CPT1 Inhibitor Etomoxir Enhances The Anti-tumor Effect Of Chemotherapeutics

Lung cancer is one of the most common human cancer in the world today,where its incidence rate and mortality rate have increased year by year.In tumor cells,abnormal lipid decomposition and anabolism play a very important role in the occurrence and development of tumors.In the process of fat ?-oxidation,the key regulatory enzyme of this process is carnitine palmitoyltransferase(CPT).And CPT1 is the rate-limiting enzyme of fatty acid(3 oxidation,for catalyzing an acyl carnitine and acylcarnitines binding form;after extensive research,it has been found that CPT1 plays an important role in the development of a variety of tumors,which probably makes CPT1 to become a molecular marker for tumor diagnosis and a new target for anti-tumor therapy.Etomoxir,an ethylene oxide carboxylic acid derivative,is an inhibitor of CPT1,inhibiting the CPT1 activity.Cisplatin is an inorganic platinum complex,one of the most widely used chemotherapy drugs.However,Cisplatin has more toxic and side effects and tumor cells are prone to drug resistance leading to disease recurrence.Through previous experiments,the knockdown of CPT1 can promote the sensitivity of tumor cells to Cisplatin.The latest gene editing technology,Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins 9(CRISPR/Cas9)system,was using in a great range of researches,including animal models construction,cell lines establishment,precise treatment and so on,with the characteristics of high efficiency,high speed,strong reproductive system transfer ability,and simple economy.The purpose of this study was to use the small-molecule inhibitor Etomoxir of CPT1 to influence and synergize with the conventional chemotherapy drug Cisplatin by affecting CPT1 activity,improve the sensitivity of tumor cells to the chemotherapeutic drugs,inhibit tumor cell proliferation and promote apoptosis,then provides a new way for improving the chemotherapy effect of non-small cell lung cancer.To this end,in our studies,we focused on the effects of chemotherapy drug Cisplatin and small molecule inhibitor of CPT1(Etomoxir)on the proliferation and migration of tumor cells in vitro;and in vivo,we also studied the effects of chemotherapy drugs Cisplatin and CPT1 small-molecule inhibitor Etomoxir on tumor proliferation and migration in mice.Meanwhile,in our experiment,CRISPR/Cas9 technology was used to perform gene editing on the non-small cell lung cancer H1299 cell line to establish a cell line targeted for knockout CPT1A.We first treated human non-small cell lung cancer cell H1299 and human non-small cell lung cancer cell A549 with different concentrations of cisplatin and etomoxir respectively,through CCK8 experiment,the effects of various drugs on the proliferation inhibition of H1299 cells and A549 cells were examined,and the working concentration of Cisplatin and Etomoxir in subsequent experiments was determined.Then CCK8 test was used to test different drug treatment methods.That is,cisplatin alone,etomoxir alone and Cisplatin in combination with Etomoxir are used to treat human non-small cell lung cancer cell H 1299 and human non-small cell lung cancer cell A549 respectively,studied and analyzed Inhibition of tumor cell proliferation by various drug treatments.Furthermore,we performed Annexin V/PI staining on the non-small cell lung cancer A549 cell line,used flow cytometry to detect the apoptosis of lung cancer A549 cell line after treatment with cisplatin alone,Etomoxir alone,and cisplatin combined with Etomoxir treatment;Simultaneously,the cell migration of lung cancer A549 cell lines treated with the above three drug treatment methods was tested by transwell assay,understand the effect of drug treatment on the migration of A549 cells.In addition,in this experiment,to explore the effect of drug treatment on the expression of sternness related genes in non-small cell lung cancer cell A549,we performed protein extraction on lung cancer A549 cells treated with Cisplatin alone,Etomoxir alone,and Cisplatin combined with Etomoxir,and used Western Blot to observe the expression of sternness genes,ie,Oct4,Sox2 and Nanog in protein levels A549 cells.Finally,in the animal model of subcutaneous tumorigenesis of nude mice,different drug treatments were tested.That is,the effect of intraperitoneal injection of cisplatin alone,intraperitoneal injection of Etomoxir alone,and intraperitoneal injection of Etomoxir and cisplatin on the growth and lung metastasis of lung cancer in mice.On the other hand,we established a H1299 cell line targeted for knockout CPT1A,used by CRISPR/Cas9 technology.We first designed the sgRNA targeting the human CPT1A gene and cloned it into the CRISPR/Cas9 vector PX335 plasmid or PLenti-Guide-puro plasmid through the website http://crispr.mit.edu/.Subsequently,after the above-mentioned plasmid was transfected into human non-small cell lung cancer H1299 cells,the H1299 cell knockout mixed clone of the CPT1A gene was knocked out,and the genomic DNA of the H1299 cell mixed clone was extracted.Then,through-surveyor method,use T7E1 enzyme detects the sgRNA cleavage activity of the above CRISPR/Cas9 system.Use Western Blot method to detect CPT1A protein and detect knockout efficiency.The results we have obtained are as follows1.The proliferation of human non-small cell lung cancer A549 cells was inhibited after treated with CPT1 inhibitor Etomoxir;the proliferation of human non-small cell lung cancer A549 cells was inhibited after treatment with cisplatin;the proliferation of human non-small cell lung cancer H1299 cells was inhibited after treatment with Ctomotaxin Etomoxir;the proliferation of human non-small cell lung cancer H1299 cells was inhibited after treatment with cisplatin.2.The growth of lung cancer cell A549 was significantly inhibited after treated by Cisplatin combining with Etomoxir;The growth of lung cancer cell H1299 was significantly inhibited after treated by Cisplatin combining with Etomoxir.3.The apoptosis of lung cancer cell line A549 was significantly increased after treated by Cisplatin combining with Etomoxir.4.The migration of A549 lung cancer cells was significantly inhibited after treated by Cisplatin combining with Etomoxir.5.After treatment with Etomoxir in lung cancer cell A549,the expression of Oct4,Sox2 and Nanog protein was significantly inhibited;after the combination of Cisplatin and Etomoxir or Cisplatin alone treatment the expression of Oct4,Sox2 and Nanog did not change.6.After Cisplatin combined with Etomoxir treatment,the growth of lung cancer tumors in the subcutaneous tumorigenesis model of nude mice was significantly inhibited.7.Through the two-plasmid system of CRISPR/Cas9 technology,the CPT1A gene of non-small cell lung cancer cell H1299 cells was stabilized knockout,and gene editing efficiency was higher.In conclusion,(1)The CPT1 inhibitor Etomoxir promotes the sensitivity of tumor cells to the chemotherapeutic drug cisplatin,which lead tumor cell proliferation and migration are inhibited and apoptosis is enhanced,and lung cancer tumor growth was inhibited in a subcutaneous tumor model of lung cancer in nude mice;Meanwhile,using Etomoxir can reduce the expression of sternness gene;(2)CPT1A knockout lung cancer cells are created using CRISPR/Cas9 double knockout plasmid which to lay an experimental foundation for subsequent gene therapy research.