Effects In Human Hepatocellular Carcinoma Research Articles

Kojic acid induces oxidative stress and anti-inflammatory effects in human hepatocellular carcinoma (HepG2) cells

The cosmetic industry makes extensive use of kojic acid (KA); however, the toxicity of KA in humans is not well known. By monitoring oxidative stress, mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signalling in human hepatoma (HepG2) cells after a 24 h exposure, this study aimed to identify the toxicity of KA. KA toxicity [4.22, 8.02 and 12.67 mM] was assessed using mitochondrial output, antioxidant responses, macromolecule damage, MAPK signalling, inflammation, and cell death markers, using spectrophotometry, luminometry, Western blot and qPCR. Apoptosis was confirmed by reduced cell viability and increased caspases −9 (p < 0.0001), −8 (p = 0.0003), and −3/7 (p < 0.0001) activities at 4.22 mM and 8.02 mM. LDH leakage was present at 12.67 mM, providing significant evidence of necrosis. Malondialdehyde (MDA) levels significantly increased at 4.22 mM (p < 0.0001). There was an increase of phosphorylated nuclear factor erythroid-2 factor-2 (p-Nrf2) at 4.22 mM and 8.02 mM, whilst at 12.67 mM decreased p-Nrf2 (p < 0.0001) was observed. KA increased p38 expression (p = 0.0011). The findings point to significant suppression of the NFκB inflammatory pathway at 8.02 mM (p < 0.0001). This study showed that KA initiated MAPK signalling due to oxidative stress and suppressed inflammation. HepG2 cells showed minimal toxicity to KA.

Resistomycin Induced Apoptosis and Cycle Arrest in Human Hepatocellular Carcinoma Cells by Activating p38 MAPK Pathway In Vitro and In Vivo.

Resistomycin, a quinone-related natural antibiotic, has shown strong inhibitory activity against human hepatocellular carcinoma (HCC) in vitro. Here, we investigated the role of p38 MAPK in the pro-apoptotic and G2/M phase arrest action of HCC HepG2 cells upon treatment with resistomycin in vitro and in vivo. Our results showed that resistomycin dose- and time-dependently reduced the viability of HepG2 cells and also showed lower cytotoxicity in normal human kidney cells (293T) and hepatocyte cells (HL-7702). Resistomycin treatment induced apoptosis and cell cycle arrest in HepG2 cells, accompanied by changes in the expression of related proteins, including Bax, Cyclin B1, etc. Surprisingly, resistomycin-mediated apoptotic cell death and cell cycle arrest were impeded by SB203580 (an inhibitor of p38 catalytic activity), suggesting that p38 MAPK signaling may play an important role that impedes eventual cell death. In this connection, data in vitro and in vivo demonstrated that resistomycin increased the phosphorylation of p38 and MAPKAPK-2 in HepG2 cells. Furthermore, we provided evidence that p38 signaling is involved in resistomycin-induced p38 MAPK pathway effects in HCC, using computer docking models. Our study indicated that resistomycin activates the p38 MAPK signaling pathway by which the growth of HepG2 cells is suppressed for apoptosis and G2/M phase arrest in vitro and in vivo, and it is a promising therapeutic leading compound for drug development in HCC treatment.

Paeonol induces antitumor effects in hepatocellular carcinoma cells through survivin via the cyclooxygenase-2/prostaglandin E2 signaling pathway.

BackgroundHepatocellular carcinoma (HCC) is one of the most life-threatening malignancies worldwide due to the lack of significant improvement in therapeutic methods. This study aimed to unravel the effects of paeonol, the main active component of Paeonia suffruticosa, on survivin, a key molecule in tumorigenesis, and elucidate the mechanisms by which paeonol exerts antineoplastic effects in human HCC cells.MethodsImmunohistochemistry (IHC) was used to study the expression levels of survivin and cyclooxygenase-2 (COX-2) in 57 human HCC tissue samples. Human HCC cell lines (HepG2 and SMMC-7721) were treated with prostaglandin E2 (PGE2). Subsequently, the cells were treated with paeonol and NS-398, and the expression levels of survivin, COX-2, and PGE2 were evaluated by Western blotting and enzyme-linked immunosorbent assay (ELISA), respectively. Fluorescence-activated cell sorting (FACS) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed to analyze cell proliferation and apoptosis.ResultsSurvivin was expressed in 47 of 57 human HCC tissue samples, as observed by IHC, and its expression was correlated with COX-2 activity. Furthermore, Western blotting showed that the expression of survivin was increased in HepG2 and SMMC-7721 cells treated with PGE2, the biosynthesis of which is mainly regulated by COX-2. Interestingly, FACS and TUNEL staining demonstrated that paeonol significantly inhibited the proliferation of HepG2 and SMMC-7721 cells and induced apoptosis, concomitant with the downregulation of survivin. The levels of COX-2 and PGE2 were also reduced by paeonol, as confirmed by Western blotting and ELISA, respectively. To determine the mechanism by which paeonol inhibited survivin in HCC cells the effects of COX-2 expression on surviving were studied. Treatment with the COX-2 selective inhibitor NS398 effectively decreased the levels of PGE2 and survivin, inducing apoptosis in a manner similar to that of paeonol. Survivin expression was increased by PGE2 treatment but was blocked by paeonol, which suggests that paeonol inhibits survivin by inhibiting the COX-2/PGE2 signaling pathway.ConclusionsTo the best of our knowledge, this is the first study to demonstrate that paeonol can exert antitumor effects on HCC cells by targeting survivin via the COX-2/PGE2 signaling pathway. Paeonol could therefore be considered as a potential therapeutic candidate for HCC.

Abstract 5312: Imeglimin alone or in combination with sorafenib showed potent anti-tumor effect in human hepatocellular carcinoma: A new kid on the block for HCC treatment

Abstract Background: Diabetes as a standalone or as part of metabolic syndrome is recognized as a major risk factor in hepatocellular carcinoma (HCC). Metformin one of the most widely used first-line drug for the treatment of type II diabetes showed antitumor activity in preclinical models of HCC, and in some clinical trials. Imeglimin, a first-in-class tetrahydrotriazine oral antidiabetic agents, is currently in phase III clinical trial in diabetes with a good safety profile. Imeglimin stands as an AMPK activator and based on historical data with metformin may be a safe potentially interesting novel therapeutic drugs for patients with HCC. Hence, the current study aims to evaluate the effects of imeglimin alone or in combination with sorafenib compared to metformin in an experimental model of HCC. Methods: All in vivo experiments were carried out with ethical committee approval and met the standards required by the UKCCCR guidelines. HepG2 HCC cells were injected subcutaneously (2 × 106cells) into the flank of female athymic nude mice. Two weeks after cell inoculation, 90% mice developed single subcutaneous palpable tumors. Mice were randomized in the groups of treatment. Mice were then treated 5 days a week by oral gavage with 75 mg/kg/day metformin (n=14-metformin75), 75 mg/kg/day imeglimin (n=14-Imeglimin75), 150 mg/kg/day imeglimin (n=14-Imeglimin150), 40 mg/kg/day sorafenib (n=14), metformin75 plus sorafenib (n=14) or imeglimin75 plus sorafenib (n=14). Body weight and tumor volumes were assessed thrice a week. Mice were sacrificed after 63 days of treatment to meet ethical requirements (tumor volume &amp;lt; 2000 mm3). Tumor were weighted, excised and OCT-embedded to prevent tissue degradation from frozen conservation. Results: Our experiments showed a 90% uptake in the engrafted mice. Regardless of the administrated treatment, no toxicity was observed as shown by the body weight follow-up. After 63 days of treatment, each treatment arm showed a slight difference with placebo (n=14). Mean tumor volumes were 1096±208 mm3 (P&amp;gt;0.05), 795±222 mm3 (P&amp;gt;0.05), 563±135 mm3 (P&amp;lt;0.05), and 495±163 mm3(P&amp;lt;0.05) for metformin75 alone, imeglimin75 alone, imeglimin150 alone or sorafenib alone, respectively, versus 1132±156 mm3 in the placebo group. Moreover, the combination of imeglimin75 plus sorafenib (375±121 mm3) displayed an increased antitumor activity compared to metformin75 plus sorafenib (515±112 mm3). In addition, at day 59 of treatment, we reached a tumor growth inhibition of 27%, 49%, and 66% in the imeglimin75 alone, imeglimin150 alone, and combination group imeglimin75 plus sorafenib, respectively. At the time of the meeting, we will be displayed IHC analysis in addition to assess tumor angiogenesis. Conclusion: This study showed the antitumor effects of imeglimin, a novel AMPK activator in human HCC. Furthermore, imeglimin potentiates the antitumor effects of sorafenib, which may represent an interesting therapeutic option for patients with HCC. Citation Format: Annemilaï Tijeras-Raballand, Matthieu Martinet, Valérie Paradis, Jean-Pierre Bizzari, Eric Janin, Eric Raymond. Imeglimin alone or in combination with sorafenib showed potent anti-tumor effect in human hepatocellular carcinoma: A new kid on the block for HCC treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5312.

Coronarin D induces human oral cancer cell apoptosis though upregulate JNK1/2 signaling pathway.

The incidence of oral cancer is increasing all over the world, with rates particularly high in Southeast Asian countries, such as Taiwan. Coronarin D (CD) has been confirmed to have anti-inflammatory, anti-bacterial effects, and anti-apoptotic effects in human hepatocellular carcinoma and nasopharyngeal carcinoma. The purpose of this study is to explore whether CD has a suppression effect on oral cancer cells and the mechanisms involved. The results of our study revealed the significantly decreased cancer cell viability and increased activation of apoptosis via increased loss of mitochondrial membrane potential, increased death receptors, leading to the activation of caspase-8, -9, -3. Moreover, the rate of apoptosis of cells treated with CD plus JNK inhibitors was decreased compared to CD-treated cells. This is the first study to demonstrate that CD induces apoptosis in human oral cancer cells and can be expected to be a promising anticancer agent for oral cancer treatment.

HUS1 checkpoint clamp component (HUS1) is a potential tumor suppressor in primary hepatocellular carcinoma.

The HUS1 checkpoint clamp component (HUS1), which is a member of an evolutionarily conserved, genotoxin-activated checkpoint complex (Rad9-Rad1-Hus1 [9-1-1] complex), is involved in cell cycle arrest and DNA repair in response to DNA damage. We conducted this study to investigate the biological significances of HUS1 expression in hepatocellular carcinoma (HCC) development. The mRNA and protein expression levels of HUS1 were determined using Real-time PCR and Western blot, respectively. One hundered and twenty four paraffin sections from HCC tissues were analyzed by immunohistochemistry to assess the association between HUS1 expression and clinicopathological characteristics of patients. The Kaplan-Meier method was performed to calculate the OS and RFS curves. Cell proliferation and colony formation assays, cell migration and invasion assays and cell cycle assays were used to determine the suppressor role of HUS1 in vitro. A mouse model was used to determine the effect of HUS1 on tumorigenesis. The expression of HUS1 was significantly decreased in HCC cell lines and tissues, and low HUS1 expression was associated with poor prognosis of HCC patients. Upregulation of HUS1 expression inhibited the cell proliferation, colony formation, migration, and invasion, as well as arrested cell cycle at G0/G1 in HCC cells in vitro. Moreover, sufficient HUS1 expression inhibited the tumor growth in nude mice. Our study revealed for the first time that HUS1 is a potential tumor suppressor that might produce an antitumor effect in human HCC. Furthermore, HUS1 may serve as a prognostic indicator and could be used for therapeutic application in HCC patients.

Anticancer effects of methanol extract of Myrmecodia platytyrea Becc. leaves against human hepatocellular carcinoma cells via inhibition of ERK and STAT3 signaling pathways.

Myrmecodiaplatytyrea Becc., a member of the Rubiaceae family, is found throughout Southeast Asia and has been traditionally used to treat cancer. However, there is limited pharmacological information on this plant. We investigated the anticancer effects of the methanol extract of Myrmecodiaplatytyrea Becc. leaves (MMPL) and determined the molecular mechanisms underlying the effects of MMPL on metastasis in human hepatocellular carcinoma(HCC) cells. MMPL dose-dependently inhibited cell migration and invasion in SK‑Hep1 and Huh7 cells. In addition, MMPL strongly suppressed the enzymatic activity of matrix metalloproteinases (MMP‑2 and MMP‑9). Diminished telomerase activity by MMPL resulted in the suppression of both telomerase activity and telomerase-associated gene expression. The levels of urokinase-type plasminogen activator receptor (uPAR) expression as well as the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) were also attenuated by MMPL. The above results collectively suggest that MMPL has anticancer effects in HCC and that MMPL can serve as an effective therapeutic agent for treating human liver cancer.

Reciprocal regulation of neutrophils and B cells elicits autoantibody-associated protumorigenic effects in human hepatocellular carcinoma

Abstract Despite recent advances in identifying autoantibody-mediated carcinogenesis in animal models, direct evidence supporting the important role of autoantibodies in the immunopathogenesis of human cancers is still lacking. We recently found that neutrophils are accumulated in hepatocellular carcinoma (HCC), and their levels positively correlated with poor survival of patients. Here we detected high titers of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic autoantibodies (ANCA) in plasma from patients with advanced stage HCCs, and that increased titers of anti-PR3 autoantibodies also correlated with poor clinical outcome of patients. Neutrophils, B cells, plasma cells all predominantly accumulated in the peritumoral stroma of HCC tumors. The neutrophils isolated from HCC tumors, but not paired blood, significantly and effectively secreted PR3 proteins; accordingly, only B cells/plasma cells derived from HCC tumors markedly produced anti-PR3 autoantibodies. Further kinetic experiments revealed addition of conditioned medium from HCC tumor neutrophils to the ex vivo induction system of plasma cells led to sequential B cell activation, somatic hypermutation, and anti-PR3 autoantibody secretion. Reciprocally, we also demonstrated that, in the presence of anti-PR3 autoantibodies, the neutrophils are more potent to produce protumorigenic factors oncostatin M and matrix metalloproteinase-9. Thus, the secretion of PR3 by tumor neutrophils may represent a novel mechanism that links the autoimmunity to disease progression in the tumor milieu.

MiR-1180 promotes apoptotic resistance to human hepatocellular carcinoma via activation of NF-κB signaling pathway.

Apoptosis resistance in human hepatocellular carcinoma (HCC) is a significant factor in carcinogenesis. Therefore, understanding the molecular mechanisms involved in apoptosis resistance is crucial for developing anticancer therapies. Importantly, small non-coding microRNAs (miRNAs) have been reported as key biomarkers for detecting tumour onset and progression. In the present study, we demonstrate that miR-1180 is upregulated in HCC. Ectopic expression of miR-1180 has an anti-apoptotic effect in HCC, while miR-1180 inhibition increases cell apoptosis, both in vitro and in vivo. Moreover, our results show that miR-1180 directly targets key inhibitors of the nuclear factor (NF)-κB signaling pathway (i.e., OTUD7B and TNIP2) and the pro-apoptotic Bcl-2 associated death promoter (BAD) protein by post-transcriptional downregulation. Therefore, the anti-apoptotic function of miR-1180 in HCC may occur through NF-κB pathway activation via downregulation of its negative regulators. In conclusion, our study reveals the critical role of miR-1180 during apoptosis resistance in HCC.

APOBEC3G exerts tumor suppressive effects in human hepatocellular carcinoma

In this study, we collected 44 hepatitis B virus surface antigen positivity HBsAg (+) tumor and nontumor hepatocellular tissues from hepatocellular carcinoma (HCC) patients during hepatectomy, and quantified the APOBEC3G (A3G) mRNA by using a real-time PCR. Our results showed higher expression of A3G mRNA in the nontumor tissues than in the tumor tissues of the HBsAg (+) HCC patients. To further investigate this phenomenon, we constructed a pLV-A3G vector and transfected it into the human HCC cell line, Hep 3B. The results of an immunofluorescence analysis showed the overexpression of A3G in the cytoplasm. We then evaluated A3G cytotoxicity by using a cell viability assay (MTS assay), the results of which showed that Hep 3B cell viability was 88 and 58% after the transfection of pLV and pLV-A3G, respectively, indicating the growth inhibitory effects of A3G on Hep 3B cells. To further evaluate the tumor suppressive effects of A3G, we used a plastic pipette tip to scratch Hep 3B cells grown on a culture dish (to 70-80% confluence) after transfection with pLV-A3G. Our data indicated a ratio of wound closure of 100% in the control cells and in the pLV-expressing cells, compared with 43% in the pLV-A3G-overexpressing cells, 72 h after the wound scratch, as observed using phase-contrast microscopy. These results indicated that A3G inhibits wound healing in Hep 3B cells. Overall, our results suggest that A3G inhibits the growth of human hepatoma cells.

Direct Association of Heat Shock Protein 20 (HSPB6) with Phosphoinositide 3-kinase (PI3K) in Human Hepatocellular Carcinoma: Regulation of the PI3K Activity

HSP20 (HSPB6), one of small heat shock proteins (HSPs), is constitutively expressed in various tissues and has several functions. We previously reported that the expression levels of HSP20 in human hepatocellular carcinoma (HCC) cells inversely correlated with the progression of HCC, and that HSP20 suppresses the growth of HCC cells via the AKT and mitogen-activated protein kinase signaling pathways. However, the exact mechanism underlying the effect of HSP20 on the regulation of these signaling pathways remains to be elucidated. To clarify the details of this effect in HCC, we explored the direct targets of HSP20 in HCC using human HCC-derived HuH7 cells with HSP20 overexpression. HSP20 proteins in the HuH7 cells were coimmunoprecipitated with the p85 regulatory subunit and p110 catalytic subunit of phosphoinositide 3-kinase (PI3K), an upstream kinase of AKT. Although HSP20 overexpression in HCC cells failed to affect the expression levels of PI3K, the activity of PI3K in the unstimulated cells and even in the transforming growth factor-α stimulated cells were downregulated by HSP20 overexpression. The association of HSP20 with PI3K was also observed in human HCC tissues in vivo. These findings strongly suggest that HSP20 directly associates with PI3K and suppresses its activity in HCC, resulting in the inhibition of the AKT pathway, and subsequently decreasing the growth of HCC.

Inhibition of hepatocellular carcinoma growth using immunoliposomes for co-delivery of adriamycin and ribonucleotide reductase M2 siRNA

The chemotherapy combined with gene therapy has received great attention. We developed targeted LPD (liposome-polycation-DNA complex) conjugated with anti-EGFR (epidermal growth factor receptor) Fab' co-delivering adriamycin (ADR) and ribonucleotide reductase M2 (RRM2) siRNA (ADR-RRM2-TLPD), to achieve combined therapeutic effects in human hepatocellular carcinoma (HCC) overexpressing EGFR. The antitumor activity and mechanisms of ADR-RRM2-TLPD were investigated. The results showed that RRM2 expression was higher in HCC than in non-HCC tissue, and RRM2 siRNA inhibited HCC cell proliferation, suggesting that RRM2 is a candidate target for HCC therapy. ADR-RRM2-TLPD delivered ADR and RRM2 siRNA to EGFR overexpressing HCC cells specifically and efficiently both in vitro and in vivo, resulting in enhanced therapeutic effects (cytotoxicity, apoptosis and senescence-inducing activity) compared with single-drug loaded or non-targeted controls, including ADR-NC-TLPD (targeted LPD co-delivering ADR and negative control siRNA), RRM2-TLPD (targeted LPD delivering RRM2 siRNA) and ADR-RRM2-NTLPD (non-targeted LPD co-delivering ADR and RRM2 siRNA). Mechanism studies showed that p21 is involved in the combined therapeutic effect of ADR-RRM2-TLPD. The average weight of the orthotopic HCC in mice treated with ADR-RRM2-TLPD was significantly lighter than that of mice treated with other controls. Thus, ADR-RRM2-TLPD represents a potential strategy for combined therapy of HCC overexpressing EGFR.

Synergistic antitumour activity of sorafenib in combination with tetrandrine is mediated by reactive oxygen species (ROS)/Akt signaling

Background:Sorafenib is a potent inhibitor against Raf kinase and several receptor tyrosine kinases that has been approved for the clinical treatment of advanced renal and liver cancer. Combining sorafenib with other agents has been shown to improve its antitumour efficacy by not only reducing the toxic side effects but also preventing primary and acquired resistance to sorafenib. We have previously observed that tetrandrine exhibits potent antitumour effects in human hepatocellular carcinoma. In this study, we investigated the synergistic antitumour activity of sorafenib in combination with tetrandrine.Methods:This was a two-part investigation that included the in vitro effects of sorafenib in combination with tetrandrine on cancer cells and the in vivo antitumour efficacy of this drug combination on tumour xenografts in nude mice.Results:Combined treatment showed a good synergistic antitumour effect yet spared nontumourigenic cells. The potential molecular mechanism may be mainly that it activated mitochondrial death pathway and induced caspase-dependent apoptosis in the cancer cells. Accumulation of intracellular reactive oxygen species (ROS) and subsequent activation of Akt may also be involved in apoptosis induction.Conclusion:The antitumour activity of sorafenib plus tetrandrine may be attributed to the induction of the intrinsic apoptosis pathway through ROS/Akt signaling. This finding provides a novel approach that may broaden the clinical application of sorafenib.

ANT2 suppression by shRNA may be able to exert anticancer effects in HCC further by restoring SOCS1 expression

Suppressor of cytokine signaling 1 (SOCS1) is a negative regulator of Janus kinase and the signal transducer and activation of transcription (Jak-STAT) pathway. SOCS-1 is known to be silenced by aberrant promoter methylation in human hepatocellular carcinoma (HCC) during early tumorigenesis, therefore, a strategy to restore SOCS1 expression can be utilized for cancer therapy. Here, we examined the influence of adenine nucleotide translocase 2 (ANT2) suppression by short-hairpin RNA (shRNA) on SOCS1 expression and its downstream effect in HCC. ANT2 shRNA treatment led to restoration of SOCS1 expression along with its promoter demethylation in Hep3B cells, which was accompanied by decreased DNA methyltransferase 1 (DNMT1) activity through the suppression of Ras/PI3K/Akt signaling. Restoration of SOCS1 by ANT2 knockdown, subsequently, inhibited STAT3 activity and downregulated the expression of miR-21, which has been reported to be an important onco-miR in HCC. Downregulation of miR-21 efficiently suppressed Hep3B cell proliferation in vitro with a comparable level to ANT2 shRNA treatment. ANT2 suppression by shRNA may be able to exert anticancer effects in HCC further by restoring SOCS1 expression.

Abstract 1781: LGI147, a novel small-molecule inhibitor of the mitotic kinesin Eg5, shows anticancer effects in human hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, for which novel therapies are urgently needed. Recently, the inhibitors of mitotic kinesin Eg5 emerge as potential anticancer therapeutics. The aim of this study was to investigate the anticancer activity of a potent Eg5 inhibitor, LGI147, as targeted therapy of HCC. Method: Antiproliferative effect of LGI147 (kindly provided by Novartis), a highly selective inhibitor of mitotic kinesin Eg5, was tested in three liver cancer cell lines (Hep3B, HepG2, PLC/PRF/5). Cell viability was measured by trypan blue exclusion assay. Mitotic perturbation was visualized by immunofluorescence staining and confocal microscopy. Cell cycle profiles were assessed by flow cytometry. Apoptosis was estimated by analysis of sub-G1 fraction, Annexin V expression and immunoblotting of apoptotic proteins. Results: LGI147 induced a dose-dependent growth inhibition with IC50s ranging from 43.47 pM to 53.59 pM in the tested HCC cell lines. LGI147 treatment for 24 hours led to inhibition of Eg5 activity and induction of abnormal mitosis in a dose-dependent manner. Polypolar spindle defects in mitosis, disturbed cell cycle progression, and induction of apoptosis were observed in the HCC cells treated with LGI147. Conclusions: These findings show that LGI147 has potent anticancer effects in human HCC. Inhibitors of mitotic kinesin Eg5 may deserve further exploration as molecular targeted agents against 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 1781. doi:1538-7445.AM2012-1781

Vitamin K enhancement of sorafenib‐mediated HCC cell growth inhibition in vitro and in vivo

The multikinase inhibitor sorafenib is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Apoptosis has been shown to be induced in HCC by several agents, including sorafenib as well as by the naturally occurring K vitamins (VKs). As few nontoxic agents have activity against HCC growth, we evaluated the activity of sorafenib and VKs, both independently and together on the growth of HCC cells in vitro and in vivo. We found that when VK was combined with sorafenib, the concentration of sorafenib required for growth inhibition was substantially reduced. Conversely, VK enhanced sorafenib effects in several HCC cell lines on growth inhibition. Growth could be inhibited at doses of VK plus sorafenib that were ineffective with either agent alone, using vitamins K1, K2 and K5. Combination of VK1 plus sorafenib induced apoptosis on FACS, TUNEL staining and caspase activation. Phospho-extracellular signal-regulated kinase (ERK) levels were decreased as was myeloid cell leukemia sequence 1 (Mcl-1), an ERK target. Sorafenib alone inhibited growth of transplantable HCC in vivo. At subeffective sorafenib doses in vivo, addition of VK1 caused major tumor regression, with decreased phospho-ERK and Mcl-1 staining. Thus, combination of VK1 plus sorafenib strongly induced growth inhibition and apoptosis in rodent and human HCC and inhibited the RAF/mitogen-activated protein kinase kinase/ERK pathway. VK1 alone activated PKA, a mediator of inhibitory Raf phosphorylation. Thus, each agent can antagonize Raf: sorafenib as a direct inhibitor and VK1 through inhibitory Raf phosphorylation. As both agents are available for human use, the combination has potential for improving sorafenib effects in HCC.

Induction of heme oxygenase-1 by acrolein mediates a cytoprotective effect in HepG2 cells

Acrolein is an alpha, beta-unsaturated aldehyde present in cigarette smoke, and is also a product of lipid peroxidation. Heme oxygenase-1 (HO-1) plays critical roles in preventing oxidative stress and other cellular functions, and induces viability and proliferation of tumor cells. Acrolein is well-known to induce HO-1, although the signal pathway has not been fully elucidated. This study elucidated the involved signaling and acrolein’s cytoprotective effects in human hepatocellular carcinoma (HepG2) cells. Cells were treated with acrolein to induce HO-1, whose expression was measured by Western blot, RT-PCR and immunofluorescence staining analyses. Low concentrations of acrolein remarkably increased HO-1 mRNA and protein levels. Acrolein-mediated HO-1 induction was notably decreased by rottlerin and SB203580, selective inhibitors of PKC-δ and p38 MAPK, respectively. Furthermore, the cells displayed an increased arrest at the G0/G1 phase of the cell-cycle. The data indicates that acrolein enhances HO-1 expression via PKC-δ and p38 MAPK signaling. Acrolein may provide a cytoprotective effect via the expression of HO-1 in HepG2 cells.

The Aurora kinase inhibitor VE-465 has anticancer effects in pre-clinical studies of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and novel therapies are urgently needed. Recently, aberrant expression of Aurora kinases has been reported in various human cancers including HCC. We sought to investigate the potential of a potent and selective Aurora kinase inhibitor, VE-465, for targeted therapy of HCC. Cytotoxicity effects of VE-465 were tested in Huh-7 and HepG2 cell lines. Inhibition of Aurora kinase activity was demonstrated by Western blotting and immunofluorescence staining. Mitotic perturbation was visualized by confocal microscopy. Cell cycle profiles and apoptosis were assessed by flow cytometry. In vivo efficacy was determined in nude mice with human HCC xenografts. We demonstrated that VE-465 induced proliferation blockade, histone H3 (Ser10) dephosphorylation, mitotic disturbance, endoreduplication, and apoptosis in Huh-7 and HepG2 cells. We also found that VE-465 suppressed Aurora kinase activity, prevented tumor growth, and induced apoptosis in a Huh-7 xenograft model. These findings show that VE-465 has potent anticancer effects in human HCC. Inhibitors of Aurora kinases may deserve further exploration as molecular targeted agents against HCC.

Chemokine receptor CCR7 enhances intrahepatic and lymphatic dissemination of human hepatocellular cancer

Despite many pathophysiological analyses, the process of tumor dissemination of hepatocellular carcinoma (HCC) remains vague. In diverse tumor entities, expression of the chemokine receptor, CCR7, has been linked to tumor dissemination and poor prognosis. Therefore, we evaluated, whether CCR7 exerts similar effects in human HCC. CCR7 expression analysis was performed in vitro on human hepatoma cell lines (Huh7, Hep3B, wt HepG2, p53 dominant negative transfected HepG2). In addition, CCR7 expression was evaluated in 39 patients with hepatocellular cancer and correlated with both, tumor and patients characteristics. Human hepatocellular carcinoma samples and hepatoma cell lines displayed variable intensities of CCR7 expression. In patients, CCR7 expression was significantly associated with progressed local tumors (P = 0.02) and lymphatic metastasis (P = 0.02). Strong expression of CCR7 promotes intrahepatic and lymphatic HCC dissemination.

Dissemination of hepatocellular carcinoma is mediated via chemokine receptor CXCR4

In different tumour entities, expression of the chemokine receptor 4 (CXCR4) has been linked to tumour dissemination and poor prognosis. Therefore, we evaluated, if the expression of CXCR4 exerts similar effects in human hepatocellular carcinoma (HCC). Expression analysis and functional assays were performed in vitro to elucidate the impact of CXCL12 on human hepatoma cells lines. In addition, expression of CXCR4 was evaluated in 39 patients with HCC semiquantitatively and correlated with both, tumour and patients characteristics. Human HCC and hepatoma cell lines displayed variable intensities of CXCR4 expression. Loss of p53 function did not impact on CXCR4 expression. Exposure to CXCL12 mediated a perinuclear translocation of CXCR4 in Huh7/Hep3B cells and increased the invasive potential of Huh7 cells. In HCC patients, CXCR4 expression significantly correlated with progressed local tumours (T-status; P=0.006), lymphatic metastasis (N-status; P=0.005) and distant dissemination (M-status; P=0.009), as well as with a decreased 3-year-survival rate (P=0.01). In summary, strong expression of CXCR4 is significantly associated with progressed hepatocellular cancer.