Apoptosis In Hep3B Cells Research Articles

Correlation with Apoptosis Process through RNA-Seq Data Analysis of Hep3B Hepatocellular Carcinoma Cells Treated with Glehnia littoralis Extract (GLE).

Glehnia littoralis is a perennial herb found in coastal sand dunes throughout East Asia. This herb has been reported to have hepatoprotective, immunomodulatory, antioxidant, antibacterial, antifungal, anti-inflammatory, and anticancer activities. It may be effective against hepatocellular carcinoma (HCC). However, whether this has been proven through gene-level RNA-seq analysis is still being determined. Therefore, we are attempting to identify target genes for the cell death process by analyzing the transcriptome of Hep3B cells among HCC treated with GLE (Glehnia littoralis extract) using RNA-seq. Hep3B was used for the GLE treatment, and the MTT test was performed. Hep3B was then treated with GLE at a set concentration of 300 μg/mL and stored for 24 h, followed by RNA isolation and sequencing. We then used the data to create a plot. As a result of the MTT analysis, cell death was observed when Hep3B cells were treated with GLE, and the IC50 was about 300 μg/mL. As a result of making plots using the RNA-seq data of Hep3B treated with 300 μg/mL GLE, a tendency for the apoptotic process was found. Flow cytometry and annexin V/propidium iodide (PI) staining verified the apoptosis of HEP3B cells treated with GLE. Therefore, an increase or decrease in the DEGs involved in the apoptosis process was confirmed. The top five genes increased were GADD45B, DDIT3, GADD45G, CHAC1, and PPP1R15A. The bottom five genes decreased were SGK1, CX3CL1, ZC3H12A, IER3, and HNF1A. In summary, we investigated the RNA-seq dataset of GLE to identify potential targets that may be involved in the apoptotic process in HCC. These goals may aid in the identification and management of HCC.

Chalcomoracin promotes apoptosis and endoplasmic reticulum stress in hepatocellular carcinoma cells.

Chalcomoracin (CMR), a Diels-Alder adduct obtained from mulberry leaves, demonstrated wide-spectrum anti-cancer activity. Herein, we aimed to explore the function of CMR and how it works in hepatocellular carcinoma (HCC). Human HCC cell lines Hep3B and SNU-387 were cultured and treated with various concentrations of CMR (1.5, 3, and 6 µM). Subsequently, the effects of CMR on cell viability, colony formation, apoptosis, migration, and invasion abilities were studied in vitro. Furthermore, the levels of endoplasmic reticulum (ER) stress-related proteins and mitogen-activated protein kinase (MAPK) pathway-related proteins in cells under CMR exposure were detected using western blot. Experiments in vivo were conducted to examine the effects of CMR on tumor growth in HCC. CMR administration inhibited the viability and clonogenic, migration, and invasion abilities, as well as promoted cell apoptosis and ER stress in Hep3B and SNU-387 cells. In addition, CMR treatment reduced the phosphorylation levels of ERK, P38, and JNK in the MAPK pathway. Moreover, an in vivo study showed that CMR administration could inhibit tumorigenesis and MAPK pathway activity in HCC. Our data indicate that CMR has the potential to inhibit the development of HCC, potentially through the inhibition of the MAPK pathway. These findings suggest that CMR may have promising applications as an anticancer agent in future therapeutics for HCC.

MRPL21 promotes HCC proliferation through TP53 mutation-induced apoptotic resistance

Background and AimsThe specific mechanisms underlying the inhibition of hepatocellular carcinoma (HCC) proliferation and metastasis by mitochondrial apoptosis are not yet fully understood. However, it plays a vital role in suppressing HCC's ability to proliferate and spread. The involvement of MRPL21, a member within the family of mitochondrial ribosomal proteins (MRPs), is well-documented in both cellular apoptosis and energy metabolism. This study aims to explore and unravel the underlying mechanisms through which MRPL21 contributes to mitochondrial apoptosis and resistance against apoptosis in HCC. MethodsTo evaluate the level of MRPL21 expression at the gene and protein expression levels, analysis was performed on human liver samples and blood using techniques for quantification. A knockdown plasmid targeting MRPL21 was constructed to investigate its impact on the growth and apoptosis of hepatocellular carcinoma (HCC). To evaluate the impact of MRPL21 knockdown on hepatocellular carcinoma (HCC) cell proliferation and apoptosis, various assays were performed including CCK-8 assays, flow cytometry analysis, detection of reactive oxygen species (ROS), and assessment of mitochondrial membrane potential (MMP). Furthermore, the role of MRPL21 in TP53 mutation was examined using Nutlin-3. ResultsIn HCC tissues and blood samples, an upregulation of MRPL21 expression was observed when compared to samples obtained from healthy individuals, and it is correlated with a poor prognosis for HCC. Silencing MRPL21 can effectively suppress Hep3B and HCCLM3 cells proliferation by modulating the mitochondrial membrane potential, it triggers the generation of reactive oxygen species (ROS), thereby leading to G0/G1 cell cycle arrest and initiation of early apoptosis. Furthermore, by inhibiting P53 activity, Nutlin-3 treatment can enhance MRPL21-deficiency-mediated apoptosis in Hep3B and HCCLM3 cells. ConclusionThrough its influence on TP53 mutation, MRPL21 promotes HCC proliferation and progression while conferring resistance to apoptosis. These findings suggest that MRPL21 holds promise as a valuable biomarker for the treatment of HCC.

Mechanistic Insight into the Autophagic and Apoptotic Activity of Kaempferol on Liver Cancer Cells.

The accumulation of poorly folded protein in the endoplasmic reticulum (ER) promotes ER stress and contributes to the pathogenesis of hepatocellular carcinoma (HCC). Current therapies have various adverse effects, therefore, laying the need for an alternative approach. Kaempferol (KP), a naturally occurring flavonoid, possesses potent anti-proliferative properties against various cancer cells. Nevertheless, its involvement in HCC remains relatively unexplored, particularly regarding its influence on apoptosis and autophagy pathways. The effect of KP on cell viability, and motility of Hep3B cells was evaluated by MTT, and scratch assay, respectively. Hoechst staining and FACS analysis were done to check the effect of KP on apoptosis and cell cycle progression. qRTPCR was used to evaluate the expression of several apoptosis and autophagy-related genes. KP was docked with several ER stress-related proteins involved in HCC to gain further insights into molecular mechanisms. The results of docking studies were validated with MD simulation and in vitro studies. Treatment with KP at different time intervals showed dose- and time-dependent growth inhibition of liver cancer cells. KP decreased motility and arrested the cell cycle at the G0/G1 phase in Hep3B cells. Additionally, in the context of HCC, the relationship between KP, apoptosis, and autophagy is significant. It induced apoptosis and autophagy in Hep3B cells by downregulating the expression of Bcl-2 and upregulated Bax and Bid, Caspase-3, Beclin-1, and LC3. KP showed a better binding affinity with Nrf2, PERK, and IRE1α among all selected proteins. Further, it reversed the protective effect of 4-PBA (ER Stress inhibitor) by inducing apoptosis and autophagy in Hep3B cells. The study suggested KP as a potential chemopreventive agent for managing HCC by effectively inducing apoptosis and autophagy in Hep3B cells.

ASTAXANTHIN INHIBITS CELL PROLIFERATION, MIGRATION, INVASION AND INDUCED APOPTOSIS VIA AMPK-mTOR SIGNALING PATHWAY IN HEPATOCELLULAR CARCINOMA HEP 3B CELLS

"Hepatocellular carcinoma (HCC) is a fatal malignancy with a poor prognosis. There is an urgent need to study the molecular mechanisms of HCC development and explore potential drugs to improve survival. This study aims to investigate the antitumor effects of astaxanthin on HCC proliferation, migration, invasion and apoptosis through regulation of adenosine-activated protein kinase (AMPK). CCK8, wound healing, transwell and flow cytometry assays were used to evaluate Hep 3B cell viability, migration, invasion and apoptosis after astaxanthin treatment. Protein expression was determined by Western blot. CCK8 assays showed that all concentrations (200 μM, 400 μM, 500 μM) of astaxanthin used in this study significantly inhibited the proliferation of Hep 3B cells (P < 0.05). Wound healing, transwell and flow cytometry showed that astaxanthin inhibited Hep 3B cell migration and invasion and induced apoptosis. Western blot showed that astaxanthin increased the expression of p-AMPK (P <0.05) and inhibited its downstream p-mTOR protein expression (P < 0.05). The effects of astaxanthin on cell proliferation, migration, invasion and apoptosis was attenuated after addition of the AMPK blocker Compound C. To conclude, astaxanthin inhibits the proliferation, migration and invasion of HCC by regulating AMPK, and promotes tumor cell apoptosis in a dose-dependent manner."

Polygonatum sibiricum Polysaccharide Inhibited Liver Cancer in a Simulated Tumor Microenvironment by Eliminating TLR4/STAT3 Pathway.

Liver cancer is one of the most aggressive tumors and one of the most common malignant tumors which seriously threatens human health. Traditional Chinese medicine (TCM) was reported to resist the proliferation and metastasis of liver cancer cells. In this study, we aimed to explore the potential anti-cancer effect of Polygonatum sibiricum polysaccharide (PSP) on the tumor immune microenvironment in liver cancer cells. HepG2 and Hep3B cells were pretreated in the absence or the presence of PSP (20, 50, 100 µg/mL) for a period of 24 h. Subsequently, dendritic cells (DCs) were co-cultured with HepG2 and Hep3B cell supernatant to investigate the effect of PSP on the tumor microenvironment. The results showed that PSP dose-dependently inhibited proliferation and promoted apoptosis of HepG2 and Hep3B cells. Meanwhile, PSP dose-dependently inhibited migration, invasion, and epithelial-to-mesenchymal transition (EMT) of liver cancer cells. In addition, PSP dose-dependently induced inflammatory response of DCs, characterized by increases of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α in DCs. Mechanically, PSP dose-dependently reduced the activation of the Toll-like receptor 4 (TLR4)/Signal transducer and activator of transcription 3 (STAT3) and noncanonical nuclear factor-kappa B (NF-κB) signaling pathways. TLR4 agonist lipopolysaccharide (LPS) reversed the anti-oncogenic effects of PSP in liver cancer cells. Taken together, PSP inhibited liver cancer in a simulated tumor microenvironment by eliminating TLR4/STAT3 pathway. PSP promises an important and useful alternative to liver cancer treatment.

Gypenosides suppress hepatocellular carcinoma cells by blocking cholesterol biosynthesis through inhibition of MVA pathway enzyme HMGCS1

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high morbidity and mortality. Targeting abnormal cholesterol metabolism is a potential therapeutic direction. Therefore, more natural drugs targeting cholesterol in HCC need to be developed. Gypenosides (Gyp), the major constituent of Gynostemma pentaphyllum, has been demonstrated to have pharmacological properties on anti-cancer, anti-obesity, and hepatoprotective. We investigated whether Gyp, isolated and purified by our lab, could inhibit HCC progression by inhibiting cholesterol synthesis. The present research showed that Gyp inhibited proliferation and migration, and induced apoptosis in Huh-7 and Hep3B cells. Metabolomics, transcriptomics, and target prediction all suggested that lipid metabolism and cholesterol biosynthesis were the mechanisms of Gyp. Gyp could limit the production of cholesterol and target HMGCS1, the cholesterol synthesis-related protein. Downregulation of HMGCS1 could suppress the progression and abnormal cholesterol metabolism of HCC. In terms of mechanism, Gyp suppressed mevalonate (MVA) pathway mediated cholesterol synthesis by inhibiting HMGCS1 transcription factor SREBP2. And the high expression of HMGCS1 in HCC human specimens was correlated with poor clinical prognosis. The data suggested that Gyp could be a promising cholesterol-lowering drug for the prevention and treatment of HCC. And targeting SREBP2-HMGCS1 axis in MVA pathway might be an effective HCC therapeutic strategy.

CDCA8 Facilitates Tumor Proliferation and Predicts a Poor Prognosis in Hepatocellular Carcinoma.

CDCA8 expression is abnormally high in a variety of cancers and involved in the biological process of tumor malignancy. In this study, we discovered that the expression of CDCA8 was up-regulated in hepatocellular carcinoma cancer (HCC) tissues and high levels of CDCA8 are associated with larger tumor size, higher AFP (α-fetoprotein) levels, and unfavorable prognosis. Cell functional experiments revealed that CDCA8 silencing remarkably inhibited proliferation and promoted apoptosis in SNU-387 and Hep-3B cells. The results of flow cytometry showed that CDCA8 regulated CDK1 and cyclin B1 expression to arrest at the S phase, inhibited proliferation, and promoted apoptosis. In addition, in vivo studies have confirmed that silencing CDCA8 could regulate CDK1/cyclin B1 signaling axis to inhibit the growth of HCC xenograft tumor. Our study demonstrated CDCA8 acts an oncogene to facilitate cell proliferation of HCC via regulating cell cycle, indicating the promising application value of CDCA8 for HCC diagnosis and clinical treatment.

Chaetocochin J exhibits anti-hepatocellular carcinoma effect independent of hypoxia

The most studied epipolythiodioxopiperazine (ETP) alkaloids, such as chetomin, gliotoxin and chaetocin, were reported to exert their antitumor effects through targeting HIF-1α. Chaetocochin J (CJ) is another ETP alkaloid, of which the effect and mechanism on cancer are not fully elucidated. Considering the high incidence and mortality of hepatocellular carcinoma (HCC) in China, in the present study, using HCC cell lines and tumor-bearing mice as models, we explored the anti-HCC effect and mechanism of CJ. Particularly, we investigated whether HIF-1α is related to the function of CJ. The results showed that, both under normoxic and CoCl2 induced-hypoxic conditions, CJ in low concentrations (<1 µM) inhibits the proliferation, induces G2/M phase arrest, leading to the disorder of metabolism, migration, invasion, and caspase-dependent apoptosis in HepG2 and Hep3B cells. CJ also showed anti-tumor effect on a nude xenograft mice model without significant toxicity. Moreover, we demonstrated that the key to CJ’s function is mainly associate with its inhibition of PI3K/Akt/mTOR/p70S6K/4EBP1 pathway independent of hypoxia, and it also could suppress the expression of HIF-1α as well as disrupt the binding of HIF-1α/p300 and subsequently inhibits the expression of its target genes under hypoxic condition. These results demonstrated that CJ possessed a hypoxia-independent anti-HCC effects in vitro and in vivo, which was mainly attributable to its inhibition on the upstream pathways of HIF-1α.

Royal jelly acid suppresses hepatocellular carcinoma tumorigenicity by inhibiting H3 histone lactylation at H3K9la and H3K14la sites

Background and purposeHuman hepatocellular carcinoma (HCC) features include enhanced glycolysis and elevated lactate concentrations. Accumulation of lactate during metabolism provides a precursor for histone lysine modification. This study was designed to determine whether royal jelly acid (RJA) acts against HCC through the lactate modification pathway. Experimental approachThe effects of RJA on Hep3B and HCCLM3 cell invasion, migration, proliferation, and apoptosis were investigated using cell scratching, colony formation assay, flow cytometry, western blotting, and real-time qPCR, gas chromatography, and RNA sequencing to determine the pathways and molecular targets involved. Tumor xenografts were used to evaluate the anti-HCC effects of RJA in vivo. In-cell Western blotting and expression correlation analysis were applied to confirm the associations between H3 histone lactylation and the antitumor effects of RJA. Key resultsRJA has good antitumor effects in vivo and in vitro. Multi-omics analysis with metabolome and transcriptome determined that the glycolytic metabolic pathway provided the principle antitumor effect of RJA. Further mechanistic studies showed that RJA inhibited HCC development by interfering with lactate production and inhibiting H3 histone lactylation at H3K9la and H3K14la sites. Conclusions and implicationsThis study first demonstrated that RJA exerts antitumor effects by affecting the glycolytic pathway. RJA could regulate the lactylation of H3K9la and H3K14la sites on H3 histone using lactate as a clue in the glycolytic pathway. Therefore, the lactylation of H3 histone is vital in exerting the antitumor effect of RJA, providing new evidence for screening and exploring antitumor drug mechanisms in the later stage.

Daphnane-type diterpenoids from Stellera chamaejasme L. and their inhibitory activity against hepatocellular carcinoma cells

Daphnane-type diterpenoids, which are scarce in nature, exhibit potent growth-inhibitory activities against various cancer cells. To identify more daphnane-type diterpenoids, the phytochemical components in the root extracts of Stellera chamaejasme L. were analysed in this study using the Global Natural Products Social platform and the MolNetEnhancer tool. Three undescribed 1α-alkyldaphnane-type diterpenoids (1–3; named stelleradaphnanes A–C) and 15 known analogues were isolated and characterised. The structures of these compounds were determined using ultraviolet and nuclear magnetic resonance spectroscopy. The stereo configurations of the compounds were determined using electronic circular dichroism. Next, the growth-inhibitory activities of isolated compounds against HepG2 and Hep3B cells were examined. Compound 3 exhibited potent growth-inhibitory activities against HepG2 and Hep3B cells with half-maximal inhibitory concentration values of 9.73 and 15.97 μM, respectively. Morphological and staining analyses suggested that compound 3 induced apoptosis in HepG2 and Hep3B cells.

Gamabufotalin Induces Apoptosis and Cytoprotective Autophagy through the mTOR Signaling Pathway in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with a high rate of recurrence and a poor prognosis. Here, we investigated the effect and the potential antitumor mechanism of Gamabufotalin (CS-6) against HCC. Our results show that CS-6 strikingly reduced cell viability, inhibited colony formation, and promoted apoptosis in Hep3B and Huh7 cells. In vivo, CS-6 inhibited HCC xenograft tumor growth with no toxicity to normal tissues. Mechanistically, we found that CS-6 could induce cytoprotective autophagy through the mTOR-ULK1 signaling pathway through downregulation of p62 and upregulation of LC3 II/LC3 I. Meanwhile, CS-6 activated caspase-3 and PARP mediated apoptosis, and the caspase inhibitor Z-VAD-FMK blocked the CS-6-induced cell death in HCC cells. Moreover, autophagy and apoptosis were found to have antagonistic effects in Hep3B and Huh7 cells. Both the autophagy inhibitor chloroquine (CQ) and the mTOR activator MHY1485 blocked autophagy and further enhanced CS-6-induced apoptosis. Taken together, we demonstrated for the first time that CS-6 promotes apoptosis and cytoprotective autophagy through the mTOR signaling pathway in HCC, which proposes a novel strategy for HCC therapy.

Nanosecond pulsed electric field ablates rabbit VX2 liver tumors in a non-thermal manner.

Liver tumor remains an important cause of cancer-related death. Nanosecond pulsed electric fields (nsPEFs) are advantageous in the treatment of melanoma and pancreatic cancer, but their therapeutic application on liver tumors need to be further studied. Hep3B cells were treated with nsPEFs. The biological behaviors of cells were detected by Cell Counting Kit-8, 5-ethynyl-20-deoxyuridine, and transmission electron microscopy (TEM) assays. In vivo, rabbit VX2 liver tumor models were ablated by ultrasound-guided nsPEFs and radiofrequency ablation (RFA). Contrast-enhanced ultrasound (CEUS) was used to evaluate the ablation effect. HE staining and Masson staining were used to evaluate the tissue morphology after ablation. Immunohistochemistry was performed to determine the expression of Ki67, proliferating cell nuclear antigen, and α-smooth muscle actin at different time points after ablation. The cell viability of Hep3B cells was continuously lower than that of the control group within 3 days after pulse treatment. The proliferation of Hep3B cells was significantly affected by nsPEFs. TEM showed that Hep3B cells underwent significant morphological changes after pulse treatment. In vivo, CEUS imaging showed that nsPEFs could completely ablate model rabbit VX2 liver tumors. After nsPEFs ablation, the area of tumor fibrosis and the expression of Ki67, proliferating cell nuclear antigen, and α-smooth muscle actin were decreased. However, after RFA, rabbit VX2 liver tumor tissue showed complete necrosis, but the expression of PCNA and α-smooth muscle actin did not decrease compared to the tumor group. nsPEFs can induce Hep3B cells apoptosis and ablate rabbit VX2 liver tumors in a non-thermal manner versus RFA. The ultrasound contrast agent can monitor immediate effect of nsPEF ablation. This study provides a basis for the clinical study of nsPEFs ablation of liver cancer.

Active compounds and potential targets of Shuganning injection in the treatment of hepatocellular carcinoma by network pharmacology and in vitro validation.

Shuganning injection (SGNI), a TCM (traditional Chinese medicine) injection with good hepatoprotective effects, exerted therapeutic effects on hepatocellular carcinoma (HCC). However, the active compounds and effects of SGNI on HCC remain unclear. The objective of this study was to investigate the active compounds and potential targets of SGNI in the treatment of HCC, and explore the molecular mechanisms of main compounds. Network pharmacology was applied to predict the active compounds and targets of SGNI on cancer. The interactions between active compounds and target proteins were validated by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and pull-down assay. The in vitro test of the effects and mechanism of vanillin and baicalein was elucidated by MTT, western blot, immunofluorescence, and apoptosis analysis. According to compound characteristics, targets, etc., two typical active ingredients (vanillin and baicalein) were selected as representatives to explore the effects on HCC. Vanillin (an important food additive) bound to NF-κB1 and baicalein (a bioactive flavonoid) bound to FLT3 (FMS-like tyrosine kinase 3) were confirmed in this study. Vanillin and baicalein both inhibited cell viability and promoted apoptosis of Hep3B and Huh7 cells. In addition, both vanillin and baicalein could enhance the activation of the p38/MAPK (mitogen-activated protein kinase) signaling pathway, which may partially explain the anti-apoptosis effects of the two compounds. In conclusion, two active compounds of SGNI, vanillin and baicalein, promoted apoptosis of HCC cells via binding with NF-κB1 or FLT3, and regulating the p38/MAPK pathway. Baicalein and vanillin may be good candidates for HCC treatment on drug development.

Circ_0124208 Promotes the Progression of Hepatocellular Carcinoma by Regulating the miR-338-3p/LAMC1 Axis.

Hundreds of circular RNAs (circRNAs) have been identified as key regulators in biological processes; however, only few of these circRNAs have been functionally described to participate in the development of hepatocellular carcinoma (HCC). The present study aimed to reveal the function and molecular mechanisms of circ_0124208 in HCC. Real-time quantitative PCR revealed the upregulation of circ_0124208 in HCC tissues and cells. Based on cell functional experiments, silencing circ_0124208 attenuated proliferation and migration, but boosted the apoptosis of Hep 3B and Huh7 cells in vitro. The in vivo experiment further validated the repression of tumor growth via circ_0124208 knockdown. RNA immunoprecipitation and dual-luciferase reporter assays showed that circ_0124208 sponged miR-338-3p and reduced its expression. miR-338-3p inhibition was found to partially reverse the tumor-suppressive effects caused by circ_0124208 in Hep 3B and Huh7 cells. Furthermore, miR-338-3p directly targeted laminin subunit gamma 1 (LAMC1). The malignancy of Hep 3B and Huh7 cell was decreased by LAMC1 knockdown, and this effect was mitigated by miR-338-3p suppression. Overall, circ_0124208 was demonstrated for the first time to play a crucial role as an oncogene in HCC, implying that it could be a useful biomarker for HCC diagnosis. Furthermore, the circ_0124208/miR-338-3p/LAMC1 axis can be used as a potential therapeutic target for HCC treatment.

Acetylated xylo-oligosaccharide from Hawthorn kernels inhibits colon cancer cells in vitro and in vivo

The effects of acetylated xylo-oligosaccharide (AcXOS) from Hawthorn kernels on tumor cells were evaluated. In vitro, AcXOS (20 mg/mL) suppressed Hep3B and RKO tumor cells migration by 28.6 % and 56.6 %, and invasion by 68.0 % and 69.2 %, respectively. It also promoted the apoptosis of Hep3B cells and RKO cells. In vivo tumor growth assay, tumor-bearing mice with colon cancer HCT116 cells were orally administrated with AcXOS. The xenograft tumor growth was inhibited by 37.2 % after treatment with 5 % AcXOS. In tumors, AcXOS repressed the PI3K-Akt signaling, enhancing the expression of Bax and cleaved caspase 3 by 89.4 % and 131.4 %, respectively. Furthermore, AcXOS increased levels of short chain fatty acids in mice feces and decreased mRNA levels of histone deacetylases of mice colonic epithelial cell. These findings suggest that AcXOS from Hawthorn kernels is a potential inhibitor of tumor cells and thus may be used for prevention and intervention of tumor growth.

Auranofin accelerates spermidine-induced apoptosis via reactive oxygen species generation and suppression of PI3K/Akt signaling pathway in hepatocellular carcinoma

Auranofin is a US Food and Drug Administration (FDA)-approved anti-arthritis medication that functions as a thioredoxin reductase inhibitor. Spermidine, a polyamine present in marine algae, can exert various physiological functions. Herein, we examined the synergistic anticancer activity of auranofin and spermidine in hepatocellular carcinoma (HCC). Combined treatment with auranofin and spermidine suppressed cell viability more efficiently than either treatment alone in HCC Hep3B cells. The isobologram plotted by calculating the half maximal inhibitory concentration (IC50) values of each drug indicated that the two drugs exhibited a synergistic effect. Based on the analysis of annexin V and cell cycle distribution, auranofin and spermidine markedly induced apoptosis in Hep3B cells. Moreover, auranofin and spermidine increased mitochondria-mediated apoptosis by promoting mitochondrial membrane potential (Δψm) loss. Auranofin and spermidine significantly increased reactive oxygen species (ROS) production in Hep3B cells, and the blocking ROS suppressed apoptosis induced by spermidine and auranofin. In addition, auranofin and spermidine reduced the expression of phosphorylated phosphatidylinositol-3 kinase (PI3K) and protein kinase B (Akt), and PI3K inhibitor accelerated auranofin- and spermidine-induced apoptosis. Using ROS scavenger and PI3K inhibitor, we revealed that ROS acts upstream of auranofin- and spermidine-induced apoptosis. Collectively, our study suggests that combination treatment with auranofin and spermidine could afford synergistic anticancer activity via ROS overproduction and reduced PI3K/Akt signaling pathway.

Inhibitory Effect of Hernandezine on the Proliferation of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) causes 830000 deaths every year and is becoming the third malignant tumor worldwide. One of the primary reasons is the lack of effective drugs. Hernandezine (HER), a bisbenzylisoquinoline alkaloid of Thalictrum simplex, has been confirmed to have antitumor activity. But there are few reports about its effect on HCC and the underlying mechanisms still remain unclear. Therefore, the antitumor effects and mechanisms of HER on HCC were evaluated in HepG2 and Hep3B cells. The in vitro experiments demonstrated that HER significantly induced G0/G1 phase arrest, inhibited the proliferation and promoted cell apoptosis in liver cancer cell lines. In the mechanisms, the antitumor effects of HER on liver cancer cells were mediated by phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) pathway and reactive oxygen species (ROS), simultaneously. In one way, HER inhibited the activities of PI3K-AKT pathway, which interrupt the dimer formation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1) and result to G0/G1 phase arrest. In another way, after HER treatment, ROS accumulated in liver cancer cells and caused mitochondria injury which further influenced the expression of apoptosis-related proteins and eventually resulted to HepG2 and Hep3B cell apoptosis. In addition, HER showed a tumor restrain function in HepG2 and Hep3B bearing nude mice. Overall, these findings indicated that HER is a promising antitumor drug, which may provide a new direction for clinical HCC treatment.

Highly Oxidized Germacranolides from Elephantopus tomentosus and the Configurational Revision of Some Previously Reported Analogues.

Highly oxidized germacranolides are mainly found in the genus Elephantopus, contain a characteristic ten-membered molecular core that is highly flexible, and exhibit potential cytotoxic properties. However, their configurations were assigned ambiguously in previous reports due to spectroscopic observation of macrocyclic systems. Herein, 17 highly oxidized germacranolides, including 12 new germacranolides (1-12), were isolated from Elephantopus tomentosus. Their structures were characterized by spectroscopic data analysis combined with X-ray crystallography and ECD calculations, and it was possible to propose configurational revisions of five previously reported analogues (13-17). Cytotoxic activities for 1-17 against two hepatocellular carcinoma cell lines (HepG2 and Hep3B) were tested, and compounds 1-10 and 13-16 generated IC50 values of 2.2-9.8 μM. Furthermore, the observed cytotoxic activity of 1 was determined as being mediated by inducing the apoptosis of HepG2 and Hep3B cells via mitochondrial dysfunction.

Apigetrin Promotes TNFα-Induced Apoptosis, Necroptosis, G2/M Phase Cell Cycle Arrest, and ROS Generation through Inhibition of NF-κB Pathway in Hep3B Liver Cancer Cells.

Apigetrin (7-(β-D-glucopyranosyloxy)-4′,5-dihydroxyflavone), a glycoside bioactive dietary flavonoid derived from Taraxacum officinale and Teucrium gnaphalodes, is known to possess anticancer, antioxidant, and anti-inflammatory effects on numerous cancers. In the present study, we examined the effect of apigetrin in Hep3B hepatocellular cancer cell line (HCC). Apigetrin inhibited cell growth and proliferation of Hep3B cells, as confirmed by MTT and colony formation assay. We used apigetrin at concentrations of 0, 50, and 100 µM for later experiments. Of these concentrations, 100 µM of apigetrin showed a significant effect on cell inhibition. In apigetrin-treated Hep3B cells, cell cycle arrest occurred at the G2/M phase. Apoptosis and necroptosis of Hep3B cells treated with apigetrin were confirmed by Annexin V/propidium iodide (PI) staining and flow cytometry results. Morphological observation through 4′,6-diamidino-2-phenylindole (DAPI) staining showed intense blue fluorescence representing chromatin condensation. Hematoxylin staining showed necroptotic features such as formation of vacuoles and swelling of organelles. Apigetrin increased reactive oxygen species (ROS) levels in cells, based on fluorescence imaging. Furthermore, the underlying mechanism involved in the apoptosis and necroptosis was elucidated through western blotting. Apigetrin up-regulated TNFα, but down-regulated phosphorylation of p-p65, and IκB. Apigetrin inhibited the expression of Bcl-xl but increased Bax levels. Up-regulation of cleaved PARP and cleaved caspase 3 confirmed the induction of apoptosis in apigetrin-treated Hep3B cells. Additionally, necroptosis markers RIP3, p-RIP3, and p-MLKL were significantly elevated by apigetrin dose-dependently, suggesting necroptotic cell death. Taken together, our findings strongly imply that apigetrin can induce apoptosis and necroptosis of Hep3B hepatocellular cancer cells. Thus, apigetrin as a natural compound might have potential for treating liver cancer.