Antiproliferative And Apoptotic Effects Of Cycloxygenase-2 Inhibitor Celecoxib On Human Tongue Squamous Cell Carcinoma Cell Line Tca8113

Carcinoma of the tongue,one of the most common oral malignant tumors, accounts for over 40%of the oral carcinomas with an incidence of about 0.5～0.6/100 000 in males,and of 0.4～0.5/100 000 in females.Recently,the occurrence of tongue carcinoma tends to be significantly increasing even in younger population.The pathogenic factors still remain unknown.Local injury,smoking, alcoholic drinking and precancerous lesion may be involved in the pathogenesis.The tongue is an active muscle organ with abundant blood supply and lymph drainage, which facilitates its infiltrative growth and lymphatic metastasis at its early stage and thus makes it more difficult in its treatment.Therefore,it becomes more and more important to elucidate the pathogenic mechanism,and to explore the efficient preventive measures of the carcinoma of tongue.Epidemiological investigation revealed that patients with a long-term administration of aspirin or other non-steroidal anti-inflammatory drugs(NSAIDs) had a significantly lower incidence of colorectal cancer with less colonic polyp size and number,which reduced the risks of colonic polyp canceration.Further investigations confirmed that NSAIDs could not only inhibit gut epithelial tumors, but also prevent the pathogenesis of epithelioma other than colorectum.Such effects of NSAIDs might be mainly associated with their inhibition of the activity of cycloxygenase-2(COX-2).Therefore,selective COX-2 inhibitor is becoming a hot spot in the prevention and treatment of malignant tumors.In the present study,we investigated the inhibitory effects of Celecoxib,a selective COX-2 inhibitor,on Tca8113 cell line of human tongue squamous cell carcinoma(HTSCC) by immunohistochemistry,molecular biological and statistical methods in view of pathology,cell biology and experimental zoology. Immunohistochemistry was used in our study to investigate the expression of COX-2 and vascular endothelial growth factor-C(VEGF-C) in normal human oral mucosa, atypical hyperplastic epithelium,HTSCC and metastatic lymph nodes,which may be of important clinical value in the diagnosis and prognosis of the tongue carcinoma. Molecular biological techniques were employed to detect the effects of Celecoxib on growth inhibition and apoptosis of Tca8113 cell line.The role of COX-2 inhibitor Celecoxib in enhancing the cytotoxicity of anticancer agents in Tca8113 cell line was also detected.We established an animal tumor model through subcutaneously implantation of Tca8113 cells so as to observe the effect of Celecoxib along with CDDP on transplanted tumor growth in nude mice and the histomorphological changes of tumor-bearing tissues and organs,which may provide us primarily clinical value for COX-2 in the prevention and treatment of oral cancers.â… .Expression of COX-2 and VEGF-C in human tongue squamous cell carcinoma and its correlation with lymph node metastasisObjective:To observe the coexpression of COX-2/VEGF-C in normal oral mucosa,atypical hyperplastic epithelium and HTSCC,and to evaluate the correlation of this coexpression with canceration of tongue,and with lymph node metastasis, respectively.Methods:46 cases(26 males and 20 females,ages ranging from 28 to 81 with an average age of 53.78±11.43) of tongue squamous cell carcinoma specimen were collected from the Department of Pathology of Nanfang Hospital from 2000 to 2006.Pathological analysis confirmed that all the cases were advanced to moderately differentiated squamous cell carcinoma;13 of the 46 cases had cervical lymph node metastasis.None of the patients received radiotherapy or chemotherapy before surgery.8 cases of oral mucosa from traumatic patients were used as control. Methods:S-P immunohistochemistry staining was performed to determine COX-2 and VEGF-C expression in normal oral mucosa,cancer tissue and lymph nodes by semi-quantitative method.Based on the intensity of immunoreaction,intensity levels were classified into(-),(+),(++),(+++).CD₃₄ marked microvessel numbers were calculated by immunostaining to study microvessel density(MVD) in the cancer tissues.Results:COX-2 and VEGF-C showed—or+in normal oral mucosa,while it showed obvious strong expression in hyperplasia and atypical hyperplastic epithelium, and overexpression in most cancer tissues and metastasis lymph nodes.Expression level of COX-2 and VEGF-C demonstrated a significant difference in primary focus cancer tissues with or without lymph nodes metastasis.The result showed a positive correlation between the expression level of COX-2 and VEGF-C in carcinoma tissues of tongue.Their correlation coefficient(r_s) was 0.874(P=0.000).CD34 staining showed that newly formed microvessels were mainly distributed in tissues surrounding the carcinoma;buffy granules were found in the cytoplasm of vascular endothelial cells,while microvessels were hardly found in cancer tissues.The expression level of COX-2 and VEGF-C showed a positive correlation with MVD. Conclusions:The coexpression of COX-2 and VEGF-C had close relationship to biological behaviors in human tongue squamous cell carcinoma.It may be an important marker in evaluating the prognosis,metastasis and invasion in HTSCC. However,the mechanism of both proteins in carcinogenesis in HTSCC needs further investigation.â…¡.The growth inhibition and apoptotic induction of COX-2 inhibitor Celecoxib on Tca8113 cell line of HTSCCObjective:To observe whether Celecoxib could inhibit the growth and induce apoptosis of Tca8113 cells.Methods:24 h after incubation of Tca8113 cells,different concentrations of Celecoxib were added into the cells,with the final concentrations being 2.5,5,10,20,40,80μmol/L,respectively.Humen hepatic carcinoma cell line Hep-G2 as a Celecoxib-sensitive cells used as a positive control.MTT was used to determine the OD values at 570 nm for calculating cell growth inhibition rate after incubation for 24,48 and 72 h,respectively.Inverted microscope,optical microscope and transmission electron microscope were used to observe cell morphological changes before and after the treatment.Cell cycle phase distribution was detected by flow cytometer(FCM);morphology of apoptosis cells was observed by fluorescence microscopy;Annexin V-FITC/PI double labeling method was employed to detect early stage cell apoptosis.Results:COX-2 protein was strongly expressed in Tca8113 cells and was suppressed by Celecoxib.The growth and proliferation of Tca8113 cells treated with Celecoxib was inhibited in a dose-dependent manner,which reached its valley value at the concentration of 40μmol/L.Celecoxib's growth inhibitory effect was associated with the blockage of cell cycle.Celecoxib blocked Tca8113 cell transmission from G₁ stage to S stage in a dose-dependent manner, resulting in the accumulation of G₀/G₁ stage cells,from(53.53±0.93)%to (66.70±0.98)%(40μmol╱L,24 h,P=0.000) and to(58.77±1.35)%(80μmol/L,24 h, P=0.001),while cells at S stage reduced from(35.73±0.76)%to(25.97±1.23)%(40μmol/L,24 h,P=0.000) and to(28.63±1.12)%(80μmol/L,24 h,P=0.000).Cells at G₂/M stages also decreased significantly in accordance with the changes of the percentage in cells at both G₀/G₁ and S stages.This blockage of cell cycles mainly occurred at about 24 h.Inverted microscopic analysis showed that Tca8113 cells treated by Celecoxib was significantly inhibited,showing with less cytoplasmic processes,cell shrinkage,increase of cytoplasmic granules,nuclear pycnosis,even dead cell fragments.In HE staining sections showes cell shrinkage,reduction of karyoplasmic ratio and nucleolus number,occurrence of cytoplasmic vacuoles,and finally apoptotic cells.In electron microscopy,after exposure to the Celecoxib, Tca8113 cells became rounded in shape with higher electron dense,degenerated organelles,and condensed nuclear chromatin,but intact cell boundary with the increase of Celecoxib levels and the incubating durations.The Tca8113 cells lost their superficial villi and became early-staged apoptotic cells.Meanwhile,we also found the co-existence of the early-and later-staged apoptotic cells.Further exposure to the agent results in mid- even later- staged apoptotic cells with many apoptotic bodies.AO/EB fluorescence double staining showed that early-staged apoptotic cells were found AO-and EB-positive in experiment groups with flavo-green fluorescence on one side of the nuclei,forming a crescent-shaped or a granular structure.With the increase of concentrations and exposure periods,apoptosis became more prominent with the nuclei being stained reddish yellow by EB,karyopyknotic and eccentric. Membranous budding and vacuolization occurred,forming a sunflower-shaped structure.Cell necrosis was found under severe conditions.FCM analysis of V-FITC/PI-double labeled Annexin revealed that Celecoxib(40,80μmol/L) treatment resulted in significant early apoptosis with cell apoptosis rates of (3.86±0.23)%,(15.25±0.87)%and(7.17±0.64)%,(20.06±1.06)%at 24,48 h of incubation,respectively,as compared with control ones of 0%and(1.057±0.10)%at 24,48 h(P=0.000).The results indicate that Celecoxib inhibit cell proliferation in a dose-dependent manner.Conclusion:Celecoxib inhibits growth and proliferation of Tca8113 cells mainly in a dose-dependent manner by inhibiting cell cycle process and by inducing cell apoptosis.â…¢.COX-2 inhibitor Celecoxib enhances the lethal effects of anticancer agents in Tca8113 cell line of HTSCCObjective:To explore the role of Celecoxib in enhancing the lethal effects of anticancer agents in Tca8113 cell line.Methods:24 h after cultivation of Tca8113 cell line,different concentrations of Celecoxib,CDDP and BLM were added into the cells, with the final concentrations being 2.5,5,10,20,40,80μmol/L(Celecoxib),0, 3.125,6.25,12.5,50 mg/L(CDDP) and 0,3.125,6.25,12.5,50 mg/L(BLM), respectively.MTT was used to calculate cell growth inhibition rate after incubation for 24,48,72 h,respectively.According to IC₅₀ of the above agents,optimal concentration for interaction of Celecoxib was selected(drug concentration＜IC₅₀). We combined Celecoxib(10μmol/L) with CDDP(3.12,6.25,12.5 mg/L) and BLM (3.12,12.5,50 mg/L) to treat Tca8113 cells,respectively.Lethal effects of different drug applications in Tca8113 cells after incubation for 24,48 and 72 h were measured by MTT.Flow cytometry(FCM) was used to evaluate the effects of combined drug applications on cell cycles.Experimental data were statistically processed by S-N-K test first.Jin Zheng Jun's Method was used to calculate q values,the criterion for evaluating the interaction of Celecoxib and anticancer drugs,q=1±0.15 indicates additive effect;q＞1.15 means synergistic effect;and q＜0.85 refers to antagonism. Results:Celecoxib(10μmol/L) combined either with CDDP or with BLM showed synergism or additive lethal effect on Tca8113 cell line.The way of interaction varied with the time elapsed.Additive effect was dominant after 24 h's and 72 h's incubation, while synergism was dominant between 48～72 h.Celecoxib with CDDP or BLM could notably enhance the effect of CDDP and BLM by blocking cell cycle after 24 h. The effect was most significant when it blocked G₁-staged cells into S stage,thus increasing G₀/G₁ cells distribution and reducing S-staged and G₂/M-staged cells.The percentage of G₀/G₁-staged cells increased from(58.53±1.00)%(CDDP only) to (61.83±0.50)%(CDDP+Celecoxib,P=0.001) and from(56.5±0.96)%(BLM only) to (60.93±0.32)%(BLM+Celecoxib,P=0.000),accompanied by the reduced cells at S and G₂/M stages.Cell at S stage reduced from(31.3±0.70)%to(29.63±0.64)% (CDDP,P=0.175) and(30.3±1.95)%to(30.57±0.78)%(BLM,P=0.849).Cells at G₂/M stage changed with cells at G₀/G₁ and S stages.Conclusions:Combination of Celecoxib with anticancer drug CDDP or BLM could obviously increase the lethal effect of CDDP and BLM on Tca8113 cells by inhibiting their growth and survival. Low doses of Celecoxib could significantly enhance the lethal effect of anticancer drugs CDDP and BLM on Tca8113 cells by inhibiting cell growth and proliferation through cell cycle blockage.The ways of these interactions on inhibiting Tca8113 cell growth were synergistic or/and additive.â…£.In vivo study of the antitumous effects of Celecoxib combined with cisplatin on Tca8113 cell xenograftsObjective To further investigate whether Celecoxib combined with anticancer drugs can inhibit the growth of Tca8113 cell line-transplanted tumor in tumor-bearing nude mice.Methods:Inoculate Tca8113 cells(2×10⁶ cells) into 32 Balb/c nude mice subcutaneously.Nude mice were divided randomly into 4 groups(8 mice each): control,Celecoxib treated,CDDP treated and Celecoxib+CDDP treated.Intragastric administration of Celecoxib,200 mg/kg·d^-1,intraperitoneal injection of CDDP 5 mg/kg·w^-1,or both was applied to mice in different groups.In control group, intraperitoneal injection of normal saline and oral administration of sterile purified water were performed.Body weights and tumor sizes were measured for calculating the volume of the nude mice every seven days.Nude mice were sacrificed at 35 d after drug administration.Tumor masses were dissected and weighed for calculating tumor inhibition rates.Histological and electron microscopic analysis were used to observe nude mice tumor tissues.Immunohistochemistry staining was employed for COX-2 and VEGF-C protein expression in tumor tissues.Fluorescent quantitive real-time RT-PCR was used to detect COX-2 and VEGF-C mRNA expression in transplanted tumor tissues of each group,t 7 days after inoculation of Tca8113 cells into nude mice,tumor growth was confirmed in the injection sites in all groups.Since 14 days after administration of drugs,transplanted tumors in drug-treated groups grew slowly as compared with other groups,of which is most significant in Celecoxib+CDDP group.The above-mentioned fact indicates that tumor growth be inhibited by either Celecoxib or CDDP,or both,and that Celecoxib might act in accordance with CDDP.When the experiment terminated,the average length and short diameter of the tumor in control group were notably larger than those of other treatment groups.Tumors in control group grew most quickly with an average weight of(1.28±0.15)g,while tumors in Celecoxib+CDDP group were lightest with an average weight of(0.22±0.09)g.Tumor inhibition rates were 15.63%,37.5%and 82.81%in groups of Celecoxib-treated,CDDP-treated and Celecoxib+CDDP-treated, respectively.In light microscopy,tumor cell ischemic necrosis and prominent apoptosis in some regions were found in Celecoxib-treated group.Typical cytotoxic phenomenon occurred in CDDP-treated group,showing cell degeneration and necrosis,cellular swelling,liquefaction,plasmarrhexis,karyolysis even nuclear deprivation.Apoptosis was rare.In Celecoxib+CDDP-treated group,coagulation necrosis of tumor cells and more apoptotic cells were seen.Under TEM,Apoptotic cells were found in Celecoxib-treated group,especially in Celecoxib+CDDP-treated group.Cell shrinkage,organdie degeneration,higher electron density,lesser cell volume with intact plasmalemma,condensed but homogenous nuclear chromatin, nuclear fragmentation and absence of nucleoli.Immunohistochemical results showed that the expression of COX-2 and VEGF-C was strongly positive in control group, but significantly weaker in both Celecoxib-treated and Celecoxib+CDDP-treated group.The factorial analysis of variance demonstrated that both Celecoxib and CDDP showed their main effect and interaction on growth inhibition of xenografts.The multiproportion of cycle threshold(Ct) of COX-2 mRNA and VEGF-C mRNA in each treat group,compared with control,was also analysised with factorial analysis of variance.The results indicated that both Celecoxib and CDDP showed their significanct main effect and interaction on suppression of VEGF-C mRNA expression, but no significance of main effect or interaction on suppression of COX-2 mRNA expression.Conclusion:COX-2 inhibitor Celecoxib could not only inhibit nude mice transplanted tumor growth,but also enhance the effect of cytotoxic agent CDDP on growth inhibition of xenografts of Tca8113 cell line.In conclusion,in the present study,we found that COX-2 and VEGF-C were strongly positiveexpression in tongue carcinoma tissues,and were closely correlated with the biological behaviors of squamous cell carcinoma of tongue.Selective COX-2 inhibitor Celecoxib could inhibit growth and proliferation of Tca8113 cells of HTSCC by blocking cell cycle proceeding and by inducing cell apoptosis.Meanwhile, Celecoxib could enhance the cytotoxicity of CDDP and BLM on Tca8113 cells. Celecoxib together with CDDP and BLM showed synergism or additive effects of cytotoxcity on Tca8113 cells;Celecoxib could inhibit Tca8113 cell transplanted tumor growth,and after combined application with CDDP,it manifested more significant inhibition of nude mice tumor growth Our results provide better basis for our further study of COX-2 and oral cancer,and for the prevention of COX-2 inhibitor in oral cancer.