Poly Adenosine Diphosphate-ribose Polymerase Cleavage Research Articles

The FGFR2c/PKCε Axis Controls MCL-1-Mediated Invasion in Pancreatic Ductal Adenocarcinoma Cells: Perspectives for Innovative Target Therapies.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy whose main characterizations are Kirsten Rat Sarcoma-activating mutations (KRAS) and a highly aggressive phenotype. Based on our recent findings demonstrating that the highly aberrant expression of the mesenchymal isoform of Fibroblast Growth Factor Receptor 2 (FGFR2c) in PDAC cells activates Protein-Kinase C Epsilon (PKCε), which in turn controls receptor-mediated epithelial to mesenchymal transition (EMT), here we investigated the involvement of these signaling events in the establishment of additional tumorigenic features. Using PDAC cell lines expressing divergent levels of the FGFR2c and stable protein depletion approaches by short hairpin RNA (shRNA), we found that FGFR2c expression and its PKCε downstream signaling are responsible for the invasive response to Fibroblast Growth Factor 2 (FGF2) and for anchorage-independent growth. In addition, in vitro clonogenic assays, coupled with the check of the amount of cleaved Poly Adenosine Diphosphate-Ribose Polymerase 1 (PARP1) by Western blot, highlighted the involvement of both FGFR2c and PKCε in cell viability. Finally, monitoring of Myeloid Cell Leukemia 1 (MCL-1) expression and Sarcoma kinase family (SRC) phosphorylation suggested that the FGFR2c/PKCε axis could control cell migration/invasion possibly via MCL-1/SRC-mediated reorganization of the actin cytoskeleton. Being PKCs RAS-independent substrates, the identification of PKCε as a hub molecule downstream FGFR2c at the crossroad of signaling networks governing the main malignant tumor hallmarks could represent an important advance towards innovative target therapies overcoming RAS.

Remimazolam Inhibits Neuronal Apoptosis in Rat Brain via Slit Guidance Ligand 2/Roundabout 4 Signaling Pathway

To investigate the effect of remimazolam on neuronal apoptosis in rat brain through slit guidance ligand 2/roundabout 4 signaling pathway. Primary cortical neurons were obtained from specific pathogen free 3 w old male Wistar rats and sub cultured in cell culture dishes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay detected cell viability; Hochest/propidium iodide double staining and flow cytometry detected cell apoptosis. Western blot detected apoptosis-related gene expression and slit guidance ligand 2/roundabout 4 signaling pathway-related protein expression. In addition, after slit guidance ligand 2 inhibitor administration, Western blot detected the changes in expression levels of key genes in cells. Relative to control group, cell viability in each remimazolam group presented elevation and apoptosis presented depletion (p<0.05) in a dose-dependent manner. With the increase of remimazolam treatment time, Bcl-2- associated X protein, cleaved caspase-3 and cleaved poly adenosine diphosphate-ribose polymerase protein levels showed a gradual decrease and anti-apoptotic protein B-cell lymphoma 2 level showed a gradual increase relative to 0 h (p<0.05). At the same time, with the increase of remimazolam treatment time (0, 3, 6, 12 and 24 h), slit guidance ligand 2 and roundabout 4 protein levels increased continuously relative to 0 h. Additionally, relative to remimazolam group, Bcl-2-associated X protein, cleaved caspase-3 and cleaved poly adenosine diphosphate-ribose polymerase showed a gradual up regulation and B-cell lymphoma 2 showed a gradual down regulation in slit guidance ligand 2 inhibitor and remimazolam co-treatment group. Remimazolam pretreatment may inhibit rat cortical neuronal apoptosis through slit guidance ligand 2/ roundabout 4 signaling pathway and providing new ideas for clinical brain protection.

Scrophularia buergeriana Extract (Brainon) Improves Scopolamine-Induced Neuronal Impairment and Cholinergic Dysfunction in Mice through CREB-BDNF Signaling Pathway

We evaluated the effectiveness of Scrophularia buergeriana extract (Brainon) on cognitive dysfunction and determined its underlying mechanisms in a scopolamine (SCO)-treated mouse model of memory impairment. Brainon treatment for 28 days ameliorated the symptoms of memory impairment as indicated by the results of both passive avoidance performance and the Morris water mazes. Brainon lowered acetylcholinesterase activity and raised acetylcholine levels in the hippocampus. The treatment elevated the protein levels of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP response element-binding (CREB). Additionally, the excessive generation of SCO-induced reactive oxygen species (ROS) and subsequent oxidative stress were suppressed by the enhancement of superoxide dismutase (SOD)-1 and SOD-2 proteins. mRNA levels of upregulated interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, as well as the apoptotic protein Bcl-2-associated X protein (Bax), cleaved caspase-9, and cleaved poly adenosine diphosphate-ribose polymerase (PARP) expression after SCO injection were downregulated by Brainon treatment. Collectively, these findings suggested that Brainon possesses anti-amnesic effects through the CREB-BDNF pathway. Moreover, it exerted antioxidant, anti-inflammatory, and anti-apoptotic effects in SCO-induced mice exhibiting cognitive impairment and memory loss.

Erythropoietin alleviates spinal neuron apoptosis via c-Jun N-terminal kinase-c-Jun pathway

Objective To study the neural protective mechanism of erythropoietin (EPO) following root avulsion (RA). Methods Sixty-three adult Wistar rats were randomly divided into two groups: control group (avulsion + 1 ml normal saline s. c. on alternate days), EPO-treated group (avulsion + 1 000 U/kg EPO s. c. on alternate days). The rats were sacrificed at 0 (after awake), 1, 2, 4, 7 and 14 days after avulsion for harvesting C5-T1 spinal samples. Immunohistochemistry was used to detect cleaved-poly adenosine diphosphate-ribose polymerase (Cleaved-PARP), c-Jun and phosphorylated c-Jun N-terminal kinase (p-JNK) in spinal neurons. Results (1) The expression of Cleaved-PARP was significantly decreased in EPO-treated group at 2, 4, and 7 days post-avulsion (3 173.3±153.1, 8 943.2±351.5 and 7 017.9±424.7) as compared with control group (10 575.1±594.3, 12 737.1±385.3 and 10 256.9±629.4) (F=4 363.938, 1 587.489, 545.925, P<0.01). (2) The expression of c-Jun in EPO-treated group was significantly lower at 1, 2, 4 and 7 day(s) (1 351.2±253.7, 2 386.8±305.8, 1 898.2±138.0 and 1 547.1±163.9) than in control group (2 165.8±205.7, 3 378.1±272.5, 2 835.2±166.8, 2 579.8±264.8) (F=186.613, 175.719, 562.005, 329.896, P<0.01). (3) The expression of p-JNK in EPO-treated group was significantly lower at 1, 2, 4 days after avulsion (1 407.1±121.7, 1 925.6±126.6 and 4 892.3±330.2) than in control group (3 041.5±139.6, 9 243.8±573.9 and 5 486.6±358.4) (F=2 336.447, 4 651.806, 44.617, P<0.01). The peak of p-JNK was postponed to 4th day in EPO-treated group as compared with 2nd day in control group. Conclusion Down-regulation of p-JNK and c-Jun is the anti-apoptosis mechanism of EPO for spinal neurons. Key words: Root avulsion injury; Erythropoietin; Spinal cord neuron; C-poly adenosine diphosphate-ribose polymerase; C-Jun N-terminal kinase; C-Jun

Study on the neuroprotection and its molecular mechanisms of erythropoietin against brachial plexus root avulsion injury in rats

Objective The neuroprotective and adverse effects, associated with low dosage erythropoietin (EPO) for the spinal cord neuron following nerve avulsion will be researched. Methods 180 Wistar rats were randomly divided into two groups: control group (avulsion + 1 ml normal saline s. c. on alternate days), EPO treated group (avulsion + 1 000 U/kg EPO s. c. on alternate days). The rats were sacrificed at day 0 (before avulsion), 1, 2, 4, 7 and 14 d after avulsion for harvesting C5-T1 spinal samples. We used Nissl, terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL), and Cleaved-poly adenosine diphosphate-ribose polymerase (c-PARP) stainings to analyze the survival and apoptotic neurons. Results (1) The percentage of survivial neurons was significantly decreased, EPO group has more survival neurons compared with C group at 7 d [(78.42±2.64)% and (67.46±3.78)%] and 14 d [(57.32±4.07)% and (46.42±3.14)%]. (2) TUNEL positive (apoptotic) neurons appeared at 2 d, followed by a gradual change until 14 d. The TUNEL in EPO group was significantly decreased at 4 d [(303.1±59.9) and (861.6±85.4)] and 7 d [(419.8±74.2) and (983.2±83.9)] compared to C group. (3) The c-PARP, which is a critical apoptotic factor, was showed a pronounced decrease in EPO group than control ones at 2 d and 4 d. (4)There are no significant differences on blood routine examination in two groups at all time points. Conclusion With administration of 1 000 U/kg EPO, the survival rate of neurons was enhanced and apoptosis was alleviated without significant erythropothesis effect. Key words: Brachial plexus root avulsion injury; Neuron; Apoptosis; Cleaved-poly adenosine diphosphate-ribose polymerase

Ursolic Acid Induces Apoptosis in Colorectal Cancer Cells Partially via Upregulation of MicroRNA-4500 and Inhibition of JAK2/STAT3 Phosphorylation.

Though ursolic acid (UA) isolated from Oldenlandia diffusa was known to exhibit anti-cancer, anti-inflammatory, and anti-obesity effects, the underlying antitumor mechanism of ursolic acid was not fully understood to date. Thus, in the present study, the apoptotic mechanism of ursolic acid was elucidated in HCT116 and HT29 colorectal cancer cells in association with STAT3 and microRNA-4500 (miR-4500) by MTT assay, Terminal deoxynucleotidyl transferase-dT-mediated dUTP nick end labelling (TUNEL) assay, cell cycle analysis, immunofluorescence, and Western blotting. Ursolic acid significantly exerted cytotoxicity, increased TUNEL positive cells and sub-G1 apoptotic portion, induced cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) and caspase 3 in HCT116 and HT29 cells. Of note, ursolic acid attenuated the expression of anti-apoptotic proteins such as Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) and also blocked nuclear translocation of STAT3 in colorectal cancer cells. Notably, ursolic acid increased the expression level of miR-4500 in HCT116 cells by qRT-PCR analysis and conversely miR-4500 inhibitor reversed cytotoxic, anti-proliferative, and apoptotic effects by increasing TUNEL positive cells, PARP cleavage and inhibiting p-STAT3 in ursolic acid treated colorectal cancer cells. Overall, our findings provide evidence that usolic acid induces apoptosis in colorectal cancer cells partially via upregulation of miR-4500 and inhibition of STAT3 phosphorylation as a potent anti-cancer agent for colorectal cancer therapy.

Nature compound alternol preferentially induce apoptosis in prostate cancer cells

Objective In this study, we evaluated the anti-cancer effect of a newly isolated natural compound Alternol in prostate cancer cells. The pharmacological mechanism was discussed preliminarily. Methods Utilizing western blotting, the indicators of apoptosis-cysteinyl aspartate-specific protease (Caspase)-3 and poly adenosine diphosphate-ribose polymerase (PARP)-were tested after Alternol incubation with different prostate cancer cell lines and normal prostatic epithelial cell lines. And then we used flow cytometer to clear the apoptosis type and apoptosis rate. Finally, we compared the anti-cancer effect between Alternol and other and traditional chemotherapy drugs in the same drug concentration. Results Western blotting revealed that Alternol-induced cell death was an apoptotic response as evidenced by the appearance of apoptosis hallmarks such as Caspase-3 processing and PARP cleavage. Flow cytometer analyses showed Alternol induced prostate cancer cell death was a late apoptosis manner, at 12 h the PC3 group late apoptosis rate increased significantly [(14.8±1.2)%] compared with BPH1 [(6.8±1.1)%] and DU145 group [(3.9±0.8)%]. Finally, we compared the anti-cancer effect between Alternol and traditional chemotherapy drugs such as CPT-11, Docetaxel and Paclitaxel. Western blotting showed that Alternol could induce more prostate cancer cell apoptosis in the same drug concentration than and traditional chemotherapy drugs. Conclusion Alternol was capable of inducing intracellular apoptotic cell death preferentially in prostate cancer cells except DU145 cell lines and normal prostate epithelial cell lines. The apoptotic cell death was a time dependent late apoptosis. Compared with traditional chemotherapy drugs, Alternol had a better anti-cancer effect. Key words: Alternol; Prostate cancer; Apoptosis; Chemotherapy drugs

Silencing Bmi1 expression suppresses cancer stemness and enhances chemosensitivity in endometrial cancer cells.

Bmi1, a polycomb group gene, is essential for self-renewal of stem cells and is frequently upregulated in various cancer cells. We aimed to investigate the effect of Bmi1 silencing on cancer stemness and chemosensitivity in endometrial cancer using targeted siRNA approach in HEC1A and Ishikawa cells. Cell viability after treatment with Bmi1 siRNA was assessed using the MTT assay, and cell apoptosis was visualized using the TdT-mediated dUTP nick-end labeling (TUNEL) method. Western blotting, migration assays and invasion assays were performed to detect changes in the stem-like properties of cancer cells. To evaluate the anticancer effect of Bmi1 silencing, HEC1A and Ishikawa cells were treated with 100 nM Bmi1 siRNA and/or 40 μM cisplatin. In the MTT assay, compared to control, viability of HEC1A and Ishikawa cells significantly decreased after Bmi1 siRNA treatment in a dose-dependent manner. Bmi1 silencing using siRNA increased the expression of cleaved caspase-3 and cleaved poly adenosine diphosphate-ribose polymerase polymerase (PARP) as observed in the western blot analysis. Apoptosis significantly increased in the HEC1A and Ishikawa cells treated with 100 nM Bmi1 siRNA for 48 h than in the control cells in TUNEL assay. SOX2 and Oct4 expression decreased in the HEC1A and Ishikawa cells treated with Bmi1 siRNA, while E-cadherin expression increased. Further, migratory and invasive properties were significantly inhibited by Bmi1 siRNA treatment in both cell lines. Notably, viability of HEC1A and Ishikawa cells decreased more when they were concurrently treated with Bmi1 siRNA and cisplatin compared to when they were treated with Bmi1 siRNA or cisplatin alone. Bmi1 silencing suppresses cancer stemness in HEC1A and Ishikawa cells. Concurrent treatment with Bmi1 siRNA and cisplatin resulted in additive anticancer effect with a cell line-specific pattern, which was higher than that shown by cisplatin treatment alone.

Reactive oxygen species-mediated phosphorylation of p38 signaling is critically involved in apoptotic effect of Tanshinone I in colon cancer cells.

Though Tanshinone I (Tan I), a phenolic compound from Salvia miltiorrhiza, is known to have anticancer activity in several cancers, its anticancer mechanisms are not fully understood in colon cancer cells. Thus, in the present study, the underlying molecular mechanism of Tan I was explored in HCT116 and HT29 colorectal cancer cells (CRCs). Here, Tan I suppressed viability in HCT116 and HT29 cells in a time- and dose-dependent manner. Also, Tan I increased the number of terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and sub-G1 population in HCT116 and HT29 cells. Consistently, Tan I cleaved poly (adenosine diphosphate-ribose) polymerase (PARP) and caspase-8, caspase-3, attenuated the expression of Bid and activated tBid as a caspase-8 substrate and activated phosphorylation of p38 MAPK in HCT116and HT29 cells. Of note, Tan I generated reactive oxygen species (ROS) and conversely an ROS scavenger, N-acetyl-L -cysteine, reversed ROS production, PARP cleavage, caspase-3 activation, and p38 MAPK phosphorylation induced by Tan I in HCT116 cells. Furthermore, p38 MAPK inhibitor SB203580 reduced cytotoxicity, increase of TUNEL-positive cells, cleavages of PARP and caspase-3 induced by Tan I in HCT116 cells. Overall, our findings for the first time suggest that ROS-dependent activation of p38 MAPK and caspase-3 is critically involved in Tan I induced apoptosis in CRCs as a potent anticancer agent.

PEST-containing nuclear protein mediates the proliferation, migration, and invasion of human neuroblastoma cells through MAPK and PI3K/AKT/mTOR signaling pathways

BackgroundPEST-containing nuclear protein (PCNP), a novel nuclear protein, is involved in cell proliferation and tumorigenesis. However, the precise mechanism of action of PCNP in the process of tumor growth has not yet been fully elucidated.MethodsShRNA knockdown and overexpression of PCNP were performed in human neuroblastoma cells. Tumorigenic and metastatic effects of PCNP were examined by tumor growth, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo.ResultsPCNP over-expression decreased the proliferation, migration, and invasion of human neuroblastoma cells and down-regulation of PCNP showed reverse effects. PCNP over-expression increased protein expressions of cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and cleaved poly adenosine diphosphate-ribose polymerase, as well as ratios of B-cell lymphoma-2 (Bcl-2)-associated X protein/Bcl-2 and Bcl-2-associated death promoter/B-cell lymphoma-extra large in human neuroblastoma cells, however PCNP knockdown exhibited reverse trends. PCNP over-expression increased phosphorylations of extracellular signal-regulated protein kinase 1/2, p38, c-Jun N-terminal kinase, as well as decreased phosphorylations of phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR), nevertheless PCNP knockdown exhibited opposite effects. Furthermore, PCNP over-expression significantly reduced the growth of human neuroblastoma xenograft tumors by down-regulating angiogenesis, whereas PCNP knockdown markedly promoted the growth of human neuroblastoma xenograft tumors through up-regulation of angiogenesis.ConclusionsPCNP mediates the proliferation, migration, and invasion of human neuroblastoma cells through mitogen-activated protein kinase and PI3K/AKT/mTOR signaling pathways, implying that PCNP is a therapeutic target for patients with neuroblastoma.

Sevoflurane-induced memory impairment in the postnatal developing mouse brain.

The aim of the present study was to confirm that sevoflurane induces memory impairment in the postnatal developing mouse brain and determine its mechanism of action. C57BL/6 mice 7 days old were randomly assigned into a 2.6% sevoflurane (n=68), a 1.3% sevoflurane (n=68) and a control (n=38) group. Blood gas analysis was performed to evaluate hypoxia and respiratory depression during anesthesia in 78 mice. Measurements for expression of caspase-3 by immunohistochemistry, cleavage of poly adenosine diphosphate-ribose polymerase (PARP) by western blotting, as well as levels of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor type 2 (Ntrk2), pro-BDNF, p75 neurotrophin receptor (p75NTR) and protein kinase B (PKB/Akt) by enzyme-linked immunosorbent assay were performed in the hippocampus of 12 mice from each group. A total of 60 mice underwent the Morris water maze (MWM) test. Results from the MWM test indicated that the time spent in the northwest quadrant and platform site crossovers by mice in the 2.6 and 1.3% sevoflurane groups was significantly lower than that of the control group. Meanwhile, levels of caspase-3 and cleaved PARP in the 2.6 and 1.3% sevoflurane groups were significantly higher than that in the control group. Levels of pro-BDNF and p75NTR were significantly increased and the level of PKB/Akt was significantly decreased following exposure to 2.6% sevoflurane. Finally, the memory of postnatal mice was impaired by sevoflurane, this was determined using a MWM test. Therefore, the results of the current study suggest that caspase-3 induced cleavage of PARP, as well as pro-BDNF, p75NTR and PKB/Akt may be important in sevoflurane-induced memory impairment in the postnatal developing mouse brain.

Long non-coding RNA BANCR indicates poor prognosis for breast cancer and promotes cell proliferation and invasion.

We investigated the expression of human long non-coding ribonucleic acid (lncRNA), BRAF-activated non-coding RNA (BANCR) in breast cancer tissues and its effects on the in vitro proliferation, apoptosis, invasion and metastasis of breast cancer cells; also, we investigated its possible mechanism. The expressions of BANCR in 65 pairs of breast cancer tissues and para-carcinoma normal breast tissues were detected by Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). The expressions of BANCR in normal breast epithelial cells (MCF10A) and breast cancer cells (MCF-7, MDA-MB-231, SKBR3 and BT-20) were further detected. The knockdown efficiency of BANCR small interfering RNA (siRNA) in MCF-7 cells was detected by qRT-PCR. The effects of BANCR knockdown on proliferation, apoptosis, invasion and metastasis capacities of MCF-7 cells were explored via methyl thiazolyl tetrazolium (MTT) proliferation assay, cell colony formation assay, fluorescence-activated cell sorting (FACS) and transwell migration assay. Western blotting was used to detect the changes in expressions of apoptosis-related proteins, epithelial-mesenchymal transition (EMT)-related proteins and matrix metalloproteinases (MMPs) after knockdown of BANCR. The expression level of lncRNA BANCR in breast cancer tissues was significantly higher than that in para-carcinoma normal tissues. The prognosis of patients in low-expression BANCR group was significantly superior to that of patients in high-expression BANCR group. After BANCR knockdown in breast cancer MCF-7 cells, the cell proliferation and colony formation capacities were significantly inhibited. Further mechanism research showed that inhibiting BANCR could promote the apoptosis of MCF-7 cells. Results of Western blotting revealed that the expressions of B-cell lymphoma 2 associated X protein (BAX), cleaved-Caspase-3 and cleaved-poly adenosine diphosphate-ribose polymerase (PARP) in knockdown group were significantly up-regulated compared with those in control group. Both wound-healing assay and transwell migration assay showed that the down-regulation of lncRNA BANCR could inhibit the invasion and metastasis capacities of MCF-7 cells, whose mechanism was related to the inhibition of EMT process and down-regulation of MMP expressions in cells. LncRNA BANCR is highly expressed in breast cancer, which is significantly correlated with the prognosis of patients; moreover, it can promote the growth, invasion and metastasis of ovarian cancer cells. The down-regulation of BANCR can inhibit the proliferation, invasion and metastasis capacities of MCF-7 cells.

Lenalidomide improvement of cisplatin antitumor efficacy on triple-negative breast cancer cells in vitro.

Lenalidomide is an immunomodulatory drug and possesses anti-angiogenic and immunomodulatory activities against multiple myeloma. The present study assessed the in vitro effect of lenalidomide combined with cisplatin on MDA-MB-231, a triple-negative breast cancer (TNBC) cell line and explored the underlying molecular mechanism of this combination. Cell viability, apoptosis and the protein expression of phosphorylated (p) and total extracellular signal-regulated kinase (ERK), B-cell lymphoma-2 (Bcl-2), caspase-3, cleaved poly-adenosine diphosphate-ribose polymerase (cPARP), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were measured in MDA-MB-231 cells treated with different concentrations of lenalidomide, cisplatin and their combination using different biochemical assays. Lenalidomide demonstrated no significant effect on the cell viability of MDA-MB-231 cells, even at high concentrations, whereas lenalidomide in combination with cisplatin, significantly reduced cisplatin IC50 from 7.8 to 3.0 µM in MDA-MB-231 cells. In addition, lenalidomide and cisplatin in combination significantly induced cell apoptosis by 1.6- and 1.38-fold, respectively compared with lenalidomide and cisplatin alone (P<0.05). The expression levels of VEGF, bFGF and Bcl-2 proteins were significantly reduced (P<0.01), whereas caspase-3 and cleaved PARP expression were significantly increased in MDA-MB-231 cells treated with the combination compared to those treated with single agents (P<0.01). Lenalidomide treatment alone significantly reduced the p-ERK level compared with the control (P<0.05) and cisplatin treatment alone significantly increased it (P<0.01), however treatment with them in combination significantly reduced the p-ERK level in MDA-MB-231 cells compared with cisplatin treatment alone (P<0.05). In conclusion, the present study provides the basis for using lenalidomide in combination with cisplatin in TNBC therapy.

Interferon-α enhances etoposide-induced apoptosis and cell cycle arrest

Objective To study effect of interferon-α (IFN-α) on etoposide-induced cell cycle arrest and apoptosis in human osteosarcoma cells with its mechanisms. Methods Osteosacoma U2OS cells were treated with IFN-α and etoposide, alone or in combination, for 72 h. Growth inhibition was determined with trypan blue exclude test. Cell cycle arrest was evaluated with FACS. Apoptosis was detected through Hoechst33258 staining. The expression of p53 was determined with reverse transcriptase-polymerase chain reaction (RT-PCR) in mRNA level. small interfering RNA (siRNA) interference was used to silence p53. Western blotting was used to evaluate the expression of p53 and poly adenosine diphosphate-ribose polymerase (PARP). Results After treatment for 72 h, the growth inhibition was (2.29±1.03)%, (23.85±3.09)% and (50.51±3.77)% in IFN-α, Etoposide and combination group. IFN-α enhanced etoposide-induced growth inhibiton. There were statistic differences (P=0.008). There were apoptotic morphological changes in etoposide and combination groups. Cells in S phase were (19.68±3.62)% and (11.33±2.94)% in Etoposide and combination group, while G2/M cells were (65.02±2.55)% and (76.09±2.87)%. There were statistic differences (P=0.037). IFN-α did not further increase the expression of p53 in mRNA level. The inhibition rate was (55.72±3.88)%, (51.63±4.36)% and (36.40±3.95)% in blank, control-siRNA and p53-siRNA group after p53 silence. There were statistic differences (P=0.023). p53 silence largely decreased PARP cleavage in response to the combination. After pan cysteinyl aspartate-specific protease (Caspase) inhibitor Z-VAD-FMK pre-treatment, the growth inhibition rate was (3.52±1.98)%, (27.11±4.06)%, (56.44±4.78)%, (21.37±3.55)% and (23.81±4.22)% in Z-VAD-FMK, Etoposide, IFN-α+ Etoposide, etoposide+ Z-VAD-FMK and IFN-α+ Etoposide+ Z-VAD-FMK group. Z-VAD-FMK decreased growth inhibition caused by combination. There were statistic defferences (P=0.015). PARP cleavage caused by combination was also inhibited by Z-VAD-FMK. Conclusion IFN-α enhances etoposide-mediated growth inhibition, cell cycle arrest and apoptosis in human osteosarcoma U2OS cells. The growth inhibiton and apoptosis are p53-dependent and Caspase-dependent. Key words: Interferon-α; Etoposide; Osteosarcoma; Cell cycle arrest; Apoptosis

Ginsenoside Rh2 induces apoptosis and inhibits epithelial-mesenchymal transition in HEC1A and Ishikawa endometrial cancer cells

BackgroundAnticancer effect of ginsenoside Rh2 has been found in various cancer cells. However, the anticancer effect of Rh2 in endometrial cancer cells is still unclear. We aimed to determine the anticancer effect of Rh2 and elucidate its mechanism in endometrial cancer cells, using HEC1A and Ishikawa cell lines, in this study. MethodsCell proliferation was assessed by MTT assay, and cell apoptosis was visualized by TdT mediated-dUTP Nick-End Labeling (TUNEL) method. Western blot were performed to detect the expression of apoptosis and epithelial-mesenchymal transition (EMT)-related proteins. Further, cell invasion and migration assays were conducted to estimate cell migration and invasion abilities. ResultsRh2 treatment significantly suppressed cell proliferation in HEC1A and Ishikawa cells, in dose-dependent manner. Levels of cleaved poly adenosine diphosphate-ribose polymerase (PARP) and cleaved caspase-3 increased in the both cell lines with Rh2 compared with control. In Western blotting analysis after Rh2 treatment, the expression of E-cadherin increased, while the expression of EMT-related proteins including vimentin, TGF-β, and Snail markedly decreased in both cell lines. The cell invasion and migration assays results indicated that Rh2 inhibited the cell invasion and migration in HEC1A cells. ConclusionsOur findings suggested that Rh2 exerts the anticancer effect in endometrial cancer cells through the apoptosis induction and EMT inhibition.

Flavonoids isolated from Citrus platymamma induced G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells.

Citrus platymamma hort. ex Tanaka belongs to the Rutaceae family and is widely used in folk medicines in Korea due to its anti-proliferative, anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities. However, the molecular mechanism of its anti-cancer effect is not well understood. The present study was conducted to elucidate the anti-cancer effect and molecular mechanism of flavonoids from Citrus platymamma (FCP) on A549 cells. FCP displayed concentration-dependent inhibition on A549 cells proliferation. Further, flow cytometry revealed that FCP significantly increased the sub-G1 (apoptotic cell population) and G2/M phase population, and the total number of apoptotic cells, in a dose-dependent manner. Nuclear condensation and fragmentation were also observed upon staining with Hoechst 33342 in FCP-treated A549 cells. Immunoblotting demonstrated a dose-dependent downregulation of cyclin B1, cyclin-dependent kinase 1, cell division cycle 25c, pro-caspases −3, −6, −8 and −9, and poly (adenosine diphosphate-ribose) polymerase (PARP) in FCP-treated A549 cells. In addition, FCP induced caspase-3 activation and subsequent PARP cleavage, and increased the B-cell lymphoma (Bcl)-2-associated X protein/Bcl-extra large ratio in A549 cells. These findings suggest that FCP induced G2/M arrest and apoptosis of A549 cells. The present study provides evidence that FCP may be useful in the treatment of human lung cancer.

Pelargonium quercetorum Agnew induces apoptosis without PARP or cytokeratin 18 cleavage in non-small cell lung cancer cell lines.

Pelargonium species have various uses in folk medicine as traditional remedies, and several of them have been screened for their biological activity, including anticancer. Pelargonium quercetorum Agnew (P. quercetorum) is traditionally used for its anthelminthic activity. However, little is known about its biological activity or its effect on cancer cells. The aim of the present study was to determine the cytotoxic activity of P. quercetorum extract on lung cancer cell lines with varying properties. Following the analyses of its chemical composition, the cytotoxic activity was screened by the adenosine triphosphate viability test. M30-Apoptosense® and M65 EpiDeath® enzyme-linked immunosorbent assays were used to determine the cell death mode (apoptosis vs. necrosis). For apoptosis, additional methods, including Annexin-V-fluorescein isothiocyanate (FITC) and Hoechst 33342 staining, were employed. The cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) was assayed by western blotting to further dissect the apoptosis mechanism. The methanol extract of P. quercetorum caused cytotoxic activity in a dose-dependent manner. The mode of cell death was apoptosis, as evidenced by the positive staining of the cells for Annexin-V-FITC and the presence of pyknotic nuclei. Notably, neither PARP cleavage nor cytokeratin 18 fragmentation were observed. P.quercetorum caused cell death by an apoptosis mechanism that is slightly different from classical apoptosis. Therefore, future in vivo experiments are required for further understanding of the effect of this plant on cancer cells.

Honey and its Phytochemicals: Plausible Agents in Combating Colon Cancer through its Diversified Actions

Cancer is one of the leading causes of death worldwide. Among them, colorectal cancer is linked with diet as the epithelial cells of colon and rectum come in direct contact with diet. Diets rich in phenolic constituents are associated in eradicating various noncommunicable diseases including cancer. This work illustrates antiproliferative effects of honey and its phytochemicals against colorectal cancer to limelight. Honey and its phytochemicals are found to inhibit the cancer growth. Changes like activation of caspase-3, caspase-8, polyadenosine diphosphate-ribose polymerase cleavage, boost in ROS level activating mitochondrial pathway, lipid layer breakage, DNA fragmentation, increase of G0/G1 phase cells, up-regulation of Bax regulators and p53 dependent apoptosis are significant. It is high time for scientists to initiate more trails using honey and its phytochemicals against some immune-deficient animal models of colon cancer and in low- and high-risk human individuals to validate honey as a mighty sword against colon cancer. This review promulgates honey and its phytochemicals as candidates in colon cancer prevention. Practical Application: Colon cancer is the third largest cause of cancer death worldwide. The standard chemotherapeutic agents are usually noneffective in the latter stages of cancer. As colorectal cancer is more prone to the diet consumed, scientists have explored many dietary compounds to treat this particular type of cancer. In our review, we highlight the effect of honey and its phenolic constituents against the colon cancer cells. Various, in vitro experiments done are enlisted. This review also emphasis the need of numerous studies that are needed to be initiated to explore the anticancer effect of crude honey specifically. As honey is one of the ingredients used in daily life, honey can also be used in chemoprevention against colon cancer.

Plumbagin-silver nanoparticle formulations enhance the cellular uptake of plumbagin and its antiproliferative activities.

Colloidal silver nanoparticles (AgNPs) have attracted much attention in recent years as diagnostics and new drug delivery system in cancer medicine. To study the effects of plumbagin (PLB), a relatively non-toxic napthaquinone isolated from the roots of Plumbago indica in human cervical cancer cell line and developed a formulation to enhance its cytotoxic activities. Silver nanoparticles were synthesised by chemical reduction method and complexed with PLB. Both the AgNPs and the complex PLB-AgNPs were characterised by dynamic light scattering, high-resolution scanning electron microscopy and transmission electron microscopy. The amount of PLB and PLB-AgNPs internalised was determined by ultra-violet-visible spectrophotometer. Cell inhibition was determined by sulphorhodamine B assay. Mitotic index was determined by Wright-Giemsa staining. Apoptosis induction was assessed by western blot using cleaved poly adenosine diphosphate-ribose polymerase antibody. The scanning electron microscope analysis indicated an average particle size of 32±8 nm in diameter. Enhanced internalisation of PLB into the HeLa cells was observed in PLB-AgNPs. PLB inhibited proliferation of cells with IC50 value of about 18±0.6 µM and blocked the cells at mitosis in a concentration-dependent manner. PLB also inhibited the post-drug exposure clonogenic survival of cells and induced apoptosis. The antiproliferative, antimitotic and apoptotic activities were also found to be increased when cells were treated with PLB-AgNPs. The authors results support the idea that AgNP could be a promising and effective drug delivery system for enhanced activity of PLB in cancer treatment.

Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways

Cholangiocarcinoma (CCA) is a tumor with poor prognosis that is resistant to all currently available treatments. Whether curcumin, a nutraceutical derived from turmeric (Curcuma longa), has potential therapeutic activity against human CCA was investigated using three CCA cell lines (KKU100, KKU-M156 and KKU-M213). Examination of mitochondrial dehydrogenase activity, phosphatidylserine externalization, esterase staining, caspase activation and poly-adenosine diphosphate ribose polymerase cleavage demonstrated that curcumin inhibited proliferation of and induced apoptosis in these biliary cancer cells. Colony-formation assay confirmed the growth-inhibitory effect of curcumin on CCA cells. When examined for the mechanism, curcumin was found to activate multiple cell signaling pathways in these cells. First, all CCA cells exhibited constitutively active nuclear factor (NF)-κB, and treatment with curcumin abolished this activation as indicated by DNA binding, nuclear translocation and p65 phosphorylation. Second, curcumin suppressed activation of signal transducer and activator of transcription-3 as indicated by decreased phosphorylation at both tyrosine(705) and serine(727) and inhibition of janus kinase-1 phosphorylation. Third, curcumin induced expression of peroxisome proliferator-activated receptor gamma. Fourth, curcumin upregulated death receptors, DR4 and DR5. Fifth, curcumin suppressed the Akt activation pathway. Sixth, curcumin inhibited expression of cell survival proteins such as B-cell lymphoma-2, B-cell leukemia protein xL, X-linked inhibitor of apoptosis protein, c-FLIP, cellular inhibitor of apoptosis protein (cIAP)-1, cIAP-2 and survivin and proteins linked to cell proliferation, such as cyclin D1 and c-Myc. Seventh, the growth inhibitory effect of curcumin was enhanced in the IκB kinase-deficient cells, the enzyme required for nuclear factor-kappaB activation. Overall, our results indicate that curcumin mediates its antiproliferative and apoptotic effects through activation of multiple cell signaling pathways, and thus, its activity against CCA should be further investigated.