To enhance the penetration of P53 into tumor cells by fusion it with the cell penetrating peptide 9R. The fusion gene of 9R-p53 was cloned into the expression vector. The fusion protein, CPPs-P53, was expressed and purified. We detected the rate of cell growth inhibition and apoptosis by MTT and Annexin-V-FITC/PI double stained method respectively for measuring its effect on tumor cells. CPPs-P53 and P53 were successfully expressed and purified, the purity of both proteins reached up to 90%. MTT assay showed that the cell growth inhibition by CPPs-P53 was more efficient than P53, and the rate of cell growth inhibition is dose-dependent. The apoptosis experiment showed that P53 could induce apoptosis of tumor cells. Compared with the P53, CPPs-P53 had a more significant effect in inducing cell apoptosis (**P < 0.01). The CPPs-P53 shows more significant effects than P53 in inhibiting cell growth and inducing apoptosis on tumor cells.
Apoptosis ; Cell Line, Tumor ; Cell-Penetrating Peptides ; pharmacology ; Humans ; Tumor Suppressor Protein p53 ; pharmacology

OBJECTIVETo investigate the synergistic antitumor effects of combined use of p14ARF gene and 5-fluorouracil (5-Fu) in pancreatic cancer.

METHODSA human pancreatic cancer cell line PC-3 was transfected with lipofectin-mediated recombinant p14ARF gene, and was then administered with 5-Fu. Cell growth, morphological changes, cell cycle, apoptosis, and molecular changes were measured using the MTT assay, flow cytometry, RT-PCR, Western blotting, and immunocytochemical assays.

RESULTSAfter transfection of p14ARF, cell growth was obviously inhibited, resulting in an accumulation of cells in the G(1) phase. The proportion of cells in the G(1) phase was significantly increased from 58.51% to 75.92%, and in the S and G(2)/M phases decreased significantly from 20.05% to 12.60%, and from 21.44% to 11.48%, respectively, as compared with those of the control groups. PC-3/p14ARF cells that underwent 5-Fu treatment had significantly greater G(2)/M phase accumulation, from 11.48% to 53.47%. The apoptopic index was increased in PC-3/p14ARF cells from 3.64% to 19.62%. The MTT assay showed p14ARF-expressing cells were significantly more sensitive to 5-Fu (0.01 - 10 mg/L) than those devoid of p14ARF expression (P < 0.01). Western blotting showed p14ARF upregulates p53 expression.

CONCLUSIONCombined use of p14ARF gene and 5-Fu acts synergistically to inhibit pancreatic cancer cell proliferation, suggesting a new anticancer strategy.

Fluorouracil ; pharmacology ; Humans ; Pancreatic Neoplasms ; genetics ; therapy ; Transfection ; Tumor Cells, Cultured ; Tumor Suppressor Protein p14ARF ; genetics ; Tumor Suppressor Protein p53 ; genetics ; Up-Regulation ; physiology

OBJECTIVETo explore the effects of bears' bile on tumor cell p53 protein expression with different gene properties.

METHODSThe effects of bears' bile on the expression of p53 protein in 6 cancer cell strains were determined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis. Results Western blot analysis showed that the expression of p53 protein in HaCaT, KUMA3, KUMA4 and KUMA6 cell strains with gene mutation were increased, but no change was found in HCT116 and KUMA5 cell strains without gene mutation. There was no quantitative change in p53 mRNA in all cell strains by analysis of p53 mRNA with

CONCLUSIONThe effects of bears' bile on p53 protein expression in cancer cell strains RT-PCR analysis system. could be different based on p53 gene properties,i. e. ,bears' bile only affect p53 protein of mutation type.

Animals ; Bile ; Biological Factors ; pharmacology ; Cell Line, Tumor ; Humans ; RNA, Messenger ; metabolism ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Ursidae

OBJECTIVE: To investigate the effect of palbociclib on cell cycle progression and proliferation of human renal tubular epithelial cells. METHODS: Human renal tubular epithelial cell line HK-2 was treated with 1, 5, 10, and 20 μmol/L of palbociclib, and the changes in cell proliferation and viability were examined by cell counting and CCK8 assay. EDU staining was used to assess the proliferation of HK-2 cells following palbiciclib treatment at different concentrations for 5 days. The effect of palbociclib on cell cycle distribution of HK-2 cells was evaluated using flow cytometry. SA-β-Gal staining and C12FDG senescence staining were used to detect senescence phenotypes of HK-2 cells after palbociclib treatment at different concentrations for 5 days. The relative mRNA expression levels of P16, P21, and P53 and the genes associated with senescence-related secretion phenotypes were detected by RT-PCR, and the protein expressions of P16, P21 and P53 were detected by Western blotting. RESULTS: Palbociclib inhibited HK-2 cell proliferation and induced cell cycle arrest in G1 phase. Compared with the control cells, HK-2 cells treated with high-dose (10 μmol/L) palbociclib exhibited significantly suppressed cell proliferation activity, and the inhibitory effect was the most obvious on day 5 ( CONCLUSIONS Palbociclib induces HK-2 cell senescence by causing cell growth arrest and delaying cell cycle progression.
Cell Cycle ; Cell Cycle Checkpoints ; Cellular Senescence ; Epithelial Cells ; Humans ; Piperazines/pharmacology* ; Pyridines/pharmacology* ; Tumor Suppressor Protein p53/genetics*

OBJECTIVETo investigate the role of p53 on ran transcription in myeloma cells.

METHODSUsing real-time fluorescence quantitative PCR, the ran transcription level was measured in 8 human myeloma cell lines such as OPM-2, RPMI-8226, U-266, KAS6/1, ANML-6, H-929, MM1.S and MOLP-8. The ran transcription level and P53 expression were detected by Q-PCR in MM1.S treated with Nutlin-3a for 24, 48 and 72 hours, respectively. The Western blot was used to detect the expression levels of ran and P53 proteins, and ran expression level after transfection of MM1.S cells using different concentration of plasmids which express the P53 luciferase reporter.

RESULTSH-929 and MM1.S cells showed the highest ran transcription level among the above-mentioned 8 cell lines (P<0.05). After treatment with Nutlin-3a, ran transcription level in MM1.S cells decreased (P<0.05), (r=-1.00, P=0.04) and P53 expression increased (r=1.00, P=0.06) in time-dependence manner. The detection by p53 luciferase reporter showed that the ran transcription decreased and the plasmid increased to 25 ng (P<0.05).

CONCLUSIONThis study demonstrated that ran is a target gene regulated by P53 in myeloma cells for the first time.

Cell Line, Tumor ; Humans ; Imidazoles ; pharmacology ; Multiple Myeloma ; genetics ; metabolism ; Piperazines ; pharmacology ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; ran GTP-Binding Protein ; genetics ; metabolism

OBJECTIVES: To determine the potential molecular mechanisms underlying the therapeutic effect of curcumin on hepatocellular carcinoma (HCC) by network pharmacology and experimental in vitro validation. METHODS: The predictive targets of curcumin or HCC were collected from several databases. the identified overlapping targets were crossed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform. Two of the candidate pathways were selected to conduct an experimental verification. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay was used to determine the effect of curcumin on the viability of HepG2 and LO2 cells. The apoptosis and autophagy of HepG2 cells were respectively detected by flow cytometry and transmission electron microscopy. Besides, western blot and real-time polymerase chain reaction (PCR) were employed to verify the p53 apoptotic pathway and adenosine 5'-monophosphate (AMP)‍-activated protein kinase (AMPK) autophagy pathway. HepG2 cells were pretreated with pifithrin-‍α (PFT-‍α) and GSK690693 for further investigation. RESULTS: The 167 pathways analyzed by KEGG included apoptosis, autophagy, p53, and AMPK pathways. The GO enrichment analysis demonstrated that curcumin was involved in cellular response to drug, regulation of apoptotic pathway, and so on. The in vitro experiments also confirmed that curcumin can inhibit the growth of HepG2 cells by promoting the apoptosis of p53 pathway and autophagy through the AMPK pathway. Furthermore, the protein and messenger RNA (mRNA) of the two pathways were downregulated in the inhibitor-pretreated group compared with the experimental group. The damage-regulated autophagy modulator (DRAM) in the PFT-‍α-pretreated group was downregulated, and p62 in the GSK690693-pretreated group was upregulated. CONCLUSIONS Curcumin can treat HCC through the p53 apoptotic pathway and the AMPK/Unc-51-like kinase 1 (ULK1) autophagy pathway, in which the mutual transformation of autophagy and apoptosis may occur through DRAM and p62.
AMP-Activated Protein Kinases/pharmacology* ; Apoptosis ; Carcinoma, Hepatocellular/pathology* ; Curcumin/pharmacology* ; Humans ; Liver Neoplasms/pathology* ; Network Pharmacology ; Tumor Suppressor Protein p53/metabolism*

This study aims to investigate the mechanism of Astragali Radix-Curcumae Rhizoma(HQEZ) in the treatment of gastric cancer based on network pharmacology. Further, the SGC7901 cell model of gastric cancer was employed to validate the efficacy and key targets of the herb pair. Firstly, the CCK-8 assay was employed to evaluate the direct effect of HQEZ on the proliferation of gastric cancer SGC7901 cells. Then, network pharmacology methods were employed to investigate the active ingredients, key targets, and key signaling pathways involved in the treatment of gastric cancer with HQEZ. The results showed that HQEZ contained 18 potential active ingredients, such as quercetin, naringenin, and curcumin. The results of gene ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment suggested that the main targets of HQEZ in treating gastric cancer were involved in the regulation of protein serine/threonine kinase activity, activation of mitogen-activated protein kinase(MAPK) activity, cysteine-type endopeptidase activity, and negative regulation of protein serine/threonine kinase activity. The hypoxia-inducible factor-1(HIF-1) signaling pathway, ATP-binding cassette(ABC) transporters, cytochrome P450-mediated metabolism of xenobiotics, p53 signaling pathway, and cell apoptosis were key signaling pathways of HQEZ in treating gastric cancer. The cell experiments demonstrated that HQEZ significantly downregulated the expression of ATP-binding cassette subfamily B member 1(ABCB1), epidermal growth factor receptor(EGFR), phosphorylated serine/threonine kinase(p-AKT), hypoxia inducible factor 1 subunit alpha(HIF1A), B-cell lymphoma 2(BCL2), breast cancer susceptibility protein 1(BRCA1), DNA polymerase theta(POLH), ribonucleotide reductase M1(RRM1), and excision repair cross-complementation group 1(ERCC1), and upregulated the expression of tumor protein P53(TP53) and cysteinyl aspartate-specific proteinase(CAPS3). Finally, a multivariate COX regression model was adopted to study the relationship between gene expression and clinical information data of gastric cancer patients in the TCGA database, which demonstrated that the key targets of HQEZ were associated with the poor prognosis in gastric cancer patients. Further feature selection using the LASSO algorithm showed that EGFR, HIF1A, TP53, POLH, RRM1, and ERCC1 were closely associated with the survival of gastric can-cer patients. In conclusion, HQEZ regulates the expression of genes involved in DNA repair, survival, and apoptosis in gastric cancer cells via multiple targets and pathways, assisting the treatment of gastric cancer.
Humans ; Stomach Neoplasms/genetics* ; Tumor Suppressor Protein p53 ; Network Pharmacology ; ErbB Receptors ; Protein Serine-Threonine Kinases ; Serine ; Adenosine Triphosphate ; Molecular Docking Simulation ; Drugs, Chinese Herbal/pharmacology*

BACKGROUNDHepatitis B virus (HBV) X protein (HBx) and p53 could mutually down-regulate at transcriptional level and HBx could bind with p53 protein within its transactivation domain and inhibit the function of p53 protein. In recent years, effects of arsenic trioxide (As2O3) on the expression of p53 protein have been widely studied, while little is known about the activity of p53 protein. This study was undertaken to delineate the effect of HBV X gene and As2O3 on p53 protein expression (level and activity) in HepG2 cells by small hairpin RNA (shRNA)-mediated RNA interference (RNAi) technique.

METHODSCell line HepG2 and cells with stable expression of HBV X gene (HepG2-X) were treated with 2 micromol/L As2O3, with corresponding untreated cells serving as controls. Cell lysates and nuclear extracts were extracted. Total level and the relative activity of p53 protein were detected by modified enzyme-linked immunosorbent assay (ELISA). HBV X gene sequence-specific shRNA expression vector (pXi-1 and pXi-2) and sequence-unrelated control (pXi-3) were transfected into HepG2-X. Single cell clone with stable expression of shRNA was selected and exposed to propagating culture. The effect of As2O3 on p53 protein expression and activity was re-observed.

RESULTSTotal p53 protein level was up-regulated and its relative activity ratio was enhanced by As2O3 in HepG2 and HepG2-X cells. The total p53 protein level induced by As2O3 was up-regulated by HBV X gene expression, while its relative activity was significantly suppressed. The suppression was removed after HBV X gene expression was repressed by shRNA.

CONCLUSIONSAs2O3 up-regulates p53 protein expression and enhance its activity. HBV X up-regulates As2O3 induced-p53 protein expression while suppresses its activity.

Arsenicals ; pharmacology ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Humans ; Oxides ; pharmacology ; RNA Interference ; Trans-Activators ; genetics ; Tumor Suppressor Protein p53 ; analysis

OBJECTIVETo investigate the effects of curcumin on the acetylation of histone H3, P53 and the proliferation of NB4 cells.

METHODSThe total protein was extracted from NB4 cells treated without or with different concentrations of curcumin ( 50, 25, 12.5, 6.25 and 3.125 micromol/L) for different time (0, 4, 8, 12, 24 h). Western blot analysis was performed to determine the levels of acetylated histone H3, P53 and acetylated P53. MTT assay was performed to examine the growth inhibition effect of curcumin on NB4 cells.

RESULTSCurcumin could inhibit the proliferation of NB4 cells in a time- and dose- dependent manner, with the IC50 at 24 h and 36 h of 40 micromol/L and 25 micromol/L, respectively. The levels of histone H3 acetylation, P53 expression and P53 acetylation were increased obviously.

CONCLUSIONCurcumin functions as a deacetylase inhibitor,which could increase the level of acetylated histone H3, enhance the expression and activity of tumor suppressor P53, and inhibit the proliferation of NB4 cells.

Acetylation ; drug effects ; Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Curcumin ; pharmacology ; Histones ; metabolism ; Humans ; Tumor Suppressor Protein p53 ; metabolism

In order to investigate the role of melatonin in inhibiting the proliferation of murine gastric cancer and the underlying molecular mechanism, we performed an in vivo study by inoculating murine foregastric carcinoma (MFC) cells in mice, and then tumor-bearing mice were treated with different concentrations of melatonin (i.p.). The changes of Bcl-2, Bax, p21 and p53 expressions in tumor tissue were detected by using real-time fluorescence quantitative RT-PCR and Western blot. We found that: (1) melatonin resulted in reductions of tumor's volume and weight in the gastric cancer-bearing mice and thus showed anti-cancer effect; (2) melatonin reduced Bcl-2 expression, but increased the expression of Bax, p53 and p21 in tumor tissue. Our results suggest that melatonin could inhibit the growth of tumors in gastric cancer-bearing mice through accelerating the apoptosis of tumor cells.
Animals ; Apoptosis ; Melatonin ; pharmacology ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism