Inhibited HepG2 Proliferation Research Articles

Anti-liver tumor ingredient exploration and validation of Elephantopus tomentosus Linn. by combining in silico and in vitro experiments

Elephantopus tomentosus (ET) Linn. was reported to be an anti-tumor plant. However, the chemical composition of ET and its anti-tumor compounds and potential mechanisms still unclear. In this paper, UPLC-Q-TOF–MS/MS was firstly used to identified the ingredients in ET and UPLC was used to determine the main compounds of ET. Network pharmacology was applied to predict the potential mechanisms of anti-liver cancer. Anti-tumor nuclear activate compounds and targets of ET were obtained and the anti-liver cancer effect was validated on HepG2. Finally, Molecule docking, RT-qPCR, and western blotting were used for verification of the relationship between nuclear activate compounds and nuclear targets and the potential anti-cancer mechanisms. The result showed that 42 compounds were identified in ET, which consisted of sesquiterpene lactones, flavonoids, and phenylpropanoid compounds. Scabertopin (ST), chlorogenic acid, Isochlorogenic acid B, Isochlorogenic acid A and Isochlorogenic acid C were identified as main compounds and were determined as 0.426%, 0.457%, 0.159%, 0.701%, and 0.103% respectively. 24 compounds showed high pharmacokinetics and good drug-likeness. 520 overlapping targets of the ET compounds and liver cancer were collected. The targets were used for KEGG and GO analysis. GO enrichment analysis suggested that the targets of 24 active compound closed related to promote apoptosis, inhibit proliferation, and regulate oxidative levels. KEGG enrichment analysis suggested that pathway in cancer was enriched most and p38 MAPK/p53 signaling pathway, which closely related to promoting apoptosis and inhibiting proliferation. Compounds-targets analysis based on the parameter of Betweenness, Closeness, Information, Eigenvector, Degree, and component content indicated that ST was the nucleus anti-tumor active compound of ET. HepG2 was first used to validated the anti-tumor effect of ST and the result showed that ST significantly inhibited HepG2 proliferation with a low IC50 less than 5 μM. Nucleus active compound targets, including TP53, CASP3, BCL2, EGFR, TNF-a, IL-1β, and IL-6 were enriched based on degree value of PPI analysis. Molecule docking suggested that ST showed a good combination to TGFBR1 with the combination energy less than − 5 kcal/mol. RT-qPCR result also suggested that ST significantly medicated the mRNA expression level of TP53, CASP3, BCL2, EGFR, TNF-a, IL-1β, and IL-6. Protein expression of p-p38/p38 and p-p53/p53 notable increased by ST treatment. In conclude, combining with UPLC-Q-TOF–MS/MS qualitative analysis, UPLC quantitative analysis, network pharmacology analysis, molecule docking, and in vitro experiments on HepG2, we suggest that ST is an anti-tumor ingredient of ET, which may target to TGFBR1 and promote apoptosis and inhibited proliferation of HepG2 by activating p38 MAPK/p53 signaling pathway. ST can be regarded as a quality marker of ET.

Cytotoxic Flavokawain B Inhibits the Growth and Metastasis of Hepatocellular Carcinoma through UCK2 Modulation of the STAT3/Hif-1α/VEGF Signalling Pathway.

Hepatocellular carcinoma (HCC) is associated with a high mortality rate due to early recurrence and its metastasis features. To this day, effective treatment options for metastatic HCC remain a major challenge to patient treatment. Flavokawain B (FKB) is a naturally occurring chalcone molecule capable of providing effective therapy against this life-threatening disease. This study investigated the anti-metastatic effects of FKB on the growth and development of metastatic HCC. HepG2 cells were used in this study and a neutral red assay was performed to determine the IC50 value of FKB. Cell scratch and exclusion zone assays were performed to assess the rate of cell migration and invasion. Relative mRNA levels of UCK2, STAT3, VEGF and HIF-1α genes were quantified using RT-qPCR. FKB inhibited the proliferation of HepG2 cells at an IC50 value of 28 μM after 72 h of incubation. Its cytotoxic effect was confirmed to induce apoptosis through the phase-contrast inverted microscope. Cell migration and invasion were significantly inhibited at 7, 14, and 28 μM of FKB as compared to untreated cells. The inhibition in the cell migration significantly increased with the increasing concentrations of the bioactive compound. The relative expression levels of the UCK2 gene and its downstream genes, STAT3, VEGF and HIF-1α, were significantly downregulated after 72 h exposure to FKB treatment. Our data suggest that FKB inhibited HepG2 proliferation and further suppressed its metastasis partly by regulating the STAT3/Hif-1α/VEGF signalling pathway. FKB could be a potential alternative and viable strategy against HCC.

Effects of Scutellaria strigillosa Hemsl. extract on HepG2 cell proliferation and apoptosis through binding to aspartate β-hydroxylase

This study aims to examine the impacts of Scutellaria strigillosa Hemsl. (SSH) on the proliferation, apoptosis of human hepatoma cell HepG2 and screen the bioactive components. We found that SSH extract inhibited HepG2 proliferation, arrested cell division prior to S phase. Additionally, SSH extract exposure induced apoptosis, and increased the proportions of late apoptotic cells. Specifically, we focus on the inhibitory effect of SSH extract on aspartate β-hydroxylase, a key therapeutic target of hepatocellular carcinoma closely related with the proliferation and apoptosis of HepG2. We found SSH extract with notable inhibitory activity against aspartate β-hydroxylase, elucidated the main bioactive constituents by HPLC-Q-TOF/MS and Molecular docking analysis. In conclusion, these results provided the antiproliferative and proapoptotic effects of SSH on HepG2 cell, elucidated the main bioactive constituents based on aspartate β-hydroxylase inhibition. These data revealed the potential value of SSH and its bioactive components for the prevention and treatment of liver cancer for the first time.

Alcohol extracts of Chinese bayberry branch induce S-phase arrest and apoptosis in HepG2 cells.

The alcohol extracts of Chinese bayberry (Myrica rubra) branches (MRBE) are rich in flavonoids which have a variety of medicinal benefits, but their effects on human HepG2 were unknown. In this study, the effects of MRBE on HepG2 cell growth and its potential for inhibiting cancer were explored. The results displayed that MRBE inhibited HepG2 proliferation both by arresting cells in S phase and promoting apoptosis. Quantitative reverse-transcription PCR (qRT-PCR), western blotting, and immunofluorescence showed that MRBE induced S-phase arrest by upregulating p21, which in turn downregulated cyclin and cyclin-dependent kinase messenger RNA (mRNA) and protein. Apoptosis was induced by blocking the expression of BCL-2 and suppression of the Raf/ERK1 signaling pathways. These results indicated that MRBE may have the potential for treatment of human liver cancer, highlighting novel approaches for developing new pharmacological tools for the treatment of this deadly type cancer. Meanwhile, it provides a new direction for the medicinal added values of Chinese bayberry, which helped to broaden the diversified development of its industry chain.

Bisimidazolium Salt Glycosyltransferase Inhibitors Suppress Hepatocellular Carcinoma Progression In Vitro and In Vivo.

Hepatocellular carcinoma is a leading cause of cancer death, and the disease progression has been related to glycophenotype modifications. Previously synthesized bisimidazolium salts (C20 and C22) have been shown to selectively inhibit the activity of glycosyltransferases in cultured cancer cell homogenates. The current study investigated the anticancer effects of C20/C22 and the possible pathways through which these effects are achieved. The therapeutic value of C20/C22 in terms of inhibiting cancer cell proliferation, metastasis, and angiogenesis, as well as inducing apoptosis, were examined with hepatic cancer cell line HepG2 and a xenograft mouse model. C20/C22 treatment downregulated the synthesis of SLex and Ley sugar epitopes and suppressed selectin-mediated cancer cell metastasis. C20/C22 inhibited HepG2 proliferation, induced cell-cycle arrest, increased intracellular ROS level, led to ER stress, and eventually induced apoptosis through the intrinsic pathway. Furthermore, C20/C22 upregulated the expressions of death receptors DR4 and DR5, substantially increasing the sensitivity of HepG2 to TRAIL-triggered apoptosis. In vivo, C20/C22 effectively inhibited tumor growth and angiogenesis in the xenograft mouse model without adverse effects on major organs. In summary, C20 and C22 are new promising anti-hepatic cancer agents with multiple mechanisms in controlling cancer cell growth, metastasis, and apoptosis, and they merit further development into anticancer drugs.

ATP citrate lyase inhibitor triggers endoplasmic reticulum stress to induce hepatocellular carcinoma cell apoptosis via p-eIF2α/ATF4/CHOP axis.

ATP citrate lyase (ACLY), a key enzyme in the metabolic reprogramming of many cancers, is widely expressed in various mammalian tissues. This study aimed to evaluate the effects and mechanisms of ACLY and its inhibitor BMS‐303141 on hepatocellular carcinoma (HCC). In this study, ACLY was highly expressed in HCC tissues, especially in HepG2 and Huh7 cells, but was down‐regulated in Hep3B and HCC‐LM3 cells. Besides, ACLY knockdown inhibited HepG2 proliferation and clone formation, while opposite result was noticed in HCC‐LM3 cells with ACLY overexpression. Moreover, ACLY knockdown impeded the migration and invasion abilities of HepG2 cells. Similarly, BMS‐303141 suppressed HepG2 and Huh‐7 cell proliferation. The p‐eIF2α, ATF4, CHOP p‐IRE1α, sXBP1 and p‐PERK were activated in HepG2 cells stimulated by BMS‐303141. In cells where ER stress was induced, ATF4 was involved in BMS‐303141‐mediated cell death procession, and ATF4 knockdown reduced HCC cell apoptosis stimulated by BMS‐303141. In a mouse xenograft model, combined treatment with BMS‐303141 and sorafenib reduced HepG2 tumour volume and weight. In addition, ACLY expression was associated with HCC metastasis and tumour‐node‐metastases staging. Survival analysis and Cox proportional hazards regression model showed that overall survival was lower in HCC patients with high ACLY expression; AFP level, TNM staging, tumour size and ACLY expression level were independent risk factors affecting their overall survival. In conclusion, ACLY might represent a promising target in which BMS‐303141 could induce ER stress and activate p‐eIF2α/ATF4/CHOP axis to promote apoptosis of HCC cells, and synergized with sorafenib to enhance the efficacy of HCC treatment.

Preparation of Liver-targeted Nano-prodrug Based on Sodium Alginate Derivative and the Study on Antitumor Activity†

The high viscosity of sodium alginate( ALG) causes its insufficient targeted ligand loading,and further influences the targeted recognition effect of nano-prodrug. Here,oligomeric ethylene glycol modified-sodium alginate( ALG-mOEG) was used as a carrier to improve the targeted-ligands loading. Results showed that ALG-mOEG significantly improved glycyrrhetinic acid( GA) loading compared with unmodified ALG( 11. 8%vs. 6. 9%,1. 97-fold increase). On this basis,the liver targeted nano-prodrug( DOX-ALG-mOEG /GA-ALGmOEG NPs) was self-assembled via dialysis method by mixing GA-ALG-mOEG and DOX-ALG-mOEG. Cell cytotoxicity experiment showed that DOX-ALG-mOEG /GA-ALG-mOEG NPs inhibited HepG2 proliferation with an half maximal inhibitory concentration( IC50) value of 58.1 ng/mL while the IC50 of control group was141. 7 ng /mL; the tumor growth inhibition rate( IR) reached to 88. 4%,improved by 11. 5% compared to that of the control group. This study show that the liver targeted nano-prodrug based on ALG-mOEG can effectively improve the drug utilization,and provide a reference for the preparation of other polysaccharide targeted nanoprodrug.

The effect and mechanism of metallothionein MT1M on hepatocellular carcinoma cell.

Liver cancer is one of the most common digestive system malignant solid tumors. Its incidence and mortality rates keep high, causing serious mental and economic burden. So far, the exact mechanism of liver cancer onset has not been fully elucidated. Metallothionein (MT) widely exists in various types of organisms with highly conserved structure. It contains the short peptide of cysteine and sulfur protein with high affinity to heavy metals, including cadmium, zinc, and copper. MT1M is an important member of MT family that has been verified to participate in regulating hepatocellular carcinoma, thyroid cancer, cervical cancer, and other tumors. However, MT1M expression and mechanism in hepatoma cells have not been fully elucidated. Hepatoma cell line HepG2 was divided into control and MT1M group. MT1M plasmid was constructed and transfected to MT1M group. Real-time PCR was used to test MT1M expression. MTT assay was applied to detect HepG2 proliferation. Flow cytometry was performed to determine HepG2 apoptosis. Caspase-3 activity was measured. Western blot was used to detect Bcl-2 and Bax protein levels. MT1M expression significantly increased after MT1M plasmid transfection compared with control (p < 0.05). MT1M group showed inhibited HepG2 proliferation, declined HepG2 apoptosis, enhanced Caspase-3 activity, reduced Bcl-2 protein level, and upregulated Bax protein compared with control (p < 0.05). MT1M can suppress HepG2 proliferation and induce HepG2 apoptosis through downregulating Bcl-2, upregulating Bax, and enhancing Caspase-3 activity.

Antioxidant and antiproliferative activities of proanthocyanidins from Chinese bayberry (Myrica rubra Sieb. et Zucc.) leaves

Chinese bayberry leaves proanthocyanidin (BLPs) contain epigallocatechin-3-O- gallate (EGCG) as its terminal unit and most of its extension units, which is unusual in the plant kingdom. The present study compared the antioxidant and anti-proliferative activities of BLPs prepared from different purification stages. Low-molecular-weight BLPs were prepared by ultrasonic treatment. Results revealed that the antioxidant activities of BLPs were strongly related to the content of total phenolics and proanthocyanidins; however, the cellular assay indicated that the mean degree of polymerization (mDP) played a vital role in the cellular antioxidant activity (CAA) of BLPs. The ultrasonic depolymerized BLPs with lower mDP (3.6 ± 0.3) exhibited a much higher CAA (EC50 = 0.042 ± 0.005 mg/mL) than other groups, which suggests that bioavailability is crucial to the cellular antioxidant potential of BLPs. Additionally, BLPs strongly inhibited HepG2 proliferation in a dose-dependent manner. Our results indicated that BLPs could be a valuable resource of bioactive compounds to promote consumer health.

Raltitrexed Inhibits HepG2 Cell Proliferation via G0/G1 Cell Cycle Arrest.

Raltitrexed (RTX) is an antimetabolite drug used as a chemotherapeutic agent for treating colorectal cancer, malignant mesothelioma, and gastric cancer. The antitumor capacity of RTX is attributed to its inhibitory activity on thymidylate synthase (TS), a key enzyme in the synthesis of DNA precursors. The current study is aimed at investigating the potential antitumor effects of RTX in liver cancer. Using the HepG2 cell line as an in vitro model of liver cancer, we evaluated the effects of RTX on cell proliferation employing both a WST-8 assay and a clone formation efficiency assay. In addition, we monitored the ultrastructure changes of HepG2 cells in response to RTX with transmission electric microscopy. To investigate the mechanism underlying the regulation of cell proliferation by RTX, we analyzed cell cycle using cell flow cytometry. Moreover, real-time PCR and Western blot analyses were conducted to examine expression levels of cell cycle regulatory proteins cyclin A and cyclin-dependent kinase 2 (CDK2), as well as their mediators tumor suppressor genes p53 and p16. Our results demonstrate that RTX inhibits HepG2 proliferation by arresting the cell cycle at G0/G1. This cell cycle arrest function was mediated via downregulation of cyclin A and CDK2. The observed elevated expression of p53 and p16 by RTX may contribute to the reduction of cyclin A/CDK2. Our study indicates that RTX could serve as a potential chemotherapeutic agent in the treatment of hepatocellular carcinoma.

Ethylene glycol oligomer modified-sodium alginate for efficiently improving the drug loading and the tumor therapeutic effect.

The high viscosity of sodium alginate (ALG) causes its insufficient drug loading and limits its application as a drug carrier. In the present work, we synthesized poly(ethylene glycol) oligomer (mOEG)-modified sodium alginate (ALG-mOEG) and characterized its properties as a drug delivery system. Our results showed that ALG-mOEG displayed a greatly reduced viscosity (0.024 Pa s at 2.0 wt%) with a significantly improved drug loading capacity (18.0% (w/w)) compared with unmodified ALG (6.9% (w/w)). In addition, DOX-ALG-mOEG nanoparticles (DOX-ALG-mOEG NPs) with high drug loading were more cytotoxic for HepG2 cells in vitro and showed a higher antitumor activity in vivo compared with the control group (DOX-ALG NPs) at the same dose of DOX. DOX-ALG-mOEG NPs inhibited HepG2 proliferation in vitro with an IC50 value of 220.0 ng mL-1 and reduced tumor growth in vivo by 75.6%, while DOX-ALG NPs showed activities of 443.9 ng mL-1 and 62.7%, respectively. Meanwhile, we did not observe the "accelerated blood clearance (ABC)" phenomenon in these mOEG-modified ALG nanoparticles. In view of most biologic and chemical molecules used for the biological function being hydrophobic, we thought the ALG-mOEG scaffold also might serve as an efficient long-duration carrier for other hydrophobic molecules with minimal immunogenicity.

Photosensitization of phycocyanin extracted from Microcystis in human hepatocellular carcinoma cells: Implication of mitochondria-dependent apoptosis

The aim of this study was to explore the possibility that Microcystis phycocyanin (MC-PC) functions as a photosensitizer and to investigate the mechanism for the apoptosis induced by Microcystis phycocyanin-mediated photodynamic therapy (MC-PC-PDT) in human hepatocellular carcinoma cells (HepG2). After incubation with MC-PC, HepG2 cells were exposed to a He–Ne Laser beam and the cell survival rate was detected by MTT and Colony forming assay. The mechanism of apoptosis was determined by ultrastructural observation, reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) assay, activity detection of caspase-3 and cytosol cytochrome c and flow cytometry (FCM) for cell cycle analysis. Our results demonstrated that MC-PC-PDT effectively inhibits HepG2 proliferation with a half-lethal dose of 100μg/mL and induces apoptosis at 24h with a dose of 200μg/mL MC-PC. MC-PC was found to localized in mitochondria, it could induce a high level of ROS accumulation at 16h after PDT treatment, cause mitochondrial damage and the release of mitochondrial cytochrome c into the cytosol. These cellular changes are accompanied by a reduction of the Δψm, activation of caspase-3 and G2/M phase arrest, finally leading to apoptosis through a mitochondria-dependent pathway after 24h. Meanwhile, necrosis was also contributed to cell death in MC-PC PDT process. The present study also identified a new source of phycocyanin from Microcystis as a safe and effective photosensitizer.

Quantitative Proteomic Analysis of HepG2 Cells Treated with Quercetin Suggests IQGAP1 Involved in Quercetin-Induced Regulation of Cell Proliferation and Migration

Quercetin, a wild distributed bioflavonoid, exhibits antitumor effects on murine models by inducing apoptosis and inhibiting growth of many cancer cell lines, while proteins involved in antitumor effects at proteomic level are still unclear. In our study, we used a quantitative proteomic strategy termed stable isotope labeling by amino acids in cell culture (SILAC)-mass spectrometry (MS) to study the differential proteomic profiling of HepG2 cells treated by quercetin. In all, there were 70 changed proteins among those quantified proteins in HepG2 cells treated by 50 microM quercetin for 48 h, and 14 proteins showed significant upregulation, whereas 56 proteins were downregulated. The functional classification of changed proteins includes signaling protein, protein synthesis, cytoskeleton, metabolism, etc. Of these, Ras GTPase-activating-like protein (IQGAP1) and beta-tubulin were found to be reduced at a large degree. The migration inhibition of HepG2 cells can be induced by quercetin, and the RNA and protein expression level of IQGAP1 and beta-tubulin were respectively decreased obviously in HepG2 cells exposed to quercetin for 48 h in the scratch migration assay. The downregulated expression of IQGAP1 and beta-tubulin by quercetin treatment correlated with cell migration ability, and quercetin probably inhibits HepG2 proliferation and migration through IQGAP1 and beta-tubulin expression changes and their interactions with other proteins.

VIP inhibits human HepG2 cell proliferation in vitro

Hepatocellular carcinoma (HCC) is an aggressive and often fatal neoplasm. HepG2 cells are a cell line derived from HCC. This investigation shows that vasoactive intestinal peptide (VIP) inhibits HepG2 cell proliferation in vitro. In addition, VIP decreases the expression of signal transducers and activators of transcription-3 (STAT-3) and phosphorylated STAT-3 (pSTAT-3). Transfection of HepG2 cells with STAT-3 siRNA also dose-dependently inhibits proliferation. These findings suggest that VIP-mediated inhibition of HepG2 proliferation may be mediated by STAT-3. Further studies demonstrate that VIP increases HepG2 cAMP levels and 8-cl-cAMP inhibits HepG2 proliferation as well as pSTAT-3 and STAT-3 levels, suggesting that cAMP is also involved in the inhibition of HepG2 proliferation. VIP also attenuates the proliferative effects of hepatocyte growth factor (HGF) and interleukin-6 (IL-6) on HepG2 cells. These preliminary studies suggest that the antiproliferative actions of VIP may offer a new and promising means of suppressing HCC.