Background: Multiple myeloma (MM) is a B-cell malignancy that is largely incurable and is characterized by the accumulation of malignant plasma cells in the bone marrow. Apigenin, a common flavonoid, has been reported to suppress proliferation in a wide variety of solid tumors and hematological cancers; however its mechanism is not well understood and its effect on MM cells has not been determined. Results: In this study, we investigated the effects of apigenin on MM cell lines and on primary MM cells. Cell viability assays demonstrated that apigenin exhibited cytotoxicity against both MM cell lines and primary MM cells but not against normal peripheral blood mononuclear cells. Together, kinase assays, immunoprecipitation and western blot analysis showed that apigenin inhibited CK2 kinase activity, decreased phosphorylation of Cdc37, disassociated the Hsp90/Cdc37/client complex and induced the degradation of multiple kinase clients, including RIP1, Src, Raf-1, Cdk4 and AKT. By depleting these kinases, apigenin suppressed both constitutive and inducible activation of STAT3, ERK, AKT and NF-κB. The treatment also downregulated the expression of the antiapoptotic proteins Mcl-1, Bcl-2, Bcl-xL, XIAP and Survivin, which ultimately induced apoptosis in MM cells. In addition, apigenin had a greater effects in depleting Hsp90 clients when used in combination with the Hsp90 inhibitor geldanamycin and the histone deacetylase inhibitor vorinostat. Conclusions: Our results suggest that the primary mechanisms by which apigenin kill MM cells is by targeting the trinity of CK2-Cdc37-Hsp90, and this observation reveals the therapeutic potential of apigenin in treating multiple myeloma.

Background: Celastrol is an active ingredient of the traditional Chinese medicinal plantTripterygium Wilfordii, which exhibits significant antitumor activity in different cancer modelsin vitroandin vivo; however, the lack of information on the target and mechanism of action of this compound have impeded its clinical application. In this study, we sought to determine the mode of action of celastrol by focusing on the processes that mediate its anticancer activity.Methods: The downregulation of heat shock protein 90 (HSP90) client proteins, phosphorylation of c-Jun NH2-terminal kinase (JNK), and cleavage of PARP, caspase 9 and caspase 3 were detected by western blotting. The accumulation of reactive oxygen species (ROS) was analyzed by flow cytometry and fluorescence microscopy. Cell cycle progression, mitochondrial membrane potential (MMP) and apoptosis were determined by flow cytometry. Absorption spectroscopy was used to determine the activity of mitochondrial respiratory chain (MRC) complexes.Results: Celastrol induced ROS accumulation, G2-M phase blockage, apoptosis and necrosis in H1299 and HepG2 cells in a dose-dependent manner. N-acetylcysteine (NAC), an antioxidative agent, inhibited celastrol-induced ROS accumulation and cytotoxicity. JNK phosphorylation induced by celastrol was suppressed by NAC and JNK inhibitor SP600125 (SP). Moreover, SP significantly inhibited celastrol-induced loss of MMP, cleavage of PARP, caspase 9 and caspase 3, mitochondrial translocation of Bad, cytoplasmic release of cytochrome c, and cell death. However, SP did not inhibit celastrol-induced ROS accumulation. Celastrol downregulated HSP90 client proteins but did not disrupt the interaction between HSP90 and cdc37. NAC completely inhibited celastrol-induced decrease of HSP90 client proteins, catalase and thioredoxin. The activity of MRC complex I was completely inhibited in H1299 cells treated with 6 μM celastrol in the absence and presence of NAC. Moreover, the inhibition of MRC complex I activity preceded ROS accumulation in H1299 cells after celastrol treatment.Conclusion: We identified ROS as the key intermediate for celastrol-induced cytotoxicity. JNK was activated by celastrol-induced ROS accumulation and then initiated mitochondrial-mediated apoptosis. Celastrol induced the downregulation of HSP90 client proteins through ROS accumulation and facilitated ROS accumulation by inhibiting MRC complex I activity. These results identify a novel target for celastrol-induced anticancer activity and define its mode of action.

Neuroinflammation caused by peripheral lipopolysaccharides (LPS) under hypoxia is a key contributor to the development of high altitude cerebral edema (HACE). Our previous studies have shown that gypenosides and their bioactive compounds prevent hypoxia-induced neural injuries in vitro and in vivo. However, their effect on neuroinflammation-related HACE remains to be illustrated. The present study aimed to investigate the effects of GP-14 in HACE mouse model. HACE mice were treated with GP-14 (100 and 200 mg/kg) for 7 days. After the treatments, the level of serum inflammation cytokines and the transcription of inflammatory factors in brain tissue were determined. The activation of microglia, astrocyte and the changes of IgG leakage and the protein levels of tight junction proteins were detected. Furthermore, the inflammatory factors and nuclear factor-κB (NF-κB) signaling pathway in BV-2 cells and primary microglia were detected. GP-14 pretreatment alleviated both the serum and neural inflammatory responses caused by LPS stimulation combined with hypobaric hypoxia exposure. In addition, GP-14 pretreatment inhibited microglial activation, accompanied by a decrease in the M1 phenotype and an increase in the M2 phenotype. Moreover, the disruption of the blood brain barrier (BBB) integrity, including increased IgG leakage and decreased expression of tight junction proteins, was attenuated by GP-14 pretreatment. Based on the BV-2 and primary microglial models, the inflammatory response and activation of the NF-κB signaling pathway were also inhibited by GP-14 pretreatment. Taken together, our results demonstrated that GP-14 exhibited prominent protective roles against neuroinflammation and BBB disruption in a mouse HACE model. GP-14 could be a potential choice for the treatment of HACE in the future.

Background: High-altitude cerebral edema (HACE) is considered an end-stage acute mountain sickness (AMS) that typically occurs in people after rapid ascent to 2500 m or more. While hypoxia is a fundamental feature of the pathophysiological mechanism of HACE, emerging evidence suggests that inflammation serves as a key risk factor in the occurrence and development of this disease. However, little is known about the molecular mechanism underlying their crosstalk. Methods: A mouse HACE model was established by combination treatment with hypobaric hypoxia exposure and lipopolysaccharides (LPS) stimulation. Lactylated-proteomic analysis of microglia was performed to reveal the global profile of protein lactylation. Molecular modeling was applied to evaluate the 3-D modeling structures. A combination of experimental approaches, including western blotting, quantitative real-time reverse transcriptionpolymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and RNA interference, were used to explore the underlying molecular mechanisms. Results: We found that hypoxia exposure increased the lactate concentration and lactylation in mouse HACE model. Moreover, hypoxia aggravated the microglial neuroinflammatory response in a lactate-dependent manner. Global profiling of protein lactylation has shown that a large quantity of lysine-lactylated proteins are induced by hypoxia and preferentially occur in protein complexes, such as the NuRD complex, ribosome biogenesis complex, spliceosome complex, and DNA replication complex. The molecular modeling data indicated that lactylation could affect the 3-D theoretical structure and increase the solvent accessible surface area of HDAC1, MTA1 and Gatad2b, the core members of the NuRD complex. Further analysis by knockdown or selectively inhibition indicated that the NuRD complex is involved in hypoxia-mediated aggravation of inflammation. Conclusions: These results revealed a comprehensive profile of protein lactylation in microglia and suggested that protein lysine lactylation plays an important role in the regulation of protein function and subsequently contributes to the neuroinflammatory response under hypoxic conditions.

Introduction: High-altitude cerebral edema (HACE) is considered to be the end-stage of acute mountain sickness (AMS); however, its pathophysiological mechanism remains unknown. Increasing evidences support that inflammation is an important risk factor for the occurrence of HACE. Including our published papers, previous studies demonstrated that the levels of IL-6, IL-1β, and TNF-α in both serum and hippocampus were increased in the mouse HACE model induced by LPS stimulation combined with hypobaric hypoxia exposure; however, the expression profile of other cytokines and chemokines remains unknown. Objective: This study was to analyze the expression profile of cytokines and chemokines in the HACE model. Methods: The mouse HACE model was established by LPS stimulation combined with hypobaric hypoxia exposure (LH). The mice were divided into the normoxic group, LH-6 h group, LH-1 d group, and LH-7 d group. Brain water content (BWC) was determined using the wet/dry weight ratio. The levels of 30 cytokines and chemokines in the serum and hippocampal tissue were detected using LiquiChip. The mRNA expression of cytokines and chemokines in hippocampal tissue were determined by q-PCR. Results: In the current study, we found that the brain water content was increased after the combinational treatment of LPS and hypobaric hypoxia. The results of LiquiChip showed that, in the serum and hippocampal tissue, most factors in all 30 cytokines and chemokines were dramatically upregulated at 6 h, and then declined at the 1st d and 7th d. Among these factors, G-CSF, M-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1β were all increased in both serum and hippocampal tissue at 6 h. In addition, the results of q-PCR showed the mRNA levels of G-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1β in hippocampal tissue were dramatically upregulated at 6 h. Conclusion: This study showed that the dynamic expression profile of 30 cytokines and chemokines in a mouse HACE model induced by LPS plus hypobaric hypoxia. The levels of G-CSF, MCP-1, KC, MIG, Eotaxin, Rantes, IP10, IL-6, MIP-2, and MIP-1β in both serum and hippocampus were significantly increased at 6 h, which may be involved in the occurrence and development of HACE.