| ObjectiveLeukemia and lymphoma are the most common malignancies of blood system. Chemotherapy and radiotherapy are the primary treatment for leukemia and lymphoma today. However, they do not distinguish leukemia cells from other rapidly dividing but non-cancerous cells. As a result, chemotherapy and radiotherapy harm healthy red and white blood cells, thus creat strong side effects. Much effort had been focused on the discovery of new anticancer drugs from traditional Chinese medicine that can effectively prevent tumor but have less toxicity and side effects. The scorpion and its venom had been used as a Chinese traditional medicine for thousands of years. Scorpion venoms are the mainly active substance of scorpion and complex mixtures of molecules, most of which are proteins exhibiting a wide range of pharmacological actions. In recent years, there were some reports about anti-cancer actions of scorpion venom, but the anti-tumor mechanism was poorly understood. In the present study, we verified the anti-proliferation and cell cycle arresting properties of scorpion venom component III(SVCIII) from Buthus martensii karsch (BmK) venom and its effects on NF-κB signaling pathway in human leukemic cell lines. Methods1. Cells (THP-1, Jurkat. MT2and IM-9) and PBMC were seeded in96-well plate at a density of2x104cells per well and treated with different concentrations of SVCII1for24h.48h and72h. Cell viability was determined by MTT assay. Index of cell viability was calculated by measuring the absorbance value at570nm. IC50values was calculated for different cell lines.2. THP-1and Jurkat cells were treated with SVCIII at1/2IC50and IC50concentration respectively for48h. DNA was labeled with propidium iodide and cell cycle stage was analyzed by a flow cytometer.3. Cells were treated with SVC III at1/2IC50and IC50concentration respectively for different time and cell proteins were extracted using Nuclear extract kit. The protein expression of cyclin D1, p65, IκBα and p-IκBα were measured by Western blotting.4. THP-1and Jurkat cells were seeded in24-well plate at a density of2x105cells per well and treated with SVCIII at1/2IC50and IC50concentration respectively for24h. Total cellular RNA was isolated using Trizol and the relatively expression of p65mRNA were determined by RT-PCR.5. THP-1and Jurkat cells were seeded in24-well plate and transiently transfected with a NF-κB luciferase reporter plasmid for42h. Transfected cells were exposed to SVCIII at1/2IC50and IC50concentration respectively for6h. Then cells were harvested and lysed. Supernatants were analyzed for firefly and Renilla luciferase activity using the luciferase reporter assay system.6. THP-1and Jurkat cells were seeded in24-well plate and treated with SVCIII at1/2IC50and IC50concentration respectively for2h. Nuclear extracts were isolated using Nuclear Extract Kit. Electrophoretic mobility shift assay (EMSA) was performed to assess NF-κB activation with Chemiluminescent EMSA Kit. Specific binding between probe and protein was confirmed by competition experiments with a100-fold excess of unlabeled or mutated oligonucleotides.Results 1. Effects of SVClII on cell viability From the observed results of the MTT assay, cell viability of THP-1, Jurkat, MT2. and IM-9was decreased by SVClII in a dose-dependent and time-dependent manner. Furthermore, the sensitivity of different cell lines to SVClII was distinct, and THP-1and IM-9cells are more sensitive to SVClII. However, SVClII had no obviously inhibiting effects on PBMCs. After cells exposed to SVClII for48h, the IC50value was calculated to be29.04μg/ml for THP-1,39.6μg/ml for Jurkat,36.44μg/ml for MT2and27.10μg/ml for IM-9cell respectively. We chose approximate1/2IC50and IC50concentration to detect the venom's effects during latter experiments.2. Effect of SVCIII on cell cycle distribution Cell cycle distribution was tested after48h of SVCIII treatment by flow cytometry. Results showed that with increasing concentrations of SVCIII the number of THP-1and Jurkat cells in the GO/G1phase increased significantly compared with control cells, while the cells in G2and S phase decreased accordingly. Exposure of SVCIII to cells resulted in cell accumulation at the GO/G1phase in a dose-dependent manner. These results suggest that SVCIII inhibites cell growth with arrest at G1phase and reduces transition to the S and G2/M phases of the cell cycle.3. Effect of SVCIII on cyclinDl protein After THP-1and Jurkat cells were treated with SVCIII at1/2IC50and IC50concentration respectively for24h, western blotting showed SVCIII significantly inhibited the expression of cyclin D1in a dose-dependent manner in both cell types. There was statistically significant difference between SVCIII treatment group and the control group. This result suggests a potential mechanism on how SVC III suppresses tumor cell proliferation.4. SVC III could inhibit the expression of p65mRNA in THP-1and Jurkat cells and there was a strong dose relationship. It was different effects on p65protein in THP-1cell. SVCIII treatment showed that with increasing concentrations of SVC III, p65protein expressions in cytoplasm decrease compared with the control group but there was no statistical significance. However, p65protein in nucleus was gradually reduced compared with the control group and dose-related.5. The treatment with SVCIII resulted in a significant decrease in NF-kB activity in THP-1and Jurkat cells. EMSA results also showed that SVCIII treatment reduced the binding between NF-κB and DNA in a dose-dependent manner. These results suggest that SVCIII inhibits NF-κB activation.6. THP-1cells were pretreated with NF-κB inhibitor Bay11-7082for1h and then subjected to SVCIII. Although the expression of p-IκBα was markedly inhibited by Bay11-7082pre-treatment. it was further decreased by the treatment with SVCIII. At the same time, NF-κB inhibitor Bay11-7082led to a decrease of the expression of cyclin D1, and it was further decreased by Bay11-7082combined with SVCIII. Therefore, treatment with SVCIII and Bay11-7082synergistically inhibited NF-κB activation.Conclusions1. SVCIII could inhibit the cell viability of THP-1, Jurkat, MT2, and IM-9cells in a dose-dependent and time-dependent manner. Furthermore, the sensitivity of different cell line to SVCIII was distinct, and THP-1cell is the most sensitive to SVCIII.2. SVCIII inhibits cell growth by arresting cell cycle at G0/G1phase and this effect may be through suppressing the cell cycle regulatory protein cyclin D1.3. SVCIII may suppress NF-κB signaling pathway via the inhibition of IκBα phosphorylation and further inhibit cell proliferation. |
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