Expression Of Metallothionein And Its Relationship With Cell Proliferation, Apoptosis, Drug Resistance Induced By Cisplatin In Laryngeal Squamous Cell Carcinoma

Laryngeal carcinoma is the most common malignancies in otolaryngology- head and neck surgery. Various factors are thought to be coefficient for resulting in laryngeal carcinoma. More and more research illustrated that the occurrence of the malignant tumor was relevant to losing control of cell cycle and apoptosis. Although the surgery is preferred to laryngeal carcinoma, its curative effect is not optimistic to advanced stage or relapse of laryngeal carcinoma. So we want to pass the pathogenesy of laryngeal carcinoma to study a new way of more valid treatment to elevate survival rate. This has become an imminent problem in the process of laryngeal carcinoma therapy. In recent years, it was researched to indicate the close relation between MT with cell proliferation and apoptosis. Now it isn't reported about the expression of MT in laryngeal carcinoma. Our study primarily explored the relationship between MT and cell cycle and apoptosis of laryngeal squamouse carcinoma, and drug resistance induced cisplatin, for more elucidating mechanism of MT in the process of development and treatment of laryngeal carcinoma. It hopefully offers a new way for curing laryngeal carcinoma.1. The expression and clinic significance of Metallothionein and isoforms in laryngeal squamous cell carcinomaImmunohistochemical technique was used to examine expression of MT in 43 cases of laryngeal cancer and 12 cases of mucosa tissues of control groups in protein level, to analyse the relationship of MT protein and clinic pathological biological action. Our results showed positive stainings of MT protein were localized in cell nuclei and plasm. The positive rates of MT in laryngeal cancer were significantly higher than those in control groups (P<0.01), and had a close relation with pathological grades (P<0.05). But no significantly different expression of MT was found in different clinic stages(T grade) and primary location of laryngeal cancer. Reverse transcription-polymerase chain reaction (RT-PCR) was for more used to test MT isoforms in molecular level. The result showed that there was an incomplete consistency among diverse isofrms expression in laryngeal carcinoma. Expression of MT isoforms MT-1f, MT-1h and MT-2a mRNA in laryngeal cancer were significantly higher than those in control groups (P<0.01). MT-1f and MT-2a isoforms had a close relation with pathological grades (P<0.05). But no significantly different expression of MT-1f and MT-2a were found in different clinic stages(T grade) and primary location of laryngeal cancer. Expression of MT-1a decreased in laryngeal cancer. Expression of MT-1x were only found in control groups. It is indicated that more poorly differentiated laryngeal carcinoma, more high MT expression. MT-1f and MT-2a isoforms have a significantly relationship with pathological grades and cellular proliferation. MT-1a isoform may be related to apoptosis. MT plays an important role in the procession of laryngeal cancer. MT could be one of biology markers of laryngeal carcinoma malignancy. MT set a value on determining prognosis of laryngeal cancer.2. The relationship among cell cycle, apoptosis and expression of MT in laryngeal squamous cell carcinomaThe immunohistochemical method and flow cytometry(FCM) parameter analyses were combined to detect the cell cycle, the apoptosis and the expression of MT in each phase of the cell cycle in 24 cases of laryngea carcinomas and l0 cases of mucosa tissues of control groups. The results showed both the SPF and the PI values in laryngeal cancer were significantly higher than those in contrl groups (P<0.01). The SPF values had a close relation with pathological grades(P<0.05). Cell percent in G0/G1 stage in laryngeal carcinoma was significantly lower than that in control groups (P<0.01). Cell percent in G2/M stage in laryngeal carcinoma was significantly higher than that in control groups(P<0.01). The both stages hadn't a close relation with pathological grades (P>0.01). MT expression in S stages in laryngeal carcinoma were significantly higher than that in control groups (P<0.01). Expression of MT upgrade mainly in S phase. In moderately and poorly differentiated, the significant increasing of MT expression in S phase was detected (P<0.01). Apoptosis rate in laryngeal carcinoma was obviously lower than that in normal tissues (P<0.01). Total MT expression were negative correlation to apoptosis in both laryngeal carcinoma and control mucosa tissues (Rs=-0.532 and Rs=-0.661, P<0.05). Our study reveals that S phase is relevant to pathological grade. Expression of MT is closely related to cell percent of S phase. MT participates in cell proliferation and apoptosis. We speculate that overexpression of MT make the S stage cells DNA largely replicate, at the same time MT could restrain apoptosis. It causes cell cycle disorder, and cell proliferation dropout of laryngeal carcinoma. It may promote tumorigenesis.3. The role of MT in the chemotherapy resistance of Hep-2 cell line induced by cisplatinThe inhibition effects of different doses of DDP on Hep-2 cell line were assayed with MTT test. The immunohistochemical method and FCM parameter analyses were combined to detect the cell cycle, the apoptosis and MT expression on Hep-2 cell line induced DDP. In this study we explored the role of MT in response to DDP durg resistance, and mechanism of the chemotherapy resistance to laryngea carcinoma. DDP obviously inhibited Hep-2 cells proliferation in a manner of time- and dose-dependent. Hep-2 cells apoptosis and S phase cells were significantly increased in a dose-dependent manner. In the low DDP concentration S phase cells were not significantly higher than those in control groups (P>0.05). Hep-2 cells treated with 0.1, 0.01 and 1mg/L of DDP demonstrated a significantly increased MT compared with control groups(P<0.01). In the high DDP concentr- ation MT protein level were decreased as the concentration of DDP got higher. In the low DDP concentration MT protein level were increased in a time-dependent manner. The study has confirmed that MT mainly expresses in S phase, and the hep-2 cells disposed with the low DDP are blocked in S phase, which is mostly able to duplicate DNA. Therefore, at the same time disposed with different DDP dose, disposal of Hep-2 cells with the low DDP dose causes an increase in MT expression, which is significantly associated with resistance to cisplatin, and which may be caused by chemotherapy resistance of laryngeal carcinoma cells induced by chemotherapy agent. We consider the mechanism of MT in the resistance to DDP. One is that upgrade of MT expression could advance DNA duplication, and reinforce DNA reparation, the other one is that MT could inhibit apoptosis to induce drug resistance through clearing free radicle etc. Innovation and significance:Immunohistochemical technique and reverse transcription-polymerase chain reaction (RT-PCR) was used to examine expression of MT and isoforms in laryngeal squamous cell carcinoma. The immunohistochemical method and flow cytometry (FCM) parameter analyses were combined to detect the cell cycle, the apoptosis and the expression of MT in each phase of the cell cycle in laryngea carcinoma, for exploring the role of MT in pathogenesis of laryngea carcinoma. In vitro studies researched the expression of MT in Hep-2 cells induced cisplatin, and relation with drug resistance, to explore mechanism of the chemotherapy resistance to laryngea carcinoma. Our studies provided the theories basis for pathogenesy of laryngea carcinoma. To repress MT synthesis of tumor cells, it could be effectively cooperated with tumorous chemotherapy. These maybe offer a new way for curing laryngeal carcinoma.