Study On The Effect And Mechanism Of Apigenin Synergistically With Dihydroartemisinin Against KRAS/PIK3CA Double Mutant Tumors

Backgrounds and Objectives:KRAS is a small G protein that binds to GTP/GDP and has GTP hydrolase activity.It activates a variety of downstream signaling pathways,such as MAPK signaling pathway and PI3K-AKT signaling pathway by binding to GTP;KRAS gene is also the most commonly activated mutant oncogene in solid tumors.Approximately 95%of pancreatic cancer and 25%of non-small cell lung cancer patients have KRAS mutations.The current treatment strategies for KRAS mutant tumors mainly target KRAS mutation directly,acting on KRAS downstream signaling pathways,acting on synthetic lethal sites,immunotherapy and so on.However,due to the structural characteristics of KRAS itself,it is difficult for KRAS to be directly targeted and inhibited;acting on KRAS downstream signaling pathways will compensate the activation of other signaling pathways and make tumors resistant;because of the complicated genetic background of KRAS-mutated tumor cells,large-scale gene screening is limited,resulting in the slow development of synthetic lethal treatment for KRAS-mutated tumors;in addition,immunotherapy has limited effects on the treatment of advanced tumors with KRAS mutations.We urgently need to find a treatment strategy that can effectively inhibit KRAS mutant tumors.Studies have shown that KRAS-mutated tumor cells are divided into KRAS-dependent tumor cells that depend on KRAS and KRAS-independent tumor cells that do not depend on KRAS,direct targeting of KRAS has a better effect on KRAS-dependent tumors and the therapeutic effect is currently in clinical trials;however,the treatment for KRAS-independent tumors still face huge challenges.Therefore,we need to explore the genetic characteristics of KRAS-independent tumors,in-depth analysis of the differences ofthe genetic characteristics between these two types of tumors and the reasons for this difference,so as to provide atheoretical basis for the treatment of KRAS-independent tumors.Because KRAS-independent tumors also contain other genes mutations,they may not depend on KRAS for survive,and due to the characteristics of KRAS itself,it is difficult to inhibit it directly,while inhibiting key molecules in a single downstream signaling pathway will induce the feedback activation of other signaling pathways,leading to tumor resistance or recurrence,in view of this,it will be possible to use the combination of traditional Chinese medicine monomers with multiple targets to combat KRAS mutant tumors.The effective monomers of traditional Chinese medicine have little side effects and usually have multiple targets in cells.In recent years,effective monomers of traditional Chinese medicine have attracted increasing attention because of their varying degrees of anti-tumor effects in various types of tumor cells.Dihydroartemisinin is a sesquiterpene lactone,studies have shown that dihydroartemisinin has good antitumor effects,it can inhibite the growth of tumor and tumor cells in vivo and in vitro by regulating MAPK signaling pathway,PI3K-AKT signaling pathway,JAK-STAT3 and other signaling pathways.In view of the fact that dihydroartemisinin has been reported to be able to simultaneously target multiple targets of downstreamof KRAS,including MAPK and AKT,which has a good effect on tumor treatment,this study intends to use dihydroartemisinin as a bait drug to explore and find monomer with synergistic effects,and study its mechanism of action in-depth.Methods and Results:1.Study the KRAS mutant tumor cells for KRAS dependence(1)Construct a KRAS-shRNA lentiviral vector,package the lentivirus,and infect various types of tumor cells to knock down the KRAS gene,Western blotting experiments confirmed that KRAS was knocked down;clone formation experiments showed that the clone formation of some cells such as H460 and HCT116 were not affected after knocked down KRAS,they are KRAS independent tumor cells.Another part of cells such as H358 and PANC1 were inhibited and they are difficult to form clones,called KRAS dependent tumor cells.(2)Analyze genesmutantof KRAS mutant tumor cells through COSMIC database and find out KRAS-independent cells mostly have PIK3CA or PTEN mutations;KRAS-dependent cells mostly have BRAF mutations.2.Study the effect of apigeninsynergizes withdihydroartemisininto suppress KRAS independent tumors(1)Use H460 and HCT116 of human source as in vitro models and MTT method for drug screening,it was found that apigenin,an effective monomer of traditional Chinese medicine,can enhance the antitumor effect of dihydroartemisinin.(2)High-content cell imaging experiments confirmed that apigeninsynergizes with dihydroartemisinininhibited the proliferation of KRAS mutant tumor cells.(3)Clone formation experiments confirmed that combination of apigenin and dihydroartemisinin inhibited the colony formation of KRAS mutant tumor cells(4)Use MTT method found that the combination of apigenin and dihydroartemisinin has different degrees of antitumor effects on tumor cells of different mutation types.Combining the COSMIC database to analyze the genes mutation oftumor cell,we confirmed that the combination had the best synergistic inhibition effect on tumor cells containing KRAS/PIK3CA double mutation tumor cells.3.Study the mechanism of drug combination against KRAS independent tumors(1)Flow cytometry experiments confirmed that the combination of two drugs blocked tumor cell cycle in G0/G1 phase.(2)Western blot experiments found that the expression of P21and P27 proteins was up-regulated after the combination of two drugs,which confirmed that the drug combination caused G0/G1 phase arrest.(3)Phosphokinase chip experiments found that the combination of two drugs led to significant inhibition of phosphorylation of ERK,HSP27,STAT3 and other proteins,suggesting that ERK,HSP27 and STAT3 may play a key role in the drug combination against KRAS mutant tumors.(4)Western blot experiments confirmed that the phosphorylation of ERK,HSP27 and STAT3 was significantly inhibited after the combination of the two drugs.(5)Use MTT and Western blot experiments proved that enhance the function of HSP27 rescued tumor cell growth inhibition caused by the combination of two drugs,while inhibited the function of HSP27 or STAT3 can enhance inhibition of tumor cell growth caused by apigenin or dihydroartemisinin.4.Study the antitumor effect and the mechanism of drug combination inhibited tumors in vivo(1)In vivo experiments showed that compared with the control group and the single drug treatment group,the combination of two drugs can significantly inhibit tumor growth in nude mice.(2)Western blot experiments showed that,in accordance with the results of in vitro experiments,the combination of two drugs in vivo inhibited the phosphorylation of ERK and STAT3.Conclusion and significance:In this study,we discovered that some KRAS mutant tumor cells can grow and proliferate independently of the presence of KRAS.For this type of tumor cells,it found that the combination of apigenin and dihydroartemisinin had a good synergistic inhibitory effect on KRAS/PIK3CA mutant tumor cells by drug screening.And the experimental results in vivo also confirmed this.Molecular mechanism study showed that the drug combination exert antitumor effects by inhibiting the phosphorylation of ERK,HSP27 and STAT3,and it has been verified in vivo and in vitro.This study proposed a multi-target synergistic antitumor treatment strategy using a combination of effective Chinese medicine apigenin and dihydroartemisinin for the treatment of KRAS/PIK3CA tumors.It provides a theoretical basis for the treatment of KRAS mutant tumors.