Antony Gomes

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.

Haemorrhagic protein of Russell's viper venom with fibrinolytic and esterolytic activities.

A haemorrhagic toxin specifically active on skin and muscle at the site of introduction in mice has been purified from Vipera russelli russelli (Indian subspecies of Russells viper) venom by CM-Sephadex C-50 ion exchange chromatography and size exclusion (SE)-HPLC. This toxic protein also has strong fibrinolytic and arginine esterolytic activities. The purified preparation was a single polypeptide chain of molecular weight 73,000, as revealed by SDS-PAGE and SE-HPLC under native and denatured conditions. It has been named as VRR-73. Atomic absorption spectrometry indicated the existence of Mg(2+) in a mol per mol ratio. Antiserum was effective in neutralizing haemorrhage when administered immediately following VRR-73 but was ineffective in inhibiting fibrinolytic and esterolytic activities. On the other hand phenylmethyl sulphonyl fluoride and EDTA inhibited fibrinolysis and esterolysis but did not affect haemorrhage. Thermal denaturation of VRR-73, after exposure at 100 degrees C for 10 min, led to inactivation of all of its activities. Fibrinolytic and esterolytic activities, but not the haemorrhagic activity, were slowly regained after cooling at 25 degrees C. Thus the two pathological activities of VRR-73 appear to be associated with two different regions of the molecule.

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.

Anti-arthritic activity of Indian monocellate cobra (Naja kaouthia) venom on adjuvant induced arthritis.

The Indian Monocellate Cobra venom (NKV) showed anti-arthritic activity over FCA induced arthritis in male albino rats. NKV treatment (1/20th & 1/10th MLD doses x 13 days, i.p.) showed significant restoration in paw & ankle volume, paw weight. Urinary hydroxyproline, glucosamine, serum ACP, ALP and IL-10 level were restored significantly, due to NKV treatment, as compared with arthritic rats. NKV also showed significant protection against arthritis induced oxidative damages. Thus this study confirmed the scientific validation behind ancient belief and use of snake venom in arthritis as mentioned in Ayurveda.

In vivo and in vitro antileishmanial activity of Bungarus caeruleus snake venom through alteration of immunomodulatory activity.

Leishmaniasis threatens more than 350 million people worldwide specially in tropical and subtropical region. Antileishmanial drugs that are currently available have various limitations. The search of new drugs from natural products (plants, animals) possessing antileishmanial activity is ventured throughout the world. The present study deals with the antileishmanial activity of Bungarus caeruleus snake venom (BCV) on in vitro promastigotes and amastigotes of Leishmania donovani parasite and leishmania infected BALB/c mice. The effect of BCV on peritoneal macrophage, release of cytokines from the activated macrophages, production of nitric oxide, reactive oxygen species and cytokines were studied in vivo and in vitro. IC50 value of BCV on L. donovani promastigote was 14.5 μg/ml and intracellular amastigote was 11.2 μg/ml. It activated peritoneal macrophages, significantly increased cytokines and interleukin production. BCV (20 μg/kg and 40 μg/kg body weight of mice) decreased parasite count by 54.9% and 74.2% in spleen and 41.4% and 60.4% in liver of infected BALB/c mice. BCV treatment significantly increased production of TNF-α, IFN-γ, ROS, NO in infected mice. Histological studies showed decreased granuloma formation in treated liver as compared with control. Liver and spleen structure was partially restored due to BCV treatment in infected mice. The present study revealed that BCV possessed antileishmanial activity against L. donovani parasite in vivo and in vitro and this activity was partly mediated through immunomodulatory activity involving macrophages.

Sequences, geographic variations and molecular phylogeny of venom phospholipases and threefinger toxins of eastern India Bungarus fasciatus and kinetic analyses of its Pro31 phospholipases A2.

Eight phospholipases A2 (PLAs) and four three‐finger‐toxins (3FTx) from the pooled venom of Bungarus fasciatus (Bf) were previously studied and sequenced, but their expression pattern in individual Bf venom and possible geographic variations remained to be investigated. We herein analyze the individual venom of two Bf specimens from Kolkata (designated as KBf) to address this question. Seven PLAs and five 3FTx were purified from the KBf venoms, and respective cDNAs were cloned from venom glands of one of the snakes. Comparison of their mass and N‐terminal sequence revealed that all the PLAs were conserved in both KBf venoms, but that two of their 3FTx isoforms were variable. When comparing the sequences of these KBf‐PLAs with those published, only one was found to be identical to that of Bf Vb‐2, and the other five were 94–98% identical to those of Bf II, III, Va, VI and XI‐2, respectively. Notably, the most abundant PLA isoforms of Bf and KBf venoms contain Pro31 substitution. They were found to have abnormally low kcat values but high affinity for Ca2+. Phylogenetic analysis based on the sequences of venom group IA PLAs showed a close relationship between Bungarus and Australian and marine Elapidae. As the five deduced sequences of KBf‐3FTx are only 62–82% identical to the corresponding Bf‐3FTx from the pooled venom, the 3FTx apparently have higher degree of individual and geographic variations than the PLAs. None of the KBf‐3FTx was found to be neurotoxic or very lethal; phylogenetic analyses of the 3FTx also revealed the unique evolution of Bf as compared with other kraits.

Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin

A protein (BMP1) was purified from common Indian toad (Bufo melanostictus, Schneider) skin through DEAE cellulose ion exchange chromatography and high performance liquid chromatography. The molecular weight of the BMP1 was found to be 79 kDa. BMP1 (0.5 and 1 mg/kg/day, i.p.) significantly decreased the number of viable Ehrlich ascites carcinoma (EAC) cells, thereby increased the lifespan of EAC bearing mice (p<0.001). MTT values reduced significantly with the treatment of BMP1 (0.5 and 1.0 mg/kg/day, i.p. for 3 days) on EAC cells indicated its antiproliferative activity. This was also supported by flow-cytometric data on the cell cycle arrest at G1 in EAC cells. BMP1 (1 mg/kg) reduced the solid tumor weight and volume of about three times further support the antiproliferative nature. Fluorescence and confocal microscopic study on EAC cells after BMP1 (0.5 mg/kg/day, i.p. for 3 days) treatment indicated certain features of apoptosis, like nuclear fragmentation, membrane blebbing, and vacuolization of cells. DNA fragmentation was clearly observed in alkaline comet assay. Apoptosis induced by BMP1 was further confirmed through flow-cytometric analysis of annexin-V binding study, sub-G1 arrest in the cell cycle and found to be mediated through caspase 3 dependent pathway. LD50 of BMP1 was found to be 12.2 mg/kg, i.p. in male Swiss albino mice. BMP1 treatment at 0.5 mg/kg and 1.0 mg/kg for 10 days did not alter any hematological and biochemical parameters in mice, but after 30 days of treatment produce significant rise in total leucocyte count, neutrophil percentage, serum urea, creatinine, GOT, LDH and decrease in lymphocyte percentage as compared to respective control. In conclusion, BMP1, a protein molecule isolated from Indian toad (B. melanostictus, Schneider) skin, showed antiproliferative and apoptogenic activity on EAC cancer cell with limited toxicity.

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.

Experimental osteoporosis induced in female albino rats and its antagonism by Indian black scorpion (Heterometrus bengalensis C.L.Koch) venom.

The present study was designed to explore the antiosteoporosis activity of the Indian black scorpion (Heterometrus bengalensis) venom on experimental osteoporosis female albino rats. Sham operated control rats were designated as Gr I, Gr II animals served as osteoporosis control, Gr III osteoporosis rats were treated with SV (1/25th of MLD), Gr IV osteoporosis rats were treated with 1/50th of MLD of SV and Gr V osteoporosis rats were treated with standard (calcium and vit-D3). As compared with the Gr I rats, the Gr II rats showed typical osteoporosis changes in increased of urinary Ca(2+), PO(4)(3-), CRE, OH-P levels, serum/plasma Ca(2+), PO(4)(3-), TRAP, IL1, IL6, TNFalpha and PTH level, bone Ca(2+), PO(4)(3-), Mg(2+), Zn(2+) and decreased level of serum/plasma ALP, EST and PTH, bone Na(+). In Gr III, Gr IV and Gr V rats, the osteoporosis changes of urine, serum and bone, were significantly restored as compared with the Gr II rats. The bone dimensions, morphology and histological changes observed in Gr II rats were restored in Gr III, Gr IV and Gr V rats. This study confirms that the Indian black scorpion venom may influence bone remodeling process by stimulating bone formation and reducing bone resorption process of osteogenesis.

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.