PC12 Apoptosis Research Articles

KUD773, a phenylthiazole derivative, displays anticancer activity in human hormone-refractory prostate cancers through inhibition of tubulin polymerization and anti-Aurora A activity

BackgroundHormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.ResultsKUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr161 (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr15 (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.ConclusionsThe data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs.

SAHA triggered MET activation contributes to SAHA tolerance in solid cancer cells

Although SAHA is approved for the treatment of cutaneous T-cell lymphoma by the U.S. Food and Drug Administration, clinical trials using SAHA as a monotherapy or in combination with other chemotherapeutic agents in solid tumors have not met with success, and the mechanisms of tolerance remain unknown. In this study, using the prostate cancer cell line PC3 and the non-small lung cancer cell line A549, which have limited sensitivity to SAHA, we found that SAHA triggered MET and AKT phosphorylation at clinical concentrations. siRNA silencing of MET enhanced SAHA induced apoptosis in PC3 and A549 cells. However, MET protein expression and HGF secretion were not affected by SAHA, suggesting that the SAHA-induced MET activation was not due to MET over-expression or HGF paracrine secretion. However, mRNA and protein expression of the laminin receptor integrin α5β1 was up-regulated by SAHA prior to MET activation. Silencing of integrin α5β1 abolished SAHA-triggered MET phosphorylation, suggesting the involvement of integrin α5β1 in MET activation. Further, the combination of SAHA and XL184 resulted in a synergistic induction of cancer cell apoptosis and a synergistic inhibition of tumor growth. These data indicate that SAHA triggered MET activation in an HGF independent manner. This effect is partially involved in the resistance to SAHA in solid cancers, warranting further clinical investigation into combining SAHA with MET inhibitors in solid cancer treatment.

Chloride intracellular channel 1 regulates prostate cancer cell proliferation and migration through the MAPK/ERK pathway.

Abstract Aims: To investigate the effect of chloride intracellular channel 1 (CLIC1) on the proliferation, migration, and apoptosis of prostate cancer cell lines PC-3 and DU145 and the possible molecular mechanisms. Using the technique of RNA interference, the expression of CLIC1 was downregulated in the PC-3 and DU145 cell lines. MTT assay, Transwell chamber, and flow cytometry were used to determine the effect of CLIC1 on the proliferation, migration, and apoptosis ability of PC-3 and DU145 cells. The levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), ERK1/2, matrix metalloproteinase (MMP)-2, and MMP-9 were examined by western blotting. The results showed that the knockdown of CLIC1 exerts inhibitory effects on the proliferation and migration of PC-3 and DU145 cells. At the same time, the authors found that the knockdown of CLIC1 has no effect on the apoptosis in PC-3 and DU145 cells. Meanwhile, the levels of p-ERK1/2, MMP-2, and MMP-9 were decreased in the CLIC1 small interfering RNA (siRNA) group compared with the control and vector groups. These results indicate that CLIC1 could regulate prostate cancer cell proliferation and migration by regulating the mitogen-activated protein kinase (MAPK)/ERK pathway and offers a candidate molecular target for prostate cancer prevention and therapy.

Marked improvement of neurocognitive impairment after treatment with compounds from Hericium erinaceum: A case study of recurrent depressive disorder

Hericium erinaceus (Bulliard: Fries) Persoon (HE), synonymously called Monkey Head mushroom or Yamabushitake, contains a huge amount of nutrients, in particular, the erinacines. This study was carried out with the aim to uncover whether erinacines can play a role as a neuroprotective adjuvant and what the related mechanism of action could be. A novel medium was developed to significantly enhance the productivity of biomass as well as erinacines to reach 16.8 g/L and 225 ± 54 mg/L, respectively. The ethanolic extract of HE (HEE) was tested on PC-12 cell line for its protective effect against the 20 mM glutamate-insult (GluI). The parameters affected were (control vs. GluI): glutathione (8.9 ± 0.8 vs. 2.5 ± 0.7 nmol/mg protein); glutathione peroxidase (11.6 ± 1.6 vs. 28.2 ± 3.2 mU/mg protein); glutathione reductase (9.2 ± 0.8 vs. 2.3 ± 0.4 mU/mg-protein); Ca2+-influx (115 ± 16 vs. 360 ± 23 nmol/L); reactive oxygen species (100 ± 3 vs. 140 ± 7%); superoxide dismutase (56.5 ± 6.3 vs. 23.2 ± 4.2 U/mg-protein); H2O2 (8.2 ± 0.7 vs. 20.4 ± 3.5 nmol/mg-protein); and TBARs (MDA) (8.1 ± 0.3 vs. 13.3 ± 2.5 μmol/μg-protein) (p < 0.05). HEE effectively suppressed the ratio Bax/Bcl-2 and caspase-3, resulting in promising inhibition on PC-12 cell apoptosis induced by Glu-insult. Conclusively, the novel medium enhances both the productivity of biomass and erinacine A and its extract can effectively prevent PC-12 apoptosis via the ROS-caspase dependent pathway.

Inhibition of growth and induction of apoptosis in PC3 and A549 cell lines by hydro alcoholic fruit extract of Morinda tinctoria roxb: in vitro analysis

The present study aimed to investigate the effect of hydroalcoholic extract of Morinda tinctoria Roxb fruit on inhibition of growth and induction of apoptosis in human prostate carcinoma cells (PC-3) and lung carcinoma (A549) cells. Morinda tinctoria Roxb hydro alcoholic fruit extract (MTHFE) was taken to in vitro studies, like phytochemical screening, MTT assay, cell induced apoptosis. Dose dependant cytotoxic activities were exhibited by PC-3 cell lines and A549 cells. The result showed that, as the dose of the extract increased, the no of viable cells were found to be decreased, which confirms the anti-cancer and cytotoxic activities of the fruit extract.

A universal gene carrier platform for treatment of human prostatic carcinoma by p53 transfection

Our previous work showed that a charge-reversal layer-by-layer nanosystem, PEI/PAH-Cit/AuNP-CS, effectively facilitates cellular uptake of siRNA and enhances the silencing efficacy of MDR1 siRNA. Here, the plasmid loading capacity of this vehicle was examined using EGFP-N1, and the plasmid release profile was determined in response to pH changes. The cytotoxicity of the EGFP-N1/PEI/PAH-Cit/AuNP-CS complex against HeLa and 293T cells was almost negligible. PEI/PAH-Cit/AuNP-CS efficaciously delivered the plasmids EGFP-N1 (encoding green fluorescent protein) and pGL3.0 (encoding luciferase) into 293T and HeLa cells, thus verifying the universality of PEI/PAH-Cit/AuNP-CS as a gene carrier. The results of an inverted fluorescence microscopy, flow cytometry (FCM) and western blotting methods demonstrated that PC-3 prostate cancer cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS expressed higher levels of GFP than cells treated with EGFP-p53/PEI. Furthermore, PC-3 cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS showed reduced cellular viability and increased nuclear fragmentation, consistent with elevated p53 expression. Propidium iodide (PI) flow cytometric assays were conducted to demonstrate that EGFP-p53/PEI/PAH-Cit/AuNP-CS elevated the level of apoptosis in PC-3 cells. Western blotting and caspase activation studies revealed that EGFP-p53/PEI/PAH-Cit/AuNP-CS complexes may induce PC-3 apoptosis via the mitochondria-mediated signaling pathway by up-regulation of Bax, down-regulation of Bcl-2, and activation of caspase-3.

Role of Notch-1 signaling pathway in PC12 cell apoptosis induced by amyloid beta-peptide (25-35).

Recent studies have demonstrated that Notch-1 expression is increased in the hippocampus of Alzheimer's disease patients. We speculate that Notch-1 signaling may be involved in PC12 cell apoptosis induced by amyloid beta-peptide (25–35) (Aβ25–35). In the present study, PC12 cells were cultured with different doses (0, 0.1, 1.0, 10 and 100 nmol/L) of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, a Notch-1 signaling pathway inhibitor, for 30 minutes. Then cultured cells were induced with Aβ25–35 for 48 hours. Pretreatment of PC12 cells with high doses of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (> 10 nmol/L) prolonged the survival of PC12 cells after Aβ25–35 induction, decreased the expression of apoptosis-related proteins caspase-3, -8, -9, increased the activity of oxidative stress-related superoxide dismutase and catalase, inhibited the production of active oxygen, and reduced nuclear factor kappa B expression. This study indicates that the Notch-1 signaling pathway plays a pivotal role in Aβ25–35-induced PC12 apoptosis.

Voltage-Gated Potassium Channel KV1.5 Protects against MPP+ Mediated Neurotoxicity in PC12 Cells

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease and afflicts almost 1.8% of over 65-year-old group in the world. Epidemiological projections showed that the incidence of PD was increasing continuously each year, with a wider age range as well. A large number of studies indicated that voltage-gated potassium channel (Kv) played significant roles in cellular signaling in both excitable and non-excitable cells. What’s more, Kv was also ubiquitously expressed in neurons and participated in signaling pathway in neurons. Kv1.5 (encoded by KCNA5) is an important voltage-gated K+ channel, which is not only necessary for critical processes such as cell proliferation and apoptosis but ubiquitously expressed in neurons. Recent studies reported that PD clinical drugs could inhibit the expression of Kv1.5. To determine the mechanisms by which Kv1.5 protects against MPP+ mediated neurotoxicity in PC12 cells. Materials and Methods: Knockdown of Kv1.5 model was established with pSINsi-hU6- Kv1.5 treated by the RNAi method in PC12. MTT, and Western Blot were used to detect the influence of Kv1.5 on PC12 proliferation, and the effect of Kv1.5 on PC12 apoptosis after MPP+ treatment in vitro. Results: 1) Knockdown and overexpression of Kv1.5 participated in PC12 proliferation. Transiently over-expressed Kv1.5 could boost the survival rate of PC12, while transiently knockdown of Kv1.5 inhibited PC12 proliferation. 2) The effect of Kv1.5 on PC12 proliferation was through PI3K/Akt signaling pathway. Over-expressed Kv1.5 could induce the activation of Akt, and Bcl-2 expression in PC12; Knockdown of Kv1.5 in PC12 inhibited the activation of Akt, Bcl-2 expression, and promoted MAPK phosphorylation. 3) Over-expressed Kv1.5 could significantly prevent PC12 from apoptosis induced by MPP+ via activating Akt pathway and increasing Bcl-2 expression; Knockdown of Kv1.5 was more sensitive than its control counterpart when treated with MPP+ for 24 h. Conclusion: Kv1.5 could hinder MPP+ neurotoxicity to PC12 by PI3K/Akt signaling pathway.

The study on the effects of apigenin on the proliferation in human androgen-independent prostate cancer cell PC-3 and the role Caspase-3 involved in

Objective To observe the effect of apigenin (API) on the proliferation of human androgen-independent prostate cancer cell line PC-3 and molecular mechanisms.Methods Clutures of PC-3 cell were exposured to API at the different concentrationa of 0,10,20 and 40 μmol/L respectively.The effect of API on growth inhibition were determinated with morphometry,cell counting kit-8 (CCK-8) assay.The expression of Caspase-3 mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).Results CCK-8 assay showed that the absorbance were 1.48 ±0.04,0.81 ±0.04,0.69 ± 0.03 and 0.33 ± 0.03 respectively when 0,10,20,40 μmol/L API treated PC-3 cells for 48 h,compared to the control group,10,20,40 μmol/L groups significantly inhibited the proliferation on PC-3 cells,the inhibition rates were 44.7％,53.5％,77.4％ (P ＜ 0.01).The Caspase-3 mRNA expression of 0,10,20,40 μmol/L group were 0.230 ± 0.036,0.410 ± 0.080,0.480 ± 0.100,0.730 ± 0.150,there was statistically significant differences between experimental and control groups (P ＜ 0.01).Conclusion The growth inhibition could be taken by API in PC-3 in a dose-dependent manner.The mechanism was that high expression level of Caspase-3 was caused by API,which led to the apoptosis of PC-3. Key words: Apigenin; Androgen-independent prostate cancer; Apoptosis; Caspase-3

Synthesis and antitumor properties of novel curcumin analogs

A series of novel curcumin (CC) analogs were synthesized by reacting substituted aldehydes with intermediates 4a and 4b. The inhibitory activities of these CC analogs were investigated on human cancer cells PC3, Bcap-37, and MGC-803 in vitro by MTT assay. The results showed that most of the title compounds displayed moderate to high levels of antitumor activities. Compound 5f, the most active CC analogs, has the IC50 values of 1.34 ± 0.28, 3.90 ± 0.36, and 0.86 ± 0.44 μM against the three human cancer cells assayed, respectively. Furthermore, subsequent fluorescence staining and flow cytometry analysis indicated compound 5f could induce apoptosis in PC3, Bcap-37, and MGC-803 cells, and the apoptosis ratio reachs the peak (27.1 %) in MGC-803 cells at 24 h after treatment at 10 μM.

Abstract C53: rVP1 inhibits prostate cancer metastasis via suppression of Jagged1

Abstract Jagged1, a NOTCH ligand, has been shown to correlate with poor prognosis and metastasis of prostate cancer (CaP). CaP is a leading cause of cancer death among men worldwide. There is currently no effective therapeutic strategy for patients with castration-resistant prostate cancer (CRPC) which is defined as continued growth of CaP despite the suppression of male hormones. We previously reported that recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus causes cell apoptosis of PC-3, a CRPC, via integrin β1 in vitro. Herein, we demonstrate that rVP1 inhibited cell migration/invasion at a non-toxic concentration and reduced the expression level of Jagged1 and phosphorylation of Akt (Ser 473) and IKKα/β (Ser 176/180) of PC-3 cells in vitro. This inhibition was abolished by integrin β1 blocking antibody, knockdown of integrin β1, or a constitutively active Akt. Furthermore, intravenous administration of rVP1 induced tumor apoptosis, suppressed tumor Jagged1 expression level, reduced primary-tumor spread to lymph nodes and bone, and prolonged the survival time of tumor-bearing mice in an orthotopic PC-3 xenograft model. Recombinant VP1 retarded tumor expansion within the bone and tumor-induced osteolysis in an experimental model of bone metastasis made by injecting PC-3 cells carrying the luciferase gene into mouse left ventricle. Finally, in vivo vascular permeability analyses showed that rVP1 specifically targeted tumor cells by penetrating leaky tumor vasculature. Recombinant VP1 may thus suppress Jagged1 expression of CaP cells by inhibiting the integrin β1/Akt/NF-κB signaling pathway to attenuate expansion of bone metastases and tumor-induced osteolysis in CRPC. Citation Format: Jui-Ling Wang, Shao-Wen Hung, Chi-Ming Liang, Pei-Wen Hsiao, Shu-Mei Liang. rVP1 inhibits prostate cancer metastasis via suppression of Jagged1. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C53.

Combinatorial antitumor effect of HDACs and the PI3K-Akt-mTOR pathway inhibition in a Pten deficient model of prostate cancer

Increased expression of histone deacetylases (HDACs) and activation of the PI3K-Akt-mTORC1 pathway are common aberrations in prostate cancer (PCa). For this reason, inhibition of such targets is an exciting avenue for the development of novel therapeutic strategies to treat patients with advanced PCa. Previous reports demonstrated that HDAC inhibitors (HDACi) increases DNA damage and induce greater apoptosis in PCa cell lines that express androgen receptor (AR). In this study we utilized the AR negative PCa cell line and observed that re-expression of AR (PC3-AR) results in greater levels of apoptosis when treated with the pan-DACi, panobinostat (PAN). PAN mediated apoptosis in PC3 and PC3-AR cells was associated with increased levels of double strand DNA breaks, indicated by p-ɣH2AX. Further, PAN treatment in PC3-AR cells resulted in moderate attenuation of the ATM-Akt-ERK DNA damage response pathway. For this reason, we combined PAN with the dual PI3K-mTOR inhibitor, BEZ235. Combination of PAN with BEZ235 resulted in significant attenuation of the DNA damage repair protein ATM and significantly increased anti-tumor activity compared to each single treatment. Overall, superior anti-tumor activity with combination of PAN with BEZ235 was independent of AR status. These findings suggest that this therapeutic strategy should be further developed in clinical trials.

Protective effect of SIRT1 on toxicity of microglial-derived factors induced by LPS to PC12 cells via the p53-caspase-3-dependent apoptotic pathway

Sirtuin1 (SIRT1) has protective effects in some neurodegenerative disease models, but it is not clear whether SIRT1 play the same role on inflammation-mediated Parkinson's disease (PD) models. In this study, we firstly established an inflammation environment by stimulating microglial BV-2 cells with the inflammatory agent lipopolysaccharides (LPS), which demonstrated by increasing of the levels of TNF-a, and IL-6 in cultured medium. Then we exposed PC12 cells (a model of catecholaminergic neuronal cells) with the supernant from LPS stimulated BV-2 cells (activated BV-2). PC12 cell apoptosis and SIRT1 involved protection were investigated. The results indicated that treatment with LPS caused significant decrease in SIRT1 expression in activated BV-2 cells, and increased the levels of TNF-a and IL-6, as measured by ELISA, whereas resveratrol (a known SIRT1 activator) suppressed this effect, which was conversely strengthened by sirtinol (a SIRT1 inhibitor), suggesting that SIRT1 may be involved in regulating proinflammatory cytokines from microglial activation. Further, we found that factors derived from activated microglia significantly decreased the level of deacetylation of p53 by reducing the expression of SIRT1, an effect that increased the apoptosis of PC12 and reduced cell viability. The addition of resveratrol could protect PC12 cells from inflammation-mediated damage above-mentioned, while nicotinamide (another SIRT1 inhibitor) treatment had the opposite effect of resveratrol. Together, these data suggests that: SIRT1 inhibits LPS-mediated proinflammatory cytokines release in microglia, and circumvents dopaminergic neurons injury induced by activated microglial-derived factors via p53-caspase-3-dependent mechanism of apoptosis. Thus, upregulation of SIRT1 provides a promising research field for therapeutic intervention in neuroinflammation diseases.

Cathepsin X promotes 6-hydroxydopamine-induced apoptosis of PC12 and SH-SY5Y cells

The cysteine carboxypeptidase cathepsin X is an important player in degenerative processes under normal ageing and pathological conditions. In the present study, we investigated the potential role of cathepsin X in 6-hydroxydopamine (6-OHDA)-induced toxicity in the pheochromocytoma cell line PC12 and neuroblastoma cell line SH-SY5Y. Cells exposed to 6-OHDA demonstrated alterations in the protein level of cathepsin X and activity of cathepsin X. Downregulation of cathepsin X expression by siRNA attenuated the neuronal death caused by 6-OHDA. Treatment with specific cathepsin X inhibitor AMS36 protected cells against 6-OHDA mediated cytotoxicity, resulting in reduced cell death and apoptosis. Furthermore, AMS36 reversed 6-OHDA-induced loss of tyrosine hydroxylase and attenuated 6-OHDA-induced activation of caspase-3, triggering apoptosis, intracellular generation of reactive oxygen species and mitochondrial dysfunction, including the release of cytochrome c and an imbalanced Bax/Bcl-2 ratio. Moreover, AMS36 interfered with NF-κB activation by blocking degradation of IκBα, preventing NF-κB translocation to the nucleus. Our data provide the first evidence that inhibition of cathepsin X protects both, PC12 and SH-SY5Y cells against 6-OHDA toxicity and indicate that cathepsin X may be responsible for dopamine neuron death, involved in the pathogenic cascade event for the neurodegenerative disorders, such as Parkinson's disease.

Ghrelin Inhibits High Glucose-Induced PC12 Cell Apoptosis by Regulating TLR4/NF-κB Pathway

Ghrelin has a protective effect on diabetic encephalopathy. To expound the protective mechanism, we investigated the effects of ghrelin on high glucose-induced cell apoptosis and intracellular signaling in cultured PC12, which is a suitable model for studying neuronal cell death. High glucose-induced PC12 apoptosis was significantly inhibited by co-treatment of ghrelin. Sustaining inflammatory response is one of the molecular mechanisms of diabetic encephalopathy and TLR4 signaling has close relationship with inflammatory response. But there is no report about the biologic role of toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling in controlling high glucose-induced PC12 apoptosis by ghrelin. In this study, we found that TLR4/NF-κB pathway was activated by high glucose stimulation in PC12 and significantly alleviated by the co-treatment of ghrelin. From these findings, we made the conclusion that ghrelin could attenuate the symptoms of diabetic encephalopathy, which alleviates inflammatory reaction of diabetic encephalopathy by regulating TLR4/NF-κB pathway.

Blocked Autophagy Using Lysosomotropic Agents Sensitizes Resistant Prostate Tumor Cells to the Novel Akt Inhibitor AZD5363

Prostate cancer development is often associated with deletion or silencing of tumor suppressor phosphatase and tensin homolog (PTEN), a negative regulator of the phosphoinositide 3 kinase (PI3K)-Akt pathway, leading to resistance to various therapies in both the preclinical and clinical setting. Therefore, the PI3K-Akt pathway plays a central role in various cellular processes promoting survival signaling that can contribute to the malignant phenotype, and, consequently, is an attractive pharmacologic target. However, as single agents, the efficacy of AKT inhibitors may be limited by resistance mechanisms that result in minimal cell death in tumor cells. We investigated the effects of the Akt inhibitor AZD5363 on cell proliferation, cell cycle, apoptosis, and Akt downstream pathway proteins. Survival mechanisms induced by AZD5363 were investigated. We then examined the impacts of inhibition of autophagy in combination with AZD5363 on cell proliferation and apoptosis. Furthermore, the anticancer activity of combination treatment of the lysosomotropic inhibitor of autophagy (chloroquine) with the Akt inhibitor AZD5363 was evaluated in PC-3 prostate cancer xenografts. Here, we show that the Akt inhibitor AZD5363 affected the Akt downstream pathway by reducing p-mTOR, p-P70S6K, and p-S6K. While AZD5363 monotherapy induced G(2) growth arrest and autophagy, it failed to induce significant apoptosis in PC-3 and DU145 prostate cancer cell lines. Blocking autophagy using pharmacologic inhibitors (3-methyladenine, chloroquine, and bafilomycin A) or genetic inhibitors (siRNA targeting Atg3 and Atg7) enhanced cell death induced by Akt inhibitor AZD5363 in these tumor prostate cell lines. Importantly, the combination of AZD5363 with chloroquine significantly reduced tumor volume by 84.9% compared with the control group and by 77.5% compared with either drug alone in PC3 xenografts. Taken together, these data show that the Akt inhibitor AZD5363 synergizes with the lysosomotropic inhibitor of autophagy chloroquine to induce apoptosis and delay tumor progression in prostate cancer models that are resistant to monotherapy AZD5363, providing a new therapeutic approach potentially translatable to patients.

Protective effect of paeoniflorin on Aβ25-35-induced PC12 cell injury

To investigate the protective effect of paeoniflorin on PC12 cell injury induced by Abeta25-35. Abeta25-35-induced PC12 cell injury model was established. The effect of cell proliferation was detected by the MTT method. We also measured the apoptosis with the FITC-Annexin V/PI staining, observed the mitochondrial membrane potential and changes in intracellular calcium using JC-1 and Fluo-3/AM staining respectively, and studied the protein expression of Bax, Bcl-2, cleaved Caspase-3 by Western blotting analysis. Paeoniflorin could inhibit Abeta25-35-induced PC12 cytotoxicity and apoptosis and show dose dependence. Its protective effect may be realized by increasing mitochondrial membrane potential Bcl-2 protein expression, decreasing Ca(2+) concentration and Bax protein expression and inhibiting activation of apoptosis-related protein Caspase-3. Paeoniflorin shows a protective effect on Abeta25-35-induced PC12 cell injury.

Zoledronic acid in combination with serine/threonine phosphatase inhibitors induces enhanced cytotoxicity and apoptosis in hormone‐refractory prostate cancer cell lines by decreasing the activities of PP1 and PP2A

What's known on the subject? and What does the study add? Prostate cancer is the second most common cancer diagnosed among elderly men. Current standard of care with surgery, chemotherapy or radiation in prostate cancer patients are of limited efficacy, especially in the androgen refractory state of the disease, and unfortunately metastatic disease remains incurable. Skeletal metastases are the most common site for metastases for prostate cancer and bisphosphonates have been widely used for the treatment of morbidity due to skeletal related events. Zoledronic acid (ZA) is the most potent member of the nitrogen containing new generation bisphosphonate (N-BPs) family. Okadaic acid (OA) and Calyculin A (CA) are the most commonly used inhibitors of PP1 and 2A. OA, extracted from common black sponge Halachondria okaddai is a potent inhibitor of protein phosphatases, PP1 and PP2A, and CA was isolated from another marine sponge, Discodermia calyx. Therapies based on combinations of chemotherapeutics with phosphatase inhibitors that target signaling pathways within the cell with different mechanisms of action, may be useful for increasing therapeutic effect and also diminish toxic side effects by decreasing the doses of conventional chemotherapeutics. Although clinically well known, the in vitro effects of ZA on cancer cells and the underlying mechanisms are not well elucidated. In our previous studies, we have already shown anticancer effect of ZA in hormone-and drug refractory prostate cancer cells, PC-3 and DU-145. In addition to this, we have also shown that this anticancer effect may be augmented with some cytotoxic agents in prostate cancer. Now, in our present study, we have investigated whether ZA induced growth inhibition and apoptosis in PC-3 and DU-145 may be enhanced by the combination with CA or OA, through inhibition of serine/threonine phosphatases in prostate cancer cells. Both ZA/CA and ZA/OA combinations inhibited the cell viability of hormone-and drug refractory prostate cancer cells at in vivo achievable therapeutic concentrations. Moreover, a potentiation of the apoptotic effects of the combinations was also observed in the same experimental conditions. This is the first report of a synergistic combination of ZA with phosphatase inhibitors CA and OA which inhibits cell viability and induces apoptosis in human hormone and drug refractory prostate cancer cells. • To investigate if the cytotoxic and apoptotic effect of zoledronic acid (ZA) can be enhanced by the addition of the serine/threonine protein phosphatase inhibitors calyculin A (CA) and okadaic acid (OA) in hormone and drug refractory prostate cancer cells, PC-3 and DU-145. • To discover the effect of these combination treatments on phosphatase 1 (PP1) and PP2A protein expression levels in prostate cancer cells. • An XTT cell viability assay was used to determine cytotoxicity. • Apoptosis was evaluated by enzyme-linked immunosorbent assay (ELISA) using a Cell Death Detection ELISA Plus Kit and verified by measuring caspase 3/7 enzyme activity. • The PP1 and PP2A enzyme activities were evaluated by serine/threonine phosphatase ELISA and expression levels of PP1 and PP2A proteins were then re-assessed by Western blot analysis. • Combination of ZA with either CA or OA showed synergistic cytotoxicity and apoptosis compared with any agent alone in both PC-3 and DU-145 prostate cancer cells. • The combination of ZA with phosphatase inhibitors resulted in enhanced suppression of both PP1 and PP2A enzyme activity and protein levels, which was more overt with the ZA/CA combination. • Results from our study increase the translational potential of our in vitro findings and offer the basic rationale for the design of new combinatory strategies with ZA and phosphatase inhibitors for the treatment of prostate cancer, which may become resistant to conventional therapy.

Roles of notch signaling in rotenone-induced apoptosis of PC12 cells

Objective To elucidate the roles of Notch Signaling in rotenone-induced apoptosis of PC12 cells.Methods PC12 ceils were treated with rotenone (5 μmol/L).Apoptosis of PC12 cells and activation of Notch signaling were observed.PC12 cells were pretreated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butvlester ( DAPT,10 μmol/L) before incubation with rotenone.The effect of Notch signaling on apoptosis of PC12 cells in response to rotenone was evaluated and the relationship between Notch signaling and rotenone-induced apoptosis of PC12 cells was analyzed.Results Rotenone significantly induced apoptosis of PC12 cells [( 35.6 ± 5.4 ) ％,P ＜ 0.05] and increased Caspase-3 activity (0.52 ±0.15,P ＜0.05 ).At the same time,rotenone activated Notch/Jagged1 signaling in PC12 cells.However,inhibition of Notch signaling by DAPT not only decreased apoptosis of PC12 cells [(9.8 ±3.1 ) ％,P ＜ 0.05],but also diminished Caspase-3 activity (0.16 ± 0.06,P ＜ 0.05 ).Conclusion Activation of Notch signaling could be one of the main mechanisms for rotenone-induced apoptosis of PC12 cells. Key words: Notch signaling; PC12 cells; Rotenone; Apoptosis

Effect of interferon-.BETA. gene engineered human bone mesenchymal stem cells on proliferation and apoptosis of PC-3 cells

Objective To investigate the effect of human interferon-β (IFN-β) gene cDNA engineered human bone marrow mesenchymal stem cells (hMSCs) on the proliferation and apoptosis of prostate cancer cell line PC-3.Methods The pCDNA3.1-IFN-β vector containing human IFN-β gene was constructed and transfected into hMSCs by lipofectamine.IFN-β-expressing mesenchymal stem cells (IFN-β-hMSCs) were incubated with PC-3 cells.PC-3 cells were stained by trypan blue and viable cells were counted by hemocytometer respectively at the 1 st,3rd,and 5 th day after co-culture.The apoptosis of PC-3 cells was analyzed by flow cytometry.Results IFN-β-hMSCs could express IFN-3 successfully.Five days after co-culture,the number of viable PC-3 cells in hMSCs,mock-hMSCs,IFN-β-hMSCs groups was ( 10.81 ± 0.33) × 105,(9.90 ± 0.41 ) × 105,and (4.63 ± 0.32 ) × 105 respectively.IFN-β-hMSCs resulted in a significant decrease in PC-3 cells survival as compared with control group (P ＜0.01 ),and apoptosis rate of PC-3 cells was (22.79 ±2.71 )％,which was increased as compared with control group.Conclusion The IFN-β-hMSCs can express IFN-β successfully and inhibit the growth of prostate cancer cells significantly. Key words: Prostate carcinoma; Interferon beta; Mesenchymal stem cell; Gene therapy