In Vitro Radiosensitization And Mechanism Of Cyclooxygenase-2 Selective Inhibitor NS-398 In Hepatoma Cell Line HepG2

Background: Hepatocarcinoma is one of the most common malignant tumors not only in China but also in the world .It is reported that there are more than one million patients dying of Hepatocarcinoma every year. Its male and female incidence ranks the third and fourth most common of malignant tumors in China respectively, China accounts for about 40 percent of new cases of the whole world. Hepatocarcinoma has poor prognosis due to its bad biological behavior such as its rapid development, associated hepatic cirrhosis and so on. The most effective therapeutic method is surgical resection currently, however, curative resection is limited due to its bad biological behavior. Therefore, it becomes urgent to seek an effective and secure therapeutic method. Medical researchers have tried to treat Hepatocarcinoma with radiotherapy and acquired some effect. Nevertheless, the liver's poor tolerance to radiation leads to high incidence of radiation-induced hepatitis. In recent years, with the development of medical image and computer technique, the advanced radiotherapy methods such as 3-dimension conformal radiotherapy(3D-CRT), intensity-modulated radiotherapy(IMRT), image-guided radiotherapy(IGRT) have been applied to clinical practice, which mostly escalated the doses to the target while decreased the doses to the normal issue, hence the incidence of hepatitis decreased and the therapeutic effect was enhanced. However, it is impossible to escalate the doses to the tumor indefinitely with the existence of adjacent dose-limited tissues, so there will be not breakthrough with the development of radiation technique solely. The next step to improve therapeutic effect may lie in the invention and application of radiosensitizers, which can enhance the radiosensitivity of tumors while has no or little affect on normal tissues.In the late 1990s, cyclooxygenase-2(COX-2) has been found to be overexpressed in a broad range of human malignancies including Hepatocarcinoma, while the normal tissues around tumors have no or low levels of COX-2 expression. The overexpression of COX-2 results in high level of PGE₂, which has been found to protect tumor cells and tissues from injuries of radiation and other cytotoxic agents, thus leading to radioresistance of these tumors and detraction of radiation effect. Inhibiting COX-2 activity or PGE2-production by selective cyclooxygenase-2 inhibitors could reverse the effect of COX-2 and enhance radioresponse of these tumors. Because normal tissues do not express COX-2, selective COX-2 inhibitors should have no appreciable effect on the radioresponse of them. All of these have been verified by a series of experiments recently, either through in vitro studies of other tumor cell lines or human tumor xenografts in nude mice. Through these studies, we can deduce that selective COX-2 inhibitors greatly increase the radiotherapeutic ratio.Thus far, there has not been any radiosensitizing report of selective COX-2 inhibitors on Hepatocarcinoma. The mechanisms by which selective COX-2 inhibitors enhance tumor redioresponse are poorly understood and only limited investigations have been undergone. In our research, we studied the radiosensitizing effect of selective COX-2 inhibitor NS-398 on Hepatoma cell line HepG2, investigated the possible radiosensitizing mechanism. Which must have profound significance both in theory and in the practice of Hepatocarcinoma treatment.Objective: Selective cyclooxygenase-2 inhibitors have been reported to enhance the tumor radioresponse in vitro, but there has not been any study of Hepatocarcinoma. In the present study, we Studied the radiosensitizing effect of NS-398 on hepatoma cell line HepG2 in vitro, and preliminarily investigated several possible radiosensitizing mechanisms.Methods: Hepatoma cell line HepG2 had been treated with various concentrations (25,50,100,200μmol/L) of NS-398 before MTT assay was used to evaluate the cytotoxicity of NS-398,clonogenic assay employed to observe its effects on the radiosensitivity of the cell survival, transmission electron microscopy(TEM) used to observe changes of apoptosis in morphology, FCM performed to quantify the apoptotic percentage, real-time PCR and western blot used to detect the expression of relevant genes, and colorimetric method provided to analyze the change of caspase-3.Results : The cytotoxicity of NS-398 increased in a time-dependent and dose-dependent manner. NS-398 significantly reduced the clonogenic activity and enhanced the radiosensitivity with a SER of 1.30 at the dose of 25μmol/L, enhanced radiation-induced apoptosis(P<0.01), increased the expression of Bax mRNA, caspase-3 mRNA and Bax protein, whereas decreased the expression of COX-2 mRNA, and enhanced the activity of caspase-3 enzyme. However,there was no significant change in Bcl-2 expression(P>0.05).Conclusions: NS-398 can enhance the radiosensitivity of hepatoma cell line HepG2. Its mechanism may be associated with down-regulation the expression of COX-2 gene ,up-regulation the expression of 53 gene and induced apoptosis of HepG2 .It suggests that selective cyclooxygenase inhibitor maybe a new kind of radiosensitizer. It has good prospects for solid tumors in radiation therapy.