The Molecular Mechanisms Of Apoptosis Pathway Induced By Celastrol In Head And Neck Squamous Cell Carcinoma

Head and neck cancer is cancer that arises in the nasal cavity, oral cavity, larynx and the upper part of esophagus. The vast majority (more than90%) are head and neck squamous cell carcinomas (HNSCCs)(1,2). Approximately500,000new cases are reported annually around the world. In the United States, about30,000new cases of HNSCCs were diagnosed every year, accounting for3.2%of all incident malignancies (3,4). In China,15.22out of every100,000people develop HNSCCs per year, accounting for4.45%of all malignancies (5). The five-year survival rate for patients with HNSCCs is one of the lowest for any major cancer and has remained unimproved over the last20years. So far the molecular mechanism of occurrence and development of HNSCCs regulation is not very clear and effective treatments for HNSCCs are very limited (6). It is very important and necessary to look for a way to effectively inhibit HNSCCs.In eukaryotic cells, the endoplasmic reticulum (ER) serves many specialized functions as a plant for protein folding and posttranslational modification. Physiological and pathological stimuli that interfere with ER function subsequently cause accumulation of unfolded or misfolded proteins in the ER lumen. To deal with the accumulation of unfolded or misfolded proteins, the cell has evolved highly specific signaling pathways collectively called the "unfolded protein response"(UPR) to restore normal ER functions.The surface of the ER has three stress sensors, including inositol-requiring enzyme1(IRE1), activating transcription factor6(ATF6) and protein kinase RNA (PKR)-like ER kinase (PERK). However, if the overload of unfolded or misfolded proteins in the ER is not resolved, the prolonged UPR will induce ER stress. The ability of cells to respond to perturbations in ER function, or ’ER stress’, is critical for cell survival.It is widely accepted that the tumor cell is always under stress due to rapid proliferation that leads to a lack of blood supply in its microenvironment in most tumors. We hypothesized that due to the inherent ER stress in HNSCC cells if we were to add a small molecule to disrupt that ER stress leading to apoptosis to kill malignant cells and the healthy cells can mount an effective adaptive UPR to overcome the stress and return to homeostasis.Celastrol, which is a natural product that comes from the thunder of god vine in Chinese medicine, has antitumor activity. In this study, we investigated the molecular mechanism of celastrol anti-tumor activities in HNSCC.Experiment Methods1. Over-expression of Bip is the one biomarker of UPR in tumor tissue. Expression of Bip was detected in neck, laryngeal and lip tumor tissue and normal tissue by immunohistochemical assay.2. Cell viability was determined by ATP-glo luminescent viability assay.3. Solid tumor xenografts with cell lines transfected with UPR reporters were used to monitor UPR activation and tumor burded reduction in real time. Mice received daily oral celastrol and tumor volume was assessed weekly.4. Apoptosis genes were detected by qRT-PCR in HNSCC cells cells were challenged with celastrol. 5. Caspase activation was measured with luminescent Caspase-Glo(?)3/7assay system. The expression of Pro-Caspase9and cleaved Caspase3in celastrol-treated HNSCC cells were detected by western blot. DNA electrophoresis was used to detect apoptotic DNA fragements.6. The expression of Ub in celastrol-treated HNSCC cells was detected by western blot. Fluorescent26S proteasome assay.7. Transcription activity of CHOP in CHO-CHOP cell and XBP1s in CHO-XBP1cells were determined by the Steady-GloTM luciferase assay after the cells were treated with celastrol. Spliced XBP1was observed by conventional PCR with specific primers design.8. Cell viability was determined in celastrol-treated UPR-genes wildtype and double knockout of MEF cells by Celltiter-glo luminescent assay. The expression of UPR-protein and apoptosis-protein in celastrol-treated MEF cells were detected by western blot.Results:1. The expression of Bip in adjacent non-malignant regions was lower than in malignant regions of neck, laryngeal and lip. It was suggested that Bip expression could be one of candidate biomarkers for HNSCC tumor and UPR might be an important characteristic of malignant phenotype.2. Celatrol significantly inhibited HNSCC cell growth in a time and dose dependent manner.3. Celastrol inhibited growth in solid tumor xenografts, The tumor inhibition rate is73.77%with celastrol-treated after27days.4. Expression of apoptotic genes which are NOXA, PUMA, TRB3, GADD34was significantly increased in celastrol-treated HNSCCs. It is suggested that celastrol might inhibit HNSCC cell grow via induction of apoptosis 5. Caspase activation was higher in celastrol-treated cell lines compared with untreated-cell lines. Western blot results show that the expression of cleaved Caspase3was much higher in HNSCC cells as the dose increased. The DNA ladders of the apoptotic DNA fragmentation appeared in the longer cellastrol treatment time point. These data indicated that celastrol could induce the apoptosis pathway to kill cells.6. Western blot results show that the expression of Ub was much higher in HNSCC cells as the dose increased. That celastrol increased unfolded protein or misfolded protein in the endoplasmic reticulum of HNSCC cells, the expression of increased Ub protein suggested that more protein degradation further increase ERS leading to apoptosis. But it is couldn’t affect to26s.7. Expression of CHOP in CHO-CHOP cells and XBPls in CHO-XBP1cells were increased significantly in the cells treated with celastrol compared with untreated control cells. The results suggest the celastrol increased UPR in HNSCC cells. The expression of Spliced XBP1was induced in a dose-dependent manner. It was confirmed that celastrol led to activate UPR genes and could lead to XBP1splicing. The result suggested celastrol might increase UPR to regulate cell survival.8. Celastrol more significantly inhibits MEF wildtype cell growth than UPR-associated-genes-knockout of cells in a dose dependent manner. The expression of UPR-protein and apoptosis-protein is increased with an increased dose. It was indicated that celastrol increased misfolded proteins, which lead to the activation of ER stress-mediated apoptosis. Conclusions:Our finding indicated the celastrol activated apoptotic pathway via inducing ER-stress in HNSCC cells. HNSCC cells had significantly elevated Bip gene expression compared to healthy cells. It is considered that Bip might be a related biomarker of HNSCCs.