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A growing number of studies have suggested that traditional Chinese medicine (TCM) plays an essential role in the development and occurrence of liver cancer. However, the function of Ruangan Lidan decoction (RLD) in liver cancer are not yet adequately identified and manifested, which attracted our attention. The key genes related to liver cancer and RLD and the upstream miRNAs of PDK4 were obtained based on bioinformatics analysis, followed by verification of the targeting relationship between miR-9-5p and PDK4. Next, Huh7 cells were treated with RLD to detect cell proliferation, colony formation, migration, invasion, and apoptosis by multiple assays with gain- and loss-of-function experiments. Moreover, subcutaneous transplanted tumor model and lung metastasis model of liver cancer in nude mice were established to further verify the functional role of RLD in liver cancer growth and metastasis via miR-9-5p/PDK4 axis. Bioinformatics analysis found that PDK4 and miR-9-5p were related to liver cancer, and PDK4 may be a downstream regulator of RLD. miR-9-5p could target and inhibit PDK4. In vitro cell experiments demonstrated that RLD suppressed liver cancer cell proliferation, invasion and migration, and promoted apoptosis by inhibiting miR-9-5p expression and promoting PDK4 expression. In vivo animal experiments further confirmed that RLD inhibited liver cancer growth and metastasis via upregulation of miR-9-5p-dependent PDK4. RLD downregulated miR-9-5p and upregulated PDK4 to inhibit the proliferation, migration, invasion, and induce apoptosis, thereby suppressing the growth and metastasis of liver cancer, highlighting a potential novel target for treatment of liver cancer.

Background or Purpose: Although human umbilical cord blood-derived mesenchymal stem cell transplantation (HUCB-MSCT) resulted in a good short-term therapeutic effect on patients with decompensated liver cirrhosis (DLC), the long-term survival remained unclear. This study aimed to evaluate the impact of HUCB-MSCT on long-term outcomes in patients with DLC., Methods: This retrospective cohort study included hospitalized patients with decompensated cirrhosis in Liuzhou Hospital of Traditional Chinese Medicine between November 2010 and February 2013. The primary outcome was overall survival (OS). The secondary outcomes were 3-year and 5-year survival rates and the occurrence rate of hepatocellular carcinoma (HCC)., Results: A total of 201 subjects were enrolled, including 36 patients who underwent HUCB-MSCT (SCT group) and 165 patients who did not (non-SCT group). After PSM (1:2), there were 36 patients in the SCT group and 72 patients in non-SCT group. The 3-year and 5-year survival rates of the two groups were 83.3% vs. 61.8% and 63.9% vs. 43.6%, and median OS time was 92.50 and 50.80 months, respectively. HUCB-MSCT treatment was found to be an independent beneficial factor for patient OS (hazard ratio = 0.47; 95% CI: 0.29-0.76; P = 0.002). There was no significant difference in the occurrence rate of HCC between the two groups (P = 0.410)., Discussion or Conclusions: HUCB-MSCT may improve long-term OS without increasing the occurrence of HCC in patients with DLC., Trial Registration: The Chinese Clinical Trial Registry (ChiCTR2100047550)., (© 2022. The Author(s).)

Inactivation of tumor suppressor genes is a major contributing alteration in the initiation or progression of cancer. The human tumor suppressor gene DLC1 (deleted in liver cancer 1) is frequently downregulated or silenced in multiple cancers, predominantly by epigenetic mechanisms. With the current considerable interest and progress in epigenetic therapy, a number of promising antineoplastic agents, particularly histone deacetylase (HDAC) inhibitors, have been developed and used successfully in clinical trials. Both DLC1 and HDAC inhibitors exert antineoplastic functions, and their combined action could be exploited for a more effective cancer therapy. To evaluate the potential benefits of this approach, we examined the antineoplastic effects of adenoviral (Ad)-DLC1-mediated transduction and exposure to suberoylanilide hydroxamic acid (SAHA), a powerful HDAC inhibitor, in two human cancer cell lines that lack intrinsic DLC1 expression, 22Rv1 prostate cancer cells and 7703K human hepatocellular carcinoma cells. Consistent with the oncosuppressive function of DLC1 in several cancers, including prostate and liver cancer, transduction of 22Rv1 and 7703K cells with an Ad-DLC1 expression vector resulted in alterations of cell morphology, induction of apoptosis, and inhibition of cell proliferation, migration, and anchorage-independent growth. A low concentration of SAHA (5 microM) efficiently restored the expression of DLC1 in 22Rv1 cells that lack DLC1 expression due to histone deacetylation but had a minimal effect in 7703K cells in which silencing of the DLC1 gene is due mainly to promoter hypermethylation. Regardless of the epigenetic mechanism of DLC1 inactivation, SAHA treatment of DLC1-transduced cells had a synergistic inhibitory effect on tumor cell proliferation and tumorigenesis in both cell lines. In 22Rv1 cells, this combination regimen nearly abolished the formation of colonies in semisolid media as a measure of tumorigenicity in vitro. Current in vitro results validate this protocol as a potentially new therapeutic option in certain cancers.

During the neoplastic process tumour cells frequently acquire resistance to the antiproliferative signals of transforming growth factor-beta (TGF-beta). Here we examined a human hepatocellular carcinoma cell line (Hep3B-TS) sensitive to TGF-beta signalling, and a derivative line (Hep3B-TR) rendered resistant to TGF-beta by stepwise exposure to TGF-beta(1). Comprehensive molecular cytogenetic analysis revealed that the acquisition of TGF-beta-resistance by Hep3B-TR cells was due to loss of TGF-beta receptor 2 (TGFbetaRII) gene. As demonstrated by spectral karyotyping and array-based comparative genomic hybridization, and in difference to Hep3B-TS cells, which have three rearranged and two normal copies of chromosome 3 that harbour the TGFbetaRII gene, Hep3B-TR cells have four rearranged and one apparently normal chromosome 3, which nonetheless underwent a critical microdeletion at the site of TGFbetaRII gene. Gene expression analysis using an oligonucleotide microarray of 21,397 genes showed that Hep3B-TR differentially expressed 307 genes, out of which 197 and 110 were up- and down-regulated, respectively, compared to Hep3B-TS. Six of differentially expressed genes were identified as downstream targets of the tumour necrosis factor (TNF) gene, suggesting that loss of TGFbetaRII triggered activation of the TNF pathway known to be regulated by TGF-beta(1) network. On the functional level, the TGF-beta-resistant Hep3B-TR cells displayed significantly enhanced capacity for anchorage independent growth and cell migration in vitro, and also increased tumorigenicity in vivo and in vitro and in vivo tumorigenicity compared with parental sensitive cells.

The process of cell dissemination from the primary tumors to distant sites is the most harmful event during cancer progression, and the leading cause of cancer death. We have previously demonstrated that restoration of DLC1 tumor suppressor gene expression in the DLC1-negative Focus and 7703K human hepatocellular carcinoma (HCC) cell lines induced caspase-3 mediated apoptosis, reduced cell growth in vitro and tumorigenicity in vivo and diminished the ability to migrate through Matrigel, a property suggestive of metastatic potential in vivo. We now show that subcutaneous tumors developing after inoculation of Focus and 7703K cells into nude mice disseminate cells to liver and lung, and this process is markedly suppressed by restoration of DLC1 expression. Inhibition of tumor cell dissemination was associated with lower levels of RhoA activity, an increase in rounded cells and a reduction in actin stress fibers and focal adhesion molecules that are of critical importance in cancer cell invasion and metastasis. In addition, DLC1 down-regulated the expression of osteopontin and matrix metalloproteinase-9, which are highly up-regulated in most primary HCC with associated metastases. These observations implicate the DLC1 gene in suppression of HCC cell dissemination and identify novel cellular and genetic alterations that contribute to prevention of metastasis, a life-threatening event in cancer progression.

Multiple regions on the chromosome arm 3p are frequently affected by loss of heterozygosity in human cancers. A candidate tumor suppressor gene is TMEM7, at 3p21.3, which encodes a transmembrane protein. TMEM7 is expressed specifically in the liver, and the encoded protein shares substantial sequence homology with human and mouse 28-kDa interferon-alpha (IFN-alpha) responsive protein. In investigation of the possible role of TMEM7 in development of hepatocellular carcinoma (HCC), we examined TMEM7 expression in 20 primary HCC and 18 HCC cell lines and found recurrent functional alterations. Although TMEM7 mRNA was expressed in normal hepatic cells, downregulation or inactivation of the gene was detected in 85% of primary HCC and 33% of HCC cell lines. To identify the mechanisms responsible, we examined genomic deletion and mutation, and also the effect of inhibitors of DNA methyltransferase and histone deacetylase on cells with low or no endogenous TMEM7 expression. Homozygous deletion of TMEM7 was not detected in 17 pairs of human HCC and corresponding noncancerous liver tissues, nor in any of the 18 HCC cell lines. TMEM7 mutation was not detected in the 18 HCC cell lines (low or normal TMEM7 expression). Treatment of two of six cell lines exhibiting downregulation or loss of TMEM7 with 5-aza-2'-deoxycytidine and trichostatin A yielded additive increase in TMEM7 expression, implicating aberrant DNA methylation and histone deacetylation in transcriptional silencing of this gene. Ectopic expression of TMEM7 in two TMEM7-deficient HCC lines suppressed cell proliferation, colony formation, and cell migration in vitro and reduced tumor formation in nude mice. Treatment of two highly invasive HCC cell lines with IFN-alpha for 7 days significantly increased TMEM7 expression and inhibited cell migration. These findings implicate loss of TMEM7 expression in hepatocarcinogenesis and suggest that modification of TMEM7 expression by IFN-alpha may have therapeutic relevance in a subset of HCC.

The gene deleted in liver cancer-1 (DLC-1) is located on human chromosome 8p21-22, a region thought to harbor tumor suppressor genes on the basis of its frequent deletion or loss of heterozygosity in a variety of human cancers, including hepatocellular carcinoma (HCC). Deletion or altered expression of DLC-1 is common in HCC. In the current study, the subcellular localization of Dlc-1 protein was determined by immunostaining with antibody to DLC-1 and the possible tumor growth suppressor activity of DLC-1 was investigated by examining the effects of of DLC-1 cDNA transfection in two human HCC cell lines lacking expression of the endogenous gene. The results show that Dlc-1protein is localized in the cell cytoplasm, and the restoration of DLC-1 expression in HCC cells resulted in caspase-3-mediated apoptosis, inhibition of cell growth and invasiveness in vitro as well as in reduction of the ability of the cells to form tumors in athymic nude mice. These observations thus support the notion that Dlc-1 protein is involved in hepatocarcinogenesis and has oncosuppressive activity in HCC.

DNA amplification in cancer cells frequently involves oncogenes whose increased expression confers a selective advantage on tumor cell growth. In an attempt to identify novel oncogenes involved in hepatocarcinogenesis, representational difference analysis (RDA) was performed using DNA from a primary human hepatocellular carcinoma (HCC) that showed high-level DNA amplifications on chromosomes 1p32 and 11q13 by comparative genomic hybridization. Ten amplification fragments were isolated by RDA, and when used to probe Southern blots of tumor DNA, there was a 5- to 50-fold increase in hybridization intensity relative to normal DNA. The sequence of one amplification product matched that of the EMS1 oncogene, which is located on chromosome 11q13 and is amplified in other cancers. We detected EMS1 amplification in 3 of 17 primary HCC. Overexpression of EMS1 mRNA was observed in 12 of 14 HCC cell lines in the absence of gene amplification or an increased copy-number of the gene. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with tumor development in HCC. EMS1 amplification and overexpresion is indicative of unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer.