Multi-Omics Analysis Revealed a Significant Alteration of Critical Metabolic Pathways Due to Sorafenib-Resistance in Hep3B Cell Lines.

Hepatocellular carcinoma (HCC) is the second prominent cause of cancer-associated death worldwide. Usually, HCC is diagnosed in advanced stages, wherein sorafenib, a multiple target tyrosine kinase inhibitor, is used as the first line of treatment. Unfortunately, resistance to sorafenib is usually encountered within six months of treatment. Therefore, there is a critical need to identify the underlying reasons for drug resistance. In the present study, we investigated the proteomic and metabolomics alterations accompanying sorafenib resistance in hepatocellular carcinoma Hep3B cells by employing ultra-high-performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). The Bruker Human Metabolome Database (HMDB) library was used to identify the differentially abundant metabolites through MetaboScape 4.0 software (Bruker). For protein annotation and identification, the Uniprot proteome for Homo sapiens (Human) database was utilized through MaxQuant. The results revealed that 27 metabolites and 18 proteins were significantly dysregulated due to sorafenib resistance in Hep3B cells compared to the parental phenotype. D-alanine, L-proline, o-tyrosine, succinic acid and phosphatidylcholine (PC, 16:0/16:0) were among the significantly altered metabolites. Ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial superoxide dismutase, UDP-glucose-6-dehydrogenase, sorbitol dehydrogenase and calpain small subunit 1 were among the significantly altered proteins. The findings revealed that resistant Hep3B cells demonstrated significant alterations in amino acid and nucleotide metabolic pathways, energy production pathways and other pathways related to cancer aggressiveness, such as migration, proliferation and drug-resistance. Joint pathway enrichment analysis unveiled unique pathways, including the antifolate resistance pathway and other important pathways that maintain cancer cells' survival, growth, and proliferation. Collectively, the results identified potential biomarkers for sorafenib-resistant HCC and gave insights into their role in chemotherapeutic drug resistance, cancer initiation, progression and aggressiveness, which may contribute to better prognosis and chemotherapeutic outcomes.

ROS-Mediated Anti-Tumor Effect of Coptidis Rhizoma against Human Hepatocellular Carcinoma Hep3B Cells and Xenografts.

Coptidis Rhizoma is the dried rhizome from the Coptis chinensis Franch. that has been shown to have a number of beneficial pharmacological properties including antioxidant, anti-inflammatory, and anti-cancer effects. However, the anti-cancer effects of Coptidis Rhizoma on hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the anti-cancer properties of Coptidis Rhizoma ethanol extract (CR) in HCC Hep3B cells and in a xenograft mouse model. Our results showed that the CR significantly inhibited cell growth and induced apoptosis in Hep3B cells through increased expression of Bcl-2 associated x-protein (Bax) and cleavage of poly-ADP ribose polymerase (PARP), reduced expression of Bcl-2, and activated caspases. CR also increased the generation of intracellular reactive oxygen species (ROS), which caused a loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of the mitochondria-mediated intrinsic apoptosis pathway. Moreover, N-acetylcysteine (NAC), a ROS inhibitor, markedly blocked the effects of CR on apoptotic pathways. CR also induced the expression of light chain 3 (LC3)-I/II, a key autophagy regulator, whereas CR-mediated autophagy was significantly suppressed by NAC. In addition, pre-treatment with NAC perfectly attenuated the inhibition of cell invasion and migration of CR-stimulated Hep3B cells. Furthermore, oral administration of CR suppressed Hep3B tumor growth in xenograft mice without toxicity, alterations to body weight, or changes in hematological and biochemical profiles. Taken together, our findings suggest that CR has anti-tumor effects that result from ROS generation, and may be a potential pharmacological intervention for HCC.

Induction of Apoptosis by Coptisine in Hep3B Hepatocellular Carcinoma Cells through Activation of the ROS-Mediated JNK Signaling Pathway.

Hepatocellular carcinoma (HCC) has a high mortality rate worldwide, and treatment is very limited due to its high recurrence and low diagnosis rate, and therefore there is an increasing need to develop more effective drugs to treat HCC. Coptisine is one of the isoquinoline alkaloids, and it has various pharmacological effects. However, the evidence for the molecular mechanism of the anticancer efficacy is still insufficient. Therefore, this study investigated the antiproliferative effect of coptisine on human HCC Hep3B cells and identified the action mechanism. Our results showed that coptisine markedly increased DNA damage and apoptotic cell death, which was associated with induction of death receptor proteins. Coptisine also significantly upregulated expression of proapoptotic Bax protein, downregulated expression of anti-apoptotic Bcl-2 protein, and activated caspase-3, -8, and -9. In addition, coptisine remarkably increased the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), and release of cytochrome c into the cytoplasm. However, N-acetylcysteine (NAC), a ROS scavenger, significantly attenuated the apoptosis-inducing effect of coptisine. It is worth noting that coptisine significantly upregulated phosphorylation of ROS-dependent c-Jun N-terminal kinase (JNK), whereas treatment with JNK inhibitor could suppress an apoptosis-related series event. Taken together, our results suggest that coptisine has an anticancer effect in Hep3B cells through ROS-mediated activation of the JNK signaling pathway.

Morin enhances auranofin anticancer activity by up-regulation of DR4 and DR5 and modulation of Bcl-2 through reactive oxygen species generation in Hep3B human hepatocellular carcinoma cells.

Evidence suggests that auranofin (AF) exhibits anticancer activity by inhibiting thioredoxin reductase (TrxR). Here, in this study, we have investigated the synergistic effects of AF and morin and their mechanism for the anticancer effects focusing on apoptosis in Hep3B human hepatocellular carcinoma cells. We assessed the anticancer activities by annexin V/PI double staining, caspase, and TrxR activity assay. Morin enhances the inhibitory effects on TrxR activity of AF as well as reducing cell viability. Annexin V/PI double staining revealed that morin/AF cotreatment induced apoptotic cell death. Morin enhances AF-induced mitochondrial membrane potential (ΔΨm) loss and cytochrome c release. Further, morin/AF cotreatment upregulated death receptor DR4/DR5, modulated Bcl-2 family members (upregulation of Bax and downregulation of Bcl-2), and activated caspase-3, -8, and -9. Morin also enhances AF-induced reactive oxygen species (ROS) generation. The anticancer effects results from caspase-dependent apoptosis, which was triggered via extrinsic pathway by upregulating TRAIL receptors (DR4/DR5) and enhanced via intrinsic pathway by modulating Bcl-2 and inhibitor of apoptosis protein family members. These are related to ROS generation. In conclusion, this study provides evidence that morin can enhance the anticancer activity of AF in Hep3B human hepatocellular carcinoma cells, indicating that its combination could be an alternative treatment strategy for the hepatocellular carcinoma.

Oxyphenbutazone promotes cytotoxicity in rats and Hep3B cellsvia suppression of PGE2 and deactivation of Wnt/ß-catenin signaling pathway.

Hepatocellular carcinoma (HCC) is the fifth leading cause of death and is generally typified by elevated liver enzyme biomarkers, antioxidants, and chronic inflammation of hepatocytes. Although currently available drugs have shown remarkable alleviation of the cancerous condition, but at the same time they present a more severe challenge of toxic effects due to chemotherapy. Therefore, in order to bring more patient-compliant therapy, we aimed to refurbish the use of a COX inhibitor, oxyphenbutazone (OPB), with low dose of methotrexate (MTX) to treat diethyl nitrosamine (DENA)-induced HCC in Wistar rats and in Hep3B cells. Hep3B cells were subjected to assays like in vitro cytotoxicity, DNA synthesis, and caspase activity. The combination index was also evaluated, succeeding the cytotoxicity assay, to analyze the possible synergism. For in vivo study, Wistar strain male rats were given single intraperitoneal dose of DENA (200 mg/kg) and were supplied with sodium phenobarbital (0.1% in tap water) for promoting tumorigenesis throughout the study. MTX (2.5 and 5.0 mg/kg/week, ip) and OPB (70 mg/kg/week, po in two divided doses) were administered to the treatment groups from 3rd week till the termination of study. Several biochemical parameters including biomarkers of liver function, antioxidant enzymes, and histopathological examination of liver cells were tested. Significant synergism was witnessed in the cytotoxicity assay when Hep3B cells received varied dose combination treatment of MTX (0.25, 0.5, or 1.0 µmol/L) and OPB (2.5, 5.0, or 7.5 µmol/L). MTX (0.5 and 1.0 µmol/L) in combination with OPB (5.0 or 7.5 µmol/L) inhibited the cell proliferation as BrdU incorporation was quite low in DNA synthesis analysis, as well as caspase-9/-3 cascade was activated which led to apoptosis of cancer cells. Co-treatment with MTX and OPB exerted potential anticancer activity in rats than either of the drugs alone. Administration of combination therapy harmonized the DENA-induced elevation of serum biochemical parameters, including but not limited to, α-fetoprotein (AFP), alanine- and aspartate-aminotransferase, alkaline phosphatase, vascular endothelial growth factor (VEGF), and antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and lipid per oxidation (LPO). All these results were optimally substantiated by histopathological examination. As evident COX-2 catalyzes the synthesis of PGE2, needed in the activation of Wnt/ß-catenin pathway, which in turn is responsible for activating the transcriptional proteins required for higher degree of cell division and thence growth. Therefore, inhibition of COX-2 by our novel combination infers that even low doses of MTX can elucidate noticeable anticancer activity when paired with OPB.

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine-induced hepatocellular carcinoma.

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long-term diseases including hepatocellular carcinoma (HCC). Melatonin has been reported to alleviate promotion and progression of HCC, but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35 mg/kg body weight ip) once a week for 8 weeks. Melatonin was given at 5 or 10 mg kg-1 d-1 ip beginning 4 weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40 weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK1ε, Rev-erbα, and Rev-erbß decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT1 receptor and reduced levels of the hypoxia-inducible factors Hif-1α and Hif-2α. An increased expression of p21, p53, and PARP1/2, a higher Bax/Bcl-2 ratio, and a lower expression of Cyclin D1, CDK6, HSP70, HSP90, and GRP78 proteins were also observed in melatonin-treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV-ERB agonist SR9009 in human Hep3B cells, and BMAL1 knocking down attenuated the pro-apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.

Mechanistic Action of Cell Cycle Arrest and Intrinsic Apoptosis via Inhibiting Akt/mTOR and Activation of p38-MAPK Signaling Pathways in Hep3B Liver Cancer Cells by Prunetrin-A Flavonoid with Therapeutic Potential.

Hepatocellular carcinoma (HCC) has a poor prognosis and a low survival rate. Drugs without side effects are desperately needed since chemotherapy has a negative effect on the host cells. Previous research has firmly established that plant-based compounds have significant bioactivities without a negative impact on the host. Flavonoids, in particular, are a class of compounds with both anti-inflammatory and anti-cancer properties. Prunetrin (PUR) is a glycosyloxyisoflavone (Prunetin 4'-O-glucoside) derived from Prunus sp., and its other form, called prunetin, showed optimistic results in an anti-cancerous study. Hence, we aimed to discover the anti-cancer ability of prunetrin in liver cancer Hep3B cells. Our cytotoxicity results showed that PUR can decrease cell viability. The colony formation assay confirms this strongly and correlates with cell cytotoxicity results. Prunetrin, in a dose-dependent manner, arrested the cell cycle in the G2/M phase and decreased the expression of cyclin proteins such as Cyclin B1, CDK1/CDC2, and CDC25c. Prunetrin treatment also promoted the strong cleavage of two important apoptotic hallmark proteins called PARP and caspase-3. It also confirms that apoptosis occurs through the mitochondrial pathway through increased expression of cleaved caspase-9 and increased levels of the pro-apoptotic protein Bak. Bak was significantly increased with the declining expression of the anti-apoptotic protein Bcl-xL. Next, it inhibits the mTOR/AKT signaling pathways, proving that prunetrin includes apoptosis and decreases cell viability by suppressing these pathways. Further, it was also observed that the activation of p38-MAPK was dose-dependent. Taken together, they provide evidence that prunetrin has an anti-cancerous ability in Hep3B liver cancer cells by arresting the cell cycle via p38 and inhibiting mTOR/AKT.

Silymarin suppresses proliferation of human hepatocellular carcinoma cells under hypoxia through downregulation of the HIF-1α/VEGF pathway.

BACKGROUND: Hypoxia is a significant feature of many solid tumors and can activate hypoxia-inducible factor 1α (HIF-1α)/vascular epidermal growth factor (VEGF) signaling pathway, which is closely related to the occurrence and development of primary liver cancer (PLC). Silymarin (SM) had been used as a traditional liver protective drug for decades. Recent studies have found that SM has chemopreventive and chemosensitizing effects on multiple cancers. In this study, we investigated the effects of SM on HIF-1α/VEGF signaling in human hepatocellular carcinoma cells under hypoxia conditions. METHODS: HepG2 and Hep3B cells were divided into different experimental groups according to different culture conditions (aerobic or anaerobic) and the concentration of SM in the culture medium. The cellular proliferation, migration, invasion, colony formation, and apoptosis were observed by using methyl thiazolyl tetrazolium (MTT) assay, cell migration assay, in vitro invasion assay, soft agar colony formation assay, and Annexin V apoptosis assay, respectively. The cellular expressions of HIF-1α and VEGF were determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot (WB) analyses. RESULTS: SM reduced cellular proliferation, migration, invasion, and colony formation, but induced apoptosis in HepG2 and Hep3B cells under hypoxia conditions. The half inhibitory concentrations (IC50) of SM on HepG2 and Hep3B cells were 58.46 and 75.13 umol/L, respectively. SM also suppressed cellular expressions of HIF-1α and VEGF in HepG2 and Hep3B cells under hypoxia conditions at the mRNA and protein levels. All these effects of SM were dose dependent. CONCLUSIONS: The inhibitory effect of SM on HepG2 and Hep3B cells under hypoxia is partially via downregulating HIF-1α/VEGF signaling, which may serve as a potential drug therapy target for liver cancer based on SM.

Induction of Apoptosis by Isoalantolactone in Human Hepatocellular Carcinoma Hep3B Cells through Activation of the ROS-Dependent JNK Signaling Pathway.

Isoalantolactone (IALT) is one of the isomeric sesquiterpene lactones isolated from the roots of Inula helenium L. IALT is known to possess various biological and pharmacological activities, but its anti-cancer mechanisms are not well understood. The aim of the present study was to investigate the anti-proliferative effects of IALT in human hepatocellular carcinoma (HCC) cells and to evaluate the potential anti-cancer mechanisms. Our results demonstrated that IALT treatment concentration-dependently suppressed the cell survival of HCC Hep3B cells, which was associated with the induction of apoptosis. IALT increased the expression of death-receptor-related proteins, activated caspases, and induced Bid truncation, subsequently leading to cleavage of poly (ADP-ribose) polymerase. In addition, IALT contributed to the cytosolic release of cytochrome c by destroying mitochondrial integrity, following an increase in the Bax/Bcl-2 expression ratio. However, IALT-mediated growth inhibition and apoptosis were significantly attenuated in the presence of a pan-caspase inhibitor, suggesting that IALT induced caspase-dependent apoptosis in Hep3B cells. Moreover, IALT activated the mitogen-activated protein kinases signaling pathway, and the anti-cancer effect of IALT was significantly diminished in the presence of a potent c-Jun N-terminal kinase (JNK) inhibitor. IALT also improved the generation of intracellular reactive oxygen species (ROS), whereas the ROS inhibitor significantly abrogated IALT-induced growth reduction, apoptosis, and JNK activation. Furthermore, ROS-dependent apoptosis was revealed as a mechanism involved in the anti-cancer activity of IALT in a 3D multicellular tumor spheroid model of Hep3B cells. Taken together, our findings indicate that IALT exhibited anti-cancer activity in HCC Hep3B cells by inducing ROS-dependent activation of the JNK signaling pathway.

Synthesis of Novel Fluorescent Carbon Quantum Dots From Rosa roxburghii for Rapid and Highly Selective Detection of o-nitrophenol and Cellular Imaging.

A novel carbon quantum dots (CQDs) were successfully synthesized by one-step hydrothermal reaction using Rosa roxburghii as a biomass-based precursor. The CQDs have an average size of 2.5 nm and a narrow size distribution. They display strong blue fluorescence with a quantum yield of 24.8% and good biocompatibility. Notably, these CQDs were capable of detecting trace o-nitrophenol in surface water and sewage with high sensitivity and specificity. The linear range is 0.08-40 µmol/L, and the limit of detection is 15.2 nmol/L. Furthermore, this CQDs was successfully applied for o-nitrophenol analysis in river water and sewage samples. Additionally, Hep3B cells, a human hepatocellular carcinoma cell line, can be easily imaged with high resolution using the as-prepared CQDs as nanoprobes. These results reveal that the as-prepared CQDs have potential applications for detecting o-nitrophenol and cell imaging.

Evidence-based analysis on the toxicity of disinfection byproducts in vivo and in vitro for disinfection selection.

Disinfection is a key step in drinking water treatment process to prevent water-borne infections. However, reactions between chlorine, one of the most common disinfectants, and natural organic matter (NOM) often lead to the formation of hazardous disinfection byproducts (DBPs). However, the cytotoxicity of some DBPs is still poorly understood. Such knowledge is critical for proper selection of disinfection processes. We investigated the effects of DBPs on mouse acute liver injury. The exacerbation of liver damage increased with the DBPs concentrations, likely due to the increased hepatic macrophages. Haloacetonitriles (HANs) and haloketones (HKs) are more toxic to Human Hepatocellular (Hep3B) cells than trihalomethanes (THMs). Cytotoxicity of DBPs were governed by the halogen type (brominated DBPs > chlorinated DBPs) and the numbers of halogen atoms per molecule. Then, we used the pilot-scale WDS to study the best conditions for reducing the formation of DBPs. The result showed that the formation of DBPs followed the order: stainless-steel (SS) > ductile iron (DI) > polyethylene (PE) pipe. Higher flowrate promoted the formation of DBPs in all three pipes. The results suggest that the formation of DBPs in chlorine disinfection can be reduced by using PE pipes and low flow rate in water distribution systems (WDS).

GDF11 exhibits tumor suppressive properties in hepatocellular carcinoma cells by restricting clonal expansion and invasion.

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features, despite some controversies in age-related studies. GDF11 has been poorly investigated in cancer, particularly in those with stemness capacity, such as hepatocellular carcinoma (HCC), one of the most aggressive cancers worldwide. Here, we focused on investigating the effects of GDF11 in liver cancer cells. GDF11 treatment significantly reduced proliferation, colony and spheroid formation in HCC cell lines. Consistently, down-regulation of CDK6, cyclin D1, cyclin A, and concomitant upregulation of p27 was observed after 24 h of treatment. Interestingly, cell viability was unchanged, but cell functionality was compromised. These effects were potentially induced by the expression of E-cadherin and occludin, as well as Snail and N-cadherin repression, in a time-dependent manner. Furthermore, GDF11 treatment for 72 h induced that cells were incapable of sustaining colony and sphere capacity in the absent of GDF11, up to 5 days, indicating that the effect of GDF11 on self-renewal capacity is not transient. Finally, in vivo invasion studies revealed a significant decrease in cell migration of hepatocellular carcinoma cells treated with GDF11 associated to a decreased proliferation judged by Ki67 staining. Data show that exogenous GDF11 displays tumor suppressor properties in HCC cells.

Saikosaponin-d Inhibits the Hepatoma Cells and Enhances Chemosensitivity Through SENP5-Dependent Inhibition of Gli1 SUMOylation Under Hypoxia.

Chemosensitivity is one of the key factors affecting the therapeutic effect on cancer, but the clinical application of corresponding drugs is rare. Hypoxia, a common feature of many solid tumors, including hepatocellular carcinoma (HCC), has been associated with resistance to chemotherapy in part through the activation of the Sonic Hedgehog (SHh) pathway. Hypoxia has also been associated with the increased SUMOylation of multiple proteins, including GLI family proteins, which are key mediators of SHh signaling, and has become a promising target to develop drug-resistant drugs for cancer treatment. However, there are few target drugs to abrogate chemotherapy resistance. Saikosaponin-d (Ssd), one of the main bioactive components of Radix bupleuri, has been reported to exert multiple biological effects, including anticancer activity. Here, we first found that Ssd inhibits the malignant phenotype of HCC cells while increasing their sensitivity to the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) drug system under hypoxia in vitro and in vivo. Furthermore, we had explored that GLI family activation and extensive protein SUMOylation were characteristics of HCC cells, and hypoxia could activate the SHh pathway and promote epithelial-mesenchymal transition (EMT), invasion, and chemosensitivity in HCC cells. SUMOylation is required for hypoxia-dependent activation of GLI proteins. Finally, we found that Ssd could reverse the effects promoted by hypoxia, specifically active sentrin/small ubiquitin-like modifier (SUMO)-specific protease 5 (SENP5), a SUMO-specific protease, in a time- and dose-dependent manner while inhibiting the expression of SUMO1 and GLI proteins. Together, these findings confirm the important role of Ssd in the chemoresistance of liver cancer, provide some data support for further understanding the molecular mechanisms of Ssd inhibition of malignant transformation of HCC cells, and provide a new perspective for the application of traditional Chinese medicine in the chemical resistance of liver cancer.

The adipokine vaspin reduces apoptosis in human hepatocellular carcinoma (Hep-3B) cells, associated with lower levels of NO and superoxide anion.

BACKGROUND: Among adipose-derived factors, adipocytokines play roles as hormones and signaling mediators for apoptotic pathway. Among of them, vaspin, regulates the metabolism of adipose tissue itself as an endocrine organ, and stimulates adipocytes to maturation, differentiation, etc. Damaged adipocytes, present in obesity and hepatocellular carcinoma (HCC) respond with over-production of inflammatory cytokines. Such pro-inflammatory stimulation remains under adipokine control. Pro-inflammatory pathways are connected to oxidative stress and apoptosis, reported as co-existing with an elevated level of some adipokines in cancer cell lines. However, some hormones, such as vaspin, reduce apoptosis, have anti-inflammatory and anti-oxidative roles in cancer cell lines. METHODS: Hep-3B cells were cytometrically evaluated under vaspin treatment for reactive oxygen species (ROS) and apoptosiss induction. The statistical significant changes to the untreated controls was calculated by T-tests (indicated at value p < 0.05). RESULTS: Here we studied the production of reactive oxygen and nitrogen species in cells of HCC line Hep-3B after vaspin treatment. A decreased level of nitric oxide and superoxide anion 24 h after vaspin addition at 5 ng/ml was correlated with restricted, to the physiological level, apoptosis. A protective role of vaspin was displayed as enhanced cell viability and proliferation, which could be a poor prognostic in liver cancer. CONCLUSIONS: Apoptosis was suppressed after vaspin treatment, together with low levels of nitric oxide and superoxide anions.

The coordinated effects of Apatinib and Tripterine on the proliferation, invasiveness and apoptosis of human hepatoma Hep3B cells.

As a novel vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitor, Apatinib has exhibited antitumor effects in a variety of solid tumors. Extracts of Chinese herbal medicines have emerged as a promising alternative option to increase the sensitivity of patients to chemotherapeutics while alleviating side effects. The present study aimed to investigate the effects of Apatinib and the traditional Chinese herb Tripterine on the proliferation, invasion and apoptosis of human hepatoma Hep3B cells. The expression of VEGFR-2 in Hep3B cells was detected by western blotting and immunofluorescence assays. Hep3B cells were then divided into four different groups: Control group, Apatinib group, Tripterine group and Apatinib plus Tripterine group. The proliferation, invasion and apoptosis of these four groups of Hep3B cells were assessed by MTS, wound healing and Transwell assays, and flow cytometry, respectively. Finally, the levels of the proliferation-associated proteins phosphorylated protein kinase B (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) and the apoptosis-associated proteins cleaved Caspase-3 and B-cell lymphoma-associated X protein (Bax) were detected by western blotting. The proliferation, migration and invasion of Hep3B cells were significantly inhibited by Apatinib and Tripterine, compared with the control group (P<0.01). The inhibitory effect of the combination group was markedly stronger than that of the Apatinib and Tripterine groups. The downregulation of p-Akt and p-ERK induced by Apatinib and Tripterine was further inhibited in the combination group (P<0.05), and the expression levels of Caspase-3 and Bax were also significantly increased in the combination group (P<0.05). The combination of Apatinib and Tripterine significantly inhibited the proliferation, migration and invasion ability and promoted the apoptosis of Hep3B cells by downregulating the expression of p-Akt and p-ERK, and upregulating the expression of Caspase-3 and Bax.

Delisheng induces antiproliferation and apoptosis effects in Hep3B cells via modulation of angiogenic proteins.

Delisheng is a widely used antineoplastic agent in China. Although previous studies revealed that Delisheng exhibits numerous pharmacological effects including the inhibition of cancer cell differentiation and enhancement of immune function with the lowest toxicity, the precise anticancer mechanisms of Delisheng in human hepatocellular carcinoma (HCC) cells remains largely unknown. The present study investigated the potential mechanisms underlying the anticancer properties of Delisheng on Hep3B cells. Delisheng demonstrated a strong anti-proliferation effect on Hep3B cells compared with normal liver HL-7702 cells, as detected by MTT assays. In addition, Delisheng arrested the cells in G/G1 phase. Furthermore, it exhibited a pro-apoptotic effect on Hep3B cells, as detected by flow cytometry. When exposed to Delisheng, Hep3B cells demonstrated decreased vascular endothelial growth factor (VEGF) and osteopontin (OPN) and increased endostatin (ES) protein expressions, as detected using immunocytochemistry staining and western blotting. These data suggest that Delisheng induces antiproliferation and apoptosis of Hep3B cells via modulation of VEGF, OPN and ES protein expression. It is hypothesized that Delisheng may be used as a novel anticancer therapeutic in HCC.

A proteomics-based investigation on the anticancer activity of alisertib, an Aurora kinase A inhibitor, in hepatocellular carcinoma Hep3B cells.

Targeted therapy may provide survival benefit for advanced hepatocellular carcinoma (HCC) and Aurora A kinase (AURKA) represents a feasible target in cancer treatment. The purpose of this study is to investigate the anticancer activity of alisertib (ALS) on Hep3B cells based on a proteomic study conducted with the stable-isotope labeling by amino acids in cell culture (SILAC). The proteomic response to ALS was obtained with SILAC-based proteomic study. Cell cycle distribution and apoptosis were assessed using flow cytometry and autophagy was determined using flow cytometry and confocal microscopy. ALS inhibited the proliferation of Hep3B cells, with IC50 values for 24- and 48-h exposure of 46.8 and 28.0 µM, respectively. Our SILAC study demonstrated that there were at least 565 proteins responding to ALS treatment, with 256 upregulated, 275 downregulated and 35 stable. Ninety-four signaling pathways, majority of which involved cell proliferation and survival, programmed cell death, and nutrition and energy metabolism, were regulated by ALS. ALS significantly inhibited the phosphorylation of AURKA at Thr288 in a concentration-dependent manner. Subsequent study showed that ALS remarkably arrested Hep3B cells in G2/M phase via regulating the expression of key cell cycle regulators, and induced a marked autophagy via the PI3K/Akt/mTOR axis. Inhibition of autophagy enhanced the anticancer activity of ALS in Hep3B cells. Overall, ALS leads to comprehensive proteomic response, inhibits cellular proliferation, and induces cell cycle arrest and autophagy in Hep3B cells. Further studies are warranted to explore the role of ALS in the treatment of HCC.

Effects of JS-K, a novel anti-cancer nitric oxide prodrug, on gene expression in human hepatoma Hep3B cells.

JS-K is a novel anticancer nitric oxide (NO) prodrug effective against a variety of cancer cells, including the inhibition of AM-1 hepatoma cell growth in rats. To further evaluate anticancer effects of JS-K, human hepatoma Hep3B cells were treated with JS-K and the compound control JS-43-126 at various concentrations (0-100µM) for 24h, and cytotoxicity was determined by the MTS assay. The compound control JS-43-126 was not cytotoxic to Hep3B cells at concentrations up to 100µM, while the LC50 for JS-K was about 10µM. To examine the molecular mechanisms of antitumor effects of JS-K, Hep3B cells were treated with 1-10µM of JS-K for 24h, and then subjected to gene expression analysis via real time RT-PCR and protein immunostain via confocal images. JS-K is a GST-α targeting NO prodrug, and decreased immunostaining for GST-α was associated with JS-K treatment. JS-K activated apoptosis pathways in Hep3B cells, including induction of caspase-3, caspase-9, Bax, TNF-α, and IL-1ß, and immunostaining for caspase-3 was intensified. The expressions of thrombospondin-1 (TSP-1) and the tissue inhibitors of metalloproteinase-1 (TIMP-1) were increased by JS-K at both transcript and protein levels. JS-K treatment also increased the expression of differentiation-related genes CD14 and CD11b, and depressed the expression of c-myc in Hep3B cells. Thus, multiple molecular events appear to be associated with anticancer effects of JS-K in human hepatoma Hep3B cells, including activation of genes related to apoptosis and induction of genes involved in antiangiogenesis and tumor cell migration.

Cyperus amuricus induces G1 arrest and apoptosis through endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells.

ETHNOPHARMACOLOGY RELEVANCE: Cyperus amuricus (C. amuricus), belongs to the family Cyperaceae, was used to exert wound healing, diuretic, astringent and other intestinal problems in oriental medicine. However, only a few studies have reported its anticancer activities. AIM OF THE STUDY: In this study, we determined the activity of C. amuricus on ER stress-induced cell death and G1 cell cycle arrest in human hepatocellular carcinoma (HCC) Hep3B cells. MATERIALS AND METHODS: The in vitro cell proliferation assay of C. amuricus was tested on Hep3B and human embryonic kidney HEK293 cells. Subsequently, the cell cycle distribution in the indicated stages using flow cytometric analysis, the expression of cell cycle-related proteins by western blot analysis and immunofluorescence detection of p21CIP1/WAF1 were determined for the comprehensive identification of cell cycle arrest in Hep3B cells. The effect of C. amuricus on the expression of apoptosis-related proteins in Hep3B cells was also performed by western blot analysis. Furthermore, induction of the ER stress mediators in C. amuricus-treated Hep3B cells were observed by western blot analysis, intracellular Ca2+ mobilization assay and immunofluorescence detection of caspase-12. RESULTS: C. amuricus strongly exhibited cytotoxic activity on Hep3B cells, but not on HEK293 cells. C. amuricus affected the phosphorylation levels of endoplasmic reticulum sensors and increased the expression of GRP78/BiP and CHOP, resulting in the accumulation of unfolded proteins in the ER and triggering the unfolded protein response. These changes occurred by the elevation of intracellular Ca2+ concentrations, which contributed to ER stress-induced apoptosis in C. amuricus-treated Hep3B cells. C. amuricus also coordinated the stimulation of ER chaperones, which initiated G1 cell cycle arrest through the induction of CDKIs and the inhibition of cyclins and CDKs. Furthermore, C. amuricus triggered apoptosis through the activation of mitochondrial-dependent pathway in Hep3B cells. CONCLUSIONS: Our results suggest that C. amuricus is an effective apoptosis inducing agent for Hep3B cells via the G1 arrest, ER stress and mitochondrial mediated apoptotic pathways.

Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells.

Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics and ranks fourth in cancer-related mortality in the world. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, has biological activities, including hepatoprotection, anti-inflammation, antihepatitis B virus activity, and anticancer activity. In the present study, the antiproliferative activity and molecular mechanisms of antcin K, the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated A. cinnamomea, were investigated using human hepatoma Hep 3B cells. The results showed that antcin K effectively reduced Hep 3B cells viability within 48 hours. Antcin K induced phosphatidylserine exposure, chromatin condensation, and DNA damage, but did not significantly increase autophagosome content or cause cell expansion and cell lysis. Thus, the principal mode of Hep 3B cells death induced by antcin K was apoptosis, rather than autophagy or necrosis. In-depth investigation of the molecular mechanisms revealed that antcin K first promoted reactive oxygen species generation and adenosine triphosphate depletion, leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes. After losing the mitochondrial membrane potential, caspase-independent and caspase-dependent apoptosis-related proteins were released, including HtrA2, apoptotic-induced factor, endonuclease G, and cytochrome c. Cytochrome c activated caspase-9 and caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced mitochondrial and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has a potential to be a complementary agent in liver cancer therapy.