Effects Of The New Hepatoprotectant SY-801 On Carcinogenesis, Drug Resistance Of Cancer Cells, Injury Of Hepatocytes And Their Mechanisms

It is known that the occurrence of primary hepatocarcinoma is closely related to viral hepatitis as well as hepatocytes injuries induced by carcinogens. If an anti-hepatitis drug has blocking or suppressing effects on the development of hepato-carcinogenesis, it would be of great value. In addition, the emergence of drug resistance of tumor cells is a major problem in cancer chemotherapy. The intrinsic and acquired resistance of carcinoma to anti-cancer agents could cause failures of chemotherapy or toxicities of many organs such as bone marrow, heart, liver, as the dosage of anti-tumor drugs increased. If a hepatoprotectant possesses activity of increasing the sensitivity of carcinoma to chemotherapy, it will improve the efficiency of chemotherapeutics and reduce their toxic and side effects. Polymorphonuclear neutrophils ( PMNs ) participate in the inflammatory lesions of chronic hepatitis. If an anti-hepatitis drug could prevent the PMNs-induced injury to hepatocytes it should contribute to the reparation of liver injuries. SY-801, a new hepatoprotectant developed by our institute, showed significantly protective action against chemical and immunological hepatic injuries in mice and rats. It also has activities of anti - duck hepatitis virus B, anti-tumor and induction of cancer cells differentiation. On the bases of these investigations, the actions of SY-801 in chemoprevention of carcinogenesis, reversal of drug resistance and protection against hepatocyte injury as well as their related mechanisms were studied in the present study so as to further theoretically explore the clinical applications of SY-801. The results were summarized in three aspects as follows:1. Chmopreventive effect on carcinogenesisThe Balb / 3T3 cells were two stage transformed by co-use of 3-methylcholanthrene ( 3-MC ) and TPA in vitro. The results showed that SY-801 at concentrations of 10^-6 - 10^-4 M could obviously inhibit the malignancy of Balb / 3T3 cells expressed in decrease of transformed foci and reduction of malignant degree of transformed cells. The potency of SY-801 to inhibit such transformation was equal to the known positive cancer chemopreventive drug all trans-retinoic acid ( RA ) at the same concentration of 10^-6 M. The experiments of cell proliferation show that SY-801 obviously inhibited the increase of cell numbers and incorporation of ³H-TdR into quiescent Balb / 3T3 cells which induced by 100 ng / ml TPA with low concentration of serum ( 2.5 % ) or 2.5μg / ml insulin in serum-free culture medium. Dot blot assay of mRNA indicated that SY-801 suppressed the TPA-induced overexpression of H-ras, c-myc and PKCαgenes, whereas, markedly improved the expression of P53.In vivo, oral administration of SY-801 (100, 200mg/kg/day) markedly suppressed diethylnitrosamine (DEN)-induced praneoplasitic lesions in rat livers. Both number and area density ofγ-glutamyltransferase positive (γ-GT⁺) foci in preneoplastic livers were obviously reduced in SY-801 treated rats. It was also observed that significant decreases of mRNA and protein expression of GST-Ï€and total GST activity as well as cytosolic GSH andα-fetal protein in SY-801 treated rats. Northern blot and dot blot assay showed that SY-801 reduced the overexpression of N-ras, c-myc and PKCαmRNA in the preneoplatic livers, whereas, P53 expression was enhanced. The Western-blot indicated that SY-801 could also inhibit the Mdr-1 protein expression of multiple drug resistance gene in DEN-induced preneoplastic livers. All the results suggested that the chemoprevantive effect of SY-801 relied on its inhibition of biochemical characteristic alternations and oncogene expressions in malignant transformation, low differentiation and hyperplastic hepatic lesions induced by DEN.In order to know the relation of DEN metabolism with its induced hepatic praneoplastic lesion, the metabolism of DEN in vitro by the hepatic microsomes from rats treated with SY-801 for three days was studied. The results indicated that the metabolism of DEN by rat hepatic microsomes was obviously enhanced as accompanied with significantly increase of hepatic microsomal P-450 content. Deethylation and particular denitrosation pathways of DEN metabolism by the hepatic microsomes from SY-801 treated rats were both enhanced. However, SY-801 showed no effect on dimethylnitrosamines (DMN) metabolism in the same conditions, although the chemical structure of DMN is similar to DEN. The increase of cytosolic and microsomal GSTs activities in SY-801 treated rat liver were also observed, especially for the GSTμclass isoenzyme as using DCNB for substrate. However, no expression of GST-Ï€by Western-blot assay in normal and SY-801 treated rat livers was found. The DNA damage and its associated unscheduled DNA synthesis (UDS) in DEN treated rat hepatocytes was also observed in vitro. Co-incubation of SY-801 for 4 and 18 hr showed inhibitory effect on UDS of rat hepatocytes induced by DEN. All the results suggest that SY-801 could increase the detoxication and elimination of DEN in inactivating forms, which result in preventing DNA damage in rat livers. In addition, SY-801 inhibited the superoxide anion (O₂^-) and hydroxyl free radicals production in Xanthane oxidase system and Fenton reaction, respectively. 2. Reversal effect on drug resistance of carcinomaThe effects of SY-801 on drug resistance of two carcinoma cell lines: vincristine (VCR)- resistant human stomatic epidermoid carcinoma KBv₂₀₀ and adriamycin (Adr)-resistant human breast carcinoma Adr^R MCF-7 and their related mechanism were investigated in vitro. The results indicated that SY-801 (2.5×10^-5～1×10^-4 M) obviously reversed the drug resistance of the two kinds of tumor cell lines in dose-dependent manner. The reversal rate of SY-801 at the concentrations of 1×10^-4, 5×10^-5 and 2.5×10^-5 M on KBv₂₀₀ resistant to VCR was 20.7, 7.3 and 2.8 folds, respectively, as compared with the untreated group. 10^-4 M SY-801 also showed reversal effect on KBv₂₀₀ cross-resistant to anti-tumor drug Taxol and Adr^R MCF-7 resistant to Adr for 13.4 and 11.1 folds, respectively. In addition, SY-801 enhanced the cytotoxic effect of VCR against sensitive KB cell line for about several times, whereas no effect on Taxol to sensitive KB cells and Adr to sensitive MCF-7 cells was demonstrated.The decrease of antitumor drugs accumulation in cells was the most outstanding characteristic of drug resistant carcinoma cell lines. It was found that the intracellular concentration of adriamycin in resistant Adr^R MCF-7 cells was just about one fifth of the sensitive MCF-7 cells. Pretreatment with SY-801 for 24-72 hr or simultaneous coincubation with SY-801 obviously elevated the Adr accumulation in Adr^R MCF-7 cells. The major mechanism for carcinoma drug resistance was postulated the reason of overexpression of P-glycoprotein in cell membrane which encode by multiple drug resistant genes (MDR). Western-blot assay indicated that treatment of KBv₂₀₀ and Adr^R MCF-7 cells with SY-801 for 12～72 hr, the Mdr-1 protein in cell membrane was markedly reduced. Immunohistochemical assay also confirmed such results. The role of GSH and GST in carcinoma drug resistance was attached importantly in recent years. The GSH content in KBv₂₀₀ and Adr^R MCF-7 cells were higher than their sensitive cells for almost about 1 fold, and the GST activity in Adr^R MCF-7 cells was about 17.4 folds as compared with MCF-7 cells. It was observed that both GSH content in KBv₂₀₀ and Adr^R MCF-7 cells as well as GST activities in Adr^R MCF-7 cells were markedly decreased after these cells were treated with SY-801 (1×10^-4 M) for 24～72 hr. Bcl-2 gene plays an important role in regulation of cell death. It could suppress the toxicity and apoptosis induced by antitumor drugs. Many drug resistant carcinoma including Adr^R MCF-7 cells show overexpression of Bcl-2 as compared with sensitive cell lines. SY-801 obviously reduced the elevation of Bcl-2 expression in Adr^R MCF-7 cells by Western blot assay. 3. Protection against polymorphornuclear neutrophils (PMNs) and hydrogen peroxide (H₂O₂) induced injuries of primary cultured rat hepatocytes, and effect on the function of PMNs.Activated PMNs show important role in acute and chronic liver inflammatory lesions. The over production of oxygen Free radicals and other inflammatory mediators from PMNs also involve in the processes of mutation and promotion of carcinogenesis. The results indicated that SY-801 protected the primary cultured rat hepatocytes from the cytotoxicity induced by A23187-stimulated rat peritoneal PMNs expressed in reduction of GPT release and morphological damages of rat hepatocytes. H₂O₂ is an important inflammatory mediator in immune and inflammatory participated cells such as PMNs. It partially mediates the cytotoxicity of PMNs toward hepatocytes. SY-801 obviously inhibited the H₂O_2- induced hepatocyte injury and lipid peroxidation in cell membrane as GPT release and MDA accumulation reduced and the cell membrane fluidity increased. The scan electromicrscopy indicated the damage of rat hepatocytes surface induced by H₂O₂, while, SY-801 showed protective effects on this damages.In addition, SY-801 influenced the activating processes and release of inflammatory mediators such as oxygen free radicals (OFRs) by PMNs. SY-801 markedly reduced the O^2- production by activating PMNs which challenged by four different kinds of stimuli FMLP,PMA,fluoride ion and PMA+A23187. The H₂O₂ production by PMA-stimulated PMNs was also inhibited. The elevation of intracellular calcium concentration of PMNs modulates many functions of PMNs such as the degranulation and OFRs production. SY-801 inhibited FMLP and A23187 induced [Ca²⁺]i elevation. All the results indicated that the protective effect of SY-801 on the PMNs induced hepatocytes injuries may through directly reducing the toxicity of H₂O₂ and suppressing the activation of PMNs and its associated over production of OFRs.In summary, it was the first time demonstrated that SY-801 with very low toxicity in vivo has actions in prevention of carcinogenesis, reversal of drug resistance and protection against hepatocyte injury mediated by PMNs. The related biochemical and molecular mechanisms of the actions of SY-801 was also elucidated in different ways and levels. The results of present study not only provide a new scientific basis for using SY-801 as a potential drug to prevent heaptocarcinogenesis and overcome drug resistance of carcinoma, but also have importantly theoretical and practical values in evaluating the clinical prospects of SY-801 in the long-term treatment of chronic viral hepatitis in the future.