The Effects And The Mechanisms Of Scorpion Venom On The Proliferation And Apoptosis Of Lymphoma Cell Lines

BACKGROUND AND OBJECTIVE:Malignant tumors are serious diseases endangering human health.E very year about 1,600,000 patients with cancers were newly increased in our country and about 1,300,000 patients died from tumors.With the growth of population and the acceleration of aging population,the incidence and mortality of tumors are on the rise.Therefore,the prevention and treatment of tumors have long been the focal work in the Chinese medicine.It is then an important subject to find medicines both safe and effective in the treatment of tumors.Recently more attentions have been attached to the drugs from animals,and antineoplastics have been developed in using fully the animal sources of our country,which has then become the important content in the research work of tumors.The scorpion Buthus martensii Karsch(BmK) had been used as a Chinese traditional medicine for thousands of years.In the first authoritative Chinese pharmacopoeia "Compendium of Material Medica" edited by Li Shizhen in 1578, dried scorpion bodies were used to treat a variety of diseases.Scorpion venoms are complex mixtures of molecules,most of which are peptides displaying various kinds of biological activity.During the past two decades,several of these compounds have been identified.Toxins modulating Na⁺,K⁺,Ca²⁺ and Ckl^- currents across cell membranes have been described.Some enzymes like proteases and hyaluronidases as well as chlorotoxin-like peptides from this venom may have therapeutic potential,including anticancer activity.Recent studies have demonstrated that BmK venom can induce apoptotic cell death,and possesses anti-tumor effects.However,the molecular mechanisms that underlie the induction of apoptosis by BmK venom are poorly understood,and require further exploration.PTEN(phosphatase and tensin homolog deleted from chromosome 10) gene is a new tumor suppressor gene proved to be a lipid phosphatase.The target lipid, called phosphatidylinositol-3,4,5-trisphosphate,PIP3,and it is a key component of the major cell growth control pathways,acting both to stimulate cell growth and to inhibit tumor cell proliferation blocking apoptosis.PTEN mutations have been linked to a variety of human cancers.Mutations of PTEN during tumorigenesis allow the mutated cells to grow unchecked when they should die.Lymphoma is a cancer in the lymphatic system.Despite recent advances in radiotherapy,chemotherapy,and stem cell transplantation,the severe morbidity from lymphoma has not been alleviated.Much effort has been focused on the discovery and development of new chemopreventive agents,especially agents targeted at mechanisms known to be involved in the process of carcinogenesis. Therefore,we have sought to identify novel agents that can effectively prevent lymphoma carcinogenesis but have minimal toxicity to healthy cells.In this study, we examined the effects of BmK venom on two human lymphoma cell lines(Raji and Jurkat) compared with peripheral blood lymphocytes(PBLs),and focused on changes in the PTEN and the PI3K/Akt signal pathway and to further investigate the molecular mechanisms involved in these effects.STUDY CONTENTS:1.Identification of apoptosis induction in Raji and Jurkat cells by BmK venom.2.Analysis of the effects of BmK venom on cell cycle distribution and p27 protein level.3.Analysis of the apoptosis pathways activated by the small molecules in Raij and Jurkat cells.METHODS:1.MTT assay for cell viability,by which small molecules were screened initially.2.Methods for apoptosis identification:2.1 Observation of cell morphological changes by Phase Contrast Microscope. 2.2 Observation of nuclear morphological changes by Hoechst 3342 staining combined with fluorescence microscopy.2.2 Analysis of the changes in apoptosis by flow cytometry.3.Analysis of the changes in cell cycle distribution by flow cytometry.4.Analysis of the changes in PTEN,Akt,p-Akt,Bad,p-Bad protein level by Western blot assay.5.Analysis of the changes in PTEN mRNA level by PT-PCR assay.RESULTS:1.BmK venom inhibited the viability of Raji and Jurkat cells in a dose-and time-dependent manner,and the IC50 value calculated to be 275.40μg/ml(Raji) and 360.60μg/ml(Jurkat).2.BmK venom-induced apoptosis:When Raji and Jurkat cells were exposed to BmK venom for 24-48 h,cell shrinkage,membrane blebbing and apoptotic bodies occurred.Ultimately,many cells underwent death;Hoechst 3342 staining combined with Fluorescence Microscopy showed that compared with the nuclei in the control group,the nuclei were stained into brightly blue after treated by BmK venom for 48h.Flow cytometry showed that in Raji cells,the levels of p-Akt and p-Bad clearly decreased in a dose-dependent manner,while the levels of total Akt and Bad protein were stable.PTEN expression was inversely correlated with phosphorylation of Akt, indicating that the PTEN was functional.3.Flow cytometry showed that with increasing concentrations of BmK venom,the number of Jurkat and Raji cells in the G₀/G₁ phase increased significantly compared with control cells,while the cells in S and G₂/M phase decreased accordingly.4.In Raji cells,BmK venom up regulated the expression of PTEN accompanied by decreased levels of Akt and Bad phosphorylation.Treatment with BmK venom and LY294002(an inhibitor of Akt) synergistically enhanced apoptosis. The expression of p27 was increased in both PYEN-positive Raji and PTEN-negative Jurkat cells exposed to BmK venom.CONCLUSION:In summary,it was demonstrated that BmK venom could inhibit cell proliferation and induces apoptosis on human NHLs cell lines,Raji and Jurkat cells. The cytostatic effect was dose- and time-dependent.One possible mechanism may be suppression of Akt signal pathway via PTEN and up regulation of p27 with affecting auxiliary pathways.These data suggest that BMK may be effective in the treatment of non-Hodgkin's lymphoma.