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Open Access Highly Accessed Research article

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

Shilpi Saha1, Dewan Md Sakib Hossain1, Shravanti Mukherjee1, Suchismita Mohanty1, Minakshi Mazumdar1, Sanhita Mukherjee2, Uttam K Ghosh1, Chaturbhuj Nayek3, Chinta Raveendar3, Anil Khurana3, Rathin Chakrabarty4, Gaurisankar Sa1* and Tanya Das1*

Author Affiliations

1 Division of Molecular Medicine, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata 700054, India

2 Department of Physiology, Calcutta National Medical College, Kolkata 700014, India

3 Central Council for Research in Homeopathy, 61-65, Institutional Area, Janakpuri, New Delhi 110058, India

4 Bholanath Chakrabarty Trust, 5, Subol Koley lane, Howrah 711101, India

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BMC Complementary and Alternative Medicine 2013, 13:230  doi:10.1186/1472-6882-13-230

Published: 21 September 2013

Abstract

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 calcerea carbonica-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 cell apoptosis, 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 during calcarea carbonica-mediated tumor apoptosis. The molecular mechanism identified may serve as a platform for involving calcarea carbonica into immunotherapeutic strategies for effective tumor regression.

Keywords:
Apoptosis; Cancer; Calcarea carbonica; Breast cancer; p53