Keyi Li

Inhibition of autophagy augments chemotherapy in human salivary adenoid cystic carcinoma

Although cisplatin (DDP)-based adjuvant chemotherapy is widely used in the treatment of salivary adenoid cystic carcinoma (SACC), SACCs have developed resistance to cisplatin, resulting in chemotherapy failure. Autophagy serves as a critical adaptive response, which was increased in tumor cells in chemotherapy. However, the function of autophagy is not clear in SACC. In this study, apoptosis induced by DDP in SACC high metastatic cell line (ACC-M) was revealed using MTT assay, flow cytometry, and caspase-3 immunoblotting. The autophagy activation induced by DDP treatment was measured by transmission electron microscopy, green fluorescent protein-light chain 3 plasmid transfection LC3 immunoblotting and p62 immunoblotting. 3-methyladenine (3-MA) or small interference RNA targeting beclin 1 (beclin 1 siRNA) inhibited autophagy and significantly enhanced DDP-induced apoptosis. ACC-M xenografts in nude mice further verified the synergistic effect of DDP and 3-MA. In conclusion, autophagy activation was caused to protect cancer cells from DDP-induced apoptosis and autophagy inhibition could be a promising strategy for adjuvant chemotherapy in SACC.

The molecular mechanisms of XBP-1 gene silencing on IRE1α-TRAF2-ASK1-JNK pathways in oral squamous cell carcinoma under endoplasmic reticulum stress

BACKGROUND Proteasome inhibitor Carbobenzoxy-Leu-Leu-leucinal (MG132) induces the unfolded protein response (UPR) in oral squamous cell carcinoma (OSCC). X-box binding protein 1 (XBP1) is a key UPR component that regulates endoplasmic reticulum stress (ER) homeostasis. This study was aimed to investigate the activation of IRE1α-TRAF2-ASK1-JNK pathway by silencing the XBP1 expression in an OSCC cell line. METHODS The XBP1 specific short hairpin RNA (shRNA) plasmid vector was constructed and then transfected into the Tca-8113 cells. The effect of XBP-1 gene silencing on IRE1α-TRAF2-ASK1-JNK pathway under MG132 induced endoplasmic reticulum stress in Tca-8113 were investigated by real-time RT-PCR or western blot. Cell apoptosis was detected by flow cytometry. RESULTS XBP1 expression was reduced in transfected groups and MG132 groups. shRNA-XBP1 induces IRE1α-TRAF2-ASK1 signaling activation to activate pro-apoptotic ASK1-JNK signaling. Moreover, combined shRNA-XBP1 with MG132 further enhanced downregulated XBP1 expression and upregulated activation of ASK1-JNK signaling. CONCLUSIONS Silencing XBP1 expression under MG132 induced ER stress block the XBP1 survival pathway and synergism with MG132 to promote Tca8113 cell apoptosis. These findings provide a therapeutic option in oral squamous cell carcinoma by inhibition of proteasome and XBP1 splicing.

Inhibition of autophagy augments apoptosis in human oral squamous cell carcinoma under nutrient depletion

There has been little research conducted regarding autophagy in oral squamous cell carcinoma (OSCC). Given the prevalence of oral cancers which are OSCC and the severe side effects of current treatments, there is a pressing need to develop effective alternative therapies. In this study, we have endeavored to explore the biological characteristics of oral squamous cell carcinoma cell line KB cells, in particular with regard to the role played by autophagy in their survival. Autophagy was activated by nutrient depletion via culturing cells in Earles balanced salts (EBSS) and was measured via indices relating to Beclin 1, microtubule-associated protein light chain 3 (MAPLC3, LC3), p62, and Green fluorescent protein-light chain 3 plasmid transfection (GFP-LC3). Cell death and apoptosis induced by nutrient depletion was measured using both MTT assay and flow cytometry (FCM). Compared to initial levels at 0 h, Beclin 1 density in EBSS-treated cells was found to have increased at 6, 12, and 18 h in a time-dependent manner and was found to have subsequently declined at 24 and 48 h. p62 levels, LC3-II/LC3-I ratio, and GFP-LC3 levels increased at 6, 12, 18, 24, and 48 h in a time-dependent manner. 3-methyladenine (3-MA) was found to inhibit autophagy and the expression of Beclin 1 and significantly enhanced nutrient depletion-induced apoptosis and death. We concluded that nutrient depletion enhances OSCC cell autophagy in time-course patterns and that the inhibition of autophagy augments apoptosis in OSCC cells. We also deduced that Beclin 1 takes part in the development and progression of autophagy, potentially playing an important role in the crosstalk between apoptosis and autophagy in OSCC cells. These findings suggest that nutrient depletion may be an effective way to explore autophagy and that autophagy inhibitors should be investigated as a potential novel agent for the adjuvant treatment of human OSCC.

Overexpression of tumor suppressor gene ZNF750 inhibits oral squamous cell carcinoma metastasis

Zinc-finger protein 750 (ZNF750) encodes a putative C2H2 zinc finger protein and is typically mutated or deleted in squamous cell carcinoma. The role of ZNF750 in oral squamous cell carcinoma (OSCC) remains unknown. The aim of the present study was to investigate the effects of ZNF750 overexpression in CAL-27 cells. Cell viability, and the expression of genes associated with proliferation, differentiation and the epithelial-mesenchymal transition were investigated in CAL-27 cells following ZNF750 overexpression, using Cell Counting kit-8, reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. In addition, scratch wound, invasion and migration assays were performed. Cell viability, matrix metalloproteinase 28 expression, cyclin B1 expression and mesenchymal marker neural cadherin expression were decreased following ZNF750 overexpression compared with the control groups. ZNF750 overexpression induced the differentiation-associated genes late cornified envelope 3A and small proline-rich protein 1A and upregulated the expression of late epidermal differentiation factor Kruppel-like factor 4. Overexpression of ZNF750 in CAL-27 cells resulted in inhibition of cell invasion and migration. Taken together, these data suggest that ZNF750 may inhibit the metastasis of OSCC.

Expression of SKA1 and MMP-9 in primary salivary adenoid cystic carcinoma: Correlation with tumor progression and patient prognosis.

ABSTRACT Conclusion The spindle and kinetochore-associated complex sub unit 1(SKA1) and matrix metalloproteinase-9 (MMP-9) are highly expressed in salivary adenoid cystic carcinoma (SACC), and SKA1 may be the novel, promising prognostic factor for clinical outcomes in head and neck adenoid cystic carcinoma patients. Objectives This study aimed to investigate the expression of SKA1 and MMP-9 in SACC tissues and the clinical significance. Methods SKA1 and MMP-9 expression in samples from 42 cases of SACC and 20 subjects with the normal tissue adjacent to carcinoma were detected by immunohistochemical analysis. Results The positive rate of SKA1 and MMP-9 staining was 78.6% and 66.7% in SACC, respectively, significantly higher than in normal salivary gland tissues (p < 0.001). Chi-square test showed that there was no significant correlation between SKA1 expression and MMP-9 expression in SACC tissues. However, SKA1 and MMP-9 expression was positively associated with advanced stage and solid histological pattern of SACC (p < 0.05). In addition, SKA1 and MMP-9 expression was positively associated with recurrence and perineural invasion, and survival time, respectively.

Betulinic acid increases radiosensitization of oral squamous cell carcinoma through inducing Sp1 sumoylation and PTEN expression

Radiotherapy is one of the most effective non-surgical treatments for oral squamous cell carcinoma. However, radioresistance remains a major impediment to radiotherapy. Although BetA (Betulinic acid) can induce radiosensitization, the underlying mechanism and whether it could induce radiosensitization in oral squamous cell carcinoma are not fully understood. In this study, we showed that BetA increased radiosensitization in CAL-27 and Tca-83 cells. Radiation-triggered Sp1 overexpression was responsible for radioresistance of OSCC (oral squamous cell carcinoma) cells. Treatment with BetA downregulated Sp1 and upregulated PTEN through inducing Sp1 sumoylation and correspondingly increased radiosensitization. Moreover, Sumoylation of Sp1 upregulated PTEN protein expression by downregulating Sp1 as well as inhibiting Sp1 DNA binding activity, thereby leading to the activation of PTEN transcription. Our results suggested that BetA was able to enhance radiosensitization at least partially by downregulating Sp1 and upregulating PTEN through inducing Sp1 sumoylation. BetA is suggested to be a promising drug for increasing radiosensitization in oral squamous cell carcinoma radiotherapy.

Pathway-focused PCR array profiling of CAL-27 cell with overexpressed ZNF750

Zinc-finger protein 750 (ZNF750) is the potential anti-cancer gene in oral squamous cell carcinoma (OSCC). The present study was to investigate the expression changes of ZNF750 in OSCC tissue and to reveal the induction of altered mRNA expression profiles caused by over-expressed ZNF750 in CAL-27 cell. The expression level of ZNF750 in tissue specimens from OSCC patients was detected by immunohistochemistry. Gene expression profiling was performed using Human Signal Transduction PathwayFinder RT2 Profiler™ PCR Array. The expression changes of 84 key genes representing 10 signal transduction pathways in human following over-expressed ZNF750 in CAL-27 cell was examined. The expression of ZNF750 protein was reduced in OSCC tissues. The R2 PCR Array analysis revealed that 39 of the 84 examined genes that changed at least a two-fold between control and ZNF750 groups. These genes related to oxidative stress, WNT, JAK/STAT, TGFβ, NF-kappaB (NFκB), p53, Notch, Hedgehog, PPAR and Hypoxia signaling. ZNF750 could inhibit the candidate genes ATF4, SQSTM1, HMOX1, CCND1, TNF-alpha, TNFSF10 and FOSL1 but activate CDKN1A and EMP1. Our studies suggest that ZNF750 can regulate signaling pathways that related to proliferation, cell cycle, inflammation and oxidative stress in CAL-27 cell.

ZNF750 inhibited the malignant progression of oral squamous cell carcinoma by regulating tumor vascular microenvironment

OBJECTIVE Squamous cell carcinoma is often associated with the deletion or mutation of zinc finger protein 750 (ZNF750), its deletion or mutation is associated with squamous epithelial malignant biological characteristics. The present study is to explore the mechanism of ZNF750 to suppress the tumor malignant process by regulation tumor microenvironment. METHODS To evaluate the changes of tumor microenvironment in oral squamous cells carcinoma cell line CAL-27 cell, the expression of angiogenin, vascular endothelial growth factor (VEGF), prolyl hydroxylase 2 (PHD2), G protein signal regulated protein 5 (RGS5), integrin A5 (ITGA5), integrin B1 (ITGB1) and CD44 were detected by Western-blot. The changes of platelet derived growth factor (PDGFB) and tumor vascular marker CD105 (Endoglin) mRNA were estimated by qPCR. The effect of over-expressed ZNF750 on cell viability and lateral migration capacity was investigated by CCK-8 and cell scratch assay in three oral squamous cells carcinoma. RESULTS ZNF750 could effectively inhibit the protein or mRNA expression of angiogenin, VEGF, RGS5 and CD105, repressed the cell adhesion molecules ITGA5, ITGB1 and CD44, but up-regulate the protein or mRNA expression of PHD2 and PDGFB. The cell viability and lateral migration ability of three oral squamous cells carcinoma were reduced by over-expression of ZNF750. CONCLUSION ZNF750 could modulate the tumor vascular microenvironment to inhibit the oral squamous cells carcinoma malignant progression.

Knockdown of USP39 by lentivirus-mediated RNA interference suppresses the growth of oral squamous cell carcinoma.

BACKGROUND Oral squamous cell carcinoma (OSCC) is a frequently diagnosed life-threatening oral cancer worldwide and has become one of the leading causes of cancer-related mortality. However, the pathogenesis of this disease is very limited. OBJECTIVE In this study, we aimed to investigate the functional relationship between OSCC and a potential tumor related gene ubiquitin-specific proteases 39 (USP39). METHODS The lentivirus-based RNA interference was utilized to knock down USP39 expression in human OSCC CAL27 cells. The effect of USP39 on cell proliferation was detected by MTT and colony formation assays. RESULTS The results uncovered that the proliferation rate was significantly decreased in specific USP39-targeting lentivirus infected cells compared to control lentivirus infected cells. The colony formation capacity was also attenuated in CAL27 cells after USP39 knockdown. Moreover, knockdown of USP39 arrested CAL27 cells in S and G1/M phases of the cell cycle. Furthermore, USP39 silencing induced apoptosis of CAL27 cells via activations of Caspase 3 and PARP. CONCLUSIONS In conclusion, the inhibition of USP39 in CAL27 cells suppressed cell growth probably via induction cell cycle arrest and apoptosis. USP39 might act as an oncogenic factor in OSCC and could be a potential molecular target for OSCC gene therapy.