Shubho Das Gupta

Comparative antimutagenic and anticlastogenic effects of green tea and black tea: a review.

Tea is the most popular beverage next to water, consumed by over two-thirds of the worlds population. It is processed in different ways in different parts of the world to give green, black or oolong tea. Experimental studies have demonstrated the significant antimutagenic and anticlastogenic effects of both green and black tea and its polyphenols in multiple mutational assays. In the present review, we have attempted to evaluate and update the comparative antimutagenic and anticlastogenic effects of green tea, black tea and their polyphenols in different test systems, based on available literature. Existing reports have suggested that the protective effects of black tea is as good as green tea, however, more studies on black tea and its polyphenols are needed before a final conclusion can be made.

Apoptosis induction in human leukemic cells by a novel protein Bengalin, isolated from Indian black scorpion venom: Through mitochondrial pathway and inhibition of heat shock proteins

Scorpion venom possesses protein toxins having numerous biological activities, some of which are potentially anticancerous. Previously we had reported antiproliferative activity of the venom of Indian black scorpion, Heterometrus bengalensis Koch. Here we have isolated and purified a novel protein named Bengalin (72kDa) from the venom, responsible for antiproliferative and apoptogenic activities against human leukemic cells U937 (histiocytic lymphoma) and K562 (chronic myelogenous leukemia). N-terminal sequence of first 20 amino acids of Bengalin was G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin induced cell growth inhibition at IC(50) values of 3.7 and 4.1 microg/ml for U937 and K562 cells respectively did not significantly affect normal human lymphocytes. Inhibition of U937 and K562 cell proliferation occurred by apoptosis as evidenced from damaged nuclei, cell cycle arrest at sub G1 phase, increase of early apoptotic cells, augmentation of DNA fragmentation and also a reduction of telomerase activity. Further insights revealed that Bax:Bcl2 ratio was elevated after Bengalin treatment. Moreover Bengalin elicited loss of mitochondrial membrane potential (MMP) which commenced cytochrome c release in cytosol, decreased heat shock protein (HSP) 70 and 90 expression, activated caspase-9, caspase-3 and induced poly(ADP-ribose) polymerase (PARP) cleavage. We have also determined that HSP70 and 90 inhibitions correlated with Bengalin induced antiproliferation, caspase-3 upregulation, apoptogenesis and increased DNA fragmentation. These results hypothesize that Bengalin might provide a putative molecular mechanism for their anticancer effect on human leukemic cells which might be mediated by mitochondrial death cascade. Inhibition of HSPs might also play a crucial role in induction of apoptosis.

A lethal cardiotoxic-cytotoxic protein from the Indian monocellate cobra (Naja kaouthia) venom.

A lethal cardiotoxic-cytotoxic protein (mol. wt. 6.76 kDa) has been purified from the Indian monocellate cobra (Naja kaouthia) venom by ion-exchange chromatography and HPLC. CD spectra indicated the presence of 23% alpha helix, 19% beta sheets and 35% coil. Complete amino acid sequence was determined by MALDI, which showed similar homology with cardiotoxins/cytotoxins isolated from venom of other Naja species. Intraperitoneal LD(50) was 2.5 mg kg(-1) in BalbC male mice. In vitro cardiotoxicity studies on isolated guinea pig auricle showed that the molecule produced auricular blockade that was abolished after trypsin treatment. Cytotoxicity studies on human leukemic U937 and K562 cells showed that it significantly inhibited cell proliferation in a dose and time dependent manner, as observed by trypan blue exclusion method and tetrazolium bromide reduction assay. IC(5)(0) on U937 and K562 cells were 3.5 microg/ml and 1.1 microg/ml respectively. Morphometry and cell sorting studies indicated apoptosis induction in toxin treated leukemic cells. Apoptosis was caspase 3 and 9 dependent and the treated leukemic cells were arrested in sub-G1 stage. There was an increase in Bax-Bcl2 ratio, decrease in HSP (Heat shock protein) 70 and HSP90 and induction of PARP cleavage after NK-CT1 treatment. The toxin showed low cytotoxic effect on normal human leukocytes as compared with imatinib mesylate. Further detailed cytotoxic and cardiotoxic effects at the molecular level are in progress.

Black tea polyphenols induce human leukemic cell cycle arrest by inhibiting Akt signaling

Tea polyphenols have potent biological activities against human cancer cells. A major causative factor in malignancies is disregulation of cell‐cycle kinetics. In this study, we observed that black tea polyphenols, theaflavins (TF) and thearubigins (TR) induced cell‐cycle arrest at the G0/G1 phase in human leukemic U937 and K562 cells. Our objective was to understand the underlying molecular mechanism of cell‐cycle inhibition by TF and TR. During elucidation, we observed that both TF and TR treatment augmented expression of p19, p21 and p27, while ablating cylcin‐dependant kinase (CDK)2, CDK4, CDK6 and cyclin D1 levels. Our experimental results further determined that Akt signaling suppression by TF and TR played a major role in this process. Moreover, suppression of glycogen synthase kinase‐3β, β‐catenin and amplification of forkhead transcription factor 1 (FOXO1) expression were associated with regulation of certain key components of the cell‐cycle machinery. In addition, depletion of heat shock protein (Hsp) 90 by TF and TR also had a pivotal role in cell‐cycle arrest. More specifically, inhibition of Akt signaling by TF and TR correlated with the depletion of its downstream targets like Wnt/β‐catenin signaling, cyclin D1 and increase of FOXO1, p27 levels. Inhibition of upstream Hsp90 by TF and TR consequently attenuated Akt signaling and reduced the level of CDK2. These results suggest possible mechanisms for the chemopreventive effect of TF and TR on human leukemic cells. To our knowledge, this is the first report of such a detailed molecular mechanism for TF and the less‐investigated polyphenol TR‐mediated cell‐cycle inhibition in human leukemic U937 and K562 cells.

Anticlastogenic effects of black tea (World blend) and its two active polyphenols theaflavins and thearubigins in vivo in Swiss albino mice

This study investigated the inhibition of cyclophosphamide (CP) and dimethylbenz(a)anthracene (DMBA) induced genetic damage by black tea (World blend) and its two active polyphenols theaflavins (TF) and thearubigins (TR) in Swiss albino mice as measured by chromosome aberrations (CA) and sister chromatid exchanges (SCE). Three different concentrations (5, 10 and 20%) of tea and a single dose of TF and TR were tested for their anticlastogenic effects against DMBA (50 mg/kg body weight) and CP (20 mg/kg for CA and 10 mg/kg for SCE). A significant decrease in CA was observed in all the three concentrations of tea extract plus DMBA treated groups when compared with the respective DMBA treated group alone. Similarly a significant decrease in CA was observed in all the three concentrations of tea extracts plus CP treated series when compared with the group treated with CP alone. In SCE assay, a significant decrease in SCE was observed in 5, 10 and 20% black tea extract plus CP and 10 and 20% tea extracts plus DMBA treated groups when compared with the CP or DMBA treated group alone. In the single dose of TF and TR treated groups a significant decrease in both CA and SCE was observed in both the TF and TR plus both the carcinogen treated groups when compared with their positive controls. The protective effects of black tea extracts were more significant than that of its two polyphenols. This study indicates that both black tea and its active polyphenols TF and TR have significant anticlastogenic effects in bone marrow cells of mice.

Cytotoxic and antioxidant property of a purified fraction (NN-32) of Indian Naja naja venom on Ehrlich ascites carcinoma in BALB/c mice.

A cytotoxic and antioxidant protein (NN-32) from the Indian spectacled cobra Naja naja venom was identified and its probable mode of action on murine Ehrlich ascites carcinoma (EAC) was established. The venom purified through ion exchange chromatography produced several peaks, among which fraction 32 produced cytotoxic-cardiotoxic properties. This fraction (NN-32) showed a single peak (retention time 38.3 min) by HPLC using C4 column. The molecular mass determined by MALDI-MS, found to be 6.7 kDa and the first ten N-terminal sequence was determined (LKCNKLVPLF) by Edmann degradation method using applied Biosystem procise sequencer. It was observed that the sequence shared 100% homology with other cytotoxin cardiotoxin identified from the venom of Naja species. NN-32 showed cytotoxicity on EAC cells, increased survival time of inoculated EAC mice, reduced solid tumor volume and weight. NN-32 increased proapoptotic protein caspase 3 and 9 activity and Bax-Bcl2 ratio. It also increased the antioxidant markers glutathione, glutathione peroxidase, glutathione transferase, superoxide dismutase and catalase activity. NN-32 increased serum IL-10 level and decreased murine keratinocyte-derived chemokine level. The cardiotoxicity of NN-32 was established on isolated guinea pig auricle, where 100% irreversible blockade of auricular contraction was observed. Thus, it may be concluded that, NN-32 induced anticancer activity in EAC mice was partly mediated through its apoptogenic - antioxidant property.

Inhibition of leukemic U937 cell growth by induction of apoptosis, cell cycle arrest and suppression of VEGF, MMP-2 and MMP-9 activities by cytotoxin protein NN-32 purified from Indian spectacled cobra (Naja naja) venom

A cytotoxin NN-32 (6.7 kDa) from Indian cobra (Naja naja) venom inhibited human leukemic U937 cell growth as observed by Trypan blue dye exclusion method and cytotoxicity was confirmed by MTT assay. NN-32 induced apoptosis of U937 cell and cell cycle arrest of sub-G1 phase were revealed by FACS analysis. Increased Bax/Bcl-2 ratio, increased caspase 3 and 9 activities, cleaved PARP, decreased VEGF, MMP-2 and MMP-9 activities were observed after NN-32 treatment of U937 cell. Antileukemic activity of NN-32 on U937 cell may be due to activation of apoptosis, arresting cell cycle and antiangiogenesis activities.

A high molecular weight protein Bengalin from the Indian black scorpion (Heterometrus bengalensis C.L. Koch) venom having antiosteoporosis activity in female albino rats

This study reports the presence of a high molecular weight protein (Bengalin) from the Indian black scorpion (Heterometrus bengalensis) venom having antiosteoporosis activity in experimental osteoporosis developed in female albino Wister rats. Bengalin was purified through DEAE-cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the Bengalin was found to be 72kDa and the first 20 amino acid sequence was found to be G-P-L-T-I-L-H-I-N-D-V-H-A-A/R-F-E-Q/G-F/G-N-T. Bengalin exhibited significant antiosteoporosis activity in experimental female rats, which was confirmed through analysis of urine Ca(2+), PO(4)(3-), CRE & OH-P. Bengalin (3 microg and 5 microg/100g rat/i.p.) antagonized osteoporosis by restoring urinary Ca(2+), PO(4)(3-), CRE and OH-P, serum/plasma Ca(2+), PO(4)(3-), ALP, TRAP, PTH, T(3), TSH, Osteocalcin, IL1, IL6 and TNF alpha and bone minerals Ca(2+), P, Mg(2+), Zn(2+), Na(+), as compared with the sham operated control rats. Bone minerals density of osteoporosis female rats was improved due to Bengalin, observed through DEXA scan. Subacute toxicity studies in male albino mice, Bengalin showed cardiotoxicity. In vivo experiments, Bengalin showed cardiotoxicity on isolated guinea pig heart, guinea pig auricle, and neurotoxicity on isolated rat phrenic nerve diaphragm preparation. Further detail studies on the toxicity, antiosteoporosis and structural identity of Bengalin are warranted.

Bengalin initiates autophagic cell death through ERK-MAPK pathway following suppression of apoptosis in human leukemic U937 cells.

AIMS The aim of this study was to assess the autophagy inducing ability of the scorpion venom toxin Bengalin in human leukemic U937 cells. The same toxin was previously shown to induce apoptosis in human leukemic cells. MAIN METHODS Bengalin was purified from Indian black scorpion (Heterometrus bengalensis) venom by ion exchange chromatography and HPLC. In human leukemic U937 cells, Bengalin associated MAPK (mitogen activated protein kinase) pathway was determined by western blotting. Downstream to MAPK, the Bengalin induced apoptosis-mediator caspase-3 was blocked by chemical inhibitor and reconfirmed by siRNA mediated gene knockdown. Subsequent to caspase-3 blocking, the autophagic response was evaluated by quantification of acidic vesicle organelles formation and modulations of Atgs, Beclin-1, LC3-1 and LC3-II expression by western blotting. KEY FINDINGS In U937 cells, Bengalin increased ERK1/2 expression to bring about cell death. However in subsequent caspase-3 blocked conditions, Bengalin downregulated p-Akt, p-mTOR and decreased apoptosis. It had also increased the percentage of acidic vesicle organelles positive cells. Bengalin could induce autophagic response by augmenting Beclin-1, Atg12, Atg7, Atg5 and Atg3 in U937 cells. Moreover a time dependant reciprocal relation was observed between LC3-I and LC3-II expression upon Bengalin treatment. The decrease in LC3-II was inhibited in the presence of lysozomal enzyme blockers thereby suggesting lysosome involvement in the autophagic response. SIGNIFICANCE We have for the first time demonstrated that scorpion venom-component could induce an alternate cell death pathway other than apoptosis in the form of autophagy in human leukemic U937 cells.