| Nitidine Chloride (NC) is one of the benzophenanthridine alkaloids isolated from the root of Zanthoxylum nitidum(Roxb.)DC. Our previous study showed that NC could inhibit the proliferation of tumor cell lines 7111,KB and SPC-A-1 in vitro. As far as we know, research on the anti-tumor effect of NC on hepatoma in vivo has never been reported in literature. In the current study, we investigated the anti-tumor effect of NC both in vivo and in vitro, and explored its mechanism on systemic, cellular and molecular level.Objective1. To evaluate the anti-tumor effect and explore the molecular mechanism of NC on human tumor cell lines in vitro.2. To study the anti-hepatoma effect of NC on mice and investigate its mechanism by cDNA microarrays technology.3. To investigate the inhibiting effect of NC on topoisomerase(Topo).Methods1. The cytotoxicity of NC on 22 human tumor cell lines and 3 multidrug resistance (MDR) cell lines were detected by MTT assay in vitro.2. The sarcoma 180 ( S180) and hepatoma 22 (H22) were transplanted to mice, human hepatoma was transplanted to nude mice to evaluate the anti-tumor effects of NC in vivo. 3. The effects of NC on apoptosis and cell cycle of HepG2 cells were investigated by the methods of acridine orange (AO) fluorescent staining, agarose gel electrophoresis and Annexin V-FITC/PI double staining FCM assay.4. The ultrastructural changes of HepG2 cells after NC treatment were examined by transmission electron microscope(TEM), the apoptosis of tumor organization was detected by TUNEL assay.5. The different expressed genes of human hepatocellular transplanted tumor in nude mice treated with NC were analyzed by cDNA microarrays, FQ-PCR was used to confirm the results of cDNA microarray.6. The effects of NC on Topo I/ II mediated-pBR322 DNA unwinding were measured by using agarose gel electrophoresis, the protein expression of Topo I/II in HepG2 cells was evaluated by immunohistochemical staining.Results1.NC decreased the survival rate of tumor cells in a concentration- dependent manner, the average of IC50 value for 48h was 5.52±1.42μM. Meanwhile, it showed similar inhibiting effect on three MDR cell lines (BEL7404/ADR, KBV200 and BEL7402/5-Fu) and their parental cell lines. The average resistance factor (RF) of NC was 1.03, which was much lower than those of reference drugs, including ADR, VCR and 5-FU.2. NC(2.5, 5, 10 mg·kg-1) could inhibit the growth of S180 tumor at the inhibitory ratio of 1.95%, 27.3% and 42.9% respectively. It also significantly prolonged the survival time of H22 ascitic-tumor mice .The rate of life prolong was 35.7%, 71.4% and 85.7% respectively. For human hepatocellular transplanted tumor in nude mice, the inhibitory ratio was 12.06%, 35.63% and 60.91% respectively. These results suggested that NC had significant antitumor effect in vivo.3. NC (0.75, 1.5, 3μM) could arrest cell cycle at G2/M phase. The results showed that NC could induce apoptosis of HepG2 cells at the apoptosis ratio of 17±1.23%, 25.2±3.52% and 35.9±3.19% after treated with NC for 24h, while the ratio in control group was 0.29±0.11%.When incubated with NC for 24 h, HepG2 cells showed morphological changes associated with the character of apoptosis under fluorescent microscope. Typical DNA ladder was found during gel electrophoresis.4. Condensation of chromatin at margins of nuclei, disintegration of nucleolus and vacuoles in cytoplasm were observed in NC (2.5mg·kg-1) group by TEM. The apoptosis index (AI) was 27.5±3.6% detected by TUNEL assay. These results suggested that apoptosis was induced by NC in vivo.5.Compared with control group, 369 genes differentially expressed deviation in HepG2 cells treated with NC, among which 183 genes were up-regulated and 186 genes were down-regulated. The biochemical function of these differential genes in expression profile are diverse, which include cell proliferation/apoptotic, cell cycle regulators, immune-related, DNA duplication/transcription and damage repair factors, as well as signal transduction regulators. FQ-PCR analysis was used to validate four randomly selected genes, and results were consistent with the data obtained from the cDNA gene chip.6. NC inhibited the Topo I/II -mediated relaxation of supercoiled pBR322 plasmid DNA effectively. The results of immunohistochemical staining indicated that NC could decrease the Topo I/II protein expression. Conclusion1. NC displays significant antitumor effect both in vitro and in vivo.2. Studies on anti-tumor effect of NC show that its mechanisms are probably related to these factors as follows:①NC can arrest the cell cycle at G2/M phase and induce the apoptosis of tumor cells;②NC can regulate the transcription and expression of tumor-related genes;③DNA damage and cleavage of NC on DNA double strands may also be a potential mechanism.3. NC can inhibit the catalytic activity of Topo I/II, which may be a target of its cytotoxicity.
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