Study Of The Effect And Mechanism Of Resistance To Cisplatin And Radio-therapy Mediated By TCRP1 In Oral Squamous Cell Carcinoma

Background and Objective:Oral squamous cell carcinoma (OSCC) is one of the most common types of the SCCHN. Currently, chemotherapy is the first line of treatment, especially for some advanced OSCC patients. In the past decades, cis-diaminedichloroplatinum (cisplatin, cDDP) was the most potent and indispensable chemotherapeutic agents used in the treatment of OSCC. However, parallel to high efficacy in the chemotherapy of the tumors, the resistance to cDDP became a major obstacle for its clinical application. Tongue cancer resistance-associated protein 1 (TCRP1) is a novel gene located on human chromosome 11q13.4, and was identified a new chemoresistance-related gene, which mediated specifically resistance to cisplatin in OSCC cells by reducing cells apoptosis and promoting DNA lesion repair. Base on forementioned results, in this study, first, we investgated the TCRP1 effect on resistance to cisplatin in OSCC patients, and then, the role and mechanisms of TCRP1 mediating resistance to cisplatin in vivo and in vitro will be investigated.Methods:(1) Paraffin-embedded OSCC specimens with complete clinical data were collected and made into tissue chip, and divided into sensitive or resistant group according to the DDP treatment effect. Immunohistoc-hemistry (IHC) method was used to detect the TCRP1 expression and the results were analyzed by semiquantitative integration. The relationship between TCRP1 expression and sex, age, tumor stage, metastasis and chemotherapeutic effect were analyzed.(2) The fresh OSCC specimens were collected in sterilitas conditions. Part of the tissue was made into cell suspension and planted in 96-well plates, and the sensitivity of OSCC cells to cisplatin was analyzed using MTs assay. The others were fixed, paraffin-embedded and made into tissue chip. Immunohistochemistry (IHC) method was used to detect the TCRP1 expression and the results were analyzed by semiquantitative integration. The relationship between TCRP1 expression and the sensitivity to cisplatin in vitro were analyzed.(3) The nude transplanted tumor model of TCRP1 over/silence expression was built. The tumor growth was observed and the growth curve was drawn after treated with equal volume isotonic Na chloride, cisplatin and 5-Fu, respectively. On the 16th day post treatment, the animal was sacrificed and the tumor was dissected. After undergoing fixation, paraffin imbedding, the tumor tissue was made into tissue chip and was detected the TCRP1 expression and the cells apoptsis using immunohistochemistry and TUNEL, respectively.(4) To determinate the effect of TCRP1 on OSCC cell lines radiotherapy, the radiotherapy sensitivity of TCRP overexpression/silence expression cell lines post treatment of irradiation was detected using clone formation assay, MTT assay, comet assay and apoptosis detection.(5) To analyze the effect of TCRP1 on cellular absorption and transport of cisplatin, the modulation of intracellular accumulation of cisplatin in Tca8113 cells was detected by HPLC. The drug resistance-relative protein include Akt、p-Akt、bcl-2、NF-κB、MT-I was detected using Western Blotting. Moreover, the probalble interaction between TCRP1 protein and Akt protein was analysed using co-immunoprecipitation assay.(6) IHC method was used to detect the expression of TCRP1, p-Akt, and MT in OSCC tissue chips.Results:(1) The results of IHC showed that TCRP1 mainly located in cellular nucleus and cytoplasm in OSCC cases. There were 26 cases negative or weakly positive expression of TCRP1 and 16 cases positive expression in 42 OSCC cases with clinical data. No correlation was found between TCRP1 expression and sex, age, tumor stage and node metastasis (p>0.05).22 cases showed sensitive in 26cases high expression of TCRP1 group (84.6 %), while 6 cases showed sensitive in 16 cases low expression of TCRP1 group (37.5%) (p<0.05). This result indicates that the expression of TCRP1 is correlated with chemotherapeutic effect.(2) MTs assay was used to detect the cisplatin chemotherapy sensitivity of OSCC in vitro.27 case available results were obtained from 32 specimens, among these 18cases are sensitive to cisplatin and 9 cases are resistant. The data match with clinical cisplatin sensitivity rate in principle. The IHC detection of TCRP1 expression in fresh OSCC specimens indicated that 17 cases negative or weakly positive expression and 10 cases positive expression.16 cases showed high expression of TCRP1 in 17 cases sensitive group (94.2%), while 2 cases cases showed high expression of TCRP1 in 10 cases sensitive group (20.0%) (p<0.05). This result indicates that the expression of TCRP1 is correlated with cisplatin chemotherapy sensitivity in vitro.(3) Treatment of 5-Fu showed significent effect on inhibition of all tumor growth comparing with treatmen of saline in the nude transplant tumor models. While treatment of cDDP only showed significent inhibited effect on growth of tumor in TCRP1-deficient transplant tumor animal models. Moreover, TUNEL detection showed that there were more apoptosis cell population in TCRP1-deficient transplant tumor tissue,(4) The surviving fraction at 2Gy (SF2) of Tca8113/TCRP1 and Tca8113/vector cells were 0.69±0.04 and 0.35±0.05, respectively. SF2 of Tca8113/PYM/siRNA and Tca8113/PYM-control cells were 0.43±0.01 versus 0.78±0.07, respectively. Comet assay showed that the mean value of tail moment was significantly higher in the TCRP1-deficient cells; in contrast, the mean tail moment was markedly decreased to basal levels in the TCRP1-proficient cells. These suggesting that the expression of TCRP1 closed correlated with the repair of radiation-induced damage. Using Hoechst33258 staining, we observed that at 24 hours post-IR at 4 Gy, TCRP1-proficient cells undergo less apoptosis population compared with TCRP1-deficient cells. Western blot analysis indicated that the basal expression levels of critical proapoptotic proteins such as caspase-3, caspase-8, and caspase-9 were significantly higher in TCRP1-deficient cells, while the antiapoptotic proteins Bcl-2 levels were elevated in TCRP1-proficient cells.(5) The results of HPLC showed that the intracellular accumulation of cDDP was not significantly different among several Tca8113 cell lines. The results of western showed that TCRP1-deficient cells accompanied with down-regulation of Akt, p-Akt, NF-κB,MT-1 and bcl-2. The further co-immunoprecipitation assay confirmed that there existed interaction in physical pattern between TCRP1 and Akt. Moreover, the positive regulated effect between TCRP1 and MT-1 was confirmed by western blotting. The IHC detection showed that Akt, NF-κB and MT-1 were up-regulation in TCRP1 up-regulation OSCC tissue.Conclusion:(1) TCRP1 could predict the chemo-radio chemotherapy sensitivity in OSCC and might be a novel molecular mark on resistance to chemo-radio chemotherapy;(2) TCRP1 might mediate resistance to cisplatin in OSCC through increasing the resistance to apoptosis induced by cisplatin;(3) The mechanism of mediating resistance to irradiation by TCRP1 might be correlation with the increasing DNA damage repairing induced by radiotherapy;(4) Expression of TCRP1 mainly located in cellular nucleus and cytoplasm and do not affect the expression of membrane protein. This means fuctions of TCRP1 is not correlation with "drug pump";(5) The Akt cell signal pathway and MT-1 might be one of the most important mechansism of mediating resistance to cisplatin by TCRP1.