Protein phosphatase 6 regulates trametinib sensitivity, a mitogen-activated protein kinase kinase (MEK) inhibitor, by regulating MEK1/2-ERK1/2 signaling in canine melanoma cells.

Melanoma is a highly aggressive and metastatic cancer occurring in both humans and dogs. Canine melanoma accounts for a significant proportion of neoplastic diseases in dogs, and despite standard treatments, overall survival rates remain low. Protein phosphatase 6 (PP6), an evolutionarily conserved serine/threonine protein phosphatase, regulates various biological processes. Additionally, the loss of PP6 function reportedly leads to the development of melanoma in humans. However, there are no reports regarding the role of PP6 in canine cancer cells. We, therefore, conducted a study investigating the role of PP6 in canine melanoma by using four canine melanoma cell lines: CMec1, CMM, KMeC and LMeC. PP6 knockdown increased phosphorylation levels of mitogen-activated protein kinase kinase 1/2 (MEK1/2) and extracellular signal-regulated kinase 1/2 (ERK1/2) but not Akt. Furthermore, PP6 knockdown decreased sensitivity to trametinib, a MEK inhibitor, but did not alter sensitivity to Akt inhibitor. These findings suggest that PP6 may function as a tumor suppressor in canine melanoma and modulate the response to trametinib treatment. Understanding the role of PP6 in canine melanoma could lead to the development of more effective treatment strategies for this aggressive disease.

MicroRNA-34c-5p exhibits anticancer properties in gastric cancer by targeting MAP2K1 to inhibit cell proliferation, migration, and invasion.

Purpose: Gastric cancer(GC)is one of the deadliest digestive tract tumors worldwide，existing studies suggest that dysregulated expression of microRNAs (miRNAs) plays an important role in the pathogenesis and progression of GC. This study aimed to investigate the expression, biological function, and downstream mechanism of miR-34c-5p in GC, provide new targets for gastric cancer diagnosis and treatment. Methods: The expression of miR-34c-5p in GC tissues and cell lines was examined by RT-qPCR. Cell wound healing, transwell and cell cloning assays were used to detect the effect of miR-34c-5p on the migration and invasion abilities, respectively, of GC cells. Western blot was performed to detect the expression of related proteins. Bioinformatics analysis was used to predict the binding of MAP2K1 to miR-34c-5p and the targeting relationship was confirmed by dual luciferase reporter assay. Results: The expression level of miR-34c-5p was significantly decreased in GC tissues and cell lines. miR-34c-5p overexpression inhibited migration, invasion, and colony formation of gastric cancer cells, the related protein E-cadherin expression was significantly increased and N-cadherin, vimentin, and PCNA expression were significantly decreased, while miR-34c-5p knockdown exerted the opposite effects. In addition, the targeting relationship between miR-34c-5p and MAP2K1 was predicted and confirmed, and further confirmed by rescue experiments that MAP2K1 alleviated the inhibitory effect of miR-34c-5p in GC. Conclusion: MiR-34c-5p is lowly expressed in GC, and it can target MAP2K1 to exert inhibitory effects on GC proliferation, invasion, and migration. These findings provide a promising biomarker and a potential therapeutic target for gastric cancer.

Dexmedetomidine suppresses the isoflurane-induced neurological damage by upregulating Heme Oxygenase-1 via activation of the mitogen-activated protein kinase kinase 1/extracellular regulated protein kinases 1/nuclear factor erythroid 2-related factor 2 axis in aged rats.

Dexmedetomidine (DEX) displays a neuroprotective role in aged rats with isoflurane (ISO)-induced cognitive impairment through antioxidant, and anti-inflammatory, and anti-apoptotic effects. Therefore, the present study was performed to define the molecular mechanism of DEX on ISO-induced neurological impairment in aged rats in relation to the MEK1/ERK1/Nrf2/HO-1 axis. The study enrolled elderly patients undergoing ISO anesthesia. Patient cognitive function following treatment with DEX was evaluated using mini-mental state examination (MMSE). The results revealed that DEX supplementation of anesthesia contributed to higher MMSE scores in patients one week post treatment. Rat model of neurological impairment was also induced in 18-month-age Wistar rats by ISO, followed by DEX treatment. Based on the results of Morris water maze experiment, ELISA, and TUNEL and hematoxylin-eosin staining, in vivo experiments confirmed that DEX could reduce the oxidative stress and neurological damage induced by ISO in rats. DEX activated the nuclear factor erythroid 2-related factor (Nrf2)/Heme Oxygenase 1 (HO-1) pathway. DEX upregulated the expression of Nrf2 and HO-1 by activating the MEK1/ERK1 pathway, whereby attenuating the ISO-caused oxidative stress and neurological damage in rats. Collectively, DEX suppresses the ISO-induced neurological impairment in the aged rats by promoting HO-1 through activation of the MEK1/ERK1/Nrf2 axis.

Focal necrotizing myopathy with 'dropped-head syndrome' induced by cobimetinib in metastatic melanoma.

Therapeutic advances derived from targeted therapy and immune checkpoint inhibitors can improve melanoma prognosis. Since 2015, cobimetinib has been approved in combination with vemurafenib in the first-line treatment for BRAF-mutated melanoma. For NRAS-mutated melanomas, MEK inhibition seems to be a therapeutic target, and association with checkpoint inhibitor provides a further therapeutic perspective. Infraclinical creatine phosphokinase (CPK) elevation is an MEK inhibitor side effect. We describe a case of focal necrotizing myopathy with 'dropped-head syndrome' induced by cobimetinib, 1 month after its introduction. The clinical presentation comprised interscapular pain, axial fatigue with cervical hypotonia, CPK elevation, intense fluorine-18-fluorodeoxyglucose uptake in cervical muscles, and necrotizing myopathy was confirmed by muscle biopsy. Cobimetinib was temporarily discontinued, resulting in CPK normalization. Re-evaluation showed partial response, motivating continuation of combination therapy with a reduced dose of cobimetinib (40 mg/day). Because prescription of targeted therapies is likely to increase, this adverse event should be known.

Allosteric small-molecule kinase inhibitors.

Small-molecule kinase inhibitors are invaluable targeted therapeutics for the treatment of various human diseases, especially cancers. While the majority of approved and developed preclinical small-molecule inhibitors are characterized as type I or type II inhibitors that target the ATP-binding pocket of kinases, the remarkable sequential and structural similarity among ATP pockets renders the selective inhibition of kinases a daunting challenge. Therefore, targeting allosteric pockets of kinases outside the highly conversed ATP pocket has been proposed as a promising alternative to overcome current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small-molecule allosteric inhibitor trametinib in 2013, the progress of more than 10 other allosteric inhibitors in clinical trials, and the emergence of a pipeline of highly selective and potent preclinical molecules, have been reported in the past decade. In this article, we present the current knowledge on allosteric inhibition in terms of conception, classification, potential advantages, and summarized debatable topics in the field. Recent progress and allosteric inhibitors that were identified in the past three years are highlighted in this paper.

The mechanism of fucoidan-induced apoptosis in leukemic cells: involvement of ERK1/2, JNK, glutathione, and nitric oxide.

Fucoidan, a sulfated polysaccharide in brown seaweed, has various biological activities including anti-tumor activity. We investigated the effects of fucoidan on the apoptosis of human promyeloid leukemic cells and fucoidan-mediated signaling pathways. Fucoidan induced apoptosis of HL-60, NB4, and THP-1 cells, but not K562 cells. Fucoidan treatment of HL-60 cells induced activation of caspases-8, -9, and -3, the cleavage of Bid, and changed mitochondrial membrane permeability. Fucoidan-induced apoptosis, cleavage of procaspases, and changes in the mitochondrial membrane permeability were efficiently blocked by depletion of mitogen-activated protein kinase (MAPK) kinase kinase 1 (MEKK1), and inhibitors of MAPK kinase 1 (MEK1) and c Jun NH2-terminal kinase (JNK). The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and JNK was increased in fucoidan-treated HL-60, NB4, and THP-1 cells, but not K562 cells. ERK1/2 activation occurred at earlier times than JNK activation and JNK activation was blocked by MEK1 inhibitor. In addition, fucoidan-induced apoptosis was inhibited by addition of glutathione and/or L-NAME, and fucoidan decreased intracellular glutathione concentrations and stimulated nitric oxide (NO) production. Buthionine-[R,S]-sulfoximine rendered HL-60 cells more sensitive to fucoidan. Depletion of MEKK1 and inhibition of MEK1 restored the intracellular glutathione content and abrogated NO production, whereas inhibition of JNK activation by SP600125 restored intracellular glutathione content but failed to inhibit NO production in fucoidan-treated HL-60 cells. These results suggest that activation of MEKK1, MEK1, ERK1/2, and JNK, depletion of glutathione, and production of NO are important mediators in fucoidan-induced apoptosis of human leukemic cells.

Inhibition of mitogen activated protein kinase activity induces parthenogenetic activation and increases cyclin B accumulation during porcine oocyte maturation.

The inhibition of mitogen activated protein kinase (MAPK) activation during porcine oocyte maturation leads to decreased maturation promoting factor (MPF) activity and to the induction of parthenogenetic activation. In the present study, in order to analyze the mechanism underlying the suppression of MPF activity in MAPK-inhibited porcine oocytes, we injected mRNA of SASA-MEK, a dominant negative MAPK kinase, or antisense RNA of c-mos, a MAPK kinase kinase, into immature porcine oocyte cytoplasm. The injection of SASA-MEK mRNA or c-mos antisense RNA inhibited the MAPK activity partially or completely, respectively, decreased the MPF activity slightly or significantly, respectively, and induced parthenogenetic activation in 17.1% or 96.6% of mature oocytes, respectively, although no parthenogenetic activation was observed in the control oocytes. Immunoblotting experiments revealed that cyclin B accumulation in these MAPK-suppressed porcine oocytes was increased significantly after 50 h of culture and that a considerable amount of MPF was converted into inactive pre-MPF by hyperphosphorylation. These results indicate that the inhibition of MAPK activity in porcine oocytes did not promote cyclin B degradation but rather suppressed it; also the decrease in MPF activity in MAPK-suppressed porcine oocytes correlated with the conversion of active MPF into inactive pre-MPF.

PKC-mediated survival signaling in breast carcinoma cells: a role for MEK1-AP1 signaling.

The ability of peptide hormones, as well as the protein kinase C (PKC)-activating phorbol ester (PMA), to protect cells from apoptosis has been demonstrated to occur through activation of cellular signaling pathways such as the mitogen-activated protein kinase (MAPK) and phosphatidyl-inositol-3 kinase (PI3K) families. Here we demonstrate that tumor necrosis factor alpha (TNF)-induced apoptosis is suppressed by treatment with PMA in MCF-7 breast carcinoma cells. Reversal of the PMA survival effect with the classical isoform-specific PKC inhibitor, Go 6976, or the selective mitogen-activated protein kinase kinase (MEK) inhibitor, PD 098059, suggested a partial requirement for PKCalpha and the Erk cascade in MCF-7 cell survival. The ability of these agents to block PMA-mediated cell survival was also correlated with a suppression of PMA-induced AP-1 activity. Some naturally occurring flavonoid compounds such as apigenin can function to block cell signaling cascades such as MAPK. The ability of apigenin to block PMA-mediated cell survival was similarly correlated with suppression of PMA-stimulated AP-1 activity. Our results strongly suggest that PKC- and Erk-dependent pathways are critical components of the cell survival cascade function in suppression of TNF-induced apoptosis in MCF-7 cells. The ability of natural dietary flavonoids such as apigenin to affect cell survival pathways may represent an important aspect of the proposed anti-tumor effects of these compounds.