The Role Of Autophagy In Salivary Adenoid Cystic Carcinoma Chemotherapy And The Correlation Analysis Of Its Associated Proteins And Clinical Prognosis In Primary Salivary Adenoid Cystic Carcinoma

Objective1. To analyze the effect of autophagy inhibitors during DDP-induced cell death and apoptosis of salivary adenoid cystic carcinoma.2. To analyze the effect of silence autophagy gene during DDP-induced cell death and apoptosis of salivary adenoid cystic carcinoma.3. To detect the change of bcl-2during DDP-induced cell autophagy and apoptosis of salivary adenoid cystic carcinoma.4. To analyze whether the P53-AMPK-mTOR related protein is involved in DDP-induced autophagy reaction or not.5. To analyze anticancer effect of DDP and autophagy inhibitor combination against ACC-M xenografts.Methods1. MTT and flow cytometry were used to detect DDP-induced death and apoptosis of adenoid cystic carcinoma after using autophagy autophagy inhibitors or silence autophagy gene. Western blot was used to analyze apoptosis protein changes.2. Electron microscopy and fluorescence microscopy to detect DDP-induced autophagy changes in adenoid cystic carcinoma.3. Western blot was used to detect Bcl-2and P53-AMPK-mTOR protein.4. To build ACC-M xenografts and then detect the volume and weight changes, immunohistochemical analysis was used to detected tumor autophagy change.Results1. DDP-induced salivary adenoid cystic carcinoma cell death by enhancing apoptosis was augmented by3-MA treatment. The IC50of ACC-M cell was11.34±0.92μg/mL for24h DDP treatment, which was examined via MTT assay. Therefore, we used10μg/mL DDP in ACC-M for24h in the subsequent experiment. By DDP treatment, viability of ACC-M decreased to 61.02%and using both3-MA and DDP increased cell death to78.73%. There was no significantly different cell death by treatment of3-MA alone in ACC-M cell.In addition, apoptosis was examined by FCM and immunoblotting of actived-caspase-3. We used AnnexinV-FITC and propidium iodide (PI) staining assay to observe the apoptotic cell death. Compared with the DDP group, the apoptosis rate of the ACC-M cells that were treated with DDP and3-MA significantly increased from19.08%to29.72%. Furthermore, actived-caspase-3was examined by immunoblotting, which is active form of capase-3in downstream apoptosis pathway. DDP with addition of3-MA made actived-caspase-3increased36.36%compared with DDP group.DDP induces autophagy and3-MA is a well known autophagy inhibitor that inhibits the activity of type I PI3K (a kinase that is essential for vesicle nucleation, the first phase of autophagosome formation), which were confirmed in our experiment. Electron microscopic analysis of DDP-induced ACC-M cells indicated autophagosome. To determine whether3-MA inhibition combined with DDP induces autophagy in ACC-M cells, we transfected the cells with GFP-LC3plasmid and detected the distribution of GFP. LC3is a microtubule-associated protein that is a critical component of the autophagosome. The use of the GFP-tagged LC3plasmid has become an effective marker for the autophagosome. Diffuse cytoplasmic localization of GFP-LC3was observed in untreated ACC-M cells, whereas DDP-treated cells showed increased punctate fluorescence. Increases in punctate fluorescence indicated the presence of autophagic cells. We observed combination (DDP plus3-MA group) reduced about64.29%compared with DDP group.The change of LC3protein was also observed by immunoblotting. Since the extent of conversion of LC3-Ⅰ to LC3-Ⅱ is correlated to the level of autophagy, LC3-Ⅰ and LC3-Ⅱ were detected by western blot, and the autophagic level was demonstrated LC3-Ⅱ band density. LC3-Ⅱ was induced by DDP treatment in ACC-M cells, but3-MA reduced them about52.83%. In addition to LC3, p62/SQSTM1as a marker is also used by immunoblotting. The p62protein serves as a link between LC3and ubiquitinated substrates. P62becomes incorporated into the completed autophagosome and is degraded in autolysosomes. Correspondingly, the level of p62protein increased about36.51%with DDP added3-MA, compared with DDP treatment.Overall, DDP treatment induced autophagy in ACC-M cells, but addition of3-MA inhibited autophagy and enhanced DDP-induced ACC-M cell death by enhancing the caspase-mediated apoptosis pathway.2. DDP-induced cell death by increasing apoptosis in salivary adenoid cystic carcinoma cell was enhanced by beclin-1siRNA.Beclin-1is a critical initial protein in the process of autophagy. To observe the influence of autophagy more directly, we used beclin-1siRNA to decrease endogenous beclin-1expression. By immunoblotting, endogenous beclin-1was reduced by56.60%in ACC-M cells, compared with the control, at24h after transfection with beclin-1siRNA or the universal control siRNA.In addition, apoptosis was examined by FCM and immunoblotting of actived-caspase-3. We used AnnexinV-PE and7AAD staining to observe the apoptotic cell death. Compared with the DDP group, the apoptosis rate of the ACC-M cells that were treated with DDP and beclin-1siRNA significantly increased from18.78%to30.41%. Furthermore, DDP with addition of beclin-1siRNA made actived-caspase-3increased35.19%compared with DDP group.DDP induces autophagy and beclin-1siRNA inhibits autophagy, which were confirmed in our experiment. We observed the autophagic cells of combination (DDP plus beclin-1siRNA group) reduced about80.32%compared with DDP group. Similarly, LC3-Ⅱ was induced by DDP treatment in ACC-M cells, but beclin-1siRNA reduced them about61.11%. Correspondingly, the level of p62protein increased about100%with DDP added beclin-1siRNA, compared with DDP treatment.Overall, DDP treatment induced autophagy in ACC-M cells, but addition of beclin1siRNA inhibited autophagy and enhanced DDP-induced ACC-M cell death by enhancing the caspase-mediated apoptosis pathway. 3. Bcl-2may be a link between apoptosis and autophagy and P53-AMPK-mTOR may participate in DDP-induced autophagy response.Since it is strongly suggested that there is crosstalk between DDP-induced apoptosis and autophagy, we checked expression of the bcl-2which is reportedly implicated in both apoptosis and autophagy. By immunoblot analysis, we found that DDP increased the expression of in ACC-M cells. Use of beclin-1siRNA combination reduced the increase of bcl-2. Therefore, bcl-2may be a link between autophagy and the apoptosis pathways in our experiment.In addition, it is necessary to examine the underlying mechanism of DDP-induced autophagy. By immunoblotting, we found that with DDP treatment, expression and phosphorylation of tumour suppressor p53and AMP-activated protein kinase (AMPK) increased significantly and the mammalian target of rapamycin (mTOR), a gatekeeper of autophagy decreased also decreased in ACC-M cells.4. Anticancer effect of DDP and3-MA combination against ACC-M xenograftsTo further verify the effects of autophagy in vivo, ACC-M xenografts in nude mice were established. There were no differences in body weight between groups. Tumor growth was significantly inhibited by DDP combined with3-MA compared with the the control, DDP treatment. There is no significant influence on tumor growth treated with3-MA. On day30, compared with the DDP group, tumor volume and tumor weight of combination group mice were significantly reduced by53.64%and59.75%. The result of immunohistochemical analysis showed that expression of LC3significantly induced in combination therapy compared with DDP group. These results indicated that DDP,3-MA combination could inhibit autophagy and the growth of xenografts.Conclusion1. DDP-induced salivary adenoid cystic carcinoma cell death by enhancing apoptosis was augmented by3-MA treatment.2. DDP-induced cell death by increasing apoptosis in salivary adenoid cystic carcinoma cell was enhanced by beclin-1siRNA.3. Bcl-2may be a link between apoptosis and autophagy and P53-AMPK-mTOR may participate in DDP-induced autophagy response.4. Anticancer effect of DDP and3-MA combination against ACC-M xenografts. Autophagy is the endogenous cellular pathway which facilitates cellular survival via maintaining energy homeostasis and macromolecular synthesis during cellular stress and nutrient deprivation. Endoplasmic reticulum(ER) stress is the process that disruption of these physiological functions leads to accumulation of unfolded proteins and induces the unfolded protein response (UPR). ER stress and autophagy are involved in human cancer. We investigated the expression of autophagic proteins (LC3and beclin1) and ER stress-related protein (GRP78) in head and neck adenoid cystic carcinoma tissue. Tissue samples from79cases of head and neck adenoid cystic carcinoma tissue were utilized for immunohistochemistry. LC3expression was significantly correlated with lymph node involvement (P=.016) and TNM (P=.021). Beclin1expression was significantly correlated with histological growth pattern (P=.002), histological grade (P<.001) and longer survivial (P<.001). GRP78expression was significantly correlated with histological growth pattern (P=.019), histological grade (P=.019) and longer survivial (P=.001). LC3expression was positively correlated with beclin1expression (P<.001); LC3and beclin1expression was positively correlated with GRP78expression respectively (P=.035)(P=.008). Our study showed that the expression of LC3, beclin1and GRP78in adenoid cystic carcinoma and its relation with clinicopathologic factors and overall survival. These results suggest that LC3, beclin1and GRP78may play an important role in tumorigenesis of adenoid cystic carcinoma and beclin1and GRP78may serve as new prognostic indicators for outcome of the patients with adenoid cystic carcinoma.