Calcarea carbonica induced tumor apoptosis in vivo but not in vitro. Swiss albino mice were intra-peritoneally injected with 1×106 EAC (Ehrlich’s ascites carcinoma). After 1 week, placebo/calcarea carbonica (1C, 6C, 12C, 30C and 200C) were administered orally for 27 days. (A) Hereafter every 3 days the viable EACs were counted from the peritoneal cavity of mice and represented graphically. (B) Kaplan-Meir plot depicting survival rates in untreated, placebo- and calcarea carbonica-treated tumor-bearing mice. Arrow heads represent the statistical significance between survival percentages of un-/calcarea carbonica-treated tumor-bearing mice (p < 0.001). (C) Graphical representation of tumor cell viability after re-treatment with calcarea carbonica for 27 days to confirm that calcarea carbonica does not induce resistance. (D) Phase contrast images showing morphological changes of EAC cells after drug treatment. Bar length in images indicate 20 μm (E) At day 21 after placebo-/calcarea carbonica-/IL2-treatment percent PBMC and tumor cell death was determined by Trypan blue dye-exclusion test. (F) Graphical representation of tumor volume from placebo-/calcarea carbonica-treated tumor-bearing mice at day 21. (G) The nature of calcarea carbonica-induced tumor cell was assayed flow cytometrically using cell cycle phase distribution assay (upper panel) and Annexin-V-PE/7-AAD double labelling assay (middle panel). DAPI staining revealed nuclear morphology of apoptotic cells as indicated by arrowheads (lower panel). Bar length in images indicate 20 μm. (H) Graphical representation of percent apoptosis from control, untreated, placebo- and calcarea carbonica-induced murine and human cancer cell death was measured flow cytometrically under both in vitro and in vivo conditions. Values are mean ± SEM of five independent experiments. *p < 0.05 and **p < 0.001 when compared with respective control/treated groups.
Saha et al. BMC Complementary and Alternative Medicine 2013 13:230 doi:10.1186/1472-6882-13-230