Experimental Study Of The Apoptosis Effect Induced By TRAIL On Laryngeal Squamous Cell Carcinoma

IntroductionThe laryngeal squamous cell carcinoma (LSCC) , with the high malignant degree, the early metastasis tendency to lymph node and a bad prognosis, is one of the most primary malignant tumor occurred in head and neck. At present, the primary treatments for LSCC include operation, radiotherapy and chemotherapy. With the development of diagnosing and treatment technique, some important progresses have been made in retention of laryngeal function, prolonging patients life after applying these treatment measures. But, however, the effect of all these measures is not as good as expected in the advanced and re-occurred cancer. Therefore, in order to improve the therapeutic effect for this type cancer, the issue need badly to be solved is to find and treat the LSCC early with new medicines and techniques to reduce the damage to the normal tissue. Recently, with the development of studying of gene transformation and apoptotic signal transduction in tumor pathogenesis, it has been discovered that apoptosis induced by anti-tumor factor may kill various tumor cells and application anti-tumor factor has acquired many exciting results in treatment of some tumors. Thus this finding may provide a new therapy for the LSCC.Apoptosis, a way of programmed cell death in our body, keep a homeostasis in balance and play an important role in cleaning tumor cells, achroacyte auto reactivity, virus infected cells. The genesis of malignant tumor is a multiple stage which involves many kinds of oncogenes and anti-oncogenes. The reasons why the tumor genesis has been formerly discussed is that un-controllable cell proliferation is the mainly cause of tumor.TRAIL (Tumor necrosis factor related apoptosis induce ligand), a new member of TNF family same as TNF-αand Fasl, plays its effect after binding with the specific receptor. Compared with Fas system, the advantage of TRAIL is that it only induces apoptosis of tumor cells without obvious toxicity to the normal cells and has been considered as an anti-tumor factor.At present, the study of TRAIL application is in exploratory stage, and the effect of TRAIL in inhibiting the growth of liver cancer, breast cancer, renal carcinoma and nasopharyngeal carcinoma has been successfully proved in nude mice experimental model. But, however, the expression of TRAIL and TRAIL-receptor in LSCC is absent and the correlation between its expression and clinical pathology is not clear. There has no report about the therapeutic effect of TRAIL in LSCC. So it is necessary to do a further study about the effect of TRAIL in treating LSCC in human.In order to illustrate the mechanism of the effect of TRAIL on LSCC, RT-PCR method (reverse transcription polymerase chain reaction) and immunohistochemistry method were used to detect the distribution and expression of TRAIL and TRAILR in 68 cases of LSCC and 30 cases of laryngeal normal tissues. In vitro the apoptosis effect induced by TRAIL in laryngeal squamous Hep-2 cells was detected by the MTT method, flow cytometry, TUNEL method and transmission electron microscope. In vivo, the nude mice model of bearing laryngocarcinoma was established by using human laryngeal squamous carcinoma cell line (Hep-2).The effect for inhibiting the LSCC was studied after given TRAIL to the animals. And also, the microstructure and ultra-micro-structural changes of carcinoma during the experiment were observed under microscope and transmission electron microscope.Materials and methods1,The 68 cases of LSCC tissue were collected from the department of otorhinolaryngology at the First Affiliated Hospital of China Medical University and Second Affiliated Hospital of Harbin Medical University during March to June 2005. RT-PCR method and Immunohistochemistry method were used to detect expression of TRAIL and TRAIL receptor DR4, DR5, DcR1, DcR2.2,Human laryngeal squamous carcinoma Hep-2 cell line was treated withdifferent concentration of TRAIL in vitro. The morphologic changes of laryngeal squamous carcinoma Hep-2 cell were observed with inverted phase contrast microscope and transmission electron microscope. The inhibition ratio of tumor cells was determined by MTT colorimetric assay, the incidence of cell apoptosis was determined by flow cytometry and TUNEL method.3,In vivo, the model of bearing human laryngocarcinoma in nude mice wasestablished by using human laryngeal squamous carcinoma cell line (Hep-2). The effect of TRAIL in inhibiting the LSCC was observed after it was administered to the animals. The ultramicrostructural changes of carcinoma were studied under microscope and transmission electron microscope during the experiment.ResultsThe results of our study showed that:1,The TRAIL and TRAIL-receptor expressed both in tissues of LSCC andlaryngeal normal tissues in the experiment of the RT-PCR and immunohistochemistry. The expression of the TRAIL, DcR1 and DcR2 in LSCC was weakly but highly in laryngeal normal tissues(P<0.01. The DR4, DR5 expressed in all tissues, there was no statistical difference in both groups (P>0.05) .2,The expression of TRAIL and DcR was higher in I and II stages, and lower in III and IV stages (P<0.05) . It was not statistically different of the expression of DR between the groups (P>0.05) . The TRAIL and DcR was expressed higher in well-differentiated LSCC, but weaker in poorly-differentiated LSCC (P<0.01), the expression of DR was not significant different in both groups (P>0.05) . All of these results were archived from the experiment of RT-PCR.3,The TRAIL and DcR was highly expressed in I and II stages but weakly in III and IV stages (TRAIL P<0.05, DcR P<0.01) . The expression of DR was not statistical different between the two groups (P>0.05) , and the TRAIL and DcR was highly expressed in well-differentiated LSCC, but weakly in poor-differentiated LSCC (P<0.01), there were no significant difference of the expression of DR detected between the two groups (P>0.05) .All of these results were obtained from the immunohistochemistry study.4,Under the inverted phase contrast microscope, we discovered that the higher concentration of TRAIL was obvious different from that of low concentration. After laryngeal squamous carcinoma cell Hep-2 was treated with TRAIL, there were some changes of the carcinoma cells, including nuclear fragmentation, the disappearance of chromatospherite, the chromatin pyknosis, the increased electronic density, the chondriosome swollen and the apoptotic body detected under electron microscope.5,MTT: In vitro, all different concentrations of TRAIL inhibited laryngeal squamous carcinoma cell's growth. The inhibited growth ratio showed significant concentration-dependence. The concentrations for inducing apoptosis-ratio (TRAIL 1, 10, 100ng/ml) determined by flow cytometry was 11.49±0.36%, 22.31±0.82%, 59.64±1.10% respectively in the study group, and 3.13±0.12% in the control group, which was significantly different between these two groups(P<0.01).6,TUNEL: After the treatment with TRAIL, apoptosis occurred in the laryngeal squamous carcinoma cell Hep-2. The concentration for inducing apoptosis (TRAIL 1, 10, 100ng/ml) was 16.66±0.80%, 42.01±2.58%, 68.93±2.12%, and 3.32±0.19% in control group (P<0.01).7,All the experiments were successfully carried on in all the animal models of nude mice. The tumor of PBS group kept on growing and the volumes of the subcutaneous tumor were increasing. The condition of nude mice was dyscrasia. In contrast to the animals were given TRAIL (100ng/ml) , tumor grew slowly and only 1 nude mice died in the in treatment group, but 8 in the control group. The difference was significant in the average body weight and volume of the tumor tissue between the treatment group and the control group (P<0.01). And the tumor restrained percentage of the study group was 53.8%.8,The necrosis and apoptosis of the tumor cell were found under microscope and electron microscope in the control group. But, however, the growth of cells, nuclear fragmentation, the disappearance of chromatospherite and chromatin pyknosis was inhibited. And also the electron density increased and the apoptotic body detected.Conclusion1,The TRAIL and TRAIL-receptors are essentially expressed in LSCC andlaryngeal normal tissues. But expression degree and extent are different in different tissues. The expression of TRAIL and DcR has significant difference.2,In LSCC tissues, the TRIAL produces a marked effect in combining with itsreceptor, and eventually induces the apoptosis. In the normal laryngeal tissues, the decoy receptors competitively combine with the TRAIL, and thus inhibits the apoptosis.3,The level of TRAIL and DcR related with different clinical staging and different pathological stage.4,Laryngeal squamous carcinoma cell Hep-2 is sensitive to TRAIL. In vitro,TRAIL inhibits the growth of cells.The TRAIL shows a marked effect on inducing the apoptosis.5,The induced apoptosis of TRAIL shows significant concentration-independence.6,In vivo, TRAIL inhibits the growth of laryngeal tumor trough inducing apoptosis.