The Experimental Study Of The Proteasome Inhibitor MG-132 On Human Laryngeal Squamous Cell Carcinoma Cell Line Hep-2

Laryngeal carcinoma is a common malignant tumor of the head and neck. Recently, the incidence and mortality of laryngeal carcinoma are increasing gradually and threatens human being's health and life seriously. Although the comprehensive treatment options including surgery, radiation therapy and chemotherapy have improved the 5-year survival rate in recent decades, the prognosis for patients with advanced, relapsed or metastatic disease is poor. Moreover, traditional radiation therapy and chemotherapy are associated with considerable adverse toxicities that can not be tolerated by patients. The effective treatment has been extremely limited. Therefore, to seek novel and valid therapeutic strategies for laryngeal cancer becomes a new challenge of the head and neck researchers.The ubiquitin-proteasome pathway (UPP) is the major system responsible for degradation of intracellular proteins in eukaryotes. By controlling the levels of key proteins, it plays an essential role in multiple cellular processes, including cell cycle progression, proliferation, apoptosis, signal transduction and transcription. It is closely related to human malignancies. Blockade of UPP creates an imbalance of various regulatory proteins that control the cell function of proliferation, differentiation and apoptosis, which induces the apoptotic pathways within the cell, indicating that the proteasome inhibitor could be used as an attractive novel anticancer drug. Bortezomib (Velcade, PS-341) was the first proteasome inhibitor to receive approval from the US Food and Drug Administration for the treatment of relapsed or refractory multiple myeloma and mantle cell lymphoma. In lung cancer and lymphoma, Clinical trials with bortezomib alone or in combination are ongoing. But little has been reported of the effect of the proteasome inhibitor on laryngeal cancer cells, and the molecular mechanism is unclear so far. Signal Transducer and Activator of Transcription-3 (STAT3) is an important factor of signal transduction pathway, and is the focus of EGFR, IL-6/JAK, Src and other tyrosine kinase signal carcinogenicity channel convergents. It is constantly activated in great quantities in human tumor cells and links closly with the occurrence, development and evolution of human malignant tumor. It has been reported that the proteasome inhibitor can activate STAT3 in colorectal cancer cells. There has been no report about the effects of the proteasome inhibitor on the STAT3 protein expression in laryngeal carcinoma by now. Our previous studies have shown that the activation of STAT3 is closely related to the occurrence and development of laryngeal cancer and the suppression factor can inhibit the malignant proliferation and induced the apoptosis of cancer cells. In this study, we used human laryngeal squamous carcinoma cell line Hep-2 cultured in vitro, and MTT, Flow cytometry (FCM), Western Blot, gene transfer techniques to explore the role and the molecular mechanisms of the proteasome inhibitor MG-132. The effect of MG-132 on STAT3 was also investigated. Further more, MG-132 was combined with pshSTAT3 or cisplatin, a conventional chemotherapy drug, to observe the changes of the proliferation and apoptosis in cells. This may be helpful to find more effective molecular targets, multi-target combination therapy strategies, as well as the clinical treatment options of laryngeal cancer. This experiment scheme was separated into three parts:Part one The proteasome inhibitor MG-132 induced apoptosis and its effect on STAT3 in laryngeal carcinoma cell line Hep-2Objective: By testing the effects of MG-132 on proliferation, cell cycle distribution, apoptosis and related protein expression at different times and concentrations, we explored the role, the molecular mechanism or the effect of the proteasome inhibitor on STAT3 in human laryngeal squamous cell carcinoma cell line Hep-2 cells.Methods:1. Hep-2 cells were treated with different doses (control, 1, 2.5, 5, 10, 20μmol/L) of MG-132 for 24h or 48h. In addition, Hep-2 cells were treated with 2.5μmol/L MG-132 for varying lengths of time (control, 12h, 24h, 48h, 72h). After treatment, the effect of growth suppression on cells was evaluated with MTT assay.2. Hep-2 cells were treated with varying doses (control, 1, 2.5, 5μmol/L) of MG-132 for 48h, FCM was used to detect cell apoptosis. In addition, the cells were treated with 2.5μmol/L MG-132 for varying lengths of time (control, 12h, 24h, 48h or 72h). After treatment, cell apoptosis and cell cycle were detected by FCM.3. Western blotting was used for detecting the protein expression level of STAT3, p-STAT3, Bcl-2, p21, cyclinD1, CDK4 in cells at control, 12h, 24h, 48h after treatment.Results:1. MTT assays showed that MG-132 can effectively inhibit the proliferation of Hep-2 cells in a dose- (24h:r=0.925,P<0.01;48h:r=0.944,P<0.01) and time-dependent manner(r=0.945,P<0.01). The 50% inhibition concentration (IC50) at 24h is 20μmol/L, and at 48h is 2.5μmol/L.2. FCM demonstrated that apoptosis rate increased with the increasing of concentration and the time, and in a concentration- (r=0.842,P<0.01) and time-dependent manner (r=0.888,P<0.01). Analysis of cell cycle showed that the percentage of cells in G0/G1 and G2/M phase were increased and decreased in S phase cells (P<0.01).3. Western blot showed that MG-132 caused marked up-regulation of p21 protein. Meanwhile, a moderate down-regulation of cyclinD1 was observed in Hep-2 cells. Cells were treated with MG-132 for 12h, resulting in significantly up-regulation of the p-STAT3 protein and maintained at a stable level. The Bcl-2 protein was also moderately up-regulated following MG-132 treatment. The expression levels of STAT3 and CDK4 were not appreciably changed.Conclusion:1. Proteasome inhibition by MG-132 decreases proliferation and increases apoptosis in human laryngeal squamous cell carcinoma cell line Hep-2. 2. Proteasome inhibition by MG-132 induces cells cycle arrest in G0/G1 and G2/M phase.3. The mechanism of MG-132-induced antitumor effects on Hep-2 cells involves in up-regulation of p21 and down-regulation of cyclinD1.4. Proteasome inhibition by MG-132 led to activate of the key signaling molecule STAT3 protein and up-regulation of the anti-apoptotic family member Bcl-2 protein, which might limit the efficacy of the anticancer agents. Part Two Antiproliferative and proapoptotic effects of combination of proteasome inhibitor with pshSTAT3 on laryngeal carcinoma cellsObjective: To explore if inhibition of activated STAT3 by transfecting plasmid expressing short hair pin RNA (shRNA) can enhance the antitumor effect of the protease inhibitor MG-132 on human laryngeal carcinoma cells.Methods:1. Recombinant plasmid expressing short hairpin RNA targeting STAT3 was constructed and identified by enzyme digestion and sequencing. 2. Experiments were divided into five groups: control group, pshNeg group, MG-132 group, combined group, pshSTAT3 group. The recombinant expression plasmid (pshSTAT3) and negative control plasmid (pshNeg) were transfected into Hep-2 cells by Lipofectamine 2000. After 6 hours, the medium was changed, and cells were observed under inverted fluorescence microscopy. Twenty-four hours after the transfection, cells were observed again. Subsequently, the cells were left untreated or treated with 2.5μmol/L MG-132 for 48h.3. After treatment, cell proliferative activity and cell apoptosis rate were detected in different groups by MTT assay and FCM.4. After treatment, the protein expression of p-STAT3 in all groups was detected by Western blot.results:1. The recombinant expression plasmid pshSTAT3 was constructed successfully and confirmed by restriction enzyme digestion and sequencing results.2. The observation of transfection efficiency after recombinant plasmid transfection: After transfection with pshSTAT3 and pshNeg for 6h, green fluorescence was observed in Hep-2 cells under fluorescence microscopy. Green fluorescence gradually increased and transfection efficiency was above 80% at 24 hour post-transfection, and the transfection efficiency was 85.76% by FCM.3. The pshSTAT3 enhanced the inhibition effect of MG-132 on proliferation in Hep-2 cells. After treatment, MTT assay showed that the proliferation ability of Hep-2 cells was not altered in control group and pshNeg group (P>0.05). The pshSTAT3 combined with 2.5μmol/L MG-132 significantly inhibited proliferation of Hep-2 cells compared with the other group (inhibition rate: 62.23±1.25%, P <0.01).4. The pshSTAT3 enhanced the proapoptotic effect in Hep-2 cells induced by MG-132. After treatment, FCM showed that apoptosis rate of the pshSTAT3 plus 2.5μmol/L MG-132 group (55.80±3.12%) was significantly higher than that of the MG-132 group (41.20±3.21%) or the pshSTAT3 group (22.13±1.84%) (P<0.01). There was no significant difference between the control group and the pshNeg group (P>0.05).5. Expression of p-STAT3 after treatment. Western blot showed that up-regulation of p-STAT3 protein expression was observed in the MG-132 group, with its down-regulation in the combined group and the pshSTAT3 group. The expression levels of p-STAT3 were not appreciably changed in the control group and the pshNeg group.Conclusion: The shRNA targeting STAT3 can inhibit MG-132-induced p-STAT3 protein expression up-regulation; thereby significantly enhance the antitumor effect of protease inhibitor MG-132 on human laryngeal carcinoma cells.Part Three Observation on the antiproliferative and proapoptotic effects of proteasome inhibitor MG-132 combined with chemotherapy in laryngeal carcinoma cells Objective: Cisplatin (DDP) is commonly used as a frontline chemotherapeutic agent. Our study is to determine if proteasome inhibitor MG-132 can enhance the cytotoxicity of DDP in human laryngeal squamous carcinoma cells.Methods: Hep-2 cells in logarithmic phase were divided into four groups: control group, MG-132 group, DDP group, MG-132+DDP group. After 48h, cell viability in each group was determined by MTT assay, and the apoptosis rate was detected by FCM.Results:1. Varying doses of DDP with or without MG-132 affected the proliferation of Hep-2 cells. The cells were treated with varying concentrations of DDP (10, 20, 40, 80μmol/L) with or without 1μmol/L MG-132 for 48h. MTT assay showed that the combination of MG-132 with DDP can significantly enhance the proliferation inhibition of the cells at the same dose level (P<0.01, P<0.05). The cellular proliferation of 10μmol/L DDP+MG-132 group was significantly lower than that of 20μmol/L DDP group (P<0.01). Similarly, there was a significant difference between 20μmol/L DDP+MG-132 group and 40μmol/L DDP (P<0.05). The IC50 value of DDP was 62.22μmol/L, and the value descended to 27.79μmol/L when combined with 1μmol/L MG-132.2. DDP plus MG-132 increased the apoptosis of Hep-2 cells. The cells were treated with 10μmol/L DDP alone and with 1μmol/L MG-132 for 48h. FCM results demonstrated that the combination of DDP plus MG-132 can markedly increased the apoptosis rate (P<0.01).Conclusion: Proteasome inhibitor can enhance the cytotoxicity of the chemotherapy drug cisplatin in Hep-2 cells. The combination of two drugs potentiated the anticancer effect on Hep-2 cells.