ABSTRACT
Protein kinase (PK) N1, also called PKC-related protein 1, participates in the proliferation, invasion and metastasis of various malignant tumors. However, the role of PKN1 in liver cancer remains to be elucidated. The present study investigated the expression of PKN1 using immunohistochemistry in surgical specimens from 36 patients and analyzed the correlation with VEGF, microvascular density (MVD), cell proliferation index (Ki67) and clinicopathological parameters. PKN1 was highly expressed in hepatocellular carcinoma (HCC) and was positively correlated with histological grading of HCC, Ki67 expression and MVD. PKN1 expression in moderately and poorly differentiated HCC was significantly higher compared with highly differentiated HCC. Expression of PKN1 was positively correlated with Ki67 and MVD, and Ki67 expression was positively correlated with MVD. The effects of PKN1 on proliferation, invasion and apoptosis of liver cancer cells were detected in vitro. Cell viability, migration and invasion were reduced and the apoptosis rate was significantly improved when PKN1 expression was silenced in liver cancer cells. Thus, PKN1 serves an important role in the development and progression of liver cancer. Inhibition of PKN1 activity may provide a promising therapeutic target for liver cancer.
ABSTRACT
Resveratrol is a common, naturally occurring polyphenol confirmed with inhibited the cellular effects of carcinogenesis. However, the molecular mechanism underlying resveratrol's action against hepatocellular carcinoma (HCC) is still unclear. In addition, MARCH1 promotes the initiation and progression of HCC, but it is unclear whether resveratrol exerts antitumor efforts by regulating MARCH1 expression. This study determined the molecular mechanisms underlying the antitumor effects of resveratrol in HCC. Resveratrol induced apoptosis and inhibited the proliferation, migration, and invasion of HCC cell lines (HepG2 and Hep3B). In addition, it inhibited MARCH1 and phospho-protein kinase B (p-AKT) expression but upregulated the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) dose-dependently both in vitro and in vivo. MARCH1 knockdown by small interfering RNA (siRNA) also increased PTEN expression. Meanwhile, MK2206 (an AKT inhibitor) and bisperoxovanadium (BPV; a PTEN inhibitor) combined with resveratrol decreased MARCH1 expression more than the single-treatment HCC group. These results suggested that resveratrol affects the biological characteristics of HCC via downregulation of MARCH1 expression.
Subject(s)
Carcinogenesis/drug effects , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Resveratrol/pharmacology , Ubiquitin-Protein Ligases/metabolism , Animals , Apoptosis/drug effects , Carcinogenesis/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Disease Progression , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockdown Techniques , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Mice , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/metabolism , Resveratrol/therapeutic use , Signal Transduction/drug effects , Signal Transduction/genetics , Ubiquitin-Protein Ligases/genetics , Xenograft Model Antitumor AssaysABSTRACT
B7-H4, as a member of the B7 superfamily, was overexpressed in various types of cancers. However, the effects of B7-H4 on the aggressiveness of HCC and the underlying mechanisms have not yet been fully explored. For this purpose, B7-H4 expression was detected by Flow cytometry and Western blotting, it was highly expressed in several HCC cell lines but not in normal LO2 cell line. Knockdown B7-H4 expression induced HCC cells apoptosis by flow cytometry and colony formation assays and increased several apoptosis-related proteins, including survivin, cleaved caspase-3, cleaved caspase-7, and Bax, while the pro-growth protein survivin was reduced. Then the proliferation and cell cycle were suppressed after treated by siB7-H4. Moreover, the level of B7-H4 was significantly correlated with cell migration. In vivo, intra-tumor injection of siRNA targeting B7-H4 can significantly inhibited the growth of HepG2 cells in nude mice. Finally, regions of interest were manually traced on T1WI, T2WI, DWI and ADC of MR images. ADC values were increased in HCC xenografts after B7-H4 siRNA treatment. These data indicated that downregulation of B7-H4 suppressed the proliferation and migration and promoted apoptosis in vitro and in vivo. Blocking the B7-H4 channel might be a potential therapeutic strategy for HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , RNA, Small Interfering/genetics , V-Set Domain-Containing T-Cell Activation Inhibitor 1/genetics , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Apoptosis/genetics , Cadherins/genetics , Cadherins/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/therapy , Caspase 3/genetics , Caspase 3/metabolism , Caspase 7/genetics , Caspase 7/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Progression , Female , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/therapy , Mice , Mice, Nude , RNA, Small Interfering/metabolism , Signal Transduction , Survivin/genetics , Survivin/metabolism , Tumor Burden , V-Set Domain-Containing T-Cell Activation Inhibitor 1/antagonists & inhibitors , V-Set Domain-Containing T-Cell Activation Inhibitor 1/metabolism , Vimentin/genetics , Vimentin/metabolism , Xenograft Model Antitumor Assays , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Membrane-associated RING-CH-1 (MARCH1) is a membrane-anchored E3 ubiquitin ligase that is involved in a variety of cellular processes. MARCH1 was aberrantly expressed as a tumour promoter in ovarian cancer, but the signalling about the molecular mechanism has not yet been fully illuminated. Here, we first determined that MARCH1 was obviously highly expressed in human hepatocellular carcinoma samples and cells. In addition, our findings demonstrated that the proliferation, migration and invasion of hepatocellular carcinoma were suppressed, but the apoptosis was increased, as a result of MARCH1 knockdown by either siRNA targeting MARCH1 or pirarubicin treatment. Conversely, the proliferation, migration and invasion of hepatocellular carcinoma were obviously accelerated, and the apoptosis was decreased, by transfecting the MARCH1 plasmid to make MARCH1 overexpressed. Moreover, in vivo, the results exhibited a significant inhibition of the growth of hepatocellular carcinoma in nude mice, which were given an intra-tumour injection of siRNA targeting MARCH1. Furthermore, our study concluded that MARCH1 functions as a tumour promoter, and its role was up-regulated the PI3K-AKT-ß-catenin pathways both in vitro and in vivo. In summary, our work determined that MARCH1 has an important role in the development and progression of hepatocellular carcinoma and may be used as a novel potential molecular therapeutic target in the future treatment of hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Disease Progression , Liver Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Ubiquitin-Protein Ligases/metabolism , beta Catenin/metabolism , Animals , Apoptosis/genetics , Carcinoma, Hepatocellular/diagnostic imaging , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Down-Regulation/genetics , Female , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Magnetic Resonance Imaging , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Up-Regulation/geneticsABSTRACT
Liver cancer is a very common and significant health problem. Therefore, powerful molecular targeting agents are urgently needed. Previously, we demonstrated that secalonic acid-F (SAF) suppresses the growth of hepatocellular carcinoma (HCC) cells (HepG2), but the other anticancer biological functions and the underlying mechanism of SAF on HCC are unknown. In this study, we found that SAF, which was isolated from a fungal strain in our lab identified as Aspergillus aculeatus, could inhibit the progression of hepatocellular carcinoma by targeting MARCH1, which regulates the PI3K/AKT/ß-catenin and antiapoptotic Mcl-1/Bcl-2 signaling cascades. First, we confirmed that SAF reduced the proliferation and colony formation of HCC cell lines (HepG2 and Hep3B), promoted cell apoptosis, and inhibited the cell cycle in HepG2 and Hep3B cells in a dose-dependent manner. In addition, the migration and invasion of HepG2 and Hep3B cells treated with SAF were significantly suppressed. Western blot analysis showed that the level of MARCH1 was downregulated by pretreatment with SAF through the regulation of the PI3K/AKT/ß-catenin signaling pathways. Moreover, knockdown of MARCH1 by small interfering RNAs (siRNAs) targeting MARCH1 also suppressed the proliferation, colony formation, migration, and invasion as well as increased the apoptotic rate of HepG2 and Hep3B cells. These data confirmed that the downregulation of MARCH1 could inhibit the progression of hepatocellular carcinoma and that the mechanism may be via PI3K/AKT/ß-catenin inactivation as well as the downregulation of the antiapoptotic Mcl-1/Bcl-2. In vivo, the downregulation of MARCH1 by treatment with SAF markedly inhibited tumor growth, suggesting that SAF partly blocks MARCH1 and further regulates the PI3K/AKT/ß-catenin and antiapoptosis Mcl-1/Bcl-2 signaling cascade in the HCC nude mouse model. Additionally, the apparent diffusion coefficient (ADC) values, derived from magnetic resonance imaging (MRI), were increased in tumors after SAF treatment in a mouse model. Taken together, our findings suggest that MARCH1 is a potential molecular target for HCC treatment and that SAF is a promising agent targeting MARCH1 to treat liver cancer patients.
