Total saponins isolated from Radix et Rhizoma Leonticis suppresses tumor cells growth by regulation of PI3K/Akt/mTOR and p38 MAPK pathways

Both PI3K/AKT/mTOR and mitogen activated protein kinase (MAPK) signaling cascades played an important role in tumorigenesis, a more complete understanding of these signaling pathways allowed the development of new therapeutic strategies. Total saponins isolated from Radix et Rhizoma Leonticis (RLTS) was recognized with anticancer properties. In a murine hepatocellular carcinoma H22 cell-bearing mouse model, RLTS exhibited significant inhibitory effect on tumor growth. Here, we investigated the role of RLTS on the PI3K/AKT/mTOR signaling pathway and MAPK pathways in liver and lung cancer cells. Results obtained showed RLTS inhibited cell proliferation and induced cell apoptosis in vitro, which attributed to the inhibition on the activation of PI3K/AKT/mTOR cascade and its related signaling molecules, such as activated VEGFR and NF-κB, and activation of p38 MAPK in tumor cells. Additional, RLTS inhibited cell migration and downregulated proteins that mediated metastasis including CXCR4, MMP2 and MMP9. Overall, these findings suggested that RLTS interfered with multiple signaling cascades involved in tumorigenesis and had potential in cancer therapy.     

抗ErbB2嵌合抗体chA21对SKBR3细胞的增殖抑制以及对PI3K/Akt和Erk1/2信号转导途径的影响

目的检测抗ErbB2嵌合抗体chA21对高表达ErbB2乳腺癌细胞SKBR3的增殖抑制作用,并分析抗体对细胞膜上ErbB2分布以及对细胞PI3K/Akt和Erk1/2信号转导途径的影响,探讨chA21的抑癌机制。方法采用MTT检测chA21对SKBR3的增殖抑制作用,流式细胞术检测经chA21处理后细胞膜上ErbB2抗原的分布变化,Western blot检测抗体作用后对细胞PI3K/Akt和Erk1/2信号转导途径的影响。结果chA21可抑制SKBR3细胞的增殖,其作用呈剂量和时间依赖性。抗体作用后可下调细胞膜上ErbB2含量,并不同程度下调细胞的PI3K/Akt和Erk1/2的活化水平。结论chA21可抑制SKBR3细胞增殖,可能通过下调细胞膜上ErbB2分布而减少其介导的PI3K/Akt和Erk1/2信号转导途径的活化来实现的。     

Cobalt oxide nanoparticles induced oxidative stress linked to activation of TNF‐α/caspase‐8/p38‐MAPK signaling in human leukemia cells

The purpose of this study was to determine the intracellular signaling transduction pathways involved in oxidative stress induced by nanoparticles in cancer cells. Activation of reactive oxygen species (ROS) has some therapeutic benefits in arresting the growth of cancer cells. Cobalt oxide nanoparticles (CoO NPs) are an interesting compound for oxidative cancer therapy. Our results showed that CoO NPs elicited a significant (P <0.05) amount of ROS in cancer cells. Co‐treatment with N‐aceyltine cystine (an inhibitor of ROS) had a protective role in cancer cell death induced by CoO NPs. In cultured cells, the elevated level of tumor necrosis factor‐alpha (TNF‐α) was noted after CoO NPs treatment. This TNF‐α persuaded activation of caspase‐8 followed by phosphorylation of p38 mitogen‐activated protein kinase and induced cell death. This study showed that CoO NPs induced oxidative stress and activated the signaling pathway of TNF‐α‐Caspase‐8‐p38‐Caspase‐3 to cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

迷迭香酸衍生物RAD-9通过PI3K/Akt和p38 MAPK信号通路诱导胃癌MGC-803细胞凋亡

目的研究迷迭香酸衍生物RAD-9诱导胃癌MGC-803细胞凋亡的作用及其机制。方法 MTT法观察RAD-9对胃癌MGC-803细胞增殖的抑制作用;流式细胞术检测细胞的凋亡;Hoechst 33258染色法观察RAD-9对MGC-803细胞核凋亡形态学的影响;Western blot检测RAD-9干预MGC-803细胞36 h后,对Akt、p-Akt、p38 MAPK、p-p38 MAPK蛋白及凋亡相关蛋白Bcl-2、Bax、caspase-3的影响。结果MTT结果显示,RAD-9呈时间、浓度依赖性抑制胃癌MGC-803细胞增殖;流式细胞术结果显示,RAD-9对胃癌MGC-803细胞有明显的促凋亡作用(P<0.01);Hoechst 33258染色实验结果显示,RAD-9干预胃癌MGC-803细胞36 h后,细胞核呈现典型凋亡形态学改变;Western blot结果显示,RAD-9干预胃癌MGC-803细胞36 h后,Bcl-2蛋白表达水平明显降低,Bax、caspase-3蛋白表达水平明显提高,Akt、p-Akt蛋白表达水平明显下调,p38 MAPK、p-p38 MAPK蛋白表达水平明显上调(P<0.01)。结论 RAD-9能抑制胃癌MGC-803细胞生长,且能诱导其凋亡,其机制可能与抑制PI3K/Akt和激活p38 MAPK信号通路相关。     

Review of the expression of peroxisome proliferator-activated receptors alpha (PPARα), beta (PPARβ), and gamma (PPARγ) in rodent and human development

The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily and there are three primary subtypes, PPARα, β, and γ. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis. These nuclear receptors have important roles in reproduction and development and their expression may influence the responses of an embryo exposed to PPAR agonists. PPARs are relevant to the study of the biological effects of the perfluorinated alkyl acids as these compounds, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), activate PPARα. Exposure of the rodent to PFOA or PFOS during gestation results in neonatal deaths, developmental delay and growth deficits. Studies in PPARα knockout mice demonstrate that the developmental effects of PFOA, but not PFOS, depend on expression of PPARα. This review provides an overview of PPARα, β, and γ protein and mRNA expression during mouse, rat, and human development. The review presents the results from many published studies and the information is organized by organ system and collated to show patterns of expression at comparable developmental stages for human, mouse, and rat. The features of the PPAR nuclear receptor family are introduced and what is known or inferred about their roles in development is discussed relative to insights from genetically modified mice and studies in the adult.     

Annosquacin B induces mitochondrial apoptosis in multidrug resistant human breast cancer cell line MCF-7/ADR through selectively modulating MAPKs pathways

Context: Multidrug resistance (MDR) is a major obstacle to efficient therapy of cancers. It is a prime concern for researchers to find compounds with anti-proliferative activity on MDR cell lines. In recent years, annonaceous acetogenins (ACGs) were reported to have anti-proliferative activity. However, the underlying mechanisms are still unknown.

Objective: This study determines the mechanisms of anti-proliferative activity induced by Annosquacin B (AB) against MCF-7/ADR cells.

Material and methods: The cytotoxicity of AB at varying concentrations (0.64, 1.6, 4, 10, 25, 62.5, 156.25?μM) on MCF-7/ADR cells was assessed using the MTT assay. Annexin V-FITC/propidium iodide staining and Acrinidine orange and ethidium bromide (AO/EB) staining were employed to investigate whether AB (14, 7, 3.5?μM) could induce apoptosis in MCF-7/ADR cells. Levels of caspase-3 and caspase-9, Bax, Bcl-2 and MAPKs kinases were evaluated by western blot assay following treatment with various concentrations of AB (3.5, 7, 14?μM) at different time points (0, 0.5, 1, 2, 4, 8, 12?h).

Results and conclusion: MTT assay showed that AB significantly decreased cell viability on MCF-7/ADR (IC₅₀ of 14.69?μM). AB-induced apoptosis in MCF-7/ADR cells through mitochondrial apoptosis pathways. It induced typical apoptosis by morphologic changes; elevate levels of caspase-3, caspase-9 as well as the ratio of Bax/Bcl-2. In addition, AB increased the expression of p-p38 MAPK and decreased the expression of p-JNK, while whether ERK1/2 had an effect on the MCF-7/ADR apoptosis remains to be determined.     

PTPL1 suppresses lung cancer cell migration via inhibiting TGF-β1-induced activation of p38 MAPK and Smad 2/3 pathways and EMT

Epithelial-mesenchymal transition (EMT) enables dissemination of neoplastic cells and onset of distal metastasis of primary tumors. However, the regulatory mechanisms of EMT by microenvironmental factors such as transforming growth factor-β (TGF-β) remain largely unresolved. Protein tyrosine phosphatase L1 (PTPL1) is a non-receptor protein tyrosine phosphatase that plays a suppressive role in tumorigenesis of diverse tissues. In this study we investigated the role of PTPL1/PTPN13 in metastasis of lung cancer and the signaling pathways regulated by PTPL1 in terms of EMT of non-small cell lung cancer (NSCLC) cells. We showed that the expression of PTPL1 was significantly downregulated in cancerous tissues of 23 patients with NSCLC compared with adjacent normal tissues. PTPL1 expression was positively correlated with overall survival of NSCLC patients. Then we treated A549 cells in vitro with TGF-β1 (10 ng/mL) and assessed EMT. We found that knockdown of PTPL1 enhanced the migration and invasion capabilities of A549 cells, through enhancing TGF-β1-induced EMT. In nude mice bearing A549 cell xenografts, knockdown of PTPL1 significantly promoted homing of cells and formation of tumor loci in the lungs. We further revealed that PTPL1 suppressed TGF-β-induced EMT by counteracting the activation of canonical Smad2/3 and non-canonical p38 MAPK signaling pathways. Using immunoprecipitation assay we demonstrated that PTPL1 could bind to p38 MAPK, suggesting that p38 MAPK might be a direct substrate of PTPL1. In conclusion, these results unravel novel mechanisms underlying the regulation of TGF-β signaling pathway, and have implications for prognostic assessment and targeted therapy of metastatic lung cancer.     

ERK and p38 MAPK signaling pathways are involved in ochratoxin A-induced G2 phase arrest in human gastric epithelium cells

Ochratoxin A (OTA) is a ubiquitous mycotoxin with potential nephrotoxic, hepatotoxic, and immunotoxic effects. Recent work from our laboratory found that OTA evoked G2 phase arrest in GES-1 cells in vitro by modulating the key factors Cdc25C, Cdc2 and cyclinB1, which were critical to the G2/M phase transmission, suggested that OTA-induced G2 arrest mediate at least in part OTA toxicity effect. However, the molecular mechanism of this effect is currently unclear. In the present study, we showed that treatment of GES-1 cells with OTA could induce the activation of MAPK family members ERK and p38. ERK inhibitor PD98059 and p38 inhibitor SB203580 significantly reversed the depression of Cdc25C/p-Cdc25C, Cdc2/p-Cdc2, cyclinB1 as well as the cyclinB1-Cdc2 complex, thereby, abolished the delay in G2 phase. In addition, silencing ERK and p38 expression with siRNA significantly inhibited OTA-induced G2 arrest in GES-1 cells as well. Collectively, these data suggest that the ERK and p38 MAPK signaling pathways play important roles in the regulation of OTA-induced G2 arrest in GES-1 cells.     

Proliferation of the human urothelium is induced by atypical β1‐adrenoceptors

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Biphasic regulation of P-glycoprotein function and expression by NO donors in Caco-2 cells

Aim:

To investigate the effects of nitric oxide (NO) donors on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells.

Methods:

Caco-2 cells were exposed to NO donors for designated times. P-gp function and expression were assessed using Rhodamine123 uptake assay and Western blotting, respectively. Intracellular reactive oxygen species (iROS) and intracellular reactive nitrogen species (iRNS) levels were measured using ROS and RNS assay kits, respectively.

Results:

Exposure of Caco-2 cells to 0.1 or 2 mmol/L of sodium nitroprusside (SNP) affected the function and expression of P-gp in concentration- and time-dependent manners. A short-term (4 h) exposure reduced P-gp function and expression accompanied with significantly increased levels of iROS and iRNS. In contrast, a long-term (24 h) exposure stimulated the P-gp function and expression. The stimulatory effects of 2 mmol/L SNP was less profound as compared to those caused by 0.1 mmol/L SNP. The other NO donors SIN-1 and SNAP showed similar effects. Neither the NO scavenger PTIO (2 mmol/L) nor soluble guanylate cyclase inhibitor ODQ (50 μmol/L) reversed the SNP-induced alteration of P-gp function. On the other hand, free radical scavengers ascorbate, glutathione and uric acid (2 mmol/L for each), PKC inhibitor chelerythrine (5 μmol/L), PI3K/Akt inhibitor wortmannin (1 μmol/L) and p38 MAPK inhibitor SB203580 (10 μmol/L) reversed the upregulation of P-gp function by the long-term exposure to SNP, but these agents had no effect on the impaired P-gp function following the short-term exposure to SNP.

Conclusion:

NO donors time-dependently regulate P-gp function and expression in Caco-2 cells: short-term exposure impairs P-gp function and expression, whereas long-term exposure stimulates P-gp function and expression. The regulation occurs via a NO-independent mechanism.     

Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serum-free medium

Silymarin was proved to have a protective effect of UV-induced A375-S2 cell apoptosis in our previous research. In this study, its pro-apoptotic and anti-apoptotic activities on human cervical cancer (HeLa) cells in vitro were investigated. Silymarin induced HeLa cell death through both apoptotic and necrotic pathways. At low doses (below 80 μmol l^? 1), it induced cell apoptosis, but caused necrosis at high dose (160 μmol l^? 1). Silymarin induced typical chromatin condensation and nuclear fragmentation as a hallmark of apoptosis. In this case, mitochondrial Bcl-2 family, Bcl-2 and Bax, were not involved in apoptotic effects; however, silymarin-induced cell death was regulated by the activation of p38 and JNK MAPKs. We also found that pan-caspase inhibitor and caspase-3 inhibitor could not antagonise silymarin-induced apoptosis. Therefore, silymarin induced and augmented HeLa cell apoptosis through p38/JNK MAPKs in the serum-free medium.     

Signalling pathways involved in p47phox‐dependent reactive oxygen species in platelets of endotoxemic rats

Thrombocytopenia during sepsis is associated with a less favourable clinical outcome. Overproduction of reactive oxygen species (ROS) by different cell types contributes to sepsis. Platelets generate ROS, but the upstream pathways of NADPH oxidase activation are not completely understood. Here, we designed experiments in washed platelets from lipopolysaccharide (LPS)‐treated rats to investigate the p47^phox activation and ROS generation, and its modulation by c‐Src family kinase (c‐Src), phosphoinositide 3‐kinase (PI3K), protein kinase C (PKC) and protein kinase G (PKG). Rats were injected intraperitoneally with LPS (1 mg/kg), and at 48 hours thereafter, arterial blood was collected and washed platelets were obtained. Washed platelets were pre‐incubated with different inhibitors and subsequently activated or not with ADP. Flow cytometry, Western blotting and ELISA were performed. We found that LPS significantly increased the p47^phox phosphorylation and ROS generation compared with the control group (P < 0.05). The enhanced ROS production in the LPS group was unaffected by the non‐selective SFKs inhibitor PP2, the PI3K inhibitor wortmannin or the Akt inhibitor PPI‐1. The cyclic GMP levels were 115% higher in activated platelets of LPS compared with the saline group (P < 0.05). Moreover, in the LPS group, the sGC inhibitor ODQ, the PKG inhibitor Rp‐8‐Br and the PKC inhibitor GF109203X abrogated the increased p47^phox phosphorylation and reduced the ROS levels. In conclusion, selective inhibitors of cGMP‐PKG and PKC‐p47^phox pathways that regulate ROS generation by LPS in platelets may help control the redox balance in sepsis improving the survival of patients.     

MAPK inhibitors and pifithrin-alpha block cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. The aim of this study was to investigate the effect of pifithrin-alpha (PFTalpha; a specific p53 inhibitor) and mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on apoptotic signaling transduction mechanism induced by Cin in human hepatoma PLC/PRF/5 (CD95-negative) cells. Using XTT assay, Cin exhibited a powerful cytotoxic effect and apoptotic induction in PLC/PRF/5 cells. Apoptosis was elicited when cells were treated with 1 microM Cin as characterized by morphological changes and the appearance of phosphatidylserine on the outer surface of the plasma membrane. Cin down-regulated the expression of Bcl-(XL), up-regulated mutant p53 and Bax proteins and promoted caspase-3 to active forms, as well as cleaving poly (ADP-ribose) polymerase (PARP) in a time-dependent pattern. This could be supported by the activation and phosphorylation of MAPKs, including JNK, ERK and p38 kinases. Pre-incubation with PFTalpha and specific MAPK inhibitors significantly diminished the effect of Cin-induced apoptosis. The activities of anti-apoptotic (Bcl-(XL)) and pro-apoptotic (Bax) proteins were remarkably affected by PFTalpha and PD98059 pre-treatment. PFTalpha effectively blocked PARP cleavage in cells treated with Cin, and also markedly prevented the phosphorylation of JNK, p38 and ERK proteins. These results suggest that p53 induction and MAPK signaling pathways are required for Cin-mediated apoptosis in PLC/PRF/5 cells.     

Involvement of the activation of Nrf2/HO-1, p38 MAPK signaling pathways and endoplasmic reticulum stress in furazolidone induced cytotoxicity and S phase arrest in human hepatocyte L02 cells: modulation of curcumin

Furazolidone (FZD) is extensively used as the antiprotozoal and antibacterial drug in clinic. The previous study has shown that curcumin pretreatment could improve FZD induced cytotoxicity by inhibiting oxidative stress and mitochondrial apoptotic pathway. The current study aimed to investigate the potential roles of endoplasmic reticulum (ER) stress, p38 mitogen-activated protein kinases (p38 MAPK) signaling pathway in curcumin against FZD cytotoxicity by using human hepatocyte L02 cells. The results showed that curcumin could markedly attenuate FZD induced cytotoxicity. Compared with FZD alone group, curcumin pretreatment significantly reduced the expression of phospho (p)-p38, cyclin D1, p-checkpoint kinase 1 (ChK1) and breast cancer associated gene 1 (BRCA1) protein, followed to attenuate S phase arrest. Meanwhile, curcumin pretreatment prevented FZD induced ER stress, evidenced by the inhibition of glucose-regulated protein 78 and DNA damage inducible gene 153/C/EBP-homologous protein (GADD153/CHOP) protein expression. Moreover, compared with the control, FZD exposure activated the protein and mRNA expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1), which were further activated by curcumin treatment. These results reveal that curcumin could prevent FZD induced cytotoxicity and S phase arrest, which may involve the activation of Nrf2/HO-1 pathway and the inhibition of p38 MAPK pathway and ER stress.     

Inositide-dependent signaling pathways as new therapeutic targets in myelodysplastic syndromes

Introduction: Nuclear inositide signaling pathways specifically regulate cell proliferation and differentiation. Interestingly, the modulation of nuclear inositides in hematological malignancies can differentially affect erythropoiesis or myelopoiesis. This is particularly important in patients with myelodysplastic syndromes (MDS), who show both defective erythroid and myeloid differentiation, as well as an increased risk of evolution into acute myeloid leukemia (AML).

Areas covered: This review focuses on the structure and function of specific nuclear inositide enzymes, whose impairment could be linked with disease pathogenesis and cancer. The authors, stemming from literature and published data, discuss and describe the role of nuclear inositides, focusing on specific enzymes and demonstrating that targeting these molecules could be important to develop innovative therapeutic approaches, with particular reference to MDS treatment.

Expert opinion: Demethylating therapy, alone or in combination with other drugs, is the most common and current therapy for MDS patients. Nuclear inositide signaling molecules have been demonstrated to be important in hematopoietic differentiation and are promising new targets for developing a personalized MDS therapy. Indeed, these enzymes can be ideal targets for drug design and their modulation can have several important downstream effects to regulate MDS pathogenesis and prevent MDS progression to AML.     

新藤黄酸诱导人鼻咽癌细胞CNE-1凋亡以及对p-p38和p-ERK1/2蛋白的影响

目的探讨新藤黄酸对人鼻咽癌细胞CNE-1凋亡的作用以及对磷酸化p38、p-ERK1/2蛋白的影响。方法倒置显微镜观察新藤黄酸不同时间和不同浓度处理CNE-1细胞形态变化;采用四甲基偶氮唑蓝(MTT)比色法检测不同浓度新藤黄酸(0.25、0.5、1.0、2.0、4.0和8.0μmol.L-1)处理24 h、48 h和72 h对人鼻咽癌细胞CNE-1增殖的抑制作用;新藤黄酸作用CNE-1细胞24 h后的IC50为2.34μmol.L-1,再结合倒置显微镜观察的结果故选用2.0μmol.L-1作为药物作用浓度。Annexin V-FITC/PI双染流式细胞仪检测细胞凋亡率,并应用透射电子显微镜检测细胞凋亡形态改变及线粒体损伤;Western blot检测p-p38、p38、ERK1/2和p-ERK1/2蛋白和线粒体凋亡相关蛋白Cytochrome C和Caspase-3蛋白的表达。结果新藤黄酸对人鼻咽癌细胞CNE-1生长和增殖有抑制作用,并随着新藤黄酸浓度的增加或作用时间的延长细胞活力明显下降。通过电镜可见2.0μmol.L-1新藤黄酸作用CNE-1细胞后细胞体积皱缩变圆,细胞核发生典型核染色质固缩等细胞凋亡形态学的变化;与control组相比包括对线粒体的损伤。Western blot表明p-p38蛋白表达有所上调,特别是在短时间(120 min)内,p-ERK1/2蛋白表达呈现时间依赖性下降;而p38和ERK1/2表达则基本不变。Cytochrome C和Caspase-3蛋白表达也呈现时间依赖性增加。结论新藤黄酸能诱导人鼻咽癌细胞CNE-1凋亡,增强Cytochrome C和Caspase-3蛋白表达,同时对p-p38和p-ERK1/2蛋白也有影响。     

Multiple pathways are responsible to the inhibitory effect of butorphanol on OGD/R-induced apoptosis in AC16 cardiomyocytes

Ischemic heart disease is the leading cause of cardiovascular mortality, which is related to cardiac myocyte apoptosis. Butorphanol is an opioid receptor agonist with potential cardioprotective function. The purpose of this work is to explore the function and mechanism of butorphanol in oxygen and glucose deprivation/reperfusion (OGD/R)-induced cardiomyocyte apoptosis. The overlapping targets of ischemic heart disease and butorphanol were analyzed according to GeneCards, ParmMapper, Cytoscape, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Human cardiomyocyte AC16 cells were incubated with butorphanol and then stimulated with OGD/R. Cell injury was investigated by Cell Counting Kit-8, lactate dehydrogenase (LDH) assay kit, TUNEL staining, caspase-3 activity assay kit, and Western blotting. The proteins in signaling pathways were measured using Western blotting. A total of 93 overlapping targets of ischemic heart disease and butorphanol were obtained. Pathway analysis exhibited that these targets might be involved in multiple signaling pathways. Butorphanol alone showed little cytotoxicity to cardiomyocytes, and it protected against OGD/R-induced viability inhibition, LDH release, cell apoptosis, and increase of caspase-3 activity and expression levels of cleaved caspase-3 and Bim. Butorphanol promoted the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O (FoxO) and hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathways and attenuated the activation of the mitogen-activated protein kinase (MAPK) signaling in OGD/R-treated cardiomyocytes. In conclusion, butorphanol prevents OGD/R-induced cardiomyocyte apoptosis through activating the PI3K/Akt/FoxO and HIF-1α/VEGF pathways and inactivating the MAPK pathway.