Hepatoma SMMC-7721 Cells Research Articles

Retraction Note: MicroRNA-302b suppresses cell proliferation by targeting EGFR in human hepatocellular carcinoma SMMC-7721 cells

Retraction Note: MicroRNA-302b suppresses cell proliferation by targeting EGFR in human hepatocellular carcinoma SMMC-7721 cells

Preparation of kakkatin derivatives and their anti-tumor activity.

Modern pharmacological studies have confirmed that plant-derived compounds from Puerariae flos (PF) has significant biological activities against liver damage, tumors and inflammation. Kakkatin is an isoflavone polyphenolic compound isolated from PF flower. However, the effect of kakkatin and its derivatives on anti-tumor has not been well explored. To design and synthesize a kakkatin derivative [6-(hept-6-yn-1-yloxy)-3-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one (HK)] to explore its anti-tumor biological activity. Hept-6-yn-1-yl ethanesulfonate was introduced to replace hydrogen at the hydroxyl position of kakkatin phenol, and the derivative of kakkatin was prepared; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was used to detect cell viability, a clone formation assay was adopted to detect cell proliferation, apoptosis, necrosis, and cell cycles were analyzed by Annexin V/propidium iodide staining and flow cytometry. Cell migration and invasion ability were evaluated by cell scratch assay and transwell assay. The potential mechanism of HK on hepatocellular carcinoma (HCC) SMMC-7721 cells was explored through network pharmacology and molecular docking, and finally real-time PCR assays was used to verify the potential targets and evaluate the biological activity of HK. Compared with kakkatin, the modified HK did not significantly increase the inhibitory activity of gastric cancer MGC803 cells, but the inhibitory activity of HCC SMMC-7721 cells was increased by about 30 times, with an IC50 value of 2.5 μM, and the tumor inhibition effect was better than cisplatin, which could significantly inhibit the cloning, invasion and metastasis of HCC SMMC-7721 cells, and induce apoptosis and G2/M cycle arrest. Its mechanism of action is mainly related to the upregulation of PDE3B and NFKB1 target proteins in the cAMP pathway. HK have a significant inhibitory effect on HCC SMMC-7721 cells, and the targets of their action may be PDE3B and NFKB1 proteins in the cAMP pathway, making it a good lead drug for the treatment of HCC.

Environmentally Friendly UV Absorbers: Synthetic Characterization and Biosecurity Studies of the Host-Guest Supramolecular Complex.

Isoamyl 4-methoxycinnamate (IMC) is widely used in various fields because of its exceptional UV-filter properties. However, due to its cytotoxicity and anti-microbial degradability, the potential eco-environmental toxicity of IMC has become a focus of attention. In this study, we propose a host-guest supramolecule approach to enhance the functionality of IMC, resulting in a more environmentally friendly and high-performance materials. Sulfobutyl-β-cyclodextrin sodium salt (SBE-β-CD) was used as the host molecule. IMC-SBE-β-CD supramolecular substances were prepared through the "saturated solution method", and their properties and biosecurity were evaluated. Meanwhile, we conducted the AOS tree evaluation system that surpasses existing evaluation approaches based on apoptosis, oxidative stress system, and signaling pathways to investigate the toxicological mechanisms of IMC-SBE-β-CD within human hepatoma SMMC-7721 cells as model organisms. The AOS tree evaluation system aims to offer the comprehensive analysis of the cytotoxic effects of IMC-SBE-β-CD. Our findings showed that IMC-SBE-β-CD had an encapsulation rate of 84.45% and optimal stability at 30 °C. Further, IMC-SBE-β-CD promoted cell growth and reproduction without compromising the integrity of mitochondria and nucleus or disrupting oxidative stress and apoptosis-related pathways. Compared to IMC, IMC-SBE-β-CD is biologically safe and has improved water solubility with the UV absorption property maintained. Our study provides the foundation for the encapsulation of hydrophobic, low-toxicity organic compounds using cyclodextrins and offers valuable insights for future research in this field.

Synthesis of stimuli-responsive copolymeric hydrogels for temperature, reduction and pH-controlled drug delivery

Stimuli-responsive copolymeric hydrogels (CH) are synthesized for controlled drug delivery. Oxidized glycyrrhizic acid is connected to acrylamide through Schiff base reaction, which is then copolymerized with N,N-bis (acryloyl) cystamine and N-isopropylacrylamide by free-radical polymerization for the loading of 5-fluorouracil with a loading efficiency of 96.7 %. The lower critical solution temperature of the CH is 39.14 °C. The N-isopropylacrylamide unit in the CH is sensitive to temperature, endowing the CH with temperature sensitivity. The carboxyl groups of oxidized glycyrrhizic acid can be deprotonated in alkaline solutions, and the electrostatic repulsions between the resultant carboxylate can cause swelling of the CH. The disulfide bond (S–S) of the N,N-bis (acryloyl) cystamine unit can be reduced to sulfhydryl (–SH) by glutathione, and thus the CH is also reduction-sensitive. Therefore, temperature, reduction and pH-controlled delivery of 5-fluorouracil from the CH is achieved. Cytotoxicity assay reveals that the drug-free hydrogels have good biocompatibility while the CH shows high cytotoxicity against human hepatoma SMMC-7721 cells, and the cell viability is only 25 % at the CH concentration of 1 mg mL−1.

Compound Taxus exerts marked anti-tumor activity and radiosensitization effect on hepatocellular carcinoma cells

BackgroundCompound Taxus capsule, as an antineoplastic Chinese patent drug, has been increasingly applied as an adjunctive treatment for the management of non-small-cell lung cancer (NSCLC) and some other malignancies, but research about its antitumor activity and radiosensitization effect on hepatocellular carcinoma (HCC) cells is very rare. PurposeTo investigate the antitumor activity and radiosensitization effect of Compound Taxus on HCC cells and to preliminarily explore the possible molecule mechanisms involved. MethodsCell viability, cell cycle distribution, apoptosis, DNA damage repair and protein expression levels were detected by CCK-8 assay, flow cytometry, immunofluorescence staining, western blotting analysis and immunohistochemical staining, respectively. The migration and invasion activities and vasculogenic mimicry (VM) formation and angiogenesis were evaluated by tube formation and VM formation assay. Radiation survival curves were obtained from the colony formation assay in human HCC cell lines, Smmc7721 and Bel7402 cells, pretreated with or without Compound Taxus before receiving X-ray irradiation. A Bel7402 tumor-bearing mouse model was established and the radiosensitization effect of Compound Taxus in vivo was evaluated by analyzing tumor volume and tumor weight in different groups receiving different treatments. ResultsCompound Taxus decreased viability, induced G2/M arrest, promoted apoptosis, suppressed migration and invasion, and inhibited VM formation and angiogenesis in Smmc7721 and Bel7402 cells. Furthermore, Compound Taxus inhibited irradiation-induced DNA damage repair, enhanced the radiosensitivity of Smmc7721 and Bel7402 cells and improved the anti-tumor therapeutic efficacy of irradiation in Bel7402 tumor-bearing mice. Radiotherapy in combination with Compound Taxus showed the best tumor inhibition compared to that of Compound Taxus alone or irradiation alone. In addition, Compound Taxus significantly down-regulated NF-κB p65, p–NF–κB p65 and Bcl-2, and up-regulated Bax in vitro and in vivo, yet NF-κB p65 overexpression reversed the proapoptotic effect of Taxus on HCC cells, indicating that the NF-κB signaling pathway might be an important signal mediator in the Compound-Taxus-modulated biological responses. ConclusionOur findings suggest that Compound Taxus shows marked antitumor activity and significant radiosensitization effect on HCC cells, making it possible for Compound Taxus to become a promising auxiliary modality for HCC management and a potential radiosensitizer of HCC in the future.

Circ_MAPK9 promotes STAT3 and LDHA expression by silencing miR-642b-3p and affects the progression of hepatocellular carcinoma

BackgroundAberrant expression and activation of circular RNAs (circRNAs) are closely associated with various cancers. The role of circ_MAPK9 (hsa_circ_0001566) in cancer progression remains unknown. This study aims to investigate the function, mechanism and clinical significance of circ_MAPK9 in hepatocellular carcinoma (HCC).MethodsCirc_MAPK9 expression on the microarray of tumor from clinical HCC patients was detected by in situ hybridization (ISH). Circ_MAPK9 knockdown was achieved with siRNAs in SMMC-7721 and SK-Hep1 HCC cell lines. The biological function of circ_MAPK9 was verified in vitro by CCK8 test, colony formation assay, transwell assay, PI-Annexin V staining, and in vivo by xenograft tumor in nude mice. Fluorescent in situ hybridization (FISH), subcellular fractionation assay, a dual-luciferase reporter assay and rescue experiments were employed for further mechanistic investigation.ResultsThe expression of circ_MAPK9 was significantly up-regulated in HCC tissues and cells, which was found to be associated with poor prognosis. Patients with high expression of circ_MAPK9 had a shorter overall survival and disease-free survival in comparison to those with low circ_MAPK9 expression. Functional assays showed that circ_MAPK9 knockdown suppressed cellular proliferation, migration, invasion and tumor growth in vivo, and promoted apoptosis in HCC cells. Moreover, we found that circ_MAPK9 knockdown could inhibit aerobic glycolysis by decreasing the production of adenosine triphosphate (ATP) and lactic acid, which was mediated by lactate dehydrogenase (LDHA). Mechanistically, circ_MAPK9 functioned as ceRNA via sponging miR-642b-3p and alleviated the inhibitory effect of miR-642b-3p on its target signal transducer and activator of transcription 3 (STAT3) and LDHA, thereby leading to STAT3 activation and LDHA expression.ConclusionsCirc_MAPK9, as an oncogene, promotes HCC growth and metastasis through miR-642b-3p/STAT3-LDHA axis. Circ_MAPK9 could serve as a potential biomarker for HCC poor prognosis and diagnosis.

Synthesis of oxidized carboxymethyl cellulose/chitosan hydrogels doped with graphene oxide for pH- and NIR-responsive drug delivery

Biological macromolecules hydrogels are synthesized via the Schiff base reaction between oxidized carboxymethyl cellulose (oxCMC) and chitosan (CS), and graphene oxide (GO) and 5-fluorouracil (5-FU) are co-doped in the oxCMC/CS hydrogels during the cross-linking process. The acylhydrazone bonds (–HC = N–) between oxCMC and CS can be easily hydrolyzed in acidic solutions, leading to the swelling of the hydrogels and facilitated delivery of 5-FU. On the other hand, the doped GO is a promising photothermal agent, and the delivery of 5-FU can also be facilitated by the generated hyperthermia upon near infrared (NIR) irradiation. Therefore, pH- and NIR-responsive delivery of 5-FU from the hydrogels can be achieved. The kinetics of drug delivery indicate that the delivery of 5-FU from the hydrogels is controlled by first-order model. The drug-free carrier (oxCMC/CS/GO) has excellent biocompatibility while the developed drug delivery system (oxCMC/CS/GO/5-FU) exhibits high cytotoxicity against human hepatoma SMMC-7721 cells.

Cytotoxic terpenes from the flowers of Inula japonica against human hepatocarcinoma cells.

Two new 1,10-seco-eudesmanolides (1 and 2) were isolated from the flowers of Inula japonica together with two eudesmanolide analogues (3 and 4) and two monoterpene derivatives (5 and 6). Their structures were established on the basis of detailed spectroscopic analyses and ECD data. All isolates were evaluated for their antiproliferative activities against human hepatocarcinoma HepG2 and SMMC-7721 cells. Compound 3 exhibited the most potent effect with the IC50 values of 14.60 ± 1.62 and 22.06 ± 1.34 μM against HepG2 and SMMC-7721 cells, respectively. Furthermore, compound 3 showed significant efficacies of arresting the cell cycle at the S/G2-M stages, inducing mitochondria-mediated apoptosis, and inhibiting cell migration in HepG2 cells.

Eriocitrin suppresses proliferation and migration of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPK pathway

To investigate the role of the ROS/MAPK signaling axis in mediating the inhibitory effect of eriocitrin on proliferation and migration of hepatocellular carcinoma SMMC-7721 cells. SMMC-7721 cells were treated with different concentrations of eriocitrin for 24 h, and the changes in cell viability were detected with CCK-8 assay. The migration and invasion abilities of the treated cells were evaluated using Transwell and scratch healing assays, the cell proliferation was assessed with colony-forming assay, and changes in nuclear morphology were observed with DAPI staining. Western blotting was performed to examine the changes in the expressions of E-cadherin, N-cadherin, MMP-2, MMP-9, PARP, Pro-caspase 3, pJNK, p-P38, and p-ERK. The effect of eriocitrin on PARP cleavage in SMMC-7721 cells pretreated with ERK, JNK and P38 inhibitors (U0126, SB203580 and SP600125, respectively) was detected using Western blotting. The effect of treatment with Nacetyl-cysteine (NAC, 30 μmol/L) and eriocitrin (100, 200, and 300 μg/mL), alone or in combination, on reactive oxygen species (ROS) levels in the cells was examined using a DCFH-DA fluorescent probe. Eriocitrin below 50 μg/mL did not produce significant effect on the viability of SMMC-7721 cells (P>0.05). Treatment with eriocitrin significantly inhibited scratch healing, migration, and colony formation of the cells (P < 0.01), reduced the protein expressions of N-cadherin, MMP-2, and MMP-9 (P < 0.01), and up-regulated E-cadherin protein expression (P < 0.05). Eriocitrin-treated SMMC-7721 cells showed obvious apoptotic morphologies with decreased Procaspase 3 expression and increased PARP cleavage (P < 0.01) and phosphorylation levels of JNK, P38, and ERK (P < 0.01); Eriocitrin-induced PAPR cleavage was obviously enhanced by U0126 and SB203580 but attenuated by SP600125. Treatment with 300 μg/mL eriocitrin for 30 min significantly increased ROS level in the cells, and this effect was obviously suppressed by NAC. Eriocitrin can suppress the proliferation and migration and promote apoptosis of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPKs signaling pathway.

Synthesis and anti-hepatocellular carcinoma evaluation of salicylic acid-modified indole trimethoxy flavonoid derivatives.

Simultaneous inhibition of tumor vasculature and the glycolysis pathway may be a targeted anti-tumor strategy to inhibit tumor nutrient supply. Flavonoids are natural products with strong biological activity, which inhibit hypoxia induction factor 1α (HIF-1α) regulating glycolysis and tumor angiogenesis, while salicylic acid can reduce the glycolysis level of tumor cells by inhibiting related rate-limiting enzymes. A series of salicylic acid-modified indole trimethoxy-flavone derivatives were designed and synthesized by introducing benzotrimethoxy-structure commonly used in blood vessel blockers, and their anti-tumor activities were evaluated. Among them, compound 8f exhibited significant anti-proliferative activity against two hepatoma cells, HepG-2 and SMMC-7721, with IC50 values of 4.63 ± 1.13 μM and 3.11 ± 0.35 μM, respectively. Colony formation experiments also further verified its excellent in vitro anti-tumor activity. In addition, compound 8f showed the ability to induce apoptosis in SMMC-7721 cells in a concentration-dependent manner. After treatment with compound 8f, the expressions of the rate-limiting enzymes PKM2, PFKM, HK2 and tumor angiogenesis-related vascular endothelial growth factor of the glycolytic pathway were all down-regulated, and the lactate level in the hepatoma cell SMMC-7721 was significantly reduced. The morphology of the nucleus and tubulin was also observed to disperse gradually with the increase of compound 8f concentration. And compound 8f showed strong binding ability to tubulin. Our results suggest that the strategy of synthesizing the salicylic acid-modified indole flavone derivative 8f is a way to obtain active anti-tumor candidate compounds that may be further developed as targeted agents to inhibit tumor vasculature and glycolytic pathways.

Cytotoxic phenyl polyketides from Hypericum curvisepalum N. Robson

(±)-Hypecurvone A (1) and B (2), two new undescribed phenyl polyketides, along with seven known analogues (3–9) were isolated from the whole plant of Hypericum curvisepalum. Chiral separation of 1 and 2 yielded two pairs of enantiomers 1a/1b and 2a/2b, respectively. The structures of these compounds were elucidated by extensive spectroscopic analyses and ECD spectra simulations. All isolates exhibited moderate cytotoxicity against human hepatocellular carcinoma SMMC-7721 cells, and compound 3 also showed weak cytotoxicity toward MGC-803 cells. The cytotoxicity of these compounds was found to be related to enhanced mitochondria-mediated apoptosis and inhibition of the G2/M phase of the cell cycle.

Levo-tetrahydropalmatine promotes apoptosis of human hepatocellular carcinoma through switching energy metabolism phenotype via upregulation of mitochondrial reactive oxygen species

Mitochondrion is an important organelle that maintains cellular homeostasis and plays a crucial role in determining cell fate. The present study investigated the effect of levo-tetrahydropalmatine(THP) on autophagic flux and energy metabolism phenotype of human hepatocellular carcinoma(HCC) SMMC-7721 and BEL-7402 cells. SMMC-7721 and BEL-7402 cells were treated with THP(100 μmol·L~(-1)) with or without N-acetyl-L-cysteine(NAC, 10 μmol·L~(-1)) for 24 h. The mitochondrial reactive oxygen species(mtROS) was detected by flow cytometry(FCM) with MitoSOX probe and fluorescence microscopy, respectively. Thereafter, autophagic flux was detected by FCM with CYTO-ID probe, and the protein levels of microtubule-associated protein 1 A/1 B-light chain 3-Ⅰ(LC3Ⅰ), LC3Ⅱ, and phosphorylated AMP-activated protein kinase(p-AMPK)/AMPK were measured by Western blot. Mitochondrial respiration was examined by Seahorse XFp assay and cell proliferation by a system. Annexin V-FITC and PI/RNase staining was employed to detect apoptosis of SMMC-7721 and BEL-7402 cells treated with THP and/or NAC. Subsequently, membrane potential was measured with MitoTracker Red CMXRos. Compared with the control group, THP promoted mtROS production and THP combined with NAC attenuated the autophagic flux increase induced by THP alone in SMMC-7721 and BEL-7402 cells. When cells were co-treated with THP and chloroquine(CQ, an autophagy inhibitor), THP further increased mtROS and apoptosis. In addition, THP significantly reduced mitochondrial respiration in terms of mitochondrial basal respiration, ATP production, and maximal respiration. Meanwhile, THP significantly reduced the proliferation index and mitochondrial membrane potential of HCC cells accompanied by the increased apoptosis. This study demonstrates that the up-regulation of mtROS by THP significantly promotes HCC cell autophagy(protective autophagy) and impairs mitochondrial respiration through reprogramming energy metabolism, ultimately inducing the mitochondria-mediated apoptosis of SMMC-7721 and BEL-7402 cells.

GPM6A expression is suppressed in hepatocellular carcinoma through miRNA-96 production

AbstractGPM6A is a glycoprotein in endothelial cells, and its biological function in the development of hepatocellular carcinoma (HCC) is unknown. Through Affymetrix gene expression microarray and bioinformatic analysis, very low GPM6A expression was found in HCC tissue. The present study aims to explore the function and regulatory mechanism of GPM6A in HCC development and progression. Levels of GPM6A expression in HCC specimens from different disorders and various hepatoma cell lines were determined, and its role on cell proliferation was evaluated in hepatoma cells stably overexpressing GPM6A. Modulation of a specific microRNA (miRNA) on its expression and function was evaluated with miRNA mimetic transfection. Herein, it is reported that much lower GPM6A levels were found in HCC tissues than pericancerous liver tissues and correlated to a poor prognosis. GPM6A overexpression inhibited cell proliferation, suppressed colony formation, migration and invasion in two hepatoma cell types. Available evidence does not support that genetic and epigenetic dysregulation contributes significantly to GPM6A inactivation in HCC. Additional findings demonstrated that miR-96-5p acted directly on the 3′-UTR of the GPM6A gene and significantly decreased its mRNA and protein levels. MiR-96-5p transfection promoted proliferation, migration and invasion of SMMC-7721 and MHCC-97H hepatoma cells; whereas the function of oncogenic microRNA-96 was significantly inhibited in GPM6A-overexpressed hepatoma cells. In conclusion, GPM6A expression in HCC is commonly suppressed regardless its base disease types, and its low expression in HCC tissues is most likely attributed to upregulated miR-96-5p. GPM6A may function as a valuable biomarker for HCC progression and prognosis.

Berberine enhances the anti-hepatocellular carcinoma effect of NK92-MI cells through inhibiting IFN-gamma-mediated PD-L1 expression

Background and aimsBerberine is one of the most promising clinically tested natural alkaloids, and immunotherapy using natural killer (NK) cells is a potentially effective treatment for hepatocellular carcinoma (HCC). This study aims to elucidate the effect of berberine on the anti-HCC effect of NK92-MI cells. Materials and methodsHuman HCC SMMC-7721 and Hep3B cells were co-incubated with NK92-MI cells, berberine (60 or 80 μmol/L), or their combination for 36 h. To evaluate the killing effect of NK92-MI cells on HCC cells, the release of lactate dehydrogenase (LDH) in HCC cells was measured. The anti-tumor effects of berberine, NK92-MI cells, and their combinations were evaluated by MTS, EdU, Tunel, and Western blot assays. A male BALB/c nude mouse subcutaneous tumor model was used to investigate the anti-HCC effect of berberine and NK92-MI cells in vivo. ResultsThe LDH assay showed that berberine enhanced the cytotoxicity of NK92-MI cells on HCC cells. The combination of berberine and NK92-MI cells demonstrated more obvious anti-HCC effect than did the berberine or NK92-MI single treatment in inhibiting cell proliferation and inducing apoptosis both in vitro and in vivo. Mechanistically, the expression of programmed cell death-ligand 1 (PD-L1) in HCC cells was upregulated after co-culture with NK-92MI cells. PD-L1 expression was knocked down, thereby inhibiting the proliferation and promoting apoptosis of HCC cells, and inhibited by berberine that blocked the secretion of interferon gamma (IFN-γ), thereby enhancing the anti-tumor effect of NK92-MI cells. ConclusionsCurrent data show that the immunomodulatory role of berberine is to enhance the cytotoxic effect of NK92-MI cells and inhibit tumor immune escape by reducing the expression of PD-L1. Our study provides a rationale for the clinical application of berberine in combination with NK cells for the treatment of HCC.

Development of icariside II loaded polymeric micelles and evaluation of anticancer activity in vitro and in vivo

Icariside II (ICA II) is a bioactive flavonoid in the traditional Chinese medicine Epimedii Folium with potential anticancer effects. However, poor water solubility and low systemic bioavailability limit its application in clinic. Therefore the present study aimed to develop an injectable polymeric micellar system delivering ICA II for cancer chemotherapy. The amphiphilic block copolymers were used as drug carrier, and the drug loaded micelles were prepared via thin-film hydration according to the optimal formulation and conditions from the response surface methodology. The physicochemical properties of micelles were characterized and the antitumor effects were investigated by the in vitro and in vivo assays. Resultantly the micelles had an average particle size of about 20 nm along with a narrow size distribution, and favorable drug loading (∼10%) and entrapment efficiency (>98%). The drug loaded micelles displayed sustained drug release over 10 days in the in vitro system. Compared with the free drug of ICA II, these micelles showed significantly enhanced cytotoxicity against in vitro proliferation of human hepatocellular carcinoma SMMC-7721 cells. The results from nude mice bearing human liver carcinoma further revealed the potent anticancer efficacy in vivo of these micelles. All the findings thus demonstrated that the micellar formulation of the natural bioactive flavonoid ICA II may serve as promising nanocarrier for anticancer treatments.

Design, Synthesis, and Apoptosis-Promoting Effect Evaluation of Chalcone Derivatives Containing Aminoguanidine Units.

Chalcones are precursors of flavonoids or isoflavonoids, and they are abundant in edible plants. Chalcones constitute an important group of natural and synthetic products with a wide range of pharmacological activities. To determine the seeds of the anti-tumor agents, we focused on the potential bioactive materials obtained from chalcone derivatives. Two series of chalcone derivatives containing aminoguanidine or bis-chalone were designed, synthesized, and screened for their cytotoxicity, proliferation inhibition, and apoptosis-promoting activity in vitro against a panel of human tumor cell lines. Among the various compounds studied in this work, 2-((E)-4-((E)-3-oxo-3-(p-tolyl)prop-1-en-1- yl)benzylidene)hydrazine-1-carboximidamide (5f) was the most potent, with IC50 values of 7.17 μM and 3.05 μM antiproliferative activity in vitro against human hepatocarcinoma HepG2 cells and SMMC-7721 cells, respectively. This result showed that the compound possessed a certain degree of selectivity for human hepatocarcinoma cells, especially for SMMC-7721. Then, Annexin V/PI flow cytometry assay was used to investigate different concentrations of compound 5f to demonstrate the ability of compound 5f in inducing apoptosis of SMMC-7721 cells in a concentrationdependent manner. Finally, these results were further verified by Western blot analysis. Based on the collective results, compound 5f may be a promising anti-cancer compound, and may play a significant role in subsequent research.

Arabinogalactan from Ixeris chinensis (Thunb.) Nakai as a stabilizer to decorate SeNPs and enhance their anti-hepatocellular carcinoma activity via the mitochondrial pathway

Arabinogalactan (ICPA) was isolated from the medicinal plant Ixeris chinensis (Thunb.) Nakai, and was used as the reducing agent to stabilize selenium nanoparticles (SeNPs). ICPA-decorated SeNPs (ICPA-SeNPs) were synthesized with Na2SeO3 and ICPA, and their average diameter was 82.6 ± 2.5 nm. ICPA-SeNPs had good dispersity in phosphate-buffered saline (PBS) with a hydrodynamic size of 156.2 ± 2.8 nm. They were stable in PBS solution with a zeta potential of −24.8 ± 0.6 mV. The anti-hepatoma activity of ICPA-SeNPs was investigated in vitro. ICPA-SeNPs significantly suppressed the growth of SMMC-7721 and HepG2 hepatoma cells. They entered SMMC-7721 cells via energy- and caveolae-dependent endocytosis. ICPA-SeNPs were able to increase reactive oxygen species levels, reduce mitochondrial membrane potential, and increase caspase-3 expression in SMMC-7721 cells, which led to the apoptosis of SMMC-7721 cells. They also arrested the cell cycle of SMMC-7721 cells in the S phase. These findings indicate that ICPA could enhance the stability of SeNPs and inhibit SMMC-7721 cell proliferation via the mitochondrial pathway. ICPA-SeNPs could be developed as an antitumor agent for hepatocellular carcinoma treatment.

Transcriptome changes in ERGIC3-knockdown hepatocellular carcinoma cells: ERGIC3 is a novel immune function related gene.

ObjectiveThe expression of ERGIC3 is increased in a variety of tumors and promotes the growth and metastasis of liver cancer, but the molecular mechanism needs to be further studied.In this study, we aimed to analyze the molecular mechanism of ERGIC3 regulating the proliferation of human hepatocellular carcinoma (HCC) SMMC-7721 cells using transcriptomics.MethodsERGIC3 was knocked down in SMMC-7721 cells by RNAi technique, and the expression of ERGIC3 was detected by Q-RT-PCR and Western Blot. RNA sequencing was performed in the Illumina HiSeq platform in the control group and the ERGIC3i group and bioinformatics methods were selected to analyze the data.ResultsThe expression of ERGIC3 was reduced to 10% in SMMC-7721 cells by RNAi technique, and 176 genes were up-regulated and 34 genes were down-regulated in ERGIC3i group compared with the control group. Analysis of the pathways and biological processes that enrich the function of differentially expressed genes showed thatthese differentially expressed genes were mainly involved in vesicular transport, growth factors, PI3K-Akt, NOD-like, Jak-STAT, NF-kappa B and other protein kinase-coupled receptors mediated signal transduction pathways, tumor immune response, collagen-integrin receptor-actin axis, and miRNA pathways. More importantly, most of the significantly altered pathways were related to immunity. ERGIC3 may be a key immune-related gene.ConclusionBased on the transcriptomic analysis, the mechanism of ERGIC3 promoting the growth of HCC is link with the transport of growth factor receptor, cytokine receptor and collagen. Then it is involved in signal transduction pathways mediated by protein kinase-coupled receptors, PI3K-Akt, NOD-like, Jak-STAT and NF-kappa B. In particular, the mechanism is also involved in the ERGIC3-dependent immune pathways. ERGIC3 is a potential target for prevention and treatment of HCC.

Knockdown of RSPH14 inhibits proliferation, migration, and invasion and promotes apoptosis of hepatocellular carcinoma via RelA

BackgroundHigh RSPH14 expression appears to be related to poor prognosis of hepatocellular carcinoma (HCC). This study aimed to investigate the possible roles of RSPH14 in the proliferation, apoptosis, and invasion of HCC cells.MethodsThe UALCAN database and Kaplan–Meier Plotter were used to evaluate the expression level and prognostic role of RSPH14 in HCC. Lentiviral vectors containing shRNA against RSPH14 were constructed to transfect the BEL-7404 and SMMC-7721 HCC cell lines. Cell proliferation was investigated by BrdU, MTT, and colony-formation assays. Apoptosis was detected using flow cytometry. Cell migration and invasion were evaluated using the scratch wound-healing and Transwell assays. Immunohistochemistry and western blot were used to determine the expression levels of the proteins. The function of RSPH14 in vivo was evaluated using a xenograft mouse model.ResultsThe expression of RSPH14 was higher in HCC tumor tissues than in adjacent normal tissues and was closely related to unfavorable prognostic factors and poorer survival (all P < 0.05). Knockdown of RSPH14 inhibited the cell proliferation, migration, and invasion of HCC cells and promoted apoptosis (all P < 0.05). Knockdown of RSPH14 inhibited tumor growth in vivo (P < 0.05). RSPH14 knockdown led to decreased expression of RelA (NF-κBp65), CDH2, and AKT1, thereby affecting the functions of the HCC cells (all P < 0.05). RelA overexpression could abate the inhibitory effect of BEL-7404 cell proliferation caused by RSPH14 depletion.ConclusionKnockdown of RSPH14 could decrease cell proliferation, migration, and invasion and increase apoptosis of HCC cells by inhibiting RelA expression. RSPH14 could be a new treatment target for HCC.

Niuhuang (Bovis Calculus)-Shexiang (Moschus) combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

ObjectiveTo investigate the effects of Niuhuang (Bovis Calculus, BC) and Shexiang (Moschus) (BC-Moschus) on human hepatocellular carcinoma (HCC) cells SMMC-7721 and a nude mouse model of subcutaneous xenografts, and to explore its anti-HCC mechanism. MethodsThe BC-Moschus combination was applied to two liver cancer models in vivo and in vitro. SMMC-7721 was divided into the BC-Moschus group and the control group, and different doses (rude drug dosage 0.625, 1.25, 2.5, and 5 mg/mL) of BC-Moschus extract were used for the intervention. The proliferation ability of HCC cells was detected using the Cell Counting Kit-8 (CCK-8) assay, and the migration ability was detected by a wound healing assay. A subcutaneous xenograft model was prepared using nude mice with human HCC. Specific pathogen-free-grade BALB/c nude mice (5-week-old) were randomly divided into the following groups (n = 6 per group): control (0.9% physiological saline 0.2 mL/d), BC-Moschus [BC 45.5 mg/(kg·d)+ Moschus 13 mg/(kg·d)], and cisplatin (DDP, intraperitoneal injection 5 mg/kg per week) groups. All groups were administered for 14 d. The volume and mass of the subcutaneous xenografts in nude mice were observed. The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, apoptosis-associated factor p70 S6 Kinase (S6K), Bax, Bcl-2, caspase-3, and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR (RT-qPCR) and Western blot. Terminal Deoxynucleotidy Transferase-Mediated dUTP Nick-End Labeling (TUNEL) was used for quantitative analysis of apoptotic cells. ResultsThe CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone, and the inhibition effect was dose- and time-dependent (P < 0.01). The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration (P < 0.01). In the subcutaneous xenograft model of nude mice with human HCC, we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group (P < 0.01). The levels of the PI3K/AKT/mTOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased (P < 0.01). The expression level of the anti-apoptotic gene Bcl-2 was downregulated (P < 0.05), and the expression of the pro-apoptotic gene Bax and apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated (P < 0.01). The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC (P < 0.01). ConclusionThe BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice. The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway, regulation of apoptosis-related protein caspase-3, caspase-9, Bcl-2, and Bax expression, and promotion of apoptosis.