SK-Hep1 Cell Lines Research Articles

Green Formulation of Descurainia sophia Aqueous Extract Mediated Ag NPs and Determination of Its Anti-Hemangioma Effects

Hemangioma is a common cancer with severe clinical signs. Finding a new anti-hemangioma supplement or drug inside of the chemotherapeutic drug is valuable. We synthesized silver nanoparticles containing Descurainia sophia leaf for investigating the anti-hemangioma, cytotoxicity, and antioxidant effects. Characterization of AgNPs was performed by Field Emission Scanning Electron Microscopy (FE-SEM), UV–Visible Spectroscopy (UV-Vis), and X-Ray diffraction analysis (XRD). The plain peak in the wavelength 449 nm revealed the AgNPs synthesis. TEM and FE-SEM analyses showed that synthesized AgNPs were found to be 16.9 to 48.43 nm mean particle size. For investigating the antioxidant efficacy, the DPPH assay was conducted. AgNPs prevented 50% of DPPH in the dilation of 40 μg/mL. To measure the anti-hemangioma efficacy of AgNPs, MTT assay was applied on SK HEP-1 and HAEND cell lines. AgNPs had high anti-hemangioma efficacy on above tumor cells without any significant toxicity on HUVEC. The best finding of anti-hemangioma properties of AgNPs was seen in the case of the HAEND cell line. This study indicated significant anti-hemangioma and antioxidant potentials of AgNPs containing Descurainia sophia.

Abstract 3284: Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma cell lines

Abstract Hepatocellular carcinoma (HCC) is the most common form of liver cancer which accounts for more than 90% of occurrences of liver cancer cases. Surgical therapies such as liver transplantation and hepatectomy may help cure hepatocellular carcinoma diagnosed in its early stages. Advanced stages of the disease have a poor prognosis and a high refractoriness because of drug resistance. Sorafenib is a tyrosine kinase inhibitor that has been approved for the systemic treatment of patients with advanced HCC. However, the efficacy of this medication has shown a limited prolongation of the overall survival in patients with advanced HCC. The mechanism of resistance to sorafenib in advanced HCC is poorly understood. The TCGA analysis of the HCC patient cohort shows an elevated expression of YBX1 in tumors as compared to normal. The expression is also correlated with progression, metastasis, and poor survival of HCC patients. Transcription factor YBX1 is involved in the regulation of drug resistance in different cancers, especially breast and colorectal cancers. Our lab is interested in understanding if YBX1 is involved in sorafenib resistance in HCC. Different HCC cell lines procured from ATCC SK-Hep1, Huh-7luc, and C3A, were analyzed for their IC50 values for sorafenib using MTT assays, at different time points. SK-Hep1 showed 8.780µM and 8.174µM, HepG2luc 4.659µM and 3.596µM at 48 and 72 hrs respectively. Overexpression of YBX1 in the SK-Hep1 cell line increased the cell survival and IC50 value of sorafenib at 48 and 72 hrs at 13.19µM and 10.64µM respectively. This supports the hypothesis which demonstrates the role of YBX1 in mediating resistance to sorafenib in advanced HCC. YBX1 could be a major therapeutic target and by inhibiting its action could improve the sensitivity of advanced HCC to sorafenib. We have developed YBX1 overexpressing and knockdown (GFP expressing) cell lines, FACS sorted and Puromycin selected for further mechanistic studies. We are also further developing the sorafenib-resistant HCC cell lines to understand the mechanism underlying sorafenib resistance in HCC. Protein and RNA analyses revealed a relative increase in YBX1 overexpression in sorafenib-resistant cell lines as compared to its parental cell lines. Understanding the mechanism of YBX1-induced drug resistance to sorafenib and its associated signaling pathways could help improve the sensitivity of advanced hepatocellular carcinoma to treatment by sorafenib and thus improve the livelihood of its patients. Citation Format: Dennis Kwabiah, Ana AyalaPazzi, Kyle Doxtater, Sophia Leslie, Manish K. Tripathi. Transcription factor YBX1 elevating the sorafenib IC50 in hepatocellular carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3284.

A Subpathway and Target Gene Cluster-Based Approach Uncovers lncRNAs Associated with Human Primordial Follicle Activation.

Long non-coding RNAs (lncRNAs) are emerging as a critical regulator in controlling the expression level of genes involved in cell differentiation and development. Primordial follicle activation (PFA) is the first step for follicle maturation, and excessive PFA results in premature ovarian insufficiency (POI). However, the correlation between lncRNA and cell differentiation was largely unknown, especially during PFA. In this study, we observed the expression level of lncRNA was more specific than protein-coding genes in both follicles and granulosa cells, suggesting lncRNA might play a crucial role in follicle development. Hence, a systematical framework was needed to infer the functions of lncRNAs during PFA. Additionally, an increasing number of studies indicate that the subpathway is more precise in reflecting biological processes than the entire pathway. Given the complex expression patterns of lncRNA target genes, target genes were further clustered based on their expression similarity and classification performance to reveal the activated/inhibited gene modules, which intuitively illustrated the diversity of lncRNA regulation. Moreover, the knockdown of SBF2-AS1 in the A549 cell line and ZFAS1 in the SK-Hep1 cell line further validated the function of SBF2-AS1 in regulating the Hippo signaling subpathway and ZFAS1 in the cell cycle subpathway. Overall, our findings demonstrated the importance of subpathway analysis in uncovering the functions of lncRNAs during PFA, and paved new avenues for future lncRNA-associated research.

ME2 Promotes Hepatocellular Carcinoma Cell Migration through Pyruvate.

Cancer metastasis is still a major challenge in clinical cancer treatment. The migration and invasion of cancer cells into surrounding tissues and blood vessels is the primary step in cancer metastasis. However, the underlying mechanism of regulating cell migration and invasion are not fully understood. Here, we show the role of malic enzyme 2 (ME2) in promoting human liver cancer cell lines SK-Hep1 and Huh7 cells migration and invasion. Depletion of ME2 reduces cell migration and invasion, whereas overexpression of ME2 increases cell migration and invasion. Mechanistically, ME2 promotes the production of pyruvate, which directly binds to β-catenin and increases β-catenin protein levels. Notably, pyruvate treatment restores cell migration and invasion of ME2-depleted cells. Our findings provide a mechanistic understanding of the link between ME2 and cell migration and invasion.

IGF-1R down regulates the sensitivity of hepatocellular carcinoma to sorafenib through the PI3K / akt and RAS / raf / ERK signaling pathways

BackgroundInsulin-like growth factor-1 receptor (IGF-1R) promotes cell proliferation and migration and inhibitsapoptosis, all of which can contribute to the development of cancers.MethodThis study investigated the effect and mechanism of IGF-1R in mediating the desensitization of hepatocellular carcinoma (HCC) to sorafenib.ResultsIGF-1R, highly expressed in the HCC cell lines SK-Hep1 and HepG2, promotes cell proliferation, migration, and anti-apoptosis through PI3K / Akt and RAS / Raf / ERK signaling pathways, resulting in HCC resistance to sorafenib. Knockdown of IGF-1R by RNA interference decreased proliferation and cell migration and upregulation of sorafenib-induced apoptosis of HCC cells. In vivo studies demonstrated that IGF-1R knockdown inhibited the growth of SK-Hep1 xenografts.ConclusionThese data are evidence that IGF-1R participates in regulating the survival and cell growth of HCC through the PI3K / Akt and RAS / Raf / ERK signaling pathways. Intervention in the expression of IGF-1R may increase the inhibitory effect of sorafenib on HCC.

COMMD3 Expression Affects Angiogenesis through the HIF1α/VEGF/NF-κB Signaling Pathway in Hepatocellular Carcinoma In Vitro and In Vivo

Background High expression of copper metabolizing MURR1 domain (COMMD3) is significantly correlated with poor prognosis in hepatocellular carcinoma (HCC) patients. Here, we explored the mechanism by which COMMD3 affects HCC angiogenesis through the HIF1α/VEGF/NF-κB signaling pathway. Methods SK-Hep1 and Hep-3B cell lines were transfected by COMMD3 overexpression and RNA interference lentivirus and verified using RT-qPCR and western blotting techniques. Using RNA sequencing, we analyzed differentially expressed genes in COMMD3-overexpressed and COMMD3-knockdown HCC cells. Altogether, colony formation assay, wound healing assay, transwell cell invasion assay, flow cytometry apoptosis experiments, HUVEC tube formation detection, phalloidin staining assay, western blotting, immunohistochemical staining, and a nude mouse xenograft model were used for experimental verification. Results Lentivirus COMMD3 overexpression and knockdown were successfully established in HCC cells. COMMD3 overexpression significantly promoted the proliferation, angiogenesis, migration, and invasion capacities of HCC cells with no obvious effect on apoptosis versus controls while COMMD3 knockdown showed the opposite trend. The expression and protein levels of COMMD3 as well as HIF1α, VEGF, and NF-κB were increased in COMMD3-overexpressing HCC cells versus control cells, while they were reduced after COMMD3 knockdown. In addition, RNA-seq indicated that COMMD3 is an indispensable gene for HCC angiogenesis through HIF1α and NF-κB signaling pathways. Conclusion This study showed that low expression of COMMD3 can inhibit HCC angiogenesis by suppressing the HIF1α/VEGF/NF-κB pathway. This implicates COMMD3 as a potential biomarker for improving the therapeutic outcome of HCC.

Evaluation of antioxidant, antimicrobial and antiproliferative activity of silver nanoparticles derived from Galphimia glauca leaf extract

ObjectiveThe bio-synthesis of silver nanoparticles (AgNPs) is regarded as environment friendly and cost effective method compared to physical and chemical synthesis methods. Silver (Ag) is an important noble metal due to its tremendous use in research and medical fields throughout the world. This research work was aimed at green synthesis and characterizations of AgNPs from G. glauca leaf extract and evaluation of their bioactive potential. MethodsIn the current research work, synthesis of AgNPs from G. glauca aqueous leaf extracts was performed and was characterized by UV–Visible (UV–Vis.), Fourier transform infrared (FTIR), Atomic force microscopy (AFM), Scanning electron microscopy (SEM), Energy dispersive spectroscopy (EDS), High-resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), Thermo gravimetric analysis (TGA) and Zeta potential exploration. Later, antioxidant, antimicrobial activity was examined against various pathogenic organisms and anticancer activities of AgNPs were carried out against SK-HEP1 cell line. ResultsThe first indication of synthesis of nanoparticles was the change in color from yellowish to brown. UV–Vis. band exhibited surface plasmon resonance (SPR) at 402 nm. FTIR analysis revealed the probable bio-molecules including alkanes, alkenes, aromatics, aromatic phosphates, imine or oxime, etc. subjected to reduction of silver ions to metallic silver. The AFM, SEM and TEM analysis reported the particles were spherical shaped, poly-dispersed, 17 to 40 nm in size. The zeta potential analysis expressed a peak at –32.0 ± 0.5 mV and suggested the particles with significant long term stability. The TGA analysis revealed the stability of AgNPs at high temperature (179 ℃). The antioxidant assay of AgNPs unveiled an effective dose dependent increase in scavenging activity. Antimicrobial activity showed efficient inhibitory activity against P. aeruginosa and C. glabrata. Lastly, the AgNPs revealed a strong anticancer activity against SK-HEP1 liver cancer cell line with an IC50 value of 19.12 μg/mL. ConclusionsTherefore, it could be concluded that a substantial in-vivo investigations are needed for antioxidant, antibacterial and anticancer activities, so that it will be useful in medical field in future.

Extracellular and intracellular intermittent magnetic-fluid hyperthermia treatment of SK-Hep1 hepatocellular carcinoma cells based on magnetic nanoparticles coated with polystyrene sulfonic acid.

The use of magnetic nanoparticles (MNPs) magnetized on applying an alternating magnetic field (AMF) to stimulate the thermal characteristics and to induce tumor apoptosis is a currently active area of research in cancer treatment. In previous work, we developed biocompatible and superparamagnetic polystyrene-sulfonic-acid-coated magnetic nanoparticles (PSS-MNPs) as applications for magnetically labeled cell trapping, but without assessment of treatment effects on tumor diseases. In the present work, we examined PSS-MNP-induced magnetic fluid hyperthermia (MFH) on SK-Hep1 hepatocellular carcinoma (HCC) cells for lethal thermal effects with a self-made AMF system; an adjustable AMF frequency generated a variable intensity of magnetic field and induced MNP relaxation. The extracellular and intracellular MFH treatments on a SK-Hep1 cell line were implemented in vitro; the result indicates that the lethal effects were efficient and caused a significantly decreased cell viability of SK-Hep1 cells. As the PSS-MNP concentration decreased, especially in intracellular MFH treatments, the MFH effects on cells, however, largely decreased through heat spreading to the culture medium. On controlling and decreasing the volume of culture medium, the problem of heat spreading was solved. It can be consequently expected that PSS-MNPs would be a prospective agent for intracellular cancer magnetotherapy.

Effect of Zebularine in Comparison to Trichostatin A on the Intrinsic and Extrinsic Apoptotic Pathway, Cell Viability, and Apoptosis in Hepatocellular Carcinoma SK-Hep 1, Human Colorectal Cancer SW620, and Human Pancreatic Cancer PaCa-44 Cell Lines.

Aberrant histone modifications or promoter region hypermethylation of tumor suppressor genes (TSGs) have been recognized as the important epigenetic molecular mechanism in cancer induction. The potential anticancer activities of histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) have been investigated in recent years. The current study was assigned to investigate the effect of trichostatin A (HDACI) in comparison to zebularine (DNMTI) on the intrinsic pro-apoptotic (Bax, Bim, and Bak) and anti-apoptotic (Bcl-2, Mcl-1, and Bcl-xL) genes and extrinsic (DR4, DR5, FAS, FAS-L, and TRAIL genes) pathways, DNA methyltransferase 1, 3a, and 3b, histone deacetylase inhibitors 1, 2, and 3, cell viability, and apoptosis in hepatocellular carcinoma (HCC) SK-Hep 1, colorectal cancer SW620, and pancreatic cancer PaCa-44 cell lines. The SK-Hep 1, SW620, and PaCa-44 cells were cultured and treated with TSA and zebularine. To determine cell apoptosis, cell viability, and the relative gene expression level, flow cytometry assay, MTT assay, and qRT-PCR were done respectively. The result indicated that zebularine and TSA changed the expression level of the Bax, Bak, Bim Bcl-2, Bcl-xL, Mcl-1, DR4, DR5, FAS, FAS-L, TRAIL, DNA methyltransferase 1, 3a, and 3b, histone deacetylase inhibitors 1, 2, and 3 by which induced cell apoptosis and inhibit cell growth in all three cell lines. Concluding, TSA induced its role through both extrinsic and intrinsic apoptotic pathways in three cell lines, whereas, zebularine played its role via both pathways in the SK-Hep 1cell line, it had no significant effect on Bcl-2, Bcl-xL, and Mcl-1 gene expression in SW620 and PaCa-44 cell lines.

ARHGEF11 promotes proliferation and epithelial-mesenchymal transition of hepatocellular carcinoma through activation of β-catenin pathway.

Rho guanine nucleotide exchange factor 11 (ARHGEF11) has been proved to promote tumor metastasis in glioblastoma and ovarian carcinoma. However, the role of ARHGEF11 in hepatocellular carcinoma (HCC) progression is largely unknown. Here, we found that ARHGEF11 was upregulated in HCC samples and highly metastatic hepatoma cell lines. Knockdown of ARHGEFF11 inhibited the cell proliferation and invasion in both HCCLM3 and SKHEP1 cell lines. Subsequent mechanistic investigation showed that downregulation of ARHGEF11 significantly attenuated β-catenin nuclear translocation, thereafter repressed the expression of ZEB1 and cyclinD1, finally contributing to inhibition of epithelial-mesenchymal transition (EMT) and cell cycle arrest. Moreover, high levels of ARHGEF11 were found to be associated with shorter disease free and overall survival. A prognostic nomogram model that integrated ARHGEF11, tumor size and BCLC classification showed good performance in predicting clinical outcomes of HCC patients. Overall, this study demonstrated that ARHGEF11 could promote proliferation and metastasis of HCC via activating β-catenin pathway, suggesting that ARHGEF11 might serve as a potential prognostic biomarker for HCC.

Mechanism of citron rho-interacting serine/threonine kinase gene regulating adriamycin resistance in hepatocellular carcinoma cell line SK-Hep1#br#

Mechanism of citron rho-interacting serine/threonine kinase gene regulating adriamycin resistance in hepatocellular carcinoma cell line SK-Hep1#br#

Follistatin Like 5 (FSTL5) inhibits epithelial to mesenchymal transition in hepatocellular carcinoma.

BackgroundEpithelial to mesenchymal transition (EMT) is a key process in determining distant metastasis and intra-hepatic dissemination of hepatocellular carcinoma (HCC). Follistatin (FST) family members are considered to be an attractive therapeutic targets and prognostic indicators in cancers. As a derivative of FST, Follistatin Like 5 (FSTL5) may play a similar role in HCC cells. This study aimed to investigate the expression and function of FSTL5 in HCC and its role in EMT.MethodsFSTL5, E-cadherin and vimentin in HCC, and paracancerous tissues were detected by immunohistochemistry. Correlation of FSTL5 expression with overall survival was assessed. The proliferation and invasion of HCC cell lines SK-Hep1 and MHCC-LM3 were analyzed by cell counting kit-8 and Transwell assays. The expression of FSTL5, E-cadherin, and vimentin in HCC cells was examined by polymerase chain reaction and Western blot analysis. T-test was used to analyze the difference in proliferation and invasion ability between groups. The Spearman rank correlation test was used to detect the correlation between the expression of FSTL5 and E-cadherin or vimentin.ResultsThe expression of FSTL5 in HCC was lower than that in paracancerous tissues (9.97% vs. 82.55%, χ2 = 340.15, P < 0.001). Patients with high FSTL5 expression had a better prognosis (χ2 = 8.22, P = 0.004) and smaller tumor diameter (χ2 = 45.52, P < 0.001), less lymph node metastasis (χ2 = 5.58, P = 0.02), earlier tumor node metastasis stage (χ2 = 11.29, P = 0.001), a reduced number of tumors (χ2 = 5.05, P = 0.02), lower alpha-fetoprotein value (χ2 = 24.36, P < 0.001), more probability of hepatitis carrying (χ2 = 40.9, P < 0.001), and better liver function grade (χ2 = 5.21, P = 0.02). Immunohistochemistry showed that FSTL5 expression in HCC tissues was positively correlated with E-cadherin expression (r = 0.38, P < 0.001) and negatively correlated with vimentin expression (r = −0.385, P < 0.001). Furthermore, over-expression of FSTL5 up-regulated the expression of E-cadherin and down-regulated the expression of vimentin in SK-Hep1 (negative control [NC] vs. FSTL5-interfering group [Lv-FSTL5]: E-cadherin [t = 45.03, P < 0.001], vimentin [t = 67, P < 0.001]) and MHCC-LM3 (NC vs. Lv-FSTL5: E-cadherin [t = 50, P < 0.001], vimentin [t = 72.75, P < 0.001]) cells at mRNA level. The same as protein level. In addition, the over-expression of FSTL5 inhibited the proliferation (NC vs. Lv-FSTL5: SK-Hep1, 3 d [t = 7.324, P = 0.018], 4 d [t = 6.23, P = 0.021], 5 d [t = 10.21, P = 0.003]; MHCC-LM3, 3 d [t = 4.32, P = 0.037], 4 d [t = 7.49, P = 0.012], 5 d [t = 9.3661, P = 0.009]) and invasion (NC vs. Lv-FSTL5: SK-Hep1, t = 21.57, P < 0.001; MHCC-LM3, t = 18.04, P < 0.001) of HCC cells.ConclusionsDown-regulation of FSTL5 may contribute to EMT of HCC, and FSTL5 is a potential target in the treatment of HCC.

Effect of nicastrin on hepatocellular carcinoma proliferation and apoptosis through PI3K/AKT signalling pathway modulation

BackgroundIncreasing evidence has proven that the γ-secretase complex plays significant roles in the carcinogenesis of malignancies. However, the independent effect of nicastrin (NCSTN), the largest constituent of the γ-secretase complex, on the progression of hepatocellular carcinoma (HCC) remains to be discovered.MethodsIn our study, we used open online databases, including the Oncomine database, GEPIA and KMPlotter, to analyse the expression of 4 genes and their correlation with prognosis in HCC. NCSTN expression in 60 HCC patients from our centre was determined by immunohistochemical staining and qRT-PCR. The clinical and prognostic significance of NCSTN expression were analysed statistically. Stable Sk-hep1 cell lines with NCSTN overexpression were established using lentivirus-based vectors, and RNAi technology was used to transiently downregulate NCSTN expression in HepG2 cell lines. Cell growth and apoptosis were assessed by using EdU, clone formation, flow cytometry and Western blotting assays.ResultsBioinformatics analysis showed that NCSTN mRNA expression was generally higher in HCC tissues than in normal tissues according to a meta-analysis of 9 HCC datasets, excluding PS-1, PEN-2 and APH-1. Moreover, NCSTN was associated with a poor prognosis in HCC patients from The Cancer Genome Atlas (TCGA). Although the relationship between NCSTN levels and the clinicopathological features of HCC patients was not significant, a survival analysis of HCC patients from TCGA indicated that overall and disease-free survival were significantly associated with NCSTN expression. NCSTN expression in HCC cell lines regulated cell growth and apoptosis in vitro. NCSTN downregulation in HepG2 cells inhibited tumour growth ability in vivo. In addition, NCSTN downregulation in HepG2 cell lines decreased p-PI3K and p-Akt expression, and IGF1, a PI3K/Akt activator, neutralized the effects on PI3K and Akt phosphorylation. Moreover, NCSTN overexpression in Sk-hep1 cells activated the PI3K/Akt signalling pathway, and MK-2206, a PI3K/Akt inhibitor, reversed this activation according to Western blotting analysis.ConclusionsWe suggest that NCSTN serves as an oncogene in HCC by promoting growth and inhibiting apoptosis via the PI3K/Akt pathway, providing a potential novel therapeutic target for HCC treatment.

Upregulation of hypoxia-inducible factor 1α mRNA expression was associated with poor prognosis in patients with hepatocellular carcinoma.

BackgroundHIF1α mRNA expression in hepatocellular carcinoma (HCC) tissues and its relationship with the prognosis in HCC patients is still unclear. We performed this study to investigate the expression of HIF1α mRNA and its correlation with the prognosis in HCC patients.Materials and methodsGSE14520 and Oncomine database were used to analyse the differential expression of HIF1α mRNA among HCC tissues and corresponding peritumour tissues or normal liver tissues. The relationship between HIF1α mRNA expression and the clinicopathological features and survival in HCC patients was analysed using the GSE14520 dataset. CCK-8 assay, wound-healing assay, transwell invasion assay, tube formation assay, and subcutaneous xenograft tumour assays using nude mice were used to confirm the function of HIF1α.ResultsExpression of HIF1α mRNA was significantly upregulated in HCC tissues (P<0.05 in all cases); this was supported by the results of the Western blotting (P=0.031) and IHC analyses. Our analysis of the clinicopathological features of HCC patients indicated that high HIF1α mRNA expression was strongly related with TNM stage III (P=0.002) and BCLC stage C (P=0.038). Survival analysis demonstrated that HCC patients with high HIF1α mRNA expression had a short overall survival (OS) (P=0.048), but showed no significant difference in recurrence-free survival (RFS) (P=0.066) compared to patients with low HIF1α mRNA expression. We further demonstrated that HIF1α promoted the proliferation, migration, invasion, and angiogenic ability of HCC cells, by using the stably transformed SK-Hep1 and Hep-3B cell lines showing HIF1α overexpression. Finally, xenograft tumour models of nude mice showed that RNA interference-mediated HIF1α silencing suppressed tumour growth and angiogenesis in HCC.ConclusionOur study suggests that the upregulation of HIF1α mRNA, which is found in HCC tissues and associated with poor prognosis in HCC patients, contributed to the proliferation, migration, invasion, and angiogenic ability of HCC cells.

Dependence of artesunate on long noncoding RNA-RP11 to inhibit epithelial-mesenchymal transition of hepatocellular carcinoma.

As a first line medicine for malaria treatment, artesunate (ART) also shows antitumor potential. However, little is known about the effect of ART on the cancer cell epithelial-mesenchymal transition (EMT). In this study, we found that ART inhibited cell growth in SK-HEP1 and SM7721 hepatocellular carcinoma cell lines. A microarray was used to identify differentially expressed protein-coding RNAs (pcRNA) and long noncoding RNAs (lncRNA) between SK-HEP1 cells with and without ART treatment. A differentially expressed lncRNA-RP11, the most related to the EMT of liver cancer cells-RP11 was identified by abioinformatics method Overexpressing and silencing assays were used to verify the role of RP11 in cancer cell EMT. The levels of RP11- and EMT-related genes in liver cancer samples from 75patients were detected by using qualitative polymerase chain reaction or immunohistochemistry. We identified 1334 pcRNAs and 1670 lncRNA with differential expression induced by ART. ART inhibits EMT, proliferation, migration, invasion, and adhesion of liver cancer cells. RP11 depresses the inhibitory effect of ART on cancer cell EMT. The level of RP11 is associated with cancer cell EMT and metastasis and survival rate of the patient. These data suggest that RP11-linking ART and cancer cell EMT are important for ART-inhibited metastasis of liver cancer.

Sphingosine kinase 1 overexpression is associated with poor prognosis and oxaliplatin resistance in hepatocellular carcinoma.

Sphingosine kinase 1 (SphK1) is a tumor-associated protein overexpressed in numerous types of cancer and is involved in the regulation of resistance to multiple chemotherapeutic agents. However, the role of SphK1 in the resistance of hepatocellular carcinoma (HCC) to oxaliplatin remains unclear. In the present study, the transcriptional levels of SphK1 were analyzed in 21 patients with HCC and the SphK1 expression levels were identified to be significantly upregulated in HCC tissue compared with that in adjacent normal tissue samples (P<0.001). High SphK1 expression correlated with shorter overall survival times in patients with HCC (P<0.05). Furthermore, SphK1 expression levels and activity were analyzed in a series of HCC cell lines and they were both demonstrated to be associated with resistance to oxaliplatin. Conversely, the knockdown of SphK1 protein expression resulted in decreased oxaliplatin resistance in SK-Hep1 and HCCLM3 cell lines. In addition, the results of the current study demonstrated that the downregulation of SphK1 decreased the levels of phosphorylated AKT serine/threonine kinase (Akt) and glycogen synthase kinase-3β (GSK3β), suggesting that SphK1 promotes oxaliplatin resistance of HCC cells via modulation of the Akt/GSK3β signaling pathway. To the best of our knowledge, the present study is the first to report that SphK1 is associated with poor prognosis and oxaliplatin resistance in HCC. Thus, the findings of the current study have provided a direction for the identification of novel therapeutic targets for the treatment of HCC.

Amentoflavone enhances sorafenib-induced apoptosis through extrinsic and intrinsic pathways in sorafenib-resistant hepatocellular carcinoma SK-Hep1 cells in vitro.

The present study aimed to evaluate the effects of amentoflavone on sorafenib-induced apoptosis in sorafenib-resistant hepatocellular carcinoma (HCC) cells. The sorafenib-resistant SK-Hep1 (SK-Hep1R) cell line was established for the present study. Initially, the differences in sorafenib-induced cytotoxicity and apoptosis between wild-type SK-Hep1 and SK-Hep1R cells were verified using the MTT assay and flow cytometry. The effects of amentoflavone on sorafenib-induced cytotoxicity and apoptosis were then investigated using MTT, flow cytometry, DNA gel electrophoresis and western blot analysis. The results demonstrated that cell viability of SK-Hep1R cells was increased compared with that of SK-Hep1 cells following treatment with different concentrations of sorafenib for 24 h. Apoptosis of SK-Hep1R cells was lower than that of SK-Hep1 cells following treatment with 20 µM sorafenib for 24 h. Amentoflavone alone did not inhibit cell viability but significantly triggered sorafenib-induced cytotoxicity and apoptosis in SK-Hep1R cells. Amentoflavone not only reversed sorafenib-induced anti-apoptotic protein levels but also enhanced sorafenib-induced pro-apoptotic protein expression in SK-Hep1R cells. In conclusion, amentoflavone may be used as a sorafenib sensitizer to enhance sorafenib-induced cytotoxicity and trigger sorafenib-induced apoptosis through extrinsic and intrinsic pathways in SK-Hep1R cells.

Abstract 5328: RNF12 inhibits the proliferation of hepatocellular carcinoma through c-Myc and p21

Abstract Purpose: RNF12/RLIM, a RING E3 ubiquitin ligase, plays an important role in the epigenetic regulation of sex-specific nurturing tissues in female mice. Recently, a few studies demonstrated a correlation between RNF12 and the proliferation of multiple cancers. The role of RNF12 in hepatocellular carcinoma is incompletely understood. The purpose of this study is to investigate the effect of RNF12 on the proliferation of human hepatocellular carcinoma cells. RNF12 is also known to interact with a number of nuclear proteins, including CLIM, HDAC2, and Smad4. We also explored whether c-Myc and p21, an oncogene and tumor suppressor essential to liver tumorigenesis, were affected by RNF12. Methods: Immunohistochemistry of RNF12 and c-Myc was performed on surgically dissected human hepatocellular carcinoma tissues. The soft-agar cloning, flow cytometry and Transwell invasion assays were carried out to investigate the role of RNF12 in SK-Hep1 cell proliferation, cell cycle and invasiveness. Real-time quantitative PCR and Western blotting were used to examine the mRNA and protein levels of RNF12 and related proteins in SK-Hep1 cells. Results: We found that the level of RNF12 was lower in hepatocellular carcinoma, compared with the surrounding non-cancerous tissues, while the c-Myc expression was higher. Using a SK-Hep1 cell line stably expressing RNF12, we showed that RNF12 over-expression inhibited cell proliferation and induced cell cycle arrest at G1 phase. Furthermore, we showed that RNF12 over-expression decreased the level of oncoprotein c-Myc, while not much affecting its mRNA level. Co-immunoprecipitation demonstrated that RNF12 and c-Myc associated with each other, suggesting a direct protein-protein interaction. RNF12 over-expression also markedly increased the mRNA and protein levels of p21, a tumor suppressor and the downstream target of p53, c-Myc and many other cancer-related regulators. Consistently, silencing of RNF12 reduced p21 mRNA and protein levels and increased c-Myc protein. Conclusion: Our study demonstrated reduced expression of RNF12 in hepatocellular carcinoma, and RNF12 over-expression inhibited the proliferation and cell cycle progression of SK-Hep1 cells. Mechanistically, RNF12 may directly regulate the level of c-Myc, similarly as other transcription factors such as CLIM and Smad4. The mechanisms underlying the increased expression of p21 is being investigated. Taken together, RNF12 may serve as a tumor suppressor in hepatocellular carcinoma, partly through oncoprotein c-Myc and tumor suppressor p21. Note: This abstract was not presented at the meeting. Citation Format: Meng Nie, Yongsheng Huang, Lin Wang. RNF12 inhibits the proliferation of hepatocellular carcinoma through c-Myc and p21 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5328. doi:10.1158/1538-7445.AM2017-5328

Single Agent and Synergistic Activity of the "First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma.

Deregulated PI3K/AKT/mTOR, Ras/Raf/MAPK, and c-Myc signaling pathways are of prognostic significance in hepatocellular carcinoma (HCC). Sorafenib, the only drug clinically approved for patients with advanced HCC, blocks the Ras/Raf/MAPK pathway but it does not inhibit the PI3K/AKT/mTOR pathway or c-Myc activation. Hence, there is an unmet medical need to identify potent PI3K/BRD4 inhibitors, which can be used either alone or in combination with sorafenib to treat patients with advanced HCC. Herein, we show that SF1126 (pan PI3K/BRD4 inhibitor) as single agent or in combination with sorafenib inhibited proliferation, cell cycle, apoptosis, and multiple key enzymes in PI3K/AKT/mTOR and Ras/Raf/MAPK pathway in Hep3B, HepG2, SK-Hep1, and Huh7 HCC cell lines. We demonstrate that the active moiety of the SF1126 prodrug LY294002 binds to and blocks BRD4 interaction with the acetylated histone-H4 chromatin mark protein and displaced BRD4 coactivator protein from the transcriptional start site of MYC in Huh7 and SK-Hep1 HCC cell lines. Moreover, SF1126 blocked expression levels of c-Myc in HCC cells. Treatment of SF1126 either alone or in combination with sorafenib showed significant antitumor activity in vivo Our results establish that SF1126 is a dual PI3K/BRD4 inhibitor. This agent has completed a phase I clinical trial in humans with good safety profile. Our data support the potential future consideration of a phase II clinical trial of SF1126, a clinically relevant dual "first-in-class" PI3K/BRD4 inhibitor in advanced HCC, and a potential combination with sorafenib. Mol Cancer Ther; 15(11); 2553-62. ©2016 AACR.

Huaier restrains proliferative and invasive potential of human hepatoma SKHEP-1 cells partially through decreased Lamin B1 and elevated NOV.

Hepatocellular carcinoma (HCC) is one of the most common cause of malignancy-related mortality worldwide. It is urgently needed to develop potential drugs with good efficacy and low toxicity for HCC treatment. The anti-tumor effect of Traditional Chinese Medicine (TCM) has received increasing attention worldwide. Trametes robiniophila Murr. (Huaier) has been used in TCM for approximately 1,600 years. Clinically, Huaier has satisfactory therapeutic effects in cancer treatment, especially in HCC. However, the mechanisms underlying the anti-cancer effect of Huaier remain ill defined. Herein we have demonstrated that Huaier dramatically inhibited cell proliferation and induced apoptosis in human hepatoma cell line SKHEP-1. Importantly, Huaier restrained the metastatic capability of SKHEP-1 cells. Mechanistically, down-regulation of Lamin B1 and up-regulation of Nephroblastoma overexpressed (NOV) were at least partially responsible for the inhibitory effect of Huaier on the proliferative and invasive capacity of SKHEP-1 cells. Our finding provided new insights into mechanisms of anti-HCC effect of Huaier and suggested a new scientific basis for clinical medication.