Tanmoy Bhowmik

Influence of gold nanoparticle tagged snake venom protein toxin NKCT1 on Ehrlich ascites carcinoma (EAC) and EAC induced solid tumor bearing male albino mice.

Earlier the conjugation of gold nanoparticle (GNP) and snake venom protein toxin NKCT1 was reported and primary characterization was performed. In the present communication, further characterizations of GNP-NKCT1 were done with SEM, EDS, XRD and Raman spectra for its physio-chemical nature and bonding. SEM showed the formation of gold nanoparticles, whereas EDS and XRD confirmed 60-90% gold nanoparticles in the solution. Raman shift corresponding to (C=O), (N-H), (C-N) confirmed the proper conjugation of GNP with NKCT1. GNP-NKCT1 showed anticancer effect both in vivo and in vitro in EAC cell and antitumor effect in EAC induced mice. In in vivo studies, GNPNKCT1 increased MST 108.30% and decreased viable EAC cell count 51.39%. Fluorescent micrograph showed signs of apoptosis (membrane blebbing, membrane disruption). Decreased level of IL-10 and low incorporation of BrdU showed decreased proliferation of EAC induced by GNP-NKCT1. With upregulation of Bax, down regulation of Bcl2 and increased expression of caspase 3/9, it was confirmed that GNP-NKCT1 induced caspase dependent apoptosis pathway in EAC cell. In in vitro studies, GNP-NKCT1 increased the late apoptotic stage of cell and arrested cell cycle division at G0/G1 state. GNP-NKCT1 also decreased the tumor volume and tumor weight in EAC induced tumor in male albino mice. It inhibited angiogenesis, which was confirmed by lower percentage of expression of VEGF. This study indicated the capability of gold nanoparticles which enhanced the tumor uptake of NKCT1 and also suggested that GNP-NKCT1 might be a good source for anti-carcinoma and anti-tumor agents.

In vivo and in vitro toxicity of nanogold conjugated snake venom protein toxin GNP-NKCT1

Research on nanoparticles has created interest among the biomedical scientists. Nanoparticle conjugation aims to target drug delivery, increase drug efficacy and imaging for better diagnosis. Toxicity profile of the nanoconjugated molecules has not been studied well. In this communication, the toxicity profile of snake venom cytotoxin (NKCT1), an antileukemic protein toxin, was evaluated after its conjugation with gold nanoparticle (GNP-NKCT1). Gold nanoparticle conjugation with NKCT1 was done with NaBH4 reduction method. The conjugated product GNP-NKCT1 was found less toxic than NKCT1 on isolated rat lymphocyte, mice peritoneal macrophage, in culture, which was evident from the MTT/Trypan blue assay. Peritoneal mast cell degranulation was in the order of NKCT1 > GNP-NKCT1. The in vitro cardiotoxicity and neurotoxicity were increased in case of NKCT1 than GNP-NKCT1. On isolated kidney tissue, NKCT1 released significant amount of ALP and γ-GT than GNP-NKCT1. Gold nanoconjugation with NKCT1 also reduced the lethal activity in mice. In vivo acute/sub-chronic toxicity studies in mice showed significant increase in molecular markers due to NKCT1 treatment, which was reduced by gold nanoconjugation. Histopathology study showed decreased toxic effect of NKCT1 in kidney tissue after GNP conjugation. The present study confirmed that GNP conjugation significantly decreased the toxicity profile of NKCT1. Further studies are in progress to establish the molecular mechanism of GNP induced toxicity reduction.

Gecko Skin Extract Induce Cytotoxicity and Apoptosis in Human Breast Cancer Cell Line

Gecko has been used against malignant tumors in medicinal practice in china, especially digestive system tumors such as esophageal cancer, gastric cancer, and liver cancer. Reduce inflammation, active against allergic responses, swelling having low toxicity are other activity of Gecko skin extract, has been mentioned in chinese folk medicine. The aim of this study was to examine the alcoholic skin extract of Gecko species induced cytotoxicity and apoptosis activity in breast cancer (MCF7) cell line. in vitro, the inhibitory effect of gecko skin alcohol extract (GSAE) on cytotoxicity of human breast cancer cells was measured by MTT assay and trypan blue exclusion assay which resulted in MCF7 cell proliferation was significantly inhibited in both dose- and time- dependent manner (P < 0.01). High percentage (71.19%) early cell apoptosis in GSAE-treated group (IC50 dose) exhibited selective cytotoxic action of GSAE confirmed by PE-Annexin V/FITC assay. Flow cytometry study showed that MCF7 cell cycle was arrested at G1 phase. Immunocytochemical study revealed that GSAE down regulated nuclear proliferative antigen Ki-67. Gecko alcohol extract decreased the potential of mitochondrial membrane and increased the expression of Bax, cytochrome c, caspase9, caspase3 and Ca2+ in cytosol. GSAE also downregulated the expression of Bcl2, HSP70 and HSP90. Although the level of VEGF did not change significantly, the expression of MMP9 was remarkably down-regulated in GSAE treated group. Thus GSAE showed significantly inhibitory effect in MCF7 cell line and acts through mitochondrial intrinsic pathway of cell apoptosis with downregulation of nuclear proliferative protein and heat shock protein.

Protection against osteoarthritis in experimental animals by nanogold conjugated snake venom protein toxin gold nanoparticle-Naja kaouthia cytotoxin 1.

Background & objectives: Increased severity of osteoarthritis (OA) and adverse side effects of its treatment led to the search for alternative therapies. It was previously reported that snake venom protein toxin Naja kaouthia cytotoxin 1 (NKCT1) and gold nanoparticle (GNP) individually have potential against excremental arthritis. In this study, we analyzed the protective activity of GNP conjugated protein toxin NKCT1 (GNP-NKCT1) against experimental OA. Methods: Gold nanoparticle conjugation with NKCT1 (GNP-NKCT1) was done and its physiochemical properties were studied. OA was induced in male albino rats by intra-articular injection of bacterial collagenase and treatment was done with NKCT1/GNP-NKCT1/standard drug (indomethacin). Physical parameter (ankle diameter), urinary markers (hydroxyproline, glucosamine, pyridinoline, deoxypyridinoline), serum and synovial membrane pro-inflammatory markers [tumour necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-17, vascular endothelial growth factor (VEGF)] and matrix metalloproteinase 1 (MMP1) were measured. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface were also done. Results: Physical parameters, urinary markers, serum and synovial membrane pro-inflammatory makers and MMP1 were increased in arthritic rats and significantly restored after GNP-NKCT1/NKCT1 treatment. Joint histopathology and scanning electron microscopy imaging of articular cartilage surface also indicated the protective effect of GNP-NKCT1 against inflammatory response and cartilage degradation in osteoarthritic rats. Interpretation & conclusions: In this study restoration of the arthritic markers and bone degradation by GNP-NKCT1 treatment indicated the anti-osteoarthritic property of GNP-NKCT1. Further studies need to be done to confirm these findings.

Russells Viper Venom Purified Toxin Drct-II Inhibits the Cell Proliferation and Induces G1 Cell Cycle Arrest in Human Leukemic Cancer Cells

The present study was an effort to establish the anticancer activity of the purified protein toxin (drCT-II) from Indian Russell’s viper (Daboia russelli russelli) venom in leukemic cell line and animal model. Isolation and purification of drCT-II was done through CM-cellulose ion exchange chromatography and RP- HPLC. SDS- PAGE molecular weight and first 20 amino acid sequence of drCT-II was done. The anti-leukemic activity using U937 and K562 cell line was established through cytotoxicity, apoptosis, cell cycle study, morphology, cancer marker proteins. The mean survival time of EAC induced male albino mice was established. Human lymphocyte cytotoxicity was done. drCT-II was eluted with 0.1 M NaCl on CM-cellulose ion exchange chromatography. On RP-HPLC, drCT-II produced single peak with retention time of 14.6 min. SDS-PAGE molecular weight was found to be 6.6 KDa and the first 20 amino acid sequence was found to be LQXNKLVPIASKTXPPGKNL. drCT-II produced time and dose dependent cell (U937 and K562) growth inhibition. The IC50 was found to be 35.5 μg/ml for U937 cell and 48.2 μg/ml for K562 cells. drCT-II produced membrane disruption, blebbing and nuclear disintegration in U937 and K562 cells observed through confocal and scanning electron microscopy. It exhibited DNA fragmentation and comet formation in leukemic cells. drCT-II produced apoptosis, cell cycle arrest at G1 phase and increased the expression of P21, P27 and P53. drCT-II induced apoptosis in leukemic cells was followed through caspase 3 and 9 pathway activation. EAC cell growth in male albino mice was significantly inhibited by drCT-II, thus increased the mean survival time. drCT-II significantly reduced the human lymphocyte count (in culture). It may be concluded that drCT-II, a 6.6 KDa protein purified from Daboia russelli russelli venom would be a novel pro-apoptotic agent that induced cancer cell killing through p53 and caspase pathway.

Caspase Pathway Activation and Reactive Oxygen Species Generation in Apoptotic Cell Death of Human Leukemic U937 and K562 Cell Line in Response to King Cobra (Ophiophagus hannah) Venom

Resistance and decreasing efficacy of current synthetic drug for chemotherapy of leukemic cancer draws attention for development of newer anticancer agent from natural resources. In the present study, king cobra venom (OHV) significantly inhibited leukemic cell growth in dose and time dependent manner. For U937 and K562 cell line, the IC 50 dose (72 h) was found to be 4.1 µg/ml and 3.9 µg/ml respectively, observed by trypan blue exclusion method and tetrazolium bromide reduction assay. OHV treated morphometry of leukemic cell showed the characteristic features of apoptosis. Both U937 and K562 cells were arrested in the G1 phase of cell cycle with most cells exhibiting the biochemical feature of early and late apoptosis. Mitochondrial membrane potential was lost and reactive oxygen species generated highly in OHV treated leukemic cell line (U937 and K562). Western blot analysis showed OHV increased expression of Bax and decreased expression of Bcl2 in OHV treated cell as compared to untreated control U937 and K562 cell. Upregulation of Cytochrome c, Bid, Bad, Caspase 3/8/9, p21 and NF-κB down regulation of Cyclin D1, CDK4 was also showed by western blot analysis which revealed the possible pathway of OHV in cellular level. The results of this study demonstrated that OHV significantly and selectively induced leukemic cell death through both extrinsic and intrinsic apoptotic pathway.