Condurango (Gonolobus condurango) Extract Activates Fas Receptor and Depolarizes Mitochondrial Membrane Potential to Induce ROS-dependent Apoptosis in Cancer Cells in vitro: CE-treatment on HeLa: a ROS-dependent mechanism.

OBJECTIVES: Condurango (Gonolobus condurango) extract is used by complementary and alternative medicine (CAM) practitioners as a traditional medicine, including homeopathy, mainly for the treatment of syphilis. Condurango bark extract is also known to reduce tumor volume, but the underlying molecular mechanisms still remain unclear. METHODS: Using a cervical cancer cell line (HeLa) as our model, the molecular events behind condurango extract's (CE's) anticancer effect were investigated by using flow cytometry, immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Other included cell types were prostate cancer cells (PC3), transformed liver cells (WRL-68), and peripheral blood mononuclear cells (PBMCs). RESULTS: Condurango extract (CE) was found to be cytotoxic against target cells, and this was significantly deactivated in the presence of N-acetyl cysteine (NAC), a scavenger of reactive oxygen species (ROS), suggesting that its action could be mediated through ROS generation. CE caused an increase in the HeLa cell population containing deoxyribonucleic acid (DNA) damage at the G zero/Growth 1 (G0/G1) stage. Further, CE increased the tumor necrosis factor alpha (TNF-α) and the fas receptor (FasR) levels both at the ribonucleic acid (RNA) and the protein levels, indicating that CE might have a cytotoxic mechanism of action. CE also triggered a sharp decrease in the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB ) both at the RNA and the protein levels, a possible route to attenuation of B-cell lymphoma 2 (Bcl-2), and caused an opening of the mitochondrial membrane's permeability transition (MPT) pores, thus enhancing caspase activities. CONCLUSION: Overall, our results suggest possible pathways for CE mediated cytotoxicity in model cancer cells.

Anti-lung cancer potential of pure esteric-glycoside condurangogenin A against nonsmall-cell lung cancer cells in vitro via p21/p53 mediated cell cycle modulation and DNA damage-induced apoptosis.

BACKGROUND: Marsdenia condurango (condurango) is a tropical woody vine native to South America. Our earlier study was limited to evaluation of anti-cancer potentials of crude condurango extract and its glycoside-rich components in vitro on lung cancer. OBJECTIVE: This study aims at evaluating the effect of the single isolated active ingredient condurangogenin A (ConA; C32H42O7) on A549, H522 and H460-nonsmall-cell lung cancer cells. MATERIALS AND METHODS: ConA was isolated by column chromatography and analyzed by mass spectroscopy, Fourier transform infrared spectroscopy and proton-nuclear magnetic resonance. diphenyltetrazolium bromide assays were conducted on three cell-types using 6%-alcohol as control. Critical studies on cellular morphology, cell-cycle regulation, reactive oxygen species, mitochondrial membrane potential, and DNA-damage were made, and expressions of related signaling markers studied. RESULTS: As IC50 doses of ConA proved to be too high and toxic to both A549 and H522 cells, all experimental studies were carried out on H460 cells with the IC50 dose (32 µg/ml - 24 h). Cellular morphology revealed typical apoptotic features after ConA treatment. At early treatment hours (2 h-12 h), maximum cells were arrested at G0/G1 phase that could be correlated with reduced level of cyclin D1-CDK with p21 up-regulation. At 18 h - 24 h, sub G0/G1 cell population was increased gradually, as revealed from cytochrome-c release and caspase-3 activation, further confirming the apoptosis-inducing ability of ConA at later phases. Gradual increase of TUNEL-positive cells with significant modulation of mitochondria-dependent apoptotic markers at longer time-points would establish apoptosis-induction property of ConA, indicating its potential as a strong candidate for anti-cancer drug formulation. CONCLUSION: Further studies are warranted against other types of cancer cells and animal models before its possible human use.

Condurango 30C Induces Epigenetic Modification of Lung Cancer-specific Tumour Suppressor Genes via Demethylation.

BACKGROUND: DNA hypermethylation induces cancer progression involving CpG island of DNA and causes inactivation of tumour suppressor genes. In this study, DNA hypermethylation status of lung cancer and ability of ultra-highly diluted Condurango 30C to modulate DNA methylation were ascertained by analysis of lung cancer-specific tumour suppressor genes in respect to placebo. MATERIALS AND METHODS: DNA methylation status, if any, was determined by PCR-SSCP analyses in lung cancer-specific tumour suppressor genes (p15, p16 and p53) using H460-NSCLC cell and BaP-induced lung cancer of rats. The ability of Condurango 30C to modulate DNA methylation, if any, was verified against placebo control in blinded manner. RESULTS: Condurango 30C-treated DNA showed significant decrease in band intensity of p15 and p53 genes especially in methylated condition in vitro, at IC50 dose (2.43µl/100µl). SSCP analysis of p15 and p53 genes in Condurango 30C-treated DNA also suggests that Condurango 30C can decrease methylation, in vitro. Inhibition of p15 hypermethylation was observed in post-cancer treatment of rats with Condurango 30C. SSCP results gave a better indication of differences in band position of p15 and p53 in Condurango 30C-treated lung samples. CONCLUSION: Condurango 30C could trigger epigenetic modification in lung cancer via modulation of DNA hypermethylation.

Evaluation of chemopreventive potentials of ethanolic extract of Ruta graveolens against A375 skin melanoma cells in vitro and induced skin cancer in mice in vivo.

OBJECTIVE: Chemopreventive approach with natural products, particularly plants and plant-derived ones, is receiving increasing attention for their effective role against cancer without any palpable side effects. In this study, efficacy of ethanolic extract of Ruta graveolens (RG) on skin melanoma cells (A375) in vitro and on 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer in vivo has been tested in Swiss albino mice. METHODS: Studies on cell viability, apoptosis and autophagy induction were conducted in vitro. To check apoptosis, assays like alteration in mitochondrial membrane potential, annexin V-fluorescein isothiocyanate/propidium iodide assay and immunoblot were performed. Fluorescence microscopic and immunoblot assays were performed to confirm autophagy induction. The effects of RG were determined by evaluating body weight, tumor incidence, tumor volume and tumor burden in mice. Enzymatic and non-enzymatic antioxidant status was assessed. The role of some relevant signaling proteins was also analyzed. RESULTS: RG caused death of A375 cells through induction of caspase 3-mediated apoptosis and Beclin-1-associated autophagy. Moreover, RG administration (75 mg/kg body weight) which showed no acute or chronic toxicity, showed significant reduction in the skin tumor burden of DMBA-painted mice. RG also demonstrated potent anti-lipid peroxidative and antioxidant functions during the course of skin cancer induction by DMBA. CONCLUSION: Chemopreventive potential of RG was demonstrated from overall results of this study, indicating its possible use in therapeutic formulation of an effective drug to treat skin cancer.

Evaluation of chemopreventive potentials of ethanolic extract of Ruta graveolens against A375 skin melanoma cells in vitro and induced skin cancer in mice in vivo / 中西医结合学报

Chemopreventive approach with natural products, particularly plants and plant-derived ones, is receiving increasing attention for their effective role against cancer without any palpable side effects. In this study, efficacy of ethanolic extract of Ruta graveolens (RG) on skin melanoma cells (A375) in vitro and on 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer in vivo has been tested in Swiss albino mice.

Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cell-proliferation in lung cancer, in vitro and in vivo.

Chemotherapeutic potential of Condurango glycoside-rich components (CGS) was evaluated in NSCLC, in vitro and in BaP-intoxicated rats, in vivo. NSCLC cells were treated with different concentrations of CGS to test their effect on cell viability. Cellular morphology, DNA-damage, AnnexinV-FITC/PI, cell cycle regulation, ROS-accumulation, MMP, and expressions of related signalling genes were critically analysed. 0.22 µg/µl CGS (IC50 dose at 24 h) was selected for the study. CGS-induced apoptosis via DNA damage was evidenced by DNA-ladder formation, increase of AnnexinV-positive cells, cell cycle arrest at subG0/G1 and differential expressions of apoptotic genes. ROS-elevation and MMP-depolarization with significant caspase-3 activation might lead to apoptotic cell death. Anti-proliferative activity was confirmed by EGFR-expression modulation. ROS accumulation and DNA-nick formation with tissue damage-repair activity after post-cancerous CGS treatment, in vivo, supported the in vitro findings. Overall results advocate considerable apoptosis-inducing potential of CGS against NSCLC, validating its use against lung cancer by CAM practitioners.

Relative Apoptosis-inducing Potential of Homeopa-thic Condurango 6C and 30C in H460 Lung Cancer Cells In vitro: -Apoptosis-induction by homeopathic Condurango in H460 cells.

OBJECTIVES: In homeopathy, it is claimed that more homeopathically-diluted potencies render more protective/curative effects against any disease condition. Potentized forms of Condurango are used successfully to treat digestive problems, as well as esophageal and stomach cancers. However, the comparative efficacies of Condurango 6C and 30C, one diluted below and one above Avogadro's limit (lacking original drug molecule), respectively, have not been critically analyzed for their cell-killing (apoptosis) efficacy against lung cancer cells in vitro, and signalling cascades have not been studied. Hence, the present study was undertaken. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) assays were conducted on H460-non-small-cell lung cancer (NSCLC) cells by using a succussed ethyl alcohol vehicle (placebo) as a control. Studies on cellular morphology, cell cycle regulation, generation of reactive oxygen species (ROS), changes in mitochondrial membrane potential (MMP), and DNA-damage were made, and expressions of related signaling markers were studied. The observations were done in a "blinded" manner. RESULTS: Both Condurango 6C and 30C induced apoptosis via cell cycle arrest at subG0/G1 and altered expressions of certain apoptotic markers significantly in H460 cells. The drugs induced oxidative stress through ROS elevation and MMP depolarization at 18-24 hours. These events presumably activated a caspase-3-mediated signalling cascade, as evidenced by reverse transcriptase- polymerase chain reaction (RT-PCR), western blot and immunofluorescence studies at a late phase (48 hours) in which cells were pushed towards apoptosis. CONCLUSION: Condurango 30C had greater apoptotic effect than Condurango 6C as claimed in the homeopathic doctrine.

Ethanolic Extract of Marsdenia condurango Ameliorates Benzo[a]pyrene-induced Lung Cancer of Rats: Condurango Ameliorates BaP-induced Lung Cancer in Rats.

OBJECTIVES: Condurango is widely used in various systems of complementary and alternative medicines (CAM) against oesophageal and stomach ailments including certain types of cancer. However, until now no systematic study has been conducted to verify its efficacy and dose with proper experimental support. Therefore, we examined if ethanolic extract of Condurango could ameliorate benzo[a]pyrene (BaP)-induced lung cancer in rats, in vivo to validate its use as traditional medicine. METHODS: Fifteen male and 15 female Sprague-Dawley (SD) rats were treated with 0.28 mg/kg of Sweet Bee Venom (SBV) (high-dosage group) and the same numbers of male and female SD rats were treated with 0.2 mL/kg of normal saline (control group) for 13 weeks. We selected five male and five female SD rats from the high-dosage group and the same numbers of male and female SD rats from the control group, and we observed these rats for four weeks. We conducted body-weight measurements, ophthalmic examinations, urinalyses and hematology, biochemistry, histology tests. RESULTS: A histological study revealed gradual progress in lung tissue-repair activity in Condurango-fed cancer-bearing rats, showing gradual tissue recovery after three months of drug administration. Condurango has the capacity to generate reactive oxygen species (ROS), which may contribute to a reduction in anti-oxidative activity and to an induction of oxidative stress-mediated cancer cell-death. Condurango-activated pro-apoptotic genes (Bax, caspase-3, caspase-9, p53, cytochrome-c, apaf-1, ICAD and PARP) and down-regulated antiapoptotic-Bcl-2 expression were noted both at mRNA and protein levels. Studies on caspase-3 activation and PARP cleavage by western blot analysis revealed that Condurango induced apoptosis through a caspase-3-dependent pathway. CONCLUSION: The anticancer efficacy of an ethanolic extract of Condurango for treating BaP-induced lung cancer in rats lends support for its use in various traditional systems of medicine.

Condurango-glycoside-A fraction of Gonolobus condurango induces DNA damage associated senescence and apoptosis via ROS-dependent p53 signalling pathway in HeLa cells.

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). ß-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.

Ethanolic extract of the Goldenseal, Hydrastis canadensis, has demonstrable chemopreventive effects on HeLa cells in vitro: Drug-DNA interaction with calf thymus DNA as target.

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-κß 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.

Antihyperglycemic drug Gymnema sylvestre also shows anticancer potentials in human melanoma A375 cells via reactive oxygen species generation and mitochondria-dependent caspase pathway.

OBJECTIVE: Ethanolic extract of Gymnema sylvestre (GS) leaves is used as a potent antidiabetic drug in various systems of alternative medicine, including homeopathy. The present study was aimed at examining if GS also had anticancer potentials, and if it had, to elucidate its possible mechanism of action. METHODS: We initially tested possible anticancer potential of GS on A375 cells (human skin melanoma) through MTT assay and determined cytotoxicity levels in A375 and normal liver cells; we then thoroughly studied its apoptotic effects on A375 cells through protocols such as Hoechst 33258, H2DCFDA, and rhodamine 123 staining and conducted ELISA for cytochrome c, caspase 3, and PARP activity levels; we determined the mRNA level expression of cytochrome c, caspase 3, Bcl2, Bax, PARP, ICAD, and EGFR signaling genes through semiquantitative reverse transcriptase polymerase chain reaction and conducted Western blot analysis of caspase 3 and PARP. We also analyzed cell cycle events, determined reactive oxygen species accumulation, measured annexin V-FITC/PI and rhodamine 123 intensity by flow cytometry. RESULTS: Compared with both normal liver cells and drug-untreated A375, the mortality of GS-treated A375 cells increased in a dose-dependent manner. Additionally, GS induced nuclear DNA fragmentation and showed an increased level of mRNA expression of apoptotic signal related genes cytochrome c, caspase 3, PARP, Bax, and reduced expression level of ICAD, EGFR, and the anti-apoptotic gene Bcl2. CONCLUSION: Overall results indicate GS to have significant anticancer effect on A375 cells apart from its reported antidiabetic effect, indicating possibility of its palliative use in patients with symptoms of both the diseases.

Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation.

OBJECTIVE: Preventive measures against skin melanoma like chemotherapy are useful but suffer from chronic side effects and drug resistance. Ethanolic extract of Phytolacca decandra (PD), used in homeopathy for the treatment of various ailments like chronic rheumatism, regular conjunctivitis, psoriasis, and in some skin diseases was tested for its possible anticancer potential. METHODS: Cytotoxicity of the drug was tested by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on both normal (peripheral blood mononuclear cells) and A375 cells. Fluorescence microscopic study of 4',6-diamidino-2-phenylindole dihydrochloride-stained cells was conducted for DNA fragmentation assay, and changes in cellular morphology, if any, were also recorded. Lactate dehydrogenase activity assay was done to evaluate the percentages of apoptosis and necrosis. Reactive oxygen species (ROS) accumulation, if any, and expression study of apoptotic genes also were evaluated to pin-point the actual events of apoptosis. RESULTS: Results showed that PD administration caused a remarkable reduction in proliferation of A375 cells, without showing much cytotoxicity on peripheral blood mononuclear cells. Generation of ROS and DNA damage, which made the cancer cells prone to apoptosis, were found to be enhanced in PD-treated cells. These results were duly supported by the analytical data on expression of different cellular and nuclear proteins, as for example, by down-regulation of Akt and Bcl-2, up-regulation of p53, Bax and caspase 3, and an increase in number of cell deaths by apoptosis in A375 cells. CONCLUSION: Overall results demonstrate anticancer potentials of PD on A375 cells through activation of caspase-mediated signaling and ROS generation.

Post-cancer Treatment with Condurango 30C Shows Amelioration of Benzo[a]pyrene-induced Lung Cancer in Rats Through the Molecular Pathway of Caspa- se-3-mediated Apoptosis Induction: -Anti-lung cancer potential of Condurango 30C in rats.

OBJECTIVES: The present investigation aimed at examining if post-cancer treatment with a potentized homeopathic drug, Condurango 30C, which is generally used to treat oesophageal cancer, could also show an ameliorating effect through apoptosis induction on lung cancer induced by benzo[a]pyrene (BaP) in white rats (Rattus norvegicus). METHODS: Lung cancer was induced after four months by chronic feeding of BaP to rats through gavage at a dose of 50 mg/kg body weight for one month. After four months, the lung-cancer-bearing rats were treated with Condurango 30C for the next one (5(th)), two (5(th)-6(th)) and three (5(th)-7(th)) months, respectively, and were sacrificed at the corresponding time- points. The ameliorating effect, if any, after Condurango 30C treatment for the various periods was evaluated by using protocols such as histology, scanning electron microscopy (SEM), annexinV-FITC/PI assay, flow cytometry of the apoptosis marker, DNA fragmentation, reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and western blot analyses of lung tissue samples. RESULTS: Striking recovery of lung tissue to a near normal status was noticed after post-cancerous drug treatment, as evidenced by SEM and histology, especially after one and two months of drug treatment. Data from the annexinV-FITC/PI and DNA fragmentation assays revealed that Condurango 30C could induce apoptosis in cancer cells after post-cancer treatment. A critical analysis of signalling cascade, evidenced through a RT-PCR study, demonstrated up-regulation and down-regulation of different pro- and anti-apoptotic genes, respectively, related to a caspase-3-mediated apoptotic pathway, which was especially discernible after one-month and two- month drug treatments. Correspondingly, Western blot and immunohistochemistry studies confirmed the ameliorative potential of Condurango 30C by its ability to down-regulate the elevated epidermal growth factor receptor (EGFR) expression, a hallmark of lung cancer. CONCLUSION: The overall result validated a positive effect of Condurango 30C in ameliorating lung cancer through caspase-3-mediated apoptosis induction and EGFR down-regulation.

Evidence of an Epigenetic Modification in Cell-cycle Arrest Caused by the Use of Ultra-highly-diluted Gonolobus Condurango Extract.

OBJECTIVES: Whether the ultra-highly-diluted remedies used in homeopathy can effectively bring about modulations of gene expressions through acetylation/deacetylation of histones has not been explored. Therefore, in this study, we pointedly checked if the homeopathically-diluted anti-cancer remedy Condurango 30C (ethanolic extract of Gonolobus condurango diluted 10(-60) times) was capable of arresting the cell cycles in cervical cancer cells HeLa by triggering an epigenetic modification through modulation of the activity of the key enzyme histone deacetylase 2 vis-a-vis the succussed alcohol (placebo) control. METHODS: We checked the activity of different signal proteins (like p21(WAF), p53, Akt, STAT3) related to deacetylation, cell growth and differentiation by western blotting and analyzed cell-cycle arrest, if any, by fluorescence activated cell sorting. After viability assays had been performed with Condurango 30C and with a placebo, the activities of histone de-acetylase (HDAC) enzymes 1 and 2 were measured colorimetrically. RESULTS: While Condurango 30C induced cytotoxicity in HeLa cells in vitro and reduced HDAC2 activity quite strikingly, it apparently did not alter the HDAC1 enzyme; the placebo had no or negligible cytotoxicity against HeLa cells and could not alter either the HDAC 1 or 2 activity. Data on p21(WAF), p53, Akt, and STAT3 activities and a cell-cycle analysis revealed a reduction in DNA synthesis and G1-phase cell-cycle arrest when Condurango 30C was used at a 2% dose. CONCLUSION: Condurango 30C appeared to trigger key epigenetic events of gene modulation in effectively combating cancer cells, which the placebo was unable to do.

Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation / 中西医结合学报

Preventive measures against skin melanoma like chemotherapy are useful but suffer from chronic side effects and drug resistance. Ethanolic extract of Phytolacca decandra (PD), used in homeopathy for the treatment of various ailments like chronic rheumatism, regular conjunctivitis, psoriasis, and in some skin diseases was tested for its possible anticancer potential.

Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro.

OBJECTIVE: To study the possible anticancer and antiproliferative activities of ethanolic leaf extract of Thuja occidentalis (TO) on A549 non-small lung carcinoma cells in vitro. METHODS: Cell viability was ascertained through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after deployment of TO in different doses. The half maximal inhibitory concentration (IC50) dose (282 µg/mL) was determined, and two other doses for dose-dependence study, one below the IC50 dose (IC35=188 µg/mL) and one above the IC50 dose (IC65=376 µg/mL) were selected. Bromodeoxyuridine (BrdU) incorporation assay and migration studies were performed to elucidate antiproliferative activity of the drug, if any. Fluorescence-activated cell sorting analysis after annexin V-fluorescein isothiocyanate and propidium iodide (annexin V-FITC-PI) dual staining method was done to ascertain early stage of apoptosis, if any. DNA fragmentation assay was done through Hoechst 33258 and acridine orange-ethidium bromide staining. DNA damage was quantified through comet assay. Bax-Bcl2 regulation and expression studies were performed through indirect enzyme-linked immunosorbent assay (ELISA). Caspase 3 activity was measured at gene level through reverse transcription-polymerase chain reaction (RT-PCR) analysis. Its activation at protein level was analyzed through indirect ELISA and Western blot analysis. RESULTS: TO demonstrated a dose-dependent decrease in viability of A549 cells after 24 h of exposure. Cell proliferation was reduced in a time-dependent manner of drug exposure as revealed from BrdU incorporation and migration studies. Annexin-V-FITC positivity of cells up to 11.72% as compared to the untreated control revealed early state of TO-induced apoptosis. Occurrence of comet tail and increased fluorescence of Hoechst after 24 h of drug exposure revealed significant DNA nick generation and chromatin condensation. Bax up-regulation and Bcl-2 down-regulation suitably altered ratio of Bax/Bcl-2 in favor of apoptosis. From RT-PCR, indirect ELISA and Western blot studies, caspase 3 activity was also found to be significantly increased along with cleaved poly ADP-ribose polymerase expression. CONCLUSION: Ethanolic leaf extract of TO demonstrated apoptotic and antiproliferative potentials against A549 cell line.

Chelidonine isolated from ethanolic extract of Chelidonium majus promotes apoptosis in HeLa cells through p38-p53 and PI3K/AKT signalling pathways.

OBJECTIVE: To evaluate the role of chelidonine isolated from ethanolic extract of Chelidonium majus in inducing apoptosis in HeLa cells and to assess the main signalling pathways involved. METHODS: Cells were initially treated with different concentrations of chelidonine for 48 h and the median lethal dose (LD50) value was selected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Morphological analysis of nuclear condensation and DNA damage and fragmentation were measured by 4',6-diamidino-2-phenylindole staining and comet assay. Further, reactive oxygen species (ROS) generation, cell cycle arrest and change in mitochondrial membrane potential were also examined and analyzed by flow cytometry. Evaluation of interaction of drug with CT DNA was investigated by circular dichroism (CD) spectral analysis to find any possible drug-CT DNA interaction. The mRNA and protein expressions of major signal proteins like p38, p53, protein kinase B (AKT), phosphatidylinositol 3-kinases (PI3K), Janus kinase 3 (JAK3), signal transducer and activator of transcription 3 (STAT3) and E6 and E7 oncoproteins as well as the pro-apoptotic genes and antiapoptotic genes were also estimated by reverse transcriptase-polymerase chain reaction and Western blotting. RESULTS: Based on LD(50) value (30 µg/mL) of chelidonine, three doses were selected, namely, 22.5 µg/mL (D1), 30.0 µg/mL (D2) and 37.5 µg/mL (D3). Results showed that chelidonine inhibited proliferation and induced apoptosis in HeLa cells through generation of ROS, cell cycle arrest at sub-G1 and G0/G1 stage, change in mitochondrial membrane potential and fragmentation of DNA. Results of CD spectra showed effective interaction between chelidonine and calf thymus DNA. Studies of signalling pathway revealed that chelidonine could efficiently induce apoptosis through up-regulation of expressions of p38, p53 and other pro-apoptotic genes and down-regulation of expressions of AKT, PI3K, JAK3, STAT3, E6, E7 and other antiapoptotic genes. CONCLUSION: Chelidonine isolated from Chelidonium majus efficiently induced apoptosis in HeLa cells through possible alteration of p38-p53 and AKT/PI3 kinase signalling pathways.

[6]-Gingerol isolated from ginger attenuates sodium arsenite induced oxidative stress and plays a corrective role in improving insulin signaling in mice.

Arsenic toxicity induces type 2 diabetes via stress mediated pathway. In this study, we attempt to reveal how sodium arsenite (iAs) could induce stress mediated impaired insulin signaling in mice and if an isolated active fraction of ginger, [6]-gingerol could attenuate the iAs intoxicated hyperglycemic condition of mice and bring about improvement in their impaired insulin signaling. [6]-Gingerol treatment reduced elevated blood glucose level and oxidative stress by enhancing activity of super oxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and GSH. [6]-Gingerol also helped in increasing plasma insulin level, brought down after iAs exposure. iAs treatment to primary cell culture of ß-cells and hepatocytes in vitro produced cyto-degenerative effect and accumulated reactive oxygen species (ROS) in pancreatic ß-cells and hepatocytes of mice. [6]-Gingerol appeared to inhibit/intervene iAs induced cyto-degeneration of pancreatic ß-cells and hepatocytes, helped in scavenging the free radicals. The over-expression of TNFα and IL6 in iAs intoxicated mice was down-regulated by [6]-gingerol treatment. iAs intoxication reduced expression levels of GLUT4, IRS-1, IRS-2, PI3K, AKT, PPARγ signaling molecules; [6]-gingerol mediated its action through enhancing the expressions of these signaling molecules, both at protein and mRNA levels. Thus, our results suggest that [6]-gingerol possesses an anti-hyperglycemic property and can improve impaired insulin signaling in arsenic intoxicated mice.

Ethanolic extract of Thuja occidentalis blocks proliferation of A549 cells and induces apoptosis in vitro / 中西医结合学报

To study the possible anticancer and antiproliferative activities of ethanolic leaf extract of Thuja occidentalis (TO) on A549 non-small lung carcinoma cells in vitro.

Chelidonine isolated from ethanolic extract of Chelidonium majus promotes apoptosis in HeLa cells through p38-p53 and PI3K/AKT signalling pathways / 中西医结合学报

To evaluate the role of chelidonine isolated from ethanolic extract of Chelidonium majus in inducing apoptosis in HeLa cells and to assess the main signalling pathways involved.