Herbs and other plant-based compounds have increasingly been recognized as useful for the prevention and treatment of cancer. There exists enormous scope for screening and evaluation of herbal/plant products to develop an effective radiosensitizer and radioprotector that is relevant for cancer therapy. Anticancer agents that can effectively trigger the process of cell death in tumor cells need to be developed. This study describes the effect of the flavonoid biochanin A (BCA), administered alone or in combination with gamma radiation, on the growth of radioresistant human colon cancer HT29 cells in vitro. Proliferation studies were carried out using MTT assay with increasing concentration of BCA (1-100 µM) followed by gamma irradiation at a dose of 2 Gy. Induced reactive oxygen species, mitochondrial membrane potential, lipid peroxidation, and caspase-3 activation were measured by fluorescence assays and the magnitude of induced apoptosis in cells was evaluated by flow cytometry. Cellular DNA damage was determined by comet assay. Combined treatment caused a significant decrease in cell proliferation, a substantial increase in the generation of reactive oxygen species, enhanced lipid peroxidation, and increased mitochondrial membrane potential in treated HT29 cells compared with controls. Significantly enhanced apoptosis and DNA damage were found with a combination of drug and radiation treatments. Furthermore, it was found that combined treatment yielded an additive increase of caspase-3 in these cells. Our findings indicate that BCA acts as a remarkable pro-oxidant, significantly enhancing the radiotoxicity of colon cancer cells in vitro.

Betulinic acid (BA) has been shown to cause apoptosis in neuroblastoma and melanoma cell lines. We evaluated the cytotoxicity of BA in two breast cancer cell lines MCF-7 and T47D differing in their p53 status. Treatment with BA resulted in a dose dependent inhibition of cell proliferation and induction of apoptosis. This indicates p53-independent apoptotic pathway, because response of both p53 mutant and wild type cell line were found unaffected after treatment with pifithrin-α, an inhibitor of p53. Cells were significantly protected when treated by tocopherol suggesting involvement of membrane centered lipid peroxidation-mediated mechanism in BA-induced apoptosis.

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To investigate the protective role of Oleic acid against the cytotoxic mediators present in the low dose irradiated cell conditioned medium (ICCM) to unirradiated normal human keratinocyte cell line (HaCaT). Cell survival was investigated using the clonogenic assay, media transfer of HaCat cells exposed to low-LET γ radiation (0.5Gy) to by stander HaCat cells treated with oleic acid. The free radical scavenging property of oleic acid was performed by DCHFDA method and magnitude of lipid peroxidation was estimated with cisparineric acid as a probe using a fluorescence spectrometer. Mitochondrial membrane potential (∆ψm) was observed using DiOC 6 as a sensitive probe visualized under a confocal microscope. Based on clonogenic assay, it was found that a significant reduction in clonogenic survival was seen in unirradiated cells exposed to low dose ICCM. Results have further shown that HaCaT cells pretreated with oleic acid before the medium transfer showed an increased percentage of clonogenic survival suggesting protection of cells against the ICCM factors. Free radical scavenging activity was also reported in cell membrane and mitochondria. A correlation between the antioxidant properties of OA and an increase in clonogenic survival was seen. These results showed that oleic acid was protecting normal cells from indirect low-dose γ irradiated cells. It seems that ROS were involved in the mechanism of bystander effect which was significantly suppressed when OA was present.