Sainte-Foy-lès-Lyon, France
Sainte-Foy-lès-Lyon, France

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Khuda-Bukhsh A.R.,Kalyani University | Bhattacharyya S.S.,Kalyani University | Paul S.,Kalyani University | Dutta S.,Kalyani University | And 2 more authors.
Evidence-based Complementary and Alternative Medicine | Year: 2011

In homeopathy, ability of ultra-high diluted drugs at or above potency 12C (diluted beyond Avogadro's limit) in ameliorating/curing various diseases is often questioned, particularly because the mechanism of action is not precisely known. We tested the hypothesis if suitable modulations of signal proteins could be one of the possible pathways of action of a highly diluted homeopathic drug, Secale cornutum 30C (diluted 1060 times; Sec cor 30). It could successfully combat DMBA + croton oil-induced skin papilloma in mice as evidenced by histological, cytogenetical, immunofluorescence, ELISA and immunoblot findings. Critical analysis of several signal proteins like AhR, PCNA, Akt, Bcl-2, Bcl-xL, NF-κB and IL-6 and of pro-apoptotic proteins like cytochrome c, Bax, Bad, Apaf, caspase-3 and -9 revealed that Sec cor 30 suitably modulated their expression levels along with amelioration of skin papilloma. FACS data also suggested an increase of cell population at S and G2 phases and decrease in sub-G1 and G1 phages in carcinogen-treated drug-unfed mice, but these were found to be near normal in the Sec cor 30-fed mice. There was reduction in genotoxic and DNA damages in bone marrow cells of Sec Cor 30-fed mice, as revealed from cytogenetic and Comet assays. Changes in histological features of skin papilloma were noted. Immunofluorescence studies of AhR and PCNA also suggested reduced expression of these proteins in Sec cor 30-fed mice, thereby showing its anti-cancer potentials against skin papilloma. Furthermore, this study also supports the hypothesis that potentized homeopathic drugs act at gene regulatory level. Copyright © 2011 Anisur Rahman Khuda-Bukhsh et al.


Khuda-Bukhsh A.R.,Kalyani University | Bhattacharyya S.S.,Kalyani University | Paul S.,Kalyani University | Boujedaini N.,Boiron Laboratory
Journal of Chinese Integrative Medicine | Year: 2010

Objective: We formulated nano-encapsulation of a naturally occurring coumarin - scopoletin (7-hydroxy-6-methoxy coumarin, HMC, C 10H 8O 4), isolated from plant Gelsemium sempervirens having anticancer potentials, with a bio-adhesive agent - polylactic-co-glycolic acid (PLGA) and tested if its cellular uptake, bioavailability and apoptotic (anticancer) potentials could thus be increased vis-à-vis unencapsulated HMC. Methods: A375 melanoma cancer cells were used for testing cellular entry and anticancer potentials of HMC and nano-7-hydroxy-6-methoxy coumarin (NHMC) through several standard protocols. Characterization of NHMC was done by dynamic light scattering for determination of particle size, polydispersity index (PDI), and zeta potential. Surface morphology of nanoparticles was determined by scanning electron microscopy and atomic force microscopy. Results: HMC was encapsulated with more than 85% entrapment efficiency, the average particle size of NHMC being less than 110 nm and a PDI 0.237, which resulted in enhanced cellular entry and greater bioavailability. NHMC showed a faster cellular uptake (15 min) than its unencapsulated counterpart (30 min). Study of signal molecules through mRNA expressions revealed that NHMC caused down-regulation of cyclin-D1, proliferating cell nuclear antigen (PCNA), survivin and Stat-3, and up-regulation of p53 and caspase-3, that in turn induced a greater number of apoptosis vis-à-vis unencapsulated HMC. Conclusion: The formulation yielded small-sized NHMC by biodegradable PLGA that took less time for cellular entry, and caused more apoptosis to cancer cells, but apparently had negligible cytotoxicity against normal skin cells. Nano-encapsulation of bioactive plant ingredients can be a strategy worth trying for designing effective chemopreventive drug products.


Bishayee K.,Kalyani University | Paul A.,Kalyani University | Ghosh S.,Kalyani University | Sikdar S.,Kalyani University | And 4 more authors.
Molecular and Cellular Biochemistry | Year: 2013

Gonolobus condurango plant extract is used as an anticancer drug in some traditional systems of medicine including homeopathy, but it apparently lacks any scientific validation. Further, no detailed study is available to suggest whether condurango-glycoside-A (CGA), a major ingredient of condurango serves as a potent anticancer compound. Therefore, we investigated apoptosis-inducing ability of CGA against cervix carcinoma cells (HeLa). β-galactosidase- activity and DNA damage were critically studied at different time points; while induced DNA-damage was observed at 9-12th hours, senescence of cells appeared at a later stage (18th hour after CGA treatment), implicating thereby a possible role of DNA damage in inducing pre-mature cell senescence. Concurrently, the number of cells undergoing apoptosis increased along with increase in reactive oxygen species (ROS) generation. Expression of p53 was also up-regulated, indicating that apoptosis could have been mediated through p53 pathway. DCHFDA (4′,6-Diamidino-2-phenylindole dihydrochloride) assay, acridine orange/ethidium bromide staining and annexin V/PI assay results collectively confirmed that apoptosis was induced by increased ROS generation. Reduction in proliferation of cells was further evidenced by the cell cycle arrest at G0/G1 stage. Expression profiles of certain relevant genes and proteins like p53, Akt, Bcl-2, Bax, cytochrome c and caspase 3 also provided evidence of ROS mediated p53 up-regulation and further boost in Bax expression and followed by cytochrome c release and activation of caspase 3. Overall results suggest that CGA initiates ROS generation, promoting up-regulation of p53 expression, thus resulting in apoptosis and pre-mature senescence associated with DNA damage. © 2013 Springer Science+Business Media New York.


Samadder A.,Kalyani University | Das S.,Kalyani University | Das J.,Kalyani University | Paul A.,Kalyani University | And 2 more authors.
JAMS Journal of Acupuncture and Meridian Studies | Year: 2013

Cancer is a disease that needs a multi-faceted approach from different systems of medicine. The purpose of this study was to evaluate whether homeopathically-potentized ultra-high dilutions of Lycopodium Clavatum (LC-5C and LC-15C, respectively) have any anti-cancer effects on HeLa cells. Cells were exposed to either LC-5C (diluted below Avogadro's limit, i.e., 10-10) or LC-15C (diluted beyond Avogadro's limit, i.e., 10-30) (drug-treated) or to 30% succussed ethanol ("vehicle" of the drug). The drug-induced modulation in the percent cell viability, the onset of apoptosis, and changes in the expressions of Bax, Bcl2, caspase 3, and Apaf proteins in inter-nucleosomal DNA, in mitochondrial membrane potentials and in the release of cytochrome-c were analyzed by utilizing different experimental protocols. Results revealed that administration of LC-5C and LC-15C had little or no cytotoxic effect in normal peripheral blood mononuclear cells, but caused considerable cell death through apoptosis in cancer (HeLa) cells, which was evident from the induction of DNA fragmentation, the increases in the expressions of protein and mRNA of caspase 3 and Bax, and the decreases in the expressions of Bcl2 and Apaf and in the release of cytochrome-c. Thus, the highly-diluted, dynamized homeopathic remedies LC-5C and LC-15C demonstrated their capabilities to induce apoptosis in cancer cells, signifying their possible use as supportive medicines in cancer therapy. © 2013.


Das S.,Kalyani University | Das J.,Kalyani University | Samadder A.,Kalyani University | Boujedaini N.,Boiron Laboratory | Khuda-Bukhsh A.R.,Kalyani University
Experimental Biology and Medicine | Year: 2012

We isolated apigenin (5,7,4′-trihydroxy flavone) from ethanolic extract of Lycopodium clavatum (LC) used as a homeopathic mother tincture for treatment of various diseases. We assessed the anticancer potentials of the compound using human malignant melanoma cell line A375 and a lung carcinoma cell line A549 and focussed on its putative molecular mechanism of action on apoptosis induction. We examined the cytotoxicity of apigenin in both cancer cells and normal peripheral blood mononuclear cells (PBMC). A375 cells were more prone to apigenin-induced apoptosis, as compared with A549 cells after 24 h of treatment, while PBMC showed little or no cytotoxicity to apigenin. We also evaluated the effects of apigenin on interaction with DNA by comparative analysis of circular dichroism spectral data and melting temperature profiles (Tm) of calf thymus DNA (CT-DNA) treated with or without apigenin. Reactive oxygen species (ROS) accumulation in mitochondria, super-oxide dismutase and total thiol group (GSH) activities were also analyzed. The apoptotic process involved mitochondrial pathway associated with apigenin-DNA interaction, DNA fragmentation, ROS accumulation, cytochrome c (cyt c) release and mitochondrial transmembrane potential depolarization, Bax, caspase 3, 9, PARP, upregulation, Bcl-2 down-regulation and down-regulation of cyt c in the mitochondrial fraction. Results of mitochondrial inner membrane swelling measurements, intracellular ADP/ATP ratio and ATPase activity showed that in A549 cells, apigenin did not appear to directly target the mitochondrial oxidative phosphorylation system but rather acted at an upstream step to activate the mitochondrial apoptotic pathway. However, apigenin could directly target and impair mitochondrial function in A375 cells by breaking down their oxidative phosphorylation system. Collectively, these results suggest that apigenin exhibits anticancer potential in A375 and A549 cells that may be mediated through DNA interaction, damage and mitochondrial dysfunction either by direct or indirect action on mitochondrial oxidative phosphorylation system. © 2008 Society for Experimental Biology and Medicine.


Mukherjee A.,Kalyani University | Das D.,Kalyani University | Kumar Mondal S.,Kalyani University | Biswas R.,Kalyani University | And 3 more authors.
Ecotoxicology and Environmental Safety | Year: 2010

The arsenate tolerance limit in wild-type Aspergillus niger was determined. Because of its high tolerance, toxic effects of arsenate concentrations ranging from 25 to 100 mg/L were investigated in regard to growth, intracellular thiols, proline and malondialdehyde (MDA) contents of wild-type A. niger. Cellular arsenate uptake was analyzed. Activities of catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and succinate dehydrogenase (SDH) were assayed. Growth of A. niger increased at 25 mg/L arsenate, and it survived up to 100 mg/L. MDA, intracellular thiol and proline contents increased up to a certain level. Activities of GR, SOD and CAT declined following a rise at low concentration(s); SDH activity decreased gradually with increased arsenate stress. Results indicated that A. niger had high arsenate uptake potential and could tolerate oxidative stress by manipulating its anti-oxidative defense mechanism, a property that may be exploited for removal of arsenate from contaminated aqua-environment. © 2009 Elsevier Inc. All rights reserved.


De A.,Kalyani University | Das D.,Kalyani University | Dutta S.,Kalyani University | Chakraborty D.,Kalyani University | And 2 more authors.
Journal of Chinese Integrative Medicine | Year: 2012

OBJECTIVE: To examine if potentized homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can reduce sodium arsenite-induced toxicity in Escherichia coli. METHODS: E. coli were exposed to low arsenite insult after they grew up to log phase in Standard Luria-Bertani medium. E. coli were treated with 1 or 2 mmol/L sodium arsenite alone (control), or Ars Alb 30C was added to the medium of a subset of sodium arsenite-treated bacteria (drug-treated), or homeopathically agitated alcohol was added to the medium containing a subset of sodium arsenite-treated bacteria (placebo-treated). A sub-set of untreated E. coli served as the negative control. Glucose uptake, specific activities of hexokinase, lipid peroxidase (LPO), Superoxide dismutase (SOD) and catalase, intra- and extra-cellular sodium arsenite content, cell growth, cell membrane potential, DNA damage, intracellular reactive oxygen species (ROS), adenosine triphosphate (ATP) and free glutathione content and expressions of arsB and ptsG gene in normal control, sodium arsenite-treated, drug-treated and placebo-treated E. coli were analyzed. Treatments were blinded and randomized. RESULTS: In sodium arsenite-treated E. coli, glucose uptake, intracellular ROS, LPO and DNA damage increased along with decrease in the specific activities of hexokinase, SOD and catalase, intracellular ATP and free glutathione contents and cell membrane potential and growth, and there were increases in expression levels of arsB gene and ptsG gene. Ars Alb 30C administration reduced arsenic toxicity in E. coli by inhibiting generation of ROS and increasing tolerance to arsenite toxicity and cell growth. CONCLUSION: Ars Alb 30C ameliorated arsenic toxicity and DNA damage, validating efficacy of ultra-highly diluted remedies used in homeopathy.


Saha S.K.,Kalyani University | Sikdar S.,Kalyani University | Mukherjee A.,Kalyani University | Bhadra K.,Kalyani University | And 2 more authors.
Environmental Toxicology and Pharmacology | Year: 2013

This study tested chemotherapeutic potential of Hydrastis canadensis (HC) extract in HeLa cells in vitro, with emphasis on its drug-DNA interaction and apoptosis induction ability. Nuclear uptake of HC by DAPI, Ao/Eb staining and internucleosomal DNA damage by comet assay was studied through fluorescence microscopy. Possible changes in MMP and apoptotic signalling events were critically analyzed. Cell cycle progression studied through FACS and fragmented DNA through "TUNEL" assay were critically analyzed. RT-PCR studies were conducted for analyzing Cyt-C and Bax translocation in mitochondrial and cytosolic extracts, and Caspase 3 in whole cell lysate. Role of p53-mediated regulation of NF-κβ and TNF-α was elucidated by Western blot analysis. Data of CD and Tm profile of CT-DNA were analyzed. Overall results indicated anti-cancer potential of HC through its ability to induce apoptosis, and interaction with CT-DNA that changed structural conformation of DNA, proving HC to be a promising candidate for chemoprevention. © 2013 Elsevier B.V.


Bhattacharyya S.S.,Kalyani University | Paul S.,Kalyani University | Dutta S.,Kalyani University | Boujedaini N.,Boiron Laboratory | Khuda-Bukhsh A.R.,Kalyani University
Journal of Chinese Integrative Medicine | Year: 2010

Objective: Anti-cancer potentials of scopoletin (7-hydroxy-6-methoxy coumarin) separated from plant extract (Gelsemium sempervirens) were demonstrated earlier from our in vitro studies. In the present study, its in vivo effects have been evaluated in mice. Methods: Mice were chronically administered 7,12-dimethylbenz[a]anthracene (DMBA) once a week and croton oil twice a week on their back, which resulted in the development of fully grown finger-like projections (papilloma) after 24 weeks. Two subgroups of mice (drug-treated) were treated with two doses of scopoletin (50 mg and 100 mg/kg body weight) respectively while control received 2% ethyl alcohol (the "vehicle" of scopoletin). After the 24-week drug administration, expressions of several key receptors such as aryl hydrocarbon receptor (AhR) and signal proteins like p53, cytochrome P450 1A1 (CYP1A1), proliferating cell nuclear antigen (PCNA), signal transducer and activator of transcription-3 (Stat-3), survivin, matrix metalloproteinase-2 (MMP-2), cyclin D1, c-myc, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and caspase-3, and some anti-oxidant markers were studied. Lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase in supernatant were also detected. Results: Carcinogens induced toxicity, and over-expression of AhR, CYP1A1, PCNA, Stat-3, survivin, MMP-2, cyclin D1 and c-myc and down-regulation of p53, caspase-3 and TIMP-2. In mice treated with scopoletin, the expressions of these proteins and toxicity biomarkers were reverted. Conclusion: Since AhR is known to be ligand-activated by DMBA to release signals for several downstream proteins initiating reactive oxygen species generation, the down-regulation of AhR by scopoletin appeared to play a significant role in subsequent down-regulation of some key signal proteins. One possible mechanism of down-regulation of AhR may be through competitive inhibition by scopoletin. Mitogen-activated protein kinases may also have some critical role. This compound can be considered as a possible candidate for chemoprevention.


Paul S.,Kalyani University | Bhattacharyya S.S.,Kalyani University | Samaddar A.,Kalyani University | Boujedaini N.,Boiron Laboratory | Khuda-Bukhsh A.R.,Kalyani University
Journal of Chinese Integrative Medicine | Year: 2011

Objective: To evaluate anticancer potentials of Polygala senega on lung cancer induced by benzo [a]pyrene (B[a]P) in mice. Methods: Swiss albino mice were divided into live groups with each containing six animals. Group 1 served as control, and the animals received olive oil as vehicle. Group 2 animals were treated with B[a]P (50 mg/kg body weight dissolved in olive oil) orally twice a week for four consecutive weeks. Group 3 animals were fed B[a]P as in group 2 and 48% alcohol (since the vehicle of the remedy was alcohol). Group 4 animals were B[a]P-intoxicated mice (as in group 2) which were additionally fed ethanolic extract of Polygala senega (EEPS) daily for 16 weeks. EEPS treatment started after the first dose of B[a]P. Group 5 animals were treated with EEPS alone for 16 weeks to test cytotoxicity of EEPS if any. Mice were sacrificed after 16 weeks and the following parameters were assessed: the anti-oxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl free radical assay, tumor incidence, lung weight and body weight, DNA damage evaluation by comet assay and enzyme-linked immunosorbent assay (ELISA); toxicity biomarkers like catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, lipid peroxidation (LPO) and total thiol content were also detected. Results: Treatment with EEPS increased the final body weight and significantly decreased the lung weight in group 4 mice (P<0.01) compared with group 3 mice. Comet assay showed that EEPS-treated mice in group 4 presented a decrease of DNA damage significantly (P<0.01) in lung tissues. There was a significant increase observed in the level of p53 in group 4 as compared with group 3 (P<0.01) detected by ELISA. A highly significant increase in tissue LPO with concomitant decrease in the activity of anti-oxidants was observed in group 2 and group 3 mice (P<0.05) compared with the control mice. These adverse changes were reversed significantly in group 1 mice (P<0.01). Conclusion: Chemopreventive potentials of Polygala senega against chemically induced lung cancer in mice are confirmed.

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