Experimental And Clinical Study On The Effect And Mechanism Of Nimesulide On Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. Although the clinical diagnosis and management of early-stage HCC has significantly improved, HCC prognosis remains poor. Only 10-20% of patients qualify for curative surgery. The median survival of patients who have unresectable tumors is only 4 months. Chemotherapy is one of the commonly used strategies in HCC treatment, especially for unresectable patients. But antineoplastic agents often achieve limited antineoplastic activity at the expense of close to unacceptable toxicity, which have been demonstrated a consistent response rate of less than 20% when used as single-agent in inoperable HCC patients. Thus it is imperative to develop more effective and low-toxic chemopreventive and chemotherapy agents for HCC.The cyclooxygenase-2 (COX-2) is highly expressed in HCC. The expression pattern of COX-2 protein in HCC is well correlated with the differentiation grade, suggesting that abnormal COX-2 expression plays an important role in hepatocarcinogenesis. Overexpression of COX-2 may increase the resistance of apoptosis in cancer cells and secretion of cytokine such as vascular endothelial growth factor (VEGF) which, one of the most potent angiogenic factors, has been shown to play a pivotal role in tumor angiogenesis, including HCC. Thus, the reduction of the COX-2 enzyme activity or protein expression may inhibit cell growth in cancer cells. Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to inhibit COX enzymes and may be employed for the chemoprevention of cancer. Some NSAIDs such as nimesulide exhibit high selectivity for COX-2 enzyme. Inhibition of COX-2 by several inhibitors including nimesulide induces growth inhibition and marked apoptosis in cultured HCC cells. There is no convincing evidence from randomized trails that the combination chemotherapy prolongs the survival of patients with unresectable HCC better than single agents. Whether COX-2 inhibitors are beneficial in the treatment of HCC if given in combination with these effective agents such as doxorubicin with complementary mechanisms remains unknown.In this study, we will firstly investigate the growth-inhibitory effect of NIM with DOX in HCC cell lines HepG₂ and SMMC-7721 and the possible mechanism. In clinical trial, we will detect whether NIM can improves the quality of life (QOL) of the patients with hepatocellular carcinoma.1. Effect of NIM on the proliferation of hepatocellular carcinoma cell lines HepG₂ and SMMC-7721The antiproliferative effect of NIM on HepG₂ and SMMC-7721 cell lines in vitro was measured by MTT assay. The results suggested that NIM at concentrations of 25,50,100,200,400μmol·L^-1 , had inhibitory effects on the proliferation of both HepG₂ and SMMC-7721 cells. The higher the concentration of NIM was, the stronger the cytotoxic effect reached, which suggested obvious dose-dependent manner of NIM. The r values of dose-effect curves for single-agent NIM on HepG₂ and SMMC-7721 cell lines were 0.9807 and 0.9715 respectively. The IC₅₀ values were 285.41μmol·L^-1 (HepG₂) and 358.99μmol·L^-1(SMMC-7721). 2. Effect of NIM combined with DOX on the proliferation of humanå’Œhepatocellular carcinoma cell lines HepG₂ and SMMC-7721To investigate the synergistically inhibitory effects of NIM and DOX on the proliferation of HepG₂ and SMMC-7721 cell lines, three doses of NIM (25,50,100μmol·L^-1) were used to combine with DOX. The Jin's formula was used to analyse the synergistically inhibitory effect of drug combination. NIM combining with different concentrations of DOX showed different extent synergistically inhibitory effect on the proliferation of the two cell lines in appropriate concentrations.3. Possible mechanisms of the synergistic antiproliferation effect of NIM in combination with DOX on human hepatocellular carcinoma cell lines HepG₂3.1 Morphological evidence of apoptosis induced by NIM combined with DOXMorphological evidence of apoptosis was demonstrated by using acridine orange (AO) fluorescence staining. The results showed that AO permeated through cell membrane and made the nuclei appear green and chromatin appear yellow. The HepG₂ cell line treated with NIM and DOX showed the typically apoptotic changes, such as chromatin condensation, membrane blebing, deformed and fragmented nuclei, and so on.3.2 Apoptosis caused by NIM combined with DOXA Flow Cytometry (FCM) assay was performed to analyze apoptotic rate of HepG₂ cells treated with NIM or DOX or a combination of the two agents for 24 h. The sub-G₁ peak appeared before G₁ phase, which represents apoptotic cell population, was observed. The apoptotic rate of combination group was higher than drugs used alone, which demonstrated that NIM in combination with DOX had synergistic apoptosis-induction effect on the HepG₂ cell line.3.3 Co-treatment with doxorubicin and nimesulide reduced production of VEGF in hepatocellular carcinoma cells.It is not known whether the inhibition of COX-2 reduces VEGF production in hepatocellular carcinoma cells. To test this possibility, we treated HepG₂ cells with 25μM or 50μM of nimesulide and measured VEGF levels in the culture supernatants by an ELISA. Ttreatment with nimesulide alone led to significant reduction in VEGF levels. VEGF production was also significantly reduced if the cells were co-treated with doxorubicin and a lower concentration (25μM) of nimesulide, suggesting the synergistic inhibition of VEGF production in HepG₂ cells by two agents.3.4 Nimesulide inhibits the COX-2 activity in HepG₂ hepatocellular carcinoma cells.We performed the Western blot analysis to examine whether COX-2 protein is expressed in HepG₂ cells. COX-2 protein was highly expressed in HepG2 cells. As treatment with 25-100 Mm nimesulide for 24 h did not significantly alter expression levels of COX-2 protein in HepG₂ cells. Next we tested whether treatment with NIM inhibits the COX-2 enzyme activity by measuring PGE₂ levels of culture supernatant. Compared with the control, the PGE₂ production was reduced by about 80%, 65% and 40% 24 h after the treatment with 25μM, 50μM or 100μM of nimesulide, respectively, indicating the dose-dependent inhibition.4. NIM improves the quality of life of the patients with hepatocellular carcinoma After the patient were treated with NIM, the response rates for function of body, role, emotion, cognition and social in 15 scales of EOREC QLQ-30 were 30 %-44.4 %. The response rates of inertia, nausea/vomiting, pain dyspnea, sleeplessness, anorexia, constipation, diarrhea, financial straits was 20%-62.5%. The response rate of total QOLwas 50%.The mean response rate for 15 scales of EOREC QLQ-30 was 44.4%. These results display that NIM can improve QOL involving body, role, emotion, symptoms and so on.When the patients were treated with NIM and DOX, the improvement of QOL was not superior to that in the patients treated with NIM alone. The reasons for this satuation might be too earlier time for questionnaire and limited cases in combined group.To sum up, this study for the first time presents evidence that a combination of COX-2 inhibitor nimesulide and doxorubicin additively inhibits growth of the human hepatocellular carcinoma cells. This effect was mainly observed owing to increased apoptosis and reduced VEGF production. In clinical trial, we found that NIM had improved the quality of life of the patients with hepatocellular carcinoma. The study provides a new strategy that the combination of COX-2 inhibitors and doxorubicin may be effective in the treatment of human hepatocellular carcinoma.