Contribution of persistent C-Jun N-terminal kinase activity to the survival of human vestibular schwannoma cells by suppression of accumulation of mitochondrial superoxides.
ABSTRACT: Vestibular schwannomas (VSs) result from inactivating mutations in the merlin tumor suppressor gene. The merlin protein suppresses a variety of progrowth kinase-signaling cascades, including extracellular regulated kinase/mitogen-activated protein kinase (ERK/MAPK), c-Jun N-terminal kinase (JNK), and phosphatidyl-inositol 3-kinase (PI3-K)/Akt. Recent studies indicate that ERKs and Akt are active in human VSs, and here we show that JNKs are also persistently active in human VS cells. With use of cultures of human VSs, we investigated the contribution of each of these signals to the proliferative and survival response of VS cells. Inhibition of ERK or Akt signaling reduced VS cell proliferation but did not increase apoptosis, whereas inhibition of JNK with SP600125, I-JIP, or siRNA knock-down reduced VS cell proliferation and survival by inducing apoptosis. By contrast, JNK activity promotes apoptosis in normal Schwann cells. Inhibition of JNK increased the fluorescence intensity of VS cells loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), a fluorescent probe for reactive oxygen species (ROS). Furthermore, ebselen, a ROS scavenger, rescued VS cells with suppressed JNK from apoptosis, suggesting that JNK activity protects VS cells from apoptosis by limiting accumulation of ROS. VS cultures treated with JNK inhibitors demonstrated significantly higher levels of MitoSOX Red fluorescence, implying that persistent JNK activity specifically suppresses superoxide production in the mitochondria. Overexpression of superoxide dismutase 2 (MnSOD; mitochondrial SOD) prevented apoptosis in VS cells with suppressed JNK signaling. Taken together, these results indicate that persistent JNK activity enhances VS cell survival, at least in part, by suppressing accumulation of mitochondrial superoxides.
Project description:Radiosurgery is increasingly used to treat vestibular schwannomas (VSs). Increasing the sensitivity of VS cells to irradiation (IR) could allow for lower and/or more effective doses of IR, improving safety and efficacy. Persistent c-Jun N-terminal kinase (JNK) activity in VS cells reduces cell death by suppressing the accumulation of reactive oxygen species (ROS), raising the possibility that JNK activity protects against IR-induced VS cell death, which is mediated by ROS.To determine the extent to which JNK signaling contributes to VS cell radiosensitivity.Primary human VS cultures, derived from acutely resected tumors, received single doses (5-40 Gy) of gamma irradiation. Histone 2AX phosphorylation, a marker of IR-induced DNA damage, was assayed by Western blot and immunostaining. ROS levels were quantified by measuring 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescence. Cell apoptosis was determined by terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling.The JNK inhibitors SP6000125 and I-JIP reduced histone 2AX phosphorylation after IR. They also increased H2DCFDA fluorescence in nonirradiated cultures and significantly increased IR-induced (5-10 Gy) H2DCFDA fluorescence 72 hours, but not 2 hours, after IR. Finally, I-JIP (50 μmol/L) significantly increased VS cell apoptosis in cultures treated with 20 to 40 Gy. I-JIP (20 μmol/L), SP600125 (20 μmol/L), and JNK1/2 short interfering RNA knockdown each increased VS cell apoptosis in cultures treated with 30 to 40 Gy, but not lower doses, of IR.Inhibition of JNK signaling decreases histone 2AX phosphorylation and increases ROS and apoptosis in VS cells after gamma irradiation. These results raise the possibility of using JNK inhibitors to increase the effectiveness of radiosurgery for treatment of VSs.
Project description:Ethyl rosmarinate (RAE) is one of the active constituents from Clinopodium chinense (Benth.) O. Kuntze, which is used for diabetic treatment in Chinese folk medicine. In this study, we investigated the protective effect of RAE on high glucose-induced injury in endothelial cells and explored its underlying mechanisms. Our results showed that both RAE and rosmarinic acid (RA) increased cell viability, decreased the production of reactive oxygen species (ROS), and attenuated high glucose-induced endothelial cells apoptosis in a dose-dependent manner, as evidenced by Hochest staining, Annexin V?FITC/PI double staining, and caspase-3 activity. RAE and RA both elevated Bcl-2 expression and reduced Bax expression, according to Western blot. We also found that LY294002 (phosphatidylinositol 3-kinase, or PI3K inhibitor) weakened the protective effect of RAE. In addition, PDTC (nuclear factor-?B, or NF-?B inhibitor) and SP600125 (c-Jun N-terminal kinase, or JNK inhibitor) could inhibit the apoptosis in endothelial cells caused by high glucose. Further, we demonstrated that RAE activated Akt, and the molecular docking analysis predicted that RAE showed more affinity with Akt than RA. Moreover, we found that RAE inhibited the activation of NF-?B and JNK. These results suggested that RAE protected endothelial cells from high glucose-induced apoptosis by alleviating reactive oxygen species (ROS) generation, and regulating the PI3K/Akt/Bcl-2 pathway, the NF-?B pathway, and the JNK pathway. In general, RAE showed greater potency than RA equivalent.
Project description:Both phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen activated protein kinase (MAPK) signaling cascades play an important role in cell proliferation, survival, angiogenesis, and metastasis of tumor cells. In the present report, we investigated the effects of licochalcone A (LA), a flavonoid extracted from licorice root, on the PI3K/AKT/mTOR and MAPK activation pathways in human gastric cancer BGC-823 cells. LA increased reactive oxygen species (ROS) levels, which is associated with the induction of apoptosis as characterized by positive Annexin V binding and activation of caspase-3, and cleavage of poly-ADP-ribose polymerase (PARP). Inhibition of ROS generation by N-acetylcysteine (NAC) significantly prevented LA-induced apoptosis. Interestingly, we also observed that LA caused the activation of ERK, JNK, and p38 MAPK in BGC-823 cells. The antitumour activity of LA-treated BGC-823 cells was significantly distinct in KM mice in vivo. All the findings from our study suggest that LA can interfere with MAPK signaling cascades, initiate ROS generation, induce oxidative stress and consequently cause BGC cell apoptosis.
Project description:Human bladder cancer is a common genitourinary malignant cancer worldwide. However, new therapeutic strategies are required to overcome its stagnated survival rate. Triterpene glycoside Actein (ACT), extracted from the herb black cohosh, suppresses the growth of human breast cancer cells. Our study attempted to explore the role of ACT in human bladder cancer cell growth and to reveal the underlying molecular mechanisms. We found that ACT significantly impeded the bladder cancer cell proliferation via induction of G2/M cycle arrest. Additionally, ACT administration triggered autophagy and apoptosis in bladder cancer cells, proved by the autophagosome formation, LC3B-II accumulation, improved cleavage of Caspases/poly (ADP-ribose) polymerase (PARP). Furthermore, reduction of reactive oxygen species (ROS) and p-c-Jun N-terminal kinase (JNK) could markedly reverse ACT-induced autophagy and apoptosis. In contrast, AKT and mammalian target of rapamycin (mTOR) were greatly de-phosphorylated by ACT, while suppressing AKT and mTOR activity could enhance the effects of ACT on apoptosis and autophagy induction. In vivo, ACT reduced the tumor growth with little toxicity. Taken together, our findings indicated that ACT suppressed cell proliferation, induced autophagy and apoptosis through promoting ROS/JNK activation, and blunting AKT pathway in human bladder cancer, which indicated that ACT might be an effective candidate against human bladder cancer in future.
Project description:The PI3K-AKT-mTOR cascade is required for renal cell carcinoma (RCC) progression. SC66 is novel AKT inhibitor. We found that SC66 inhibited viability, proliferation, migration and invasion of RCC cell lines (786-O and A498) and patient-derived primary RCC cells. Although SC66blocked AKT-mTORC1/2 activation in RCC cells, it remained cytotoxic in AKT-inhibited/-silenced RCC cells. In RCC cells, SC66 cytotoxicity appears to occur via reactive oxygen species (ROS) production, sphingosine kinase 1inhibition, ceramide accumulation and JNK activation, independent of AKT inhibition. The ROS scavenger N-acetylcysteine, the JNK inhibitor (JNKi) and the anti-ceramide sphingolipid sphingosine-1-phosphate all attenuated SC66-induced cytotoxicity in 786-O cells. In vivo, oral administration of SC66 potently inhibited subcutaneous 786-O xenograft growth in SCID mice. AKT-mTOR inhibition, SphK1 inhibition, ceramide accumulation and JNK activation were detected in SC66-treated 786-O xenograft tumors, indicating that SC66 inhibits RCC cell progression through AKT-dependent and AKT-independent mechanisms.
Project description:<h4>Background</h4>Celastrol, a triterpene compound derived from the traditional Chinese medicine Tripterygium wilfordii, has been reported to possess potential antitumor activity towards various malignancies. However, the effect of celastrol on glioma cells and the underlying molecular mechanisms remain elusive.<h4>Methods</h4>Glioma cells, including the U251, U87-MG and C6 cell lines and an animal model were used. The effects of celastrol on cells were evaluated by flow cytometry, confocal microscopy, reactive oxygen species production assay and immunoblotting after treatment of celastrol. Fisher's exact test, a one-way ANOVA and the Mann-Whitney U-test were used to compare differences between groups. All data were analyzed using SPSS version 21.0 software.<h4>Results</h4>Here, we found that exposure to celastrol induced G2/M phase arrest and apoptosis. Celastrol increased the formation of autophagosomes, accumulation of LC3B and the expression of p62 protein. Celastrol-treated glioma cells exhibited decreased cell viability after the use of autophagy inhibitors. Additionally, autophagy and apoptosis caused by celastrol in glioma cells inhibited each other. Furthermore, celastrol induced JNK activation and ROS production and inhibited the activities of Akt and mTOR kinases. JNK and ROS inhibitors significantly attenuated celastrol-trigged apoptosis and autophagy, while Akt and mTOR inhibitors had opposite effects.<h4>Conclusions</h4>In conclusion, our study revealed that celastrol caused G2/M phase arrest and trigged apoptosis and autophagy by activating ROS/JNK signaling and blocking the Akt/mTOR signaling pathway.
Project description:In this study, we investigated the functional role of Akt and c-jun-NH(2)-kinase (JNK) signaling cascades in apigenin-induced apoptosis in U937 human leukemia cells and anti-leukemic activity of apigenin in vivo. Apigenin induced apoptosis by inactivation of Akt with a concomitant activation of JNK, Mcl-1 and Bcl-2 downregulation, cytochrome c release from mitochondria, and activation of caspases. Constitutively active myristolated Akt prevented apigenin-induced JNK, caspase activation, and apoptosis. Conversely, LY294002 and a dominant-negative construct of Akt potentiated apigenin-induced apoptosis in leukemia cells. Interruption of the JNK pathway showed marked reduction in apigenin-induced caspase activation and apoptosis in leukemia cells. Furthermore, in vivo administration of apigenin resulted in attenuation of tumor growth in U937 xenografts accompanied by inactivation of Akt and activation of JNK. Attenuation of tumor growth in U937 xenografts by apigenin raises the possibility that apigenin may have clinical implications and can be further tested for incorporating in leukemia treatment regimens.
Project description:Neurofibromatosis type 2 [NF2; MIM # 101000] is an autosomal dominant disorder characterised by the occurrence of vestibular schwannomas (VSs), schwannomas of other cranial, spinal and cutaneous nerves, cranial and spinal meningiomas and/or other central nervous system (CNS) tumours (e.g., ependymomas, astrocytomas). Additional features include early onset cataracts, optic nerve sheath meningiomas, retinal hamartomas, dermal schwannomas (i.e., NF2-plaques), and (few) café-au-lait spots. Clinically, NF2 children fall into two main groups: (1) congenital NF2 - with bilateral VSs detected as early as the first days to months of life, which can be stable/asymptomatic for one-two decades and suddenly progress; and (2) severe pre-pubertal (Wishart type) NF2- with multiple (and rapidly progressive) CNS tumours other-than-VS, which usually present first, years before VSs [vs. the classical adult (Gardner type) NF2, with bilateral VSs presenting in young adulthood, sometimes as the only disease feature]. Some individuals can develop unilateral VS associated with ipsilateral meningiomas or multiple schwannomas localised to one part of the peripheral nervous system [i.e., mosaic NF2] or multiple non-VS, non-intradermal cranial, spinal and peripheral schwannomas (histologically proven) [schwannomatosis]. NF2 is caused by mutations in the NF2 gene at chromosome 22q12.1, which encodes for a protein called merlin or schwannomin, most similar to the exrin-readixin-moesin (ERM) proteins; mosaicNF2 is due to mosaic phenomena for the NF2 gene, whilst schwannomatosis is caused by coupled germ-line and mosaic mutations either in the SMARCB1 gene [SWNTS1; MIM # 162091] or the LZTR1 gene [SWNTS2; MIM # 615670] both falling within the 22q region and the NF2 gene. Data driven from in vitro and animal studies on the merlin pathway [e.g., post-translational and upstream/downstream regulation] allowed biologically targeted treatment strategies [e.g., Lapatinib, Erlotinib, Bevacizumab] aimed to multiple tumour shrinkage and/or regression and tumour arrest of progression with functional improvement.
Project description:OBJECTIVES:SL4, a chalcone-based compound, exhibits clearly inhibitory effects on HIF-1 and has been shown to effectively suppress tumour invasion and angiogenesis in vitro and in vivo. Here, studies were conducted to determine SL4's anti-apoptotic effects and its underlying mechanisms, in human cancer cells. MATERIALS AND METHODS:Cytotoxicity, apoptotic induction and its involved mechanisms of SL4 were investigated using normal cells, cancer cells and mouse xenograft models. The role of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signalling in SL4-induced apoptosis was explored by manipulating specific scavenger or signalling inhibitors, in cultured cells. RESULTS:SL4 significantly inhibited cell population growth of human cancer cell lines but exhibited lower cytotoxicity against normal cells. In addition, SL4 effectively induced apoptosis of Hep3B and MDA-MB-435 cells by activating procaspase-8, -9 and -3, and down-regulating expression levels of XIAP, but did not affect HIF-1 apoptosis-related targets, Survivin and Bcl-XL. Further study showed that SL4 also reduced mitochondrial membrane potential and promoted generation of ROS. ROS generation and apoptotic induction by SL4 were blocked by NAC, a scavenger of ROS, suggesting SL4-induced apoptosis via ROS accumulation. We also found that MAPKs, JNK and p38, but not ERK1/2, to be critical mediators in SL4-induced apoptosis. SP600125 and SB203580, specific inhibitors of JNK kinase and p38 kinase, significantly retarded apoptosis induced by SL4. Moreover, anti-oxidant NAC blocked activation of JNK and p38 induced by SL4, indicating that ROS may act as upstream signalling of JNK and p38 activation. It is noteworthy that animal studies revealed dramatic reduction (49%) in tumour volume after 11 days SL4 treatment. CONCLUSIONS:These data demonstrate that SL4 induced apoptosis in human cancer cells through activation of the ROS/MAPK signalling pathway, suggesting that it may be a novel lead compound, as a cancer drug candidate, with polypharmacological characteristics.
Project description:Apoptosis and autophagy are the two prominent forms of developmental cell death, and researches have shown that crosstalk exists between these two processes. A prior study demonstrated that triptolide inhibited the proliferation of malignant glioma cells. However, whether apoptosis and autophagy participate in the inhibitory effect of triptolide in glioma cells has not been clarified. In the present study, we demonstrated that triptolide potently inhibited the growth of glioma cells by inducing cell cycle arrest at the G2/M phase. Additionally, the treatment with triptolide induced apoptosis and autophagy in various glioma cell lines. Triptolide-induced autophagy may have tumor-supporting effects. Autophagy and apoptosis could cross-inhibit each other in glioma cells treated with triptolide. Moreover, we found that triptolide induced ROS production and JNK activation and inhibited the activity of Akt and mTOR. Finally, we demonstrated that triptolide suppressed tumor growth in an orthotopic xenograft glioma model. Collectively, these data indicated that triptolide induced G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK and blocking the Akt/mTOR signaling pathways in glioma cells. Triptolide may be a potential anti-tumor drug targeting gliomas.