JNK Inhibitor SP600125 Research Articles

Abstract 1469: T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase

Abstract T-oligo, a guanine-rich oligonucleotide (GRO) homologous to the 3′ overhang of telomeres, elicits potent DNA-damage responses (DDRs) in cancer cells. However, the detailed molecular mechanism of T-oligo in cancer cells remains elusive. Recent studies confirmed that T-oligo can form intermolecular G-quadruplexes (G4), which are stabilized by hydrogen bonding of guanine residues, and cause disruption of the protective shelterin complex of telomeres. We hypothesize that single-stranded (SS) T-oligo and G4 T-oligo may modulate the shelterin proteins TRF2 and POT2 and thus induce DDRs. In this study, we utilized a pull-down assay using T-oligo, showing that T-oligo is co-localized with TRF2 and POT1 and indicating that T-oligo may interact with these telomeric proteins. We further investigated the modulation of these proteins by western blotting, showing that T-oligo treatment upregulates TRF2 by 2.2 and 3.0-fold (p<0.01) at 48h and 72h, respectively, and POT1 by 3.0-fold (p<0.02) both at 48h and 72h in melanoma cells (MM-AN). Immunofluorescence studies confirmed upregulation of TRF2 (2.4-fold) and POT1 (2.0-fold). Additionally we found that T-oligo can co-localize with telomere binding proteins TRF2 (88.4±4.5%) and POT1 (84.5±8%) using immunofluorescence. Using qRT-PCR, we found that T-oligo inhibited mRNA expression of hTERT, a catalytic subunit of telomerase, by 50%. It has been suggested that JNK activation may lead to downregulation of hTERT, hence we investigated the effect of the JNK inhibitor SP600125 on hTERT expression and found that treatment with SP600125 in presence of T-oligo partially reversed the downregulation of hTERT. We found a 16% decrease in hTERT expression in comparison to 50% reduction by T-oligo treatment alone. Recently, it has been reported that the novel drug 6-Thio-2'-Deoxyguanosine (6-thio-dG), a nucleoside analogue of the approved drug 6-thioguanine, inhibits growth of cancer cells by its incorporation into the telomere via telomerase followed by subsequent shelterin disruption. Furthermore, this study aims to investigate the potential of 6-thio-dG in combination with T-oligo as an anticancer therapeutic. We first studied the concentrations of 6-thio-dG that are effective in inhibiting the growth of melanoma cells. We found that 1.25μM and 2.5μM 6-thio-dG significantly inhibited growth of melanoma cells by 1.6 and 3.8-fold, respectively (p<0.01). However, treatment of melanoma cells in combination with both 10μM T-oligo and 6-thio-dG (1.25μM or 2.5μM) did not significantly inhibit cell growth in comparison to T-oligo alone. T-oligo may downregulate hTERT, which is required for telomerase activity, and/or disrupt the shelterin complex, both of which are necessary for 6-thio-dG mediated inhibition of growth of melanoma cells. These results indicate that T-oligo and 6-thio-dG may induce their effects by a similar mechanism of action. Citation Format: Nabiha Haleema Khan, Zachary Schrank, Joseph Kellen, Sanjana Singh, Chike Osude, Neelu Puri, Gagan Chhabra. T-oligo mediates DNA damage responses by modulating telomere associated proteins and telomerase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1469.

Abstract 271: Diterpenoid pseudolaric acid B induces M-phase arrest and apoptosis primarily through JNK stress signaling in LNCaP prostate cancer cells

Abstract Pseudolaric acid B (PAB) is a major diterpenoid component isolated from the root bark of the traditional Chinese medicinal plant Pseudolarix kaempferi Gordon (Pinaceae). PAB has shown potential as an anticancer agent via inducing cell cycle arrest, apoptosis and DNA damage in several cancer models. To explore potential use of PAB for prostate cancer chemoprevention, we used human LNCaP prostate cancer cells that harbor wild-type p53, functional androgen receptor (AR), and defective PTEN that are characteristic of early stage prostate adenocarcinomas to study cell fates and signaling mechanisms upon PAB exposure in cell culture. Our results show that PAB inhibited LNCaP cell proliferation in a dose- and time-dependent manner (IC50 ~5.4 µM at 24 h and ~ 0.2 µM at 72 h). PAB induced M phase cell cycle arrest as confirmed by increased phosphorylation of histone H3 (p-H3 Ser10). PAB also induced apoptosis evidenced by Annexin V staining and cleavage of the canonical caspase substrate PARP. PAB at lethal exposure concentration decreased AR protein and its best-known target prostate-specific antigen (PSA). Timewise, PAB treatment increased p-H3 (mitosis marker) and the expression of another mitosis/prosurvival protein survivin several hours ahead of PARP cleavage. PAB induced p-H2A.X, which would mark DNA double-strand breaks, and the best-known DNA damage response protein P53 and its target P21Cip1 preceding cPARP. PAB stimulated phosphorylative activation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 decreased PAB-induced p-H2A.X, P53/P21Cip1 and cleavage of PARP as well as M phase arrest. Furthermore, PAB significantly increased 2',7'-dichlorofluorescin diacetate (DCF)-reactive oxygen species (DCF-ROS) in LNCaP cells and addition of antioxidant N-acetyl-L-cysteine (NAC) attenuated P53/P21Cip1 cascade and cPARP/apoptosis, but did not affect M phase arrest. Knocking-down of P53 or P21Cip1 by siRNA attenuated cPARP and apoptosis without affecting p-H2A.X. Taken together, these results suggest a rapid induction of M phase arrest by PAB that was primarily mediated by JNK stress signaling, either parallel to or upstream of ROS-DNA damage-driven P53-P21Cip1 apoptosis cascade in LNCaP cells. Citation Format: Kartick C. Pramanik, Nehal Gupta, Min Ye, Junxuan Lu, Cheng Jiang. Diterpenoid pseudolaric acid B induces M-phase arrest and apoptosis primarily through JNK stress signaling in LNCaP prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 271.

Abstract 4381: Cordycepin promotes apoptosis in renal carcinoma cells by activating the MKK7-JNK signaling pathway through inhibition of cFLIPL expression

Abstract Cellular FLICE inhibitory protein (c-FLIP) is a key anti-apoptotic regulator that associates with the signaling complex downstream of NF-κB, negatively interfering with apoptotic signaling. The role of c-FLIP downregulation by negative regulation of NF-κB signaling during apoptosis is poorly understood. Here, we demonstrate that NF-κB-mediated c-FLIPL negatively regulates the JNK signaling pathway, and that cordycepin treatment of human renal cancer cells leads to apoptosis induction through c-FLIPL inhibition. TNF-α-induced inflammatory microenvironments stimulated NF-κB signaling and the c-FLIP long form (c-FLIPL) in TK-10 cells. Specifically, cordycepin inhibited TNF-α-mediated NF-κB activation, which induced renal cancer cell apoptosis. Cordycepin downregulated GADD45B and c-FLIPL, but upregulated MKK7 and phospho-JNK, by preventing nuclear mobilization of NF-κB. Furthermore, siRNA-mediated knockdown of GADD45B in cordycepin-treated TK-10 cells considerably increased MKK7 compared to cordycepin alone. siRNA-mediated knockdown of cFLIPL prevented TNF-α-induced JNK inactivation, whereas c-FLIPL overexpression inhibited cordycepin-mediated JNK activation. The JNK inhibitor SP600125 strongly inhibited Bax expression. In nude mice, cordycepin significantly decreased tumor volume. Taken together, the results indicate that cordycepin inhibits TNF-α-mediated NF-κB/GADD45B signaling, which activates the MKK7-JNK signaling pathway through inhibition of c-FLIPL expression, thus inducing TK-10 cell apoptosis. Citation Format: Ik-Soon Jang, Hyun-Jin Jang, Eunbi Jo. Cordycepin promotes apoptosis in renal carcinoma cells by activating the MKK7-JNK signaling pathway through inhibition of cFLIPL expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4381.

PO-076 HMNQ induces apoptosis and autophagy dependent on reactive oxygen species through activation of JNK signalling pathway

Introduction8-hydroxy-2-methoxy-1,4-naphthoquinone (HMNQ), a natural compound isolated from bark of Juglans sinensis Dode, has been previously reported to possess cytotoxic activity toward various human cancer cells. However, the molecular mechanism of its anticancer effect remains unknown.Material and methodsIn this study, the anticancer activity and molecular mechanism of HMNQ were investigated using cell viability/colony formation assay and wound healing assay. In addition, apoptosis analysis and measerement of mitochondria membrane potential were performed.Results and discussionsOur results showed that HMNQ reduced cell viability, decreased colony formation, and inhibited cell migration in breast, lung, and colon cancer cells. HMNQ effectively induced apoptosis by upregulating the expression of pro-apoptotic protein Bax, cleaved PARP, and downregulating the expression of anti-apoptotic protein Bcl-2 in A549 and MCF7 cells. In addition, HMNQ also induced reactive oxygen species (ROS) production through the decreased mitochondrial-membrane potential and this effect was attenuated by ROS scavengers, N-acetylcysteine (NAC) and l-glutathione (GSH). Furthermore, HMNQ increased the expression of JNK phosphorylation and JNK inhibitor SP600125 suppressed HMNQ-induced decrease in cell viability.ConclusionTaken together, our findings suggest that HMNQ exhibits anti-proliferative activity through induction of ROS-mediated apoptosis in human cancer cells, indicating that HMNQ as a potential anticancer agent.

Epidermal-specific deletion of TC-PTP promotes UVB-induced epidermal cell survival through the regulation of Flk-1/JNK signaling

UVB exposure can contribute to the development of skin cancer by modulating protein tyrosine kinase (PTK) signaling. It has been suggested that UVB radiation increases the ligand-dependent activation of PTKs and induces PTP inactivation. Our recent studies have shown that T-cell protein tyrosine phosphatase (TC-PTP) attenuates skin carcinogenesis induced by chemical regimens, which indicates its critical role in the prevention of skin cancer. In the current work, we report that TC-PTP increases keratinocyte susceptibility to UVB-induced apoptosis via the downregulation of Flk-1/JNK signaling. We showed that loss of TC-PTP led to resistance to UVB-induced apoptosis in vivo epidermis. We established immortalized primary keratinocytes (IPKs) from epidermal-specific TC-PTP-deficient (K14Cre.Ptpn2fl/fl) mice. Immortalized TC-PTP-deficient keratinocytes (TC-PTP/KO IPKs) showed increased cell survival against UVB-induced apoptosis which was concomitant with a UVB-mediated increase in Flk-1 phosphorylation, especially on tyrosine residue 1173. Inhibition of Flk-1 by either its specific inhibitors or siRNA in TC-PTP/KO IPKs reversed this effect and significantly increased cell death after UVB irradiation in comparison with untreated TC-PTP/KO IPKs. Immunoprecipitation analysis using the TC-PTP substrate-trapping mutant TCPTP-D182A indicated that TC-PTP directly interacts with Flk-1 to dephosphorylate it and their interaction was stimulated by UVB. Following UVB-mediated Flk-1 activation, the level of JNK phosphorylation was also significantly increased in TC-PTP/KO IPKs compared to control IPKs. Similar to our results with Flk-1, treatment of TC-PTP/KO IPKs with the JNK inhibitor SP600125 significantly increased apoptosis after UVB irradiation, confirming that the effect of TC-PTP on UVB-mediated apoptosis is regulated by Flk-1/JNK signaling. Western blot analysis showed that both phosphorylated Flk-1 and phosphorylated JNK were significantly increased in the epidermis of TC-PTP-deficient mice compared to control mice following UVB. Our results suggest that TC-PTP plays a protective role against UVB-induced keratinocyte cell damage by promoting apoptosis via negative regulation of Flk-1/JNK survival signaling.

Study on effect and mechanism of Honokiol enhancing tumor necrosis factor-related apoptosis inducing ligand against hepatocellular carcinoma HepG2 cells via activating JNK signaling pathway

Objective: To investigate the effect and intrinsic mechanism of Honokiol (HNK) enhanced tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on hepatocellular carcinoma HepG2 cells. Methods: HepG2 cells were routinely cultured. Firstly, a concentration gradient of HNK was given to observe its effect on the vitality of tumor cells. Western blot were used to detect change in the expression levels of c-jun N-terminal kinase (JNK), death receptor 4 (DR4), and DR5.Secondly, we observed the effect of combined drugs (HNK and TRAIL) on the vitality of tumor cells. Apoptosis-related protein expression levels were detected to determine the apoptosis condition. Thirdly, JNK inhibitor SP600125 was used to block the JNK pathway, and it was evaluated whether JNK signaling pathway mediated the DR4 and DR5 levels and finally, the subcutaneous tumor model of nude mice was constructed, and enhancement effect of HNK on TRAIL was confirmed in vivo. Results: Cell vitality was decreased (P < 0.05) in a dose-dependent manner after treatment with gradient HNK. Combined effect of TRAIL and HNK was superior to that of single drug administration (P < 0.05). Western blot analysis showed that pJNK level increased after HNK treatment and TRAIL receptor DR4 and DR5 expression were up-regulated. Combined application of HNK and TRAIL, B lymphocyte tumor factor 2 (BCL2) decreased significantly while Bcl2 related X protein (Bax) increased significantly. Blocking JNK pathway by SP600125, the expression of DR4 and DR5 decreased (P < 0.05), Bax expression decreased and Bcl2 expression increased compared with HNK+TRAIL group. In vivo results showed that TRAIL inhibited the growth of subcutaneous tumors, and enhanced inhibition effect in combination with TRAIL and HNK. Conclusion: HNK may enhance the inhibitory effect of TRAIL on HepG2 cells by activating JNK pathway and then upregulating the expression of DR4 and DR5.

Novel role of PKR in palmitate-induced Sirt1 inactivation and endothelial cell senescence.

Endothelial cell senescence is regarded as a vital characteristic of cardiovascular diseases. Elevated palmitate (PA) is an independent risk factor of cardiovascular diseases, but its role in endothelial cell senescence is currently unknown. During the course of studying the prosenescent role of PA, we discovered a key role of dsRNA-dependent protein kinase [protein kinase R (PKR)] in endothelial senescence. Exposure of human umbilical vein endothelial cells (HUVECs) to PA-induced cell senescence is characterized by increased levels of senescence-associated β-galactose glucosidase activity, excessive production of reactive oxygen species production, impaired cellular proliferation, and G1 phase arrest. This phenomenon is associated with an increase of PKR autophosphorylation and decreased activity of sirtuin 1 (Sirt1), a pivotal antisenescent factor. PKR inactivation by PKR siRNA or its phosphorylation inhibitor 2-aminopurine significantly attenuated PA-induced HUVEC senescence by reversing Sirt1 activity and its downstream signaling. Moreover, to study the regulatory mechanism between PKR and Sirt1, we found that PKR promotes JNK activation to inhibit Sirt1 activity and that this effect could be reversed by the JNK inhibitor SP600125. These findings provide evidence that PKR mediates PA-induced HUVEC senescence by inhibiting Sirt1 signaling. Our study provides novel insights into the actions and mechanisms of PKR in endothelial senescence. NEW & NOTEWORTHY This study first provides a novel observation that dsRNA-dependent protein kinase (PKR) mediates palmitate-induced sirtuin 1 inactivation and subsequent human umbilical vein endothelial cell senescence. Most importantly, these new findings will provide a potential therapeutic strategy to improve free fatty acid-induced endothelial senescence by targeting PKR in cardiovascular diseases.

Synergistic Inhibition of ERK1/2 and JNK, Not p38, Phosphorylation Ameliorates Neuronal Damages After Traumatic Brain Injury.

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that play a critical role in signal transduction and are activated by phosphorylation in response to a variety of pathophysiology stimuli. While MAP kinase signaling has a significant role in the pathophysiology of several neurodegenerative diseases, the precise function of activation of MAP kinase in traumatic brain injury (TBI) is unknown. Therefore, it is important to study the role of MAP kinase signaling in TBI-associated neurological ailments. In this study, using an in vitro stretch injury model in rat embryo neuronal cultures and the in vivo fluid percussion injury (FPI) model in rats, we explored the role of MAP kinase signaling in the mechanisms of cell death in TBI. Our study demonstrated that the stretch injury in vitro and FPI in vivo upregulated the phosphorylation of MAP kinase proteins ERK1/2 and JNK, but not p38. Using ERK1/2 inhibitor U0126, JNK inhibitor SP600125, and p38 inhibitor SB203580, we validated the role of MAP kinase proteins in the activation of NF-kB and caspase-3. By immunofluorescence and western blotting, further, we demonstrated the role of ERK1/2 and JNK phosphorylation in neurodegeneration by analyzing cell death proteins annexin V and Poly-ADP-Ribose-Polymerase p85. Interestingly, combined use of ERK1/2 and JNK inhibitors further attenuated the cell death in stretch-injured neurons. In conclusion, this study could establish the significance of MAP kinase signaling in the pathophysiology of TBI and may have significant implications for developing therapeutic strategies using ERK1/2 and JNK inhibitors for TBI-associated neurological complications.

LPS-induced MMP-9 expression is mediated through the MAPKs-AP-1 dependent mechanism in BEAS-2B and U937 cells

Aim of the Study: Matrix metalloproteinases (MMPs) play a critical role in chronic obstructive pulmonary disease (COPD). This study investigated the role of mitogen-activated protein kinases (MAPKs) in MMP-9 secretion of BEAS-2B cells, a human bronchial epithelial cell line and U937 cells, a human myeloid leukaemia cell line, which could differentiate into macrophage, after LPS stimulation, and some details of involved signaling. Materials and Methods: MTT assay was used to measure cell viability. U937 cells were incubated for 48h with 100ng/ml PMA, and had a resting period of 24h with culture medium without PMA for differentiation of U937 cells into macrophages. For the experiments, U937 cells or BEAS-2B cells were pretreated with several inhibitors and then stimulated by LPS. Western blotting, quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and DNA binding activity assay were used for measuring the protein expression, RNA expression, cytokine production and DNA binding activity, respectively. Results: We found LPS induced MMP-9 expression and secretion were completely blocked by stress-activated protein kinase/jun kinase (SAPK/JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors but not by p38 inhibitor. LPS-induced transactivation of AP-1 was also inhibited by JNK inhibitor SP600125 and ERK1/2 inhibitor PD98059. Conclusions: The present study suggests that in BEAS-2B cells and U937 cells, LPS probably activates ERK1/2 pathway and JNK pathway, which in turn initiate AP-1 activity, and leading to MMP-9 expression. Thus the ERK1/2 inhibitor and JNK inhibitor may have potential clinical value in treating COPD.

CD137-CD137L signaling influences the autophagy via JNK pathway in mouse vascular smooth muscle cells

Objective: To investigate whether CD137-CD137L signaling can affect the autophagy of mouse vascular smooth muscle cells(VSMCs) through JNK signal pathway. Methods: Primary culture of C57BL/6J mouse thoracic aorta VSMCs was performed by tissue block adherence method. VSMCs between the third to fifth passages were isolated and cultured. VSMCs were divided into 4 groups: control group, CD137 agonist group, JNK inhibition group, and DMSO group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 μg/ml), VSMCs in JNK inhibition group were treated with JNK inhibitor SP600125 (10 μmol/L) for 30 minutes followed by recombinant protein of CD137L (10 μg/ml) and DMSO group was treated with the same amount of DMSO in JNK inhibition group for 30 minutes, then added recombinant protein of CD137L (10 μg/ml). Western blot was used to detect the protein expression of p-JNK, LCⅡ and p62 in each group. Fluorescence microscopy was used to track the changes of autophagy in cells which was infected with adenovirus expressing tandem mRFP-GFP-LC3. Transmission electron microscope (TEM) was used to observe intracellular autophagosomes and autolysosomes. Results: (1) Compared with the control group, stimulating CD137-CD137L axis by recombinant protein of CD137L significantly upregulated the expression of p-JNK, LCⅡ and p62 (1.15±0.19 vs. 0.72±0.21, P<0.05;1.03±0.13 vs. 0.59±0.15, P<0.05, and 1.10±0.19 vs. 0.76±0.15, P<0.05). These effects could be reduced by JNK inhibitor (0.61±0.21 vs. 1.15±0.19, P<0.05;0.74±0.11 vs. 1.03±0.13, P<0.05, and 0.21±0.12 vs. 1.10±0.19, P<0.05). The expression of these proteins in DMSO group remained unchanged compared with CD137 agonist group (P>0.05). (2) Changes of autophagy in cells of various group: the number of total fluorescent spots and yellow fluorescent spots in CD137 agonist group was significantly increased compared to control group (total fluorescent spots:(93.00±14.11)/cell vs. (52.33±9.61)/cell, P<0.05, and (64.33±6.81)/cell vs. (25.67±3.51)/cell, P<0.05), moreover, the number of yellow fluorescent spots was higher than the red fluorescent spots fluorescent spots in CD137 agonist group. Compared with CD137 agonist group, pretreatment with JNK inhibitor significantly reduced the number of total fluorescent spots and yellow fluorescent spots ((53.00±3.17)/cell vs. (93.00±14.11)/cell, P<0.05,and (15.33±4.51)/cell vs. (64.33±6.81)/cell, P<0.05). The red fluorescent spots were higher than the yellow fluorescent spots in JNK inhibition group. The number of total fluorescent spots and yellow fluorescent spots in CD137 agonist group was not affected by pretreatment with DMSO (P>0.05). (3) The number of intracellular autophagosomes and autolysosomes was significantly higher in CD137 agonist group than in control group((17.67±6.03)/cell vs. (5.67±2.52)/cell, P<0.05), and the number of autophagosomes was higher than that of autolysosomes in CD137 agonist group((14.00±4.00)/cell vs. (3.67±2.08)/cell, P<0.05). The number of intracellular autophagosomes and autolysosomes was significantly lower in JNK inhibition group compared to CD137 agonist group((5.67±4.04)/cell vs. (17.67±6.03)/cell, P<0.05) and the number of autophagosomes was lower than that of autolysosomes in JNK inhibition group((1.33±1.53)/cell vs. (4.33±2.52)/cell, P<0.05). The number of intracellular autophagosomes and autolysosomes was similar between DMSO group and CD137 agonist group (P>0.05). Conclusion: CD137-CD137L signal may influence autophagy of mouse VSMCs via JNK pathway.

Peroxiredoxin 2 deficiency accelerates age-related ovarian failure through the reactive oxygen species-mediated JNK pathway in mice

Reactive oxygen species (ROS) produced in biological reactions have been shown to contribute to ovarian aging. Peroxiredoxin 2 (Prx2) is an antioxidant enzyme that protects cells by scavenging ROS; however, its effect on age-related, oxidative stress-associated ovarian failure has not been reported. Here, we investigated its role in age-related ovarian dysfunction and 4-vinylcyclohexene diepoxide (VCD)-induced premature ovarian failure using Prx2-deficient mice. Compared to those in wildtype (WT) mice, serum levels of anti-Müllerian hormone, 17β-estradiol, and progesterone and numbers of follicles and corpora lutea were significantly lower in 18-month-old Prx2−/− mice. Moreover, levels of Bax, cytochrome c, cleaved caspase-3, and phosphorylated JNK proteins were higher and numbers of apoptotic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cells were considerably greater in 18-month-old Prx2−/− ovaries than WT ovaries. Furthermore, the effects of the ovarian toxicant VCD in significantly enhancing ROS levels and apoptosis through activation of JNK-mediated apoptotic signaling were more pronounced in Prx2−/− than WT mouse embryonic fibroblasts. Expression of the steroidogenic proteins StAR, CYP11A1, and 3β-HSD and serum levels of 17β-estradiol and progesterone were also reduced to a greater extent in Prx2−/− mice than WT mice after VCD injection. This reduced steroidogenesis was rescued by addition of the Prx mimic ebselen or JNK inhibitor SP600125. This constitutes the first report that Prx2 deficiency leads to acceleration of age-related or VCD-induced ovarian failure by activation of the ROS-induced JNK pathway. These findings suggest that Prx2 plays an important role in preventing accelerated ovarian failure by inhibiting ROS-induced JNK activation.

Oxymatrine Causes Hepatotoxicity by Promoting the Phosphorylation of JNK and Induction of Endoplasmic Reticulum Stress Mediated by ROS in LO2 Cells.

Oxymatrine (OMT) often used in treatment for chronic hepatitis B virus infection in clinic. However, OMT–induced liver injury has been reported. In this study, we aim to investigate the possible mechanism of OMT-induced hepatotoxicity in human normal liver cells (L02). Exposed cells to OMT, the cell viability was decreased and apoptosis rate increased, the intracellular markers of oxidative stress were changed. Simultaneously, OMT altered apoptotic related proteins levels, including Bcl-2, Bax and pro-caspase-8/-9/-3. In addition, OMT enhanced the protein levels of endoplasmic reticulum (ER) stress makers (GRP78/Bip, CHOP, and cleaved-Caspase-4) and phosphorylation of c-Jun N-terminal kinase (p-JNK), as well as the mRNA levels of GRP78/Bip, CHOP, caspase-4, and ER stress sensors (IREI, ATF6, and PERK). Pre-treatment with Z-VAD-fmk, JNK inhibitor SP600125 and N-acetyl-l-cysteine (NAC), a ROS scavenger, partly improved the survival rates and restored OMT-induced cellular damage, and reduced caspase-3 cleavage. SP600125 or NAC reduced OMT-induced p-JNK and NAC significantly lowered caspase-4. Furthermore, 4-PBA, the ER stress inhibitor, weakened inhibitory effect of OMT on cells, on the contrary, TM worsen. 4-PBA also reduced the levels of p-JNK and cleaved-caspase-3 proteins. Therefore, OMT-induced injury in L02 cells was related to ROS mediated p-JNK and ER stress induction. Antioxidant, by inhibition of p-JNK or ER stress, may be a feasible method to alleviate OMT-induced liver injury.

The Anti-neuroinflammatory Activity of Tectorigenin Pretreatment via Downregulated NF-κB and ERK/JNK Pathways in BV-2 Microglial and Microglia Inactivation in Mice With Lipopolysaccharide.

The activation of microglia is decisively involved with the neurodegeneration observed in many neuroinflammatory pathologies, such as multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease. Tectorigenin (TEC) is an isoflavone isolated from various medicinal plants, such as Pueraria thunbergiana Benth, Belamcanda chinensis, and Iris unguicularis. In the present study, the neuroinflammatory effects of TEC were evaluated in both lipopolysaccharide (LPS)-treated BV-2 microglial and mouse models. TEC remarkably inhibited reactive oxygen species (ROS) generation. TEC also inhibits the production and expression of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in LPS-stimulated BV-2 cells. In addition, TEC suppressed the LPS-induced activation of nuclear factor-κB (NF-κB), phosphorylation of extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) to regulate the inflammatory mediators, such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, and IL-6. These results indicate that TEC may inhibit neuronal inflammation through the downregulation of inflammatory mediators, including iNOS, COX-2, TNF-α, and IL-6 by suppressing NF-κB/ERK/JNK-related signaling pathways. Furthermore, cotreatment with TEC and ERK inhibitor SCH772984 or JNK inhibitor SP600125 suppressed the overproduction of LPS-induced NO production in BV-2 cells. Consistent with the results of in vitro experiments, an LPS-induced brain inflammation mouse model, administration of TEC effectively decrease the levels of malondialdehyde, iNOS in hippocampus, and prevented increases in the levels of TNF-α and IL-6 in the serum. TEC showed marked attenuation of microglial activation. Finally, TEC inhibited protein expression of toll-like receptor 4 and myeloid differentiation factor 88 in LPS-activated BV-2 microglia and mouse models. Taken altogether, the cumulative findings suggested that TEC holds the potential to develop as a neuroprotective drug for the intervention of neuroinflammatory disorders.

Luteolin and sorafenib combination kills human hepatocellular carcinoma cells through apoptosis potentiation and JNK activation.

Sorafenib is a small-molecule multi-kinase inhibitor approved by FDA as an oral agent for the treatment of hepatocellular carcinoma (HCC) and renal cell carcinoma. However, unresponsiveness and acquired resistance are commonly observed, which hinder the clinical use of sorafenib. As combination therapy is a promising approach to improve its efficacy, we investigated if sorafenib and luteolin combination is effective in killing human HCC cells. Cell death was examined by lactate dehydrogenase (LDH) releasing assay. Apoptosis was detected by flow cytometric. The activation of apoptotic pathway and c-Jun N-terminal kinase (JNK) signaling pathway was measured by western blot. The results showed that sorafenib and luteolin combination synergistically induced cytotoxicity in HCC cells, which was accompanied by potentiation of apoptosis as demonstrated by increased apoptotic cell populations, caspase activation, and suppression of cell death by the pan-caspase inhibitor z-VAD-fmk. Furthermore, the combination of both agents enhanced expression of phosphorylated form of JNK, and the JNK inhibitor SP600125 effectively attenuated cell death induced by the combination treatment. Thus, sorafenib and luteolin combination synergistically kills HCC cells through JNK-mediated apoptosis, and luteolin may be an ideal candidate for increasing the activity of sorafenib in HCC therapy.

Functional cooperation between HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR

ObjectiveHypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are important transcription factors regulating expression of genes involved in cell survival. HIF-1α and c-Jun are key components of HIF-1 and AP-1, respectively, and are regulated by epidermal growth factor receptor (EGFR)-mediated cell signaling and tumor microenvironmental cues. The roles of HIF-1α and c-Jun in development of resistance to EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) with activating mutation of EGFR have not been explored. In this study, we investigated the roles of HIF-1α and c-Jun in mediating primary and acquired resistance to gefitinib in NSCLC cells with activating mutation of EGFR. Materials and methodsChanges in HIF-1α protein and in total and phosphorylated c-Jun levels in relation to changes in total and phosphorylated EGFR levels before and after gefitinib treatment were measured using Western blot analysis in NSCLC cells sensitive or resistant to gefitinib. The impact of overexpression of a constitutively expressed HIF-1α (HIF-1α/ΔODD) or a constitutively active c-Jun upstream regulator (SEK1 S220E/T224D mutant) on cell response to gefitinib was also examined. The effect of pharmacological inhibition of SEK1-JNK-c-Jun pathway on cell response to gefitinib was evaluated. ResultsDownregulation of HIF-1α and total and phosphorylated c-Jun levels correlated with cell inhibitory response to gefitinib better than decrease in phosphorylated EGFR did in NSCLC cells with intrinsic or acquired resistance to gefitinib. Overexpression of HIF-1α/ΔODD or SEK1 S220E/T224D mutant conferred resistance to gefitinib. There exists a positive feed-forward regulation loop between HIF-1 and c-Jun. The JNK inhibitor SP600125 sensitized gefitinib-resistant NSCLC cells to gefitinib. ConclusionsHIF-1α and c-Jun functionally cooperate in development of resistance to gefitinib in NSCLC cells. The translational value of inhibiting HIF-1α/c-Jun cooperation in overcoming resistance to EGFR TKI treatment of NSCLC cells with activating mutation of EGFR deserves further investigation.

Low-dose tributyltin exposure induces an oxidative stress-triggered JNK-related pancreatic \u03b2-cell apoptosis and a reversible hypoinsulinemic hyperglycemia in mice

Tributyltin (TBT), an endocrine disrupting chemical, can be found in food (particular in fish and seafood) and drinking water by contamination. Here, we elucidated the effects and possible mechanisms of low-dose TBT on the growth and function of pancreatic β-cells and glucose metabolism in mice. Submicromolar-concentration of TBT significantly induced β-cell cytotoxicity and apoptosis, which were accompanied by poly (ADP-ribose) polymerase cleavage and mitogen-activated protein kinases-JNK and ERK1/2 phosphorylation. TBT could also suppress the glucose-stimulated insulin secretion in β-cells and isolated mouse islets. TBT increased reactive oxygen species production. TBT-induced β-cell cytotoxicity and apoptosis were significantly prevented by antioxidant N-acetylcysteine (NAC) and JNK inhibitor SP600125, but not ERK1/2 inhibitor PD98059 and p38 inhibitor SB203580. Both NAC and SP600125 inhibited JNK phosphorylation and reduced cell viability in TBT-treated β-cells. Four-week exposure of TBT (0.25 mg/kg) to mice revealed the decreased plasma insulin, increased blood glucose and plasma malondialdehyde, suppressed islet insulin secretion, and increased islet caspase-3 activity, which could be reversed by NAC treatment. After removing the TBT exposure for 2 weeks, the TBT-induced glucose metabolism alteration was significantly reversed. These results suggest that low-dose TBT can induce β-cell apoptosis and interfere with glucose homeostasis via an oxidative stress-related pathway.

Thrombin promotes PAI-1 expression and migration in keratinocytes via ERK dependent Smad linker region phosphorylation

Keratinocyte proliferation and migration is essential during re-epithelialisation for the restoration of the epithelial barrier during skin wound healing. Numerous growth factors are involved in the stimulation of keratinocyte proliferation and migration. The signalling pathways that drive these processes during wound healing are not well defined. This study investigated thrombin-mediated signalling in keratinocytes. The thrombin receptor, protease-activated receptor 1 (PAR-1) is a seven transmembrane G-protein coupled receptor that is known to transactivate the epidermal growth factor receptor (EGFR). Immortalized human keratinocytes (HaCaT cells) were treated with thrombin and selective inhibitors to EGFR and MAP kinases. Whole cell lysates were separated on SDS-PAGE and analysed by Western blot using antibodies against transcription factor Smad2. Quantitative real-time polymerase chain reaction was used to measure the mRNA expression of PAI-1 while scratch wound assays were used to measure keratinocyte migration. Western blot data showed that thrombin mediates PAR-1 transactivation of EGFR and the downstream phosphorylation of the transcription factor Smad2 linker (Smad2L) region. ERK1/2 inhibition by UO126 caused a decrease in Smad2L phosphorylation while the p38 inhibitor SB202190 and JNK inhibitor SP600125 did not. Smad2L Ser250 was specifically phosphorylated by this thrombin mediated pathway while Ser245 and Ser255 were not. Thrombin increased PAI-1 mRNA expression and keratinocyte migration and this was reduced when either EGFR or ERK1/2 were blocked. Taken together these results show that thrombin mediated mRNA expression of PAI-1 in keratinocytes and migration occurs via EGFR transactivation and involves signalling intermediates ERK1/2 and Smad2 and may be a key pathway in skin wound healing.

RBBP6 promotes human cervical carcinoma malignancy via JNK signaling pathway

Previous studies have shown that retinoblastoma binding protein 6 (RBBP6) was overexpressed in malignant tumors and was correlated with poorer prognosis in various cancers. However, its role in cervical carcinoma has not been elucidated. This study was to investigate the relationship between RBBP6 and cervical carcinoma. Cervical carcinoma cell lines SiHa and C33a were used to assess the effect of RBBP6 on cell viability, migration, and proliferation. RBBP6 mRNA and protein levels in cervical cancer tissues increased at least three times as that in the adjacent non-cancerous tissues. Overexpression of RBBP6 in SiHa and C33a cell lines resulted in increased phosphorylated c-Jun NH2-terminal kinase (p-JNK) as well as increased cell viability, migration, and proliferation. Moreover, this effect was suppressed by specific JNK inhibitor SP600125. RBBP6 might potentiate cervical carcinoma cell viability, migration and proliferation through JNK signaling pathway. RBBP6 and JNK inhibitor may be beneficial as a novel preventive and therapeutic target for cervical cancer.

Remifentanil preconditioning confers cardioprotection via c-Jun NH2-terminal kinases and extracellular signal regulated kinases pathways in ex-vivo failing rat heart

Remifentanil preconditioning (RPC) exerts protection in normal hearts, but has not been investigated in heart failure. The aim of the present study was to evaluate the effect of RPC in a chronic failing rat heart model and the mechanisms involving mitogen-activated protein kinases (MAPK) and Bcl-2 protein family. The doxorubicin induced failing rat hearts were subjected to 30 min ischemia / 120 min reperfusion (IR) with or without RPC by using Langendorff apparatus. RPC was induced by three cycles of 5 min remifentanil / 5 min drug-free perfusion before IR, with three different concentrations: 25, 50 and 100 μg/l. An extracellular signal regulated kinases (ERK) inhibitor PD98059, p38MAPK inhibitor SB203580, c-Jun NH2-terminal kinases (JNK) inhibitor SP600125 were perfused at 10 min before RPC. Infarct size, cardiac function and protein kinase activity were determined. RPC significantly reduced infarct size and the rise in lactate dehydrogenase (LDH) level caused by IR injury in failing heart. The JNK inhibitor SP600125 and ERK inhibitor PD98059 abolished the RPC mediated reduction effect on the infarct size and LDH activity after reperfusion. In addition, RPC increased the phosphorylation of JNK, ERK1/2 and the downstream GSK-3β, as well as the Bcl-2/Bax ratio, while, these changes were completely reversed by SP600125 and PD98059. And of note, SB203580 had no effect. In conclusion, our results suggested that the activation of JNK and ERK pathways, by leading to inhibition of GSK-3β and regulating Bcl-2 protein family, is a major mechanism that RPC confers cardioprotection in failing rat heart.

Wedelolactone Enhances Osteoblastogenesis through ERK- and JNK-mediated BMP2 Expression and Smad/1/5/8 Phosphorylation.

Our previous study showed that wedelolactone, a compound isolated from Ecliptae herba, has the potential to enhance osteoblastogenesis. However, the molecular mechanisms by which wedelolactone promoted osteoblastogenesis from bone marrow mesenchymal stem cells (BMSCs) remain largely unknown. In this study, treatment with wedelolactone (2 μg/mL) for 3, 6, and 9 days resulted in an increase in phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal protein kinase (JNK), and p38. Phosphorylation of mitogen-activated protein kinases (MAPKs), ERK and JNK started to increase on day 3 of treatment, and p38 phosphorylation was increased by day 6 of treatment. Expression of bone morphogenetic protein (BMP2) mRNA and phosphorylation of Smad1/5/8 was enhanced after treatment of cells with wedelolactone for 6 and 9 days. The addition of the JNK inhibitor SP600125, ERK inhibitor PD98059, and p38 inhibitor SB203580 suppressed wedelolactone-induced alkaline-phosphatase activity, bone mineralization, and osteoblastogenesis-related marker genes including Runx2, Bglap, and Sp7. Increased expression of BMP2 mRNA and Smad1/5/8 phosphorylation was blocked by SP600125 and PD98059, but not by SB203580. These results suggested that wedelolactone enhanced osteoblastogenesis through induction of JNK- and ERK-mediated BMP2 expression and Smad1/5/8 phosphorylation.