Cytotoxicity In Hepatocellular Carcinoma Cells Research Articles

Comparative analysis of sorafenib and lenvatinib on HepG2 cells and human umbilical vein endothelial cells: Involvement of transforming growth factor-β signaling in their molecular effects.

This study aimed to compare the effects of the molecular targeted drugs,sorafenib and lenvatinib, on the survival, invasion, and angiogenesis of hepatocellular carcinoma cells. Additionally, we investigated the involvement oftransforming growth factor beta (TGF-β) signaling in their molecular mechanisms. To investigate the effects of sorafenib and lenvatinib, we conducted cell viability, invasion, and angiogenesis assays, as well as western blotting analyses. In human hepatocellular carcinoma cells (HepG2), sorafenib demonstrated potent inhibitory effects on cell proliferation, but induced cell invasion similar to TGF-β. In contrast, lenvatinib showed weaker cytotoxicity compared with sorafenib, but suppressed cell invasion induced by TGF-β. The actions of these two molecular targeted drugs were suggested to involve the regulation of the TGFβR2/ERK pathway. Moreover, in human umbilical vein endothelial cells, Sorafenib showed weaker cytotoxicity and enhanced the effects of TGF-β on angiogenesis. Conversely, lenvatinib showed potent cytotoxic abilities and suppressed angiogenesis induced by TGF-β. The actions of these two molecular targeted drugs were suggested to involve the regulation of the crosstalk between TGF-β signaling and vascular endothelial growth factor signaling. Our findings indicate that both sorafenib and lenvatinib possess anticancer abilities by inducing the cytotoxicity of hepatocellular carcinoma cells. Furthermore, they show opposing effects on TGF-β-induced cell invasion and angiogenesis, thereby enhancing the understanding of the multifaceted functions of molecular targeted drugs in treating hepatocellular carcinoma.

Leonurine ameliorates the STAT3 pathway through the upregulation of SHP-1 to retard the growth of hepatocellular carcinoma cells

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directs the transcription of genes involved in the promotion of cell survival and proliferation, inflammation, angiogenesis, invasion, and migration. Overactivation of STAT3 is often witnessed in human cancers, thereby making it a good target in oncology. Herein the efficacy of Leonurine (Leo), a bioactive alkaloid present in Herba leonuri, was investigated for its STAT3-inhibitory potential in hepatocellular carcinoma (HCC) cells. Leo downregulated the persistent as well as IL-6-driven activation of STAT3. Leo abrogated the nuclear localization and DNA interacting ability of STAT3. Leo was also found to impart STAT3 inhibition by mitigating the activation of upstream kinases such as JAK1, JAK2, and Src both in constitutive and IL-6 inducible systems. Leo curbed the STAT3-driven luciferase gene expression and the depletion of STAT3 resulted in the reduced responsiveness of HCC cells to Leo. Pervanadate exposure counteracted Leo-induced STAT3 inhibition suggesting the involvement of a protein tyrosine phosphatase. SHP-1 was significantly elevated upon Leo exposure whereas the depletion of SHP-1 was found to revert the effect of Leo on STAT3. Leo induced apoptosis and also significantly potentiated the cytotoxic effect of paclitaxel, doxorubicin, and sorafenib. Leo was found to be non-toxic up to the dose of 10 mg/kg in NCr nude mice. In conclusion, Leo was demonstrated to induce cytotoxicity in HCC cells by mitigating the persistent of activation of STAT3 pathway.

Discovery of β-cyclocitral-derived mono-carbonyl curcumin analogs as anti-hepatocellular carcinoma agents via suppression of MAPK signaling pathway

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high recurrence and mortality rate. In this study, a series of β-cyclocitral-derived mono-carbonyl curcumin analogs were synthesized and their anticancer properties were evaluated. Among the series, A19 exhibited the strongest cytotoxic activity by inhibiting cell viability and colony formation, inducing cell cycle G2/M phase arrest and cell apoptosis of HCC HepG2 and Huh-7 cells, while having almost no cytotoxicity on normal liver MIHA cells. Mechanistically, our results demonstrated that A19 triggered intense DNA damage via suppression of the ERK/JNK/p38 MAPK signaling pathway. Additionally, a combination of A19 with sorafenib significantly induced synergistic cytotoxicity in HCC cells. Overall, our results indicate the potential of A19 as a novel chemotherapeutic drug administered either separately or in combined therapy for HCC treatment.

Enhancement of Radiation-induced Cytotoxicity in Hepatocellular Carcinoma Cells by Some Plant Extracts In vitro

Enhancement of Radiation-induced Cytotoxicity in Hepatocellular Carcinoma Cells by Some Plant Extracts In vitro

Involvement of adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 in diallyl trisulfide-induced cytotoxicity in hepatocellular carcinoma cells.

It has been demonstrated that APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) is involved in the regulation of several growth-related signaling pathways and thus closely associated with the development and progression of some cancers. Diallyl trisulfide (DAT), a garlic-derived bioactive compound, exerts selective cytotoxicity to various human cancer cells through interfering with pro-survival signaling pathways. However, whether and how DAT affects survival of human hepatocellular carcinoma (HCC) cells remain unclear. Herein, we tested the hypothesis of the involvement of APPL1 in DAT-induced cytotoxicity in HCC HepG2 cells. We found that Lys 63 (K63)-linked polyubiquitination of APPL1 was significantly decreased whereas phosphorylation of APPL1 at serine residues remained unchanged in DAT-treated HepG2 cells. Compared with wild-type APPL1, overexpression of APPL1 K63R mutant dramatically increased cell apoptosis and mitigated cell survival, along with a reduction of phosphorylation of STAT3, Akt, and Erk1/2. In addition, DAT administration markedly reduced protein levels of intracellular TNF receptor-associated factor 6 (TRAF6). Genetic inhibition of TRAF6 decreased K63-linked polyubiquitination of APPL1. Moreover, the cytotoxicity impacts of DAT on HepG2 cells were greatly attenuated by overexpression of wild-type APPL1. Taken together, these results suggest that APPL1 polyubiquitination probably mediates the inhibitory effects of DAT on survival of HepG2 cells by modulating STAT3, Akt, and Erk1/2 pathways.

Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 are Involved in Ritonavir‐induced Cytotoxicity in Hepatoma Cells

Ritonavir (RTV), a pharmacoenhancer used in anti‐HIV regimens, can induce liver damage. RTV is mainly metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A‐AS1) and HNF1A antisense RNA 1 (HNF1A‐AS1) are long noncoding RNAs (lncRNAs) that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and molecular mechanisms of HNF4A‐AS1 and HNF1A‐AS1 in RTV‐induced hepatotoxicity. Small hairpin RNAs were used to knock down HNF4A‐AS1 and HNF1A‐AS1 in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV‐induced hepatotoxicity. Chromatin immunoprecipitation qPCR was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A‐AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV‐induced cytotoxicity, whereas HNF1A‐AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A‐AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A‐AS1 knockdown. Alterations in RTV‐induced hepatotoxicity caused by decreasing HNF4A‐AS1 or HNF1A‐AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV‐induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A‐AS1 overexpression or HNF1A‐AS1 knockdown. Taken together, these results revealed that HNF4A‐AS1 and HNF1A‐AS1 modulated RTV‐induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter.

Konjac glucomannan enhances 5-FU-induced cytotoxicity of hepatocellular carcinoma cells via TLR4/PERK/CHOP signaling to induce endoplasmic reticulum stress.

5-Fluorouracil (5-FU) is a commonly used chemotherapeutic agent for various cancers. However, the drug resistance developed by tumor cells hinders the therapeutic effect. Konjac glucomannan (KGM) is indicated to sensitize 5-FU-resistant hepatocellular carcinoma (HCC) cells to 5-FU. In our study, we found that KGM or 5-FU treatment alone did not affect the malignant cell behaviors and endoplasmic reticulum (ER) stress of 5-FU-resistant HCC cells or HepG2/5-FU and Bel-7402/5-FU cells, while cotreatment with KGM and 5-FU significantly facilitated HCC cell apoptosis and ER stress and suppressed cell proliferation potential and migration abilities. Moreover, we explored the underlying mechanism by which KGM induces 5-FU cytotoxicity in HCC cells. We found that Toll-like receptor 4 (TLR4) was downregulated in KGM- and 5-FU-treated HCC cells. TLR4 overexpression reversed the KGM and 5-FU cotreatment-induced inhibition of the malignant behaviors of 5-FU-resistant HCC cells. Furthermore, KGM enhanced 5-FU-induced ER stress by inhibiting TLR4 to activate PERK/ATF4/CHOP signaling. Xenograft mouse models were established using HepG2/5-FU cells, and KGM was demonstrated to reverse 5-FU resistance in HCC tumors in vivo by suppressing TLR4 to enhance ER stress and activate PERK/ATF4/CHOP signaling. In conclusion, KGM combined with 5-FU treatment significantly promoted apoptosis and reduced cell proliferation, migration and ER stress in 5-FU-resistant HCC cells compared with KGM or 5-FU treatment alone by downregulating TLR4 to activate PERK/ATF4/CHOP signaling.

Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 Are Involved in Ritonavir-Induced Cytotoxicity in Hepatoma Cells.

Ritonavir (RTV), a pharmacoenhancer used in anti-HIV regimens, can induce liver damage. RTV is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A-AS1) and HNF1A antisense RNA 1 (HNF1A-AS1) are long noncoding RNAs that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and underlying mechanisms of HNF4A-AS1 and HNF1A-AS1 in RTV-induced hepatotoxicity. HNF4A-AS1 and HNF1A-AS1 were knocked down by small hairpin RNAs in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV-induced hepatotoxicity. Chromatin immunoprecipitation quantitative real-time polymerase chain reaction was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A-AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV-induced cytotoxicity, whereas HNF1A-AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A-AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A-AS1 knockdown. Alterations in RTV-induced hepatotoxicity caused by decreasing HNF4A-AS1 or HNF1A-AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV-induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A-AS1 overexpression or HNF1A-AS1 knockdown. Taken together, these results revealed that HNF4A-AS1 and HNF1A-AS1 modulated RTV-induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter. SIGNIFICANCE STATEMENT: HNF4A-AS1 and HNF1A-AS1, transcribed separately from neighboring antisense genes of the human transcription factor genes HNF4A and HNF1A, were identified as long noncoding RNAs that can affect RTV-induced hepatotoxicity and susceptibility to RTV-induced hepatotoxicity caused by rifampicin exposure, mainly by affecting the expression of CY3A4 via alterations in PXR enrichment and histone modification status in the CYP3A4 promoter. This discovery provides directions for further research on the mechanisms of RTV-induced liver injury.

Synthetic Tryptanthrin Derivatives Induce Cell Cycle Arrest and Apoptosis via Akt and MAPKs in Human Hepatocellular Carcinoma Cells

Trytanthrin, found in Ban-Lan-Gen, is a natural product containing an indoloquinazoline moiety and has been shown to possess anti-inflammatory and anti-viral activities. Chronic inflammation and hepatitis B are known to be associated with the progression of hepatocellular carcinoma (HCC). In this study, a series of tryptanthrin derivatives were synthesized to generate potent anti-tumor agents against HCC. This effort yielded two compounds, A1 and A6, that exhibited multi-fold higher cytotoxicity in HCC cells than the parent compound. Flow cytometric analysis demonstrated that A1 and A6 caused S-phase arrest and downregulated the expression of cyclin A1, B1, CDK2, and p-CDC2. In addition to inducing caspase-dependent apoptosis, A1 and A6 exhibited similar regulation of the phosphorylation or expression of multiple signaling targets, including Akt, NF-κB, and mitogen-activated protein kinases. The anti-tumor activities of A1 and A6 were also attributable to the generation of reactive oxygen species, accompanied by an increase in p-p53 levels. Therefore, A1 and A6 have potential clinical applications since they target diverse aspects of cancer cell growth in HCC.

Pyrimidine-2,4-dione targets STAT3 signaling pathway to induce cytotoxicity in hepatocellular carcinoma cells

Signal transducer and activator of transcription 3 (STAT3) is a tumorigenic transcription factor that is persistently activated in various human cancers including hepatocellular carcinoma (HCC). Therefore, STAT3 is considered as a prominent target to counteract the uncontrolled proliferation of cancer cells. In the present report, pyrimidine-2,4-diones (N-methyluracil derivatives) (MNK1-MNK14) were synthesized in an ionic liquid (BMIm PF6) medium employing a ligand-free Suzuki-Miyaura cross-coupling process. Among the 14 derivatives, compound MNK8 showed good cytotoxicity towards both the tested cell lines and did not display a toxic effect against normal hepatocytes (LO2). MNK8 significantly increased the Sub-G1 cell count in both cell lines and the cytotoxic effect of MNK8 was found to be mediated through the suppression of constitutive phosphorylation of STAT3Y705. It also decreased the DNA interaction ability of nuclear STAT3 in HCC cells. MNK8 downregulated the levels of apoptosis-related proteins (such as Bcl-2, cyclin D1, survivin) and increased cleaved caspase-3 inferring the apoptogenic effect of MNK8. It also reduced the CXCL12-triggered cell migration and invasion in in vitro assay systems. Overall, MNK8 has been demonstrated as a new inhibitor of STAT3 signaling cascade in HCC cells.

A Novel mRNA-Mediated and MicroRNA-Guided Approach to Specifically Eradicate Drug-Resistant Hepatocellular Carcinoma Cell Lines by Se-Methylselenocysteine.

Despite progress in the treatment of non-visceral malignancies, the prognosis remains poor for malignancies of visceral organs and novel therapeutic approaches are urgently required. We evaluated a novel therapeutic regimen based on treatment with Se-methylselenocysteine (MSC) and concomitant tumor-specific induction of Kynurenine aminotransferase 1 (KYAT1) in hepatocellular carcinoma (HCC) cell lines, using either vector-based and/or lipid nanoparticle-mediated delivery of mRNA. Supplementation of MSC in KYAT1 overexpressed cells resulted in significantly increased cytotoxicity, due to ROS formation, as compared to MSC alone. Furthermore, microRNA antisense-targeted sites for miR122, known to be widely expressed in normal hepatocytes while downregulated in hepatocellular carcinoma, were added to specifically limit cytotoxicity in HCC cells, thereby limiting the off-target effects. KYAT1 expression was significantly reduced in cells with high levels of miR122 supporting the concept of miR-guided induction of tumor-specific cytotoxicity. The addition of alpha-ketoacid favored the production of methylselenol, enhancing the cytotoxic efficacy of MSC in HCC cells, with no effects on primary human hepatocytes. Altogether, the proposed regimen offers great potential to safely and specifically target hepatic tumors that are currently untreatable.

Methyl gallate, gallic acid-derived compound, inhibit cell proliferation through increasing ROS production and apoptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a global health problem. Currently, there is no effective therapeutic strategy for HCC. Methyl gallate (MG), from plant-derived phenolic gallic acid, has exhibited antitumor efficacy. However, the effect of MG on HCC is unclear. In vitro growth activity was detected by a sulforhodamine assay. A zebrafish xenotransplantation was applied to evaluate the inhibitory effect of MG. Reactive oxygen species (ROS) production, autophagy, and lysosome formation were detected by specific dyes. Finally, apoptosis was examined using annexin V-FITC/PI staining and western blot was performed to determine the molecular mechanism. It was demonstrated that MG treatment inhibited the proliferation of Hep3B, Mahlavu, and HepJ5 cells. Xenotransplantation also showed that MG inhibited the growth of Hep3B and HepJ5 cells. MG treatment increased cellular levels of superoxide and oxidative stress. Increases in autophagy and lysosome formation were found after MG treatment. The western blot analysis showed that MG activated cleavage of caspase-3 and poly (SDP ribose) polymerase (PARP), modulated levels of the Bcl2, Bax, and Bad ligands, and induced apoptosis. MG induced autophagy with notable activation of beclin-1, autophagy related 5+12 (ATG5+12), and conversion of light chain 3-I (LC3-I) to II. Our study showed that MG exposure inhibited HCC proliferation both in vitro and in vivo. And blocking autophagy enhanced MG-induced cytotoxicity in HCC cells. These findings suggested MG might serve as a powerful therapeutic supplement for human HCC patients.

Amination of Graphene Oxide Leads to Increased Cytotoxicity in Hepatocellular Carcinoma Cells.

Clinically, there is an urgent need to identify new therapeutic strategies for selectively treating cancer cells. One of the directions in this research is the development of biocompatible therapeutics that selectively target cancer cells. Here, we show that novel aminated graphene oxide (haGO-NH2) nanoparticles demonstrate increased toxicity towards human hepatocellular cancer cells compared to pristine graphene oxide(GO). The applied novel strategy for amination leads to a decrease in the size of haGO-NH2 and their zeta potential, thus, assuring easier penetration through the cell membrane. After characterization of the biological activities of pristine and aminated GO, we have demonstrated strong cytotoxicity of haGO-NH2 toward hepatic cancer cells—HepG2 cell line, in a dose-dependent manner. We have presented evidence that the cytotoxic effects of haGO-NH2 on hepatic cancer cells were due to cell membrane damage, mitochondrial dysfunction and increased reactive oxygen species (ROS) production. Intrinsically, our current study provides new rationale for exploiting aminated graphene oxide as an anticancer therapeutic.

Olaparib and enzalutamide synergistically suppress HCC progression via the AR‐mediated miR‐146a‐5p/BRCA1 signaling

Hepatocellular carcinoma (HCC) is one of most common cancers worldwide, however, the treatment for advanced HCC remains unsatisfactory. We focused on the function of the androgen receptor (AR) in HCC and tried to find new treatment strategy based on antiandrogen enzalutamide (Enz). Here, we found that olaparib, a FDA-approved PARP inhibitor, could enhance the cytotoxicity in HCC cells with a lower BRCA1 expression, and suppressing the AR with either Enz or AR-shRNA could further increase the olaparib sensitivity to better suppress the HCC cell growth via a synergistic mechanism that may involve suppressing the expression of BRCA1 and other DNA damage response (DDR) genes. Mechanism studies revealed that Enz/AR signaling might transcriptionally regulate the miR-146a-5p expression via binding to the Androgen Response Elements on its 5' promoter region, which could then lead to suppress the homologous recombination-related BRCA1 expression via direct binding to the mRNA 3'UTR. Preclinical studies using an in vivo mouse model also demonstrated that combining Enz plus olaparib led to better suppression of the HCC progression. Together, these in vitro/in vivo data suggest that combining Enz and olaparib may help in the development of a novel therapy to better suppress the HCC progression.

Ethanol extract of Ginger Zingiber officinale Roscoe by Soxhlet method induces apoptosis in human hepatocellular carcinoma cell line

Introduction: Ginger has had a long history of use in healthcare in Asian countries, including Vietnam. Internationally, there have been a few studies done to assess the anticancer activity and potency of ginger. In this study, we investigated the activity (cytotoxicity) of various ginger root extracts that were produced by different preparation methods on human hepatocellular carcinoma cell line (HepG2 cell line). Methods: Ginger (Zingiber officinale Roscoe) root extract were extracted from the fresh ginger roots by three methods, which included maceration (G1), Soxhlet (G3), and ultrasonic (G5) methods. The extracts were then evaluated for cytotoxic activity on HepG2 hepatocellular carcinoma cell line based on IC50 calculation. The most potent extract was then evaluated for its cytotoxicity by Propidium iodide (PI) staining method and microscopic examination by fluorescence microscopy for both 2D and 3D culture conditions. After treatment, cells were also stained with Annexin-V to assess the apoptosis-inducing ability of the extract. Results: The results showed that the ginger root extract prepared by the Soxhlet method (G3) had the strongest activity, and showed significant differences compared to extracts from methods G1 and G5, on the hepatocellular carcinoma cell lines cultured in both 2D and 3D conditions. Indeed, the IC50 value of ginger root extract of the G3 was 83.3 +/- 0.9189 ug/ml, compared to 159 +/- 7.6 ug/ml of G1, and 284 +/- 5.116 ug/ml of G5 in 2D culture latform; the IC50 value was 228 +/- 33.52 ug/ml for G3 compared to 341+/- 3.93 ug/ml for G1, and 603.7 +/- 56.33 ug/ml for G5 in 3D culture platform. The results of PI staining of cells in 2D and 3D culture conditions showed that ginger root extract killed HepG2 cells in a concentration-dependent manner. The nuclei of HepG2 cells in 2D culture showed nuclear fragmentation when treated with ginger root extract; this is one of the apoptosis indicators. Moreover, Annexin-V staining results showed that HepG2 cells underwent apoptosis when treated with ginger root extract. Conclusion: The results of the study show that ginger root extract prepared by the Soxhlet method induced strong cytotoxicity of hepatocellular carcinoma cells cultured in both 2D and 3D conditions, through induction of apoptosis. The evidence suggests that ginger is a potential agent for anticancer therapy. It is necessary to perform further research to isolate compounds from fresh ginger root extract by the Soxhlet method to evaluate the mechanism of anti-tumor activity.

Luteolin and sorafenib combination kills human hepatocellular carcinoma cells through apoptosis potentiation and JNK activation.

Sorafenib is a small-molecule multi-kinase inhibitor approved by FDA as an oral agent for the treatment of hepatocellular carcinoma (HCC) and renal cell carcinoma. However, unresponsiveness and acquired resistance are commonly observed, which hinder the clinical use of sorafenib. As combination therapy is a promising approach to improve its efficacy, we investigated if sorafenib and luteolin combination is effective in killing human HCC cells. Cell death was examined by lactate dehydrogenase (LDH) releasing assay. Apoptosis was detected by flow cytometric. The activation of apoptotic pathway and c-Jun N-terminal kinase (JNK) signaling pathway was measured by western blot. The results showed that sorafenib and luteolin combination synergistically induced cytotoxicity in HCC cells, which was accompanied by potentiation of apoptosis as demonstrated by increased apoptotic cell populations, caspase activation, and suppression of cell death by the pan-caspase inhibitor z-VAD-fmk. Furthermore, the combination of both agents enhanced expression of phosphorylated form of JNK, and the JNK inhibitor SP600125 effectively attenuated cell death induced by the combination treatment. Thus, sorafenib and luteolin combination synergistically kills HCC cells through JNK-mediated apoptosis, and luteolin may be an ideal candidate for increasing the activity of sorafenib in HCC therapy.

RETRACTED: Hispidulin inhibits hepatocellular carcinoma growth and metastasis through AMPK and ERK signaling mediated activation of PPARγ

Hispidulin, a phenolic flavonoid, exerts potent cytotoxicity towards a variety of human cancers. However, the effects of hispidulin on hepatocellular carcinoma (HCC) and underlying molecular mechanisms of its action remain elusive. The present study investigated the effect of hispidulin on HCC in experimental models, including tumor cell lines and mouse tumor xenograft. Results demonstrated that hispidulin was cytotoxic and anti-proliferative to HCC cell lines (SMMC7721 and Bel7402). Hispidulin activated caspase-3 and triggered apoptosis in HCC cells. Moreover, hispidulin inhibited cell migration and invasion by inhibiting the expression of matrix metalloproteinases (MMP-2, MMP-9) and by inducing tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. Hispidulin activated peroxisome proliferator-activated receptor γ (PPARγ) signaling which mainly contributed to its cytotoxicity in HCC cells. Remarkably, GW9662 (a PPARγ inhibitor) or PPARγ targeting siRNA significantly abrogated the anti-proliferative, pro-apoptotic, and anti-metastatic effects of hispidulin in HCC cells. Furthermore, hispidulin induced activation of PPARγ which was associated with increased phosphorylation of AMPK, ERK, JNK in HCC cells. Compound C (an AMPK inhibitor) or PD98059 (a MEK inhibitor) partly reversed the effects of hispidulin on PPARγ signaling in HCC cells. In contrast, no significant changes in PPARγ signaling were observed in HCC cells pretreated with SP600125 (a JNK inhibitor), while SP6000125 significantly inhibited the anti-cancer effects of hispidulin in HCC cells. Hispidulin administration effectively suppressed Bel7402 xenograft tumor growth and lung metastasis in vivo. Our findings indicate that PPARγ activation by hispidulin effectively suppressed HCC cell growth and metastasis both in vitro and in vivo.

Synergistic antitumor effects of tanshinone IIA and sorafenib or its derivative SC-1 in hepatocellular carcinoma cells.

IntroductionHepatocellular carcinoma (HCC) is the most common form of hepatic malignancy in the world. We aimed to determine the effect of tanshinone IIA (Tan-IIA) in combination with sorafenib or its derivative SC-1 on cytotoxicity, apoptosis, and metastasis in human HCC cells.Materials and methodsCytotoxicity was detected by MTT assay. Apoptosis and sub-G1 populations were analyzed by flow cytometry. Cell migration and invasion were evaluated by Transwell assay. Protein expression was detected by Western blot.ResultsTan-IIA combined with sorafenib or SC-1 exerted synergistic cytotoxicity in HCC cells. Elevated proportions of sub-G1 and caspase activation were observed in the combinative treatments; in addition, marked inhibition of cell migration and invasion, which could be mediated by the modulation of epithelial–mesenchymal transition was observed. pSTAT3 levels were significantly reduced as well.ConclusionA combination therapy using Tan-IIA and sorafenib or SC-1 could be a promising approach to target HCC, and further preclinical investigations are warranted to establish their synergetic advantage.

Abstract B73: The association of caffeine, trigonelline, and chlorogenic acid, active components from coffee, enhances caffeine-induced cytotoxicity in hepatocellular carcinoma cells

Abstract Background: A cup of traditional coffee is a complex mixture of biologically active components, including caffeine (CAF), trigonelline (TRI), chlorogenic acid (CGA), and many others (1). Daily coffee consumption reduces by ~40% the risk of fibrosis/cirrhosis and hepatocellular carcinoma (HCC) in humans while, in general, decaffeinated coffee consumption does not (2-4). However, it is not clear whether this beneficial effect is attributed to the CAF alone or its association with other coffee constituents. Aim: This work evaluated the modifying effects of CAF alone or associated to CGA and/or TRI in human HCC and hepatic stellate cells (HSC). Experimental Procedures: Human C3A (clonal derivative of HepG2 cells) and LX2 (HSC) cells were grown in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37oC. C3A and LX2 cells were seeded in 96-well plates at a density of 7 x 104 and 1 x 105 cells/mL, respectively, and treated with CAF alone (20, 40, 80, or 160 μM) or combined to TRI (10, 20, 40, or 80 μM) and/or CGA (10, 20, 40, or 80 μM) for 24 and 48 h. To select the CAF concentration, we considered the human serum peak (~35 µM) after the ingestion of ~450 mg of CAF, corresponding to 2-3 cups of coffee, and the toxic limit established for plasma concentration in humans (~400 μM) (5,6). For CGA and TRI, abundant in coffee beverages, we considered the 1:2 proportion (CGA or TRI:CAF) found in traditional coffee (1). We evaluated the cell viability (MTT assay) of LX2 and the cytotoxicity (lactate dehydrogenase [LDH] assay) of C3A cells. Results: All tested CAF concentrations significantly increased cytotoxicity in C3A cells at 48 h compared to untreated cells (p<0.001). The CAF+TRI+CGA combinations (20, 10, and 10 µM and 40, 20, and 20 µM, respectively) enhanced cytotoxicity at 24 and 48 h when compared to the control, CAF alone, and other associations (p<0.001). Notably, these combinations enhanced caffeine-induced cytotoxicity at 48 h (p<0.001). All treatments did not alter the viability of LX2 cells at both 24 and 48 h. Conclusion: In general, the combination of CAF with other common compounds found in filtered and espresso coffee, TRI and CGA, enhanced CAF-mediated cytotoxicity on HCC cells at physiologically applicable concentrations. These findings suggest a synergistic effect of coffee compounds in exerting a chemopreventive effect and reducing the risk for HCC. The potential mechanisms related to these modifying effects will be further evaluated. Financial support: CNPq (140251/2016-2) and FAPESP (16/14420-0).

MicroRNA-889 is downregulated by histone deacetylase inhibitors and confers resistance to natural killer cytotoxicity in hepatocellular carcinoma cells.

Major histocompatibility complex class I chain-related gene B (MICB) is expressed on tumor cells and participates in natural killer (NK) cell-mediated antitumor immune response through engagement with the NKG2D receptor. This study was undertaken to identify novel microRNA (miRNA) regulators of MICB and clarify their functions in NK cell-mediated cytotoxicity to hepatocellular carcinoma (HCC) cells. Bioinformatic analysis and luciferase reporter assay were conducted to search for MICB-targeting miRNAs. Overexpression and knockdown experiments were performed to determine the roles of candidate miRNAs in the susceptibility of HCC cells to NK lysis. miR-889 was identified as a novel MICB-targeting miRNA and overexpression of miR-889 significantly inhibited the mRNA and protein expression of MICB in HepG2 and SMMC7721 HCC cells. miR-889 expression had a negative correlation with MICB mRNA levels in HCC specimens (r=-0.392, P=0.0146). NK cell-mediated cytotoxicity was reduced in miR-889-overexpressing HCC cells, which was reversed by restoration of MICB expression. In contrast, knockdown of miR-889 led to more pronounced NK cell-mediated lysis in HCC cells. HCC cells exposed to the histone deacetylase (HDAC) inhibitor sodium valproate showed downregulation of miR-889. Enforced expression of miR-889 prevented the upregulation of MICB and enhancement of NK cell-mediated lysis by HDAC inhibitors. In conclusion, miR-889 upregulation attenuates the susceptibility of HCC cells to NK lysis and represents a potential target for improving NK cell-based antitumor therapies.