Yubo Tang1, Jie Chen2, Jiaqi Li2, Yifan Zheng2, Xiuxiu Zhong3, Shuai Huang4, Bin Chen5, Baogang Peng5, Xuenong Zou6, Xiao Chen7. 1. Department of Pharmacy, The First Affiliated Hospital, Sun Yat-sen University, 510080 Guangzhou, China. Electronic address: tangyb6@mail.sysu.edu.cn. 2. Department of Pharmacy, The First Affiliated Hospital, Sun Yat-sen University, 510080 Guangzhou, China. 3. Department of Pharmacy, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, 510623 Guangzhou, China. 4. Department of Orthopaedic Surgery, The Second Affiliated Hospital, Guangzhou Medical University, 510260 Guangzhou, China. 5. Department of Liver Surgery, The First Affiliated Hospital, Sun Yat-sen University, 510080 Guangzhou, China. 6. Guangdong Provincial Key Laboratory of Orthopedics and Traumatology, Department of Spinal Surgery, The First Affiliated Hospital, Sun Yat-sen University, 510080 Guangzhou, China. Electronic address: zouxuen@mail.sysu.edu.cn. 7. Department of Pharmacy, The First Affiliated Hospital, Sun Yat-sen University, 510080 Guangzhou, China. Electronic address: chenx5@mail.sysu.edu.cn.
Abstract
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. Sorafenib (SORA), as a first-line therapeutic drug, has been used to treat HCC, but resistance poses a major limitation on the efficacy of SORA chemotherapy. Pristimerin (PRIS), a natural bioactive component isolated from various plant species in the Celastraceae and Hippocrateaceae families, has been reported to exhibit outstanding antitumor effects in several types of cells in vitro. PURPOSE: The aim of this study was to investigate whether PRIS can exert synergistic anti-tumor effects with the combination of SORA, and if so, through what mechanism. METHODS: Conditionally reprogrammed patient derived-primary hepatocellular carcinoma cells (CRHCs) were isolated from human liver cancer tissues and treated with SORA and PRIS. Cell proliferation, apoptosis, migration and tube formation ability were detected by DNA content quantification, flow cytometry, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively. RESULTS: Initially, we observed that the combination of the two drugs had a much stronger inhibitory effect on CRHCs growth than either drug alone. Moreover, the combination of 2 µM SORA and 1 µM PRIS exhibited a significant anti‑migrative and anti-invaded effect on CRHCs, and remarkably inhibited capillary structure formation of Human Umbilical Vein Endothelial Cells (HUVECs). Furthermore, the combined treatment with SORA and PRIS synergistically induced intrinsic apoptosis in CRHCs, involving a caspase-4-dependent mechanism paralleled by an increased Bax/Bcl-xL ratio. These activities were mediated through ROS generation and the induction of endoplasmic reticulum (ER) stress and mitochondrial dysfunction. GRP78 silencing or ER stress inhibitor 4-phenylbutyric acid administration was revealed to abolish the anticancer effects of PRIS, indicating the critical role of GRP78 in mediating the bioactivity of PRIS. The present study also provides mechanistic evidence that PRIS modulated the Akt/FoxO1/p27kip1 signaling pathway, which is required for mitochondrial-mediated intrinsic apoptosis, activation of ER stress, and stimulation of caspase-4 induced by PRIS, and, consequently resulting in suppressed cell viability, migration and angiogenesis co-treated with SORA in CRHCs. CONCLUSION: Our results suggest the use of PRIS as sensitizers of chemotherapy paving the way for innovative and promising targeted chemotherapy-based therapeutic strategies in human HCC.
BACKGROUND:Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-associated mortality worldwide. Sorafenib (SORA), as a first-line therapeutic drug, has been used to treat HCC, but resistance poses a major limitation on the efficacy of SORA chemotherapy. Pristimerin (PRIS), a natural bioactive component isolated from various plant species in the Celastraceae and Hippocrateaceae families, has been reported to exhibit outstanding antitumor effects in several types of cells in vitro. PURPOSE: The aim of this study was to investigate whether PRIS can exert synergistic anti-tumor effects with the combination of SORA, and if so, through what mechanism. METHODS: Conditionally reprogrammed patient derived-primary hepatocellular carcinoma cells (CRHCs) were isolated from humanliver cancer tissues and treated with SORA and PRIS. Cell proliferation, apoptosis, migration and tube formation ability were detected by DNA content quantification, flow cytometry, transwell assay and Matrigel-based angiogenesis assay. Gene and protein expression were assessed by qRT-PCR and Western blot respectively. RESULTS: Initially, we observed that the combination of the two drugs had a much stronger inhibitory effect on CRHCs growth than either drug alone. Moreover, the combination of 2 µM SORA and 1 µM PRIS exhibited a significant anti‑migrative and anti-invaded effect on CRHCs, and remarkably inhibited capillary structure formation of HumanUmbilical Vein Endothelial Cells (HUVECs). Furthermore, the combined treatment with SORA and PRIS synergistically induced intrinsic apoptosis in CRHCs, involving a caspase-4-dependent mechanism paralleled by an increased Bax/Bcl-xL ratio. These activities were mediated through ROS generation and the induction of endoplasmic reticulum (ER) stress and mitochondrial dysfunction. GRP78 silencing or ER stress inhibitor 4-phenylbutyric acid administration was revealed to abolish the anticancer effects of PRIS, indicating the critical role of GRP78 in mediating the bioactivity of PRIS. The present study also provides mechanistic evidence that PRIS modulated the Akt/FoxO1/p27kip1 signaling pathway, which is required for mitochondrial-mediated intrinsic apoptosis, activation of ER stress, and stimulation of caspase-4 induced by PRIS, and, consequently resulting in suppressed cell viability, migration and angiogenesis co-treated with SORA in CRHCs. CONCLUSION: Our results suggest the use of PRIS as sensitizers of chemotherapy paving the way for innovative and promising targeted chemotherapy-based therapeutic strategies in human HCC.