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Objective:To investigate the effect of S-phase kinase-associated protein 2 (SKP2) expression level on radiosensitivity of human hepatocellular carcinoma (HCC) cells and the correlation of SKP2 expression with clinical prognosis of patients with HCC.Methods:The expression levels of SKP2 gene in liver cancer tissues and normal tissues were validated and its correlation with clinical prognosis of HCC patients was analyzed based on the TCGA database. Western blot was used to determine the SKP2 protein levels in HCC cell lines before and after radiation. CRISPR/Cas9 technology was employed to delete the promoter and first exon of SKP2 gene in PLC/PRF/5 (PLC) and Hep3B HCC cells for generating the SKP2 knockout cell lines. The difference of radiosensitivity and cell survival rate between normal (SKP2 +/ +) and SKP2 knockout (SKP2 -/ -) HCC cells was determined by using cell clonogenic assay and CCK8 kit. Results:Compared with normal tissues, the expression levels of SKP2 gene in HCC were increased based on the results of TCGA database analysis. K-M analysis showed that the HCC patients with high SKP2 expression had relatively poor prognosis. The 5-year overall survival (OS) was 34.6% in high SKP2 expression HCC patients and 50.6% in low SKP2 expression HCC patients, respectively ( HR=2.18, 95% CI=1.46-3.27, P<0.001). In vitro experiment showed that the expression levels of SKP2 were significantly increased after radiation in HCC cells. Simultaneously, deletion of SKP2 significantly increased the radiosensitivity of HCC cells. Conclusion:The expression level of SKP2 gene is increased in HCC patients, and patients with high SKP2 expression have worse prognosis than those with low expression. Radiation can upregulate the SKP2 expression levels in HCC cells, while the radiosensitivity of the cells is significantly increased after SKP2 deletion.
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Objective To evaluate the tolerance of preoperative neoadjuvant chemoradiotherapy (neoCRT) plus esophagectomy, as well as the short-term outcome, tumor resection rate, incidence of postoperative complications, and perioperative mortality, in patients with locally advanced esophageal cancer.Methods This study included 74 patients with thoracic esophageal cancer who were admitted to our hospital from May 2011 to June 2015.Chemotherapy and radiotherapy were performed concurrently.The chemotherapy consisted of vinorelbine (25 mg/m2 on days 1, 8, 22, and 29) and cisplatin (25 mg/m2 on days 1-4 and 22-25).The radiotherapy was conventionally fractionated with a total dose of 40 Gy (2.0 Gy/d).At 4-8 weeks after chemoradiotherapy, esophagectomy was performed (neoCRT+surgery group);definitive chemoradiotherapy (DCRT) was performed in the patients who refused surgery (DCRT group);follow-up was performed in the patients who refused any anti-cancer therapies after neoCRT (neoCRT group).Results Forty-four patients underwent neoCRT+surgery, with a radical resection (R0) rate of 100% and a pathological complete response (pCR) rate of 43%;17 patients received DCRT;13 patients received neoCRT alone.For the neoCRT+surgery group, DCRT group, and neoCRT group, the 2-year overall survival (OS) rates were 79%, 75%, and 17%, respectively, and the 2-year disease-free survival (DSF) rates were 75%, 55%, and 17%, respectively.There were significant differences in OS between the neoCRT group and the neoCRT+surgery group (P=0.000) and between the neoCRT group and the DCRT group (P=0.001), but no significant difference was observed between the neoCRT+surgery group and the DCRT group (P=0.415).There were significant differences in DFS between the neoCRT group and the neoCRT+surgery group (P=0.000) and between the neoCRT group and the DCRT group (P=0.002), but no significant difference was observed between the neoCRT+surgery group and the DCRT group (P=0.416).The rate of clinical response to preoperative neoCRT was 87% for all patients.Fifty-six patients (76%) developed grade ≥3 myelosuppression due to preoperative neoCRT.The incidence rates of postoperative pulmonary infection, anastomotic leakage, and anastomotic stenosis were 21%, 12%, and 7%, respectively, and the perioperative mortality rate was 2%.Conclusions For patients with locally advanced esophageal cancer, preoperative neoCRT plus surgery can increase the clinical response rate and pCR rate, reduce the tumor stage, and improve the survival, but chemoradiotherapy toxicities and perioperative complications cannot be ignored.
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To investigate the effect of recombinant human growth hormone (rhGH) on JAK2-STAT3 pathway and the growth of gastric cancer cell lines at different GHR expression status, the eukaryotic expression vector targeting human GHR (pGPU6/GFP/Neo-shGHR and pGPU6/GFP/Neo-scramble) was constructed and transfected into MGC803 cells by Lipofectamine 2000. Stable expressive cell lines were obtained by G418 screening. The expression of GHR was analyzed by Western blotting. After being stimulated with rhGH, cell growth was detected by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. The components of JAK2/STAT3 signaling pathway were detected by Western blotting. There is no significant difference of GHR expression between MGC803 and pGPU6/GFP/Neo-scramble-transfected cells (named as MGC803-NC) (P > 0.05). Compared with MGC803, the GHR expression in pGPU6/GFP/Neo-shGHR-transfected cells (named as MGC803-shGHR) decreased significantly (protein decreased 50%). The cells were treated with rhGH at 0, 150 and 300 ng x mL(-1), the growth rate of MGC803 and MGC803-NC increased significantly, PI and the number of G2/M phase cells all increased significantly, and apoptosis decreased significantly. Western blotting revealed that the expression of pJAK2 and pSTAT3 was up-regulated after being treated with rhGH in MGC803 and MGC803-NC cells. In contrast, similar change was not observed in MGC803-shGHR cells. Knockdown of GHR gene may decrease the sensitivity of gastric cancer cells to rhGH, and down-regulating of components of the expression of JAK2/STAT3 signaling pathway may be the potential mechanisms.
Subject(s)
Humans , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Human Growth Hormone , Genetics , Pharmacology , Janus Kinase 2 , Metabolism , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , Receptors, Somatotropin , Genetics , Metabolism , Recombinant Proteins , Genetics , Pharmacology , STAT3 Transcription Factor , Metabolism , Signal Transduction , Stomach Neoplasms , Metabolism , Pathology , TransfectionABSTRACT
To investigate the effect of recombinant human growth hormone (rhGH) on JAK2-STAT3 pathway and the growth of gastric cancer cell lines at different GHR expression status, the eukaryotic expression vector targeting human GHR (pGPU6/GFP/Neo-shGHR and pGPU6/GFP/Neo-scramble) was constructed and transfected into MGC803 cells by Lipofectamine 2000. Stable expressive cell lines were obtained by G418 screening. The expression of GHR was analyzed by Western blotting. After being stimulated with rhGH, cell growth was detected by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. The components of JAK2/STAT3 signaling pathway were detected by Western blotting. There is no significant difference of GHR expression between MGC803 and pGPU6/GFP/Neo-scramble-transfected cells (named as MGC803-NC) (P > 0.05). Compared with MGC803, the GHR expression in pGPU6/GFP/Neo-shGHR-transfected cells (named as MGC803-shGHR) decreased significantly (protein decreased 50%). The cells were treated with rhGH at 0, 150 and 300 ng x mL(-1), the growth rate of MGC803 and MGC803-NC increased significantly, PI and the number of G2/M phase cells all increased significantly, and apoptosis decreased significantly. Western blotting revealed that the expression of pJAK2 and pSTAT3 was up-regulated after being treated with rhGH in MGC803 and MGC803-NC cells. In contrast, similar change was not observed in MGC803-shGHR cells. Knockdown of GHR gene may decrease the sensitivity of gastric cancer cells to rhGH, and down-regulating of components of the expression of JAK2/STAT3 signaling pathway may be the potential mechanisms.
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<p><b>OBJECTIVE</b>To investigate the role of methylation on E-cadherin inactivation in nasopharyngeal carcinoma (NPC) cell line HNE1 and CNE2, as well as evaluate the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-dC) on cell abilities of proliferation and invasion.</p><p><b>METHODS</b>The expression level of E-cadherin was measured by RT-PCR, Western blot and immunohistochemistry (polymer method), the methyaltion status was analyzed by methylation-specific PCR (MSP), and cell proliferation and invasion were examined by MTT and invasion assay, separately before and after treatment with demethylating agent 5-Aza-dC.</p><p><b>RESULTS</b>The expression level of E-cadherin was down-regulated compared with the normal tissue, simultaneously partially methylated in gene promoter. Treatment with 20 µmol/L 5-Aza-dC increased the expression of E-cadherin and reduced the methylation degree. Moreover, it also significantly suppressed cell growth (27.6% for HNE1 cells and 34.3% for CNE2 cells, P < 0.05) and invasiveness (37.2% for HNE1 cells and 29.7% for CNE2 cells, P < 0.05).</p><p><b>CONCLUSIONS</b>Aberrant methylation around gene promoter region may play an important part in down regulation of E-cadherin in NPC, suggesting a potential therapeutic strategy for demethylating agents such as 5-Aza-dC.</p>