Caudatin Induces Autophagy Via The ROS/JNK Signaling Pathway In Testicular Cancer

Objective:With the gradual development of traditional Chinese medicine research,the integrated treatment method combining traditional Chinese medicine and western medicine has achieved good efficacy,and has become the preferred treatment for a variety of tumors.Caudatin is a C21 steroidal aglycon extracted from the rhizomes of Cynanchum auriculatum Royle ex Wight(Cynanchum bungei),which has a long history of medicinal use in China and a wide range of pharmacological effects.Clinical practice and experimental studies have found that caudatin plays an important role in anti-inflammation,anti-oxidation,anti-tumor,anti-mutation and other aspects.In this study,NCCIT and Tcam-2 were used as the research objects to explore the occurrence and m olecular mechanism of caudatin-induced autophagy in testicular cancer,so as to provide experimental basis for further study on the anti-tumor effect of caudatin.Methods:In this paper,the NCCIT and Tcam-2 were treated with caudatin at gradient concentration,and effects of caudatin on the proliferation of testicular cancer cells were studied by MTT and clone forming experiments;Apoptosis was detected by hoechst 33258 staining;GFP-LC3 puncta assay was used to detect the autophagy;The levels of reactive oxygen species(ROS)were determined by 2’,7’-dichlorofluorescin diacetate(DCFH-DA)staining.Results:The experimental results showed that compared with the control group,the testicular cancer cells were treated with caudatin at a concentration gradient of 0～75 μM for 0～72h,respectively,caudatin could significantly inhibit testicular cancer cell proliferation,and observed time and concentration gradient dependence.Hoechst 33258 staining assay was used to detect apoptosis,which showed that caudatin induced apoptotic bodies in testicular cancer cells,and apoptosis occurred.It was found that treatment of testicular cancer cells with gradient concentration of GFP-LC3 could significantly increase the production of autophagosomes.Caudatin promoted the production of reactive oxygen species(ROS)in testicular cancer cells by 2’,7’-Dichlorodihydrofluorescein diacetate(DCFH-DA)staining.Western blot results showed that caudatin could promote the expression of autophagy proteins such as beclin1,LC3-Ⅱ and JNK signaling pathway protein such as p-JNK,p-ERK,p-p38 and so on,and the promotion effect of caudatin on autophagy-related proteins LC3-II and Beclin1 was weakened in testicular cancer cells under the action of SP600125(JNK inhibitor)or N-acetylcysteine(NAC,ROS inhibitor).Conclusion:Caudatin can inhibit the proliferation and migration of testicular cancer cells,promote their apoptosis,and induce the occurrence of autophagy in testicular cancer cells by activating the ROS/JNK signaling pathway.