Connexin 32 downregulation is critical for chemoresistance in oxaliplatin-resistant HCC cells associated with EMT

Cancer Management and Research Dovepress open access to scientific and medical research Cancer Management and Research downloaded from https://www.dovepress.com/ by 88.198.20.149 on 26-Sep-2019 For personal use only. Open Access Full Text Article Connexin 32 downregulation is critical for chemoresistance in oxaliplatin-resistant HCC cells associated with EMT This article was published in the following Dove Press journal: Cancer Management and Research Yan Yang 1 Jing-Hao Yao 1 Qian-Yu Du 1 Yong-Chun Zhou 2 Ting-Jing Yao 3 Qiong Wu 4 Jing Liu 1 Yu-Rong Ou 4 1 Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, People’s Republic of China; 2Department of Radiation Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, People’s Republic of China; 3Department of Surgical Oncology, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, People’s Republic of China; 4Department of Pathology, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, People’s Republic of China Correspondence: Yan Yang Department of Medical Oncology, The First Affiliated Hospital of Bengbu Medical College, No. 287 Changhuai Road, Longzihu District, Bengbu, Anhui 233004, People’s Republic of China Tel +86 552 307 4480 Fax +86 552 307 4480 Email Yu-Rong Ou Department of Pathology, The First Affiliated Hospital of Bengbu Medical College, No. 287 Changhuai Road, Longzihu District, Bengbu, Anhui 233004, People’s Republic of China Tel +86 552 307 0209 Fax +86 552 307 0209 Email Background: Oxaliplatin (OXA)-based chemotherapy is critical in the management of advanced hepatocellular carcinoma (HCC); however, acquired drug resistance has largely restricted its clinical efficacy. This study aims to explore the key mechanisms and regulatory factors determining chemosensitivity in HCC. Methods: We developed OXA-resistant (OR) HCC cells and used multiple methods, including real-time RT-PCR, Western blot, immunofluorescence, transwell invasion assay, wound-healing assay, MTT assay, gene transfection, and immunohistochemistry to achieve our goals. Results: We found that OR HCC cells showed a typical epithelial–mesenchymal transition (EMT) phenotype. Meanwhile, the expression of Cx32, a major member of the liver connexin (Cx) family, was lowly expressed in OR HCC cells. Downregulation of Cx32 in parental HCC cells led to EMT induction and thereby reduced OXA cytotoxicity, while Cx32 upregulation in OR HCC cells could reverse the EMT phenotype and partially restore chemosensitivity to OXA. Finally, in human HCC tissue samples, Cx32 was positively correlated with the expression of the EMT marker E-cadherin and negatively correlated with the expression of Vimentin. Conclusion: Our findings demonstrated that downregulation of Cx32 may be an important determinant for HCC cells to acquire EMT-related acquired drug resistance to OXA, and targeting Cx32 could be a novel strategy to overcome OXA resistance in HCC. Keywords: hepatocellular carcinoma, oxaliplatin chemoresistance, connexin32, epithelialmesenchymal transition Introduction Hepatocellular carcinoma (HCC) is the fourth common malignancy worldwide,1 with characteristics of high aggressivity and poor prognosis. Hepatocarcinogenesis is concealed, and early diagnosis of HCC is difficult. Most HCC patients cannot undergo curative surgery due to locally extensive invasion or distant metastasis at the time of diagnosis. In recent years, targeted therapy and immunotherapy have improved the prognosis of patients with advanced HCC.2 However, their clinical benefits remain moderate, especially with the lack of effective predictive indicators, resulting in unsatisfactory efficacy in clinical practice. With the application of third-generation cytotoxic drugs, systemic chemotherapy is making a breakthrough in the treatment of advanced HCC. Specifically, oxaliplatin (OXA)-based systemic chemotherapy has become one of the most important options for advanced HCC.3,4 However, patients 5133 submit your manuscript | www.dovepress.com Cancer Management and Research 2019:11 5133–5146 DovePress © 2019 Yang et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). http://doi.org/10.2147/CMAR.S203656 Powered by TCPDF (www.tcpdf.org) ORIGINAL RESEARCH Dovepress Cancer Management and Research downloaded from https://www.dovepress.com/ by 88.198.20.149 on 26-Sep-2019 For personal use only. Yang et al using platinum drugs often suffer from drug resistance during the clinical application,5 and the mechanisms underlying drug resistance remain largely unknown. Epithelial–mesenchymal transition (EMT) refers to the process by which epithelial cells switch to mesenchymal cells through specific processes and is associated with chemoresistance, including to platinum drugs.6 Studies have revealed that cancer cells acquire the mesenchymal phenotype in the process of generating acquired drug resistance, and tumor cells in the mesenchymal differentiation state often exhibit the characteristics of primary drug resistance.7 Our previous study confirmed phenotypic changes consistent with the EMT phenotype in gemcitabine (GEM)-resistant HCC cells.8 It is generally recognized that during EMT, epithelial cells lose epithelial properties and acquire the characteristics of mesenchymal cells, resulting in decreased intercellular adhesion and increased cell motility and invasion capacities. At the molecular level, cells lowly express or lose epithelial markers such as E-cadherin and highly express mesenchymal molecular markers, including Vimentin and N-cadherin, with upregulation of transcription factors such as Snail, Slug, and Twist.9 Connexin (Cx) is the basic unit of gap junction (GJ), which communicates the cytoplasm of two adjacent cells by directly mediating the transmission of electrical, chemical, and metabolic substances, thereby playing important roles in a series of life events, including cellular activity synchronization, tissue homeostasis maintenance, and cell proliferation and apoptosis.10 Abnormal Cx and GJ levels are closely related to the occurrence and development of various tumors, including HCC.11 Cx32, forming 90% of hepatic GJs, is the major Cx isoform expressed in the liver.12 This gene was originally considered a tumorsuppressor gene13,14 and has been demonstrated to be implicated in multiple hepatocellula (...truncated)