Cancer Biology and Signal Transduction Doxycycline as an Inhibitor of the Epithelial-to-Mesenchymal Transition and Vasculogenic Mimicry in Hepatocellular Carcinoma

This study was conducted to examine the effects of doxycycline on the survival time and proliferation of hepatocellular carcinoma (HCC) in vivo and on the biologic functions of HCC in vitro. This study was also designed to evaluate the effects of doxycycline on epithelial-to-mesenchymal transition (EMT)– and vasculogenic mimicry (VM)–related protein expression and on matrix metalloproteinase (MMP) and DNA methyltransferase (DNMT) activity in vitro. Human MHCC97H cells were injected into BALB/c mice, which were divided into treatment and control groups. Doxycycline treatment prolonged the mouse survival time and partly suppressed the growth of engrafted HCC tumor cells, with an inhibition rate of 43.39%. Higher amounts of VM and endothelium-dependent vessels were found in the control group than the treatment group. IHC indicated that epithelial (E)-cadherin expression was increased in the doxycycline-treated mice compared with the control group. In in vitro experiments, doxycycline promoted HCC cell adhesion but inhibited HCC cell viability, proliferation, migration, and invasion. Western blot analysis, semiquantitative RT-PCR, qRT-PCR, and immunofluorescence demonstrated that doxycycline inhibited the degradation of the epithelial marker E-cadherin and downregulated the expression levels of EMT promoters, the mesenchymal marker vimentin, and the VM-associated marker vascular endothelial (VE)-cadherin. Furthermore, the activities of MMPs and DNMTs were examined in different groups via gelatin zymography and a DNMT activity assay kit. A methylation-specific PCR was performed to assess the promoter methylation of CDH1 (the gene encoding E-cadherin). Doxycycline prolonged the mouse survival time by inhibiting EMT progression and VM formation. Mol Cancer Ther; 13(12); 3107–22. 2014 AACR. Introduction Hepatocellular carcinoma (HCC) is one of the most common causes of cancermorbidity andmortality worldwide (1). Despite advancements in treatment options, the 5-year survival rate of patients with HCC has not improved. This negative result is primarily attributed to early-stage metastasis, which involves a series of rare, stochastic events. During the stages, the loss of epithelial (E)-cadherin expression and the subsequent loss of homotypic cellular adhesion may be a unique and important step that enables malignant epithelial cells to invade and metastasize normal cells. This mechanism characterizes a developmental regulatory program, the epithelial-tomesenchymal transition (EMT). HCC is a hypervascular solid tumor that primarily exhibits aberrant angiogenesis. Previous studies indicated that only endothelial cells could form blood vessels to support the growth of malignant tumors; however, recent studies have revealed several new patterns by which tumor tissues nourish themselves. These patterns include the formation of a pattern of mosaic vessels from endothelium and tumor cells (2) and the generation of channels lined exclusively with tumor cells. The latter is also known as vasculogenic mimicry (VM; ref. 3). The molecular mechanisms underlying VM remain unclear; nevertheless, tumor cells lining VM networks secrete matrix metalloproteinases (MMP) and express vascular endothelial (VE)-cadherin to induce extracellular matrix (ECM) remodeling (4, 5). Our previous studies also Department of Pathology, Tianjin Medical University, Tianjin, China. Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin, China. Department of Pathology, Tianjin General Hospital, Tianjin Medical University, Tianjin, China. Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). J. Meng, B. Sun, and Xiulan Zhao contributed equally to this article. Corresponding Author: Baocun Sun, Department of Pathology and Cancer Hospital and General Hospital of Tianjin Medical University, No. 22 Qixiangtai Road, Heping District, Tianjin 300070, China. Phone: 8613602111192; Fax: 86-22-83336813; E-mail: [email protected] or [email protected] doi: 10.1158/1535-7163.MCT-13-106