Purification And Study Of Anti-tumor Activity Of A New Phospholipase A2 Homologue From Agkistrodon Halys Pallas

Snakes are extensively distributed worldwide and with species numerous. Since ancient times, snake venoms have been one of the most extensively used medicines in the practice of traditional Chinese medicine. Therefore, study and development of bioactive components in snake venoms has become an important research area in basic medicine and pharmaceutical industry. China is rich in Snakes. More than 200 species and subspecies of snakes have been found in mainland China, but up to now only a few crude venoms or partially purified protein components of the venoms have been studied. Moreover, the anti-tumor components in majority of the venoms have not been identified yet.Phospholipase A2 (PLA2) catalyses the hydrolysis of the sn-2 ester bond of glycerophospholipids, leading to the generation of NEFAs and lysophospholipids (lysoPLs). This enzyme exists in venoms of almost all the snakes. PLA2 exhibits a lot differences in its structure and function as a result of its sources of snake species. And the places where the snakes inhabit also contribute to the differences. There are many PLA2 isoenzymes which exist in snake venoms. The homology of amino acid sequence is above ninety percent and the constitutions are very similar, but they exhibit diverse pharmaco-physiological function. Based on this background, we took the venoms of Agkistrodon halys Pallas, a snake which is abundant in South of China, as the source of samples in this paper, established a one step method for rapid isolation and purification of the snake venom extracts, and made the screening and evaluation of the anti-tumor activity of these protein components, for the purpose of the discovery of new polypeptide with anti-tumor activity in snake venom.The principal results and conclusions are as follows:1. The establishment of rapid, one step isolation of the protein components in snake venom from the crude extract by high performance liquid chromatography (HPLC).In methods of isolation of the protein components from crude extracts of snake venom, a two-step or even more than three steps method is generally used now. To perform rapid one-step isolation of the protein components from venom crude extracts, we used C18 reverse-phase silica gel particles with large diameter (300(?)) as the stationary phase of HPLC column, and eluted with line-gradient trifluoroacetic acid/acetonitrile within 0-120min. By using this new method, we have isolated 16 protein components from the crude extracts of Agkistrodon venom by only one step procedure.2. Determination of the purity of the 16 protein components isolated with HPLC.The purity of the 16 protein components isolated from the crude extract of Agkistrodon venom was determined with HPLC. Reverse-phase C18 column was used as the chromatography column, trifluoroacetic acid/acetonitrile as the flow phase, and the elution time is 0-40min. Under this condition, the purity of all the 16 protein components isolated has reached more than 95%. This shows that our new method is more rapid for the isolation of snake venom and more efficient in obtaining the products with high purity. 3. Determination of the molecular weight of the 16 protein components isolated with HPLC.The molecular weight of the 16 protein components is determinated with Electrospray ionization mass spectrum (ESI-MS) and Matrix assisted laser desorption /ionozation mass spectrum (MALDI-TOF-MS). The molecular weight of component No.1,2,3,4,5,6,8,9,10,12,15,and 16 is 1234.615,1234.575,7480,7554,13900,14504,13912,13962,13974,24748,23337and 22695Da respectively.4. Determination of the protein concentration of the 16 protein components isolated with HPLC.The protein concentration of the 16 protein components is determinated with Bradford method, Bovine serum albumin is used to make a standard curve. The protein concentration of the 16 protein components, from No.1 to No.16 sequentially is 0.631,2.653,5.78,0.983,10.545,4.586,10.68,15.32,14.46,14.84,54.25,106.958,104.36,69.59,64.858 and 137.608μg/ml. The protein concentration in each component differs largely, this indicates that their contents are different in the crude extract of the Agkistrodon venom.5. The effect of the anti-tumor activity of the 16 protein components isolated with HPLC on human hepatoma Hep3B cells.The anti-tumor activity of the 16 protein components isolated was measured and evaluated. The cultured human hepatoma Hep3B cell line was used as the model. Among the 16 protein components, we found component 3, component 4, and component 5 have anti-tumor activity against Hep3B cells. We named component 3 AHP-3 (the abbreviation of Akgistrodon halys Pallas),component 4 AHP-4,component 5 AHP-5.We have examined the morphological changes of Hep3B cells treated with AHP-5 involved in cancer chemopreventive activity.In cultures without AHP-5, Hep3B cells grew normal in monolayer and were distributed evenly with spindle shape. After treatment with 139μg/ml of AHP-5 for 24 h, cells exhibited shrinkage and shedding under phase contrast microscope. Transmission electron microscope examination revealed chromatin condensation, nuclear fragmentation, and shrinkage of the cells. These results show that AHP-5 possesses remarkable anti-tumor activity by inhibiting cell growth and inducing cell apoptosis.6. AHP-5 molecule was of a single peptide chain structure(proved by electrophoresis)We tried to identify whether AHP-5 is of single or double peptide chain by means of SDS reducing and non-reducing electrophoresis. It is shown that under the condition of either reducing or non-reducing condition, AHP-5 appears as a single band. This shows that AHP-5 is a protein composed of a single peptide chain.7. Sequence determination of the amino acids at the N-terminal of AHP-5The 1st to the 15th amino acids at the N-terminal of AHP-5 were sequenced with the Edman degradation method. It was shown that the 15 amino acids at the N-terminal end were HLLQFRKMIKKMTKK in sequence. After comparison of the amino acid sequence with that of the known protein sequence of protein-protein BLAST in GenBank, we have not found any amino acid sequence which is identical to the 1st through 15th amino acids of AHP-5. So it is proved that AHP-5 is a new snake venom polypeptide. Meanwhile, 15 amino acids at the N-terminal end of AHP-5 are highly homology with other known PLA2. So we suppose that this composition is the PLA2 homologue.8. A preliminary study of the molecular mechanism of the anti-tumor activity of AHP-5 against Hep3B cells.The molecular mechanism of the anti-tumor activity of AHP-5 is not very clear now as it is a new snake venom polypeptide. Therefore, we use SuperArray gene chips to evaluate the effect of AHP-5.After exposure to 140μg/ml PLA2 homologue for 12h, 19 genes were down-regulated, and 20 genes were up-regulated. ICAM-1, Integrinα1 and LFA1b, whose expressions were down-regulated exceeding ten-fold, are mainly involved in cell adhesion. Bcl-2 and Fas, whose expressions were up-regulated over ten-fold, is mainly involved in cell apoptosis and aging. Integrinαv is involved in cell adhesion. Thrombospondin2 is involved in vascularization. MTS1 and PAI-2 is involved in tumor cell invasion and metastasis. PLA2 homologue has very broad targets of action, especially in tumor cell metastasis and growth.9. Effect of AHP-5 on the changes of cell cycle and [Ca²⁺]i in Hep3B cellsThe effect of AHP-5 on cell cycle distribution is analyzed by flow cytometry and the results showed that after 24h treatment, AHP-5 caused an increase of cells in the S phase and a corresponding decrease of cells in the G0/G1. It indicated that AHP-5 could inhibit the growth of Hep3B cells and cause cell cycle arrested in S phase. Furthermore, sub-G1 cell populations were found, which indicated that APH-5 could induce Hep3B cell apoptosis. And the apoptosis-inducing effect was also demonstrated by the fact that phosphatidyl serine flipped across the plasma membrane from inside to outside and the increase in cytosolic [Ca²⁺] examined with confocal microscope.Conclusion:1,A HPLC method was established to rapidly isolate the protein components in the crude extract of snake venom. Large diameter reverse phase silica gel C18 was used to prepare the chromatographic column, and acetonitrile/triflroroacetic acid was used as the flow phase. Gradient elution was made within a time-interval of 0-120 minutes. Sixteen protein components (each with a purity of more than 95%) have been isolated by one-step procedure from the crude extract of Agkistrodon halys Pallas venom.2,Among the 16 protein components in snake venom, three of them have anti-tumor activity against human hepatoma Hep3B cells in vitro. The cells treated with the venom exhibited cell shrinkage and shedding under phase contrast microscope and displayed chromatin condensation and nuclear fragmentation under transmission electron microscope. These results show that AHP-5 possesses remarkable anti-tumor activity by inhibiting cell growth and inducing cell apoptosis.3. With electrophoresis analysis, molecular weight determination and amino acid sequence, it was shown that AHP-5 is a protein composed of a single peptide chain with molecular weight of 13900Da. The 15 amino acids (the 1st through the 15th) at the N-terminal end are HLLQFRKMIKKMTKK in sequence. After comparison of protein-protein BLAST in GenBank, we have not found any amino acid sequence which is identical to the 1st through 15th amino acids of AHP-5. AHP-5 is highly homology with other known PLA2s. So we preliminarily suppose that this composition is a new PLA2 homologue.4. The expression of a great number of genes was influenced by treatment with PLA2 homologue and the main functions of influenced genes are involved in growth and metastasis of cancer cells. Our study provided elementary clues and foundation for further study of the possible mechanism of PLA2 homologue on Hep3B cells.