Study Of Anti-tumor Mechanism Of The Compounds Indole Amide And Benzothiazole

As we all know,the blood vessels supply the nutrient for the development of tumors,hence,targeting vessels of tumors has the chance to inhibit the tumor growth.The PI3K/Akt/mTOR signaling pathway affect angiogenesis in tumor by regulating cell proliferation,survival and migration.Thus,blocking the PI3K/Akt/mTOR pathway will inhibit angiogenesis and supress the tumor growth.The PI3K/Akt/mTOR pathway is an important regulator in protein translation and actived in malignant tumor.Phosphorylated translation initiation factors on the PI3K/Akt/mTOR pathway downstream control the translation of related tumor mRNA(Cyclin D1,c-myc,Bcl-2,VEGF,et al)and take part in tumorigenesis.Tumor suppressor gene 4E-BP1 on the downstream of the mTOR pathway binds with eIF4E and decreases eIF4E-eIF4G(Erk promotes to form eIF4E-eIF4E),then the translation starts.During the research of anti-tumor drugs,the proliferation of tumor cells were inhibited and caused cellapoptosis by blocking the proteins on the downstream of the PI3K/Akt/mTOR pathwaytranslation related proteins,or supressing the interaction between eIF4E and eIF4G.During the reseaches of anti-angiogenesis and anti-tumor drugs,besides targeting angiogenesis,it is necessary to affect the proliferation and growth of tumor by inhibiting the PI3K/Akt/mTOR pathway and targeting critical regulatory or translation related proteins.Previous research have chosen ZJQ-24 using the chick embryo chorioallantoic membrane(CAM)and further proved that shows its anti-angiogenes activity by inhibiting the proliferation and migration of HUVEC cells.But the anti-tumor of ZJQ-24 is unclear.First of the research,MTT assay the cytotoxicity of ZJQ-24.The results showed that ZJQ-24 caused a strong reduction in growth of liver cancer cell HepG2,breast cancer cell MCF-7,prostate cancer cell PC-3 and lung cancer cell A549,with IC50 values of 3.54±0.38?M,7.83±0.44?M,8.05±0.12?M and 17.63±1.08?M respectively.These datas told us ZJQ-24 has the greatest cell killing effect in HepG2 and HepG2 was choosed to study the anti-tumor mechanism of ZJQ-24.Secondly,we study the cell cycle of HepG2.ZJQ-24 has treated HepG2 cells for 24h by the concentration gradient(1?M,5?M and 10?M),and 5?M ZJQ-24 caused the G2/M arrest of the cell cycle.So,5?M ZJQ-24 was chosed to study the cell cycle in time gradient(6h,12h,24h,48h).And ZJQ-24 has induced G2/M arrest at 12h,but when the time has been prolonged(24h,48h),it showed G1 arrest.Annexin V/FITC and Western Blot were used to test the HepG2 apoptosis in ZJQ-24.The results displayed that ZJQ-24(5?M,24h)began to lead to early apoptosis,while ZJQ-24(1?M,5?M,10?M;24h)didn't effect HUVEC apoptosis.These point out ZJQ-24 can inhibit cell proliferation,promote apoptosis and has the great drug selective.ZJQ-24 has treated HepG2 cell for 24h at 1?M,5?M,10?M does and it reduces proteins expression like p-Akt(Ser473),p-mTOR(Ser2448),Raptor(mTORC1),p-PRAS40(Thr246),p-PRAS40(Serl83),p-P70S6K(Thr389),p-S6(Ser240/244).It speculated that ZJQ-24 supressed HepG2 proliferation,protein translation and induced cell apoptosis.ZJQ-24(1?M,5?M,10?M)has treated HepG2 for 24h,it didn't affect translational controlled genes like Bcl-2 and Cyclin D1 on the transcription level unconspicuously,but it can decrease their protein levels,which means ZJQ-24 only affect the proteins expression of translational controlled genes and may has the function of inhibiting translation.What's more,p-Erk1/2(Thr202/Tyr204)and translational related proteins p-4E-BP 1(Thr70),p-eIF4E(Ser209),p-eIF4G(Serl108)in HepG2 have decreased the protein expression by 5?M ZJQ-24,it proved ZJQ-24 can inhibit protein translation.In addition,4E-BP1 and eIF4G combine with eIF4E competitively,Co-IP was used to test the influence of ZJQ-24(1?M,5?M,10?M;24h)in the interaction of 4E-BP1,eIF4G and eIF4E in HepG2.The results reflected that the protein expression of eIF4G and 4E-BP1 decreased by the increasing concentration of ZJQ-24.It reminders the drug target of ZJQ-24 likely sites the peptide mold structure of eIF4E which is in common for eIF4G and 4E-BP1.To sum up,the recent study discovers that ZJQ-24 can inhibit cell HepG2 proliferation and promote apoptosis in HepG2.Besides,ZJQ-24 arrest G2/M in HepG2 and suppress the PI3K/Akt/mTOR signaling pathway.AhR(Aryl hydrocarbon receptor,AhR)is highly expressed in gastric cancer and closely related to the genesis of gastric cancer.Some researches have showed that 5F-203,a sort of benzothiazole compounds,can bind AhR and induce DNA injury in some cancer cell of breast cancer and ovarian cancer,leading to cell death finally.But this phenomenon is less researched in gastric cancer.In this paper,we explored the effect of 5F-203 and its possible mechanism in anti-gastric cancer.Tracey D.Bradshaw,et al have studied 5F203 cytotoxicity of breast cancer cell MCF-7 and ovarian cancer cell IGROV-1,and found that 5F203 cytotoxicity is AhR dependent.5F203 plays a role in anti-tumor by inducing CYP1A1 expression and DNA single/double chain broken.The CYP family(CYP1A1,CYP1B1,CYP2S1,CYP2W1)participates the metabolism of tumor cells.The expressions of CYP1A1,CYP1B1,CYP2S1 are induced by AhR ligands,that will cause DNA damage and the death of tumor cell.As CYP2W1 is constitutive expression in many tumors and lack of expression in normal tissue,AhR ligands may be anti-tumor by suppressing CYP2W1 expression?In the early phase of research the project,we have tested the cytotoxicity of 5F-203 on gastric cancer cells,and found that the value of IC50 in SGC-7901?BGC-7901?KATO ?isgreater than 50?M.But IC50 in NCI-N87 and MKN-45 are apparently 22.63±4.28?M and 0.090±0.0076?M.So,we studied the effect of 5F-203 on MKN-45 in this paper.Frist of all,the protein expression of AhR and CYP1A1 in cytoplasm and cell nucleus of MNK-45,MCF-7,SGC-7901,HepG2,colon cancer cell HCT116 and prostate cancer cell PC-3were tested by Western Blot.The results showed that AhR in cytoplasm and cell nucleus of MKN-45 is high expression,but it is lower expression in MCF-7.In order to study the mechanism of 5F-203 in MKN-45,the protein and mRNA levels of AhR and CYP1A1 in MKN-45 under 5F-203 treated were tested.MKN-45 was treated by 1?M 5F-203 of time gradient(1h,3h,6h;2h,6h,24h;24h,48h,72h),AhR and CYP1A1 reflect the same trend both in nuclear and cytoplasmic,that means AhR has decreased while CYP1A1 has incresed either in nuclear or cytoplasmic and 5F-203 can active CYP1A1.During the DNA damaged research,p-H2A.X has increased in MKN-45 while 5F-203(24h,48h,72h;1?M)treated and 5F-203 will induce DNA damage happened in MKN-4,that may connect with the level of CYP1A1 mRNA and the metabolism of the drug.So,considering CYP1A1,CYP1B1,CYP2S1 and CYP2W1 are the members of CYP family.We detected the transcriptional levels by RT-PCR in MKN-45 cells as the cells were treated by 1?M 5F-203 for 3h,6h and 12h.The result showed the transcriptional levels of CYP1A1,CYP1B1,CYP2S1 increased at 3h and 6h,decreased at 12h.On the contrary,CYP2W1 decreased at 3h and 6h,increased at 12h.It has further confirmed that 5F-203 actives the CYP family.After that,the cell cycle of MKN-45 was G2/M arrest by Flow Cytometer as 1?M 5F-203 treated MKN-45 for 3h,6h,12h,24h and 48h.Moreover,PARP is the apoptosis marker,1?M 5F-203(1h,3h,6h,12h,24h,48h and 72h)caused cleaved PARP band in MKN-45 at 48h.It is speculated that 5F-203 can induce cell apoptosis of MKN-45.In conclusion,5F-203 shows the significant killing effect on gastric cancer cell MNK-45.It is speculated that 5F-203 inhibits MKN-45 growth by inducing AhR and CYP1A1 expression on the protein level and transcriptional levels of CYP1A1,CYP1B1,CYP2S1,CYP2W1,and leads to arrest G2/M and cell apoptosis in MKN-45.