Line HepG2 Research Articles

IFI44L is a novel tumor suppressor in human hepatocellular carcinoma affecting cancer stemness, metastasis, and drug resistance via regulating met/Src signaling pathway

BackgroundHepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. The disease recurrent rate is relatively high resulted in poor 5-year survival in advanced HCC. Cancer stem cells (CSCs) have been considered to be one of the main mechanisms for chemoresistance, metastasis, and recurrent disease. Interferon-induced protein 44-like (IFI44L) gene is a type I interferon-stimulated gene (ISG) and belongs to the IFI44 family. Previous reports indicated antiviral activity against HCV in IFI44L, however, its precise role and function in HCC has not been unveiled.MethodsTo explore the characteristics of hepatic CSCs, we successfully enriched hepatic cancer stem-like cells from three established liver cancer cell lines (Hep3B, HepG2, and PLC lines). Parental Hep3B and HepG2 cells and their sphere cells were treated with doxorubicin for 48 h and cell viability was measured by MTT assay. HCC tissue blocks from 217 patients were sampled for tissue microarray (TMA). Follow-up information and histopathological and clinical data including age, gender, tumor grade, advanced stages, HBV, HCV, tumor number, tumor size, relapse-free survival, and overall survival were obtained from the cancer registry and medical charts. The liver TMA was evaluated for IFI44L expression using immunohistochemical staining and scores.ResultsThese hepatic cancer stem-like cells possess important cancer stemness characteristics including sphere-forming abilities, expressing important HCC cancer stem cell markers, and more chemoresistant. Interestingly, we found that overexpression of IFI44L decreased chemoresistance towards doxorubicin and knockdown of IFI44L restored chemoresistance as well as promoted sphere formation. Furthermore, we found that depletion of IFI44L enhanced migration, invasion, and pulmonary metastasis through activating Met/Src signaling pathway. Clinically, the expression level of IFI44L significantly reduced in HCC tumor tissues. Low expression of IFI44L levels also correlated with larger tumor size, disease relapse, advanced stages, and poor clinical survival in HCC patients.ConclusionTaken together, we first demonstrated that IFI44L is a novel tumor suppressor to affect cancer stemness, metastasis, and drug resistance via regulating Met/Src signaling pathway in HCC and can be serve as an important prognostic marker.

The effect of microRNA-194 on the proliferation and migration of hepatoma cells

Objective To detect the expression of microRNA (miRNA, miR)-194 in hepatocellular carcinoma tissues and hepatoma cell lines, and further explore the effect of miR-194 on the proliferation and migration of hepatoma cells. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-194 in 20 cases of hepatocellular carcinoma and 20 cases of adjacent tissues and hepatocellular carcinoma cell line HepG2 and normal liver cell line L02. The hepatocellular carcinoma cell line overexpressing or low expressing miR-194 was established The cell proliferation assay, cell cycle assay, scratch assay, and Western blotting were used to detect the cancer cell migration and proliferation change and its related mechanism. Results The relative expression of miR-194 in hepatocellular carcinoma tissues was significantly higher than that in the tissue adjacent to carcinoma (0.364±0.040 vs. 0.198±0.028, t=3.176, P=0.003). The relative expression of miR-194 in HepG2 cells was also significantly higher than that in the normal liver cell line (0.517±0.069 vs. 0.181±0.025, t=4.557, P=0.010). In miR-194 overexpressed cell lines, the cell proliferation (1.857±0.170 vs. 1.019±0.019, t=7.065, P=0.000) and migration (0.483±0.084 vs. 0.927±0.030, t=5.181, P=0.008) significantly increased as compared with the control group of hepatocellular carcinoma cells, and in miR-194 low expression group the cell proliferation ability decreased significantly as compared with the control group (0.529±0.095 vs. 1.019±0.019, P=0.006) in hepatocellular carcinoma cells. the expression of Wnt protein in the over expressed miR-194 was significantly higher than the control group (0.589±0.194 to 0.302±0.013, t=3.035, P=0.039); and the expression of β-catenin protein was significantly higher than the control group (0.941±0.101 to 0.220±0.115, t=9.434, P=0.000), the differences were statistically significant, and the low expression of miR-194 cells Wnt protein (0.310±0.028 to 0.260±0.026, t=1.308, P=0.261) and β-catenin protein (0.179±0.028 to 0.080±0.034, t=2.242, P=0.088) compared with the control group was no statistically significant difference. Conclusion The overexpression of miR-194 in hepatocellular carcinoma is associated with the increased proliferation and invasion of hepatocellular carcinoma cells, probably by upregulating the Wnt/β-catenin signaling pathway. Key words: Carcinoma, hepatocellular; MicroRNA-194; Cell proliferation; Cell migration; Wnt/β-catenin signaling pathway

Microbial transformation of pseudoprotodioscin by Gibberella fujikuroi

Three new (6, 9, and 12) and nine known steroidal saponins were obtained from the fermentation broth of pseudoprotodioscin (PPD) incubated with a fungus Gibberella fujikuroi CGMCC 3.4663. Structures of the metabolites were elucidated by 1-D (1H, 13C), 2-D (HMBC, HSQC, NOESY) NMR, and HR-MS analyses. The biotransformation pathway of pseudoprotodioscin by Gibberella fujikuroi CGMCC 3.4663 was proposed. Compounds 1–11 were tested in vitro for their cytotoxic activities against two human cancer cell lines (HepG2 and Hela). Compounds 1, 6, 9, and 10 exhibited cytotoxic activity against HepG2 cells. Compound 10 exhibited cytotoxicity to Hela cells.

Abstract 033: Differential Effects of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Production and Function on Microsomal Triglyceride Transfer Protein

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein that plays a key role in the regulation of plasma low-density lipoprotein (LDL) cholesterol levels. PCSK9 binding to the LDL receptor (LDLR) leads to receptor-mediated endocytosis and lysosomal degradation of LDLR. Prior studies have shown that PCSK9 increases production of triglyceride-rich apoB-lipoproteins via up-regulation of lipogenic genes, and that PCSK9 inhibition reduces plasma triglyceride levels. However, the effect of PCSK9 inhibition on hepatic lipogenic genes expression remain unclear. Using a human hepatocellular carcinoma cell line (HepG2) we show that overexpression of PCSK9 upregulated the expression of several lipogenic genes, including a 1.5-fold increase in ATP citrate lyase (ACLY), a 1.2-fold increase in Fatty Acid Synthase (FAS), a 1.2-fold increase in HMG-CoA-reductase, and a 1.8-fold increase in microsomal triglyceride transfer protein (MTTP), when compared with untransfected HepG2 cells. Overexpression of a gain-of-function mutant of PCSK9 (PCSK9-D374Y) had stronger effects in the same direction: a 2.3-fold increase in ACLY, a 1.8-fold increase in FAS, a 1.6-fold increase in HMG-CoA-reductase, and a 2.1-fold increase in MTTP. In HepG2 cells overexpressing either normal PCSK9 or PCSK9-D374Y, inhibition of PCSK9 function using a monoclonal antibody blocked PCSK9-mediated degradation of LDLR, and reduced expression of SREBP-dependent genes (ACLY, FAS and HMG-CoA-reductase) to levels of untransfected HepG2 cells. Interestingly, while inhibition of PCSK9 function via monoclonal antibody did not affect MTTP gene expression in these cells, inhibition of PCSK9 production via RNA interference reduced MTTP gene expression by 66%. We conclude that inhibition of PCSK9 production decreases MTTP gene expression, whereas blockade of PCSK9 function (and cellular re-entry) only decreases SREBP-dependent genes expression, but does not affect MTTP gene expression. This study shows that while PCSK9 exerts an influence on genes related to both lipogenesis and lipoprotein assembly, any inhibition of PCSK9 controls SREPB-dependent genes but only inhibiting PCSK9 production controls MTTP gene expression.

Synthesis of New α-Amino Phosphonates Containing 3-Amino-4(3H) Quinazolinone Moiety as Anticancer and Antimicrobial Agents: DFT, NBO, and Vibrational Studies

Aim and Objective: Synthesis of new .-aminophosphonates containing quinazoline moiety through Kabachnik-Fields reaction in the presence of copper triflate catalyst [32], followed by studying their antimicrobial activities and in vitro anticancer activities against liver carcinoma cell line (HepG2) with the hope that new anticancer agents could be developed. Also, the quantum chemical calculations are performed using density functional theory (DFT) to study the effect of the changes of molecular and electronic structures on the biological activity of the investigated compounds. Materials and Method: The structures of the synthesized compounds are confirmed by FT-IR, 1H NMR, 13C NMR, 31P NMR and MS spectral data. The synthesized compounds show significant antimicrobial and also remarkable cytotoxicity anticancer activities against liver carcinoma cell line (HepG2). Density functional theory (DFT) was performed to study the effect of the molecular and electronic structure changes on the biological activity. Results: It was found that the electronic structure of the substituents affects on the reaction yield. The electron withdrawing substituent, NO2 group 3b, on the aromatic aldehydes gave a good yield more than the electron donating substituent, OH group 3c. The electron deficient on the carbon atom of the aldehydic group may increase the interaction of the Lewis acid (Cu(OTf)2) and the Lewis base (imine nitrogen), and accordingly, facilitate the formation of imine easily, which is attacked by the nucleophilic phosphite species to give the α- aminophosphonates. Conclusion: The newly synthesized compounds exhibit a remarkable inhibition of the growth of Grampositive, Gram-negative bacteria and fungi at low concentrations. The cytotoxicity of the synthesized compounds showed a significant cytotoxicity against the liver cancer cell line (HepG 2). Also, it was shown from the quantum chemical calculations that the electron-withdrawing substituent increases the biological activity of the α-aminophosphonates more than the electron donating group which was in a good agreement with the experimental results. Also, a good agreement between the experimental FT-IR and the calculated one was found.

Synthesis of anthraquinone-capped TiO2 nanoparticles using R. emodi roots: preparation, characterization and cytotoxic potential

Rheum emodi is a Himalayan herb of family Polygonaceace, known for its medicinal properties owing to the presence of chrysophanol, emodin, physcion, aloe emodi anthraquinones as major constituents. The present investigation aims to synthesize TiO2 nanoparticles (TiO2 NPs) using R. emodi root extract. The synthesized nanoparticles were characterized using UV–Vis, FTIR, FESEM, EDX, AFM, TEM and XRD techniques. UV–visible spectra indicated the surface plasmon resonance (SPR) at 336 nm. The size distribution profiles were analyzed through XRD which revealed the crystalline nature of nanoparticles with average size of 45 nm. TEM and SEM micrographs showed that the particles are spherical and uneven. FTIR spectra reflected the evidence of the presence of anthraquinones, possible bio-molecules responsible for stabilization, reduction and capping of TiO2 NPs. The cytotoxic potential of TiO2 NPs was tested against liver cancer cells (HepG2) lines which was found very effective and the formulation can be used in nano-drug to treat cancer cells in future.

Starch-templated bio-synthesis of gold nanoflowers for in vitro antimicrobial and anticancer activities

We describe an in situ method of synthesizing highly branched gold nanoflower (AuNFs) using aqueous seed extract of Syzygium cumini (L.) Skeels as reductant in the presence of 0.3% starch. Surprisingly, when the same reaction was carried out in the absence of starch or with starch at a lower concentration (0.15%), instead of flower-like morphology quasi-spherical or polyhedral nanoparticles (AuNPs) are obtained. The nanomaterials were extensively characterized by HRTEM, FESEM, UV–Vis, FTIR, XRD, XPS and TGA analysis. The biological activities of the materials were investigated for antimicrobial activities against four bacterial strains that include one Gram positive (Staphylococcus aureus MTCC 121), two Gram negative (Escherichia coli MTCC 40 and Pseudomonas aeruginosa MTCC 4673) and one fungi (Candida albicans MTCC 227). The nanoparticles functioned as effective antimicrobial and anti-biofilm agents against all the strains under study. Controlled study revealed that, the AuNFs showed improved efficacy over conventional polyhedral AuNPs against all the microbes under study which might be attributed to the larger surface-to-volume ratio of the nanoflowers. The AuNFs also showed effective in vitro anticancer activity against a human liver cancer cell line (HepG2) with no significant cytotoxicity. Our data suggest that the AuNFs can significantly reduce the cancer cell growth with IC50 value of 20 µg mL−1.

On reprogramming of tumor cells metabolism: detection of glycogen in the cell lines of hepatocellular origin with various degrees of dedifferentiation.

The reprogramming of cancer cells includes shifts in glucose and glycogen metabolism. The aim of our work was to check the ability of forming glycogen grains in hepatocellular tumor cell lines of various dedifferentiation levels. We studied the monolayer culture established in vitro after explanting cells from rat ascites Zajdela hepatoma strain C (ZH-C) as a "parental" line and its five daughter clonal sublines: the holoclonal sublines 3H, 5F, 6H and the meroclonal ones 1E, 9C, which possess, respectively, the properties of cancer stem-like cells (CSLCs) and cancer progenitor-like cells(CPLCs). Besides, we studied four permanent cell lines of a rat hepatoma HTC, two murine hepatomas BWTG3 and MH-22a,and human hepatoblastoma HepG2. We used normal rat hepatocytes as positive control cells that form glycogen. We estimated relative cell dedifferentiation levels of the studied lines via analysis of cell morphology, morphometry and motility character on stained cell preparations and lifetime video files. Glycogen in the cells was detected using a Schiff type Au-SO2 reagent. All studied hepatocellular tumor lines were not of equal dedifferentiation level as manifested by different nucleus-to-cytoplasm ratio, by epithelium-like or fibroblast-like morphology, by tight or loosen intercellular contacts, by cell migration of collective or individual types. Glycogen fluorescence of uneven intensity was observed in all normal rat hepatocytes, but only in some cell groups or in single cells of hepatocellular tumor lines. The large or small fluorescent grains were found not only in relatively less dedifferentiated parental ZH-C line, BWTG3 and HepG2 lines, but also in moderately dedifferentiated 1E and HTC lines and even in severely dedifferentiated 3H, 5F and 6H sublines, as well as in the islets of the rat ascites hepatoma induced in vivo by the injection of 3H cells (the tumor-initiating cells). On the other hand, MH-22 and 9C lines, being relatively less and moderately dedifferentiated, showed no glycogen fluorescence. Thus,in 10 tumor cell lines of hepatocellular origin, an ability to reserve glycogen manifested no obvious dependency on their dedifferentiation level. Glycogen grains were detected in some cells even of the severely dedifferentiated lines: in single CSLCs of holoclonal ZH sublines grown in vitro and in a majority of tumor-initiating cells derived from ascites hepatoma in vivo. We suggest that dynamic changes in glycogen formation in CSLCs and tumor-initiating cells might be of importance for their dedifferentiation, self-renewal in vitro, survival and metastasis in vivo. The role of glycogen in maintaining viability and metastasis of tumor cells is to be further studied.

Differential Cell Death In Hepatocellular Cell Lines Induced By Non-Thermal Plasma

Cancer recurrence, which is frequently accompanied by chemotherapy, has been a challenge in cancer treatment. Non-thermal plasma (NTP) has been recognized as a promising tool across a vast variety of biomedical applications including as a potential new therapeutic modality for cancer, with the potential of creating novel therapeutic methods. However, its mechanism of action remains unclear. In this study we examined the influence of non-thermal air-plasma on human hepatocellular carcinoma cell lines. We show how air non-thermal plasma induces cell death in two hepatocellular carcinoma cell lines (HepG2 and Huh7) via the formation of multiple intracellular reactive oxygen/nitrogen species. Interestingly, NTP demonstrated greater selective anti-proliferative activity against Huh7 cells relative to HepG2. Our results showed a discrepancy in the superoxide accumulation and lysosomal activity in response to plasma in these cell lines, suggesting that plasma-triggered signaling cascades might be grossly different between HepG2 and Huh7. Additionally, NTP induced distinct reactive oxygen species compartmentalization in two different hepatocellular carcinoma cell lines. In Huh7 plasma treatment resulted in nuclear accumulation of superoxide, whereas in HepG2 superoxide was diffusively distributed in cytosol. Our findings offer novel insight into plasma-induced cellular responses, and provide a basis for better controlled biomedical applications.

肝癌细胞系与正常细胞系中的组蛋白修饰差异分析

肝癌细胞系与正常细胞系中的组蛋白修饰差异分析

New cycloartanes and new iridoids from Dolichandrone spathacea collected in the mangrove forest of Soc Trang province, Vietnam

Two new cycloartanes, named dolichandrone A (1) and dolichandrone B (2), as well as two new iridoids, named [6-O-[(E)-4-methoxycinnamoyl]-1β-hydroxy-dihydrocatalpolgenin (3) and 6-O-[(E)-4-methoxycinnamoyl]-1α-hydroxy-dihydrocatalpolgenin (4), together with four known iridoids (5–8), were isolated from the leaves and barks of Dolichandrone spathacea. Their structures were elucidated by means of extensive analysis of their HRESIMS, 1D and 2D NMR spectroscopic data. All of these compounds have been isolated for the first time from this plant. Compounds 1, 2, 5, and 7 were evaluated for their cytotoxic activity in vitro against four human cancer cell lines KB, Lu, HepG2, and MCF7. The results showed that only compound 2 exhibited a good cytotoxicity against KB cell line with IC50 of 18.77 μM.

Four New Glycosides from the Rhizoma of Anemarrhena asphodeloides.

Four new compounds, aneglycoside A–C (1–3) and timosaponin U (4), were isolated from the rhizomes of Anemarrhena asphodeloides. Their structures were determined through extensive spectroscopic analysis, chemical characteristics, and high-resolution mass spectrometry (HRMS). All the isolations were evaluated for cytotoxicity against HepG2, Hela, and SGC7901 human cancer lines. Compounds 1, 2, and 4 showed weak antiproliferative activities on HepG2, Hela, and SGC7901 cells.

Genotoxic effects of food contact recycled paperboard extracts on two human hepatic cell lines

ABSTRACTFood contact paperboards may be a potential source of food contamination as they can release chemicals (intentionally added or not), especially recycled paperboards. This study assessed the in vitro genotoxicity of food contact paperboard samples from a manufacturer, collected at the beginning and at the end of a recycling production chain. Samples were extracted in water to mimic a wet food contact. Different genotoxic endpoints were evaluated in two human hepatic cell lines (HepG2 and HepaRG) using bioassays: γH2AX and p53 activation, primary DNA damage with the comet assay and micronucleus formation. It was found that the samples from the beginning and the end of the production chain induced, with the same potency, γH2AX and p53-ser15 activation and DNA damage with the comet assay. The micronucleus assay was negative with the paperboard extract from the beginning of the chain, whereas positive data were observed for the end paperboard extract. These results indicate that samples from recycled food contact paperboard can induce in vitro genotoxic effects in this study’s experimental conditions.

Sporadic PCDH18 somatic mutations in EpCAM-positive hepatocellular carcinoma

BackgroundThe relationship between specific genome alterations and hepatocellular carcinoma (HCC) cancer stem cells (CSCs) remains unclear. In this study, we evaluated the relationship between somatic mutations and epithelial cell adhesion molecule positive (EpCAM+) CSCs.MethodsTwo patient-derived HCC samples (HCC1 and HCC2) were sorted by EpCAM expression and analyzed by whole exome sequence. We measured PCDH18 expression level in eight HCC cell lines as well as HCC1 and HCC2 by real-time quantitative RT-PCR. We validated the identified gene mutations in 57 paired of HCC and matched non-cancerous liver tissues by Sanger sequence.ResultsWhole exome sequencing on the sorted EpCAM+ and EpCAM− HCC1 and HCC2 cells revealed 19,263 nonsynonymous mutations in the cording region. We selected mutations that potentially impair the function of the encoded protein. Ultimately, 60 mutations including 13 novel nonsense and frameshift mutations were identified. Among them, PCDH18 mutation was more frequently detected in sorted EpCAM+ cells than in EpCAM− cells in HCC1 by whole exome sequences. However, we could not confirm the difference of PCDH18 mutation frequency between sorted EpCAM+ and EpCAM− cells by Sanger sequencing, indicating that PCDH18 mutation could not explain intracellular heterogeneity. In contrast, we found novel PCDH18 mutations, including c.2556_2557delTG, c.1474C>G, c.2337A>G, and c.2976G>T, were detected in HCC1 and 3/57 (5.3%) additional HCC surgical specimens. All four HCCs with PCDH18 mutations were EpCAM-positive, suggesting that PCDH18 somatic mutations might explain the intertumor heterogeneity of HCCs in terms of the expression status of EpCAM. Furthermore, EpCAM-positive cell lines (Huh1, Huh7, HepG2, and Hep3B) had lower PCDH18 expression than EpCAM-negative cell lines (PLC/PRL/5, HLE, HLF, and SK-Hep-1), and PCDH18 knockdown in HCC2 cells slightly enhanced cell proliferation.ConclusionsOur data suggest that PCDH18 is functionally suppressed in a subset of EpCAM-positive HCCs through somatic mutations, and may play a role in the development of EpCAM-positive HCCs.

Determination of genotoxicity of aqueous extracts of <em>Flueggea leucopyrus</em> willd. (Katupila) using the optimized “Alkaline Comet Assay”

Purpose: The comet assay is a simple, rapid, versatile, sensitive and cost effective method used to measure DNA damage caused by a suspected genotoxic agent. In this study, genotoxic potential of Flueggea leucopyrus Willd., which is used in traditional medicine to treat diseases, including cancers was evaluated using the optimized alkaline comet assay. Methods: Aqueous extracts of Flueggea leucopyrus Willd. leaves (from two different samples isolated from Anuradhapura (FLA) and Maharagama (FLM) was used to evaluate genotoxicity on White Blood Cells (WBC) and Hepato cellular carcinoma cell line (HepG2) cells, using the comet assay. The parameter used to measure the level of genotoxicity in the above cell types was the % tail DNA after treatment with aqueous extract of Flueggea leucopyrus Willd. Different concentrations of FLA and FLM samples (0.001 mg ml-1, 0.005 mg ml-1, 0.01 mg ml-1, 0.02 mg ml-1) were prepared and the effect on WBC and HepG2 cells was evaluated separately, and compared to a negative control (HBSS for WBC and culture media for HepG2) and positive control (200 μM H2O2 for WBC and 50 μM H2O2 for HepG2). The results were analyzed by one way ANOVA followed by Dennett post hoc test using SPSS software. Results: As the concentration of the aqueous extract was increased, the % tail DNA of both WBC and HepG2 cells increased gradually, compared to the negative controls (p Conclusion: Aqueous extracts of Flueggea leucopyrus Willd. appear to be moderately genotoxic at high concentrations, but the effect is insignificant at lower concentrations.

Paclitaxel-loaded nanoparticles decorated with bivalent fragment HAb18 F(ab’)2 and cell penetrating peptide for improved therapeutic effect on hepatocellular carcinoma

Hepatocellular carcinoma (HCC) shows low response to most conventional treatment strategies. Therefore, there is an urgent need for new and effective chemotherapies. Nanotechnology gives a dramatic impact on medicine. In this work, paclitaxel loaded nanoparticles (NPs) decorated with bivalent fragment HAb18 F(ab’)2 and/or cell penetrating peptide (CPP) were developed and evaluated. NPs were prepared by emulsification-solvent evaporation method and decorated by carbodiimide chemistry. The physicochemical characteristics of NPs (i.e. encapsulation efficiency, particle size distribution, morphology, release in vitro) were investigated. Cellular uptake and accumulation in tumor tissue of NPs were determined. To assess anti-tumor activity of NPs in vitro and in vivo, cell survival assay and tumor regression study were carried out using HCC cell lines (HepG2 and Huh7) and their xenografts. Average particle size of all NPs was between 100 and 200 nm. Drug-loaded NPs possessed spherical morphology and higher encapsulation efficiency. The accumulation of NPs decorated with HAb18 F(ab’)2 and CPP depended on dual effects of passive and active targeting. Drug loaded nanoparticles showed cytotoxicity on the tumor cells in vitro and in vivo. NPs decorated with HAb18 F(ab’)2 and CPP showed maximization of therapeutic action for targeting and effective endocytosis. These results suggest that the nano-drug delivery system could be a promising candidate with excellent therapeutic efficacy for HCC therapy.

Oncolyic adenovirus SG600-IL24 expressing human melanoma differentiation associated gene-7/interleukin-24 selectively induces growth arrests and apoptosis in hepatocellular carcinoma cell lines

Objective To investigate the effect of oncolytic adenovirus vector SG600-IL24 expressing human melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) on hepatocellular carcinoma cell lines with different metastatic potentials (HepG2, Hep3B, SMMC7721 and normal liver cell line L02). Methods The oncolytic adenovirus SG600-IL24 carrying MDA-7/IL-24 gene was transfected into hepatocellular carcinoma cell lines and normal liver cell line. The mRNA and protein expression of MDA-7/IL-24 in HepG2, Hep3B, SMMC7721 and L02 cells was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA) and Western blotting respectively. Methyl thiazol tetrazolium (MTT) assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst 33258 and flow cytometry were studied to indicate the apoptosis effects. Results It was confirmed by RT-PCR, ELISA and Western blotting that the exogenous MDA-7/IL-24 gene was highly expressed in HepG2, Hep3B, SMMC7721 and L02 cells. MTT and apoptosis detection indicated that MDA-7/IL-24 could induce the growth suppression [inhibition rate: (75.0±2.5)%, (85.0±2.0)%, and (86.0±3.5)% respectively. for HepG2: F=5.860; for Hep3B: F=7.210; for SMMC7721: F=7.980, P=0.000], promote apoptosis [apoptosis rate: (56.5±4.0)%, (34.4±2.0)% and (43.3±2.5)% respectively. For HepG2: F=203.400; for Hep3B: F=313.200; for SMMC7721: F=130.500, P=0.000], and block cancer cells in G2/M phase [the blocking rate: (35.4±4.2)%, (47.3±6.2)% and (40.5±5.0)%] but not L02 cells. Conclusion Oncolytic adenovirus vector SG600-IL24 can selectively induce growth suppression and promote apoptosis in hepatocellular carcinoma lines in vitro but not in L02 cells. Key words: Carcinoma, hepatocellular; Oncolytic viruses; Melanoma differentiation associated gene-7/interleukin-24 gene; Gene therapy

Abstract 3201: Therapeutic potential of anti-angiogenic multimodal biomimetic peptide in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) related mortality ranks second worldwide and stands one step behind lung cancer. Due to the asymptomatic nature of the cancer, early diagnosis is a major problem. Currently only liver transplantation is curative for early stage hepatocellular cancer. There are other multidisciplinary treatment options like radiation and chemotherapy available for advanced patients. However, chemotherapy resistance is a common problem in the treatment of liver cancer. Newer therapeutics are needed for better management of advanced HCC. Angiogenesis and lymphangiogenesis are processes that are vital for tumorigenesis and metastasis. HCC is known to be a hypervascular tumor and many pro-angiogenic proteins are found significantly overexpressed in HCC. We explored the therapeutic potential of the anti-angiogenic and anti-lymphangiogenic, biomimetic peptide SP2043 developed by our group in HCC. Hepatocellular carcinoma cell lines HuH-7, Hep3b and HepG2 showed significant disruption of cell adhesion and migration following treatment. Furthermore, SP2043 was found to impair microvascular endothelial cell (MEC) tube formation induced by HepG2 tumor conditioned media and to significantly inhibit HepG2 tumor xenograft growth compared to untreated controls. The peptide drug was also found to improve the survival of autochthonous Myc induced HCC in a transgenic mouse model from two weeks to four weeks. We discovered the peptide to be a multi-modal anti-angiogenic drug that also inhibits IGF1R and MET signaling pathways in HCC cell lines. We also found that SP2043 treatment reduced the microvascular density in both subcutaneous and autochthonous liver tumors with reduced tumor cell proliferation and increased apoptosis. This study shows the therapeutic potential of SP2043 in the treatment of hepatocellular carcinoma. Note: This abstract was not presented at the meeting. Citation Format: Mustafa A. Barbhuiya, Adam C. Mirando, Brian W. Simons, Ghali Lemtiri-Chlieh, Jordan J. Green, Aleksander S. Popel, Niranjan B. Pandey, Phuoc T. Tran. Therapeutic potential of anti-angiogenic multimodal biomimetic peptide in hepatocellular carcinoma [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 3201. doi:10.1158/1538-7445.AM2017-3201

Synthesis and cytotoxic activities of spin-labeled derivatives of Cinobufagin

Two series of novel spin-labeled derivatives of Cinobufagin(compounds 5 and 8a―8f in series 1 with five-membered ring nitroxyl free radical and compounds 6 and 9a―9f in series 2 with six-membered ring nitroxyl free radical) were synthesized. The cytotoxic activities in vitro against two tumor cell lines(HepG2 and HeLa) were evaluated, and the results indicate that all compounds display potent cytotoxicity against HepG2 and HeLa cells, and most compounds show better activities on HeLa cells than on HepG2 cells except for compounds 8a and 9d. Generally, the compounds in series 2 have more potent cytotoxic activity against HepG2 than the compounds in series 1. Especially, compounds 6 and 9f in series 2 exhibit even more potent activities against the two tumor cell lines than Cinobufagin. Thus incorporation of different L-amino acids as the linker changed the cytotoxic profile of the spin-labeled Cinobufagin. In addition, the representative compound 9f significantly changed the cell cycle distribution and led to HeLa cell cycle arrested at G2/M phase.

Yangonindimers A-C, three new kavalactone dimers from Piper methysticum (kava)

Three new kavalactone dimers, designated as yangonindimers A-C (1–3), along with one known analogue were isolated from the roots of Piper methysticum. Their structures were elucidated via extensive analysis of their 1D, 2D NMR and mass spectroscopic data. All these dimers possess a skeleton featuring a cyclobutane ring connecting two kavalactone units. Compounds 1–4 were evaluated for their cytotoxic activities against human tumour cell lines NCI-H46, SW480 and HepG2, but none showed significant activity.