In Vitro And In Vivo Antiproliferative Activity Of Fenretinide On Human Heptoma And Mechanism Study

Hepatoma is one of the main complications of liver cirrhosis and a serious health problem worldwide, it is usually treated by surgical resection or liver transplantation with curative options for the patients when the disease is caught at an early stage. However, about 70% of patients are inoperable because of advanced tumor growth or liver cirrhosis and in the other hand hepatoma belongs to the group of cancers that are resistant to systemic chemotherapy. Therefore, an alternative hepatocellular carcinoma treatment strategy focused on the development of chemotherapy approaches.N-(4-hydroxyphenyl) retinamide (fenretinide, 4-HPR) is a synthetic derivative of all-trans-retinoic acid that induces apoptosis in cancer cell lines and is in clinical trials for adult and pediatric cancers. It is well tolerated during long-term oral administration, as shown in chemoprevention studies and in recent phase I trials against solid tumors. 4-HPR has been demonstrated to have cytotoxicity against various cell lines and have much fewer negative effects. Previous reports have indicated that 4-HPR induced apoptosis in vitro in several solid tumors by retinoid receptor-dependent and retinoid receptor-independent pathways and in part due to the generation of free radicals and activation of the ceramide pathway, but the molecularmechanisms of 4-HPR still remains poorly understood.Purposes: our purpose was to determine the anticancer ability of 4-HPR both in vitro and in vivo against hepatoma, elucidate its mechanism, and to expand its indications in the clinical trail.Methods:1. Cancer cell killing ability of 4-HPR was measured by MTT method/ flow cytometry;2. Tumor growth inhibiton activity of 4-HPR was measured by human xenograft tumor models in vivo;3. Mitochondria membrane potential (JCl stain) and Reactive oxygen species (ROS, DCFDA stain) were detected by flow cytometry;4. Protein (p53, Bcl-2, Bax, Pro-caspase, XIAP, PARP) expression was detected by Western blotting;5. Ceramide was detected by HPLC-MS/MS.Results:1. Cytotoxicity AssayUsing the MTT method, we determined the cytotoxic activity of 4-HPR towards three human heptoma cell lines (Bel-7402, Smmc-7721 and HepG2). All three cells exhibited dose-dependent sensitivity to 72-hours exposure with 4-HPR (0 - 20.0 jiM) and the IC50 value ranged from 12.5 -13.9 fj,M.2. Apoptosis Assay of 4-HPR against Bel-7402.Hepatoma cell Bel-7402 was treated with 15 uM 4-HPR for 12, 24 and 48 hours. Collect the treated cells and the control cells, and then use the flow cytometry to detect the apoptosis rate. It indicated that the increase of the apoptosis rate is in a dose-dependent manner. The percentages of apoptotic Bel-7402 cells induced by 15.0 uM 4-HPR for 0, 12,24 and 48 hour were 3%, 7%, 24% and 40% respectively.3. Effect of 4-HPR on Tumor Growth in vivo in Bel-7402 Xenografted Models.On the basis of the encouraging in vitro data, the antiproliferation activity of 4-HPR was evaluated in human tumor models xenografted in athymic mice. 4-HPR induced tumor regression and inhibited tumor growth in a dose-dependent manner. Compared with the control group, the tumor volumes were significantly inhibited {P < 0.05 - 0.01) from day 12 to day 21 in the groups treated with 4-HPR (100.0 mg/kg). From day 0 to day 21, the tumor volumes in the control group achieved a 5.6-fold increase, while tumor volumes in 4-HPR treatment groups obtained 2.0-fold (100.0 mg/kg), 2.7-fold (50.0 mg/kg) and 3.2-fold (25.0 mg/kg) increase respectively. While there was no significant difference on athymic mice body weight during the experiment.The ratio of tumour inhibition of 4-HPR were 58.3%(100.0 mg/kg),45.8%(50.0mg/kg)and 38.9% (25.0mg/kg) respectively.4. Determination of the Expression of Apoptosis-related Proteins.Incubate the Bel-7402 cells with 15.0 uM 4-HPR for 12, 24, 48 hours, collect thetreated cells and the cells without exposure to 4-HPR, followed by extracting proteins from these cells. It showed that, with the time of Bel-7402 cells incubated with drug prolonging, the expression of p53 increased visibly, accompanied by the decrease of the proportion of Bcl-2/Bax. After exposure to fenretinde for 48 hours, the expression of procaspase-3 and XIAP decreased obviously and cleaved fragment of PARP appeared.5. The effect of piceatannol on 4-HPR-induced apoptosis in Bel-7402 cells.Piceatannol (2 ng/ml) could inhibit 4-HPR-caused ceramide and ROS production, induce a loss of mitochondria membrane potential, and induced cleav of PARP, finally suppress 4-HPR triggered apoptosis in Bel-7402 cells.6> The effect of Boc-d-fmk on 4-HPR-induced apoptosis in Bel-7402 cells.Boc-d-fmk (40 \iM) could antagonize 4-HPR-triggered apoptosis through inhibiting caspase activity and the cleavage of PARP which induced by caspases..Discuss and Conclusion:4-HPR is a synthetic derivative of all-trans-retinoic acid that induces apoptosis in various cancer cell lines including human neuroblastoma cells, cervical, breast, human ovarian carcinoma cells, head and neck, lung, and human malignant hematopoietic cells, etc . Our results showed that 4-HPR exerted potent antiproliferative action on human hepatoma cells in vitro and in vivo. We also identified that apoptosis waseffectively induced by 4-HPR in human hepatoma Bel-7402 cells, which indicated that its mechanism of anti-hepatoma effect was through apoptosis pathway.In previous study, it has been reported that 4-HPR induced apoptosis by several pathways simultaneously, including RAR-dependent pathway, the Bcl-2 family pathway and the activation of caspase cascade. Bax is a Bcl-2 family member that promotes apoptosis and counters the protective effect of Bcl-2. It has been demonstrated that Bax is a downstream effector of p53-induced apoptosis and is transcriptionally regulated by p53. In our experiment, the results of Western Blotting indicated that the expression of p53 and Bax protein enhanced greatly by 4-HPR with a time-dependent manner, while the amount of Bcl-2 slightly decreased. As we all know, Bax has the ability to heterodimerize with the Bcl-2 protein, therefore, the ratio of Bcl-2 to Bax is an important symbol relative with the measurement of the occurrence of apoptosis. We analyzed the ratio and it indicated that the proportion decreased as the exposure time extended, which was convincible to make such a presumption that 4-HPR induced apoptosis of Bel-7402 through the p53 pathways. Basing on warranted evidence that p53 is an upstream regulator of Bcl-2 family and Bax is an effector of p53, we determined the expression of p53 and it displayed a similar trend as the Bax, further indicated that p53 pathways were involved in the 4-HPR-mediated apoptosis in Bel-7402 cell lines.Activation of the caspase cascade is another crucial gateway that involved in theexecution of apoptosis in a variety of cellular systems, among which, procaspase-3 is an ultimate executioner of caspase-family that is essential for the nuclear changes associated with apoptosis, including chromatin condensation. PARP is a highly conserved nuclear enzyme that binds tightly to DNA and plays a role in DNA repair, recombination, proliferation, and genomic stability. It is also known that when the caspase cascade is activated, procaspase-3 will disassemble PARP into cleaved fragments. Therefore, the emergence of the cleaved PARP is another proof to demonstrate the activation of caspase family. Our observation suggested that the expression of procaspase-3 decreased visibly at 48 hours, and cleaved PARP appeared at 48 hours too, which revealed the possible fact that the activation of caspase family is related to the 4-HPR-induced apoptosis. The inhibitors of apoptosis (IAP) family, including cIAP-1, cIAP-2, XIAP, neuronal apoptosis inhibitory protein, and surviving, were initially identified in baculovirus and highly conserved across species. These proteins act directly on caspases, distal to mitochondrial perturbation. We found that the expression of XIAP obviously decreased at 48 hours, which further demonstrated the postulation that the involvement of caspases exists in the process of 4-HPR caused apoptosis.Piceatannol, an antioxidant. was reported to exert anti-cancer activity through decrease ROS production. In the present study, we reported that piceatannol could inhibit 4-HPR-caused ceramide and ROS production, induce a loss of mitochondria membrane potential, induced uncleavage of PARP, finally suppress 4-HPR triggeredapoptosis in BeI-7402 cells. Therefore, mitochondria-related ROS pathway also did contribution to the antiproliferation of 4-HPR in Hepatoma cells.In conclusion, we have shown that 4-HPR could induce apoptosis in human heptoma cell lines both in vivo and in vitro and its mechanism may be associated with p53, caspase and mitochondria related apoptotic pathways.