer cell lines. I would greatly appreciate any insights in this field.
Hello Mansha Saxena
You may test the anti-proliferative activity of a natural plant extract compound in vitro against bone/osseous cancer on MG-63 osteosarcoma human cell line by using MTT assay at 570nm.
In MTT assay, try using various concentration of the extract say in the range of 25ug/ml to 500ug/ml for various time points (24, 48 and 72 hours).
You may follow the MTT protocol as provided below.
1. Plate the MG-63 cells in the 96-well plate at the desired cell density such that they are 80% confluent on the day of readout. If cells are overconfluent on the final day of the assay, you may end up with inaccurate results.
2. After 24 hours, when the cells have attached to the substratum, treat the cells with the extract by changing the media with the treatment media at various concentration of the extract as mentioned above.
3. Incubate the cells with the treatment media for 24, 48 and 72 hours.
4. After the treatment period, aspirate the treatment media and add MTT to a final concentration of 0.2 to 0.5mg/ml and incubate in the dark for 1 to 4 hours for the formation of formazan crystals.
5. After incubation, solubilize the crystals into a colored solution with the solubilizing solution which may include either dimethyl sulfoxide or acidified isopropanol solution, or a solution of 10% SDS in 0.01M hydrochloric acid.
6. Measure the absorbance at a wavelength of 570 nm.
You may also use live & dead assay, which is a quick two-color assay, used to determine the viability of cells based on plasma membrane integrity and esterase activity in live cells.
Also, you may use LDH assay which is a method used for the evaluation of cellular toxicity since LDH release is considered as an early event of necrosis.
For in vivo experiment, a xenograft for MG-63 may be created in nude mice and treated with the extract.
You may follow the protocol as provided below.
1. You may generate a human osteosarcoma tumor xenograft by subcutaneous (s.c.) in vivo injection of human MG-63 osteosarcoma cell line (10 -15×10^6 cells/flank/0.2 ml) in the right flank of adult athymic female nude mice.
2. After the tumor inoculation (say approximately 5- 7 days later) the animals may be randomly divided into four groups of say six mice each:
Group 1 (Negative control) receiving the vehicle control orally once daily for say 15 days.
Group 2 (positive control) receiving a standard drug for bone cancer orally once daily for 15 days.
Group 3 (Experimental group A) receiving a low dose of the extract dissolved in an appropriate solvent orally once daily for 15 days.
Group 4 (Experimental group B) receiving a high dose of the extract dissolved in an appropriate solvent orally once daily for 15 days.
The mice may be euthanized at the end of 15 days of treatment period and the tumors may be excised and weighed. The tumor volumes may be measured according to the formula given below:
Tumor volume = A × B^2 × 0.5
where A: length
B: width
You may also use the excised tumor fixed in formalin (37%) for histological analyses by a trained pathologist.
I hope this may be helpful.
Good Luck!
Hello Malcolm Nobre. Thank you for taking the time to provide such a comprehensive and informative response to my query.
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