Human Tongue Squamous Cell Research Articles

Abstract 4222: Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway

Abstract Oral cancer is the sixth most prevalent malignancy in the world and oral squamous cell carcinoma accounts for majority of all oral malignancies. Upregulated NLRP3 inflammasome innate immune pathway is of importance to tumor development. Current efforts are being focused on identifying small molecules that exhibit anti-cancer activity as inflammasome pathway inhibitors. Our previously published work on phenstatin based indole linked chalcone scaffold 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring revealed 9a as an anti-oral cancer compound. 9a was found to act through inhibiting tubulin polymerization at protein level, using in vitro models oral cancer cell line/spheroid cells and in vivo animal oral cancer xenograft model. 9a had also shown significant reduction in radiolabeled-glucose uptake in xenograft mice model. Current study was undertaken to evaluate if small molecule inhibitor 9a acts through regulating the NLRP3 pathway. Using computation approach, we predicted the binding of 9a with NLRP3NACHT domain, which revealed stable interaction as similar to that exhibited by NLRP3 inhibitors MCC950 and ADP. Further, we checked immune mechanistic activity of 9a on NLRP3 pathway intermediates in oral cancer cells. AW13516 cell line which was human tongue squamous tumor-derived cell line; indigenously developed at our department previously, was activated for NLRP3 inflammasome pathway using LPS and activator Nigericin in presence of 9a. MCC950 treated cells and only LPS or LPS/Nigericin treated cells served as controls. NLRP3, caspase-1 and mitochondrial protein expression was analyzed in these cells by immunofluorescence (IF) and found to be increased upon LPS/NIG activation and reduced significantly upon MCC950 and 9a treatment. Activation led to puncta formation which was found diffused after MCC950/9a treatment. Similarly treated AW13516 cells were also validated using western blotting experiments. Expression of 118kDa NLRP3 protein was found increased upon inflammasome activation that was significantly reduced in 9a treated cells and reduction was dose dependent. 9a had shown significant reduction in oral cancer xenograft of AW13516 in NOD-SCID mice model. We tested formalin-fixed paraffin sections of these tumors by immunohistochemistry. Tumor areas were assessed for expression of NLRP3 pathway markers and there was significant reduction in NLRP3, Caspase-1, GSDMD and IL-1β in 9a treated tumors compared to control tumors. This reduction was at par with that shown by Adriamycin. Summarizingly, 9a has been found to be regulating inflammatory immune mechanisms and can be developed further as immunomodulatory anti-cancer agent. Since macrophages are major resource immune cells of NLRP3, further studies are underway to test 9a on macrophages from nlrp3-/- and caspase-/- mice in comparison with normal mice. Citation Format: Jyoti Kode, Jitendra Maharana, K. Nirmal Kumar, Trupti Pradhan, Arvind Ingle, Madan Barkume, Meena Patkar, Namitha Thampi, Ankita Patil, Anand Vaibhaw, Jeshma Kovvuri, Ahmed Kamal. Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4222.

Anlotinib is effective in patients with advanced oral cancer?

Oral cancer patients with recurrence or distant metastasis often present poor prognosis. Majority of advanced oral cancer patients suffer from treat-related adverse events and drug resistance. For those patients, the survival time and quality of life are urgent to be improved. Anlotinib, as a multi-targets tyrosine kinase inhibitor (TKI), has been demonstrated to be effective in many refractory tumors by inhibiting tumor angiogenesis and partial functions of tumor cells. In this paper, we performed CCK-8 assay, wound healing assay and transwell assay to explore the effect of anlotinib on human tongue squamous carcinoma Tca8113 cell line. Preliminary data indicated that anlotinib significantly inhibited Tca8113 cells proliferation, migration and invasion in vitro. Together, we proposed a hypothesis that anlotinib might be effective in prolonging survival time of patients with advanced oral cancer.

Suppression on invasion and migration of human tongue squamous cell carcinoma after knocking down EZH2 expression

Suppression on invasion and migration of human tongue squamous cell carcinoma after knocking down EZH2 expression

XIAP inhibitor embelin induces autophagic and apoptotic cell death in human oral squamous cell carcinoma cells.

Embelin is an active ingredient of traditional herbal remedies for cancer and other diseases. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, little data are available regarding the role of autophagy in oral cancers. Therefore, we conducted this study to examine whether Embelin modulates autophagy in Ca9‐22. Our results showed that Embelin had anticancer activity against the Ca9‐22 human tongue squamous cell, and we observed that autophagic vacuoles were formed by MDC and AO. We also analyzed Embelin‐treated Ca9‐22 cells for the presence of biochemical markers and found that it directly affected the conversion of LC3‐II, the degradation of p62/SQSTM1, full‐length cleavage formation of ATG5‐ATG12 complex and Beline‐1, and caspase activation. Rescue experiments using an autophagy inhibitor showed Embelin‐induced cell death in Ca9‐22, confirming that autophagy acts as a pro‐death signal. Furthermore, Embelin exhibited anticancer activity against Ca9‐22 via both autophagy and apoptosis. These findings suggest that Embelin may potentially contribute to oral cancer treatment and provide useful information for the development of a new therapeutic agent.

Signal transducer and activator of transcription-3 inhibitor WP1066 affects human tongue squamous cell carcinoma proliferation and apoptosis in vitro and in vivo

To investigate the antitumour molecular mechanisms of WP1066 (STAT-3 inhibitor ) to human tongue squamous cell carcinoma in vitro and in vivo. WP1066 was used to inhibit the p-STAT-3 expression in Tscca human tongue squamous cell carcinoma cell line .Real-time PCR was used to detect the microRNA-21 expression after treatment with DMSO and WP1066. Methyl thiazolyl tatrozolium (MTT) assay was employed to determine cell survival. Flow cytometry (FCM) was used to measure apoptosis. The expression level of STAT-3/p-STAT-3, programmed cell death protein 4 (PDCD-4), antigen 67 (Ki-67), B cell lymphoma 2 (Bcl-2) and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) was examined by Western blotting. Luciferase reporter gene assay was conducted to verify the regulation of STAT-3 to microRNA-21. Tscca xenograft tumor model was established in BALB/c nude mice and the tumors were divided into control, DMSO and WP1066 treated groups. The tumor tissues were measured by immunohistochemistry stain and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) assay. STAT-3/p-STAT-3 protein was suppressed after treatment with WP1066 (STAT-3:F = 15.336, P = 0.004, p-STAT-3: F = 52.837, P = 0.000). MicroRNA-21 relative expression level was down-regulated (F = 8.197, P = 0.019). Cell survival rate was significantly reduced after treatment with WP1066 than control groups (F = 94.388, P = 0.000). Early apoptosis rate increased after treatment with WP1066 (F = 217.080, P = 0.000) . PDCD-4 and cleaved cysteinyl aspartate specific proteinase-3 (CCASP-3) protein expression was increased after treatment with WP1066 (PDCD-4:F = 8.771, P = 0.017; CCASP-3: F = 26.611, P = 0.001) .Ki-67 and Bcl-2 protein was down regulated (Ki-67:F = 5.854, P = 0.039; Bcl-2:F = 125.502, P = 0.000). Luciferase reporter gene assay proved that STAT-3 combined with specific promoter region of microRNA-21.In vivo, the tumor volume after treatment with WP1066 was significantly smaller than control groups by the end of observation (F = 15.390, P = 0.000) .Immunological histological chemistry indicated that PDCD-4 and CCASP-3 protein expression was up-regulated simultaneously while Ki-67 and Bcl-2 protein of tumor tissue was down-regulated after treatment with WP1066 than control groups. TUNEL assay suggested that apoptosis index rose after treatment with WP1066 than control groups (F = 133.368, P = 0.000) . WP1066 affected Tscca cancer cell proliferation and apoptosis via inhibiting STAT-3/microRNA-21.WP1066 provided new direction and possibility to human tongue squamous cell carcinoma therapy.

Antimutagenic and antioxidative activities of sandy tea and its in vitro anticancer effect on TCA8113 human tongue squamous carcinoma cells (647.7)

Sandy tea is a traditional and popular drink in China. The aim of this study was to determine the antioxidant activity, antimutagenic activity of sandy tea and its in vitro anticancer effect on TCA8113 human tongue squamous carcinoma cells. The antioxidant effects were determined by measuring the 1,1‐diphenyl‐2‐picryhydrazyl (DPPH) free radical舉 and hydroxyl radical舉scavenging activity. The sandy tea extract demonstrated high antioxidant activity in the assays of DPPH and hydroxyl radical舉scavenging activity. At concentrations of 1.25 and 2.5 mg/plate, sandy tea exhibited anti‐mutagenicity with N‐methyl‐N'‐nitro‐N‐nitrosoguanidine (MNNG) in the Salmonella typhimurium TA100 strain. MTT assay and RT‐PCR assay were employed to check the anticancer effects on TCA8113 cancer cells. At the concentration of 200 µg/ml, sandy tea (81%) showed the best inhibitory effect on TCA8113 human tongue squamous carcinoma cells compared to the other concentrations. Compared with 100 µg/ml and 50 µg/ml of sandy tea extracts, 200 µg/ml of sandy tea induced apoptosis, up‐regulated mRNA expressions of Bax, caspase‐3 and caspase‐9, and down‐regulated Bcl‐2 expression. These results suggest that sandy tea possesses good antioxidant activity and anticancer effect on TCA8113 human tongue squamous carcinoma cells.

English

English

Signal transducers and activators of transcription-3 modulates human squamous cell carcinoma invasion via targeting mircoRNA-21 in vitro

To investigate the effect and mechanism of signal transducers and activators of transcription 3 (STAT-3) modulates human tongue squamous cell carcinoma invasion ability via targeting mircoRNA-21. Tscca and Tca8113P160 human tongue squamous cell carcinoma cell lines were used.WP1066 (STAT-3 inhibitor) , the small molecule inhibitor of STAT-3 was used to suppress the STAT-3 expression. The half maximal inhibitory concentration (IC50 value) of WP1066 in the two cell lines was determined by methyl thiazolyl tetrazolium (MTT) assay. The expression level of STAT-3 and phosphorylation of STAT-3 (pSTAT-3) was examined by Western blotting. Real-time PCR was used to detect the mircoRNA-21 expression after treated with WP1066. Matrigel matrix and transwell assay were used to determine cancer cell colony formation and invasion ability after treated with WP1066. Tumor invasion related proteins in Tscca and Tca8113P160 cell lines were measured by Western blotting. Luciferase reporter gene assay was conducted to detect the relationship between STAT-3 and mircoRNA-21. The IC50 to WP1066 in Tscca cell was 3.1 and 3.5 µmol/L for Tca8113P160 cell respectively. STAT-3/pSTAT-3 protein level was suppressed significantly (Tscca: STAT-3: F = 887.154, P = 0.000; pSTAT-3: F = 332.212, P = 0.000; Tca8113P160: STAT-3: F = 322.895, P = 0.000; pSTAT-3:F = 788.357, P = 0.000). mircoRNA-21 expression was down-regulated (Tscca:F = 32.157, P = 0.000; Tca8113P160: F = 11.349, P = 0.007). The diameters of culture clone in cell treated with WP1066 were less than control groups (Tscca:F = 15.751, P = 0.004; Tca8113P160: F = 12.964, P = 0.007). The number of tongue cancer cell migrating through the transwell membrane in WP1066 treated group was less than in control groups (Tscca: F = 1688.926, P = 0.000; Tca8113P160: F = 327.528, P = 0.000). In addition, MMP-2/9 protein expression was decreased in both of the cell lines treated with WP1066, while TIMP-3 was up regulated dramatically. STAT-3 could modulate mircoRNA-21 directly. Reduction of STAT-3 can inhibit tongue cancer cell invasion ability via targeting mircoRNA-21.

环氧合酶-2抑制剂对人舌鳞癌Tca8113/BLM 细胞MDR1/P-gp表达的影响

Objective To investigate the effect of celecoxib,a selective COX-2 inhibitor,on the expression of multidrug resistance gene(MDR1) and P-glycoprotein(P-gp) in human tongue cancer drug-resistance cell line Tca8113/BLM.Methods Human tongue squamous cell carcinoma cell line Tca8113 cells were repeatedly treated with BLM(30μg/ml) to establish drug-resistant cell line Tca8113/BLM.The inhibitory effect of celecoxib on Tca8113/BLM cell line was determined by MTT assay.The expression of P-gp and MRP were detected by flow cytometry and RT-PCR,respectively.Results The MTT results suggested that celecoxib inhibited the growth and proliferation of Tca8113/BLM cell lines in dose-dependent manner.Celecoxib treatment also resulted in significant down-regulated expression of MDR1 mRNA and P-gp.Conclusion Celecoxib shows a significant effect on inhibiting expression of MDR1 mRNA and P-gp in Tca8113/BLM cells,this is probably related to enhancing the lethal effects of anticancer agents against carcinoma cells.

Cross-talk between Notch1 and epidermal growth factor receptor signalling in regulating cell proliferation of human tongue squamous carcinoma cells

To investigate the cross-talk between Notch1 and epidermal growth factor receptor (EGFR) signaling in regulating the cellular proliferation of human tongue squamous cell carcinoma (SCC). Human tongue SCC cell line Tca8113 cells was transiently transfected with the vector encoding exogenous intracellular fragment of Notch1 and the vector encoding the specific short hairpin RNA (shRNA) targeting EGFR respectively and were treated by AG1478, an inhibitor of receptor tyrosine kinases, for elucidating the effects of constitutive activation, EGFR gene silencing and blocking EGFR signaling upon cellular proliferation and expression of Notch1 and EGFR. The mRNA and protein levels of Notch1 and EGFR were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. The cellular proliferation was evaluated by methyl thiazolyl tetrazolium (MTT) assay. Constitutive activation of Notch1 resulted in inhibition of cellular proliferation, and up-regulation of Notch1 (1.102 +/- 0.135, 0.243 +/- 0.032, P < 0.05) but down-regulation of EGFR (0.083 +/- 0.009, 0.605 +/- 0.075, P < 0.05) at the the mRNA and protein levels. Silencing of EGFR gene resulted in inhibition of cell proliferation, and down-regulation of EGFR (0.148 +/- 0.019, 1.175 +/- 0.132, P < 0.05) but up-regulation of Notch1 (0.978 +/- 0.115, 0.083 +/- 0.009, P < 0.05) at the mRNA and protein levels. Blocking EGFR signaling had no significant effect upon EGFR expression (P > 0.05), but resulted in inhibition of cellular proliferation and up-regulation of Notch1 (P < 0.05) at the mRNA and protein levels. There might be a cross-talk of bi-directional control between Notch1 and EGFR signaling in regulating the cellular proliferation of human tongue SCC cells.

EMMPRIN expression in tongue squamous cell carcinoma

Extracellular matrix metalloproteinase inducer (EMMPRIN) is identified as a tumor-cell membrane protein that stimulates matrix metalloproteinases (MMPs) production. Several studies have shown that higher EMMPRIN expression is associated with shorter survival time and correlated significantly with more advanced clinico-parameters of cancer. The aim of this study was to investigate the relationship between clinico-pathologic characteristics and EMMPRIN, and prognostic significance of EMMPRIN expression in human tongue squamous cell carcinoma. Extracellular MMP inducer expression was examined immunohistochemically on paraffin-embedded tissue specimens from 68 patients with tongue squamous cell carcinoma and who underwent radical surgeries from 1996 to 2006. The 68 patients were followed up from 1 to 119 months, with an average of 27.5 months. Nonparametric tests were performed for the comparison of EMMPRIN expression between two independent groups. Survival analysis was performed to find the prognostic significance of EMMPRIN expression. We found that EMMPRIN expression in tongue squamous cell carcinoma is significantly higher than that in non-cancerous epithelium adjacent to carcinoma of tongue. In addition, EMMPRIN expression is significantly associated with tumor diameter and clinical stage in the samples, but did not correlate with gender, age, tumor metastasis, and pathological grade. Finally, survival analysis indicates that EMMPRIN overexpression correlates significantly with poor overall survival in the patient cohort. These results suggest that EMMPRIN might represent an attractive target for immunotherapeutic approaches in a subgroup of patients with tongue squamous cell carcinoma.

Effects of epidermal growth factor receptor gene silencing mediated by short hairpin RNA on proliferation and apoptosis of human tongue carcinoma cells

To investigate the effects of epidermal growth factor receptor (EGFR) gene silencing mediated by short hairpin RNA (shRNA) on proliferation and apoptosis of human tongue carcinoma cells. shRNA eukaryotic expression vector targeting the specific sequence of human EGFR gene was constructed and termed shEGFR. The control vector targeting the unrelated sequence was also constructed and termed shNC. The vectors were transiently transfected into Tca8113 cells of human tongue squamous cell carcinoma by Lipofectamine 2000, respectively. The mRNA and protein levels of EGFR in Tca8113 cells were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The cell proliferation of Tca8113 cells was evaluated by methyl thiazolyl tetrazolium (MTT) assay. The apoptosis of Tca8113 cells was assessed by flow cytometry. EGFR expression in Tca8113 cells transfected with shEGFR were obviously decreased at mRNA level (81.6%) and protein level (72.0%) (P < 0.05) 48 h after transfection of shEGFR compared with untransfected Tca8113 cells. The proliferation activity of Tca8113 cells transfected with shEGFR was significantly lower than that of Tca8113 cells transfected with shNC and untransfected Tca8113 cells (P < 0.05). The early apoptotic rate of Tca8113 cells transfected with shEGFR was significantly higher than that of Tca8113 cells transfected with shNC and untransfected Tca8113 cells [(39.4 +/- 7.7)%, (4.3 +/- 1.2)%, (2.5 +/- 0.9)%, P < 0.05] 48 h after transfection of shEGFR. EGFR gene silencing mediated by shRNA may inhibit cell proliferation and induce apoptosis in human tongue carcinoma cells.

Celecoxib enhances the lethal effects of bleomycin in human tongue squamous carcinoma cell line Tca8113

To investigate the role of COX-2 inhibitor celecoxib in enhancing the lethal effects of bleomycin in Tca8113 cell line. Tca8113 cells were treated with different concentrations of celecoxib and bleomycin for 24, 48, 72 h. Methyl thiazolyl tetrazolium assay was used to calculate cell growth inhibition rate and Jin Zheng Jun's method was used to evaluate the interaction of celecoxib and bleomycin on Tca8113 cells. Flow cytometry was used to evaluate the effects of combined use of celecoxib and bleomycin on cell cycle progress and apoptosis. Low dose of celecoxib (10 micromol/L, < IC(50)) combined with bleomycin showed synergism or additive lethal effect on Tca8113 cell line. Celecoxib could notably enhance the inhibitory effect of bleomycin on Tca8113 cells by blocking cell cycle progress and thus resulting in the increasing G(0)/G(1) cells [(60.93 +/- 0.32)%] distribution and inducing apoptosis [(1.87 +/- 0.11)%]. Low doses of celecoxib could significantly enhance the lethal effect of bleomycin on Tca8113 cells by inhibiting cell growth and proliferation through blocking cell cycle progress and inducing apoptosis. The ways of these interactions on inhibiting Tca8113 cell growth were synergistic or/and additive.

Anticancer drug induced apoptosis of human tongue squamous carcinoma cells: Activation of the FAS pathway following carboplatin treatment

Anticancer drug induced apoptosis of human tongue squamous carcinoma cells: Activation of the FAS pathway following carboplatin treatment