Effect Of Combined Cyclooxygenase-2 Selective Inhibitor NS-398 And Lipoxygenase Inhibitor NDGA On Proliferation And Apoptosis In Hepatocellular Carcinoma Cell Line HepG2

Hepatocellular carcinoma (HCC) is one of the most common malignancies in China, which tends to present at an advanced stage before it is detected clinically. It is not amenable to curative therapies and the prognosis of such patients is extremely poor, with a 5-year survival of less than 10%. Cylooxygenases (COX) and lipoxygenase (LOX) are two key enzymes in the metabolism of arachidonic acid (AA). Phospholipid (PHL) in cellular membrane released AA under the effect of phosphatidolipase. AA are catalyzed to a series of biotic activators such as PG, TXA₂, HETES, LTS etc. through COX and LOX. COX and LOX effect on cell signal transduction and metabolism through these biotic activators and play an important role in many kinds of disease including tumor and inflammation. NS-398 is a selective COX-2 inhibitor, different from the traditional nonsteroidal anti-inflammatory drugs (NSAIDS), its inhibition effect on COX-1 is poor, so palliate adverse reaction of gastrointestinal tract. NS-398 can inhibit the proliferation and induce cell life cycle arrest in human hepatoma carcinoma cell. NDGA is a LOX inhibitor, also can inhibit the proliferation, induce apoptosis and cell cycle arrest at the G₁/S phase in human hepatoma carcinoma cell HepG2. The present study was designed to explore the effect of NS-398 in combination with NDGA on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2, in order to develop an effective combination therapy for HCC.OBJECTIVETo explore the effect of NS-398 in combination with NDGA or alone on the proliferation, apoptosis and expression of bcl-2 mRNA in human hepatocellular carcinoma cell line HepG2.METHODS(1) The inhibitory effect of NS-398 in combination with NDGA or alone on the proliferation of HepG2 in vitro was measured by MTT assay. The Jin's formula was used to analyze the synergic inhibitory effect.(2) Morphological evidence of apoptosis induced by NS-398 in combination with NDGA or alone of HepG2 cells was detected by TUNEL. The apoptosis index (AI) was also calculated.(3) Apoptosis of HepG2 cells caused by NS-398 in combination with NDGA or alone was detected by flow cytometry (FCM).(4) The expression of bcl-2 mRNA of HepG2 treated with NS-398 in combination with NDGA or alone was detected by RT-PCR.RESULTS(1) The MTT results suggested that NS-398 or NDGA had inhibitory effect on the proliferation of HepG2. The higher the concentration of NS-398 or NDGA was, the stronger the cytotoxic effect reached, which suggested obvious dose-dependent manner of NS-398 or NDGA. And the inhibitory effects were augmented with the prolongation of culture time (24h, 48h to 72h), which had time-dependence. NS-398(20,40,80, 160μmol/L) in combination with NDGA(25,50,100μmol/L) showed synergic effect on the proliferation of human cell line HepG2 (q >1.15), and others were addition(0.85< q <1.15 )effect. There was significant difference between the combined group and the NDGA group alone(*P<0.05,**P<0.01).(2) Results of TUNEL suggested that treatment with NS-398 (160μmol/L) or NDGA (200μmol/L) or a combination of the two agents for 48h. The AI of combination group was higher than drugs used alone, which demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.(3) FCM assay showed that treatment with NS-398 (160μmol/L) or NDGA (200μmol/L) or a combination of the two agents for 48h, the sub-G1 peak appeared before G1 phase, which represents apoptotic cell population, was observed clearly in the HepG2 cells, and the apoptotic rate of combination group was higher than drugs used alone. The results demonstrated that NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells.(4) The results of RT-PCR showed that the expression of bcl-2 mRNA of HepG2 cells in combined group or alone all decreased. There was significant difference between combined group than alone(**P<0.01).CONCLUSIONS(1) NS-398 or NDGA used alone has antiproliferative effect on the human hepatoma cell line HepG2, with an obvious dose- and time-dependent manner. NS-398 in combination with NDGA had synergistic growth-inhibitory effect on the HepG2 cells.(2) NS-398 or NDGA used alone could induce apoptosis on the human hepatoma cell line HepG2. NS-398 in combination with NDGA had synergistic apoptosis-induction effect on HepG2 cells and correlated with potentialize reducing the expression of bcl-2 mRNA probably.