Growth Of Hepatocellular Cancer Cells Research Articles

Cytotoxic Activity of Phytoconstituents Isolated from Monotheca buxifolia against Hepatocellular Carcinoma Cell Line HepG2: In Vitro and Molecular Docking Studies.

Natural products and conventional chemotherapeutic drugs are believed to enhance anticancer treatment efficacy while lowering toxicity. The current study investigates the cytotoxic and apoptogenic effects of Monotheca buxifolia bioactive compounds on HepG2 cell lines. MTT assay was used to assess the effect on the viability of HepG2 cells. Morphological changes were investigated. Annexin-V-FITC/PI was used to demonstrate apoptotic activity. A molecular dynamics simulation study was carried out to investigate the compound binding pattern in the active site of the PPRAδ protein. MTT and annexin V-FITC/PI assays revealed that the isolated compounds lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate inhibited the growth of hepatocellular cancer cells. The IC50 value for lauric acid was 56.46 ± 1.20 μg/mL, 31.94 ± 1.03 μg/mL for oleanolic acid, and 83.80 ± 2.18 μg/mL for bis(2-ethylhexyl) phthalate. Apoptosis was observed in 29.5, 52.1 and 22.4% of HepG2 cells treated with lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate, respectively, after 24 h of treatment. Morphological assays and Hoechst staining microscopy revealed that the treatment caused morphological changes in the cell membrane and nuclear condensation. The high fluctuation indicates that various interactions were highly potent and widely adopted, and vice versa. Oleanolic acid displayed high residue fluctuation, remaining stable in the active site of the PPRAδ protein and involved in various interactions while remaining locally fluctuating in the binding sites of the other two compounds. These findings concluded that lauric acid, oleanolic acid, and bis(2-ethylhexyl) phthalate have a significant apoptogenic effect against HepG2 cells in inducing apoptosis. Our findings suggest that these bioactive compounds could be used as adjuvant therapies.

Circular RNA circBNC2 facilitates glycolysis and stemness of hepatocellular carcinoma through the miR-217/high mobility group AT-hook 2 (HMGA2) axis

Hepatocellular cancer (HCC) accounts for approximately 90% of primary liver carcinoma and is a significant health threat worldwide. Circular RNA basonuclin 2 (circBNC2) is implicated with the progression of several cancers. However, its roles in carcinogenesis and glycolysis are still unclear in HCC. In this study, the levels of circBNC2 and high mobility group AT-hook 2 (HMGA2) were highly expressed, while these of miR-217 were poorly expressed in HCC tissues and cells. Upregulation of circBNC2 was related to poor prognosis and tumor node metastasis (TNM) stage. Knockdown of circBNC2 inhibited the HCC progression. Moreover, knockdown of circBNC2 suppressed the levels of Ras, ERK1/2, PCNA, HK2, and OCT4. Notably, circBNC2 functioned as a molecular sponge of microRNA 217 (miR-217) to upregulate the HMGA2 expression. The inhibitory effects of the circBNC2 silence on the growth and stemness of HCC cells, and levels of PCNA, HK2 and OCT4 were aggravated by the miR-217 overexpression, but neutralized by the HMGA2 overexpression. Besides, silencing of circBNC2 blocked the tumor growth through upregulating the expression of miR-217 and downregulating the levels of HMGA2, PCNA2, HK2 and OCT4 in vivo. Thus, the current data confirmed that circBNC2 sponged miR-217 to upregulate the HMGA2 level, thereby contributing to the HCC glycolysis and progression. These findings might present novel insight into the pathogenesis and treatment of HCC.

MicroRNA-30a-5p suppresses proliferation, invasion and tumor growth of hepatocellular cancer cells via targeting FOXA1.

[This retracts the article DOI: 10.3892/ol.2017.6745.].

Expression of miR-145-3p in hepatocellular cancer and its regulatory effect on growth of hepatocellular cancer cells

Expression of miR-145-3p in hepatocellular cancer and its regulatory effect on growth of hepatocellular cancer cells

AP-2α inhibits hepatocellular carcinoma cell growth and migration.

Transcription factor AP-2α is involved in many types of human cancers, but its role in hepatocellular carcinogenesis is largely unknown. In this study, we found that expression of AP-2α was low in 40% of human hepatocellular cancers compared with adjacent normal tissues by immunohistochemical analysis. Moreover, AP-2α expression was low or absent in hepatocellular cancer cell lines (HepG2, Hep3B, SMMC-7721 and MHHC 97-H). Human liver cancer cell lines SMMC-7721 and Hep3B stably overexpressing AP-2α were established by lentiviral infection and puromycin screening, and the ectopic expression of AP-2α was able to inhibit hepatocellular cancer cell growth and proliferation by cell viability, MTT assay and liquid colony formation in vitro and in vivo. Furthermore, AP-2α overexpression decreased liver cancer cell migration and invasion as assessed by wound healing and Transwell assays, increasing the sensitivity of liver cancer cells to cisplatin analyzed by MTT assays. Also AP-2α overexpression suppressed the sphere formation and renewed the ability of cancer stem cells. Finally, we found that AP-2α is epigenetically modified and modulates the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), β-catenin, p53, EMT, and CD133 expression in liver cancer cell lines. These results suggested that AP-2α expression is low in human hepatocellular cancers by regulating multiple signaling to affect hepatocellular cancer cell growth and migration. Therefore, AP-2α might represent a novel potential target in human hepatocellular cancer therapy.

TM-06 * MYC AND MYCN DISRUPTION OF THE MOLECULAR CLOCK IN CANCER CELLS

Cancer cells are known to have a metabolic advantage, but it is not known whether the circadian clock, which couples metabolic and cell cycles, restrains cancer cell metabolism and growth. We report that the deregulated expression of the MYC or MYCN oncogene disrupts the molecular clock by inducing NR1D1 (Rev-erbα) to diminish expression and dampen oscillation of ARNTL (Bmal1) that could be rescued by knockdown of Rev-erb. The expression of Rev-erbα is highly correlated with MYC expression in human B cell lymphoma, human T-cell acute lymphoblastic leukemia, murine hepatocellular carcinoma cells and human neuroblastoma cells. MYC or MYCN directly upregulates Rev-erbα expression by binding to its promoter. Suppression of Rev-erbα diminished hepatocellular cancer cell growth, illustrating Rev-erbα's role in MYC-induced tumorigenesis. Furthermore, Rev-erbα expression is a predictor of poor clinical outcome for human neuroblastoma and correlates with MYCN expression. Our results demonstrate an unsuspected link between oncogenic transformation and circadian arrhythmia, which may be metabolically advantageous for cancer cells. Having observed that Myc and N-Myc disrupt circadian rhythm in tumor cells, suggesting a therapeutic opportunity could exist in the window between circadian regulation of the ebb-and-flow of normal cell metabolism and the sustained, not oscillate non-circadian cancer metabolism. The understanding of these basic mechanisms from our work should lead to better cancer treatment strategies that reduce side effects and increase effectiveness.

Abstract 2784: Targeting TGF-β regulated E3 ligases: a novel therapeutic approach for primary liver cancer

Abstract Background: Hepatocellular cancer (HCC) is the 3rd leading cause of cancer death in the world, that is difficult to treat due to late diagnosis and few viable targeted therapeutics. A large number of studies have demonstrated that the TGF-β pathway plays a fundamental role in the biology of the GI tract, and as such, several components of the pathway are commonly targeted in various GI cancers. TGF-β signaling is regulated by the ubiquitin-proteasome pathway, in which E3 ubiquitin ligases recognize and target proteins for degradation by the proteasome. Numerous E3 ubiquitin ligases have been identified as negative regulators of different components of the TGF-β pathway. More recently, Praja and Keap1 have been identified as E3 ligases that ubiquitinates β2-spectrin (β2SP) in a TGF-β-dependent manner. Accordingly, Praja E3 ligase activity regulates TGF-β signaling by controlling β2SP abundance through ubiquitin-mediated degradation. Hypothesis: Specific E3 ligases, which disrupt TGF-β/β2SP/Smad3 tumor suppressor pathway lead to uncontrolled activation of chromatin and tumor formation. Therefore, small molecule inhibitors that specifically target these E3 ligases could present a novel therapeutic approach for liver cancer. Materials & Methods: Broad genome datasets including 110 cases of HCC patients were retrieved from The Cancer Genome Atlas (TCGA) and analyzed. 23 HCC and normal liver tissue specimens were immunostained with anti-Praja1 antibody. Whole mount in situ hybridization histochemistry (ISHH) assay was used to determine knock down Praja1in zebrafish embryos. A small chemical compound library was screened for E3 ligase inhibitors targeting Praja1. Results: (1) Broad genome analysis reveals that E3 ubiquitin ligases regulating the TGF-β pathway are altered in GI cancers mainly by increase in mRNA and that they negatively correlate with patient survival. (2) Praja1 expression is dramatically raised in human HCCs with loss of TGF-β signaling. (3) Praja1 inhibits TGF-β/Smad3 tumor suppressor function and increases c-Myc activities. (4) Inhibition of Praja1 leads to apoptosis and suppresses its oncogenic activities. (5) Small chemical inhibitors suppress Praja1 activities and restores TGF-β tumor suppressor function in HCC cells. (6) Small chemical inhibitors induce apoptosis and inhibit HCC cell growth and tumorigenesis. Conclusions: E3 ubiquitin ligases that modulate the TGF-β pathway are frequently increased in HCC. Praja1 presents a therapeutic target- it disrupts the TGF-β tumor suppressor pathway, and its overexpression promotes cancer cell survival and proliferation. Small molecule inhibitors such as triterpenoids that specifically target Praja1 could be very useful in HCC therapy, through targeting c-Myc, restoring TGF-β tumor suppressor function. This study may lead to new therapeutics targeting this lethal cancer and potentially a Phase I clinical trial in HCC. Citation Format: Jian Chen, Jiun-Sheng Chen, YoungJin Gi, Lior H Katz, Ji-Hyun Shin, Liem Phan, Wilma Jogunoori, Vivek Shukla, Bibhuti Mishra, Shulin Li, Milind Javle, Mien-Chie Hung, Lopa Mishra. Targeting TGF-β regulated E3 ligases: a novel therapeutic approach for primary liver cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2784. doi:10.1158/1538-7445.AM2014-2784

Rat hepatic stellate cells alter the gene expression profile and promote the growth, migration and invasion of hepatocellular carcinoma cells.

The aim of the present study was to examine the effects of activated hepatic stellate cells (HSCs) and their paracrine secretions, on hepatocellular cancer cell growth and gene expression in vitro and in vivo. Differentially expressed genes in McA-RH7777 hepatocellular carcinoma (HCC) cells following non-contact co-culture with activated stellate cells, were identified by a cDNA microarray. The effect of the co-injection of HCC cells and activated HSCs on tumor size in rats was also investigated. Non-contact co-culture altered the expression of 573 HCC genes by >2-fold of the control levels. Among the six selected genes, ELISA revealed increased protein levels of hepatic growth factor, matrix metalloproteinase-2 (MMP-2) and −9 (MMP-9). Incubation of HCC cells with medium conditioned by activated HSCs significantly increased the proliferation rate (P<0.001), migration rate and the number of invasive HCC cells (P=0.001). Co-injection of HCC cells and activated HSCs into rats significantly increased the weight of the resulting HCC tumors (P<0.01). The paracrine activity of activated HSCs markedly altered the gene expression profile of HCC cells and affected their growth, migration and invasiveness. The results from the present study indicate that the interaction between the activated HSCs and HCC has an important role in the development of HCC.

Abstract 5198: Targeting E3 ligase PRAJA1 in hepatocellular cancer .

Abstract Background: Hepatocellular Cancer (HCC) is a rising and lethal disease, that is difficult to treat due to late diagnosis and few viable targeted therapeutics. Recent studies support a key role for TGF-β signaling in suppressing these tumors. Expression of the E3 ubiquitin ligase PRAJA, a negative regulator of TGF-β signaling via Smad3 and its adaptor β2-spectrin (β2SP), is dramatically raised in up to 70% of human HCCs. This indicates that PRAJA1 overexpression suppresses Smad3 and β2SP mediated TGF-β tumor suppressor signaling hence promoting HCC progression. Hypothesis: Disruption of TGF-β/β2SP tumor suppressor pathway by PRAJA1 leads to uncontrolled activation of chromatin and tumor formation. Therefore, small molecule inhibitors that specifically target PRAJA1 could be very useful in HCC therapy. Materials &amp; Methods: Cancer patient datasets were retrieved from Gene Expression Omnibus and analyzed with Oncomine analysis tools. We analyzed 3 group-arrays of liver tumor tissues (total 298 cancer tissues vs. 249 normal tissues, p-value&amp;lt;1E-4). We also analyzed 55 arrays for other cancers (total 2850 cancer tissues vs. 779 normal tissues, p-value&amp;lt;1E-4). To validate protein expression, specimens from 13 HCC and normal liver tissues were immunostained with anti-PRAJA1. Whole mount in situ hybridization histochemistry (ISHH) assay was used to determine knock down PRAJA1in zebrafish embryos. Soft agar assay and colony formation assay were performed to elucidate PRAJA1 oncogenic activity in HCC cells. Additionally, we screened a compound library for E3 ligase inhibitors targeting PRAJA1. Results: (1) PRAJA1 expression is dramatically raised in human HCCs with loss of TGF-β signaling. (2) PRAJA1 interacts with β2SP/Smad3 and inhibits TGF-β tumor suppressor target gene activation. (3) Inhibition of PRAJA1 in the developing Zebrafish embryo and HCCs leads to high levels of apoptosis. (4) Overexpression of PRAJA1 leads to increased HCC growth; conversely knock down of PRAJA1 suppresses its oncogenic activities. (5) TGF-β promotes complex of CTCF and β2SP in nucleus. (6) RTA402/RTA405 inhibits PRAJA1 and restores TGF-β tumor suppressor function in HCC cells. (7) RTA402/RTA405 induces apoptosis and inhibits HCC cell growth and tumorigenesis. Conclusions: PRAJA1 disrupts the TGF-β tumor suppressor pathway, and its overexpression promotes cancer cell survival and proliferation. We have identified 2 novel triterpenoids that exhibit PRAJA1 inhibitory activity, which have demonstrated that suppression of PRAJA1 by specific small molecule inhibitors restores TGF-β tumor suppressor function and suppresses growth of HCC cells. Our results provide a better understanding of the mechanisms that regulate HCC development. Importantly, this study may lead to new therapeutics targeting this lethal cancer and potentially to a Phase I clinical trial in HCC. Citation Format: Jian Chen, Jiun-Sheng Chen, Vivek Shukla, Zhixing Yao, Hai-Long Piao, Wilma Jogunoori, Bibhuti Mishra, Lopa Mishtra. Targeting E3 ligase PRAJA1 in hepatocellular cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2013-5198

Abstract 3490: Targeting E3 ligases in liver cancer stem cells with disruption of TGF-β signaling

Abstract Introduction: Hepatocellular Cancer (HCC) remains the 4th common cause of cancer deaths worldwide. Because it is often diagnosed at an advanced stage in a fragile cirrhotic patient, effective therapies whether they are chemotherapy or biologically based are disappointingly few, with a narrow therapeutic index. Recent studies support a key role for TGF-β signaling in suppressing these tumors. We have previously found that deletion of Smad3/4 adaptor β2SP results in a dramatic and spontaneous formation of liver (HCC) with exon 15 mutations in 11% of human HCC. β2SP+/− and β2SP+/−/Smad3+/− mice develop visceromegaly and multiple cancers (70% of mice), including HCC spontaneously presenting a strong model of the hereditary human cancer syndrome, Beckwith-Wiedemann (BWS) as well as the stages of human HCC. High levels of E3 ligases KEAP1 and PRAJA are observed in human HCCs and in tumors of these mutant mice. Our hypothesis is that disruption of the TGF-β tumor suppressor pathway (through β2SP, Smad3 and Smad4) leads to a proliferative potential in cells that then acquire secondary events such as activation of E3 ligases (KEAP 1, PRAJA and others), presenting these as potentially powerful new targets for HCC. Methods &amp; Results: (1) β2SP+/− and β2SP+/−/Smad3+/− mice are ideal models for a human stem cell disorder (BWS) and HCC progression. (2) Inhibition of PRAJA in the developing Zebrafish embryo leads to high levels of apoptosis. (3) PRAJA is a key E3 Ligase that modulates β2SP and Smad3. (4) PRAJA levels markedly over- expressed in human HCC cell lines and tumor tissues and promotes HCC cell growth. (5) KEAP1 levels are raised in β2SP knockout tissues and human HCCs (6) Knocking-down KEAP1 inhibits HCC tumor cell growth. (7) Seven tumor suppressors are discovered to be potential targets of KEAP1 in HCC. (8) Triterpenoid derivates (Oleanolic Acid/RTA 402/RTA 405) inhibit KEAP1/Nrf2 pathway, as well as PRAJA and inhibit HCC tumor cell growth. Conclusions: Our studies demonstrate that E3 Ligases KEAP1 and PRAJA are dramatically raised in human and mouse hepatocellular cancers when TGF-β signaling is inactivated, inhibiting KEAP1 or PRAJA reduces HCC growth. Determining the synergistic therapeutic effects by targeting both KEAP1 and PRAJA will provide us important insights into development of new therapeutic targets for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3490. doi:1538-7445.AM2012-3490

Intercellular nanovesicle-mediated microRNA transfer: A mechanism of environmental modulation of hepatocellular cancer cell growth

Hepatocellular carcinoma (HCC) is characterized by a propensity for multifocality, growth by local spread, and dysregulation of multiple signaling pathways. These features may be determined by the tumoral microenvironment. The potential of tumor cells to modulate HCC growth and behavior by secreted proteins has been extensively studied. In contrast, the potential for genetic modulation is poorly understood. We investigated the role and involvement of tumor-derived nanovesicles capable of altering gene expression and characterized their ability to modulate cell signaling and biological effects in other cells. We show that HCC cells can produce nanovesicles and exosomes that differ in both RNA and protein content from their cells of origin. These can be taken up and internalized by other cells and can transmit a functional transgene. The microRNA (miRNA) content of these exosomes was examined, and a subset highly enriched within exosomes was identified. A combinatorial approach to identify potential targets identified transforming growth factor β activated kinase-1 (TAK1) as the most likely candidate pathway that could be modulated by these miRNAs. Loss of TAK1 has been implicated in hepatocarcinogenesis and is a biologically plausible target for intercellular modulation. We show that HCC cell-derived exosomes can modulate TAK1 expression and associated signaling and enhance transformed cell growth in recipient cells. Exosome-mediated miRNA transfer is an important mechanism of intercellular communication in HCC cells. These observations identify a unique intercellular mechanism that could potentially contribute to local spread, intrahepatic metastases, or multifocal growth in HCC.

Expression and functional role of a transcribed noncoding RNA with an ultraconserved element in hepatocellular carcinoma

Although expression of non-protein-coding RNA (ncRNA) can be altered in human cancers, their functional relevance is unknown. Ultraconserved regions are noncoding genomic segments that are 100% conserved across humans, mice, and rats. Conservation of gene sequences across species may indicate an essential functional role, and therefore we evaluated the expression of ultraconserved RNAs (ucRNA) in hepatocellular cancer (HCC). The global expression of ucRNAs was analyzed with a custom microarray. Expression was verified in cell lines by real-time PCR or in tissues by in situ hybridization using tissue microarrays. Cellular ucRNA expression was modulated with siRNAs, and the effects on global gene expression and growth of human and murine HCC cells were evaluated. Fifty-six ucRNAs were aberrantly expressed in HepG2 cells compared with nonmalignant hepatocytes. Among these ucRNAs, the greatest change was noted for ultraconserved element 338 (uc.338), which was dramatically increased in human HCC compared with noncancerous adjacent tissues. Although uc.338 is partially located within the poly(rC) binding protein 2 (PCBP2) gene, the transcribed ncRNA encoding uc.338 is expressed independently of PCBP2 and was cloned as a 590-bp RNA gene, termed TUC338. Functional gene annotation analysis indicated predominant effects on genes involved in cell growth. These effects were experimentally demonstrated in both human and murine cells. siRNA to TUC338 decreased both anchorage-dependent and anchorage-independent growth of HCC cells. These studies identify a critical role for TUC338 in regulation of transformed cell growth and of transcribed ultraconserved ncRNA as a unique class of genes involved in the pathobiology of HCC.

GLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

A survivin double point mutant has potent inhibitory effect on the growth of hepatocellular cancer cells

Survivin is an attractive target in cancer therapy. Previous studies have demonstrated that survivin dominant-negative mutants T34A and C84A were able to induce apoptosis in cancer cells. Given that they had different mechanisms in inducing apoptosis, our study was undertaken to determine whether a survivin double point mutant (TC34,84AA) could achieve more potent inhibitory effect on the growth of hepatocellular cancer cells. Adenoviruses expressing survivin mutants were constructed and transduced into hepatocellular cancer cells. The inhibitory effect of the survivin mutants on cancer cell growth was measured. Transduction of cancer cells with all three survivin mutants resulted in significant apoptosis. Compared with survivin mutants T34A or C84A alone, the cancer killing effect of survivin TC34,84AA was much stronger. In addition, the survivin mutants were more sensitive than wild type survivin to the degradation in the ubiquitin-proteasome pathway. Our results suggest that adenovirus-delivered dominant-negative survivin TC34,84AA promotes apoptosis-mediated hepatocellular carcinoma suppression, and could potentially be a promising candidate for cancer therapies.