Developing and identifying effective medications and targets for treating hepatic fibrosis is an urgent priority. Our previous research demonstrated the efficacy of artesunate (ART) in alleviating liver fibrosis by eliminating activated hepatic stellate cells (HSCs). However, the underlying mechanism remains unclear despite these findings. Notably, endocytic adaptor protein (NUMB) has significant implications for treating hepatic diseases, but current research primarily focuses on liver regeneration and hepatocellular carcinoma. The precise function of NUMB in liver fibrosis, particularly its ability to regulate HSCs, requires further investigation. This study aims to elucidate the role of NUMB in the anti-hepatic fibrosis action of ART in HSCs. We observed that the expression level of NUMB significantly decreased in activated HSCs compared to quiescent HSCs, exhibiting a negative correlation with the progression of liver fibrosis. Additionally, ART induced senescence in activated HSCs through the NUMB/P53 tumor suppressor (P53) axis. We identified NUMB as a crucial regulator of senescence in activated HSCs and as a mediator of ART in determining cell fate. This research examines the specific target of ART in eliminating activated HSCs, providing both theoretical and experimental evidence for the treatment of liver fibrosis.
Hepatic Stellate Cells/cytology* ; Liver Cirrhosis/genetics* ; Artesunate/pharmacology* ; Cellular Senescence/drug effects* ; Membrane Proteins/genetics* ; Animals ; Humans ; Nerve Tissue Proteins/genetics* ; Tumor Suppressor Protein p53/genetics* ; Male ; Mice

Cancer represents a significant disease that profoundly impacts human health and longevity. Projections indicate a 47% increase in the global cancer burden by 2040 compared to 2020, accompanied by a further rise in the associated economic burden. Consequently, there is an urgent need to discover and develop new alternative drugs to mitigate the global impact of cancer. Natural products (NPs) play a crucial role in the identification and development of anticancer therapeutics. This study identified ustusolate E (UE) and its analog 11α-hydroxy-ustusolate E (HUE) from strain Aspergilluscalidoustus TJ403-EL05, and examined their antitumor activities and mechanisms of action. The findings demonstrate that both compounds significantly inhibited the proliferation and colony formation of AGS (human gastric cancer cells) and 786-O (human renal clear cell carcinoma cells), induced irreversible DNA damage, blocked the cell cycle at the G₂/M phase, and further induced apoptosis in tumor cells. To the best of the authors' knowledge, this is the first report on the anticancer effects of UE and HUE and their underlying mechanisms. The present study suggests that HUE and UE could serve as lead compounds for the development of novel anticancer drugs.
Humans ; Apoptosis/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Cell Line, Tumor ; Phosphatidylinositol 3-Kinases/genetics* ; Signal Transduction/drug effects* ; Tumor Suppressor Protein p53/genetics* ; Cell Proliferation/drug effects* ; Antineoplastic Agents/pharmacology* ; Sesquiterpenes/pharmacology* ; Aspergillus/chemistry*

This study aimed to explore the mechanism of Zhongfeng Xingnao Decoction(ZFXN) in intervening microcirculatory di-sorders in cerebral hemorrhage by network pharmacology and molecular docking techniques. The information on the components of ZFXN was obtained through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) database, and the predicted targets of chemical components were obtained from PubChem and SwissTargetPrediction. The relevant targets of cerebral hemorrhage and microcirculatory disorders were collected from the GeneCards database, and the common targets of the components and diseases were analyzed by the Database for Annotation, Visualization, and Integrated Discovery(DAVID) for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses. Visualization of the correlation network was carried out using Cytoscape software to further screen important chemical components for molecular docking prediction with disease targets. The animal experiment validation was performed using modified neurological severity score(mNSS), enzyme-linked immunosorbent assay(ELISA), quantitative real-time polymerase chain reaction(qRT-PCR), immunofluorescence, and Western blot to detect the effects of ZFXN intervention in mice with cerebral hemorrhage. The results showed that there were 31 chemical components and 856 targets in the four drugs contained in ZFXN, 173 targets for microcirculatory disorders in cerebral hemorrhage, and 57 common targets for diseases and components. The enrichment analysis showed that common targets were mainly involved in biological processes, such as cell proliferation and apoptosis, and signaling pathways, such as tumor pathway, viral infection, phosphoinositide-3-kinase/protein kinase B(PI3K/AKT) signaling pathway, and mitogen-activated protein kinase(MAPK) signaling pathway. Molecular docking results revealed that the common components β-sitosterol of Rhei Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Ginseng Radix et Rhizoma Rubra showed good docking with proto-oncogene tyrosine-protein kinase(SRC), signal transducer and activator of transcription 3(STAT3), phosphoinositide-3-kinase catalytic alpha polypeptide gene(PIK3CA), recombinant protein tyrosine phosphatase non receptor type 11(PTPN11), AKT1, epidermal growth factor receptor(EGFR), calcium adhesion-associated protein beta 1(CTNNB1), vascular endothelial growth factor A(VEGFA), and tumor protein p53(TP53). Moreover, sennoside E of Rhei Radix et Rhizoma showed good docking with MAPK1. The results revealed that the ZFXN relieved the neural injury in mice with cerebral hemorrhage, decreased the expression of S100 calcium-binding protein B(S100β), neuron specific enolase(NSE), matrix metalloproteinase 9(MMP9), tumor necrosis factor α(TNF-α), interleukin 1β(IL-1β), SRC, EGFR, CTNNB1, VEGFA, TP53, glial fibrillary acidic protein(GFAP), and leukocyte differentiation antigen 86(CD86), and increased the expression of p-PI3K, p-AKT, and zona occludens 1(ZO-1). The results indicate that ZFXN may inhibit neuronal apoptosis and inflammatory response through PI3K/AKT/p53 pathway to protect the blood-brain barrier, thereby slowing down microcirculatory impairment in cerebral hemorrhage.
Animals ; Mice ; Tumor Suppressor Protein p53 ; Proto-Oncogene Proteins c-akt ; Molecular Docking Simulation ; Network Pharmacology ; Vascular Endothelial Growth Factor A ; Microcirculation ; Phosphatidylinositol 3-Kinases/genetics* ; Tumor Necrosis Factor-alpha ; ErbB Receptors ; Cerebral Hemorrhage/drug therapy* ; Neoplasms ; Phosphatidylinositols ; Drugs, Chinese Herbal/pharmacology*

Platycodon grandiflorum (Jacq.) A. DC. is a famous medicinal plant commonly used in East Asia. Triterpene saponins isolated from P. grandiflorum are the main biologically active compounds, among which polygalacin D (PGD) has been reported to be an anti-tumor agent. However, its anti-tumor mechanism against hepatocellular carcinoma is unknown. This study aimed to explore the inhibitory effect of PGD in hepatocellular carcinoma cells and related mechanisms of action. We found that PGD exerted significant inhibitory effect on hepatocellular carcinoma cells through apoptosis and autophagy. Analysis of the expression of apoptosis-related proteins and autophagy-related proteins revealed that this phenomenon was attributed to the mitochondrial apoptosis and mitophagy pathways. Subsequently, using specific inhibitors, we found that apoptosis and autophagy had mutually reinforcing effects. In addition, further analysis of autophagy showed that PGD induced mitophagy by increasing BCL2 interacting protein 3 like (BNIP3L) levels.In vivo experiments demonstrated that PGD significantly inhibited tumor growth and increased the levels of apoptosis and autophagy in tumors. Overall, our findings showed that PGD induced cell death of hepatocellular carcinoma cells primarily through mitochondrial apoptosis and mitophagy pathways. Therefore, PGD can be used as an apoptosis and autophagy agonist in the research and development of antitumor agents.
Humans ; Mitophagy ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Cell Line ; Autophagy ; Apoptosis ; Membrane Proteins ; Proto-Oncogene Proteins/genetics* ; Tumor Suppressor Proteins/pharmacology*

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.
Humans ; Doublecortin-Like Kinases ; Hippo Signaling Pathway ; Verteporfin/pharmacology* ; Cell Line, Tumor ; Protein Serine-Threonine Kinases/metabolism* ; Pancreatic Neoplasms/pathology* ; YAP-Signaling Proteins ; Transcription Factors/metabolism* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Tumor Suppressor Proteins/genetics*

Lnc-HUR1 is an HBV-related long non-coding RNA, which can promote the proliferation of hepatoma cells and the occurrence and development of liver cancer. In this study we explored the effect of lnc-HUR1 on the apoptosis of hepatocellular carcinoma cells by taking the approach of immunoblotting, quantitative real time PCR, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and flow cytometry. We found that overexpression of lnc-HUR1 significantly reduced the activity of caspase3/7 and the cleavage of PARP-1, while knocking down of lnc-HUR1 significantly increased the activity of caspase3/7 and promoted the cleavage of PARP-1 in HepG2 cells treated with TGF-β, pentafluorouracil or staurosporine. Consistently, the data from Annexin-V/PI staining showed that overexpression of lnc-HUR1 inhibited apoptosis, while knockdown of lnc-HUR1 promoted apoptosis. Moreover, overexpression of lnc-HUR1 up-regulated the apoptosis inhibitor Bcl-2 and down-regulated the pro-apoptotic factor BAX at both RNA and protein levels. In the CCL4-induced acute liver injury mice model, the expression of Bcl-2 in the liver tissue of lnc-HUR1 transgenic mice was higher than that of the control mice. The data from ChIP assay indicated that lnc-HUR1 reduced the enrichment of p53 on Bcl-2 and BAX promoters. All these results indicated that lnc-HUR1 inhibited the apoptosis by promoting the expression of apoptosis inhibitor Bcl-2 and inhibiting the expression of apoptosis promoting factor BAX. Further studies showed that lnc-HUR1 regulated the transcription of Bcl-2 and BAX in HCT116 cells, but had no effect on the expression of Bcl-2 and BAX in HCT116 p53^-/- cells, indicating that lnc-HUR1 regulates the transcription of Bcl-2 and BAX dependent upon the activity of p53. In conclusion, HBV upregulated lnc-HUR1 can inhibit the apoptosis of hepatoma cells. Lnc-HUR1 inhibits apoptosis by inhibiting the transcriptional activity of p53. These results suggest that lnc-HUR1 plays an important role in the occurrence and development of HBV-related hepatocellular carcinoma.
Animals ; Annexins/pharmacology* ; Apoptosis ; Carcinoma, Hepatocellular/genetics* ; Cell Proliferation ; Hep G2 Cells ; Hepatitis B virus/metabolism* ; Humans ; Liver Neoplasms/genetics* ; Mice ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology* ; Proto-Oncogene Proteins c-bcl-2/pharmacology* ; RNA, Long Noncoding/metabolism* ; Staurosporine/pharmacology* ; Transforming Growth Factor beta/pharmacology* ; Tumor Suppressor Protein p53/pharmacology* ; bcl-2-Associated X Protein/pharmacology*

OBJECTIVE: To investigate the effect of SPARC gene overexpression on the chemotherapeutic sensitivity of AML-MDS cell line SKM-1 to Ara-C and to further explore its mechanism. METHODS: Subjects were divided into 6 groups: SKM-1 cells (Control), Negative control (LV-NC), SPARC overexpression (LV-SPARC), SKM-1 cells+30 ng/ml Ara-C (30 ng/ml Ara-C), LV-NC+30 ng/ml Ara-C and LV-SPARC+30 ng/ml Ara-C. Cell activity was detected by CCK-8 assay, cell cycle distribution and apoptosis were detected by flow cytometry, mRNA expression levels of SPARC, CPBP and MLKL were detected by RT-qPCR, and the expression levels of related protein were detected by Western blot. RESULTS: After co-treatment with SPARC overexpression and Ara-C, the cell viability decreased and apoptosis increased significantly, with obvious up-regulation of Bax and down-regulation of BCL-2 (P<0.05). Compared with the control group, the cell cycle of LV-SPARC+30 ng/ml Ara-C group was significantly arrested in S phase with obvious down-regulation of CDK2 and up-regulation of p27^KIP1 (P<0.05). Compared with LV-SPARC group and 30 ng/ml Ara-C group, the mRNA and protein expression levels of CPBP and MLKL (p-MLKL) were significantly elevated in LV-SPARC+30 ng/ml Ara-C group (P<0.05). In addition, after co-treatment with SPARC overexpression and Ara-C, the protein expression level of p-AKT decreased and the protein expression level of p53 increased (P<0.05). CONCLUSION SPARC overexpression enhanced the sensitivity of SKM-1 cells to Ara-C and promoted cell cycle arrest and apoptosis, the mechanism of which may be related to the regulation of CPBP/MLKL pathway.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cytarabine ; Humans ; Kruppel-Like Factor 6/metabolism* ; Osteonectin/pharmacology* ; Protein Kinases/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger ; Sincalide/pharmacology* ; Tumor Suppressor Protein p53 ; bcl-2-Associated X Protein/pharmacology*

OBJECTIVE: To investigate the expression of cytokine signal suppressor 3 (SOCS3) in colon cancer tissue and the mechanism by which SOCS3 regulates the proliferation and invasion of colon cancer. METHODS: We collected the specimens of tumor tissues and paired adjacent tissues from 80 patients with colon cancer undergoing radical resection in our hospital between July, 2014 and May, 2017, and the expression of SOCS3 in the tissue samples was analyzed using Western blotting. We also transfected colon cancer cell line SW480 with a SOCS3-overexpressing plasmid or a small interference RNA (siRNA) for SOCS3 knockdown, and the changes in the cell proliferation and invasion capacity were evaluated using CCK-8 assay and Transwell assay, respectively. The effect of demethylation and IL-6 treatment on SOCS3 expression and the proliferation and invasion of SW480 cells were observed. RESULTS: Colon cancer tissues showed a lowered expression of SOCS3 compared with the adjacent tissues. Over-expression of SOCS3 significantly inhibited while SOCS3 knockdown obviously promoted the proliferation and invasion of SW480 cells . Demethylation treatment up-regulated SOCS3 expression and inhibited the proliferation and invasion capacity of SW480 cells; IL-6 treatment of the cells caused the reverse changes. CONCLUSIONS SOCS3 participates in the development and progression of colon cancer and serves as a potential target for colon cancer treatment. In patients with colon cancer, the low expression of SOCS3 possibly as a result of methylation may promote the proliferation and invasion of the cancer cells.
Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; etiology ; pathology ; Cytokines ; Demethylation ; Disease Progression ; Humans ; Interleukin-6 ; pharmacology ; Neoplasm Invasiveness ; Neoplasm Proteins ; metabolism ; RNA, Small Interfering ; Signal Transduction ; Suppressor of Cytokine Signaling 3 Protein ; genetics ; metabolism ; Transfection

OBJECTIVE: To investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. METHODS: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. RESULTS: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL (P<0.05). CONCLUSION The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Hepatic Stellate Cells ; drug effects ; physiology ; Humans ; Injections ; Liver Cirrhosis ; drug therapy ; Tumor Suppressor Proteins ; genetics

OBJECTIVETo investigate the effect of oridonin on the human acute lymphocytic leukemia cell line Jurkat and its mechanism.

METHODSJurkat cells were cultured in vitro and treated with various concentrations (0, 1.25, 2.5, 5, and 10 μmol/L) of oridonin for different lengths of time (24, 48, and 72 hours). The proliferation of Jurkat cells was analyzed by MTT assay. The changes in nuclear morphology were evaluated by fluorescence microscopy at 12 hours after treatment with various concentrations of oridonin. The expression levels of Brg1, P53, and C-myc were determined by semi-quantitative Western blot in Jurkat cells treated with various concentrations of oridonin for 24 hours or 5 μmol/L oridonin for various lengths of time (0, 2, 6, 12, and 24 hours). The expression levels of P53 and C-myc and proliferation of Jurkat cells were evaluated after Brg1 expression was knocked down by Brg1-specific siRNA.

RESULTSCompared with the control group, the proliferation of oridonin-treated Jurkat cells was significantly inhibited in a concentration- and time-dependent manner (P<0.05). According to the florescence microscopic analysis, oridonin treatment led to nuclear pyknosis in Jurkat cells. Compared with the control group, Jurkat cells treated with 5 μmol/L oridonin had reduced expression of Brg1 and C-myc but elevated expression of P53. Brg1 knock-down led to a significant reduction in proliferation of Jurkat cells (P<0.05), up-regulated expression of P53, and down-regulated expression of C-myc.

CONCLUSIONSOridonin can inhibit the proliferation of Jurkat cells, probably via the Brg1 signaling pathway.

Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Proliferation ; drug effects ; DNA Helicases ; analysis ; physiology ; Diterpenes, Kaurane ; pharmacology ; Dose-Response Relationship, Drug ; Down-Regulation ; Humans ; Jurkat Cells ; Nuclear Proteins ; analysis ; physiology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; Proto-Oncogene Proteins c-myc ; analysis ; Signal Transduction ; physiology ; Transcription Factors ; analysis ; physiology ; Tumor Suppressor Protein p53 ; analysis