| Up to date, cancer therapy is still one of the serious problems that puzzle the scientistsin the world. It is an important topic to seek an efficient way to treat cancer in the study ofcancer therapy. In this study, scorpion venom was purified by chromatography. Theanti-tumor effects of scorpion venom and its purified fractions were observed in cellularand molecular biology respectively. Results showed that the scorpion venom and itspurified fractions had some inhibitory effects on tumor growth and the possiblemechanisms were related to the induction of apoptosis in tumor cells. It will provide aneffective way to cancer therapy with scorpion venom for clinical use in future.1. Separation and purification of scorpion venomScorpion venom (SV) was purified by chromatography and five fractions wereobtained. According to the results of protein electrophoresis, the first three fractions, whichwere about 6-100 kD, were collected in this study. After desaltification and concentration,the purified fractions were gotten and stored at -20℃. The purified fractions of scorpionvenom were expressed as PFs , including PF-I, PF-II and PF-III respectively in this study.2. Inhibitory effects of SV and PFs on tumor cells in vitro2.1 Inhibitory effects of SV and PFs on tumor cell viabilityMTT assay was applied to observe the inhibitory effects of scorpion venom (SV) andthe PFs. Five concentrations (10 μg/ml, 50 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml.)were used.2.1.1 Effects of SV and PFs on B16 melanoma cell viabilityResults showed that the viability of B16 cells declined significantly by SV with theconcentrations of 400 μg/ml and 800 μg/ml compared with control (P<0.01, P<0.05,respectively). And the viability of B16 cells also decreased significantly by PF-I, PF-II andPF-III with the concentrations of 200 μg/ml -800 μg/ml compared with control (P<0.05 -P<0.01). It indicated that SV and PFs could inhibit the growth of B16 cells obviously.2.1.2 Effects of SV and PFs on HCT-8 cell viabilityResults showed that the viability of HCT-8 cells declined significantly by PF-I andPF-II with the concentrations of 200 μg/ml -800 μg/ml compared with control (P<0.01,respectively). It indicated that PFs could inhibit the growth of HCT-8 cells.2.1.3 Effects of SV and PFs on SKOV-3 cell viability Results demonstrated that the viability of SKOV-3 cells decreased significantly by SVat 800 μg/ml compared with control (P<0.01). The viability of SKOV-3 cells declinedsignificantly by PF-I, PF-II and PF-III with the concentrations of 400 μg/ml and 800 μg/mlcompared with control ( P<0.05 -P<0.01). It indicated that SV and PFs had someinhibitory effects on the growth of SKOV-3 cells.2.1.4 Effects of SV and PFs on EJ cell viability Results showed that the viability of EJ cells decreased significantly by SV with theconcentrations of 200 μg/ml -800 μg/ml compared with control (P<0.01, respectively).And the viability of EJ cells decreased significantly by PF-I at 400μg/ml and 800 μg/ml, byPF-II at 200 μg/ml, 400μg/ml and 800 μg/ml and by PF-III at 200 μg/ml compared withcontrol ( P<0.05 -P <0.01). It indicated that SV and PFs had some inhibitory effects on thegrowth of EJ cells.2.2 Inhibitory effects of SV and PFs on colony formation of tumor cells Colony formation assay was applied to observe the inhibitory effects of SV and PFs.And only two concentrations (400 μg/ml and 800 μg/ml) were chosen according to theabove results.2.2.1 Effects of SV and PFs on colony formation of B16 cells Results showed that the number of colonies of B16 cells reduced significantly by SV,PF-I, PF-II and PF-III with the concentrations of 400 μg/ml and 800 μg/ml compared withcontrol (P<0.01, respectively). It indicated that SV and PFs could inhibit the proliferationof B16 cells.2.2.2 Effects of SV and PFs on colony formation of HCT-8 cells Results showed that the number of colonies of HCT-8 cells reduced significantly byPF-II at 400 μg/ml compared with control (P<0.01). The number of colonies of HCT-8cells decrease significantly by SV and PF-II at 800 μg/ml compared with control (P<0.01,respectively). It indicated that SV and PFs could inhibit the proliferation of HCT-8 cells.2.2.3 Effects of SV and PFs on colony formation of SKOV-3 cells Results showed that the number of colonies of SKOV-3 cells was reducedsignificantly after treatment by PF-I, PF-II and PF-III with the concentrations of 400 μg/mland 800 μg/ml compared with control (P<0.01, respectively). It indicated that PFs couldinhibit the proliferation of SKOV-3 cells.3. Mechanisms of anti-tumor effects of SV and PFs3.1 Effects of SV and PFs on tumor cell cycle Two concentrations (400 μg/ml and 800 μg/ml) of SV and PFs were used to study thechanges in cell cycle of tumor cells by FCM assay.3.1.1 Effects of SV and PFs on B16 cell cycle Results showed that the percentage of B16 cells in G2+M phase was increasedsignificantly 72 h after treatment by PF-I, PF-II and PF-III with concentrations of 400μg/ml and 800 μg/ml compared with control (P<0.05 -P<0.01). It indicated that G2arrestcould be induced by PFs in B16 cells.3.1.2 Effects of SV and PFs on HCT-8 cell cycle Results showed that the percentage of HCT-8 cells in S phase was increasedsignificantly 72 h after treatment by PFs with concentrations of 400 μg/ml and 800 μg/mlcompared with control (P<0.01, respectively). We could deduce that the inhibitory effectsof PFs on HCT-8 cells might be an induction of S phase arrest.3.1.3 Effects of SV and PFs on SKOV-3 cell cycle Results showed that the percentage of SKOV-3 cells in G0/G1 phase was increasedsignificantly by PF-II and PF-III with concentrations of 400 μg/ml compared with control(P<0.01, respectively). And the percentage of SKOV-3 cells in G2+M phase was increasedsignificantly by PF-I, PF-II and PF-III with concentrations of 400 μg/ml compared withcontrol (P<0.05 -P<0.01) and by SV, PF-I and PF-III with concentrations of 800 μg/mlcompared with control (P<0.01, respectively). It indicated that G1 and G2 arrest could beinduced by SV and PFs in SKOV-3 cells.3.1.4 Effects of SV and PFs on EJ cell cycle Results showed that the percentage of EJ cells in G2+M phase was increasedsignificantly by PF-I, PF-II and PF-III with concentrations of 400 μg/ml compared withcontrol (P<0.01, respectively). And the percentage of EJ cells in G0/G1 phase was increasedsignificantly by PF-I and cells in G2+M phase was increased significantly by SV and PF-IIIat 800 μg/ml compared with control (P<0.05 -P<0.01). It indicated that G1 and G2 arrestcould be induced by SV and PFs in EJ cells.3.2 Induction of apoptosis in tumor cells with SV and PFs Two concentrations (400 μg/ml and 800 μg/ml) of SV and PFs were used to study thechanges in apoptosis in tumor cells with TUNEL or FCM assay.3.2.1 Effects of SV and PFs on apoptosis of B16 cells TUNEL assay was used to detect the apoptosis of B16 cells. Results showed that thenumber of apoptosis increase significantly by SV, PF-I and PF-III at 400 μg/ml comparedwith control (P <0.05 -P <0.01). It indicated that SV and PFs could inhibit the growth ofB16 cells by inducing apoptosis.3.2.2 Effects of SV and PFs on apoptosis of HCT-8 cells TUNEL and FCM were applied to detect the apoptosis of HCT-8 cells. The resultsshowed that the number of apoptosis increased significantly by PF-I, PF-II and PF-III at400 μg/ml compared with control (P <0.01, respectively) with TUNEL assay. The sameresults were also showed with FCM assay. It indicated that PFs could inhibit the growth of...
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