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Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit

Silpi S, Hossain DS, Mukherjee S, Mohanty S, Mazumdar M, Mukherjee S, Ghoah UK, Nayek C, Raveendar C, Shurana A, Chakrabarty R, Sa G, Das T. Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immune-modulatory circuit. BMC Comp Alt. 2013; 13:230.

BACKGROUND: Complementary medicines, including homeopathy, are used by many patients with cancer, usually alongside with conventional treatment. However, the molecular mechanisms underneath the anti-cancer effect, if any, of these medicines have still remained unexplored. To this end we attempted to evaluate the efficacy of calcarea carbonica, a homeopathic medicine, as an anti-cancer agent and to delineate the detail molecular mechanism(s) underlying calcereacarbonica-induced tumor regression.

METHODS:

To investigate and delineate the underlying mechanisms of calcarea carbonica-induced tumor regression, Trypan blue dye-exclusion test, flow cytometric, Western blot and reverse transcriptase-PCR techniques were employed. Further, siRNA transfections and inhibitor studies were used to validate the involvement of p53 pathway in calcarea carbonica-induced apoptosis in cancer cells.

RESULTS:

Interestingly, although calcarea carbonica administration to Ehrlich's ascites carcinoma (EAC)- and Sarcoma-180 (S-180)-bearing Swiss albino mice resulted in 30-35% tumor cellapoptosis, it failed to induce any significant cell death in ex vivo conditions. These results prompted us to examine whether calcarea carbonica employs the immuno-modulatory circuit in asserting its anti-tumor effects. Calcarea carbonica prevented tumor-induced loss of effector T cell repertoire, reversed type-2 cytokine bias and attenuated tumor-induced inhibition of T cell proliferation in tumor-bearing host. To confirm the role of immune system in calcarea carbonica-induced cancer cell death, a battery of cancer cells were co-cultured with calcarea carbonica-primed T cells. Our results indicated a "two-step" mechanism of the induction of apoptosis in tumor cells by calcarea carbonica i.e., (1) activation of the immune system of the host; and (2) induction of cancer cell apoptosis via immuno-modulatory circuit in p53-dependent manner by down-regulating Bcl-2:Bax ratio. Bax up-regulation resulted in mitochondrial transmembrane potential loss and cytochrome c release followed by activation of caspase cascade. Knocking out of p53 by RNA-interference inhibited calcarea carbonica-induced apoptosis thereby confirming the contribution of p53.

CONCLUSION:

These observations delineate the significance of immuno-modulatory circuit duringcalcarea carbonica-mediated tumor apoptosis. The molecular mechanism identified may serve as a platform for involving calcarea carbonica into immunotherapeutic strategies for effective tumor regression.