“Determination of the chemo-protective effects of specific Phyto-compounds in normal cells against the adverse effects of Breast Cancer chemotherapy”
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Amygdalin, a naturally occurring glycoside used in traditional Chinese medicine, can mostly be isolated from the kernels of apricot, cherries, almonds and plums. Even though the anti-cancer properties of Amygdalin are well known, its effect on normal cells has not been thoroughly investigated. The aim of the present study was to investigate and unveil a possible chemo-protective role of Αmygdalin against the cytotoxic effects of chemotherapy for normal human cells. Specifically, it was tested in combination with a strong chemotherapeutic drug used for breast cancer treatment, known as Cisplatin, cisplatinum, or cis-diamminedichloroplatinum (II). Human non-cancer immortalized breast cells MCF-12F, human fibroblasts cells, human breast cancer MCF-7 and MDA MB-231 cells were treated with Cisplatin in a dose- and time-depended manner in the absence or presence of Amygdalin. The effect of Amygdalin and Cisplatin treatment on normal (MCF-12F) and cancer (MCF-7, MDA-MB-231) breast cells as well as in fibroblasts (FBS) was investigated using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, Flow Cytometry with Annexin V/PI double staining, Western Blotting, Real Time-PCR and by calculation of Combination Index (CI). When MCF-12F cells and fibroblasts underwent pre-treatment with Amygdalin followed by Cisplatin treatment (24 hrs Amygdalin + 24 hrs Cisplatin), the cell viability was increased (22%, p < 0.001) as indicated using MTT assay. Using Flow Cytometry, the proportion of Annexin V/PI positive cells following combination treatment was found to be significantly lower: 1) when 15μM Cisplatin treatment was used alone or with 10mM Amygdalin when in combination (from 16% reduced to 10%, p < 0.01) and 2) when 20μM Cisplatin was used alone or with 20μM Amygdalin when in combination (from 23% reduced to 9%, p < 0.01). Moreover, the levels of PUMA (pro-apoptotic gene), P53 and BAX mRNA were significantly downregulated (~83%, ~66% and ~44%, respectively) compared with Cisplatin alone (p < 0.05, p < 0.01, p < 0.001 respectively), while the mRNA levels of B-cell lymphoma 2 (BCl-2) and B-cell lymphoma-extra-large (Bcl- XL) were upregulated (~44.5% and ~51%, respectively), again compared with Cisplatin alone (p < 0.05, p < 0.001 respectively ) after 24h of combination treatment. The study on the Combination index (CI) assay indicated that Amygdalin could be possibly considered as an antagonist to Cisplatin with a score of 2.2 and 2.3 (>1) for MCF-12F and Fibroblast cells respectively. In contrast, for the breast cancer MCF-7 and MDA MB-231 cells, Amygdalin and Cisplatin indicated a synergistic effect with a CI score of 0.8 and 0.65 (<1), respectively. Concluding, our findings suggest that when Amygdalin is used in combination with chemotherapeutic agents and especially Cisplatin can possibly significantly protect the non-cancerous (normal breast cells) as well as the fibroblasts during chemotherapy treatment; thus indicating a strong potential selective chemoprotective ability and role. Nowadays, chemoprotective agents are very limited and inventions associated with such characteristics are of major importance and medical need. Our results, for first time, possibly provide scientifically new insights of Amygdalin as a natural strong chemoprotective agent and potentially able to contribute to a better quality of life for cancer patients.