| Head and neck cancer is one of the six most common malignancy worldwidely, in which human tongue squamous cell carcinoma (TSCC) is the most common histological type. Most advanced squamous cell carcinoma showed infiltrative growth and accompaning cervical lymph node metastasis, whith is not suitable for sugery threatment. Cis-platinum (DDP) is widely used in the treatment of human tongue squamous cell carcinoma for chemotherapy, but adverse reactions and chemodrug resistance is major obstacle to clinical usage. Thus, reversing its chemodrug resistance should be the most important issue for tongue cancer resaerch.STAT3was over-expressed in heptocelluar carcinoma, breast cancer, lung cancer, colon cancer, head and neck squamous cell carcinoma and other epithelial-origined cancers, and participate in a variety of cell transcription of proliferation and apoptosis related genes, regulation of invasion and metastasis, and etc. It was found that STAT3could regulate the transcription of miR-21directly in multiple myeloma and glioma. TCA8113and TCA8113/DDP human tongue squamous cell carcinoma cell lines were chosen, WP1066, the functional inhibitor of STAT3, was used to suppress the STAT3phosphorylation. DDP is the chemotherapy drug in the clinical use we chose. We investigate the effects and mechanisms WP1066combined with DDP treating human tongue squamous cell carcinoma cells in vitro and in vivo. Research is divided into three parts:Part Ⅰ:The half maximal inhibitory concentration (IC50value) of WP1066and DDP in the two cell lines was determined and the expression level of STAT3and p-STAT3was examined. MTT assay and plate clone formation assay were performed to measure the effect of WP1066and DDP on cell proliferation. Annexin V/PI staining was performed and apoptosis was evaluated by flow cytometry analysis. Transwell and3D Matrigel matrix assay were conducted to measure invasiveness. The migration of TCA8113and TCA8113/DDP was measured with scratch assay.Part Ⅱ:Real-time PCR and fluorescence in situ hybridization (FISH) were deployed to detect the mirR-21expression, the expressions of PTEN, PDCD4and TIMP3were detected at mRNA and protein level.CHIP and Luciferase reporter gene assay was conducted to detect the relationship between STAT-3and miR-21.Part III:Tumor-bearing nude mouse models were established by subcutaneous implantation of TCA8113/DDP cells into25mice, and were randomly divided into groups. After two weeks, each group received corresponding treatment every3days for21days. Mice were sacrificed after treatment, and tumor volumes were compared among groups. Immunohistochemistry (IHC) was performed to detect expressions of p-STAT3, PTEN, PDCD4, TIMP3, mTOR, Ki-67, Caspase-3, Bcl-2and MMP2/9. FISH on paraffin embedded section was used to detect the miR-21expression. Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) assay was applied to investigate active cell death (apoptosis).Results:The ICsos to WP1066and DDP in TCA8113cell were3.1μmol/L and5.4μmol/L, while3.5μmol/L and45.1μmol/L for TCA8113/DDP cell respectively. The expression of p-STAT3protein was significantly suppressed by WP1066, instead of DDP. WP1066or DDP could inhibit tumor cell’s invasion, migration and proliferation capability, while in combination the effect were remarkably enhanced. WP1066or DDP caused a significantly increased amount of apoptotic death. Bcl-2, mTOR, Ki-67, MMP-2/9were reduced by treating with WP1066, while Caspase-3was increased, while DDP-treated group remained unchanged. The miR-21level was down-regulated by WP1066, instead of DDP. No statisitical difference of PTEN, PDCD4and TIMP3were validated at mRNA level, while at protein level their expressions were obviously elevated by WP1066. A luciferase reporter gene assay and CHIP assay both indicated that STAT3could modulate mirR-21directly. The xenograft with WP1066or DDP treating showed a decreased growth rate, and more significantly in combination. The expression of miR-21was also deregulated by WP1066in vivo. There was high apoptosis rate in WP1066or DDP groups, higher rate in WP1066+DDP group. The expressions of STAT3/p-STAT3were declined by WP1066, as well as Bcl-2, mTOR, Ki-67and MMP2/9, while Caspase3, PTEN, PDCD4and TIMP3were elevated. The in vivo results were equal to the in vitro ones. Conclusions:1. STAT3/miR-21axis was overexpressed in DDP-resistant human tongue squamous cell carcinoma and cell lines.2. WP1066treatment inhibited STAT3phosphorylation, whitch led to miR-21transcriptional repression, thus miR-21’s targets (PTEN, PDCD4, TIMP3) were suppressed at protein level. Accordingly, WP1066treatment cells showed attenuated proliferation, invasion and migration ability, and induced cancer cell apoptosis.3. The effect of combined-therapy (WP1066and DDP) in human tongue squamous cell carcinoma cells and animal model was much more enhanced than a single process (WP1066or DDP) significantly. STAT3can serve as a target with therapeutic potential, because its inhibitor (WP1066) could reverse DDP chemoresistance in tongue squamous cell carcinoma. |
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