395-403.qxd

16/12/2009

02:02 ÌÌPage 395

INTERNATIONAL JOURNAL OF ONCOLOGY 36: 395-403, 2010

395

Cytotoxic effects of ultra-diluted remedies on breast cancer cells
MOSHE FRENKEL1, BAL MUKUND MISHRA2, SUBRATA SEN2, PEIYING YANG1, ALISON PAWLUS1,
LUIS VENCE3, AIMEE LEBLANC2, LORENZO COHEN1, PRATIP BANERJI4 and PRASANTA BANERJI4
1

Integrative Medicine Program, 2Department of Molecular Pathology, 3Department of Melanoma
Medical Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA;
4P. Banerji Homeopathic Research Foundation, Kolkata, India
Received May 28, 2009; Accepted July 23, 2009
DOI: 10.3892/ijo_00000512

Abstract. The use of ultra-diluted natural products in the
management of disease and treatment of cancer has generated
a lot of interest and controversy. We conducted an in vitro
study to determine if products prescribed by a clinic in India
have any effect on breast cancer cell lines. We studied four
ultra-diluted remedies (Carcinosin, Phytolacca, Conium andThuja) against two human breast adenocarcinoma cell lines(MCF-7 and MDA-MB-231) and a cell line derived fromimmortalized normal human mammary epithelial cells(HMLE). The remedies exerted preferential cytotoxic effects
against the two breast cancer cell lines, causing cell cycle
delay/arrest and apoptosis. These effects were accompaniedby altered expression of the cell cycle regulatory proteins,
including downregulation of phosphorylated Rb andupregulation of the CDK inhibitor p27, which were likely
responsible for the cell cycle delay/arrest as well as inductionof the apoptotic cascade that manifested in the activation of
caspase 7 and cleavage of PARP in the treated cells. The
findings demonstrate biological activity of these natural
products when presented at ultra-diluted doses. Further indepth studies with additional cell lines and animal models are
warranted to explore the clinical applicability of these agents.
Introduction
The use of herbs, minerals, vitamins, homeopathic remedies
and other complementary and alternative medicine (CAM) ison the rise worldwide, and patients with cancer are increasingly_________________________________________
Correspondence to: Dr Moshe Frenkel, Integrative Medicine
Program-Unit 145, The University of Texas M.D. Anderson Cancer
Center, 1515 Holcombe Blvd., Houston, TX 77030-4009, USA
E-mail: frenkelm@netvision.net.il; moshefrenkelmd@gmail.com
Dr Prasanta Banerji and Dr Pratip Banerji, PBH Research Foundation,
10/3/1 Elgin Road, Kolkata-700 020, India
E-mail: info@pbhrfindia.org; pbhrf@vsnl.com

Key words: breast cancer, breast cancer cells, cell cycle, apoptosis,
homeopathy, cancer care, alternative medicine, complementary
medicine, integrative medicine, integrative oncology

opting to be treated with CAM therapeutic regimens (1-3). The
safety and efficacy of many CAM approaches have not been
well studied, especially in cancer care. Therefore, the US
National Cancer Institute (NCI) developed the Best Case Series
program inviting CAM practitioners worldwide to present
their clinical experience and ‘ best cases’ in the use ofalternative medicine in the treatment of cancer, with theobjective to develop further research toward rigorous scientificvalidation.
In 1999, the NCI evaluated a cancer treatment protocol
developed at the P. Banerji Homeopathic Research Foundation(PBHRF) in Kolkata, India. The ‘Banerji protocol’ used
specific ultra-diluted natural substances to treat patients withdifferent cancers. The NCI reviewed 10 patients treated on the
Banerji protocol. In four of the cases with lung and esophageal
cancers, the NCI confirmed partial responses (4). All patients
reviewed had appropriate pathology and imaging studies toconfirm diagnosis and response. The patients only received
the remedies prescribed at the PBHRF clinic and did notreceive any additional conventional treatment, such as surgery,radiation, or chemotherapy. After rigorous evaluation, theNCI concluded that there was sufficient evidence of efficacy
to warrant further research of the Banerji protocol.
As documented by the clinic, 21,888 patients with malignant tumors who were treated only on the Banerji protocol
were followed at PBHRF between 1990 and 2005. Of thepatients, 941 had breast cancer. Clinic physicians reported
that in 19% of the patients, the malignant tumors completely
regressed, and in 21% the tumors were stable or improved
with treatment. For patients with stable tumors, follow-up
continued for at least 2 years and for as long as 10 years (5).
In 2003, Pathak et al reported that an ultra-diluted doseof the homeopathic remedy Ruta graveolens, commonlyprescribed as the standard Banerji protocol therapeutic agent
for brain cancer, selectively induced death in glioblastoma
multiforme cells while promoting the proliferation of normal
peripheral blood lymphocytes (6).
Since those findings were reported, we have noticed thatpatients who come to M.D. Anderson's Integrative Medicine
Clinic already use homeopathy or have a marked interest in
integrating this treatment with their conventional therapies
because the agents have no toxicity and are easy to use. Mostof the information on the use of these agents is available topatients on the Internet and through support groups, and395-403.qxd

16/12/2009

02:02 ÌÌ396

Page 396

FRENKEL et al: ULTRA-DILUTED REMEDIES ON BREAST CANCER

many of the remedies can be purchased at health food stores,
supermarkets, or from online vendors.
Considering the growing interest in Banerji protocol
remedies among patients at our clinic, most of whom haveadvanced breast cancer, we decided to evaluate the in vitro
effect of those remedies. In this study, we compared the effects
of four ultra-diluted remedies in two well-characterized humanbreast cancer cell lines and an immortalized normal humanmammary epithelial cell line grown in vitro.

and SV40 large-T and small-T antigens, was kindly provided
by Dr Sendurai Mani of M.D. Anderson. The HMLE cells
were cultured in a 1:1 ratio of Dulbecco's modified Eagle's
medium: Ham's F12 (Mediatech, Manassas, VA) andMammary Epithelial Cell Growth Medium (Lonza, Allendale,
NJ); the culture was supplemented with 2.5 μg/ml insulin(Sigma), 5 ng/ml human Epidermal Growth Factor (Sigma),and 250 ng/ml hydrocortisone (Sigma). All the cell lines wereincubated at 37˚C in an atmosphere of 5% CO2 and 95% air.

Materials and methods

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay.
Cell lines were analyzed for cell viability by MTT assay.
Approximately 5,000 cells grown in triplicate in 96-well plates
for 24 h were treated with the remedies for 24, 48, 72 and
96 h. To determine cell viability, 50 μl of MTT (5 mg/ml)
was added in the dark in sterile conditions to the cells for 2 h
at 37˚C. After 2 h, the medium was removed and 100 μl
dimethyl sulfoxide was added. The plates were incubated atroom temperature for 8-10 min. The absorbance at OD 570 nm
was then measured by an enzyme-linked immunosorbent
assay plate reader.

Drugs. The ultra-diluted remedies used by PBHRF wereobtained from Sharda Boiron Laboratories Ltd India andadditional similar remedies obtained from the Standard
Homeopathic Company (King of Prussia, PA, USA). The
experiments were conducted in triplicate and repeated at least
twice in each case of remedy. The remedies used and theirdilutions were the same as those prescribed by the PBHRF
for the treatment of human breast cancer: Carcinosin, 30C;
Conium maculatum, 3C; Phytolacca decandra, 200C andThuja occidentalis, 30C. The remedies were diluted with
87% extra-neutral alcohol, referred to as the ‘solvent’. The
solvent was also evaluated for its effects on the cells bothby itself and after being processed through a process of
succussion method similar to the way used for making thepotency dilutions of the remedies.
High-performance liquid chromatography (HPLC) for
detection of chemical components in ultra-diluted remedies.
Ultra-diluted remedies may contain many chemical constituents, since they are derived from plant and animal cell
extracts. Fingerprinting of the remedies was therefore
performed by HPLC to identify constituents. Fingerprinting
was performed with Waters Delta 600 HPLC systems(Waters Co., Milford, MA) equipped with a solvent delivery
pump unit, a Waters 717 plus autosampler, and a Waters
2996 photodiode array detector. Chromatographic separation
was achieved using a C18 column (5.0 μm, 250x4.6 mm)(Agilent) with a guard cartridge (5.0 μm, 20x3.9 mm I.D.).
The mobile phase consisted of methanol (A) and water (B),and separation was achieved using a linear gradient of 10-90%
methanol with an injection-to-injection time of 60 min. The
flow rate was 1 ml/min. The detection wavelengths were
254 and 220 nm. All samples were run under the same
conditions and had the same scale of 0.2 astronomical units.
Cell lines. Two human breast adenocarcinoma cell lines,
MCF-7 and MDA-MB-231, were obtained from the American
Tissue Culture Collection (Manassas, VA). The MCF-7 cells
harbor the wild-type p53 gene and are estrogen- andprogesterone-receptor positive, while the MDA-MB-231
cells harbor a mutant p53 gene and are estrogen- andprogesterone-receptor negative. The cells were cultured in
Dulbecco's modified Eagle's medium supplemented with
10% heat-inactivated fetal bovine serum (Atlanta Biologicals,
Atlanta, GA), 1% L-glutamine (Invitrogen, Carlsbad, CA)
and 1% pen-strep (Invitrogen). A control cell line, HMLE,
which was derived form normal human mammary epithelial
cells immortalized with the catalytic subunit of telomerase

Cytologic preparations and fluorescence in situ hybridization(FISH). Remedy-treated and control cell cultures were treatedwith 0.04 μl/ml of colcemid (Gibco/Invitrogen, Carlsbad,CA) for about an hour at 37˚C and then processed forchromosome preparation following a brief hypotonic treatment
in 0.9% sodium citrate and fixation in a 3:1 solution of
methanol and acetic acid. The slides were air-dried andstained with Giemsa stain, and the cultures were evaluatedfor their mitotic indices. The slides were also processed for
FISH using a Cy-3-labeled peptide nucleic acid telomericprobe (Dako Corporation, Carpinteria, CA) according to the
manufacturer's protocol. At least 200 cells from treated anduntreated samples were analyzed for mitotic index andtelomeric DNA signals with a Nikon Eclipse 80i microscope
equipped with fluorescence attachment and a Photometrics
CoolSNAP HQ2 monochrome digital camera.
Flow cytometry for cell cycle distribution and subdiploid
population assay. The cell cycle progression and apoptosis
were analyzed by fluorescence activated cell sorting (FACS)
analysis. Approximately 1x10 6 each of the control andremedy-treated cells were trypsinized, washed twice withcold phosphate-buffered saline (1X PBS) and fixed in 2 ml of
ice-cold ethanol (70%) overnight at 4˚C. Fixed cells were
washed twice with 1X PBS and then incubated with 1 ml of
PBS containing 20 μg/ml RNAse and 50 μg/ml propidium
iodide (PI) for 30 min at 37˚C. The stained cells were analyzed
using a Coulter Epics XL cell counter (Beckman Coulter,
Brea, CA).
Apoptosis detection by annexin V labeling and flow cytometry.
Remedy-treated and control cells (~1x106 cells each) were
cultured in 60 mm plates for 72 h. Both floating and adherent
cells were collected by centrifugation at 1,200 rpm for 5 min
at 4˚C. The cell pellet was washed once with cold 1X PBS.
The pellet was re-suspended in 500 μl of TBS/Ca2+ [20 mM
Tris plus 150 mM NaCl (pH 7.4) and 2 mM Ca2+]. Then, 25 μlof annexin V/PI solution (Trevigen, Gaithersburg, MD) was

395-403.qxd

16/12/2009

02:02 ÌÌPage 397

INTERNATIONAL JOURNAL OF ONCOLOGY 36: 395-403, 2010

added into the solution and incubated for 5 min at room
temperature. The reaction was analyzed by FACS at anemission wavelength of about 525 nm with a blue laser forannexin V-fluorescein isothiocyanate (FITC) staining and at
an emission wavelength of about 620 nm with a red laser for
PI staining.
Immunoblotting. Expression of proteins associated with cell
cycle regulation and apoptosis was detected by Western blot
analysis of the control and remedy-treated cells. For cell
cycle analysis, we used antibodies against the cell cycle
regulatory proteins cyclin D1, cyclin D3, cyclin-dependent
kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), p27 (aCDK inhibitor) and the phosphorylated form of Rb protein
while to analyze apoptotic response, antibodies against theapoptosis-associated proteins poly (ADP-ribose) polymerase(PARP) and caspase 7 (Cell Signaling Technology, Danvers,
MA) were used. To isolate total proteins, cell pellets were
washed once with ice-cold 1X PBS, and then resuspended in
100 μl of cold lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM
NaCl, 0.1 mM phenylmethylsulfonyl fluoride 1 mM ethylene
diamine tertaacetic acid, 1% Triton X-100, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.5 mg ofcomplete Protease inhibitor cocktail), homogenized andcentrifuged. Protein concentrations were determined using a
Bio-Rad (Richmond, CA) protein assay kit. Equal amounts
of total cellular protein (50 μg) were suspended in 1X sample
buffer (125 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol,
0.1% bromophenol blue and 1% ß-mercaptoethanol) anddenatured by boiling for 5 min. The prepared samples wereresolved by 10% SDS-polyacrylamide gel electrophoresis
and transferred onto a Hybond-ECL nitrocellulose membrane(Amersham Biosciences, Piscataway, NJ). The membranes
were then incubated with the desired antibodies overnight at
4˚C. The membranes were subsequently incubated for 1 h
with anti-rabbit immunoglobulin G antibody conjugated tohorseradish peroxidase and visualized using enhanced
chemiluminescence kits (GE Healthcare, Bucks, UK).
Results
Chromatographic fingerprinting. The solvent showed only
one distinct peak, eluted at about 2 min, in the chromatogram.
To determine whether succussion caused any chemicalchanges in the solvent, we compared the fingerprinting
profiles of the solvent before and after succussion. The
chromatogram of the untreated and treated solvents appeared
identical, indicating that succussion did not cause chemicalchanges in the solvent. All four remedies had very similar
HPLC chromatograms to each other, with only trace amountsof limited number of peaks. They were not significantly
distinct from the solvent and they lacked the distinct peak seenin the solvent.
Ultra-diluted remedies reduce viability of human breast
adenocarcinoma cells. As shown in Fig. 1A, the solvent
reduced the viability of all three cell types; the overall
reduction in cells at different doses of solvent was about 30%
for MCF-7, 20-30% for MDA-MB-231 and 20% for HMLE
cells. Interestingly, the inhibitory effects on cell viability of397

the remedies in both the MCF-7 and MDA-MB-231 cells
were distinctly greater for each of the doses tested than thoseseen in cells treated only with solvent. MCF-7 cells were
found to be more sensitive to all four remedies than the
MDA-MB-231 cells (Fig. 1A, panels 2 and 3). The inhibitory
effects of the remedies were also dose-dependent for both
MCF-7 and MDA-MB-231 cells, with progressively more
inhibition seen at higher doses (50-75% loss of viability inthe two cell lines). To investigate whether the inhibitory
effects increased with time of treatment, we treated the cellswith the solvent and remedies at concentrations of 5 and10 μl/ml for 48 and 72 h. The results revealed that the
inhibitory effects were higher for the longer period of treatment and greater inhibition was caused by the remedies thanby the solvent. Among the four remedies investigated,
Carcinosin and Phytolacca, reproducibly revealed relatively
higher inhibitory effects in replicate experiments. These two
remedies reduced viability of the MCF-7 cells by 60-75% at
5 μl/ml and by 70-80% at 10 μl/ml doses after 48 and 72 htreatments, respectively. For the MDA-MB-231 cells, thereductions were 50-65% at 5 μl/ml and 65-70% at 10 μl/ml atthese times. The solvent, on the other hand, caused reduction
in survival of the two cell lines by 30-35% under the same
conditions (Fig. 1B). Strikingly, the effects of the four
remedies on the viability of the HMLE cells were nearly
indistinguishable from the effects of the solvent alone(Fig. 1A and B). In view of the fact that Carcinosin andPhytolacca appeared relatively more potent inhibitors ofcancer cell survival, we decided to investigate these two
remedies further to understand the mechanisms responsible
for their observed effects on cell proliferation and survival.
Induction of cell cycle arrest and cell death by the remedies.
We next examined cell cycle progression profiles of MCF-7,
MDA-MB-231 and HMLE cells treated with 5 μl/ml dose ofCarcinosin and Phytolacca. As shown in Fig. 2A, at 24 h oftreatment with the remedies, MCF-7 cells showed G1 phasearrest with a concomitant decrease in the S and G2/M phase
populations. At 48 h and later, distinct sub-G0 populations,representing cell death, appeared and G1 phase cells decreased.
The sub-G0 population showed a marginal increase at 96 h
post-treatment, while S phase cells had increased and G2/M
phase cells were almost completely absent. In the HMLE
cells, a relatively less pronounced G1 phase arrest wasevident after 24 h of treatment with both the solvent and theremedies, while a relatively greater G2/M phase delay orarrest was seen following treatment with the remedies thanwith the solvent treated and untreated cells. A sub-G0
population became evident at 72 and 96 h in the treatedsamples, though the peak was distinctly smaller than in theremedy-treated MCF-7 cells. The cell cycle profiles of theremedy-treated MDA-MB-231 cells appeared different fromthose of the remedy-treated MCF-7 cells. While a G1 arrestin cells treated for 24 h with solvent and Carcinosin was
similar to that seen in HMLE cells, the same was not observed
after Phytolacca treatment. The Phytolacca-treated cells hadgreater G2/M phase accumulation following 24, 48 and 72 hof treatment than the MCF-7 cells. Sub-G0 peaks were visible
at 48, 72 and 96 h, but those peaks were not seen in theuntreated controls and were higher than those seen in the395-403.qxd

398

16/12/2009

02:02 ÌÌPage 398

FRENKEL et al: ULTRA-DILUTED REMEDIES ON BREAST CANCER

A

B

Figure 1. (A) Methylthiazolyldiphenyl-tetrazolium (MTT) bromide viability assay of HMLE, MCF-7 and MDA-MB-231 cells following treatments with thesolvent, Carcinosin, Phytolacca, Conium, or Thuja for 48 h at various concentrations (0, 1.25, 2.5, 5 and 10 μl/ml). The values were obtained in independent
experiments performed in triplicate and were represented as mean ± standard error vs. control (bars). (B) MTT assay for viability of the cells treated with 5
and 10 μl/ml solvent, Carcinosin, Phytolacca, Conium, or Thuja for 48 and 72 h. The values were obtained in independent experiments performed in triplicate
and are presented as mean ± standard error (bars) vs. control.

395-403.qxd

16/12/2009

02:02 ÌÌPage 399

INTERNATIONAL JOURNAL OF ONCOLOGY 36: 395-403, 2010

399

A

B

Figure 2. (A) Cell cycle progression analyses of the HMLE, MCF-7 and
MDA-MB-231 cells treated with 5 μl/ml solvent, Carcinosin or Phytolacca
for various time points (24, 48, 72 and 96 h). Fluorescence-activated cellsorting (FACS) was used to develop a DNA histogram and determine thepercentages of cells in each cell cycle phase. (B) Mitotic indices of control
and Carcinosin-treated MCF-7 and MDA-MB-231 cells.

telomeric DNA signals in the treated HMLE cells were muchless affected than signals in the adenocarcinoma cells. Thispreferential reduction of telomeric DNA signals indicatedthat Carcinosin and Phytolacca caused loss of genomic
integrity, which possibly contributed to induction of cell
death.

solvent-treated samples. Preferential growth delay or arrest of
the MDA-MB-231 cells at the G2/M phase following treatment with the remedies was revealed with microscopic
analysis of the remedy-treated cells. The number of mitotic
cells was clearly elevated in the treated MDA-MB-231 cells
compared to the treated MCF-7 cells and the untreated controls(Fig. 2B).
Preferential loss of telomeric DNA in remedy-treated adenocarcinoma cells. The MCF-7, MDA-MB-231 and HMLE cells
treated with Carcinosin and Phytolacca were analyzed by
FISH to quantify telomeric DNA. As shown in Fig. 3, there
was significant reduction in telomeric DNA signals in theremedy-treated cells compared to the untreated controls. The

Altered expression profiles of the cell cycle regulatory proteinsin Carcinosin- and Phytolacca-treated cells. Since the cells
treated with the remedies displayed cell cycle delay/arrest,
we analyzed the expression profiles of the cell cycle regulatory
proteins in Carcinosin- and Phytolacca-treated HMLE,
MCF-7 and MDA-MB-231 cells (Fig. 4). The cell cycle
proteins analyzed in these experiments included cyclins D1
and D3, CDK4, CDK6, phosphorylated Rb (pRb) and p27.
Each of the three cell types revealed both unique and common
changes in the expression profiles of the cell cycle regulatory
proteins, reflecting the cycle pathway-associated molecularalterations that occurred in response to the remedy treatments. In the HMLE cells, the levels of cyclins D1 and D3
decreased significantly, but those cyclins did not show any
perceptible change in expression in the MCF-7 and MDAMB-231 cells. The levels of CDK4 and CDK6 declined at 24 hand then were marginally elevated at later time points in the395-403.qxd

400

16/12/2009

02:02 ÌÌPage 400

FRENKEL et al: ULTRA-DILUTED REMEDIES ON BREAST CANCER

Figure 3. (A) Representative telomere DNA fluorescence in situ hybridization images of untreated MCF-7 cells. (B) MCF-7 cells treated with Carcinosin. (C)Untreated MDA-231 cells. (D) MDA-MB-231 cells treated with Carcinosin. Treated cells were exposed to 5 μl/ml Carcinosin for 72 h.

Figure 4. Western blot analyses of cell cycle regulatory proteins in HMLE, MCF-7 and MDA-MB-231 cells, treated with 5 μl/ml Carcinosin or Phytolacca
for various lengths of time (24, 48 and 72 h). Protein loading was verified by detection of ß-actin in the same gels.

HMLE cells. The lowered expression levels of the CDKs
coincided with elevated levels of p27 and lowered levels ofpRb, likely reflecting the mechanism of cell cycle delay/arrest in these cells. In the MCF-7 and MDA-MB-231 cells,
while the levels of cyclins D1 and D3 remained almost
unchanged in the treated cells, the levels of CDK4 and CDK6
showed contrasting expression patterns following remedy

treatments. In MCF-7 cells, CDK4 and CDK6 remained
elevated at 24 h, had decreased at 48 h, and had increased at
72 h, whereas the levels showed a gradual decline in MDAMB-231 cells. These minimal changes in the G1 phase CDKs
in MCF-7 cells coincided with their delay/arrest at this cell
cycle stage, and their decline in MDA-MB-231 cells reflected
uninhibited progression through this phase of the cell cycle.

395-403.qxd

16/12/2009

02:02 ÌÌPage 401

INTERNATIONAL JOURNAL OF ONCOLOGY 36: 395-403, 2010

401

Figure 5. Density plot of apoptotic response in HMLE, MCF-7 and MDA-MB-231 cells treated with 5 μl/ml Carcinosin or Phytolacca for 72 h. Apoptotic
response was determined by fluorescence-activated cell sorting. Viable cells are in the lower left quadrant, and early- and late-stage apoptotic cells are in thelower right and upper right quadrants. Note the increase in late apoptotic MCF-7 and MDA-MB-231 cells.

Interestingly, despite maintenance of the elevated CDK4 and
CDK6 levels at 24 h in the treated MCF-7 cells, the level of
its phosphorylated substrate, Rb, showed a sharp decline at
24 h. This coincided with elevated expression of p27,
indicating that induction of p27 following remedy treatment
played an important role in regulating the proliferation andviability of these MCF-7 cells through the cell cycle. A
progressive decline of pRB in MDA-MB-231 cells from 24
to 72 h of remedy treatment, on the other hand, demonstrated
that the remedies had minimal effect during transition throughthe G1 phase of these cells. However, the increasing level of
p27 through 72 h of treatment suggested that induction ofthis kinase inhibitor is responsible for eliciting the cytostaticand cytotoxic responses to the remedies in MDA-MB-231
cells.
Induction of apoptosis and activation of the apoptotic
cascade in Carcinosin and Phytolacca-treated cells. To
confirm that the ultra-diluted remedies induced apoptosis,
HMLE, MCF-7 and MDA-MB-231 cells treated with
Carcinosin and Phytolacca for 72 h were stained with PI andannexin V-Alexa Fluor 488 and analyzed with FACS.
Fig. 5 shows that Carcinosin and Phytolacca markedly
increased the late apoptotic population, as evidenced by theincreased number of MCF-7 cells in the upper right quadrantof the FACS histogram; late apoptosis was also higher in

MDA-MB-231 than in the controls. In contrast, the numbersof such late apoptotic cells were only marginally elevated in
the HMLE cells. Quantitatively, Carcinosin and Phytolaccaincreased the population of apoptotic cells only from 5 to
10% in solvent-treated control cells. The increase in theapoptotic MCF-7 cell population was much larger, ranging
from 7% in solvent-treated cells to 27% and 28%, respectively,
in Carcinosin- and Phytolacca-treated cells. The increase in
the apoptotic cell population in Carcinosin- and Phytolaccatreated MDA-MB-231 cells was less pronounced, ranging
from 10% in control cells to 18 and 16%, respectively, in
Carcinosin- and Phytolacca-treated cells.
As initiation of the apoptotic cascade is associated withthe generation of activated cleaved caspases as well as
cleavage of PARP, we analyzed these molecular markers of
apoptosis in the Carcinosin- and Phytolacca-treated HMLE,
MCF-7 and MDA-MB-231 cells. As shown in Fig. 6, treatment of MCF-7 cells enhanced PARP degradation, resulting
in the appearance of the 85-kDa PARP fragment in Western
blot analyses. Surprisingly, in the MDA-MB-231 cells, theamount of intact PARP progressively declined with increasingtime of treatment but the cleaved 85-kDa band was notevident. HMLE cells showed minimal cleavage of PARP
after treatment with the remedies. Furthermore, activation of
the apoptotic cascade in the remedy-treated cells was evident
from the appearance of the activated cleaved caspase 7 in both

395-403.qxd

16/12/2009402

02:02 ÌÌPage 402

FRENKEL et al: ULTRA-DILUTED REMEDIES ON BREAST CANCER

Figure 6. Western blot analyses of poly (ADP-ribose) polymerase cleavage and activated cleaved caspase 7 in Carcinosin- and Phytolacca-treated HMLE,
MCF-7 and MDA-MB-231 cells. Protein loading in the gel was verified with the detection of ß-actin.

the MCF-7 and MDA-MB-231 cells. Whereas activation of
caspase 7 was minimal in the treated HMLE cells, the activatedform of the enzyme was evident from 24 h onward during
Carcinosin treatment and at 72 h of Phytolacca treatment in
the MCF-7 cells. Activation of caspase 7 in MDA-MB-231
cells, on the other hand, was clearly detectable after 72 h oftreatment with Carcinosin and Phytolacca. The results
therefore demonstrate that apoptotic cascade was preferentially
activated in the remedy-treated carcinoma cells compared to
the normal cells and that the activation in the cancer cells
was temporally regulated in a cell-type-specific manner.
Discussion
Our findings suggest that ultra-diluted homeopathic remedies
prescribed in the ‘Banerji protocol’ exert preferential cytotoxic effects against the human breast carcinoma cell lines
MCF-7 and MDA-MB-231. Further, we found that theseeffects resulted from altered expression of cell cycle regulatory
proteins, which causes cell cycle delay/arrest as well as
induction of cell death by activation of the apoptotic cascade.
Preferential inhibition of survival and induction of cell deathin presence of the remedies compared with the solvent andalso the dose-dependence of these effects validate ourconclusion. Importantly, the study also revealed that these
remedies have relatively less toxic effects on the survival ofcells derived from normal mammary epithelium andmoderately inhibits proliferation of peripheral blood mononuclear cells (PBMC). Proliferation of CD3 and IL-2
stimulated PBMCs, assayed by 3H-thymidine incorporation,
were inhibited by about 10-15% in presence of the remedies
and by about 5% in presence of the solvent (data not shown).
Interestingly, the cytotoxic effect of two of the remedies
investigated in this study, Carcinosin and Phytolacca,
appeared similar to the activity of 0.12 μM paclitaxel (Taxol),
the most commonly used chemotherapeutic drug for breast
cancer, when it was tested in the two adenocarcinoma celllines investigated in this study in parallel experiments (datanot shown). While it is significant that the remedies
negatively affected the survival of the wild-type p53 andestrogen-responsive MCF-7 cells as well as p53-mutated andestrogen-independent MDA-MB-231 cells, the findings ofdifferential susceptibility in these two cell types also indicatethat the cytotoxic activities of the remedies are dependent on
the genetic background of the treated cancer cells.
In this context, it is intriguing that a publication hasreported lack of in vitro growth-inhibitory activity of theremedies investigated in this study in the breast adenocarcinoma cell line MDA-MB-231 and three additional
prostate cancer cell lines, even though the same investigators
had earlier reported significant anti-tumor activity of theremedies in an in vivo model of prostate cancer in Copenhagenrats (7). While these apparently paradoxical in vitro and in vivo
results are somewhat puzzling, the results of this study provide
a mechanistic explanation supporting the previously observedin vivo antitumor effects of these remedies reported by theother group. It is, however, worth mentioning that the in vitro
study published by this group analyzed for gene expression
alterations at the transcript level by RNAse protection assay,
which is not expected to detect the changes in protein
expression profiles of the genes regulating cell death, observedin this investigation.
Transition through the cell cycle phases is regulated by
CDKs in complex with the specific cyclins (8). Cyclin D1 andD3 interact with their regulatory subunits, CDK4 and CDK6,
and the active kinases phosphorylate the Rb protein to release
the transcription factors of the E2F family, which transactivate the genes regulating G1-S phase transition. The
fact that the remedy-treated HMLE and MCF-7 cells showedmarked downregulation of phosphorylated Rb with accompanying changes in the cyclin and CDK levels demonstrates
that the remedies interfere with the cell cycle regulatory pathways, causing G1 phase delay/arrest in these cells.
Regulation of cell cycle progression is also controlled bywell-coordinated inactivation of the CDKs (9,10). The
members of the CDK inhibitor family of proteins, such as
p21 and p27, are known to inactivate the cyclin/CDK
complexes to regulate G1-S and G2-M phase transitions(8,11). Both p21 and p27 physically interact with the CDKs
at the amino terminal domain to inhibit their kinase activity
and in the event of genomic damage activate the checkpoint
response pathways by preventing the cells from initiating
DNA replication and mitosis. This helps maintain genomic
integrity (12,13). Additionally, there are published reports of
p27 overexpression inducing apoptosis in various epithelial
cancer cell lines (14). It is relevant in this context that cell
cycle delay/arrest and the induction of the apoptotic cascade,evident from the presence of reduced telomeric DNA (15)and PARP cleavage as well as activated caspase 7 in theremedy-treated carcinoma cells, were accompanied byupregulation of p27. Inactivation of CDK inhibitors,commonly observed in cancer, is believed to endow the tumor
cells with endless survival and proliferation potential. Thus,
it seems logical that upregulation of CDK inhibitors would

395-403.qxd

16/12/2009

02:02 ÌÌPage 403

INTERNATIONAL JOURNAL OF ONCOLOGY 36: 395-403, 2010activate the checkpoint response mechanisms and control
survival and proliferation in cancer cells (16). Chemopreventive agents have also been described as having similareffects on the regulation of CDK inhibitors to induce proapoptotic response (17). Consistent with these findings and inview of our current observations, the ultra-diluted natural
homeopathic remedies investigated in this study offer thepromise of being effective preventive and/or therapeutic
agents for breast cancer and worthy of further study.
In summary, our study demonstrates that the ultra-diluted
natural product remedies prescribed in the ‘Banerji Protocol’
induce cell cycle delay/arrest with subsequent apoptosis inbreast adenocarcinoma cells. Though the degree of the antisurvival effect appeared to correlate with the presence of thewild-type p53 gene, overall susceptibility to the inhibitory
effects of the remedies appeared independent of the functional
p53 and estrogen-receptor status of the breast carcinoma
cells. Finally, the preferentially elevated cytotoxic effects onbreast adenocarcinoma cells compared with cells derived
from normal mammary epithelium raises the exciting
possibility of a window of therapeutic opportunity for
preferentially eliminating breast cancer cells with minimal
damage to the surrounding normal mammary tissue by using
the ultra-diluted remedies investigated in this report. The
findings of this study should encourage further preclinicaland animal investigation of these remedies as preventive and/or therapeutic treatments for breast cancer.
Acknowledgements
The study was wholly supported by internal funds of MD
Anderson Cancer Center. We also acknowledge Zhijun Liu,
Ph.D at Louisana State University for the constituent analysisof the ultra diluted remedies.
References
1. Barnes P, Bloom B and Nahin R: Complementary andalternative medicine use among adults and children: UnitedStates, 2007. National Health Statistics Reports; no. 12 National
Center for Health Statistics, Hyattsville, MD, 2008.
2. Johannessen H, von Bornemann Hjelmborg J, Pasquarelli E,
Fiorentini G, Di Costanzos F and Miccinesi G: Prevalence in theuse of complementary medicine among cancer patients in
Tuscany, Italy. Tumori 94: 406-410, 2008.

403

3. Trager-Maury S, Tournigand C, Maindrault-Goebel F, Afchain P,
de Gramont A, Garcia-Larnicol ML, Gervais H and Louvet C:
Use of complementary medicine by cancer patients in a French
oncology department. Bull Cancer 94: 1017-1025, 2007.
4. Banerji P and Campbell DR: Cancer patients treated with the
Banerji protocols utilising homoeopathic medicine: A Best Case
Series Program of the National Cancer Institute USA. Oncol
Rep 20: 69-74, 2008.
5. Banerji P: P. Banerji Homeopathic Research Foundation(PBHRF) in Kolkata, India, 2008.
6. Pathak S, Multani AS and Banerji P: Ruta 6 selectively inducescell death in brain cancer cells but proliferation in normal
peripheral blood lymphocytes: a novel treatment for humanbrain cancer. Int J Oncol 23: 975-982, 2003.
7. Thangapazham RL, Gaddipati JP, Rajeshkumar NV, Sharma A,
Singh AK, Ives JA, Maheshwari RK and Jonas WB: Homeopathic medicines do not alter growth and gene expression in
prostate and breast cancer cells in vitro. Integr Cancer Ther 5:356-361, 2006.
8. Sherr CJ and Roberts JM: CDK inhibitors: positive and negative
regulators of G1-phase progression. Genes Dev 13: 1501-1512,
1999.
9. Shapiro GI: Cyclin-dependent kinase pathways as targets forcancer treatment. J Clin Oncol 24: 1770-1783, 2006.
10. Lee YM and Sicinski P: Targeting cyclins and cyclin-dependent
kinases in cancer: lessons from mice, hopes for therapeutic
applications in human. Cell Cycle 5: 2110-2114, 2006.
11. Spankuch B, Kurunci-Csacsko E, Kaufmann M and Strebhardt K:
Rational combinations of siRNAs targeting Plk1 with breast
cancer drugs. Oncogene 26: 5793-5807, 2007.
12. Schwartz GK and Shah MA: Targeting the cell cycle: a new
approach to cancer therapy. J Clin Oncol 23: 9408-9421, 2005.
13. Enders GH and Maude SL: Traffic safety for the cell: influenceof cyclin-dependent kinase activity on genomic stability. Gene
371: 1-6, 2006.
14. Wang X, Gorospe M, Huang Y and Holbrook NJ: p27Kip1
overexpression causes apoptotic death of mammalian cells.
Oncogene 15: 2991-2997, 1997.
15. Pathak S, Multani AS, Narayan S, Kumar V and Newman RA:
Anvirzel, an extract of Nerium oleander, induces cell death inhuman but not murine cancer cells. Anticancer Drugs 11: 455-463,
2000.
16. Roy S, Gu M, Ramasamy K, Singh RP, Agarwal C, Siriwardana S,
Sclafani RA and Agarwal R: p21/Cip1 and p27/Kip1 areessential molecular targets of inositol hexaphosphate for its
antitumor efficacy against prostate cancer. Cancer Res 69:
1166-1173, 2009.
17. Singh RP and Agarwal R: Natural flavonoids targeting
deregulated cell cycle progression in cancer cells. Curr Drug
Targets 7: 345-354, 2006.