Shikonin Induces Mitochondria-mediated Apoptosis And Enhances Chemotherapeutic Sensitivity In Gastric Cancer

BackgroundGastric cancer is one of the most common digestive system neoplasms, which ranks the fifth in the incidence of malignant tumors and the third in the tumor related mortality. The new cases and deaths of China account nearly half of the world. In China, 80% gastric cancer patients are diagnosed in the advanced stages and the 5-year overall survival of the total gastric cancer population is less than 30%. The high level incidence, late stage, high heterogeneity and limited comprehensive treatments lead poor overall survival of gastric cancer patients, which makes great importance to discover and develop novel anti-tumor agents. The traditional Chinese medicine is a potent source for discovering novel anti-tumor agents. Shikonin, a natural naphthoquinone, has been reported to have anti-tumor effects in some tumors. However, the anti-tumor efficiency and mechanism of shikonin in gastric cancer are still unclear.ObjectiveTo analyze the anti-tumor efficiency of shikonin in gastric cancer and preliminarily investigate the anti-tumor mechanism. To explore the potential inhibition effect of shikonin in different gastric cancer cell lines. The apoptosis mechanisms induced by shikonin in gastric cancer were studied and the Caspase-dependent and -independent pathways in regulating apoptosis were further investigated. The role of RIPK1 in regulating shikonin induced apoptosis was detected and the upstream role of ROS in mitochondria-mediated apoptosis was measured. To analyze the chemotherapy sensitization of shikonin in gastric cancer. We established the xenograft models to further explore the effects of shikonin-induced apoptosis and chemotherapy sensitization.MethodsMTT assays were performed for detecting cell viability and Annexin V/PI staining was performed to analyze the type and proportion of cell death. Western blot analysis was used for detection of the activity of apoptosis related proteins. DCFH-DA and JC-1 probes were used to measure the level of intracellular ROS and the mitochondrial membrane potential.The caspases inhibitors, RIPK1 inhibitor and ROS scavengers were explored to measure apoptosis related effects of the key protein blockage. SiRNA, lentivirus and plasmid were used to down or over express the key apoptosis related proteins, in order to detect their roles in shikonin induced apoptosis in gastric cancer. Isolation and extraction of cytoplasmic and nuclear proteins, mitochondrial protein extraction and immunofluorescence localization were applied to detecting the translocation of apoptosis related proteins. Subcutaneous xenograft animal models were established and the TUNEL staining and MDA detection were used to analyze the proportion of cell apoptosis and intracellular ROS in vivo.Results(1) Shikonin suppresses proliferation and triggers cell death of gastric cancer cells but leads minor damage to gastric epithelial cells. The increasing concentrations of SHK caused S-phase cell cycle arrest in gastric cancer cells. (2) We stained nuclei to detect karyopyknosis,chromatin condensation or nuclear fragmentation characteristic of apoptosis. We confirmed that SHK induces apoptosis of gastric cancer cells not only in a caspase-dependent manner which induced Caspase cleavage and triggered the caspase cascade, but also in a caspase-independent manner which mediated the nuclear translocation of apoptosis-inducing factor and Endonuclease G. (3) RIPK1 promotes survival of gastric cancer cells in shikonin-induced apoptosis. Shikonin induced the generation of intracellular ROS which played an upstream role. Shikonin depolarized the mitochondrial membrane potential and ultimately triggered mitochondria-mediated apoptosis through ROS. (4) We demonstrated that SHK enhanced the chemotherapeutic sensitivity of 5-fluorouracil and oxaliplatin, which was related to ROS accumulation and mitochondrial membrane potential depolarization. (5) In xenograft animal models, we found that shikonin inhibited the growth of xenografts and confirmed that shikonin also induced apoptosis, increased ROS accumulation and enhanced the chemotherapeutic sensitivity in vivo.ConclusionIn vitro, shikonin induces Caspase-dependent and -independent apoptosis and enhances the chemotherapeutic sensitivity in gastric cancer and ROS plays an upstream role in regulating shikonin-induced apoptosis. Meanwhile, we demonstrate that shikonin also induces apoptosis and enhances the chemotherapeutic sensitivity in gastric cancer in vivo.Our studies suggest that shikonin is a potent anti-tumor agent in gastric cancer, which lays the groundwork for future clinical studies.