The Broad Spectrum Effect Of One Anti-neoplastic Peptide In Cancers

Cancer is a serious disease threatening human health.At present,the treatment of cancer is mainly based on small molecule drugs,and there are significant adverse reactions such as toxicity and drug resistance.Polypeptide drugs have the advantages of low toxicity,targeted and high bioavailability,and become a new way for the development of tumor drugs.As a tumor suppressor gene,PIAS3 is an inhibitor of STAT3 signaling,which plays a significant role in tumor suppression.Our previous study showed that short PIAS3isoforms expression a deletion of amino acids 87-121 in various human tumor cells,lost the inhibitory function of tumor,PIAS3-deta segment,which is PIAS3 sequence difference of two isomers,play a key domain STAT3/AR signal suppression.These results suggest the possibility of the differential peptide PIAS3-deta as an anticancer drug.In the present paper,we synthesized（PIAS3-deta）polypeptide of 35 amino acids（named PIAS3-deta peptide）using synthetic peptide technique PIAS3 87-121 amino acids,through cytological experiments to study the inhibitory characteristics of PIAS3-deta peptide on tumor cells and screen the sensitive variety of tumor cells;prove its anti-tumor spectrum and reveals the minimum domain;highlighting the security features of this peptide drugs without cytotoxicity on normal cells.Moreover,the three kinds of animal model proves that the inhibitory effect of PIAS3-deta peptide on tumor in vivo and in vivo drug concentration;set up a special cisplatin drug for comparison,emphasizing the non-toxic safety features of the peptide drugs within the object;provide a theoretical basis for the depth of research and development of peptide drugs.Firstly,we investigated the inhibitory effect of PIAS3-deta peptide on the proliferation of tumor cells.The PIAS3-deta peptide and the control peptide on 11 human tumor cells and two normal cells.MTT method detected cell proliferation and found different concentrations of no cytotoxicity on tumor cells in the control peptide,PIAS3-deta peptide inhibit tumor cell and has no inhibitory effect on both normal cells,the concentration of10^-3g/L（0.228uM）which showed broad-spectrum anti-tumor effect,especially through the comparison table of chemical drugs and cisplatin.The PIAS3-deta peptide specifically inhibited the growth of tumor cells,and cisplatin have the significant inhibition on tumor and normal cells,suggesting that the security of PIAS3-deta peptide drugs.Furthermore,we compared the inhibitory effects of PIAS3-deta full-length peptide（35aa）and four different truncated PIAS3-deta peptides on tumor cells in order to find the minimal functional domain of PIAS3-deta.We found that compared with the control peptide and four different truncated.The inhibitory effect of PIAS3-deta peptide on the length of the 11 kinds of tumor cells were significantly higher than the truncated PIAS3-deta peptide,revealing that the full-length 35aa is essential for the anti-tumor effect of the peptide.There is closely relation between the abnormal activation of STAT3 signal and the development of tumor.We further investigated the effect of PIAS3-deta peptide on STAT3signaling pathway.We found that PIAS3-deta peptide can inhibit the expression of STAT3transcription activation of STAT3-Luc luciferase report gene by the luciferase report gene laboratory tests.Western-Blot experiments also showed that PIAS3-deta peptide could reduce the expression of P-STAT3 but no obvious effect on the expression of STAT3ontology,suggesting that PIAS3-deta peptide could inhibit the activation of STAT3.STAT3downstream target genes and tumor cell proliferation and metastasis are closely related,and the Western-Blot assay showed that compared with the control peptide,PIAS3-deta peptide reduced the expression of STAT3 target genes cyclinD1,BCL-2,signal PCNA,c-myc,survivin.Above all,PIAS3-deta peptide plays an important role in antitumor activity by inhibiting the STAT3 signaling pathway.Moreover,we used three animal models to investigate the anti-tumor effect,dosage and safety of PIAS3-deta peptide in vivo.First of all,we investigated the antitumor effect of PIAS3-deta peptide in vivo by using four kinds tumor cells in a tumor-burdened model.We transplanted the mouse prostate cancer cell RM1,rat breast cancer cell 4T1,rat lung cancer cells LLC and mouse melanoma cell B16 into white mice,1 x 10⁷ cells per point injection,after 8 days,the tumor size of the tumor-bearing mice is about 0.2cm³.The three doses of PIAS3-deta（1mg/kg,3mg/kg and 10mg/kg）and the control peptide（10mg/kg）were injected rats twice a week for a total of 36 days.The results showed that compared with the control group,the tumor size of 10mg/kg peptide group decreased significantly,but there was no significant difference in the size of the tumor 1mg/kg and 3mg/kg peptide group.The result shows that the effective dose of PIAS3-deta peptide to inhibit tumor growth in vivo is 10mg/kg.To this end,we use three kinds of people cancer cells（human gastric cancer SGC-7901cells,prostate cancer DU145 cells,MDA-MB-231 breast cancer cells）in nude mice tumor-bearing model experiment,we verify the 10 mg/kg of the effectiveness of PIAS3-deta peptide in nude mice in vivo.We transplant human cancer cells into nude mice,and inject 1 x 107 cells per point.After 10 days we found the tumor size of tumor-bearing mice is about 0.2 cm³,using 10 mg/kg PIAS3-deta peptide and control peptide,intraperitoneal injection of nude mice respectively,injection once a day,a total of injection for 30 days.Results show that the 10 mg/kg of tumor size PIAS3-deta peptide injection group was obviously less than the normal saline control group.It proves the Antagonism of human tumors of 10 mg/kg of the effectiveness of PIAS3-deta peptide in nude mice in vivo.In addition,we use another three kinds of human cancer cells（HEPG-2 cancer cells,A375 melanoma cells,non-small cell lung cancer A549 cells）in nude mice tumor-bearing model experiment to test the security of PIAS3-deta peptide.We transplant them into nude mice,and inject 1 x 10⁷ cells per point.After 13 days we found the tumor size of mice tumor-bearing is about 0.2 cm³,using three doses of PIAS3-deta peptide of 3 mg/kg,10mg/kg,and 30 mg/kg as experimental group and control group of 10 mg/kg at the same time,we set chemotherapy drugs cisplatin group of 1 mg/kg and cisplatin of 1 mg/kg with10 mg/kg PIAS3-deta peptide group,injecting the nude mice intraperitoneal once a day,31days totally.The results showed that although the two groups’tumor size is less than the control group,the nude mice’s weight at least markedly reduced about 20%with the symptom of thin,anorexia,and many other adverse reactions,however,the two PIAS3-deta peptide injection group（10 mg/kg and 30 mg/kg）,not only reduce the tumor size significantly but also the weight without falling,fur burnish and good feeding.All of these illustrates the PIAS3-deta peptide drugs is better than the medicine,which is safety and no adverse reaction in nude mice.Moreover,the immunohistochemical experiment showed that PIAS3-deta peptide group can significantly surpress the expression of STAT3,P-STAT3,Bcl-2,CyclinD1,PCNA and c-myc.In conclusion,we found PIAS3-deta peptide can inhibit proliferation of multiple tumor cells effectively and arrest them in S phase.We select dosage of 10mg/kg in animal experi ment,we found transplanted tumor model in mice.it has a good treatment without toxic an-d side effect to inhibit cancer through inhibit signaling pathway of STAT3.This study prov-ide a primary experimental evidence for the application of PIAS3-deta peptide drug’s broad spectrum effect.