| Background and ObjectivesLiver cancer is a severe condition that is found worldwide. Among the diverse, histologically distinct primary hepatic neoplasms, hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for83%of all cases. The prevalence of this disease is especially severe in China due to high rate of hepatitis B virus infection in the population. Liver transplantation, surgical resection, and local-regional therapy such as transarterial chemoembolization have made great progress and play a dominant role in HCC management. However, tumor metastasis at early stage and the high frequency of tumor recurrence and/or metastasis after those treatments acquires systematic drug intervention. The approval of sorafenib, an agent that targets receptor tyrosine kinases (RTKs), as the first effective drug for systemic treatment of HCC represents a milestone in treatment of this disease. As a typical member of the RTK family, c-Met represents an intriguing target for cancer therapy. However, the role of the c-Met signal transduction pathway is less unambiguous in HCC pathology, giving rise to concerns about the feasibility of utilizing c-Met targeting approaches for HCC treatment. Recently, basic studies on des-y-carboxy prothrombin, an abnormal cytokine secreted by HCC cells, by the current authors and other researchers have highlighted the critical role of c-Met signaling in HCC progression. Second review of c-Met signal pathway and further investigation of the unclear points in this signal pathway in HCC pathology could increase the understanding of mechanisms of HCC initiation and progression and facilitate development of molecule targeted therapies for HCC.Aminopeptidase N (APN/CD13) plays a critical role in tumor invasion and metastasis and represents one of the most important molecule targets for development of anticancer drugs. Intrahepatic metastasis in early stage and the resulting high tumor occurrence after the first treatments are the most prominent characteristics of HCC. Therefore, it would be of great importance to control the invasion and metastasis of HCC. Previous studies indicated that HCC cells expressed high levels of APN/CD13, which might be related with the metastatic behavior of HCC. Thus APN/CD13inhibitors may theoretically exert anti-metastatic effects on HCC. Liver, which is the largest solid viscera with abundant blood supply, is the second predilection site to lymph nodes for metastatic tumors. Secondary liver cancer due to metastasis of ovarian carcinoma is among the most frequent types of metastatic carcinomas. Treatment would be rather difficult when multiple hepatic metastases of ovarian carcinoma exist and are resistant to the current chemotherapeutic regimens. Therefore, it is urgently needed to develop novel and effective drugs for treatment of ovarian carcinoma. APN/CD13is highly expressed in ovarian carcinoma cells and is testified to be one of the molecular targets for drug development. One of the objectives of the current project is to investigate the effectiveness of APN/CD13inhibitors in suppressing invasion and metastasis of HCC and in inhibiting the growth of ovarian carcinoma.Volume1.DCP/c-Met expression profiles and their clinical significances in HCC patientsChapter1.Tissue expression profiles and serum concentration of DCP and their clinical significances in HCC patientsObjectives:To determine the tissue expression profiles and serum levels of DCP and analyze their relationships with clinicopathological characteristics of HCC, therefore examining the possible role of DCP in initiation and progression of human HCC.Methods:The study consisted of92consecutive patients with a single primary HCC nodule who underwent surgical resection. Clinicopathological characteristics of patients were collected, including age, sex, hepatitis virus infection, liver cirrhosis, tumor stage, differentiation grade, tumor size, vascular invasion, and intrahepatic metastasis. Tissue expression features and serum levels of DCP were determined using immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA), respectively. A x2test was used to evaluate the relationship between the serum DCP levels and/or the expression of DCP in the cancer tissues of HCC patients and the clinicopathological parameters. Survival curves were calculated using the Kaplan-Meier method and compared with the results of the log-rank test. A stepwise multivariate survival analysis was performed according to the Cox regression hazards model.Results:DCP staining was positive in cancer tissues in68(68/92,73.9%) patients. The presence of DCP in HCC tissues was related with tumor growth types. Positive DCP staining in cancer tissues was more frequent in cases of infiltrative growth than in cases of expansive growth, and was more frequent in cases where no capsule formation was noted than in cases of capsule formation. These results implied that expression of DCP in cancer tissues might enhance the malignancy of HCC and promote tumor invasion to adjacent non-tumorous tissues. ELISA demonstrated that forty-one of92HCC (44.6%) showed serum DCP levels that were higher than the cut-off value of0.0625AU/ml. High serum DCP levels were significantly frequent in patients who were positive for the HBsAg than in patients who were positive for the HCVAb. The serum DCP level of the moderately differentiated HCC group was significantly higher than that of the well-differentiated HCC group. There were also significant associations between high serum levels of DCP with vascular invasion, intrahepatic metastases, stages of III and IV, and large tumor size. HCC patients with high serum DCP levels showed significantly poorer prognoses than those with low serum DCP levels.Conclusion:DCP is usually present in HCC tissues and in serum of HCC patients and is correlated with multiple clinicopathological characteristics. Expression of DCP in HCC tissues or presence of high serum levels of DCP indicated high malignancy of HCC. Results obtained in the current study suggested that DCP might play a critical role in HCC progression.Chapter2. Co-expression of DCP and c-Met in HCC tissues and its clinical significance in HCC patientsObjective:To explore whether DCP and c-Met are consistently expressed in HCC tissues and its clinical significance in predicting tumor recurrence after surgical resection.Methods:The study consisted of153consecutive patients with a single primary HCC nodule who underwent surgical resection. Clinicopathological characteristics of patients were collected, including age, sex, tumor recurrence. Tissue expressions of DCP and c-Met were determined using immunohistochemical staining, respectively. A X2test was used to evaluate the relationship between DCP/c-Met expression and tumor recurrence.Results:DCP and c-Met staining were observed in tumor areas in63.4%(97/153) and66.7%(102/153) of patients, respectively, and these figures are markedly higher than the rates at which adjacent nontumorous areas tested positive of13.1%(20/154) and28.8%(44/153). Furthermore, DCP and c-Met were consistently present or absent in HCC regions in51.0%(78/153) and20.9%(32/153) of patients, in adjacent nontumorous regions in7.2%(11/153) and65.4%(100/153) of patients, and in whole regions including HCC and adjacent nontumorous regions in58.2%(89/153) and19.6%(30/153) of patients. These results indicate that DCP and c-Met usually appeared or disappeared in HCC in a parallel manner. c-Met was found to be related to tumor recurrence in patients with HCC. When combined with DCP, c-Met is more effective at predicting non-recurrence of HCC than c-Met alone. Expression of neither DCP nor c-Met in HCC regions and adjacent regions signified a low rate of tumor recurrence after surgical resection.Conclusion:Results of the current study suggested that DCP and c-Met are commonly and concurrently expressed in HCC and their absence is associated with a low risk of tumor recurrence.Volume2. Studies on antitumor activities of c-Met inhibitor against HCCObjective:To examine the growth inhibitory effects of c-Met inhibitor SU11274on HCC cellsMethods:Two well-differentiated HCC cell strains HepG2and HuH-7and one poorly-differentiated HCC cell strain HLE were employed in this study since they were reported to express c-Met. MTT assay was used to examine the stimulatory effects of DCP, inhibitory of effects of SU11274, and comprehensive effects of co-administration of DCP and SU11274on the growth of three kinds of cells mentioned above, respectively. Western blotting assay was used to determine the effects of SU11274on the levels of c-Met, phosphorylated c-Met, phosphorylated ERK, respectively.Results:c-Met specific inhibitor SU11274inhibited the proliferation of all three kinds of cells employed in this study. The IC50values determined in both well-differentiated and poorly-differentiated cells are similar at6-9μM. DCP was demonstrated to stimulate HCC cell growth. The relative extent of growth in the presence of DCP differed among the HCC cell lines analyzed, and this difference may be due to their sensitivity to DCP. The activation of HCC cell growth by DCP was neutralized by the simultaneous addition of SU11274. Western blot analysis was performed to clarify the changes in c-Met expression or its activation profile as a result of SU11274. All HCC cell lines analyzed were positive for c-Met expression, and this level of expression in those HCC cell lines did not change significantly with the addition of SU11274. In contrast, the expression of phosphorylated-c-Met gradually decreased in a dose-dependent manner with SU11274. In addition, the expression of phosphorylated-ERK, the downstream kinase of the signal transduction pathway activated by c-Met, also decreased in a dose-dependent manner with SU11274.Conclusion:Inhibition of c-Met by the small-molecular inhibitor SU11274arrested HCC cell growth. In addition, DCP’s action as a growth factor might be neutralized by the inhibition of c-Met activation. Results obtained in the current study suggest that the functional inhibition of c-Met might be a strategy for the development of chemotherapeutic agents for HCC, and especially those that are positive for expression of DCP.Volume3. Studies on the antitumor activities of APN/CD13inhibitors against HCC and ovarian carcinomaChapter1. Studies on the effects of APN/CD13inhibitor in suppressing HCC invasion and angiogenesisObjectives:To determine the effects of APN/CD13inhibitor24F in suppressing HCC invasion and angiogenesis.Methods:HCC cells HuH-7and human umbilical vein endothelial cells (HUVECs) were employed in this study. APN/CD13was expressed in these two cell lines. The enzyme activity was measured using a spectrophotometric method with L-leucine-p-nitroanilide as a substrate of APN/CD13. The inhibitory effects of24F on the growth of HuH-7cells were determined using MTT assay. The cell invasion assay was performed using a Transwell invasion chamber. Tube formation assay was used to examine the anti-angiogenic effects of24F on HUVECs.Results:IC50of24F (the volume of24F that displayed50%inhibition of enzyme activity) was calculated to be1.88mM. HuH-7cell growth was inhibited by incubation with24F, but there was no significant difference in the inhibition rate between0-200μg/mL of24F. No acute cytotoxic effect was confirmed using microscopic observation in those analyzed concentrations of24F. The number of invading cells in24F treated group was significantly lower than that in negative control group, suggesting24F has an ability to inhibit the invasion of HuH-7cells. The number of tube-like structures of HUVECs on the surface of Matrigel was decreased by incubating HUVECs with24F, indicating that migration and tube formation of vascular endothelial cell can be inhibited by incubation with24F.Conclusion:24F can inhibit the activity of the targeted enzyme APN/CD13and suppress the invasive capacity of HCC cells. Furthermore, it was also suggested that24F functions to suppress the angiogenic phenomenon of vascular endothelial cells, which are essential events for cancer progression. Novel APN/CD13inhibitor24F is expected to serve as a multi-functional anti-cancer chemotherapeutic agent.Chapter2.Studies on the effects of APN/CD13inhibitor in suppressing the growth and angiogenesis of ovarian carcinomaObjective:To determine the effects of APN/CD13inhibitor LYP in suppressing the growth and angiogenesis of ovarian carcinoma.Methods:Human ovarian carcinoma cell lines ES-2and SKOV-3and human umbilical vein endothelial cells HUVEC were used in the current study. All these three cell lines express APN/CD13. The levels of APN/CD13on the cell surface of ES-2, SKOV-3, and HUVEC range from high to low as determined by flow cytometry. The inhibitory effects of LYP on cell growth were determined by MTT assay. Cytotoxic effects of LYP were evaluated by trypan blue assay. The enzyme activity was measured using a spectrophotometric method with L-leucine-p-nitroanilide as a substrate of APN/CD13. Flow cytometry and Western blot assay were performed to examine the effects of LYP on APN/CD13expression. Scratch assay and Transwell invasion chamber assay were conducted to measure the effects of LYP on cell migration and invasion. Tube formation assay was used to determine the anti-angiogenic effects of LYP on HUVECs. Nude mice bearing ES-2xenografts were intravenously administered with LYP for consecutive two weeks, aiming to examine the inhibitory effects of LYP on tumor growth and the tolerance to therapeutic regimens by mice. ES-2tumors were removed from the mice and then subjected to immunohistochemistry assay for CD34expression and Western blot assay for APN/CD13, VEGF, bFGF, and TGF-a expression.Results:LYP effectively inhibited ES-2cell growth as estimated by MTT assay and the trypan blue dye-exclusion test. LYP significantly suppressed APN/CD13activity on the surface of ES-2cells as measured by quantifying the enzymatic cleavage of the substrate L-leucine-p-nitroanilide. The inhibitory effects of LYP were greater than those of bestatin at the same concentrations. In contrast, LYP was a relatively weak inhibitor of SKOV-3cell growth, suggesting that LYP may inhibit ES-2cell growth via suppression of APN/CD13.Inhibition of APN/CD13expression was also demonstrated with immunofluorescent flow cytometry and Western blot analysis. Inhibitory effects of LYP were confirmed by using a mouse model in which LYP delayed the growth of ES-2xenografts in mice after2weeks of LYP injections. Inhibition of APN/CD13expression was demonstrated in the ES-2xenografts using Western blot analysis. The inhibitory effects of LYP on the ES-2xenografts were stronger than those of bestatin. No obvious side effects of LYP were observed in animals and the mean body weights of LYP treated group did not varied significantly from that of untreated group.We further examined the effects of LYP in suppressing angiogenesis. LYP did not possess cytotoxicity to HUVEC proliferation at a concentration range of5-80uM according to the MTT assay and trypan blue exclusion assay. However, APN/CD13activity on cell surface of HUVECs was suppressed in the presence of LYP as measured by quantifying the enzymatic cleavage of the substrate L-leucine-p-nitroanilide. The assays of scratch and transwell chamber showed that LYP significantly inhibited HUVEC migration and invasion through Matrigel coated polycarbonate filters. Capillary tube formation assay revealed that the number of branch points formed by HUVECs on Matrigel was reduced after incubation with LYP. The antiangiogenesis of LYP was verified in ES-2xenografts in mice. The mean vascular density (MVD) and mean vascular luminal diameter (MVLD) were markedly reduced by LYP after two weeks of intravenous injection as evaluated by CD34immunohistochemical staining. LYP suppression of cancer angiogenesis was greater than that of bestatin. The inhibition of angiogenic molecules may involve in anti-angiogenesis of LYP. The levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF-a) were decreased in ES-2xenografts after treatment with LYP as determined by Western blot analysis.Conclusion:These results indicated that the high efficacy of LYP against human ovarian carcinoma may partially relate to its abilities to inhibit APN/CD13and angiogenesis. |
PDF Full Text Request