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

RESEARCH ARTICLE Open Access
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*
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
* Correspondence: gauri@jcbose.ac.in; tanya@jcbose.ac.in
1
Division of Molecular Medicine, Bose Institute, P1/12, CIT Scheme VIIM,
Kolkata 700054, India
Full list of author information is available at the end of the article
© 2013 Saha et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Saha et al. BMC Complementary and Alternative Medicine 2013, 13:230
http://www.biomedcentral.com/1472-6882/13/230

Background
Despite significant advances toward targeted therapy and
screening techniques, breast cancer continues to be the
leading cause of cancer-related deaths and the most fre-
quently diagnosed cancer among women worldwide [1].
Although chemotherapy plays an important role in the
treatment of breast cancer, the high percentage of failures
after initial responses and the adverse toxic side effects [2]
of chemotherapeutic drugs highlight the necessity of the
identification of novel agents that can suppress growth of
human breast cancers and are still relatively safe. In this re-
gard, the use of complementary and alternative medicine
(CAM) including homeopathic remedies is on the rise
worldwide, and patients with cancer are increasingly opting
to be treated with CAM therapeutic regimens [3-5]. A few
reports describe the anti-cancer effect of homeopathic
remedies and on their mechanism of action in experimen-
tal cancers and cell cultures [6-10]. Among conventionally
used homeopathic medicine, calcarea carbonica, which is
derived from the soft white middle layer of the oyster shell
that is composed of fine crystalline calcium carbonate
(CaCO3) with traces of other minerals, such as magnesium
carbonate, has been reported to have in vitro and in vivo
anti-cancer properties in a murine melanoma model [11].
However, the detail mechanistic studies affirming the anti-
cancer effect of calcarea carbonica are still inadequate.
It is now acknowledged that the multifaceted defect in
the immune capacity of patients with advanced malig-
nancy contributes not only to disease progression but also
constitutes a barrier to therapeutic interventions. Both hu-
man patients and experimental animals with advanced
cancer often exhibit a poorly functioning immune system
[12-15], manifested by decreased T cell proliferation [16],
alteration in signal-transducing molecules [17,18], reduced
CD4+
:CD8+
ratios, and deficient production of Th-1 cyto-
kines [16,19,20]. These alterations correlate with the sever-
ity of disease and with poor survival. On the other hand,
activation of tumor-suppressed immune system has been
observed to regress tumor via immuno-modulatory cir-
cuit. For example Das et al., have demonstrated that sol-
uble immune mediators like TNF-α and NO (Nitric oxide)
released from spleenic cells resulted in tumor apoptosis.
Importantly, many of the cancer drugs in use suppress im-
mune system [21] thereby adding to the causes of failure
of cancer therapeutic regimens. A few reports have shown
that calcarea carbonica, on the other hand, possessed
immuno-potentiating effects [11,22] and improved the im-
mune response against tumor cells or even induce direct
dormancy in malignancies [11]. All these information raise
a possibility that calcarea carbonica may regress cancer by
correcting the suppressed immune system of the tumor-
bearer.
Multiple pathways have been proposed by which im-
mune system can be stimulated to recognize and trigger
cancer cell apoptosis. Cytotoxic T lymphocytes (CTL) are
antigen-specific effector cells of the immune system with
the ability to lyse target cells in a contact-dependent
manner. Most CTL expressing antigen specific receptors
(TCRs) mediate the elimination of tumor cells by recogni-
tion of antigen in the form of individual peptides bound to
MHC molecules [23,24]. Operationally, apoptosis is initi-
ated by “death receptors” (TNF receptor, Fas, DR3, DR4,
and DR5), by p53-dependent and -independent cellular
stress pathways that induce permeability transition in
mitochondria and release of cytochrome c, and by the se-
cretion of granules that contain perforin and granzymes
from CTLs [25-28]. Studies by Dorothee et al. [29] suggest
that lung carcinoma-specific CTLs use mainly a granule
exocytosis-dependent pathway to lyse autologous target
cells and that these effectors are able to circumvent alter-
ation of the Fas-triggered intracellular signalling pathway
via activation of a caspase-independent cytoplasmic death
mechanism. Similarly Kawasaki et al. [30] have helped to
understand the intracellular trafficking events during the
very early stages of target cell apoptosis induced by CTLs.
The report that calcarea carbonica improves the immune
response against tumor cells [11] tempted us to hypothe-
size that calcarea carbonica regresses tumor via immuno-
modulatory circuit, the molecular basis of which needs to
be explored for future translational research.
In the present study we delineated the detail molecular
mechanisms underlying the anti-cancer effect of calcarea
carbonica. Interestingly our results indicate that although
calcarea carbonica (6C) resulted in 30-35% tumor cell
apoptosis when administered to Ehrlich’s ascites carcin-
oma (EAC) and S-180 bearing Swiss albino mice, it failed
to induce any significant cell death in ex vivo conditions.
Importantly, since calcarea carbonica 6C lessened tumor
burden significantly while 12C, 30C and 200C failed to
impart any decrease in tumor cell number, further studies
were performed using this dose of calcarea carbonica.
Moreover, while in tumor-bearing mice, there was pro-
found depletion of CD4+
and CD8+
cells in peripheral
circulation, dominance of T helper cell type-2 (Th2) that
dampened T cytotoxic cell type-1 immune responses, and
inhibition of T cell proliferation, calcarea carbonica pro-
tected the immune system from such tumor-insult. These
results tempted us to hypothesize that calcarea carbonica
might adopt a “two-step” mechanism of the induction of
apoptosis in tumor cells, i.e., (1) activation of the immune
system of the host, and (2) induction of cancer cell apop-
tosis via immuno-modulatory circuit. In an attempt to
confirm the role of calcarea carbonica-activated immune
system in cancer cell death, tumor cells were co-cultured
with T cells from calcarea carbonica-administered tumor-
bearing mice. Our results indicated that in comparison to
untreated T cells, calcarea carbonica-activated T cells in-
duced cancer cell apoptosis in p53-dependent manner by
Saha et al. BMC Complementary and Alternative Medicine 2013, 13:230 Page 2 of 19

down-regulating Bcl-2/Bax ratio that finally culminated at
the activation of mitochondrial death cascade. In sum-
mary, these observations for the first time delineate the
molecular mechanism underlying immuno-therapeutic ac-
tivity of calcarea carbonica against cancer that can be
exploited in future to achieve efficient tumor regression
via immuno-modulatory circuit.
Methods
(A) in vivo experiments
Placebo and drug details
The placebo (potentized hydroalcoholic solution) and dif-
ferent strengths (1C, 6C, 12C, 30C and 200C) of calcarea
carbonica were purchased from Hahnemann Publishing
Co. Pvt. Ltd., authorized manufacturing house certified by
GMP and ISO. The drugs procured were colorless, odor-
less, pre-sterilized and endotoxin free. The remedies were
stored in brown coloured glass containers at room
temperature, away from sunlight.
Treatment of animals
Swiss albino mice (NCLAS, Hyderabad, India) weighing
20-25 g were maintained in temperature-controlled room
with light–dark cycle. All animal experiments were per-
formed following ‘Principles of laboratory animal care’
(NIH publication No. 85–23, revised in 1985) as well as
Indian laws on ‘Protection of Animals’ under the provision
of the Ethics Committee for the purpose of control and
supervision of experiments on animals (Reg. No. 95/99/
CPCSEA), Bose Institute. The experimental sets were as
follows- 1) normal set (non-tumor bearing mice), 2)
tumor-bearing set which were intra-peritoneally injected
with 1×106
exponentially grown p53-wild-type-Ehrlich’s
ascites carcinoma (EAC), 3) placebo 6C-treated EAC-
bearing set, 4) calcarea carbonica 1C-treated EAC-bearing
set, 5) calcarea carbonica 6C-treated EAC-bearing set,
6) calcarea carbonica 12C-treated EAC-bearing set, 7)
calcarea carbonica 30C-treated EAC-bearing set and 8)
calcarea carbonica 200C-treated EAC-bearing set, 9) tumor-
bearing set which were intra-peritoneally injected with 1×106
exponentially grown p53-wild-type-Sarcoma-180 (S-180)
and 10) placebo 6C-treated S-180-bearing set, 11) calcarea
carbonica 6C-treated S-180-bearing set and 12) IL2-
treated EAC-bearing set. For experiments, the results of
which have been furnished in Figure 1A and 1C, each
group comprised of 3 mice for each time point. For rest of
the experiments, each group comprised of 5 mice.
The experiments were blind performed. Standard pub-
lished protocol [11] with few modifications was followed
for drug administration. To delineate the efficacy of
calcarea carbonica 6C to provide survival benefit to
tumor-bearing mice and to understand the refractory
nature of tumors harvested from calcarea carbonica 6C-
treated animals to re-treatment with the drug, calcarea
carbonica was orally administered by pipetting 1 ml/kg
of body-weight into the mouth of mouse, twice daily for
27 days (treatment started seven days after tumor inocu-
lation). Besides the above mentioned experiments, for
rest of the in vivo experiments calcarea carbonica treat-
ment was done twice daily for 21 days. Immediately be-
fore each treatment remedies were vigorously shaken
(succussed) by manually tapping on palm ten times.
Care was taken to give similar pressure at each stroke.
To delineate the efficacy of calcarea carbonica 6C to
provide survival benefit to tumor-bearing mice, the mice
were divided into 4 groups of 10 animals each including
normal set (non-tumor-bearing), tumor-bearing set (which
were intra-peritoneally injected with 1 × 106
exponentially
grown p53-wild-type-Ehrlich’s ascites carcinoma (EAC),
placebo 6C-treated tumor-bearing set and calcarea car-
bonica 6C-treated tumor-bearing set.
The end-point of the mice in the experiments was de-
cided by measuring the tumor burden. The experiment
was ended on day 28 of the tumor inoculation when the
tumor burden was less than one-fifth of the original
body-weight of mice. IL2 (300 IU/kg body-weight) was
administered to tumor-bearing mice for 21 days as posi-
tive control for the anti-tumor and immune-modulating
properties of calcarea carbonica 6C.
The efficacy of treatment on liquid tumors in mice
was examined by measuring the changes in the peri-
toneal ascites volume of un-treated, placebo-/calcarea
carbonica-treated tumor-bearing mice after completion
of treatments.
Transplantation and Re-treatment experiment
To transplant the tumor into new mice, 1 × 106
viable
Ehrlich’s ascites carcinoma (EAC) cells were inoculated
into the peritoneal cavity of the mice. The EACs were
sorted by negative selection using anti-CD3 and anti-
CD16 antibody coated micro-beads (Milteny Biotech).
Before inoculation, more than 98% of the CD16-/CD3-
negative cells were morphologically characterized as
EAC by Wright staining [19,20].
To understand the refractory nature of tumors to re-
treatment, EACs were isolated from the peritoneal cavity
of mice that had undergone 27 days of treatment with
calcarea carbonica. Mice were sacrificed on the specified
day and EACs were then subjected to sorting and cha-
racterization as described above. Viability was assessed
by Trypan blue dye exclusion. Viable 1 × 106
EAC cells
were then transplanted into the peritoneal cavity of nor-
mal mice. The experimental sets included un-/placebo
6C-/calcarea carbonica 6C-treated tumor-bearing mice.
Peripheral blood mononuclear cells (PBMC) isolation
Peripheral blood collected from mice was centrifuged
over Ficoll-Hypaque (Ammersham Pharmacia) density-

gradient to obtain total lymphocytes [15-17]. T cells were
purified by positive selection from total lymphocytes using
micro-beads coated with mouse/human anti-CD3 anti-
bodies (Milteny Biotech).
Phenotypic analysis of helper and cytotoxic T cells
For the determination of helper and cytotoxic T lympho-
cytes, T cells from thymus, spleen, lymph node and per-
ipheral blood from normal (non-tumor bearing) mice,
Figure 1 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.

control, placebo- and calcarea carbonica/IL2-treated
tumor-bearing mice were isolated after 21 days of treat-
ment, and labeled with PerCP-conjugated CD4, PE-
conjugated CD8 antibodies (BD Bioscience). Cells were
then analyzed in FACS (BD Bioscience) equipped with
488 nm argon laser light source and a 675/20-nm band
pass filter for PerCP-fluorescence and 575 nm band
pass filter for PE-fluorescence. Cells were properly ac-
quired, gated and analyzed using CellQuest Software
(BD Bioscience). To purify CD4+
and CD8+
T cells for
co-culture experiments, total T cell population isolated
from normal human blood was stained with anti-CD4-
PerCP and anti-CD8-PE antibodies. Stained cells were
then subjected to high speed cell sorting (FACS-Aria; BD
Bioscience) under sterile condition to obtain CD4+
-
depleted T cells and CD8+
-depleted T-cell populations.
For the determination of apoptosis, total T cell popula-
tion isolated after 21 days of treatment, from the periton-
eal cavity of un-/placebo-/calcarea carbonica-treated mice
were divided into two equal parts, one part was labeled
with PerCP-CD4 and Annexin-V-FITC and propidium
iodide, the other part was labeled with PE-CD8, 7-amino-
actinomycin D (7-AAD) and Annexin-V-FITC (BD Bio-
science) and analyzed on flowcytometer. Annexin-V+
cells
were regarded as apoptotic cells [19,20]. To prevent the
overlapping of fluorescent emission spectra of 7-AAD -
PE and PerCP - PI, the spectral patterns of respective
fluorochrome pairs were compensated during acquisition
of flowcytometric data.
Proliferation assay
The CD3+
cells isolated from peripheral blood of normal
mice (non-tumor bearing), control (un-treated), placebo
treated- and calcarea carbonica-treated tumor bearing mice
after 21 days of treatment, were loaded with 5-(and-6)-
carbonicaoxy fluorescein succinimidyl ester (CFSE; Mo-
lecular Probe) and proliferation was assessed by stimulating
CD3+
cells (1 × 106
cells/ml) in combination with cross-
linked anti-CD3 antibody and soluble anti-CD28 antibody
for 72 h. Decrease in CFSE-fluorescence as marker of cell
proliferation was assayed flow cytometrically [16].
Cell cycle phase distribution and apoptosis assay
For the determination of cell cycle phase distribution of
DNA content, EAC cells harvested from the peritoneal
cavity of un-/placebo-/calcarea carbonica-treated mice
tumor-bearing mice were permeabilized and nuclear DNA
was labelled with propidium iodide (PI) using Cycle TEST
PLUS DNA reagent kit. Cell cycle phase distribution of
nuclear DNA was determined on FACS, fluorescence de-
tector equipped with 488 nm argon laser light source and
623 nm band pass filter (linear scale) using CellQuest soft-
ware (Becton Dickinson). A total of 10, 000 events was ac-
quired and analysis of flowcytometric data was performed
using ModFit software. A histogram of DNA content
(x-axis, PI fluorescence) versus counts (y-axis) has been
displayed [21].
For DAPI staining cell were fixed in 3% p-formalde-
hyde/Triton-×100 and stained with 4’,6-diamidino-2-
phenylindole (DAPI; Pharmingen). A Leica fluorescent
microscope DM 900 was used to visualize the fluores-
cent images. Digital images were captured with a highly
sensitive cool (−25°C) charged coupled device camera
(Princeton Instruments) controlled with the MetaMorph
software (Universal Imaging).
Flow cytometric detection of intracellular cytokine
T cells isolated from peripheral blood, spleen, lymph node
and thymus of non-tumor bearing normal mice, control
(un-treated) and placebo-/calcarea carbonica-treated tumor
bearing mice after 21 days of placebo-/drug-treatment were
stimulated with phorbol-12-myristate-13-acetate (PMA;
10 ng/ml) and ionomycin (1 μM) (Sigma). After incubation
for 4 h at 37°C cells were washed with PBS and half of the
cells were labeled with PerCP-CD4 or PerCP-CD8 anti-
bodies. Cells were permeabilized with saponin and intracel-
lular IFN-γ, and IL-4 (10 μl, dilution 1:30; BD Bioscience)
were labeled with PE-/FITC-tagged antibodies and were
analyzed in FACS. Type-2 bias is defined as the ratio of
cells producing type-2 cytokine (IL-4) divided by the pro-
portion of cells producing type-1 cytokine (IFNγ) [16].
(B) in vitro experiments
Cell culture
p53-wild-type-MCF-7, -HBL-100 and p53-mutated-MDA-
MB-231, human breast cancer cells were obtained from
NCCS and routinely maintained in complete RPMI 1640
medium at 37°C in humidified incubator containing 5%
CO2 [31,32]. Furthermore, to determine the role of p53 in
calcarea carbonica-induced apoptosis, EAC-p53-deficient
cells and p53-silenced MCF-7 cells were utilized. The
p53-silencing was done by transfecting EAC cells with p53-
shRNA (small hairpin) and permanent clones (EAC-p53-
deficient cells) were selected by culturing the transfectants
with G418 (400 μg/ml) for 2 weeks with passaging after
each 3rd day [33,34]. The p53-deficient-EAC cells were
maintained in 200 μg/ml of G418 and then injected in the
peritoneal cavity of mice. Transient p53-silencing was done
by transfecting MCF-7 cells with p53-siRNA (small inter-
fering) following manufacturer’s instructions. The p53-
siRNA/shRNA transfection efficacy in EAC/MCF-7 cells
was validated by Western blot analysis.
Co-culture experiments
For co-culture experiments isogenic conditions were
maintained, i.e., T cells isolated from peripheral blood of
mice were co-incubated with cancer cells of mice origin,
EAC and S-180 cells and human peripheral T cells were

co-cultured with breast cancer cells of human origin
(MCF-7, HBL-100, MDA-MB-231). Prior to incubation
with target cells, T cells isolated from normal human
donors were cultured in anti-CD3/anti-CD28 coated
culture plates in media alone (control), tumor spent
medium (un-primed) placebo-treated-(placebo-primed)
and calcarea carbonica-treated-tumor spent medium
(calcarea carbonica-primed). For priming T cells, tumor
spent medium were treated with 20 μl/ml of placebo-/
calcarea carbonica 6C. Tumor spent medium is 72-hour
old cell-free tumor supernatants used for co-culture ex-
periments to mimic the tumor-bearing condition in
which tumor shed mediators influence the circulating T
cell repertoire. After 3 days these control, un-/placebo-/
calcarea carbonica-primed T cells were co-cultured with
breast cancer cells (MCF-7, HBL-100, MDA-MB-231)
for 48 hrs.
To examine the effect of varying effector-to-target ra-
tio, CD3+
T cells isolated from peripheral blood of con-
trol or placebo-/drug-treated mice were incubated with
EAC cells for 48 hrs at effector-to-target ratio of 5:1,
10:1 and 50:1 and percent apoptosis was scored by
Annexin-V/7-AAD assay. In another experiment, human
T cells and T cell-free supernatants after priming were
co-incubated with MCF-7 cells to understand the re-
quirement of T cell-tumor cell contact during T cell-
mediated tumor killing. After 48 hrs, MCF-7 cells from
both the sets were scored for percent apoptosis employing
Annexin-V/7-AAD assay.
Treatment of cells
During all in vitro experiments, cancer cells were
treated with 20 μl/ml calcarea carbonica 6C. To under-
stand the sequence of events leading to apoptosis, can-
cer cells were treated with mitochondrial pore inhibitor
CsA (25 μM; Merck, Germany) for 1 h prior to calcarea
carbonica treatment and with 50 μM of caspase-3 in-
hibitor Z-DEVD-FMK and caspase-9 inhibitor Z-LEHD-
FMK (Calbiochem), 3 h prior to incubation with calcarea
carbonica.
Flow cytometric measurement of mitochondrial
membrane potential
For measurement of mitochondrial transmembrane po-
tential (MTP) loss, cells were loaded with potential-
sensitive dye Dihexyloxacarbonicao cyanine (DiOC6,
Merck, Germany) during the last 30 min of treatment at
37°C in the dark. Fluorescence of retained DiOC6 was
determined flow cytometrically using logarithmic amp-
lification by CellQuest software (Becton Dickinson).
Western blot
Cell lysates were prepared in lysis buffer [20 mMTris–
HCl (pH 7.4), 100 mM NaCl, 1% NP40, 0.5% sodium
deoxycholate, and 1 mM EGTA] containing protease inhib-
itors. Mitochondrial and cytosolic fractions were prepared
according to Yamaguchi and Wang [35]. A total of 50 μg of
protein was separated by SDS-PAGE and transferred to
nitrocellulose filter paper for Western blotting using spe-
cific antibodies e.g., anti-p53 (DO-1), anti-Bcl-2 (N-19),
anti-Bax (N-20), anti-cytochrome c (C-20), caspase-3 (E-8),
caspase-9, anti-MnSOD (N-20) from Santa Cruz. The blots
were developed by chemiluminescence (1:1) [33,34]. In par-
allel experiment equivalent amount of protein was Western
blotted with anti-α-actin antibody (C-2; Santa Cruz) to
confirm equal protein leading.
siRNA, transfections and RT-PCR
Cells were transfected with 300pmole of p53-/caspase-
3-/control-ds-siRNA or p53-shRNA (Santa Cruz) and
lipofectamine-2000 separately for 12 h. The protein
levels of p53-/caspase-3-were estimated by Western blot-
ting. For RT-PCR assay, 2 μg of total RNA, extracted with
TRIzol reagent, was reverse-transcribed and then subjected
to PCR with enzymes and reagents of the RTplusPCR
system (Eppendorf, Hamburg, Germany) using GeneAmp
PCR system 2720 (Applied Biosystems; Foster City)
[31-34]. Primers for Bcl-2 were 5′-CTGGCATCTTCTCCT
TCCAG-3′ and 5′-GACGGTAGCGGACGAG-AGAAG-
3′; Bax were 5′-TTTGCTTCAGGGTTTCATCC-3′ and
5′-CAGTTGAAGTTGCCGTCAGA-3′; and GAPDH (in-
ternal standard) were 5′-CAGAACATCATCCCTGCC
TCT-3′ and 5′-GCTTGACAAAGTGGTCGTTGA G-3′.
Explant assay
Isolation and culture of primary breast cancer cells
Normal breast tissue or primary lesions of breast cancer
were obtained from patients with localized disease after
prior written informed consent under the provision of Eth-
ics committee, Calcutta National Medical College, Kolkata,
India (Approval letter No: CNMC/ETHI/162/P) and Hu-
man Ethics Committee, Bose Institute (Approval letter No:
BIHEC/2010-11/2). The selected cases consisted of 5 pri-
mary breast cancer patients that had not been treated with
chemotherapy or radiation. Normal mammary epithelial
tissue of the same patient was used as the control. The
specimens were washed with phosphate buffered saline,
cut into small pieces, 5×5 mm in size, and immersed in a
mixture of colloagenase (10%, Calbiochem) and hyaluroni-
dase (0.5 mg/ml, Calbiochem) for 12-16 h at 37°C on or-
bital shaker. The contents were then centrifuged at 80×g
for 30 sec at room temperature. The supernatant compris-
ing mammary fibroblasts were discarded and in the pellet
pre-warmed 0.125% trypsin-EDTA was added. The mix-
ture was gently pipetted and kept for 30 min at 37°C. Fi-
nally the pellet obtained was washed with cold Hank’s
buffer saline with 2% fetal bovine serum and centrifuged at
450×g for 5 min at room temperature. Then the single

cells were seeded on poly-L lysine coated dishes and cul-
tured in a serum-free medium containing growth factors,
0.1 ng/ml human recombinant epidermal growth factor,
5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 50 μg/ml
gentamycin, 50 ng/ml amphotericin-B, and 15 μg/ml bo-
vine pituitary extract at 37°C. Medium was replaced every
4 days and passages were done when the cells reached,
80% confluence. Peripheral blood was obtained from
healthy volunteers and patients after prior written in-
formed consent under the provision of Ethics committee,
Calcutta National Medical College, Kolkata, India (Ap-
proval letter No: CNMC/ETHI/162/P) and Human Ethics
Committee, Bose Institute (Approval letter No: BIHEC/
2010-11/2).
Statistical analysis
Values are shown as standard error of mean (SEM) except
otherwise indicated. Data were analyzed and, when appro-
priate, significance of the differences between mean values
was determined by a Student’s t test. Results were consid-
ered significant at p <0.05.
Results
Calcarea carbonica inhibited tumor growth and increased
survival rates of tumor-bearing mice
To identify the optimal strength of calcarea carbonica,
EAC-bearing mice were administered with different
strengths of drug (1C, 6C, 12C, 30C and 200C) for 27 days
and anti-tumor efficacy was determined by examining any
change in viable EAC cell number (Figure 1A). Non-
tumor bearing untreated- and placebo 6C treated EAC-
bearing mice served as control. It was witnessed that
calcarea carbonica 1C and 6C lessened tumor burden sig-
nificantly while 12C, 30C and 200C failed to impart any
decrease in tumor cell number. Since calcarea carbonica
at 1C formulation manifested problems related to solubil-
ity therefore further experiments were performed with
calcarea carbonica 6C. In fact, at day 28 a total of 487 ×
106
tumor cells were measured in the peritoneal fluid of
untreated or placebo-treated mice, whereas in calcarea
carbonica-treated group tumor cell count reduced to 75 ×
106
(Figure 1A). Next we examined the survival benefit of
calcarea carbonica to EAC-bearing mice orally fed with
the same for 27 days, twice daily. The results furnished in
Figure 1B depict that the tumor burden decreased the sur-
vival rates of the mice to 12.5% at day 28. Conversely,
when compared to untreated or placebo-treated sets,
calcarea carbonica provided a survival benefit of 88%
thereby suggesting the role of calcarea carbonica adminis-
tration in improving the survival rates of tumor-bearing
mice (Figure 1B).
To further understand the refractory nature of tumors
harvested from calcarea carbonica-treated animals to re-
treatment with the drug, if any, we collected EAC cells
from mice that had undergone 27 days treatment with
calcarea carbonica, and transplanted them into the peri-
toneal cavity of other mice. After 7 days of tumor inocu-
lation, these mice were orally administered with calcarea
carbonica, twice daily for another 27 days. Change in
EAC cells number was scored using trypan-blue dye ex-
clusion assay. The findings deciphered that calcarea
carbonica-treated tumors were sensitive to further treat-
ment with this drug without development of any resist-
ance (Figure 1C).
Calcarea carbonica induced apoptosis in tumor cells
in vivo
Role of calcarea carbonica in increasing the survival rate
of tumor-bearing mice prompted us to explore the mech-
anism of its action. Our results depicted that calcarea
carbonica treatment significantly depleted tumor cell
number. Importantly, calcarea carbonica treatment did
not show any toxicity to peripheral blood mononuclear
cells (PBMCs) (Figure 1E) indicating that cytotoxic effect
is specific for EAC cells. To compare cell death between
PBMCs and EACs, percent cell death was calculated from
the numbers of viable and dead cells in each case and rep-
resented graphically in Figure 1E. This decrease is consist-
ent with reduced ascetic fluid volume over untreated or
placebo-treated mice (Figure 1F). The decrease in EAC
cell number was at equivalence with that observed in IL2-
treated tumor-bearing mice (Figure 1E). IL2-fed tumor-
bearing mice served as positive control to estimate the
anti-tumor property of calcarea carbonica. Further mor-
phological changes depicting cell shrinkage and blebbing
in EAC cells (Figure 1D) compelled us to explore the na-
ture of cell death. Our results revealed significant increase
in the hypoploid (sub-G0/G1) DNA content of tumor cells
isolated from the peritoneal cavity of EAC-bearing mice
that underwent 21 days of calcarea carbonica-treatment
(Figure 1G) suggesting nuclear DNA breakdown that oc-
curs during apoptosis. Next, to confirm the nature of cell
death as apoptosis, we utilized double labeling techniques
using Annexin-V-PE/7-AAD to distinguish between apop-
totic and necrotic cells. Our flow cytometric data revealed
Annexin-V-PE-binding in EAC cells after 21 days of cal-
carea carbonica treatment when compared with placebo-
treated EAC cells (Figure 1G) indicating that the mode of
cell death is apoptosis and not necrosis. It was observed
that this remedy induced apoptosis in more than 40% of
tumor cells as compared to 12.5% apoptosis in placebo-
treated tumor-bearing mice. In addition, tumor cells iso-
lated from calcarea carbonica-treated EAC-bearing mice
after 21 days of drug treatment when stained by DAPI, re-
vealed a significant nuclear membrane blebbing, typical
characteristic feature of apoptotic cells, when compared
with tumor cells isolated from placebo-treated EAC-
bearing mice (Figure 1G). These findings supported the

notion that calcarea carbonica, asserts apoptogenic effect
in cancer cells. Interestingly, calcarea carbonica did not
furnish any significant toxic effect on peripheral circula-
tory immune cells of the tumor-bearing mice. Importantly,
this apoptogenic effect of calcarea carbonica was not cell
line-specific since S-180-bearing mice also manifested sig-
nificant tumor cell apoptosis (30%) when treated with
calcarea carbonica (Figure 1H).
Calcarea carbonica failed to induce cancer cell apoptosis
ex vivo
Our observation that calcarea carbonica induced signifi-
cant apoptosis when administered in vivo to EAC-bearing
mice model, prompted us to unveil the molecular mech-
anism of such anti-tumor effect of this homeopathic medi-
cine. For the same, the effect of calcarea carbonica in
different tumor cells (wild-type-p53 expressing cancer
cells EAC, S-180, MCF-7, HBL-100 and p53-mutated
MDA-MB-231) was determined in in vitro conditions by
exposing tumor cells with 20 μl/ml of calcarea carbonica
for a period of 96 h. Surprisingly, calcarea carbonica failed
to induce cancer cell apoptosis in ex vivo models as
evidenced by flow cytometric determination of cell cycle
phase distribution and Annexin-V-PE/7-AAD double la-
beling assays (Figure 1H).
These results demonstrated that calcarea carbonica-
mediated tumor apoptosis was restricted to in vivo condi-
tions thereby indicating that the anti-cancer effect of
calcarea carbonica may not be a direct one. We hypothe-
sized that calcarea carbonica may be exploiting cell-
mediated immune system to indirectly target cancer cells.
Calcarea carbonica restored tumor-induced depletion of T
cells in tumor-bearing host
To test whether calcarea carbonica targets immune sys-
tem to regress tumor burden, we next aimed to examine
the effect of this drug in immune system of the tumor-
bearing host. It is known that T cells play a pivotal role
in cell-mediated tumor immunity, and tumors induce T
cell apoptosis as a mechanism to evade the host defense
system [12-15]. Thymus is the major organ where the T
cell maturation process takes place, whereas spleen and
draining lymph nodes are the secondary sites where
antigen presentation process occurs and subsequently
mature effector T cell repertoire enter the peripheral cir-
culation. Closer scrutiny of thymus, spleen and lymph
node revealed decreased percentage of CD4+
CD8+
double
positive as well as CD4+
or CD8+
single positive effector
populations in tumor-bearing animals (Figure 2A & 2B).
Loss of the CD4+
CD8+
double positive populations may
cause a decrease in the number of mature effector cells
emerging from the thymus while loss of single positive
cells may result in decrease of potent T cells evolving from
lymph node and spleen. Supporting this notion, CD4+
or
CD8+
circulatory effector T cell populations were also se-
verely decreased in peripheral blood of the tumor-bearing
mice (Figure 2B). These results indicate that tumor-
induced T cell depletion encompasses primary, secondary
and effector immune compartments of the host, as de-
creased thymic, spleenic and lymph node output results in
disruption of the circulating T cell repertoire. Interestingly,
21 days of calcarea carbonica-treatment ameliorated
CD4+
-/CD8+
-positive effector T cell populations of thy-
mus, spleen, lymph node as well as circulatory effector
T cell populations to normal level (Figure 2A & 2B). To
estimate the immune-modulating effect of calcarea
carbonica, we included know immunostimulatory cyto-
kine IL2-treated tumor-bearing mice in the study. Over-
all our results indicate that calcarea carbonica protected
the effector T lymphocytes in primary and secondary
immune compartments, thereby normalizing the pool
of peripheral T cell repertoire in tumor-bearer. Interest-
ingly, calcarea carbonica showed almost similar effect
when compared with IL2 (Figure 2B).
The massive depletion of T cell populations in tumor
condition prompted us to investigate the underlying
cause. Our flow cytometry data revealed a significant in-
crease in Annexin-V-FITC/PI-positive CD4+
T-helper
and Annexin-V-FITC/7-AAD-positive CD8+
T cytotoxic
cells isolated from all the compartments of EAC-bearing
mice (Figure 2C), which indicates apoptotic cell death as
one of the major causes behind tumor-induced depletion
of T cell repertoire. Interestingly, calcarea carbonica
administration prevented apoptosis of both CD4+
and
CD8+
T cells (Figure 2C). All these results suggested that
calcarea carbonica has immunoprotective ability during
carcinogenesis.
Calcarea carbonica normalized Th1/Tc1-type cytokine-
producing T-effector cell populations in tumor-bearing host
It is known that Th1 and Th2 play important role in regu-
lating cell-mediated immune system among which Th1
pathways typically produce activation of cytotoxic T lym-
phocytes (Tc) via helper T cells (Th). Cytotoxic T lympho-
cytes attack cancer cells to defend against tumors while
Th2-mediated immunity favors tumor growth, both by
promoting angiogenesis and by inhibiting cell-mediated
immunity [36]. The capacity of the CD4+
lymphocytes iso-
lated from lymph-node of normal and EAC-bearing mice
to produce cytokines after PMA/ionomycin activation was
analyzed (Figure 3A). We observed reduced proportion of
Th1/Tc1-type cytokine (IFN-γ) secreting CD4+
/CD8+
T
cell (Figure 3B) and increased proportion of Th2/Tc2-type
cytokine (IL-4) secreting CD4+
/CD8+
T cell (Figure 3C)
populations amongst the thymus, lymph node, spleen and
peripheral circulation of EAC-bearing mice in comparison
to that from their normal counterparts. Administration
of calcarea carbonica to the tumor-bearers for 21 days

however restored such alterations of T cells in all the com-
partments (Figure 3B & 3C). Therefore, establishment of
type-2 cytokine bias in primary and secondary immune
compartments of tumor-bearing host could be efficiently
reversed by calcarea carbonica (Figure 3D).
Calcarea carbonica prevented down-regulation of T cell
proliferation in tumor condition
Our results till now demonstrated the loss of effector Tcells
and increase in type-2 cytokine bias in tumor-bearing ani-
mals, which was successfully reverted back to its normal
levels on calcarea carbonica administration to tumor-
bearing mice. Optimal T-cell mediated anti-tumor activity
likely occurs through proliferation and expansion of ef-
fector T cells. Thus, to verify if tumor also influenced the
proliferative capacity of effector T cells to TCR stimulus,
CD3+
peripheral T cells from normal/tumor-bearing/
placebo-/calcarea carbonica-fed mice were loaded with
CFSE for various periods of time. A CFSE division profile of
anti-CD3/anti-CD28 antibody-stimulated T cells isolated
from control and tumor-bearing mice is shown in Figure 4A.
Discrete division cycles could be visualized by means of dif-
ferent CFSE signal peaks in case of T cells isolated from
healthy mice, whereas T cells from tumor-bearing mice
failed to proliferate in response to anti-CD3/anti-CD28
antibody. Remarkably, 21 days of calcarea carbonica-
Figure 2 Calcarea carbonica restores tumor-induced depletion of T cell populations. Lymphocytes from thymus, spleen, lymph-node and
peripheral circulation of untreated or placebo-/calcarea carbonica-treated tumor-bearing mice were analyzed flow cytometrically to quantitate
percentages of CD4+
/CD8+
populations. (A) Flow cytometric dot plot shows CD4+
and CD8+
T cell population in the draining lymph node of
untreated or placebo-/calcarea carbonica-treated tumor-bearing mice. (B) Graphical representation of flow cytometric data showing percentages
of CD4+
and CD8+
T cells in thymus, spleen, lymph-node and peripheral circulation of untreated or placebo-/calcarea carbonica-/IL2-treated
tumor-bearing mice. (C) Percent CD4+
and CD8+
T-cell apoptosis (Annexin-V-positivity) from the same experimental sets were determined flow
cytometrically at 21 day of drug treatment. The values are mean ± SEM of five independent experiments. *p < 0.05 and **p < 0.001 when
compared with respective non-tumor/tumor-bearing control sets and placebo/drug-treated sets.

treatment prevented tumor-induced inhibition of Tcell pro-
liferation (Figure 4A & 4B), which suggests that apart from
preventing tumor-induced loss of effector T cells, calcarea
carbonica also restored T cell proliferative capacity.
Calcarea carbonica potentiated T cell-mediated tumor cell
killing in vitro
In the light of the results obtained so far, we further vali-
dated the hypothesis that calcarea carbonica induces
Figure 3 Calcarea carbonica normalizes cytokine-producing effector T cell populations in tumor-bearing host. (A) Typical flow cytometric
patterns for IFN-γ and IL-4 producing CD4+
T cells in draining lymph nodes of mice. Graphical representation of flow cytometric data of
intracellular IFN-γ (B), IL-4 (C) and type-2 cytokine bias ratio (D) in CD4+
/CD8+
T cells of thymus, spleen, lymph node and peripheral circulation of
untreated or placebo-/calcarea carbonica-treated tumor-bearing and normal mice at 21 days of drug administration. Values are mean ±SEM of
five independent experiments. *p < 0.05 and **p < 0.001 when compared with respective non-tumor/tumor bearing control sets and tumor
bearing placebo/drug-treated sets.

tumor killing via immuno-modulatory circuit. For the
same we mimicked in vivo conditions by isolating per-
ipheral CD3+
T cells from control, un-/placebo- and
calcarea carbonica-treated EAC-bearing mice after com-
pletion of 21 days treatment and co-culturing them with
EAC cells in in vitro conditions, at an effector to target
ratio of 5:1, 10:1 and 50:1 for 48 hrs. It was observed
that percent apoptosis of target tumor cells as scored by
Annexin-V-PE/7-AAD double labeling assay was directly
proportional to the number of effector cells (Figure 5A).
We performed further co-culture experiments with 10:1
effector to target ratio as it was feasible and efficient to
induce cell death in target tumor cells. Our flow cyto-
metry data revealed that T cells from un-treated and
placebo-treated EAC-bearing animals were inefficient
mediators of tumor cell killing while significant increase
in numbers of hypoploid and Annexin-V-positive cancer
cells, co-cultured with CD3+
T cells isolated from
calcarea carbonica-treated EAC-bearing mice, were ob-
served (Figure 5B). Interestingly CD3+
T cells isolated
from calcarea carbonica-treated EAC-bearing mice when
co-cultured with EAC-p53-shRNA cells (p53-deficient-
EAC) for 48 hrs, failed to induce apoptosis, signifying
p53-dependent cell killing. To further reinstate the above
hypothesis we evaluated the anti-tumor effects of control
(media-alone) and tumor-supernatant pre-exposed human
anti-CD3/CD28-stimulated T cells upon placebo-/calcarea
carbonica-treatment for 72 hrs. Our results demonstrate
that in comparison to T cells incubated with untreated
and placebo-treated tumor supernatant, T cells primed
with calcarea carbonica-treated tumor supernatant were
efficient inducers of apoptosis in wild-type p53-expressing
human breast cancer cells like MCF-7 and HBL-100
(Figure 5B) when co-cultured for 48 h. Interestingly, p53-
mutated MDA-MB-231 cells resisted T cell-mediated apop-
tosis even in the presence of calcarea carbonica (Figure 5B).
These findings further reinforced the opinion that calcarea
carbonica contributes to immunocyte-mediated tumor
elimination specifically in a p53-dependent manner.
To understand whether T cell-tumor cell contact is re-
quired for effective anti-tumorigenic potential of drug-
primed T cells we undertook two approaches in co-culture
experiments. First, to validate contact-dependent tumor
killing, T cells isolated from peripheral blood of human
volunteers after stimulation with anti-CD3/CD28, were
subjected to four experimental sets comprising of (a) Tcells
cultured in media alone (control), (b) T cells cultured
in untreated tumor supernatant (un-primed), (c) T cells
Figure 4 Calcarea carbonica attenuates tumor-induced inhibition of T cell proliferation. Flow cytometric histogram display of CFSE-
fluorescence dilution (A) and its graphical representation (B) to determine CD3/CD28-stimulated effector T cell proliferation from untreated and
placebo-/calcarea carbonica-treated tumor-bearing mice at 21 days of drug administration. Values are mean ±SEM of five independent
experiments. **p < 0.001 when compared with respective non-tumor/tumor-bearing control sets and placebo/drug-treated sets.

cultured in placebo-treated tumor supernatant (placebo-
primed) and (d) T cells cultured in calcarea carbonica-
treated tumor supernatants (calcarea carbonica-primed) for
3 days. After 3 days, control T-cells, un-primed, placebo-
primed and calcarea carbonica-primed T cells were co-
cultured with breast cancer cells, MCF-7, for 48 hrs. Sec-
ondly, to examine the effect of contact-independent mech-
anisms during T cell-mediated tumor killing, T cells
isolated from all the four experimental set were stimulated
for 4 h using PMA (10 ng/ml) and ionomycin (1 μM). On
the 3rd
day cells were centrifuged to obtain T cell-free
supernatants and MCF-7 cells were co-incubated with
these supernatants. After 48 hrs MCF-7 cells from both the
sets were scored for percent apoptosis using Annexin-V/7-
AAD assay (Figure 5C). Our findings revealed that calcarea
carbonica-primed T-cells when co-cultured with breast
cancer cells resulted in significant cell death, whereas
calcarea carbonica-treated T cell free supernatants failed to
reflect the same effect when compared with placebo data
sets. Altogether these results manifest that T cell-tumor cell
contact is definitely required for efficient tumor killing
upon calcarea carbonica treatment.
Figure 5 Calcarea carbonica potentiates T cell-mediated cancer cell killing in vitro. (A) Percent apoptosis of EAC cells co-cultured with T
cells at multiple effector to target ratios (5:1, 10:1 and 50:1), isolated from untreated or placebo-/calcarea carbonica-treated tumor-bearing mice.
Further for all co-culture experiments effector to target ratio was kept as 10:1. (B) Graphical representation of sub-G0/G1 populations and
Annexin-V-positive populations amongst EAC and p53-silenced EACs co-cultured with or murine T cells from untreated or placebo-/calcarea
carbonica-treated tumor-bearing mice. Similarly human breast cancer cell lines, MCF-7/HBL-100/MDA-MB-231 cells were co-cultured with human
T cells for 48 h to determine tumor cell apoptosis. Prior to co-culture, these human T cells were primed with placebo-/calcarea carbonica-treated
tumor supernatant for 72 h. The transfection efficacy of p53-shRNA was determined by Western blot (inset). (C) MCF-7 cell apoptosis when co-
incubated with placebo-/calcarea carbonica-primed human T cells (contact-dependent) or with supernatants of placebo-/calcarea carbonica
primed T cells (contact-independent) was determined flow cytometrically. (D) Percent apoptosis of MCF-7/HBL-100 cells when co-cultured with
placebo-/calcarea carbonica-primed CD4+
and/or CD8+
T cells. Values are mean ±SEM of five independent experiments. *p < 0.05 and **p < 0.001
when compared with respective tumor-bearing control/drug-treated sets and placebo-treated/drug-treated sets.

To further delineate the therapeutic potential of T cell
subsets, CD4+
-depleted and CD8+
-depleted T cells were
utilized. To this end, human T cells isolated from periph-
eral blood were sorted for CD4+
T cells (helper T cells) and
CD8+
T cells (cytotoxic T cells) under sterile condition.
Both T cell fractions were then cultured in untreated and
placebo-/calcarea carbonica-treated cell free tumor super-
natants for 3 days. Tumor cells (MCF-7/HBL-100) were
then co-incubated with unprimed- and placebo-/calcarea
carbonica-primed CD4+
and CD8+
T cells for 48 hrs. To
exclude the possibility of loss of T cells function due to the
sorting procedure enriched CD4+
and CD8+
T cell subsets
were mixed and incubated with tumor cells. Remarkably
mixed populations showed restored anti-tumor functions.
Percent apoptosis induced by CD4+
TH cells, CD8+
TC
cells and mixed CD4+
and CD8+
sorted T cells were scored
by Annexin-V/7-AAD positivity (Figure 5D). These results
demonstrate that as compared to the total T cells reper-
toire, percent apoptosis induced by individual CD4+
and
CD8+
T cells was significantly less. These observations sig-
nified the importance of both populations for efficient
tumor cell killing.
Calcarea carbonica-primed T cells induced cancer cell
apoptosis in p53-dependent manner
After establishing the mode of calcarea carbonica-
induced cancer cell apoptosis, next we aimed at delineat-
ing the underlying mechanism. In previously described
co-culture experiments maintaining isogenic conditions,
we verified by Western blot analysis the changes in p53
expression in cancer cells alongside with its transcription
target Bax, also a major effector of mitochondria-mediated
death. Cancer cells with functional p53, upon exposure
with calcarea carbonica-primed T cells, demonstrated in-
crease in the expression of p53. Moreover, increase in the
levels of p53 transcription target, Bax, was perceived both
at protein and mRNA levels (Figure 6A) in functional
p53-expressing cells, thereby leading towards the possibil-
ity of Bax transactivation by p53 under such conditions.
Moreover, these cancer cells upon treatment with calcarea
carbonica-primed T cells displayed decrease in Bcl-2 levels
both at transcriptional and translational levels (Figure 6A)
and thereby decreasing Bcl-2: Bax protein ratio (Figure 6B),
thus creating a pro-apoptotic environment. Cancer cells
co-cultured with control T cells failed to show any signifi-
cant change in p53 as well as in Bax (Figure 6A). However,
p53-mutated cancer cells, even in the presence of calcarea
carbonica-primed T cells failed to induce p53 and thus no
changes in the levels of Bax was evident (Figure 6A).
Moreover, in p53-silenced (Figure 6C) or p53-mutated
(Figure 5B) cancer cells, calcarea carbonica-primed T cells
failed to induce apoptosis thereby confirming the involve-
ment of p53 in calcarea carbonica-induced cancer cell
apoptosis via immunomodulatory circuit.
Calcarea carbonica-primed T cells induced apoptosis by
triggering mitochondrial death cascade in cancer cells
Our attempt to map the down-stream signalling pathways
of p53-mediated activity, revealed that in tumor cells co-
cultured with calcarea carbonica-primed T cells, Bax mi-
grated from cytosol to mitochondria, accompanied by a sig-
nificant decrease in cytochrome c level in mitochondria
and simultaneous increase in the cytosol (Figure 6D). These
results suggested that the mitochondrial translocation of
Bax might have led to initiation of the death cascade, with
the release of cytochrome c in cancer cells as a result of T
cell activation by calcarea carbonica. To determine the level
of contamination both mitochondrial and cytosolic frac-
tions isolated from tumor cells were run on the same gel
with same exposure time (Figure 6D). Involvement of mito-
chondrial pathway in p53-mediated apoptosis was further
confirmed by measuring DiOC6 retention of control and
calcarea carbonica-primed T cell co-cultured cancer cells in
flow cytometry. Calcarea carbonica-primed T cells pro-
duced a significant mitochondrial membrane potential
(MTP) loss in cancer cells while pre-treatment of the latter
with 25 μM cyclosporine A (CsA) that blocks mitochon-
drial pore formation, abrogated this effect (Figure 6E).
Mapping of the execution phase of apoptosis demon-
strated activation of the critical executioner caspase-3 in
EAC and HBL-100 cells (caspase-3-wild-type) and caspase-
9 in MCF-7 cells (caspase-3 knockout), as was evident from
the substantial decrease in pro-caspase-3/9 and increase in
caspase-3/9 at protein levels (Figure 6F) in tumor cells co-
cultured with calcarea carbonica-primed T cells for 48 hrs.
CsA completely blocked activation of executioner caspases
in cancer cells as was manifested by decrease in protein
levels of active-caspase-3 and 9 in EAC and MCF-7 cells,
respectively. Moreover, significant reduction in apoptosis
was also evident after CsA pre-treatment in tumor cells co-
cultured with calcarea carbonica-primed Tcells (Figure 6G).
Interestingly, EAC and HBL-100 cells could significantly
overcome calcarea carbonica-insult when transfected with
caspase-3-siRNA or treated with the pharmacological in-
hibitor of caspase-3, Z-DEVD-FMK (Figure 6H), as deter-
mined by scoring Annexin-V/7-AAD positivity. Similarly
significant decrease in calcarea carbonica-induced apop-
tosis was observed in MCF-7 cells pre-exposed with
caspase-9 inhibitor, Z-LEHD-FMK. All these results to-
gether indicate that calcarea carbonica treatment switched
over the tumor micro-environment towards apoptosis via
immuno-restoration, thereby culminating in tumor cell
killing.
Validation of results in patient’s biopsy samples
Studies performed in in vivo or ex-vivo system were con-
firmed by reiterating our findings in primary mammary
carcinoma and normal mammary tissue samples which
were obtained from surgical biopsies with prior consent of

Figure 6 (See legend on next page.)

patients. The biopsy samples procured were digested to
obtain single cells following protocol described in mate-
rials and methods section. Each patient’s peripheral blood
was also collected to isolate CD3+
T cells. Isogenic condi-
tions were maintained throughout co-incubation experi-
ments. Our results demonstrated that T cells primed with
calcarea carbonica-treated tumor supernatant induced
12% cell death in carcinoma sample, compared to 4%
death by untreated-T cells (Figure 7A). Our previous find-
ings demonstrated that calcarea carbonica failed to exert
direct apoptogenic effect. To further validate this in hu-
man mammary carcinoma we treated normal and
(See figure on previous page.)
Figure 6 Calcarea carbonica triggers T cell-mediated tumor killing via p53-Bax-caspase-3 cascade. (A) EAC, MCF-7 and MDA-MB-231 cells
were co-cultured with untreated-/placebo-/calcarea carbonica-primed T cells and subjected to Western blot/RT-PCR analysis to determine the
expression profile of p53/Bax/Bcl-2 at protein and Bax/Bcl-2 at mRNA levels (left panels). Right panels represent quantitative data for Western blot.
(B) Graphical representation of Bcl-2/Bax protein ratio in tumor cells co-cultured with calcarea carbonica-primed T cells. (C) Wild-type p53-
expressing cells were transfected with p53-siRNA (inset) and scored for percent apoptosis when co-cultured with calcarea carbonica-primed T
cells. (D) Bax and cytochrome c levels were determined in cytosolic and mitochondrial fractions of tumor cells co-cultured with placebo-/calcarea
carbonica-primed T cells by Western blot analysis (left panels). Middle panels represent quantitative data. α-Actin and MnSOD were used as
internal protein markers (right panels). (E) Graphical representation of mitochondrial trans-membrane potential of tumor cells co-cultured with
calcarea carbonica-primed T cells pre-treated with cyclosporine-A. (F) Expression profiles of pro-/active- forms of caspase-3 in EAC and HBL-100
cells and pro-/active caspase-9 in caspase-3-null MCF-7 cells co-cultured with calcarea carbonica-primed T cells. Right panels represent
quantitative data. (G) Expression profiles of active caspase-3 in EAC and caspase-9 in MCF-7 cells co-cultured with calcarea carbonica-primed T
cells pre-treated with cyclosporine-A (left panel). Middle panel represent quantitative data. In parallel set, cells were scored for percentage
apoptosis (right panel). (H) Percent apoptosis of EAC and HBL-100 cells co-cultured with calcarea carbonica-primed T cells in the presence of
caspase-3 inhibitor (Z-DEVD-FMK) or transfected with caspase-3-siRNA and percent apoptosis of MCF-7 cells co-cultured with calcarea carbonica-
primed T cells in the presence of caspase-9 inhibitor (Z-LEHD-FMK). Values are mean ±SEM of five independent experiments. *p < 0.05 and
**p < 0.001 when compared with respective placebo/drug-treated sets.
Figure 7 Calcarea carbonica induces T cell-mediated apoptosis of primary mammary tumor. (A) T cells isolated from patient’s peripheral
circulation were primed with media-/placebo-/calcarea carbonica-treated tumor supernatant for 72 h and then co-cultured with primary mammary
tumor for 48 h. In parallel, primary mammary tumor cells was directly exposed to placebo-/calcarea carbonica for 48 h in the absence of T cells. Tumor
cell apoptosis was then scored by Annexin-V-PE/7-AAD-positivity and represented graphically. (B) T cells isolated from normal and cancer patient’s
peripheral blood were co-cultured with control mammary tissue and tumor mammary tissue explants, respectively for 48 h and percent T cell
apoptosis was scored by Annexin-V-PE/7-AAD-positivity. (C) The same experimental set was analysed for percentage of CD4+
and CD8+
T cells
flow cytometrically. (D) Lysates of primary breast cancer cells co-cultured with or without placebo-/calcarea carbonica-primed
T cells of same patient’s blood were Western blotted for the analysis of p53, Bax, Bcl-2 and active-caspase-3. α-Actin was used as loading
control. *p < 0.05 and **p < 0.001 when compared with respective control/mammary tissue explant sets and placebo/drug-treated sets.

mammary carcinoma cells with calcarea carbonica for
48 h and scored percent apoptosis by Annexin-V/7-AAD
assay. Results of Figure 7A re-confirmed that calcarea
carbonica induces apoptosis in cancer cells not directly
but via T cells. We have already shown that calcarea
carbonica potentiates depressed immune system of the
host and employ it to induce apoptosis in tumor cells. To
validate these results next we verified the status of T cell
apoptosis when cultured in explants (spent-medium) of
control or human mammary carcinoma cells that were un-
treated or exposed to placebo or calcarea carbonica for
72 h (Figure 7B). In parallel, percentages of CD4+
and
CD8+
T cells were also analyzed (Figure 7C). Results of
Figure 7B revealed significant apoptosis in T cells cultured
in tumor explants as compared to that in normal tissue ex-
plants. Interestingly, calcarea carbonica-exposed tumor ex-
plants not only failed to induce T cell apoptosis
(Figure 7B) but also increased the percent of CD4+
and
CD8+
T cells (Figure 7C). Our previous findings revealed
that calcarea carbonica induced apoptosis in in vitro breast
cancer cells via p53 pathway. To further confirm this in
human mammary carcinoma, we co-incubated normal
and mammary carcinoma cells with placebo-/calcarea
carbonica-primed T cells for 48 h and determined the ex-
pressions of p53 and its downstream targets. Our Western
blot analysis revealed significant increase in the levels of
p53, bax and caspase-3, whereas significant reduction in
the levels of bcl-2 was perceived signifying pro-apoptotic
environment induced by calcarea carbonica-primed T cells
in mammary carcinoma cells when compared to un-/pla-
cebo-primed T cells (Figure 7D). Our results therefore
support the role of calcarea carbonica in protecting im-
mune cells from tumor insult and to mediate p53-
dependent cancer cell apoptosis via immumo-modulatory
circuit.
Discussion
Mechanisms that suppress tumorigenesis often involve
modulation of signal transduction pathways, leading to al-
teration in gene expression, arrest in cell cycle progression
or apoptosis. There has been extensive research where sev-
eral homeopathic formulations have been tested for their
anti-tumor effects in different cancers describing their dir-
ect apoptotic effects on cancer cells [6-10]. Yet another ex-
citing way of amplifying the anti-tumorigenic response is
to amplify the immuno-modulatory circuit which as such
is severely depressed in tumor conditions but if stimulated
can form a strong defense against tumor progression.
Tumor progression induces rigorous immunosuppression
by inducing apoptosis in T cells [12-14,18,19] and reducing
Th1/Tc1 response [20] thereby leading to decrease in acti-
vated T cell repertoire [15-17], which includes the abolish-
ment of effective cell-mediated immune response of the
host. Cancer-induced immune cell apoptosis as well as
block in maturation from CD4-
8-
to CD4+
8+
and finally to
CD4+
and CD8+
effector T cells were also reported [17,20].
Shift of cytokine balance from Th1/Tc1 to Th2/Tc2 has
been observed in tumor-bearing mice and in human can-
cer patients [17,20]. Considering all these information, an
approach that has received attention recently provides op-
portunities to explore the probability of manipulating im-
mune responses of the host against the disease. The prime
goal of cancer immunotherapy is to induce apoptosis in
tumor cells by recruitment of the host’s immune effector
repertoire. However, most of the cancer drugs in use add
to such tumor-induced immuno-suppression and concur-
rently exert toxic manifestations including oxidative stress,
liver damage, hepatotoxicity and immunosuppression in
the tumor-bearer [36-38]. On the other hand, reports sug-
gest that cancer patients using complementary and alter-
native medicines (CAM) strengthen immune system [39],
alleviate side-effects of chemotherapy, improve quality of
life, and help to overcome pain and stress; 62% of them
reported subjective beneficial effects [40]. Beside this, im-
mune stimulation by natural products has been attempted
in various animal models and in human cancer patients as
an adjunct to chemotherapy [41]. Pre-treatment with vary-
ing potencies of cadmium has been found to significantly
increase lymphocyte viability after toxic challenge com-
pared to control cells [42]. Oliveira et al. [43] have recently
revealed that highly diluted tinctures can efficiently de-
crease tumour necrosis factor-alpha (TNF-α) release and
IFN-γ production in lipopolysaccharide (LPS)-stimulated
macrophages. Application of highly diluted homeopathic
medicines to macrophages has been shown to suppress
previously elevated levels of TNF-α, increase the activity of
NADPH oxidase and the expression of inducible nitric
oxide synthase (iNOS), and induce differential gene ex-
pression in tumor conditions [11]. Many studies have de-
monstrated the role of different high diluted complexes in
cancer therapy via immunomodulation [44-46]. In this
context our study for the first time describes how calcarea
carbonica by re-arranging the dismantled cell-mediated
immune system brings forth active tumor killing. This
study, therefore, indicates that calcarea carbonica can be
exploited for regression of tumor burden by rejuvenating
host’s depressed immune system without inducing sys-
temic toxicity.
Boosting the cell-mediated immune system against
tumor cells requires both increasing and activating CTL
population. Depleted populations of CD8+
cells due to
tumor-induced apoptosis have been manifested in differ-
ent cancers, amelioration of which has revived the anti-
tumor potential of CTLs [47]. Consistently we observed
that calcarea carbonica ameliorated tumor-induced loss of
CD8+
T cells. Also the Canova Method, composed of
conitum napellus, arsenicum album (arsenic trioxide),
bryonia alba, lachesis muta venom and thuja occidentalis,

was found to stimulate the depressed immune system of
cancer patients by activating macrophages that in turn
stimulate lymphocytes for asserting their cytotoxic action
against cancer cells [48]. Apart from CD8+
cells there is
evidence of increased apoptosis among CD4+
T cells in
peripheral blood lymphocytes from cancer patients and
animal models [17]. Concurrently, Bhattacharyya et al.
[17,18] have reported that tumor derived factors induce
helper T cell apoptosis which could be reverted by natural
remedies. Because CD4+
cells are essential for activation
of CTL response, it is noteworthy that calcarea carbonica,
in the present study, normalized CD4+
cells in different
immune organs of tumor-bearing host. Moreover, the
immune-boosting efficacy of calcarea carbonica was found
to be comparable to the established immune-modulating
cytokine, IL2 [17,18]. Reports have also stated that cal-
carea carbonica and its associations had a promising cap-
acity to stimulate immune cells against melanoma cells
both in vitro and in vivo on melanoma metastasis mouse
model [11]. In another study, in vitro treatment with
calcarea carbonica significantly increased macrophages/
lymphocyte interaction and effectiveness against melan-
oma cells [11].
Other than inducing T cell apoptosis, blocking T cell
proliferation is also accountable for reduced T cell popula-
tions in tumor patients [49]. Numerous tumor-derived
suppressive factors have been reported to prohibit cell sig-
naling responsive for T cell proliferation. Studies showed
that tumor condition suppressed expression of JAK3 and
tyrosine phosphorylation of STAT5 [50]. Tumor superna-
tants also partially blocked induction of IL-2R beta and
gamma chains expression [50]. Interestingly natural rem-
edies like curcumin and theaflavin were found to restore
IL-2 signaling, suggesting that these compounds may
inhibit tumor-induced inhibition of T cell proliferation
[15-18]. Convincingly we observed that calcarea carbonica
restored T cell population by normalizing T cell prolife-
ration. Though T lymphocyte count is a major factor
governing anti-tumor responses, it should be remembered
that a proper cytokine environment is a must for T cell sur-
vival, proliferation and activation. Numerous studies have
reported that a type-2 cytokine bias is conducive to tumor
growth both by inhibiting production of Th1 cytokines and
reduction of CTL function [51]. Calcarea carbonica, how-
ever, was found to skew the bias towards type-1 cytokines
which implies that inclusion of this drug may necessarily
improve the process of tumor immunotherapy.
Tumor sensitivity to cell-mediated immunity often de-
pends on the status of tumor suppressor genes. Import-
antly the tumor suppressor p53 plays multiple roles in
cell cycle control, differentiation, angiogenesis, genomic
stability, and apoptosis [31-34]. Mutations of the p53
gene are frequently found in >50% of all human tumors,
suggesting that loss of this gene represents an important
step in the formation of human cancers. Thiery et al.
[28] reported that the restoration of wild-type p53 ex-
pression in p53-mutant tumor cells increases tumor
susceptibility to CTL-mediated cytolysis. CTL-targeting
results in p53 accumulation and activation at very early
times. They further showed that p53 is a key determin-
ant in anti-tumor CTL response that regulates induction
of Fas receptor expression, cellular FLICE/caspase 8 in-
hibitory protein short-form degradation, and Bid trans-
location to target mitochondria [28]. The balance between
pro-apoptotic and anti-proliferative genes, activated by
p53, is believed to control the choice between apoptosis
and growth arrest. It has been shown that p53 triggers
apoptosis by inducing mitochondrial outer membrane
permeablilization through transcription-dependent and -
independent mechanisms [52]. Transcriptional target of
p53 include the pro-apoptotic Bcl-2 family member Bax,
which translocate to the mitochondria from the cytosol in
response to apoptotic signals, permeabilize the outer
membrane, resulting in release of mitochondrial proteins
such as cytochrome c, AIF etc. in the cytosol or nucleus
where they are actively involved in the process of caspase
activation and protein/DNA degradation [53]. However,
there was still dearth of information regarding whether
immuno-modulatory circuit is involved in cancer reverting
action of calcarea carbonica and, if any, the underlying
molecular mechanisms. We shed light on the molecu-
lar mechanism underlying calcarea carbonica-induced
immune-therapy of tumor by showing that calcarea-
primed T cells executed p53-dependent tumor apoptosis
via Bax activation and loss of mitochondrial membrane
potential that led to augmentation of cytosolic cytochrome
c and caspase-3 activation. These results altogether justify
the candidature of calcarea carbonica as an anti-cancer
agent that induces apoptosis in cancer cells via immuno-
modulatory circuit.
In the future, experimental as well as clinical studies
e.g., using the combination of calcarea carbonica and other
homeopathic remedies, will further elucidate its thera-
peutic value in treating different cancers.
Conclusion
In summary, our work for the first time indicated an
apoptosis-enhancing capability of calcarea carbonica in
cancer cells by strengthening the immune system as well
as the cross-talk of various pro- and anti-apoptotic factors.
Even though further investigations and clinical trials are
needed there is an indication that the unique properties of
calcarea carbonica can be exploited for regression of
tumor burden by rejuvenating host’s depressed immune
system without inducing systemic toxicity.
Abbreviations
Bax: Bcl-2 associated X protein; Bcl-2: B cell lymphoma-2;
CAM: Complementary and alternative medicine; CTL: Cytotoxic T

lymphocyte; CFSE: Carbonicaoxyfluorescein succinimidyl ester;
CsA: cyclosporine A; DAPI: 4’,6-diamidino-2-phenylindole dihydrochloride;
DiOC6: 3,3 dihexyloxacarbonicaocyanine iodide; EAC: Ehrlich’s ascites
carcinoma; IFN-γ: Interferon gamma; IL-2: Interleukin-2; IL-4: Interleukin-4;
MTP: Mitochondrial membrane potential; PBL: Peripheral blood lymphocytes;
PCR: Polymerase chain reaction; siRNA: Short-interfering RNA; shRNA: Small
hairpin RNA; TCR: T cells expressing antigen specific receptor; Th1: T helper
cell type-1; Th2: T helper cell type-2; TNF: Tumor necrosis factor.
Competing interests
The authors declare that they have no competing interests.
Authors’ contribution
SS conceived the study, performed in vivo experiments, cell culture studies,
Western blot experiments, analyzed the data and drafted the manuscript.
DMSH performed in vivo experiment and participated in flow cytometric
studies during immuno-phenotyping and analyzed the data. SM participated
in cell culture and Western blot experiments. SM participated in cell culture
experiments and analyzed the results. MM, SM and UKG assisted in in vivo
experiments. CR, CN, AK and RC analysed the results and reviewed the
manuscript. GS and TD conceived the study, supervised the experiments,
analyzed the data and helped to draft the manuscript. All authors have read
and approved the manuscript.
Acknowledgements
Authors are thankful to Ms. R. Sarkar for editing the manuscript. Thanks are
also due to Mr. U. Ghosh, Mr. R. Dutta, Mr. S. Samanta and Ms. S. Das for
technical help. This work was supported by the grants from Central Council
for Research in Homoeopathy (CCRH), Govt. of India.
Author details
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.
Received: 24 January 2013 Accepted: 12 September 2013
Published: 21 September 2013
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doi:10.1186/1472-6882-13-230
Cite this article as: Saha et al.: Calcarea carbonica induces apoptosis in
cancer cells in p53-dependent manner via an immuno-modulatory
circuit. BMC Complementary and Alternative Medicine 2013 13:230.
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