Human Hepatoma Cell Line HepG2 Research Articles

Bioactivity Profiles of Cytoprotective Short-Chain Quinones.

Short-chain quinones (SCQs) have been investigated as potential therapeutic candidates against mitochondrial dysfunction, which was largely thought to be associated with the reversible redox characteristics of their active quinone core. We recently reported a library of SCQs, some of which showed potent cytoprotective activity against the mitochondrial complex I inhibitor rotenone in the human hepatocarcinoma cell line HepG2. To better characterize the cytoprotection of SCQs at a molecular level, a bioactivity profile for 103 SCQs with different compound chemistries was generated that included metabolism related markers, redox activity, expression of cytoprotective proteins and oxidative damage. Of all the tested endpoints, a positive correlation with cytoprotection by SCQs in the presence of rotenone was only observed for the NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent reduction of SCQs, which also correlated with an acute rescue of ATP levels. The results of this study suggest an unexpected mode of action for SCQs that appears to involve a modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone itself. This finding presents a new selection strategy to identify and develop the most promising compounds towards their clinical use.

Reversing the cytotoxicity of uric acid by supramolecular encapsulation with acyclic cucurbit[n]uril

Supramolecular encapsulation, which removes harmful substances from organisms, has evolved into a new strategy. In this paper, three supramolecular complexes of acyclic cucurbit[n]urils (ACBs) with uric acid (UA) were prepared, and the inclusion behavior of ACBs and UA was studied by fluorescence spectroscopy, UV–vis spectroscopy and nuclear magnetic resonance. Furthermore, the effect of the complexes of UA with ACBs on the expression of inflammatory biomarkers in human hepatoma HepG2 cell lines was characterized through C-reactive protein (CRP) western blot. The results showed UA molecules can be recognized by three ACBs with different binding constants, and ACBs successfully blocked the inflammatory stimulation of UA on HepG2 cell lines and inhibited the expression of the major inflammatory factor CRP by the formation of complexes between UA and ACBs. This article proves that ACBs can efficiently reverse the cytotoxicity of UA, which provides a new method for treating hyperuricemia disease.

H Long Terminal Repeat-Associating 2 (HHLA2) is a Biomarker of Advanced Stage Hepatocellular Carcinoma and Promotes Tumor Cell Development In Vitro.

BackgroundSeveral risk factors contribute to the inflammation promoting hepatocellular carcinoma (HCC) development, but the underlying mechanisms are unknown. Human endogenous retrovirus H long terminal repeat-associating 2 (HHLA2), a B7 family member, is highly expressed in various malignant tumor tissues and is related to tumor progression and metastasis.Material/MethodsBioinformatics analysis was used to analyze the gene expression chip GSE33006 of HCC tissue in the GEO database, draw a heat map of differentially expressed genes, and analyze the GO pathway of gene function annotation. Then, we compared HCC tissues with para-carcinoma liver tissues from 55 patients for expression patterns and associations with HHLA2. Effects of HHLA2 knockdown were examined in the human HCC cell line HepG2 to explore effects of HHLA2 on HepG2 cells.ResultsA significantly higher expression of HHLA2 at the mRNA and protein levels was detected in HCC tissues than in para-carcinoma liver tissues, which was similar to HHLA2 expression in the GSE33006 data. A higher expression of HHLA2 protein was associated with advanced cancer stage, tumor differentiation, and invasion of adjacent structures. In vitro knockdown of HHLA2 expression significantly increased HepG2 cell adhesion, promoted cell apoptosis, induced cell cycle arrest in the G1/S phase, and inhibited cell proliferation, migration, and invasion.ConclusionsOur data indicated there was a higher expression of HHLA2 in HCC tissues than in para-carcinoma liver tissues, and HHLA2 plays a major role in the development and progression of HCC. Owing to its higher expression, HHLA2 is a potential prognostic biomarker for HCC.

Sulfasalazine synergistically enhances the inhibitory effects of imatinib against hepatocellular carcinoma (HCC) cells by targeting NFκB, BCR/ABL, and PI3K/AKT signaling pathway-related proteins.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related fatalities worldwide. Identification of second-line therapies for patients with progressive HCC is urgently required as the use of sorafenib and/or regorafenib remains unsatisfactory. Imatinib, a small-molecule kinase inhibitor, is used to treat certain types of cancer, and nuclear factor κB (NFκB) is a positive regulator of cancer cell expansion. The combined use of tyrosine kinase and NFκB inhibitors may have potential for treating HCC. The aim of this work was to assess the potential anticarcinogenic effects of imatinib and sulfasalazine alone or in combination on the human HCC cell lines HEPG2 and Huh-7. Both drugs were shown to affect the phosphoinositide 3-kinase/protein kinase B, phosphorylated signal transducer and activator of translation (p-STAT-3), breakpoint cluster region protein/Abelson proto-oncogene and NFκB pathways. At the transcriptional level, imatinib and sulfasalazine were found to synergistically down-regulate c-MET gene expression. When compared with the activities of either medication alone, combined use of imatinib and sulfasalazine enhanced inhibition of HCC cell proliferation and extended induction of apoptosis. In summary, the presented data suggest that sulfasalazine synergistically potentiates the antitumor effects of imatinib.

Network pharmacology and molecular docking reveal the mechanism of Scopoletin against non-small cell lung cancer

AimsScopoletin is a natural anticarcinogenic and antiviral coumarin component. Many studies have proved its anti-cancer effect, and after the preliminary screening of this study, Scopoletin had the best inhibitory effect on Non-small cell lung cancer (NSCLC). But its mechanism for treating NSCLC is still unclear. Therefore, network pharmacology and molecular docking technology were used to explore the potential anti-NSCLC targets and pathways of Scopoletin. The results were verified in vitro. Main methodsFirst, Scopoletin was isolated from Fennel and screened to conduct cell proliferation assay on Human lung cancer cell line A549, Human colon cancer cell line HCT-116 and Human hepatoma cell line HepG2 respectively, through the MTT test. Then, the key targets and related pathways were screened through Protein-protein Interaction (PPI) network and “component-target-pathway” (C-TP) network constructed by network pharmacology. And the key targets were selected to dock with Scopoletin via molecular docking. A549 and Human normal lung epithelial cell BEAS-2B were used to verify the results, finally. Key findingsThrough MTT, A549 was chosen as the test cancer cell. From network pharmacology, 16 targets, 27 signaling pathways and 16 GO items were obtained (P < 0.05). The results of PPI network and molecular docking showed that EGFR, BRAF and AKT1 were the key targets of Scopoletin against NSCLC, which were consistent with the western-blot results. SignificanceThrough network pharmacology, molecular docking and experiments in vitro, Scopoletin was verified to against NSCLC through RAS-RAF-MEK-ERK pathway and PI3K/AKT pathway.

Muscari comosum L. Bulb Extracts Modulate Oxidative Stress and Redox Signaling in HepG2 Cells.

Muscari comosum L. bulbs are commonly used as food in South Italy and also in folk medicine. By evaluating in vitro antioxidant activity and biological activities of their aqueous and methanol extracts, we shed light on the potential role, including both the nutraceutical and health benefits, of this plant. Total polyphenol content (TPC) and total flavonoid content (TFC) were evaluated by the Folin–Ciocalteu method and by the aluminum chloride method, respectively. Antioxidant activity was investigated by three in vitro assays and relative antioxidant capacity index (RACI) was calculated to compare results obtained by different tests. The extracts were tested to evaluate their possible involvement in redox homeostasis, using the human hepatoma (HepG2) cell line used as model. The extracts exhibited concentration/solvent dependent radical scavenging activity, as well as dysregulation of some genes involved in redox pathways by promoting Nrf2, SOD-2, GPX1, ABCC6 and ABCG2 expression. NMR metabolomics analysis suggests that HepG2 cells treated with Muscari comosum extracts experience changes in some metabolites involved in various metabolic pathways.

Genotoxicity of selected pyrrolizidine alkaloids in human hepatoma cell lines HepG2 and Huh6

IntroductionPyrrolizidine alkaloids (PAs) are found in many plant species as secondary metabolites which affect humans via contaminated food sources, herbal medicines and dietary supplements. Hundreds of compounds belonging to PAs have been identified. PAs undergo hepatic metabolism, after which they can induce hepatotoxicity and carcinogenicity. Many aspects of their mechanism of carcinogenicity are still unclear and it is important for human risk assessment to investigate this class of compounds further. Material and methodsHuman hepatoma cells HepG2 were used to investigate the genotoxicity of different chemical structural classes of PAs, namely europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine and lasiocarpine, in the cytokinesis-block micronucleus (CBMN) assay. The different ester type PAs europine, seneciphylline, and lasiocarpine were also tested in human hepatoma Huh6 cells. Six different PAs were investigated in a crosslink comet assay in HepG2 cells. ResultsThe maximal increase of micronucleus formation was for all PAs in the range of 1.64–2.0 fold. The lowest concentrations at which significant induction of micronuclei were found were 3.2 μM for lasiocarpine and riddelliine, 32 μM for retrorsine and echimidine, and 100 μM for seneciphylline, europine and lycopsamine. Significant induction of micronuclei by lasiocarpine, seneciphylline, and europine were achieved in Huh6 cells at similar concentrations. Reduced tail formation after hydrogen peroxide treatment was found in the crosslink comet assay for all diester type PAs, while an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration. ConclusionThe widely available human hepatoma cell lines HepG2 and Huh6 were suitable for the assessment of PA-induced genotoxicity. Selected PAs confirmed previously published potency rankings in the micronucleus assay. In HepG2 cells, the crosslinking activity was related to the ester type, which is a first report of PA mediated effects in the comet assay.

Hepatitis B virus X protein regulates lipid metabolism and promotes the proliferation of liver cancer cells via the C/EBPa/SREBP-1 pathway

Objective: To investigate the role and mechanism of hepatitis B virus (HBV)-encoded X protein (HBx) on the regulation of lipid metabolism and proliferation of human hepatoma cell line HepG2. Methods: HepG2 cells were transiently transfected with HBx expressing plasmid, and the cell proliferation was detected by MTT assay. Lipid droplet accumulation condition was stained by Oil Red O. Western blot was used to detect the protein levels of lipid metabolism-related genes, such as CCAAT/enhancer binding protein α (C/EBPα), sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthetase (FASN) and acetyl-CoA carboxylase (ACC1). Methyl thiazolyl tetrazolium (MTT), Oil Red O staining and western blot were used to detect the effect of HBx on the regulation of lipid metabolism and proliferation of HepG2 cells under the conditions of overexpression and low expression of C/EBPα. Results: HBx had significantly promoted the proliferation of hepatoma cell line HepG2 in dose-and time-dependent manner (F = 32.82, P < 0.001; F = 58.21, P < 0.001). HBx had significantly promoted the lipid accumulation in HepG2 cells (F = 22.65, P < 0.001). Additionally, the protein levels of C/EBPα and SREBP-1 (key regulatory factors of lipid metabolism), and the rate-limiting enzymes FASN and ACC1 were significantly increased. C/EBPα overexpression had further strengthened the effect of HBx on HepG2 cell proliferation, lipid droplet accumulation, and lipid production-related gene expression. On the contrary, C/EBPα low expression had weakened HBx's promotional effect on cell proliferation, lipid droplet accumulation and lipid production-related gene expression. Conclusion: HBx may affect the lipid production and promote the proliferation of human hepatoma cell line HepG2 via the C/EBPa/SREBP-1 signaling pathway.

Three new sesquineolignans from the fruits of Crataegus pinnatifida

Chemical investigations of the 75% EtOH extract from the fruits of Crataegus pinnatifida led to the isolation of three undescribed naturally occurring sesquineolignans with two 8-O-4'-type neolignan moieties, cratapinnatifidas A-C (1–3). Their chemical structures were elucidated by the comprehensive spectroscopic analyses (HRESIMS, 1D and 2D-NMR). In the bioactivity assay, their cytotoxic activities against two human hepatoma cell lines (HepG2 and Hep3B) were evaluated and no significant activity was seen with IC50 values ranging from 12.5 to 50.0 μM.

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation, migration and malignant signalling events in hepatoma cells.

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1‐6 and HepG2, respectively. To establish a dose‐dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA‐ or siRNA‐mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro‐migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of β1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen‐activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up‐regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up‐regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro‐migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.

Functional Doxorubicin-Loaded Omega-3 Unsaturated Fatty Acids Nanoparticles in Reversing Hepatocellular Carcinoma Multidrug Resistance.

BackgroundThis study investigated a nanoparticle drug delivery system to reverse multidrug resistance (MDR) and assessed its anticancer efficacy in hepatocellular carcinoma (HCC).Material/MethodsDocosahexaenoic acid (DHA) was used as the functional excipient and doxorubicin (DOX) as the chemotherapeutic drug to synthesize DOX nanoparticles (DOX-nano). The human HCC cell line HepG2 was used for experiments. HepG2/DOX, HepG2+DOX, HepG2+DOX-nano, HepG2/DOX+DOX, and HepG2/DOX+DOX-nano groups cells were treated with DOX or DOX-nano (5 μg/mL). Nude mice bearing a HepG2/DOX xenograft were divided into model, DOX, vector-nano, and DOX-nano groups and injected with saline, DOX reagent, vector-nano, and DOX-nano (2 mg/kg), respectively. Next, cytotoxicity, cellular uptake, cell apoptosis and migration, fluorescence imaging, TUNEL assay, and tumor inhibition effects were assessed in vitro and in vivo. Furthermore, expression of MDR-related proteins was also detected using western blotting.ResultsFluorescence imaging showed that the DOX uptake in the DOX-nano-treated group was the strongest in the HCC cells or tumors. Cell apoptosis was significantly increased in DOX-nano-treated HepG2/DOX cells and tumors, and cell migration was significantly inhibited in the DOX-nano-treated HepG2/DOX cells compared with the other groups. The tumor inhibitory rate in DOX-nano-injected tumors was also significantly higher than in other groups. The expression of breast cancer resistance protein, B-cell lymphoma 2, lung resistance protein, multidrug resistance protein, and protein kinase C alpha was significantly decreased in DOX-nano-treated HepG2/DOX cells and xenograft tumors. Significantly better antitumor and MDR-reversing effects were also observed in the HepG2+DOX group compared with the HepG2/DOX group.ConclusionsThis study revealed the potential efficacy of a DOX-nano drug delivery system for the treatment of HCC, using HepG2/DOX cells and nude mice bearing HepG2/DOX xenografts.

Effect of lupeol on invasion and metastasis of human hepatoma HepG2 and SK-HEP-1 cells and its mechanism

Epithelial-mesenchymal transformation(EMT) exists in embryonic development and is closely related to cell migration and invasion. The increased EMT level in tumors showed that E-cadherin was replaced by N-cadherin, and the expression of interstitial markers such as α-SMA and vimentin was up-regulated. It has been reported that lupeol can reduce the expression of matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9) and N-cadherin to inhibit the metastasis of osteoma cells. However lupeol has been less studied in liver cancer. Therefore, this paper investigated the effect of lupanol on invasion and metastasis of human hepatoma cell line HepG2 and SK-HEP-1 and its possible mechanism. MTT assay and Annexin V/PI double staining were used to investigate the effect of lupeol on activity and apoptosis of HepG2 cells and SK-HEP-1 cells. Moreover, the effect of lupeol on the invasion of HepG2 cells and SK-HEP-1 cells were evaluated by Transwell assay. The expressions of E-cadherin, N-cadherin, α-SMA, vimentin and MMP-9 were measured by Western blot. The model of subcutaneous transplantation of nude mice and the lung metastasis model of H22 hepatocellular carcinoma cells were established to evaluate the efficacy of lupeol in vivo on tumor growth and lung metastasis by HE staining combined with immunohistochemical assay. The results showed that lupeol inhibited the activity and invasion of HepG2 cells and SK-HEP-1 cells in a dose-dependent manner and induced apoptosis. Western blot showed that the expression of E-cadherin, a landmark protein for EMT, was induced by lupeol, and the expressions of N-cadherin, α-SMA, vimentin and MMP-9 were decreased. In vivo experiments showed that lupeol inhibited tumor growth in mice bearing xenograft. In addition, immunohistochemical experiments confirmed that lupeol could up-regulate the expression of E-cadherin in tumor tissues of nude mice, reduce the expression of N-cadherin, and inhibit the metastasis of liver cancer H22 cells in the lungs of mice. The above results indicated that the mechanism of lupeol inhibiting the invasion and metastasis of HCC cells may be related to the regulation of EMT process.

Antivirus activity of Xiyanping Injection against Dengue virus serotype 2 in vitro and in vivo

Objective To evaluate the antivirus activity of Xiyanping Injection against Dengue virus serotype 2 (DENV2) in vitro and in vivo. Methods A human hepatoma cell line HepG2 was treated with Xiyanping Injection through different ways, including the direct inactivation of virus, the inhibition of virus entry, administration after virus exposure and combination of pre- and post-exposure administration. The supernatant and cells were harvested in different time point. Then the virus antigen positive cells were detected by indirect immunofluorescence assay (IFA), and the viral RNA was measured by quantitative real-time PCR (qPCR). Moreover, antiviral activity of Xiyanping Injection was further detected with the HepG2 transplanted SCID mice. The mice were intraperitoneally injected with different doses (1,2,4 mg/mouse) of Xiyanping Injection for 4 days following the challenge of DENV2. The organs and sera in different groups were sampled on the sixth day post infection. Then, the numbers of virus antigen positive cells in spleen, liver and sera of mice were detected by IFA, and the survival rate of each group was monitored for 14 days. Results Xiyanping Injection can inactivate DENV2 and inhibit the entry of DENV2 into cells. The replication of DENV2 was significantly inhibited by Xiyanping Injection with the aforementioned medication in a dose-dependent manner. Especially, the antivirus activity of Xiyanping Injection is superior to ribavirin injection in the early and late stage of virus infection. Attractively, Xiyanping can reduce the viral load in organs and sera, and improve the survival rate of experimental mice. Conclusion Xiyanping Injection, as a traditional Chinese herba. displayed obviously antivirus activity and wide safety range in vitro and in vivo . Our results indicate Xiyanping Injection might be a new candidate of antiviral drug for DENV infection. 摘要：目的 分别通过体外和体内实验研究喜炎平注射液对H型登革病毒( Dengue virus serotype 2, DENV2)的抗病 毒作用。 方法 体外实验以利巴韦林注射液作为阳性对照药物, 分别以灭活、抑制进人、治疗性给药以及预防联合维 持治疗的方式处理人肝癌细胞株HepG2细胞, 在不同时相点收获上清和细胞, 分别利用间接免疫荧光(indirect immunofluorescence assay, IFA)和实时荧光定量PCR(quantitative real-time PCR, qPCR)检测DENV2的病毒载量, 从而评价喜炎 平注射液的体外抗DENV2作用。体内实验以SCID小鼠为实验鼠, 腹腔注射HepG2细胞, 建立模型小鼠, 腹腔注射感染 DENV2,次日给予不同剂量(1、2、4mg/只）的喜炎平注射液, 连续给药4d,部分小鼠于感染第6日取材, 利用IFA检测实 验鼠脾、肝及血清中病毒载量, 并观察实验鼠的生存率。 结果 体外实验显示, 喜炎平注射液能够灭活DENV2,并抑制 DENV2进入细胞, 同时无论是治疗还是治疗联合预防给药, 均可对DENV2的复制具有良好的抑制作用, 即在病毒感染 早期及晚期, 喜炎平均可发挥抗病毒作用, 且优于利巴韦林。进一步在体内实验中同样显示喜炎平具有较好的抗病毒 作用, 可以降低实验鼠脾、肝及血清中病毒载量, 并可提高实验鼠的生存率。 结论 喜炎平注射液具有明显的抗 DENV2病毒的作用 , 可为DENV感染的治疗提供新的候选药物

Integrated Transcriptomic and Proteomic Analyses Reveal Role of the Hexosamine Biosynthetic Pathway in Invasion and Metastasis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a major global cause of mortality. The epithelial-mesenchymal transition (EMT) transcription factor TWIST1 has been implicated in the invasion and metastasis of HCC, but the mechanism is unclear. Integrated transcriptomic and proteomic analyses on primary liver tumors and lung metastases from a spontaneous Twist1-dependent metastasis mouse model of MYC-induced HCC identified the hexosamine biosynthetic pathway (HBP) as a cancer cell autonomous mechanism for EMT-mediated invasion and metastasis, which was further validated in vitro and ex vivo. RNA-sequencing and mass spectrometry-based proteomics were carried out on primary liver tumors, lung metastases, normal livers and normal lungs from a Twist1-dependent metastasis mouse model of MYC-induced HCC (LMT); a metastatic MYC-induced HCC mouse model dependent on Twist1 phosphorylation mimetic mutant (LMT-DQD); a non-metastatic mouse model of MYC-induced HCC (LM); a non-metastatic MYC-induced HCC mouse model with the Twist box mutant (LMT-F191G); and wildtype mice. Data was analyzed in R. Organoids were derived from LM liver tumors, embedded into collagen and imaged using time-lapse DIC microscopy. Cell lines were derived from LM and LMT liver tumors, and along with human HCC cell lines Huh7, Hep3B and HepG2, were utilized for in vitro migration and invasion assays. 91 genes were differentially expressed between lung metastases versus primary liver tumors; primary liver tumors versus normal liver; and normal lung versus normal liver with p<0.05. Gene Ontology and KEGG Pathway Enrichment analyses revealed an enrichment of metabolic processes. Gfpt2, the gene encoding for the rate-limiting enzyme of the hexosamine biosynthetic pathway, was overexpressed >2 fold in metastases compared to primary tumor (p = 0.007). Kaplan-Meier estimates from cBioPortal show that HCC patients with genetic alterations in GFPT2 and/or its isoform GFPT1 have significantly worse overall median survival compared to patients without these alterations (27.5 months vs 83.24 months, p = 0.006). Functional validation studies demonstrated that increased O-GlcNAcylation by pharmacologic treatment with TMG or genetic manipulation by GFPT2 overexpression was sufficient to increase migration and invasion in vitro in a panel of human and murine HCC cell lines. Decreased HBP-O-GlcNAcylation by pharmacologic treatment with DON or genetic manipulation by GFPT2 shRNA knockdown inhibited migration and invasion in vitro. Similarly, using a novel organoid invasion assay, TMG treatment increased invasion of organoids derived from LM tumors ex vivo 2-fold (p<0.03) while DON treatment inhibited invasion 2-fold (p<0.03). The hexosamine biosynthetic pathway is required and sufficient for invasion and metastasis in vitro in HCC cell lines and ex vivo in HCC tumor-derived organoids. The HBP may be a potential therapeutic target for advanced HCC patients.

Chloroquine Potentiates Primaquine Activity against Active and Latent Hepatic Plasmodia Ex Vivo: Potentials and Pitfalls.

For a long while, 8-aminoquinoline compounds have been the only therapeutic agents against latent hepatic malaria parasites. These have poor activity against the blood-stage plasmodia causing acute malaria and must be used in conjunction with partner blood schizontocidal agents. We examined the impacts of one such agent, chloroquine, upon the activity of primaquine, an 8-aminoquinoline, against hepatic stages of Plasmodium cynomolgi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium falciparum within several ex vivo systems-primary hepatocytes of Macaca fascicularis, primary human hepatocytes, and stably transformed human hepatocarcinoma cell line HepG2. Primaquine exposures to formed hepatic schizonts and hypnozoites of P. cynomolgi in primary simian hepatocytes exhibited similar 50% inhibitory concentration (IC50) values near 0.4 μM, whereas chloroquine in the same system exhibited no inhibitory activities. Combining chloroquine and primaquine in this system decreased the observed primaquine IC50 for all parasite forms in a chloroquine dose-dependent manner by an average of 18-fold. Chloroquine also decreased the primaquine IC50 against hepatic P. falciparum in primary human hepatocytes, P. berghei in simian primary hepatocytes, and P. yoelii in primary human hepatocytes. Chloroquine had no impact on primaquine IC50 against P. yoelii in HepG2 cells and, likewise, had no impact on the IC50 of atovaquone (hepatic schizontocide) against P. falciparum in human hepatocytes. We describe important sources of variability in the potentiation of primaquine activity by chloroquine in these systems. Chloroquine potentiated primaquine activity against hepatic forms of several plasmodia. We conclude that chloroquine specifically potentiated 8-aminoquinoline activities against active and dormant hepatic-stage plasmodia in normal primary hepatocytes but not in a hepatocarcinoma cell line.

Potent aneugenicity of 1-methylpyrene in human cells dependent on metabolic activation by endogenous enzymes.

1-Methylpyrene (1-MP) is a common environmental pollutant and animal carcinogen. After sequential activation by cytochromes P450 and sulfotransferases, it induced gene mutations and micronuclei in mammalian cells. The type of micronuclei formed, entire chromosomes or fragments, was not analysed. In this study, 1-MP and its primary metabolite, 1-hydroxymethylpyrene (1-HMP), were investigated for the induction of centromere-positive and -negative micronuclei in the human hepatoma cell line HepG2 and its derivative C3A, expressing relevant enzymes at higher levels. Under a short-exposure (9h)/long-recovery regime (2 cell cycles in total), 1-MP and 1-HMP provided negative test results in HepG2 cells. However, they induced micronuclei in C3A cells, the effect being blocked by 1-aminobenzotriazole (inhibitor of cytochromes P450s) and reduced by pentachlorophenol (inhibitor of sulfotransferases). Immunofluorescence staining of centromere protein B in the micronuclei revealed purely clastogenic effects under this regime. Unexpectedly, 1-MP and 1-HMP at concentrations 1/5-1/4 of that required for micronuclei formation led to mitotic arrest and spindle aberrations, as detected by immunofluorescence staining of β- and γ-tubulin. Following extended exposure (72h, 2 cell cycles, no recovery), damage to the spindle apparatus and centrosomes was detected at even lower concentrations, with concurrent formation of micronuclei. At low concentrations (1-8µM 1-MP, 0.25-0.5µM 1-HMP), the micronuclei induced were unexceptionally centromere-positive. Thus, the chromosome-damaging mechanism of 1-MP was regime and concentration dependent: potently aneugenic under persistent exposure, while clastogenic at higher concentrations following a short-exposure/long-recovery regime. This is a convincing evidence for the existence of metabolic activation-dependent aneugens.

Evaluation of In Vitro Anti-Cancer Activity of Kukkutanakhi Guggula on Liver, Prostrate, Ovary and Renal Cancer

The medicines prepared by using exudates of Commiphora mukul (Stocks) Hook. are described in Ayurveda under Guggula Kalpana which are among such valuable dosage forms. According to retrospective literary review, the combination of Kukkutnaki (Aspidium cicutarium Sw.) and purified Guggula(Commiphora mukul (Stocks) Hook.) was first mentioned in the book Chikitsa pradeep named as Kukkutnaki Guggula. Since last 3 decades, it was documented as an herbal drug which is used for cysts, goiter, tumors, tonsillitis, abscess, mansvaha strotas ailments, which are burning issues worldwide. Due to its observed clinical efficacy in Arbuda (~Cancer), the current in-vitro anticancer study was conducted with an aim to check its anticancer effect on human hepatoma cell line-HEPG2 of Liver; PC-3 and DU145 cancer cell lines of Prostate; Ovcar-3, A2780, SK-OV-3, PA-1 cancer cell lines of Ovary and ACHN renal cancer cell line of Kidney. The current in vitro study was conducted at ACTREC, Kharghar, Navi Mumbai. The selected cancer cell lines were procured from ATCC, USA and NCCS Pune. The Sulforhodamine B (SRB) Assay protocol was followed to observe the activity of the study drug. The positive control was Adriamycin in the study. The growth curve graphs were plotted and LC50, GI50, TGI values were calculated. Kukkutnakhi Guggula was found safe for oral administration, non- toxic at cellular level (LC50 values were &gt; 160) and have moderate activity on HEPG2, Ovcar-3, DU145, ACHN cancer cell lines and had shown negligible activity on A2780, SK-OV-3, PA-1 and PC-3 cell lines. This work provides scope to study its effect on targeted cancers, specific in vivo scientific studies and human clinical trials for further researchers.

Study on chemical bibenzyls in Dendrobium gratiosissimum

Nineteen compounds were isolated and structurally characterized from an ethanol extract of Dendrobium gratiossimum, including dendrogratiol A(1), DDB-1(2), 3,4-dihydroxyl-5,3',4'-trimethoxybibenzyl(3), amoenylin(4), chrysotoxine(5), DTB(6), 3,4,4'-trihydroxyl-5,3'-dimethoxybenzyl(7), 3-methylgiga(8), aloifol(9), gigantol tetramethyl ether(10), batatasin Ⅲ(11), moscatilin(12), moniliformine(13), gigantol(14), DMB(15), flavanthrinin(16), cannithrene-2(17), 3,4-dihydroxyl-5,4'-dimethoxystilbene(18) and 4-hydroxy-3,5,4'-trimethoxystilbene(19). 1 was a new compound, and 2-10, 16, 18 and 19 were obtained from this plant species for the first time. In vitro cytotoxic and antiviral activities of these isolates were evaluated, which displayed that 4 showed moderate cytotoxicity against human hepatoma cell line HepG2 with the IC_(50) of 10.15 μmol·L~(-1); 7 and 12 exhibited moderate inhibitory activity towards HIV virus with the IC_(50) of 9.35 and 9.15 μmol·L~(-1), respectively; and 10 displayed inhibitory activity against IAV virus with the IC_(50) of 8.90 μmol·L~(-1).

Suppression of inflammation during cell-free concentrated ascites reinfusion therapy using a blood purification device.

In recent years, cell-free concentrated ascites reinfusion therapy has been used to treat patients with malignant ascites. However, concentrated ascites reinfusion therapy involves enrichment and reinfusion of useful proteins and inflammatory cytokines. Therefore, fever is a primary side effect and significant problem for patients with ascites. We removed IL-6, an inflammatory cytokine, by mixing malignant ascites and the hexadecyl group adsorbent from a β2 -microglobulin-adsorbing column (Lixelle S-15). As a result, the hexadecyl group adsorbent did not adsorb the albumin of malignant ascites but adsorbed 43% of IL-6. To investigate the effect of the hexadecyl group adsorbent on hepatocytes, the adsorbed ascites was added to a human hepatoma cell line (HepG2), and the gene expression levels of albumin and serum amyloid A protein were examined. After absorption, ascites showed significantly suppressed serum amyloid A protein expression and significantly increased albumin gene expression compared to before adsorption. Our results suggest that incorporation of Lixelle to filter and concentrate malignant ascites can suppress inflammatory responses and reduce the inhibition of albumin synthesis in the liver after reinfusion.

Induction of cell cycle arrest and apoptosis by tomentosin in hepatocellular carcinoma HepG2 and Huh7 cells.

Tomentosin, a sesquiterpene lactone, is known to possess various biological activities. However, its anticarcinogenic activity against human hepatocellular carcinoma (HCC) cells has not been investigated in detail. Thus, this study aimed to elucidate the cytotoxic mechanism of tomentosin in human HCC cell lines HepG2 and Huh7. WST-1, cell counting, and colony formation assay results showed that treatment with tomentosin decreased the viability and suppressed the proliferation rate of HepG2 and Huh7 cells in a dose- and time-dependent manner. Cell cycle analysis revealed increased population of cells at the SubG1 and G2/M stage, and decreased population of cells at the G0/1 stage in HepG2 and Huh7 cells treated with tomentosin. Annexin V/propidium iodide double staining and TUNEL assay results showed increased apoptotic cell population and DNA fragmentation in HepG2 and Huh7 cells treated with tomentosin. Western blotting analysis results showed that tomentosin treatment significantly increased the expression level of Bax, Bim (short form), cleaved PARP1, FOXO3, p53, pSer15p53, pSer20p53, pSer46p53, p21, and p27, but decreased the expression of Bcl2, caspase3, caspase7, caspase9, cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclinB1, cyclinD1, cyclinD2, cyclinD3, and cyclinE in a dose-dependent manner. Taken together, this study revealed that tomentosin, which acted through cell cycle arrest and apoptosis, may be a useful therapeutic option against HCC.