Human Hepatoma Cell Line Research Articles

Transmembrane protein 88 suppresses hepatocellular carcinoma progression and serves as a novel prognostic factor.

Transmembrane protein 88 (TMEM88) is known to be involved in the canonical Wnt signaling pathway and is implicated in several malignancies. However, the expression, function, and prognostic significance of TMEM88 in hepatocellular carcinoma (HCC) remain unclear. In this study, we analyzed mRNA levels of TMEM88 in HCC specimens from the TCGA dataset (n=374) to explore the correlation between TMEM88 and HCC. We also overexpressed TMEM88 in the Huh7 human HCC cell line to investigate its tumor-related role in HCC. Additionally, we conducted in vivo experiments using a mouse model to further validate the critical function of TMEM88 in modulating HCC growth. Our results showed that TMEM88 is negatively correlated with the T stage, TNM stage, and pathological grade of HCC. Higher levels of TMEM88 can help predict better overall survival of HCC in both univariate and multivariate analyses. Similarly, higher TMEM88 is a novel prognostic factor for better disease-specific survival of HCC. Overexpression of TMEM88 in Huh7 cells led to a decreased cell proliferation capacity. Xenograft experiments in a mouse model showed that TMEM88 overexpression can remarkably suppress HCC progression. Transmembrane protein 88 suppresses HCC growth both in vitro and in vivo, which can act as a potential prognostic factor with clinical application potential.

Abstract 2743: Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma

Abstract Background: The telomerase-specific oncolytic adenovirus Telomelysin along with the histone deacetylase inhibitor AR42 have both demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies has been reported to be a viable approach for treating various cancers. We investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Method: HCC cell lines (PLC5 and Hep3B) were tested and median effect analysis was performed to estimate potential synergistic antiproliferative effects. Apoptosis was assessed by flow cytometry and Western blotting. The expression of coxsackievirus and adenovirus receptor (CAR) was measured through flow cytometry and immunofluorescence staining. Human telomerase reverse transcriptase (hTERT) mRNA expression was assessed by reverse transcription polymerase chain reaction. Western blotting was conducted to examine the activation of pivotal signaling pathways and the interferon (IFN) response. HCC cells constitutively overexpressing Akt were cloned for molecular mechanism verification. MK-2206 was used to inhibit the activation of Akt signaling. In vivo efficacy was determined through xenograft models of human HCC. Results: Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the CAR and the mRNA levels of hTERT, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Conclusions: Combination therapy with Telomelysin and AR42 demonstrates synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted. Keywords: Hepatocellular carcinoma, oncolytic adenovirus, Telomelysin, HDAC inhibitor, Akt Citation Format: Zhong-Zhe Lin, Mickey C-T. Hu, Chiun Hsu, Yao-Ming Wu, Yen-Shen Lu, Ja-An Annie Ho, Shiou-Hwei Yeh, Pei-Jer Chen, Ann-Lii Cheng. Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2743.

Abstract 5870: T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells

Abstract Background: We previously identified 14 isoforms of T-cell factor (TCF)-4, a key transcriptional factor in the Wnt signaling pathway, from human hepatocellular carcinoma (HCC) cell lines (Exp Cell Res 2011). The TCF-4J and K pair has been characterized based on the presence (K) or absence (J) of a conserved SxxSS motif at the head of exon 9. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012); however, resistance to anti-cancer agents was more prominent in K cells than J cells. Another analysis demonstrated that loss of exon 4 increased resistance to chemotherapeutic agents and induced epithelial-mesenchymal transition (EMT) (Liver Int 2013). EMT and the expression of programmed cell death 1-ligand 1 (PD-L1) is closely associated with each other, and our preliminary study revealed that EMT was regulated by TCF-4 variants, suggesting possible involvement of the variants in the regulation of PD-L1 expression. In general, PD-L1 is known to be upregulated by hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Therefore, if the TCF-4 variants regulate PD-L1 expression under normoxia, it would be a unique mechanism bridging the Wnt signaling pathway and the tumor immune microenvironment (TIME). Aim: To assess whether or not the TCF-4 variants regulate the expression of PD-L1 in cancer cells under normoxia. Methods: HAK-1A, a well-differentiated HCC cell line (Hepatology 1993), was used in this study. TCF-4K mutants (S269A, S272A, and S273A) were prepared with the conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (S269A, S272A, and S273A cells, respectively) were established. Empty vector-transfected mock cells (EV cells) were used as a control. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Results: J cells and K cells expressed PD-L1 mRNA 2-fold and 4-fold of that in the EV cells, respectively. Of note, the PD-L1 expression mRNA was enhanced up to 11-fold in S269A cells, 11-fold in S272A cells, and 17-fold in S273A cells. The increased mRNA expressions were verified in protein levels. Conclusion: PD-L1 expression was regulated by TCF-4 variants in liver cancer cells under normoxic conditions. The finding suggests that the Wnt signaling pathway fine-tunes the TIME by regulating the expression levels of PD-L1 in a HIF-1α-independent manner. Citation Format: Hironori Koga, Yasuko Imamura, Toshimitsu Tanaka, Hideki Iwamoto, Toru Nakamura, Atsutaka Masuda, Takahiko Sakaue, Hiroyuki Suzuki, Hirohisa Yano, Takumi Kawaguchi. T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5870.

Abstract 5985: Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression

Abstract Background: PD-1 blockade has been approved as treatment for advanced HCC. We previously demonstrated the efficacy of CDK9 inhibition in reducing the expression of inducible proteins and as a treatment for HCC (Shao et al. Oncology 2022). We thus examined the influence of CDK9 inhibition on the expression of PD-L1 in HCC and the potential of improving the efficacy of PD-1 blockade with the combination of CDK9 inhibitors. Methods: We first examined the influence of specific CDK9 inhibitors, AZD4573 and atuveciclib, on interferon-γ (IFN-γ) induced PD-L1 expression of human HCC cell lines HuH7 and Hep3B. Overexpression of CDK9 and siRNA to CDK9 were used to confirm the findings. Similar experiments were repeated in the mouse HCC cell line BNL. To test the in vivo efficacy, we orthotopically implanted BNL cells in the subcapsular area of BALB/c mice, which were then treated with vehicle, CDK9 inhibitors, anti-PD-L1 antibodies, or their combination. To compare with tumor sizes, all mice were sacrificed after 14 days of treatment. To compare survival, the mice were only sacrificed if they met the criteria of animal euthanasia. Results: Both HuH7 and Hep3B cells had minimal PD-L1 expression, which however could be induced using IFN-γ. AZD4573 and atuveciclib decreased the IFN-γ induced PD-L1 expression of HuH7 and Hep3B cells in a dose dependent manner. The flow cytometry results confirmed that membrane PD-L1 expression was also decreased by CDK9 inhibition. Overexpression of CDK9 decreased the influence of these CDK9 inhibitors on PD-L1 expression. CDK9 knockdown using siRNA reduced the IFN-γ induced PD-L1 expression. AZD4573 and atuveciclib also reduce the PD-L1 expression in BNL cell lines. In the orthotopic animal model, the tumors of the sacrificed mice were significantly smaller in mice treated with atuveciclib and an anti-PD-L1 antibody than mice treatment with either alone. The survival of mice treated with the combination was also longer than mice treated with each therapy alone. Conclusion: We demonstrated that CDK9 inhibition could reduce the IFN-γ induced PD-L1 expression of HCC cells. Combination of CDK9 inhibitors and anti-PD-L1 antibodies was more effective than either therapy alone. Citation Format: Ching-Tso Chen, Yu-Yun Shao, Hung-Wei Hsu, Rita R. Wo, Han-Yu Wang, Ann-Lii Cheng, Chih-Hung Hsu. Cyclin dependent kinase 9 (CDK9) inhibition increased efficacy of programmed cell death protein 1 (PD-1) blockade for hepatocellular carcinoma (HCC) through decreasing programmed death-ligand 1 (PD-L1) expression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5985.

Yupingfeng San exhibits anticancer effect in hepatocellular carcinoma cells via the MAPK pathway revealed by HTS2 technology.

Yupingfeng San (YPFS) is a classic rousing prescription in Chinese medicine, with widly clinical application and remarkably curative effect. It consists of three herbs named Astragalus mongholicus Bunge (Huangqi), Atractylodes rubra Dekker (Baizhu) and Saposhnikovia divaricata (Turcz.) Schischk. (Fangfeng), and has a variety of pharmacological activities including immune regulation, antioxidant, anti-tumor, regulation of cytokines, etc. AIM OF THE STUDY: It has been proved that YPFS exerts its anti-tumor effect through enhancing the systemic and local immune responses in tumor patients, moreover, it has the direct tumor-suppressing effect and can reduce the adverse reactions caused by radiotherapy and chemotherapy drugs. Therefore, in this study, we explored the potential anti-HCC mechanism of YPFS based on HTS2 technology and system pharmacology, aiming to provide a scientific basis for the clinical application of YPFS and a new strategy for Chinese medicine research. In this study, systems pharmacology plus high throughput sequencing-based high throughput screening(HTS2) technology, and experimental validation were used to investigate the therapeutic mechanisms and the chemical basis of YPFS in HCC treatment. Firstly, the potential therapeutic targets and signaling pathways of YPFS in the treatment of HCC were obtained through systems pharmacology. Subsequently, HTS2 technology combined with PPI network analysis were used to reveal potential therapeutic targets. Finally, the anti-HCC effects and underlying mechanisms of YPFS were further verified in vitro in human hepatocellular carcinoma cell lines. Moreover, the possible chemical basis was explored by drug target verification and molecular docking technology. In total, 183 active ingredients were predicted by YPFS screening and 49 anti-HCC targets were further identified. Most of these targets were enriched into the "MAPK pathway", and the expression of 37 genes was significantly changed after herb treatment. Among them, 5 key targets, including VEGFA, GRB2, JUN, PDGFRB and CDC42, were predicted by protein-protein interaction (PPI) network analysis. According to our results, YPFS inhibited the proliferation, induced the apoptosis and caused cell cycle arrest of HCC cells. In addition, YPFS significantly reduced P38 gene expression. Fangfeng, one of the three herbs in YPFS, significantly down-regulated the expression of more target genes than that of the other two herbs. Lastly, as revealed by molecular docking analysis, 4'-O-glucosyl-5-O-methylvisamminol, an active ingredient identified in Fangfeng, showed a high affinity for P38. Taken together, this study shows that YPFS possesses the activities of anti-proliferation and pro-apoptosis in treating HCC, which are achieved by inhibiting the MAPK signaling pathway. P38 is one of the critical targets of YPFS in treating HCC, which may be directly bound and inhibited by 4'-O-glucosyl-5-O-methylvisamminol, a compound derived from YPFS.

The Relative Quantification of Hedgehog Signalling Components of Hepatocellular Carcinoma (Hepg-2) Cells Treated With Sorghum bicolor Leaf Stalk Ethanolic Extract

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Patients with advanced HCC have a dismal prognosis due to the limited therapy choices available. Hence, this present study evaluates the cytotoxic potential of the ethanolic crude extract of Sorghum bicolor leaf stalk (SBELSE) against human hepatocellular carcinoma (HEPG2) cell line, and its effect in the expression of its Hh signaling component-GLI1 and PTCH. The leaf stalk of Sorghum bicolor (SB) was pulverized and extracted with 70% ethanol. HEPG2 cells were treated with different concentration of ethanol extract of SBELSE which was subjected to 2-fold serial dilution to achieve a graded concentration (9.77-5000 µg/ml). Cell viability was performed using CCK-8 assay. The cells were treated with 8 µg/mL and 23 µg/mL concentration of SBELSE for 48 hour, after which RNA was isolated for downstream gene expression studies using RT-qPCR. The expression level of GLI1 and PTCH genes in cell line was examined relative to the untreated group. SBELSE exerted a cytotoxic effect on HEPG-2 cells with an inhibitory concentration (IC50) of 150 µg/mL. After 48 hours of treatment and evaluation, Expression of GLI1 and PTCH genes was seen in all cell group but was increased in groups treated with 8µg/mL concentration of S. bicolor extract than in the control group. The findings of this study suggest the possibility of the extract to contain agonistic component that might support cancer cell proliferation.

The anti-immune dengue subgenomic flaviviral RNA is present in vesicles in mosquito saliva and is associated with increased infectivity.

Mosquito transmission of dengue viruses to humans starts with infection of skin resident cells at the biting site. There is great interest in identifying transmission-enhancing factors in mosquito saliva in order to counteract them. Here we report the discovery of high levels of the anti-immune subgenomic flaviviral RNA (sfRNA) in dengue virus 2-infected mosquito saliva. We established that sfRNA is present in saliva using three different methods: northern blot, RT-qPCR and RNA sequencing. We next show that salivary sfRNA is protected in detergent-sensitive compartments, likely extracellular vesicles. In support of this hypothesis, we visualized viral RNAs in vesicles in mosquito saliva and noted a marked enrichment of signal from 3'UTR sequences, which is consistent with the presence of sfRNA. Furthermore, we show that incubation with mosquito saliva containing higher sfRNA levels results in higher virus infectivity in a human hepatoma cell line and human primary dermal fibroblasts. Transfection of 3'UTR RNA prior to DENV2 infection inhibited type I and III interferon induction and signaling, and enhanced viral replication. Therefore, we posit that sfRNA present in salivary extracellular vesicles is delivered to cells at the biting site to inhibit innate immunity and enhance dengue virus transmission.

Antioxidant properties and cytotoxic effects of Oxalis corniculata on human Hepatocarcinoma (Hep-G2) cell line: an in vitro and in silico evaluation

BackgroundOxalis corniculata is a well-known medicinal plant used in folk medicine for the management of many diseases. The aim is to determine the physico-chemical properties, microscopic study, phytochemical properties, in-silico, in-vitro antioxidant and anticancer activity on human Hepatocarcinoma (Hep-G2) cell line of O. corniculata plant extract.ResultsMicroscopical study reaveals that presence of pericyclic fibres, starch gains, trichomes etc, and phytochemical screening is carried to find out secondary metabolites. The molecular docking study concluded that some of the phytocompounds showed inhibition of epidermal growth factor receptor tyrosine kinase domain (PDB ID: 1M17) inhibitor. Furthermore, ADMET and drug likeness study hints some of phytocompounds may act as lead for anticancer drug discovery and development. Among selected phytocompounds, compound apigenin possesses − 7.90 kcal/mol as compared to standard drug doxorubicin possesses − 7.63 kcal/mol against the epidermal growth factor receptor tyrosine kinase. The plant extract shown antioxidant activities based on the different tests were performed. The hydroalcoholic plant extracts were found to be selectively cytotoxic in vitro to human Hepatocarcinoma (Hep-G2) cell line with IC50values 34.494 ± 0.42 µg/ml and EAF showed at IC50 value 30.245 ± 0.58 µg/ml of the cells were inhibited at the concentration of 50 µg/ml as compared with standard doxorubicin at IC50 value 24.8939 ± 0.25 µg/ml, respectively.ConclusionThe present study concluded that O. corniculata possesses potential antioxidant and cytotoxic properties based upon the computer aided drug design models and in-vitro activity.

Synthesis, structural characterization, DNA/HSA binding, molecular docking and anticancer studies of some D-Luciferin complexes

Several species, such as bacteria, fungi, fish, and insects, produce light through biochemical processes. Firefly D-luciferin has been studied extensively since it possesses both a high quantum yield and a wide emission wavelength. Five transition metal complexes of D-luciferin (LN) with Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) were synthesized using a 1:2 metal to ligand ratio. The structure of the synthesized complexes was confirmed utilizing spectroscopic techniques (FTIR, 1H NMR, EPR, and UV–Vis), elemental analysis, thermogravimetric analysis, molar conductivity, and magnetic susceptibility. Density functional theory (DFT/B3LYP) calculations were also used to confirm the structural characteristics and provide the fully optimized geometries of the ligand and its complexes. The results revealed that luciferin is bidentately coupled to the relevant metals in each of these complexes through two sulfur atoms of thiazole rings. Molar conductance values showed the non-electrolytic character of the synthesized complexes. Diverse techniques were employed to examine the complexes' binding affinity to calf thymus DNA, including UV–Vis, fluorescence, viscosity measurements and molecular docking. The results revealed that they bind non-covalently with DNA via groove binding. Furthermore, the interaction of these complexes with human serum albumin (HSA) was investigated via UV–Vis, fluorescence and molecular docking. The binding susceptibly of the complexes toward breast cancer (PDB: 3eqm) and liver cancer (PDB: 4mf9) proteins was assessed using molecular docking studies. Finally, human hepatocellular carcinoma cell line (HepG-2) and human breast cancer cell line (MCF-7) were used to investigate the cytotoxic activity of ligand and metal complexes. Among the five synthesized complexes, [Zn(LN)2Cl2]H2O complex has the best anticancer activity against MCF-7 and HepG-2 cell lines with IC50 values of 20 and 37.39 µM, respectively. The molecular docking studies and in vitro cytotoxicity assay showed a significant correlation.

Highly selective butyrylcholinesterase inhibitors related to Amaryllidaceae alkaloids - Design, synthesis, and biological evaluation

Butyrylcholinesterase (BChE) is one of the most frequently implicated enzymes in the advanced stage of Alzheimer's disease (AD). As part of our endeavors to develop new drug candidates for AD, we have focused on natural template structures, namely the Amaryllidaceae alkaloids carltonine A and B endowed with high BChE selectivity. Herein, we report the design, synthesis, and in vitro evaluation of 57 novel highly selective human BChE (hBChE) inhibitors. Most synthesized compounds showed hBChE inhibition potency ranging from micromolar to low nanomolar scale. Compounds that revealed BChE inhibition below 100 nM were selected for detailed biological investigation. The CNS-targeted profile of the presented compounds was confirmed theoretically by calculating the BBB score algorithm, these data were corroborated by determining the permeability in vitro using PAMPA-assay for the most active derivatives. The study highlighted compounds 87 (hBChE IC50 = 3.8 ± 0.2 nM) and 88 (hBChE IC50 = 5.7 ± 1.5 nM) as the top-ranked BChE inhibitors. Compounds revealed negligible cytotoxicity for the human neuroblastoma (SH-SY5Y) and hepatocellular carcinoma (HepG2) cell lines compared to BChE inhibitory potential. A crystallographic study was performed to inspect the binding mode of compound 87, revealing essential interactions between 87 and hBChE active site. In addition, multidimensional QSAR analyses were applied to determine the relationship between chemical structures and biological activity in a dataset of designed agents. Compound 87 is a promising lead compound with potential implications for treating the late stages of AD.

LncRNA miR4458HG modulates hepatocellular carcinoma progression by activating m6A-dependent glycolysis and promoting the polarization of tumor-associated macrophages

Long non-coding RNAs (lncRNAs) play significant roles in different biological functions of cancers. However, their function in the metabolism of glucose in patients with human hepatocellular carcinoma (HCC) remains largely unknown. In this study, HCC and paired intact liver tissues were utilized to examine the miR4458HG expression using qRT-PCR and human HCC cell lines to examine cell proliferation, colony formation, and glycolysis after transfection of siRNAs targeting miR4458HG or miR4458HG vectors. The molecular mechanism of miR4458HG was clarified through in situ hybridization, Western blotting, qRT-PCR, RNA pull-down, and RNA immunoprecipitation analysis. The results showed that the miR4458HG affected HCC cell proliferation, activated the glycolysis pathway, and promoted the polarization of tumor-associated macrophage in vitro and in vivo models. Mechanistically, miR4458HG bound IGF2BP2 (a key RNA m6A reader) and facilitated IGF2BP2-mediated target mRNA stability, including HK2 and SLC2A1 (GLUT1), and consequently altered HCC glycolysis and tumor cell physiology. At the same time, HCC-derived miR4458HG could be wrapped in the exosomes and promoted the polarization of tumor-associated macrophage by increasing ARG1 expression. Hence, miR4458HG is oncogenic in nature among patients with HCC. To develop an effective treatment strategy of HCC patients presenting with high glucose metabolism, physicians should focus on miR4458HG and its pathway.

Novel Steroidal[17,16-d]pyrimidines Derived from Epiandrosterone and Androsterone: Synthesis, Characterization and Configuration-Activity Relationships.

Two series of novel steroidal[17,16-d]pyrimidines derived from natural epiandrosterone and androsterone were designed and synthesized, and these compounds were screened for their potential anticancer activities. The preliminary bioassay indicated that some of these prepared compounds exhibited significantly good cytotoxic activities against human gastric cancer (SGC-7901), lung cancer (A549), and hepatocellular liver carcinoma (HepG2) cell lines compared with 5-fluorouracil (5-FU), epiandrosterone, and androsterone. Especially the respective pairs from epiandrosterone and androsterone showed significantly different inhibitory activities, and the possible configuration-activity relationships have also been summarized and discussed based on kinase assay and molecular docking, which indicated that the inhibition activities of these steroidal[17,16-d]pyrimidines might obviously be affected by the configuration of the hydroxyl group in the part of the steroidal scaffold.

Liver cancer antiproliferative activity of a new nor-cucurbitacin from Mareya micrantha Müll. Arg.

Mareya micrantha Müll. Arg. (Euphorbiaceae) is a plant used in the Ivorian traditional medicinal system for various medical properties such as laxative, oxytocic, intestinal infectious diseases, malaria, etc. Six cucurbitacin derivatives (tetracyclic triterpenoids) are isolated from the leaves, stem barks or root barks of M. micrantha. Among these compounds, 29-nor-2β,15α,20β-trihydroxy-16α-acetyl-3,1,22-trioxo-cucurbita-4,23-diene (1) is a new nor-cucurbitacin isolated from the leaves; 29-nor-1,2,3,4,5,10-dehydro-3,15α,20β-trihydroxy-16α-acetyl-11,22-dioxo-cucurbita-23-ene 2-O-β-D-glucopyranoside (2) and 29-nor-2β,15α,20β-trihydroxy-16α-acetyl-3,11,22 trioxo-cucurbita-4,23-diene 2-O-β-D glucopyranoside (3) are new nor-cucurbitacins recently discovered by us in leaves and isolated again for this study while dihydro-epi-isocucurbitacin D (4), tetrahydro-cucurbitacin I (5) and cucurbitacin L (6) are known cucurbitacins but newly isolated from the stem barks and the root barks of M. micrantha. Their chemical structures are established according to spectral data (UV, IR, MS and 1H, 13C NMR). Their antiproliferative activity is explored in vitro on the chemo-resistant human hepatocarcinoma cell line Hep3B. Compound 1 showed a strong and selective antiproliferative activity against this cancer cell line (IC50 value of 0.12 ± 0.05 μM) when compared to normal hepatocytes HepaRG.

Use of Ag-Au-ICG to increase fluorescence image of human hepatocellular carcinoma cell lines

Indocyanine green (ICG) is effective for a variety of applications including liver tumour imaging and operates in the near-infrared window. Agents for near-infrared imaging are, however, still in clinical development. The present study aimed to prepare and investigate fluorescence emission properties of ICG in combination with Ag-Au in order to enhance their specific interactions with human hepatocellular carcinoma cell lines (HepG-2). The Ag-Au-ICG complex was prepared via physical adsorption, and hence evaluated for fluorescence spectra using a spectrophotometer. Ag-Au-ICG at an optimised dosage (Ag-Au:ICG = 0.0147:1 molar ratio) in Intralipid medium was added to HepG-2 to observe the maximum fluorescence signal intensity, which further enhanced HepG-2 contrast fluorescence. Ag-Au-ICG served as a fluorescence enhancer bound onto the liposome membrane, whilst free Ag, Au, and pure ICG induced low levels of cytotoxicity in HepG-2 and a normal human cell line. Thus, our findings provided new insights for the liver cancer imaging. Highlights Concentration-dependent fluorescence peaking in the near-infrared window revealed ICG aggregation in Ag-Au molecules. Ag-Au-ICG fluorescence intensity depended strongly on the environmental media. Human hepatocellular carcinoma cell lines treated with Ag-Au-ICG in Intralipid enhanced the contrast of fluorescence microscopy images by decreasing the level of scattering in the cell lines with the contrast values being approximately five times those observed in pure ICG in Intralipid.

SPRED2: A Novel Regulator of Epithelial-Mesenchymal Transition and Stemness in Hepatocellular Carcinoma Cells.

The downregulation of SPRED2, a negative regulator of the ERK1/2 pathway, was previously detected in human cancers; however, the biological consequence remains unknown. Here, we investigated the effects of SPRED2 loss on hepatocellular carcinoma (HCC) cell function. Human HCC cell lines, expressing various levels of SPRED2 and SPRED2 knockdown, increased ERK1/2 activation. SPRED2-knockout (KO)-HepG2 cells displayed an elongated spindle shape with increased cell migration/invasion and cadherin switching, with features of epithelial-mesenchymal transition (EMT). SPRED2-KO cells demonstrated a higher ability to form spheres and colonies, expressed higher levels of stemness markers and were more resistant to cisplatin. Interestingly, SPRED2-KO cells also expressed higher levels of the stem cell surface markers CD44 and CD90. When CD44+CD90+ and CD44-CD90- populations from WT cells were analyzed, a lower level of SPRED2 and higher levels of stem cell markers were detected in CD44+CD90+ cells. Further, endogenous SPRED2 expression decreased when WT cells were cultured in 3D, but was restored in 2D culture. Finally, the levels of SPRED2 in clinical HCC tissues were significantly lower than those in adjacent non-HCC tissues and were negatively associated with progression-free survival. Thus, the downregulation of SPRED2 in HCC promotes EMT and stemness through the activation of the ERK1/2 pathway, and leads to more malignant phenotypes.

PAK4-relevant proliferation reduced by cell autophagy via p53/mTOR/p-AKT signaling.

P21-activated kinase 4 (PAK4) involves in cell proliferation in cancer and mutually regulates with p53, a molecule is demonstrated to control cell autophagy by mammalian target of rapamycin (mTOR)/protein kinase B (AKT) signaling. Since the signaling exhibits an association with PAK family members in cell autophagy, it implies that PAK4-relevant proliferation may be impacted by autophagy via p53 with a lack of evidence in cancer cells. In this research, transient and stable PAK4-knockdown human hepatocarcinoma cell lines (HepG2) were constructed by transfection of PAK4-RNA interference (RNAi) plasmid and lentivirus containing PAK4-RNAi plasmid, respectively. We investigated cell proliferation using methyl thiazolyl tetrazolium (MTT) and Cell Counting Kit 8 (CCK8) assays, cell cycle by flow cytometry (FCM) and cell autophagy by monodansylcadaverine (MDC) staining and autophagic biomarker's expression, and detected the expressions of p53, mTOR, phosphorylated-AKT (p-AKT) and AKT by immunofluorescence and western blot to explore the mechanism. We successfully constructed transient and stable PAK4-knockdown HepG2 cell lines, and detected dysfunction of the cells' proliferation. An increased expression of p53, as a molecule of cell-cycle-surveillance on G1/S phase, was demonstrated in the cells although the cell cycle blocked at G2/M. And then, we detected increased autophagosome and autophagic biomarker LC3-II, and decreased expressions in p-AKT and mTOR. The proliferation is reduced in PAK4-knockdown HepG2 cells, which is relative to not only cell cycle arrest but also cell autophagy, and p53/mTOR/p-AKT signaling involves in the cell progress. The findings provide a new mechanism on PAK4 block in cancer therapy.

Discovery of canine drug toceranib phosphate as a repurposed agent against human hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is an aggressive malignancy with poor clinical outcomes. There are limited therapeutic options for those diagnosed with terminal HCC and therefore incorporating novel agents into standard-of-care regimens is urgently needed. In contrast to de novo drug discovery, the strategy of repurposing compounds initially designed to treat animals might yield substantial advantages in terms of efficacy and safety. Given the evidence for the clinical efficacy of toceranib phosphate (TOC) against canine carcinomas, we aimed to investigate its therapeutic effect on human HCC. The antitumor effects of TOC were evaluated using human HCC celllines and cell-line-derived xenograft models. Changes in autophagic response upon TOC exposure were quantified through immunoblotting and immunofluorescence analysis. The role of TOC-triggered autophagy was addressed via pharmacological and genetic inhibition. We demonstrated TOC exhibited potent antitumor activity against human HCC cells by stimulating apoptosis in vitro and in vivo by a concomitant increase in autophagic flux. Blocking the TOC-triggered autophagy inhibited cellular proliferation and decreased tumour burden, indicating a protective role of autophagy against TOC-mediated HCC cell death. This role played by TOC-induced autophagy was further linked to the inactivation of the Akt/mTOR pathway that could be attributed to the upregulation of Cyr61. Moreover, treatment with sorafenib plus TOC resulted in pronounced synergistic effects on HCC cells. Our results elucidate a newly identified therapeutic potential of TOC in treating HCC, sparking a growing interest in repurposing such canine drugs for human use.

Autophagy-Related Gene WD Repeat Domain 45B Promotes Tumor Proliferation and Migration of Hepatocellular Carcinoma through the Akt/mTOR Signaling Pathway

Hepatocellular carcinoma (HCC) is a highly aggressive malignant tumor. It has been found that autophagy plays a role both as a tumor promoter and inhibitor in HCC carcinogenesis. However, the mechanism behind is still unveiled. This study aims to explore the functions and mechanism of the key autophagy-related proteins, to shed light on novel clinical diagnoses and treatment targets of HCC. Bioinformation analyses were performed by using data from public databases including TCGA, ICGC, and UCSC Xena. The upregulated autophagy-related gene WDR45B was identified and validated in human liver cell line LO2, human HCC cell line HepG2 and Huh-7. Immunohistochemical assay (IHC) was also performed on formalin-fixed paraffin-embedded (FFPE) tissues of 56 HCC patients from our pathology archives. By using qRT-PCR and Western blots we found that high expression of WDR45B influenced the Akt/mTOR signaling pathway. Autophagy marker LC3- II/LC3-I was downregulated, and p62/SQSTM1 was upregulated after knockdown of WDR45B. The effects of WDR45B knockdown on autophagy and Akt/mTOR signaling pathways can be reversed by the autophagy inducer rapamycin. Moreover, proliferation and migration of HCC can be inhibited after the knockdown of WDR45B through the CCK8 assay, wound-healing assay and Transwell cell migration and invasion assay. Therefore, WDR45B may become a novel biomarker for HCC prognosis assessment and potential target for molecular therapy.

Use of Apatinib as a Bait to Fish its Unexpected Kinase Targets from the Hepatocellular Carcinoma Druggable Kinome

Apatinib is a tyrosine kinase inhibitor that cognately blocks the kinase activity of vascular endothelial growth factor receptor (VEGFR) signaling for the treatment of advanced gastric cancer (GC). However, the drug is also clinically found to reposition a significant suppressing potency on hepatocellular carcinoma (HCC). In this study, we reported the successful use of Apatinib as a bait to fish its potential kinase targets from the HCC druggable kinome pool. In the procedure, cell viability assays observed that the Apatinib has a potent cytotoxicity on human HCC cell lines. Dynamics simulations and affinity scoring systematically created an intermolecular interaction profile of Apatinib with ontologically enriched kinases in the HCC druggable kinome, from which the top-hit kinases were considered as potential candidates. It is revealed that the inhibitor has a weak potency on the well-established HCC target of ErbB pathway, but exhibits potent activity against some known targets or regulators of HCC. In particular, kinase assays substantiated that Apatinib can effectively inhibit four FGFR family members with moderate or high activity. In addition, the clinical FGFR1 gatekeeper mutation V561M was also observed to considerably impair the inhibitory activity, thus causing a drug resistance. Molecular modeling suggested that the Apatinib adopts two distinct binding modes to separately interact with wild-type and gatekeeper-mutant FGFR1 kinase domain.

Anti-Cancer Effects of Queen Bee Acid (10-Hydroxy-2-Decenoic Acid) and Its Cellular Mechanisms against Human Hepatoma Cells

Hepatocellular carcinoma (HCC) is the most common form of liver cancer that occurs in hepatocytes. Although many chemical drugs, e.g., cisplatin, methotrexate, taxis, and doxorubicin are used to treat HCC, there have been numerous reports related to the side effects of these drugs (e.g., emerging drug resistance, bone marrow failure, and gastrointestinal disorders). These issues led scientists to search for the novel anti-cancer drugs, mainly in natural products with greater efficiency and less toxicity. The current survey was intended to assess the anti-cancer effects of queen bee acid (10-Hydroxy-2-Decenoic Acid, 10-HDA) and its cellular mechanisms against the human hepatoma cell line HepG2. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to evaluate the effect of 10-HDA on the viability of HepG2 cells. The initial and late apoptosis in the HepG2 cells treated with 10-HDA were assessed by the Annexin-V (AV) assay. The level of the gene and protein expression of some apoptosis genes (e.g., caspase-3, Bcl-2-associated X protein (BAX), and B-cell lymphoma protein 2 (Bcl-2)), Poly (ADP-ribose) polymerases (PARP), and miRNA-34a (miR-34a), were measured by real-time PCR and Western blot. The obtained findings revealed that HepG2 cell viability was markedly reduced (p < 0.01) following exposure to 10-HDA in a dose-dependent matter. The calculated half maximal cytotoxic concentration (CC50) value of 10-HDA was 59.6 µg/mL for HepG2 cells, while this value for normal THLE-3 cells was 106.4 µg/mL. We found that 10-HDA markedly elevated (p < 0.01) the percentage of necrotic and apoptotic cells from 0.94 to 9.7 and 27.6%, respectively. The real-time PCR results showed that the expression levels of the caspase-3, Bax, and miR-34a genes were significantly (p < 0.001) elevated. Contrary to these results, a significant (p < 0.01) reduction in the expression level of the Bcl2 gene was observed. The levels of protein expression of Caspase-3, PARP, and Bax were markedly elevated following exposure of HepG2 cells to 10-HDA at ¼ CC50, ½ CC50, and CC50. The level of protein expression of Bcl-2 was markedly reduced following exposure of HepG2 cells to 10-HDA at ¼ CC50, ½ CC50, and CC50 (p < 0.01). The current results confirmed the potent in vitro cytotoxic effects of 10-HDA on HepG2 cells with no significant cytotoxic effects on normal cells. Although its mechanisms of action have not been fully studied, the induction of apoptosis via different pathways was determined as one of the principle mechanisms of action of 10-HDA against HepG2 cells. Nevertheless, additional surveys must be performed to clearly understand the mechanisms of action and safety of this fatty acid.