In my opinion, to properly assess the in vitro anticancer activity of any compound or extract you need cancer cells, nonmalignant cells, standard anticancer drugs, a simple cytotoxicity test (MTT, SRB, resazurin, etc), and an answer to the question: Can my compound improve the ability of the standard drugs to kill cancer cells without significantly affecting nonmalignant cells from appropriate tissues? http://www.amazon.com/dp/B00MMO25NM/
The minimum requirement is a human cancer cell line, a human nonmalignant cell line from the same tissue origin than that of the selected cancer cell line, and an anticancer drug commonly used in patients with the selected cancer. Both the cancer cell line and the nonmalignant cell line should be of human origin and of the same tissue origin to ensure that the selective anticancer effect is not caused by species or tissue differences in sensitivity (http://www.ncbi.nlm.nih.gov/pubmed/23752179).
The simplest method to assess whether a compound can improve the ability of the standard drugs to kill cancer cells selectively consists of
1) exposing a cancer cell line and a nonmalignant cell line originated from the same tissue to the experimental drug and to the standard anticancer drug,
2) calculating one or several cytotoxicity parameters (e.g., IC50, IC90 and LC50) for each drug in both cell lines after estimating cell viability with a cytotoxicity test (e.g., SRB assay or MTT assay, resazurin assay),
3) calculating a selectivity index for each drug, e.g., by dividing the IC50 value in the nonmalignant cell line by that in the cancer cell line, and
4) comparing the selectivity index of the experimental drug with that of the standard drug.
If the selectivity index of the experimental drug is higher than that of the standard anticancer drug, one can conclude that the experimental drug may have therapeutic potential.
This simple method is suitable to rapidly screen the in vitro therapeutic potential of large series of compounds against a particular cancer. It is suitable to show that a compound has low therapeutic potential for the selected cancer (when its selectivity index is lower than that of the standard anticancer drug). However, additional nonmalignant cell lines should be used to more robustly predict the cancer therapeutic potential of compounds that improve the selectivity index of the standard drugs. Using more nonmalignant cell lines (or primary cells) is essential to detect possible toxicity towards normal tissues that may limit the therapeutic potential of the experimental drug. For example, a compound that kills liver cancer cells at 1 micromolar and liver nonmalignant cells at 100 micromolar will have a low therapeutic potential if it also kills normal cells from other tissues (e.g., skin cells or blood cells) at 1 micromolar. If the study is focused on a particular cancer, the in vitro therapeutic potential of a compound can be robustly assessed by using one or several cancer cell lines originated from a particular cancer and several nonmalignant cell lines (or primary cells) originated from a variety of normal tissues (including the tissue from which the cancer cell lines are originated). The selectivity indices can be calculated by dividing the mean IC50 value obtained for each drug in the nonmalignant cell lines by that obtained in the cancer cell lines. In addition to comparing the selectivity index of the experimental drug with that of the standard drug, it is important to observe that none of the nonmalignant cell lines is highly sensitive to the cytotoxicity of the experimental compound. One should remember that a high toxicity to normal cells from just one particular tissue may cause that the maximum doses tolerated by the patients are insufficient to reach the drug concentrations required to efficiently kill the cancer cells of their bodies. If the study is not focused on a particular cancer, one should use further pairs of well-characterized cancer cell lines and nonmalignant cell lines, as well as standard drugs used to treat patients with the selected cancers.
Yes,,, in Cancer Chemoprevention Research Center, we usually used MTT or MTS assay for screening anticancer plant extract.. you can see our protocol here..
http://www.ccrc.farmasi.ugm.ac.id/
Thanks 4 your advise, but unfortunately i didn't find the suggested protocol ?
Hi Aditya, Really i find many protocols but the problem is the language, protocols are not written in English, Can you help me please?
Hi Ahmed,
The MTT cell proliferation assay protocols are quite commons and can be found at this web site http://www.protocol-online.org/prot/Cell_Biology/Cell_Growth___Cytotoxicity/MTT_Cell_Proliferation_Assay/index.html. Please, let me know if you have addituonal questions.
Bests
Rolando
In my opinion, to properly assess the in vitro anticancer activity of any compound or extract you need cancer cells, nonmalignant cells, standard anticancer drugs, a simple cytotoxicity test (MTT, SRB, resazurin, etc), and an answer to the question: Can my compound improve the ability of the standard drugs to kill cancer cells without significantly affecting nonmalignant cells from appropriate tissues? http://www.amazon.com/dp/B00MMO25NM/
The minimum requirement is a human cancer cell line, a human nonmalignant cell line from the same tissue origin than that of the selected cancer cell line, and an anticancer drug commonly used in patients with the selected cancer. Both the cancer cell line and the nonmalignant cell line should be of human origin and of the same tissue origin to ensure that the selective anticancer effect is not caused by species or tissue differences in sensitivity (http://www.ncbi.nlm.nih.gov/pubmed/23752179).
The simplest method to assess whether a compound can improve the ability of the standard drugs to kill cancer cells selectively consists of
1) exposing a cancer cell line and a nonmalignant cell line originated from the same tissue to the experimental drug and to the standard anticancer drug,
2) calculating one or several cytotoxicity parameters (e.g., IC50, IC90 and LC50) for each drug in both cell lines after estimating cell viability with a cytotoxicity test (e.g., SRB assay or MTT assay, resazurin assay),
3) calculating a selectivity index for each drug, e.g., by dividing the IC50 value in the nonmalignant cell line by that in the cancer cell line, and
4) comparing the selectivity index of the experimental drug with that of the standard drug.
If the selectivity index of the experimental drug is higher than that of the standard anticancer drug, one can conclude that the experimental drug may have therapeutic potential.
This simple method is suitable to rapidly screen the in vitro therapeutic potential of large series of compounds against a particular cancer. It is suitable to show that a compound has low therapeutic potential for the selected cancer (when its selectivity index is lower than that of the standard anticancer drug). However, additional nonmalignant cell lines should be used to more robustly predict the cancer therapeutic potential of compounds that improve the selectivity index of the standard drugs. Using more nonmalignant cell lines (or primary cells) is essential to detect possible toxicity towards normal tissues that may limit the therapeutic potential of the experimental drug. For example, a compound that kills liver cancer cells at 1 micromolar and liver nonmalignant cells at 100 micromolar will have a low therapeutic potential if it also kills normal cells from other tissues (e.g., skin cells or blood cells) at 1 micromolar. If the study is focused on a particular cancer, the in vitro therapeutic potential of a compound can be robustly assessed by using one or several cancer cell lines originated from a particular cancer and several nonmalignant cell lines (or primary cells) originated from a variety of normal tissues (including the tissue from which the cancer cell lines are originated). The selectivity indices can be calculated by dividing the mean IC50 value obtained for each drug in the nonmalignant cell lines by that obtained in the cancer cell lines. In addition to comparing the selectivity index of the experimental drug with that of the standard drug, it is important to observe that none of the nonmalignant cell lines is highly sensitive to the cytotoxicity of the experimental compound. One should remember that a high toxicity to normal cells from just one particular tissue may cause that the maximum doses tolerated by the patients are insufficient to reach the drug concentrations required to efficiently kill the cancer cells of their bodies. If the study is not focused on a particular cancer, one should use further pairs of well-characterized cancer cell lines and nonmalignant cell lines, as well as standard drugs used to treat patients with the selected cancers.
Hi Dr. Lazaro,
you mentioned:
... 3) calculating a selectivity index for each drug, e.g., by dividing the IC50 value in the nonmalignant cell line by that in the cancer cell line ...
If we use a primary cells, e.g. breast cancer and lung cancer; then what non-malignant cell to be tested?
thanks, anthony
it is found that the SRB assay is sensitive, simple, reproducible and more rapid than the formazan- based assays and gives better linearity, a good signal-to-noise ratio and has a stable end-point that does not require a time-sensitive measurement, as do the MTT or XTT assay. Keepers, Y., Pizao, P., Peters, G., van Ark-Otte, J., Winograd, B. and Pinedo, H. (1991). Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing. European Journal of Cancer and Clinical Oncology, 27(7), pp.897-900.
I want assay anticancer property of plants extracts, but not using animal cells lines, if anyone suggest to me, such type of protocol or prototype , it's my way to change in research interests
You can use MTT or MTS or SRB methods to test the anticancer activity of a pant extract in cell culture.
regards
Is there any other way(s) to screen anti cancer activity of plant extract/isolated compound from the extract apart from using cell line?
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