Differential cytotoxicity, ER/oxidative stress, dysregulated AMPKα signaling, and mitochondrial stress by ethanol and its metabolites in human pancreatic acinar cells.

BACKGROUND: Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disorder of the exocrine pancreatic gland. A previous study from this laboratory showed that ethanol (EtOH) causes cytotoxicity, dysregulates AMPKα and ER/oxidative stress signaling, and induces inflammatory responses in primary human pancreatic acinar cells (hPACs). Here we examined the differential cytotoxicity of EtOH and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters; FAEEs) metabolites in hPACs was examined to understand the metabolic basis and mechanism of ACP. METHODS: We evaluated concentration-dependent cytotoxicity, AMPKα inactivation, ER/oxidative stress, and inflammatory responses in hPACs by incubating them for 6 h with EtOH, acetaldehyde, or FAEEs at clinically relevant concentrations reported in alcoholic subjects using conventional methods. Cellular bioenergetics (mitochondrial stress and a real-time ATP production rate) were determined using Seahorse XFp Extracellular Flux Analyzer in AR42J cells treated with acetaldehyde or FAEEs. RESULTS: We observed concentration-dependent increases in LDH release, inactivation of AMPKα along with upregulation of ACC1 and FAS (key lipogenic proteins), downregulation of p-LKB1 (an oxidative stress-sensitive upstream kinase regulating AMPKα) and CPT1A (involved in ß-oxidation of fatty acids) in hPACs treated with EtOH, acetaldehyde, or FAEEs. Concentration-dependent increases in oxidative stress and ER stress as measured by GRP78, unspliced XBP1, p-eIF2α, and CHOP along with activation of p-JNK1/2, p-ERK1/2, and p-P38MAPK were present in cells treated with EtOH, acetaldehyde, or FAEEs, respectively. Furthermore, a significant decrease was observed in the total ATP production rate with subsequent mitochondrial stress in AR42J cells treated with acetaldehyde and FAEEs. CONCLUSIONS: EtOH and its metabolites, acetaldehyde and FAEEs, caused cytotoxicity, ER/oxidative and mitochondrial stress, and dysregulated AMPKα signaling, suggesting a key role of EtOH metabolism in the etiopathogenesis of ACP. Because oxidative EtOH metabolism is negligible in the exocrine pancreas, the pathogenesis of ACP could be attributable to the formation of FAEEs and related pancreatic acinar cell injury.

[Ruyi Jinhuang Plaster suppresses BPH by reducing the expressions of P38, JNK2, NF-кBP65 and STAT3 in the prostate tissue of rats].

OBJECTIVE: To investigate the effect of Ruyi Jinhuang Plaster (RJP) on testosterone propionate-induced BPH in the rat model and its action mechanisms. METHODS: Forty-eight SD male rats were randomly divided into six groups of equal number: normal control, BPH model control, finasteride, and high-, medium- and low-dose RJP. The BPH model was made in the latter five groups by hypodermic injection of testosterone propionate. From the first day of modeling, the rats of the normal control and BPH model control groups were treated with blank plasters and those of the high-, medium- and low-dose RJP groups with RJPs at 42.0, 21.0 and 10.5 cm2/kg applied to the dehaired area of the back, and those of the finasteride group by gavage of finasteride at 4.5 mg/kg, all once a day for 30 successive days. Then the prostates of the animals were harvested for observation of histopathological changes by HE staining, measurement of the areas of interstitial and epithelial cells and prostatic glandular cavity, and determination of the expressions of P38, JNK2, NF-кBP65 and STAT3 proteins in the prostate tissue by Western blot. RESULTS: Compared with the BPH model controls, the high-dose RJP group showed significantly decreased proliferation and area proportion of prostatic epithelial cells (P < 0.05), increased area proportion of the prostatic glandular cavity (P < 0.05), and reduced expressions of P38, p-P38, NF-кBP65, P-NF-кBP65, STAT3, P-STAT3 and JNK2 in the prostate tissue (P < 0.05); the medium-dose RJP group exhibited markedly down-regulated expressions of JNK2 and NF-кBP65 (P < 0.05) but an up-regulated level of p-JNK (P < 0.05); while the low-dose RJP group displayed a remarkably reduced expression of JNK2 (P < 0.05) but an elevated level of p-JNK (P < 0.05). CONCLUSIONS: RJP suppresses BPH in the model rat by down-regulating the expressions of P38, p-P38, NF-кBP65, P-NF-кBP65, STAT3, P-STAT3 and JNK2 or up-regulating that of p-JNK in the prostate tissue.

Modulation of the response of prostate cancer cell lines to cisplatin treatment using small interfering RNA.

Cisplatin is one of the most effective and widely used chemotherapeutic agents against several types of human cancers. However, the underlying mechanisms of action are not fully understood. We aimed to investigate the possible molecular mechanism(s) of acquired chemoresistance observed in prostate cancer cells treated with cisplatin. Human LNCaP cells (bearing wild-type p53) and PC-3 cells (lacking p53) were used. The expression levels of protein were determined by western blotting, and the mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cell viability was measured by MTT assay, and the transcriptional effect of small interfering RNA (siRNA) was measured by luciferase reporter gene. We showed that cisplatin treatment increased JNK-1 and JNK-2 activity and expression in both LNCaP and PC-3 cells. In addition, the knockdown of JNK-1 expression by siRNA-JNK-1 or siRNA-JNK-2 significantly impaired the upregulation of AP-1 luciferase reporter gene, but failed to decrease the levels of AP-1 reporter gene expression induced by TPA treatment. Our observations indicate that JNK-1 and JNK-2 may be involved in the chemoresistance observed in prostate cancer cells treated with cisplatin and that blocking the stimulation of Jun kinase (JNK) signaling may be important for regulating the susceptibility to cisplatin of prostate cancer.

Doxycycline reduces lipopolysaccharide-induced inflammatory mediator secretion in macrophage and ex vivo human whole blood models.

BACKGROUND: Tetracyclines have been extensively used as adjuncts in the treatment of some forms of periodontitis. The aim of this study was to evaluate the capacity of doxycycline to influence the secretion of inflammatory mediators in macrophage and ex vivo human whole blood models stimulated with periodontopathogen lipopolysaccharides (LPS). METHODS: Monocyte-derived macrophages were treated with various concentrations of doxycycline prior to being stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) LPS. The capacity of doxycycline to mediate the inflammatory response was also tested in an ex vivo whole blood model (whole blood isolated from periodontitis patients and healthy subjects) stimulated with Porphyromonas gingivalis LPS. The secretion of interleukin (IL)-1beta, -6, and -8 and tumor necrosis factor-alpha (TNF-alpha) in both models was assessed by enzyme-linked immunosorbent assays (ELISA). Changes in phosphorylation state of kinases induced by A. actinomycetemcomitans LPS and doxycycline in the macrophage model were characterized by a multiplex ELISA analysis. RESULTS: The secretion of IL-1beta and -8 and TNF-alpha by macrophages decreased significantly (P <0.05) when they were pretreated with 2 microM doxycycline, whereas a concentration of 10 microM was required to significantly reduce IL-6 secretion. Pretreatment of macrophages with 10 microM doxycycline prior to A. actinomycetemcomitans LPS stimulation resulted in a marked decrease in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (-76%). In the whole blood model, doxycycline, more particularly at 10 microM, was also a potent inhibitor of the proinflammatory cytokine response. CONCLUSION: These two models provided clear evidence that some of the clinically proven benefits of doxycycline may be related to its ability to regulate inflammatory mediator release by host cells.

Disruption of JNK2 decreases the cytokine response to Plasmodium falciparum glycosylphosphatidylinositol in vitro and confers protection in a cerebral malaria model.

Host inflammatory responses to Plasmodium falciparum GPI (pfGPI) anchors are believed to play an important role in the pathophysiology of severe malaria. However, relatively little is known about the signal transduction pathways involved in pfGPI-stimulated inflammatory response and its potential contribution to severe malaria syndromes. In this study, we investigated the role of MAPK activation in pfGPI-induced cytokine secretion and examined the role of selected MAPKs in a model of cerebral malaria in vivo. We demonstrate that ERK1/2, JNK, p38, c-Jun, and activating transcription factor-2 became phosphorylated in pfGPI-stimulated macrophages. A JNK inhibitor (1,9-pyrazoloanthrone) inhibited pfGPI-induced phosphorylation of JNK, c-Jun, and activating transcription factor-2 and significantly decreased pfGPI-induced TNF-alpha secretion. pfGPI-stimulated JNK and c-Jun phosphorylation was absent in Jnk2(-/-) macrophages but unchanged in Jnk1(-/-) and Jnk3(-/-) macrophages compared with wild-type macrophages. Jnk2(-/-) macrophages secreted significantly less TNF-alpha in response to pfGPI than macrophages from Jnk1(-/-), Jnk3(-/-), and wild-type counterparts. Furthermore, we demonstrate a role for JNK2 in mediating inflammatory responses and severe malaria in vivo. In contrast to wild-type or Jnk1(-/-) mice, Jnk2(-/-) mice had lower levels of TNF-alpha in vivo and exhibited significantly higher survival rates when challenged with Plasmodium berghei ANKA. These results provide direct evidence that pfGPI induces TNF-alpha secretion through activation of MAPK pathways, including JNK2. These results suggest that JNK2 is a potential target for therapeutic interventions in severe malaria.

Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2.

The up-regulation of phase II detoxifying and stress-responsive genes is believed to play an important role in cancer prevention, and many natural compounds have been shown to be potent inducers of these genes. Previous studies showed that the antioxidant responsive element (ARE), found in these genes, can be bound by the transcription factor Nrf2, and is responsive to the activation by chemopreventive compounds and by oxidative stress. In the present study, we investigated the roles of extracellular signal-regulated kinase (ERK) and c-Jun-NH(2)-kinase (JNK) in the regulation of phenethyl isothiocyanate (PEITC)-induced and Nrf2-dependent ARE activity and ARE-driven heme oxygenase-1 (HO-1) gene expression in PC-3 cells. ARE activity and HO-1 expression were strongly increased after treatment with PEITC. PEITC also increased the phosphorylation of ERK1/2 and JNK1/2 and caused release of Nrf2 from sequestration by Keap1, and its subsequent translocation into the nucleus. Importantly, Nrf2 was also translocated into the nucleus after transfection with ERK or JNK and that these activated ERK and JNK colocalized with Nrf2 in the nucleus. Activation of ERK and JNK signaling also resulted in the elevation of ARE activity and HO-1 expression. Importantly, PEITC-induced ARE activity was attenuated by inhibition of ERK and JNK signaling. In vitro kinase assays showed that both ERK2 and JNK1 could directly phosphorylate glutathione S-transferase-Nrf2 protein. Taken together, these results strongly suggest a model in which PEITC treatment of PC-3 cells activates ERK and JNK, which, in turn, phosphorylate Nrf2 and induce its translocation to the nucleus. Nuclear Nrf2 activates ARE elements and induces expression of stress-responsive genes, including HO-1.

Hyperphosphorylation of extracellular regulated kinase 2 (ERK2) and inhibition of JNK2 phosphorylation are associated with increased S-phase during transformation of Syrian hamster embryo cells by Malachite Green.

Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.