Study Of New Sansalvamide A Cyclic Peptide Derivatives On Antitumor Activity

As there is an upward trend in the incidence and mortality, tumorbecomes a serious threat to human health. At present, the efficacy ofchemotherapy is not ideal. The main reason is the side effects of traditionalantitumor drugs in the clinical treatment. Therefore, looking for new highefficiency and low toxicity of antitumor drugs is a major subject in the field ofbiomedicine. Natural product chemistry has been the important way indevelopment of new pharmaceutical industry. Natural products have theadvantages of novel structure, high curative effect and side effects, so thenatural products chemistry and its research results have been widely used inpharmaceutical.Sansalvamide A is a cyclic depsipeptide produced by a marine fungus,and it is first reported in1999. It is a natural part of five peptides has the veryhigh lipotropy and significant anti-tumor ability. Sansalvamide A cyclicpeptides are compounds by the transformation of Sansalvamide A. After yearsof research, more than200new derivatives of Sansalvamide A cyclic peptideshave been reported. The activity of any derivatives has been improvedsignificantly. In this study,9new synthetic Sansalvamide A cyclic peptidederivatives were provided by the State Key Laboratory Breeding Base-HebeiProvince Key Laboratory of Molecular Chemistry for Drug (Hebei Universityof Science and Technology). Malignant melanoma B16cells in mice, humanbreast cancer MCF-7cells and human breast cancer MDA-MB-231cells wereselected to observe the antitumor activity of these new compounds. At thesame time, peripheral blood mononuclear cells and SD rat aortic vascularsmooth muscle cells were prepared by primitive culture for experiment. B16cell was selected as the tumor model and the compound H-10and H-15werein further research on the mechanism of inhibition of cell proliferation.The research contents and results are as follows: Part I The study of antitumor activity and the toxicity of normal cell onSansalvamide A cyclic peptide derivativesObjective: to observe the activity and toxicity of the nine new cyclicpeptide derivatives of Sansalvamide A.Methods: malignant melanoma B16cells in mice, human breast cancerMCF-7cells and human breast cancer MDA-MB-231cells were cultured astumor cell models for antitumor activity screening. Peripheral bloodmononuclear cells and SD rat aortic vascular smooth muscle cells wereprepared by primitive culture for observing the toxicity of these compounds.The results were measured by sulforhodamine B (SRB) colorimetric assay.The changes and the growth states of B16cells, lymphocytes, and smoothmuscle cells (VSMC) of rats were observed by inverted phase contrastmicroscope.Results:1Nine Sansalvamide A cyclic peptide derivatives have inhibiting effecton malignant melanoma B16cells in mice, human breast cancer MCF7cellsand human breast cancer MDA-MB-231cells. After treated48h, blank groupand1%DMSO group didn’t reach statistical significance, P>0.05. Except thecompound6,8and9in50μM for MDA-MB-231, all compounds showedantitumor activity on the three kinds of tumor cells. For B16cells, compound2,5, and6have the strongest inhibition rate,82.43%,81.79%and83.19%, forMCF-7cell, the strongest inhibition rate of three kinds of compounds werecompound5,64.19%, compound9,51.08%and compound4,50.89%, forMDA-MB-231, strongest were compound4and5, and the inhibition rate were68.04%and66.56%.29compounds on human peripheral blood mononuclear cells and SD rataortic vascular smooth muscle cell proliferation did have inhibitory effect.3Compound H-10have the function to make B16cells apoptosis anddifferentiation of morphology change, compound H-15can make B16cellsappear obvious differentiation changes. Conclusion:9Sansalvamide A cyclic peptide derivatives have antitumoractivity and less toxicity to normal cells, Compound H-10have a wide varietyof tumor cells with activity, and have the function to induce apoptosis of B16cells, compound H-15could induce differentiation of B16cells and inhibit theproliferation.Part II The mechanism of H-10inhibit proliferation of B16cellsObjective: to study the mechanism of H-10on inhibiting proliferation ofB16cells.Methods: sulforhodamine B (SRB) colorimetric method was used tocalculate Percentage Growth (PG) of different concentration of H-10treatedB16cells for48h and cell counting method to draw growth curve study H-10inhibit the proliferation of B16dependencies. Flow cytometric analysis todetect the apoptotic cell death. The expression of caspase-8, caspase-9andcaspase-3were detected by western-blot.Results:1H-10has concentration-dependent effect on B16cell growth:Compared with blank group, the proliferation rate of1%DMSO group has nosignificant change, P>0.05. After the treatment of different concentrations ofH-10(0.1,1,10,50and100μM) for48h, proliferation rate of B16cell weregradually decreased. Compared with control group, the proliferation rate of100μM,50μM and10μM were significantly decreased (100μM P<0.01,50μMP<0.01and10μM P<0.05). The morphological changes could be seen throughlight microscopy.2H-10has time-dependent effect on B-16cell growth: Time-dependenteffect of H-10on cell proliferation was measured by cell number. After treatedby50μM H-10for24h,48h,72h,96h,120h and144h, cell numbers werecounted and compared with control group. H-10caused time-dependentinhibition of B-16cell lines.3H-10induced apoptosis of B16cells: Flow cytometric analysis of cellsamples has demonstrated the ability of H-10induce apoptosis. Apoptoticcells were defined as those with subdiploid DNA content and presented as the percentage of all counted cells per sample. The proportion of apoptotic cells ofuntreated B-16cells was (0.96±0.22)%. Treated with50uM H-10for24h, thepercentage of apoptotic cells was (9.21±4.62)%and the difference wasstatistically significant. Caspase-3plays a key role in cell apoptosis and is theimportant molecular in apoptosis initiation. In this study, the expression ofcaspase-3was determined by Western Blot and this further confirmed thatH-10has the function of inducing apoptosis. After treated by10,25and50μMH-10, the expression of caspase-3increased with the concentration.4H-10induced apoptosis of B16cells via mitochondrial pathway: Theresults of western blot showed ascendant trend of the expression of caspase-3and capsase-9. And the expression of caspase-8didn’t change significantlyamong the control and the test group. These results suggest H-10inducedapoptosis of B16cells via mitochondrial pathway.Conclusion: H-10has strong inhibition on the proliferation of B16cells,and the effect has significant concentration dependence and time dependence.The IC50of H-10on B16cells in48hours is39.68μM. H-10has the functionof induce apoptosis of B16cells, and H-10induces B16cells apoptosis bymitochondrial pathway.Part III The mechanism of H-15inhibit proliferation of B16cellsObjective: to study the mechanism of H-15on inhibiting proliferation ofB16cells.Methods: sulforhodamine B (SRB) colorimetric method was used tocalculate Percentage Growth (PG) of different concentration of H-15treatedB16cells for48h and cell counting method to draw growth curve study H-15inhibit the proliferation of B16dependencies. Detect melanin production ofB16cells after treated by H-15. The expression of stem cell gene Foxd3, Klf4,Sox2and Nanog were detected by RT-PCR. The expression of TYR, Sox2and Foxd3were detected by Western Blot.Results:1H-15has concentration-dependent effect on B16cell growth:Compared with blank group, the proliferation rate of1%DMSO group has nosignificant change, P>0.05. After the treatment of different concentrations of H-15(0.1,1,10,50and100μM) for48h, proliferation rate of B16cell weregradually decreased. Compared with control group, the proliferation rate of50uM and10uM were significantly decreased, P<0.05. The morphologicalchanges could be seen through light microscopy. The IC50of H-15on B16cells in48hours is69.44μM.2H-15has time-dependent effect on B-16cell growth: Time-dependenteffect of H-15on cell proliferation was measured by cell number. After treatedby50μM H-15for24h,48h,72h,96h,120h and144h, cell numbers werecounted and compared with control group. H-15caused time-dependentinhibition of B16cell lines.3The melanin content is higher than control after treated by50μM H-15for48hours: the OD value of B16cells after treated by50μM H-15for48hours was0.1743±0.0227compared with the control0.0788±0.0039, therewas statistically significant between the two groups, P<0.01.4The expression of tyrosine (TYR) enzyme was increased after treatedby50μM H-15for48hours: detected by Western Blot, the expression of TYRwas increased.5H-15has the function of inhibit the expression of Sox2and Foxd3:according to the results of RT-PCR, after treated by H-15for24h, theexpression of Sox2and Foxd3had a downward trend, Klf4expression was notsignificantly change, and Nanog expression was not regularity. It suggestedthat H-15has the function of inhibit the stem cell properties of B16cells.Conclusion: H-15has strong inhibition on the proliferation of B16cells,and the effect has significant concentration dependence and time dependence.The IC50of H-15on B16cells in48hours is69.44μM. H-15may have thefunction of the induced differentiation of B16cells, and the ability of secretionmelanin in B16cells treated by H-15was stronger than control. H-15couldinhibit the expression of Sox2and Foxd3, and inhibit the stem cell propertiesof B16cells.