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


CONCLUSIONS
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.
Division of Molecular Medicine, Bose Institute, P1/12, CIT Scheme VIIM, Kolkata 700054, India. gauri@jcbose.ac.in.
Shilpi
S
Saha
Dewan Md Sakib
DM
Hossain
Shravanti
S
Mukherjee
Suchismita
S
Mohanty
Minakshi
M
Mazumdar
Sanhita
S
Mukherjee
Uttam K
UK
Ghosh
Chaturbhuj
C
Nayek
Chinta
C
Raveendar
Anil
A
Khurana
Rathin
R
Chakrabarty
Gaurisankar
G
Sa
Tanya
T
Das
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms; D002119:Calcium Carbonate / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D005260:Female; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000187:drug effects; D006377:T-Lymphocytes, Helper-Inducer / Q000187:drug effects; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D016159:Tumor Suppressor Protein p53; D002119:Calcium Carbonate