This research investigated the impact of Ganoderma lucidum polysaccharides(GLP) on hepatoblastoma HepG2 and Huh6 cell models, as well as KM mouse model with in situ transplanted tumors, so as to provide a theoretical basis for the clinical application of GLP. Cell viability was assessed through the CCK-8 assay, whereas cell proliferation was evaluated by using the BeyoClick~(TM)EdU-488 test. Cell apoptosis was visualized via Hochest 33258 staining, and autophagy was detected through Mrfp-GFP-LC3 dual fluorescence staining. An in situ tumor transplantation model was created by using HepG2 cells in mice, and mice were treated with normal saline and GLP of 100, 200, and 300 mg·kg~(-1) for tumor count calculation and size assessment. Hematoxylin-eosin(HE) staining was used to observe pathological changes in tumor tissue and vital organs(liver, kidney, lung, spleen, and heart). Western blot analysis was conducted to measure the protein expressions of tumor protein P53(P53), B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cleaved-caspase-3, Beclin-1, autophagy related protein-5(Atg-5), microtubule-associated protein-light chain-3Ⅰ(LC3Ⅰ)/LC3Ⅱ, autophagy adapter protein 62(P62), protein kinase B(Akt), p-Akt, mammalian target of rapamycin(mTOR), and p-mTOR. The in vitro experiment revealed that compared with the control group, after GLP treatment, tumor cell viability decreased significantly; apoptosis rate increased in a dose-dependent manner, and autophagic flux was inhibited. The in vivo experiments showed that compared with the model group, mice treated with GLP exhibited significantly fewer and smaller tumors. Western blot results showed that compared with the control group or model group, levels of P53, Bax, cleaved-caspase-3, Beclin-1, Atg-5, and LC3-Ⅱ/LC3-Ⅰ were significantly increased after GLP treatment, and the levels of Bcl-2, P62, p-Akt/Akt, and p-mTOR/mTOR were significantly decreased. These outcomes suggest that GLP promotes apoptosis and autophagy in hepatoblastoma cells by regulating the Akt/mTOR pathway.
Animals ; Humans ; Autophagy/drug effects* ; Reishi/chemistry* ; Mice ; Apoptosis/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Liver Neoplasms/genetics* ; Hepatoblastoma/genetics* ; Polysaccharides/pharmacology* ; Cell Line, Tumor ; Signal Transduction/drug effects* ; Male ; Cell Proliferation/drug effects* ; Hep G2 Cells

Bufalin(BF)has a significant anti-tumor effect, but its clinical application is severely restricted by its high toxicity and poor water solubility. In this study, Scrophularia ningpoensis polysaccharide(SNP)and ursodeoxycholic acid(UDCA) were synthesized into an SNP-UDCA conjugate. BF was encapsulated to prepare BF/SNP-UDCA nanoparticles(NPs). The amphiphilic compound SNP-UDCA was synthesized via the one-step method, and its structure was characterized by Fourier-transform infrared spectroscopy(FT-IR)and proton nuclear magnetic resonance(~1H-NMR). The preparation process of BF/SNP-UDCA NPs was optimized through single-factor investigations. The encapsulation efficiency and drug-loading capacity of BF/SNP-UDCA NPs were determined by high-performance liquid chromatography(HPLC). The molecular form of BF/SNP-UDCA NPs was characterized by using a transmission electron microscope, X-ray diffraction(XRD), and differential scanning calorimeter(DSC). Additionally, the stability of BF/SNP-UDCA NPs was evaluated. The release behavior of BF/SNP-UDCA NPs at different pH values was determined by dialysis. The in vitro anti-tumor effect of BF/SNP-UDCA NPs was evaluated by MTT cytotoxicity assay, flow cytometry for apoptosis, and cellular uptake. The in vitro liver targeting was evaluated by measuring cellular uptake by laser confocal microscopy. The results demonstrated that the SNP-UDCA conjugate was successfully synthesized through an esterification reaction between SNP and UDCA. The preparation process of BF/SNP-UDCA NPs was as follows: the feed ratio of SNP-UDCA to BF was 2∶1, the ultrasonic time was 30 minutes, and the stirring time was two hours. The prepared BF/SNP-UDCA NPs were spherical in shape, with a particle size of(252.74±6.05)nm, an encapsulation efficiency of 65.00%±2.51%, and a drug-loading capacity of 6.80%±0.44%. The XRD and DSC results indicated that BF was encapsulated within the NPs and existed in a molecular or amorphous state. The short-term stability of BF/SNP-UDCA NPs and stability in DMEM medium are good, and their in vitro release behavior followed the first-order equation and was pH-dependent according to the in vitro experiment. Compared with BF, BF/SNP-UDCA NPs at the same concentration showed significantly stronger cytotoxicity and apoptotic effects on HepG2 cells(P<0.05, P<0.01). The uptake of coumarin 6(C6)/SNP-UDCA NPs in HepG2 cells was time-dependent and higher than that in HeLa cells at the same concentration of C6/SNP-UDCA NPs. Moreover, after treatment with SNP, the uptake of C6/SNP-UDCA NPs in HepG2 cells decreased. In conclusion, the preparation process of BF/SNP-UDCA NPs was simple and feasible. BF/SNP-UDCA NPs could enhance the targeting ability and inhibitory effect of BF on liver cancer cells. This study will provide a foundation for liver-targeting nanoformulations of BF.
Bufanolides/pharmacology* ; Nanoparticles/chemistry* ; Humans ; Drug Carriers/chemistry* ; Ursodeoxycholic Acid/chemistry* ; Antineoplastic Agents/pharmacology* ; Polysaccharides/chemistry* ; Scrophularia/chemistry* ; Liver Neoplasms/physiopathology* ; Hep G2 Cells

OBJECTIVE: To evaluate the anti-hepatocellular carcinoma (HCC) activity of total alkaloids from Gelsemium elegans Benth. (TAG) in vivo and in vitro and to elucidate their potential mechanisms of action through transcriptomic analysis. METHODS: TAG extraction was conducted, and the primary components were quantified using high-performance liquid chromatography (HPLC). The effects of TAG (100, 150, and 200 µg/mL) on various tumor cells, including SMMC-7721, HepG2, H22, CAL27, MCF7, HT29, and HCT116, were assessed. Effects of TAG on HCC proliferation and apoptosis were detected by colony formation assays and cell stainings. Caspase-3, Bcl-2, and Bax protein levels were detected by Western blotting. In vivo, a tumor xenograft model was developed using H22 cells. Totally 40 Kunming mice were randomly assigned to model, cyclophosphamide (20 mg/kg), TAG low-dose (TAG-L, 0.5 mg/kg), and TAG high-dose (TAG-H, 1 mg/kg) groups, with 10 mice in each group. Tumor volume, body weight, and tumor weight were recorded and compared during 14-day treatment. Immune organ index were calculated. Tissue changes were oberseved by hematoxylin and eosin staining and immunohistochemistry. Additionally, transcriptomic and metabolomic analyses, as well as quatitative real-time polymerase chain reaction (RT-qPCR), were performed to detect mRNA and metabolite expressions. RESULTS: HPLC successfully identified the components of TAG extraction. Live cell imaging and analysis, along with cell viability assays, demonstrated that TAG inhibited the proliferation of SMMC-7721, HepG2, H22, CAL27, MCF7, HT29, and HCT116 cells. Colony formation assays, Hoechst 33258 staining, Rhodamine 123 staining, and Western blotting revealed that TAG not only inhibited HCC proliferation but also promoted apoptosis (P<0.05). In vivo experiments showed that TAG inhibited the growth of solid tumors in HCC in mice (P<0.05). Transcriptomic analysis and RT-qPCR indicated that the inhibition of HCC by TAG was associated with the regulation of the key gene CXCL13. CONCLUSION TAG inhibits HCC both in vivo and in vitro, with its inhibitory effect linked to the regulation of the key gene CXCL13.
Animals ; Apoptosis/drug effects* ; Liver Neoplasms/genetics* ; Carcinoma, Hepatocellular/genetics* ; Humans ; Alkaloids/therapeutic use* ; Gelsemium/chemistry* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Mice ; Xenograft Model Antitumor Assays

OBJECTIVES: To investigate the active ingredients in Hedyotis diffusa-Scutellaria barbata D. Don and the main biological processes and signaling pathways mediating their inhibitory effect on primary hepatocellular carcinoma (HCC). METHODS: The core intersecting genes of HCC and the two drugs were screened from TCMSP, Uniport, Genecards, and String databases using Cytoscape software, and GO and KEGG enrichment analyses of the intersecting genes were conducted. Molecular docking between the active ingredients of the drugs and the core genes was carried out using Pubcham, RCSB and Autoduckto to identify the active ingredients with the highest binding energy, whose inhibitory effect on HepG2 cells was verifies using CCK-8 assay, flow cytometry and Western blotting. RESULTS: TP53 and ESR1 were identified as the core genes of HCC and the two drugs. GO and KEGG analyses showed that the two genes were mainly involved in regulation of apoptotic signaling pathway, cell population proliferation, methane raft, and protein kinase activity, and participated in the signaling pathways of apoptosis, proteoglycans in cancer, PI3K Akt signaling pathway, and hepatitis B. Molecular docking studies showed that the active ingredients of the drugs could be docked with TP53 and ESR1 genes under natural conditions, and ursolic acid had the highest binding energy to ESR1 (-4.98 kcal/mol). The results of CCK-8 assay, flow cytometry and Western blotting all demonstrated significant inhibitory effect of ursolic acid on HepG2 cells. CONCLUSIONS The inhibitory effect of Hedyotis diffusa-scutellariae barbatae on HCC is mediated by multiple active ingredients in the two drugs.
Humans ; Molecular Docking Simulation ; Liver Neoplasms/drug therapy* ; Hep G2 Cells ; Network Pharmacology ; Carcinoma, Hepatocellular/drug therapy* ; Hedyotis/chemistry* ; Signal Transduction/drug effects* ; Cell Proliferation/drug effects* ; Tumor Suppressor Protein p53/metabolism* ; Apoptosis/drug effects* ; Estrogen Receptor alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology*

Curcuma kwangsiensis root tuber is a widely used genuine medicinal material in Guangxi, with the main active components of terpenoids and curcumins. It has the effects of promoting blood circulation to relieve pain, moving Qi to relieve depression, clearing heart and cooling blood, promoting gallbladder function and anti-icterus. Modern research has proved its functions in liver protection, anti-tumor, anti-oxidation, blood lipid reduction and immunosuppression. Considering the research progress of C. kwangsiensis root tubers and the core concept of quality marker(Q-marker), we predicted the Q-markers of C. kwangsiensis root tubers from plant phylogeny, chemical component specificity, traditional pharmacodynamic properties, new pharmacodynamic uses, chemical component measurability, processing methods, compatibility, and components migrating to blood. Curcumin, curcumol, curcumadiol, curcumenol, curdione, germacrone, and β-elemene may be the possible Q-markers. Based on the predicted Q-markers, the mechanisms of the liver-protecting and anti-tumor activities of C. kwangsiensis root tubers were analyzed. AKT1, IL6, EGFR, and STAT3 were identified as the key targets, and neuroactive ligand-receptor interaction signaling pathway, nitrogen metabolism pathway, cancer pathway, and hepatitis B pathway were the major involved pathways. This review provides a basis for the quality evaluation and product development of C. kwangsiensis root tubers and gives insights into the research on Chinese medicinal materials.
China ; Curcuma/chemistry* ; Humans ; Liver ; Neoplasms ; Terpenes/pharmacology*

Ultra-high-performance liquid chromatography-Q exactive orbitrap tandem mass spectrometry(UHPLC-QEOrbitrap-MS/MS) was used to explore the inhibitory effect and mechanism of ginkgo flavone aglycone(GA) combined with doxorubicin(DOX) on H22 cells. The effects of different concentrations of GA and DOX on the viability of H22 cells were investigated, and combination index(CI) was used to evaluate the effects. In the experiments, control(CON) group, DOX group, GA group, and combined GA and DOX(GDOX) group were constructed. Then the metabolomics strategy was employed to explore the metabolic markers that were significantly changed after combination therapy on the basis of single medication treatment, and by analyzing their biological significance, the effect and mechanism of the anti-tumor effect of GA combined with DOX were explained. The results revealed that when 30 μg·mL~(-1) GA and 0.5 μmol·L~(-1) DOX was determined as the co-administration concentration, the CI value was 0.808, indicating that the combination of GA and DOX had a synergistic anti-tumor effect. Metabolomics analysis identified 23 metabolic markers, including L-arginine, L-tyrosine and L-valine, mostly amino acids. Compared with the CON group, 22 and 17 metabolic markers were significantly down-regulated after DOX treatment and GA treatment, respectively. Compared with the DOX and GA groups, the treatment of GA combined with DOX further down-regulated the levels of these metabolic markers in liver cancer, which might contribute to the synergistic effect of the two. Five key metabolic pathways were found in pathway enrichment analysis, including glutathione metabolism, phenylalanine metabolism, arginine and proline metabolism, β-alanine metabolism, and valine, leucine and isoleucine degradation. These findings demonstrated that the combination of GA and DOX remarkably inhibited the viability of H22 cells and exerted a synergistic anti-tumor effect. The mechanism might be related to the influence of the energy supply of tumor cells by interfering with the metabolism of various amino acids.
Arginine/therapeutic use* ; Doxorubicin/therapeutic use* ; Flavones/therapeutic use* ; Ginkgo biloba/chemistry* ; Glutathione ; Humans ; Isoleucine/therapeutic use* ; Leucine/therapeutic use* ; Liver Neoplasms/drug therapy* ; Metabolomics/methods* ; Phenylalanine/therapeutic use* ; Proline ; Tandem Mass Spectrometry/methods* ; Tyrosine/therapeutic use* ; Valine/therapeutic use* ; beta-Alanine/therapeutic use*

This study aimed to explore the main active ingredients and potential targets of Solanum nigrum(SN), so as to reveal the potential molecular mechanism of SN in the treatment of hepatocellular carcinoma(HCC) based on network pharmacology and molecular docking. First,the main active ingredients and predictive targets of SN were collected in the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP). Then,the targets relating to HCC were collected through retrieval of integrated bio-pharmacological network database for traditional Korean medicine(PharmDB-K), oncogenomic database of hepatocellular carcinoma(OncoDB.hcc). The common targets of disease-drug component were selected through intersection between predictive targets and disease targets. Next, based on the String platform, protein-protein interaction network(PPI) model of the potential anti-HCC targets was constructed using the software Cytoscape 3.7.1. ClueGO and CluePedia APP in Cytoscape were used to analyze the gene function of SN in the treatment of HCC, and construct the main active ingredients-potential targets-signal pathways topology network of SN. Finally,DISCOVERY STUDIO software was applied in verifying the molecular docking between the key active ingredient and potential protein target. The results showed that there were 4 main active ingredients of SN, involving 22 potential targets relating to HCC and 7 signal pathways relating to potential anti-HCC targets of SN. Network analysis showed that SN may play a therapeutic role in HCC by acting on key targets, such as EGFR, TP53, MYC, CCND1 and CTNNB1. Molecular docking results showed that quercetin and EGFR could bind stably and interact through amino acid residues LEU718, LYS745 and GLN791. This study revealed the potential active ingredients and the possible molecular mechanism of SN for treatment of HCC, providing scientific basis for follow-up exploration of the molecular mechanism of SN against HCC.
Carcinoma, Hepatocellular/drug therapy* ; Drugs, Chinese Herbal/pharmacology* ; Humans ; Liver Neoplasms/drug therapy* ; Molecular Docking Simulation ; Solanum nigrum/chemistry*

OBJECTIVE: To evaluate the effects of Celastrus Orbiculatus extracts (COE) on metastasis in hypoxia-induced hepatocellular carcinoma cells (HepG2) and to explore the underlying molecular mechanisms. METHODS: The effect of COE (160, 200 and 240 µ g/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT), scratch-wound and transwell assays, respectively. CoCl was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immunofluorescence analysis, respectively. RESULTS: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner (P<0.01). Furthermore, the expression of epithelial-mesenchymal transition (EMT) related markers were also remarkably suppressed in a dose-dependent manner (P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1 α (Hif-1 α) and Twist1 were suppressed by COE. Additionally, the Hif-1 α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced HepG2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect (P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced HepG2 cells. CONCLUSIONS COE significantly inhibited the tumor metastasis and EMT by suppressing Hif-1 α/Twist1 signaling pathway in hypoxia-induced HepG2 cell. Thus, COE might have potential effect to inhibit the progression of HepG2 in the context of tumor hypoxia.
Biomarkers, Tumor ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Celastrus ; chemistry ; Cell Hypoxia ; drug effects ; Cell Proliferation ; drug effects ; Cell Shape ; drug effects ; Cobalt ; Down-Regulation ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins ; metabolism ; Plant Extracts ; pharmacology ; therapeutic use ; Signal Transduction ; drug effects

The research of anti-hepatocellular carcinoma(HCC) drug has attracted more and more attention. Natural products are the important source of active compounds for cancer treatment. A biflavonoid HIS-4 was isolated from Resina draconis in our previous study. MTT assay, hoechst staining, and flow cytometry analysis were used to investigate the effects of HIS-4 on the proliferation and apoptosis of human hepatoma HepG2 and SK-HEP-1 cells. Moreover, the effects of HIS-4 on the migration and invasion ability of HepG2 and SK-HEP-1 cells were evaluated by wound healing assay and Transwell assay. In addition, MTT assay, flow cytometry analyses, Hoechst staining, wound healing assay, Transwell assay, and tube formation assay were used to explore the anti-angiogenic activity of HIS-4 in human umbilical vein endothelial cells(HUVECs). Mechanistically, the HIS-4 regulatory of signal pathways in H9 epG2 and SK-HEP-1 cells were analyzed by Western blot. This results showed that HIS-4 suppressed the proliferation of human hepatoma HepG2 and SK-HEP-1 cells. Moreover HIS-4 induced their apoptosis of HepG2 and SK-HEP-1 cells. HIS-4 inhibited the migration and invasion of HepG2 and SK-HEP-1 cells. Additionally, HIS-4 exhibited angiogenesis effects. Mechanistically, up-regulation of MAPK signaling pathway and down-regulation of mTOR signaling pathway may be responsible for anti-hepatoma activity of HIS-4. Therefore, HIS-4 may be a promising candidate drug for HCC treatment.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; Biflavonoids ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Cell Movement ; Cell Proliferation ; Dracaena ; chemistry ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Phytochemicals ; pharmacology

MicroRNAs (miRNAs) are conserved small non-coding RNAs that play an important role in the regulation of gene expression and participate in a variety of biological processes. The biogenesis of miRNAs is tightly controlled at multiple steps, such as transcription of miRNA genes, processing by Drosha and Dicer, and transportation of precursor miRNAs (pre-miRNAs) from the nucleus to the cytoplasm by exportin-5 (XPO5). Given the critical role of nuclear export of pre-miRNAs in miRNA biogenesis, any alterations of XPO5, resulting from either genetic mutation, epigenetic change, abnormal expression level or posttranslational modification, could affect miRNA expression and thus have profound effects on tumorigenesis. Importantly, XPO5 phosphorylation by ERK kinase and its cis/trans isomerization by the prolyl isomerase Pin1 impair XPO5's nucleo-to-cytoplasmic transport ability of pre-miRNAs, leading to downregulation of mature miRNAs in hepatocellular carcinoma. In this review, we focus on how XPO5 transports pre-miRNAs in the cells and summarize the dysregulation of XPO5 in human tumors.
Carcinoma, Hepatocellular ; genetics ; metabolism ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Humans ; Karyopherins ; chemistry ; metabolism ; physiology ; Liver Neoplasms ; genetics ; metabolism ; MicroRNAs ; chemistry ; metabolism ; NIMA-Interacting Peptidylprolyl Isomerase ; Neoplasms ; genetics ; metabolism ; RNA Precursors ; chemistry ; metabolism ; RNA Transport