Avinaba Mukherjee

Quercetin induces cytochrome‐c release and ROS accumulation to promote apoptosis and arrest the cell cycle in G2/M, in cervical carcinoma: signal cascade and drug‐DNA interaction

Small aromatic compounds like flavonoids can intercalate with DNA molecules bringing about conformational changes leading to reduced replication and transcription. Here, we have examined one dietary flavonoid, quercetin (found in many fruit and vegetables), for possible anti‐cancer effects, on HeLa cells originally derived from a case of human cervical cancer.

[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 β-cells and hepatocytes in vitro produced cyto-degenerative effect and accumulated reactive oxygen species (ROS) in pancreatic β-cells and hepatocytes of mice. [6]-Gingerol appeared to inhibit/intervene iAs induced cyto-degeneration of pancreatic β-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.

Quercetin Down-regulates IL-6/STAT-3 Signals to Induce Mitochondrial-mediated Apoptosis in a Nonsmall- cell Lung-cancer Cell Line, A549.

Objectives: Quercetin, a flavonoid compound, has been reported to induce apoptosis in cancer cells, but its anti-inflammatory effects, which are also closely linked with apoptosis, if any, on non-small-cell lung cancer (NSCLC) have not so far been critically examined. In this study, we tried to determine if quercetin had any demonstrable anti-inflammatory potential, which also could significantly contribute to inducing apoptosis in a NSCLC cell line, A549. Methods: In this context, several assays, including cytotoxicity, flow cytometry and fluorimetry, were done. Gene expression was analyzed by using a western blot analysis. Results: Results revealed that quercetin could induce apoptosis in A549 cells through mitochondrial depolarization by causing an imbalance in B-cell lymphoma 2/ Bcl2 Antagonist X (Bcl2/Bax) ratio and by down-regulating the interleukine-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway. An analysis of the data revealed that quercetin could block nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity at early hours, which might cause a down-regulation of the IL-6 titer, and the IL-6 expression, in turn, could inhibit p-STAT3 expression. Down-regulation of both the STAT3 and the NF-κB expressions might, therefore, cause down-regulation of Bcl2 activity because both are major upstream effectors of Bcl2. Alteration in Bcl2 responses might result in an imbalance in the Bcl2/Bax ratio, which could ultimately bring about mitochondria mediated apoptosis in A549 cells. Conclusion: Overall, the finding of this study indicates that a quercetin induced anti-inflammatory pathway in A549 cells appeared to make a significant contribution towards induction of apoptosis in NSCLC and, thus, may have a therapeutic use such as a strong apoptosis inducer in cancer cells.

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.

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). 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.

Flavonol isolated from ethanolic leaf extract of Thuja occidentalis arrests the cell cycle at G2‐M and induces ROS‐independent apoptosis in A549 cells, targeting nuclear DNA

The K‐ras gene mutation commonly found in lung adenocarcinomas contributes to their non‐invasive expansion. Our main objective here was to develop a chemopreventive agent against K‐ras‐mutated lung adenocarcinoma cell line like‐A549.

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.

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.

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 (5th), two (5th-6th) and three (5th-7th) 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.

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.