Celastrol Treatment Ameliorated Acute Ischemic Stroke-Induced Brain Injury by Microglial Injury Inhibition and Nrf2/HO-1 Pathway Activations.

Background: Stroke is the third main reason of mortality, which is the leading reason for adult disability in the globe. Poststroke inflammation is well known to cause acute ischemic stroke- (AIS-) induced brain injury (BI) exacerbation. Celastrol (CL) has exhibited anti-inflammatory activities in various inflammatory traits though underlying mechanisms remain unknown. So, the present investigation is aimed at studying CL protective mechanism against AIS-induced BI. Methods: A mouse model regarding middle cerebral artery occlusion and an oxygen-glucose deprivation (OGD) cell model with or not CL treatment were constructed to study CL protective effects. NF-E2-related factor 2 (Nrf2) was then silenced in BV2 microglia cells (BV2) to study Nrf2 role regarding CL-mediated neuroprotection. Results: The results showed that CL treatment suppressed AIS-induced BI by inhibiting NLRP3/caspase-1 pathway activations and induction of apoptosis and pyroptosis in vivo and in vitro. NLRP3/caspase-1 pathway blocking activation suppressed OGD-induced cell pyroptosis and apoptosis. Also, CL treatment reversed OGD-induced microglial injury by promoting Nrf2/heme oxygenase-1 (HO-1) pathway activations. Nrf2 downregulation reversed CL protective effects against OGD-induced microglial injury, pyroptosis, and apoptosis. Conclusion: The findings advise that CL treatment ameliorated AIS-induced BI by inhibiting microglial injury and activating the Nrf2/HO-1 pathway.

The role of T2E mediated CBF-1/RBP-Jκ signaling in metastatic thyroid cancer.

OBJECTIVE: Cancer has been shown to be an independent risk factor for 2019-nCoV. Expression of transmembrane serine protease 2 (TMPRSS2) is abnormal in many cancers. Nevertheless, system analysis of TMPRSS2-ERG (T2E) abnormalities in metastatic thyroid cancer remains to be elucidated. METHOD: Using genomic and chromatin data, we demonstrate a unique cis-regulatory landscape between non-T2E and T2E-positive metastatic thyroid cancers, including clusters of regulatory elements (COREs). We attempt to describe the effect of T2E silencing on the cis-regulatory structure in metastatic thyroid cancers and its phase with the obvious phenotype characteristics of T2E-positive metastatic thyroid cancers. RESULTS: These differences were linked by the ERG (erythroblast transformation-specific related gene) co-opts of FoxA1 and HOXB13, which realized T2E specific transcription profile. The study also demonstrated the T2E-specific CORE in an ERG site of structural rearrangement, which is due to the expansion of the T2E locus and contributes to its up-expression. Ultimately, we demonstrate that T2E-specific transcription profile is the basis of vulnerability of CBF-1/RBP-Jκ pathway. In fact, CBF-1/RBP-Jκ pathway inhibits the invasion and growth of T2E-positive thyroid tumors. CONCLUSION: This study indicates that the overexpression of ERG co-option has a unique cis-regulatory structure in T2E positive thyroid tumors, which induces drug dependence on CBF-1/RBP-Jκ signal. Our study solved the genetic and epigenetic variation of T2E in metastatic thyroid cancer for the first time. It is worth noting that further functional and clinical validation is needed as our study is a bioinformatics analysis.

SIRT2 modulates VEGFD-associated lymphangiogenesis by deacetylating EPAS1 in human head and neck cancer.

Sirtuin 2 (SIRT2) is one of seven mammalian homologs of silent information regulator 2 (Sir2) and an NAD+ -dependent deacetylase; however, its critical role in lymphangiogenesis remains to be explored. We investigate SIRT2 mediated regulation of vascular endothelial growth factor D (VEGFD) expression and lymphangiogenesis by deacetylating endothelial PAS domain protein 1 (EPAS1) in head and neck cancer (HNC) in vitro and in vivo. In this study, we report that SIRT2, rather than other members of the Sir2 family, reduces the expression of VEGFD and lymphangiogenesis in hypoxia-induced HNC cells and transplanted HNC mice models by reducing EPAS1 acetylation at Lys674 and decreasing the transcriptional activity of EPAS1 target genes. The expression of SIRT2 was closely related to the expression of VEGFD, lymphangiogenesis in subcutaneously transplanted mice models, and lymphangiogenesis in patients with HNC. Our results suggest that SIRT2 plays a central role in tumor lymphangiogenesis via deacetylating EPAS1 protein. Reagents targeting the NAD+ -dependent deacetylase activity of SIRT2 would be beneficial for inhibiting tumor lymphangiogenesis and treating other hypoxia-related diseases.

Single-cell RNA sequencing reveals the regenerative potential of thyroid follicular epithelial cells in metastatic thyroid carcinoma.

Thyroid stimulating hormone deficiency is the cornerstone of treatment for metastatic thyroid cancer. Due to the loss of follicular epithelial cells in thyroid cancer, the thyroid gland degenerates to 85% of its original size. When thyroid stimulating hormone is restored, follicular epithelial cells in thyroid cancer regenerate, which is postulated to be related to stem-like cells. By single cell RNA seq, we found a group of rare thyroid follicular epithelial cells in mouse metastatic thyroid cancer, which expressed stem-like genes (CD44V6+ and CD133+) and a large number of differentiated cells (CD44V6+ and CD24+). In mouse and in organoids, the two subsets contribute equally to metastatic thyroid cancer regeneration. The analysis of human metastatic thyroid cancer revealed that the differentiated thyroid follicular epithelial cell subpopulation was similar to that of the stem like epithelial cell subpopulation, and the regeneration potential was also enhanced after thyroid stimulating hormone ablation. Accordingly, we propose that the regeneration of metastatic thyroid cancer is driven by almost all persistent thyroid follicular epithelial cells, not only by few stem-like cells.

Hsa_circ_0017639 expression promotes gastric cancer proliferation and metastasis by sponging miR-224-5p and upregulating USP3.

Gastric cancer (GC) is a common malignant tumor having poor prognosis globally. Circular RNA (circRNA) is a circular endogenous RNA generated by special selective splicing that occurs in various traits. Studies show that hsa_circ_0017639 is abnormally expressed and involved in tumorigenesis. Nevertheless, the hsa_circ_0017639 role in GC is unknown. This study detected hsa_circ_0017639 expression in a GC cell line using RT-qPCR. Subcellular localization of hsa_circ_0017639 was verified via FISH. We assessed correlations amongst miRNA, hsa_circ_0017639 and relative protein levels using luciferase reporter assays and RNA pulldown assays. The data illustrated that in hsa_circ_assays, expression was enhanced in GC cell. Downregulation of hsa_circ_0017639 decreased GC cell proliferation and migration in in vitro and in vivo experiments. RNA pulldown and RT-qPCR analysis verified that hsa_circ_0017639 sponged miR-224-5p. Bioinformatic and luciferase reporter assays validated that miR-224-5p and USP3 were downstream targets of hsa_circ_0017639. Upregulation of USP3 or downregulation of miR-224-5p restored proliferation and migration by MKN-28 and MGC-803 cells after hsa_circ_0017639 silencing. Upregulation of USP3 restored MKN-28 and MGC-803 cell proliferation and migration after overexpression of miR-224-5p. Our collective findings advised that hsa_circ_0017639 takes part in GC progression through regulating the miR-224-5p/USP3 axis, highlighting its potential as an effective GC therapeutic target.

Celastrol Reverses Palmitic Acid-Induced Insulin Resistance in HepG2 Cells via Restoring the miR-223 and GLUT4 Pathway.

OBJECTIVES: The natural triterpenoid compound celastrol ameliorates insulin resistance (IR) in animal models, but the underlying molecular mechanism is unclear. In this study, we investigated how celastrol regulates IR. METHODS: The HepG2 cellular IR model was initially established with palmitic acid (PA). The expression and activity of glucose transporter 4 (GLUT4), insulin receptor substrate-1 (IRS1) and 9 microRNAs (miRNAs) (miR-7, -34a, -96, -113, -126, -145, -150, -223 and -370) were detected before and after celastrol treatment using the PA-induced HepG2 IR model. RESULTS: The results showed that 250 µM PA for ≥2 days was optimal for inducing IR in HepG2 cells; 600 nM celastrol significantly attenuated the PA-induced IR in HepG2 cells. The PA-induced GLUT4 and IRS1 downregulation and Ser307 phosphorylation on IRS1 was reversed by subsequent treatment with 600 nM celastrol for 6 h. We next investigated which IR-related miRNAs were possible upstream regulators of celastrol-mediated reversal of PA-induced HepG2 IR. Two miRNAs, miR-150 and -223, were significantly downregulated by PA and were re-raised by subsequent celastrol treatment; and miR-223 was upstream of miR-150. Moreover, knocking down miR-223 abolished celastrol's anti-IR effects in the PA-induced model. CONCLUSIONS: Collectively, our results demonstrated that celastrol reverses PA-induced IR-related alterations, in part via miR-223 in HepG2 cells. Further investigation is warranted for establishing the clinical potential of celastrol in treating IR-related disorders.

Celastrol improves the therapeutic efficacy of EGFR-TKIs for non-small-cell lung cancer by overcoming EGFR T790M drug resistance.

The development of resistance to therapy continues to be a serious clinical problem in lung cancer management. Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is one of the most common chemotherapy drugs to treat non-small-cell lung cancer. However, almost all treatments fail after ∼1 year of treatment because of drug tolerance, probably occurring from the threonine 790 mutation (T790M) of the EGFR, resulting in overactivation of the EGFR. Celastrol is a natural compound that exhibits antiproliferative activity. In this study, we showed that celastrol combined with EGFR-TKIs significantly suppressed cell invasion of lung cancer cells with a T790M mutation by suppressing the EGFR pathway. Combined therapy with celastrol and EGFR-TKIs inhibited tumor growth in vivo. Together, these results suggested that combined therapy with EGFR-TKIs and celastrol may be a more effective treatment of patients with non-small-cell lung cancer with T790M mutations of the EGFR.

Celastrol reverses palmitic acid (PA)-caused TLR4-MD2 activation-dependent insulin resistance via disrupting MD2-related cellular binding to PA.

Elevated plasma statured fatty acids (FFAs) cause TLR4/MD2 activation-dependent inflammation and insulin tolerance, which account for the occurrence and development of obesity. It has been confirmed that statured palmitic acid (PA) (the most abundant FFA) could bind MD2 to cause cellular inflammation. The natural compound celastrol could improve obesity, which is suggested via inhibiting inflammation, yet the detailed mechanism for celastrol is still unclear. As celastrol is reported to directly target MD2, we thought disrupting the binding between FFAs and MD2 might be one of the ways for celastrol to inhibit FFAs-caused inflammation and insulin resistance. In this study, we found evidence to support our hypothesis: celastrol could reverse PA-caused TLR4/MD2 activation-dependent insulin resistance, as determined by glucose-lowering ability, cellular glucose uptake, insulin action-related proteins and TLR4/MD2/NF-κB activation. Bioinformatics and cellular experiments showed that both celastrol and PA could bind MD2, and that celastrol could expel PA from cells. Finally, celastrol could reverse high fat diet caused hyperglycemia and obesity, and liver NF-kB activations. Taking together, we proved that celastrol could reverses PA-caused TLR4-MD2 activation-dependent insulin resistance via disrupting PA binding to MD2.

Celastrol treatment protects against acute ischemic stroke-induced brain injury by promoting an IL-33/ST2 axis-mediated microglia/macrophage M2 polarization.

BACKGROUND: Acute ischemic stroke (AIS) is the most common type of cerebrovascular disease and is a leading cause of disability and death worldwide. Recently, a study suggested that transformation of microglia from the pro-inflammatory M1 state to the anti-inflammatory and tissue-reparative M2 phenotype may be an effective therapeutic strategy for ischemic stroke. Celastrol, a traditional oriental medicine, may have anti-inflammatory and neuroprotective effects. However, the underlying mechanisms remain unknown. METHODS: We first determined the expression levels of inflammatory factors in patients and rodent models associated with AIS; we then determined the anti-inflammatory effects of celastrol in AIS, both in vivo and in vitro, using animal models of middle cerebral artery occlusion (MCAO) and cell models of oxygen-glucose deprivation (OGD) treatment with or without celastrol, respectively. RESULTS: The results indicated that expression of both inflammatory (interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α) cytokines, as well as the anti-inflammatory cytokine, IL-33, and IL-10, were increased following AIS in patients and in animal models. Furthermore, in vitro experiments confirmed that celastrol treatment decreased inflammatory cytokine expression induced by OGD through an IL-33/ST2 axis-mediated M2 microglia/macrophage polarization. Finally, celastrol is protected against ischemic-induced nerve injury, both in vivo and in vitro. CONCLUSIONS: Taken together, these data suggest that celastrol post-treatment reduces ischemic stroke-induced brain damage, suggesting celastrol may represent a novel potent pharmacological therapy.

Roles of circulating endothelial progenitor cells and endothelial cells in gastric carcinoma.

The present study aimed to investigate the role of endothelial progenitor cells (EPCs) and endothelial cells (ECs) in the peripheral blood of patients with gastric cancer (GC), and to investigate vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in GC tissues. First, 6 ml peripheral blood with added anticoagulant was collected from each of the 42 patients with GC, followed by determination of the number of EPCs and ECs by flow cytometry using the surface markers cluster of differentiation (CD)34brightCD133+CD31+CD45dim and CD34dimCD133-CD31brightCD45-, respectively. VEGF expression in patients with GC was detected by the streptomycin avidin-peroxidase immunohistochemical method, and MVD was calculated using the marker CD34. EPC and EC levels were positively associated with VEGF expression level, as well as with MVD. VEGF expression was positive in 66.67% GC cases, and its level was significantly associated with tumor-node-metastasis (TNM) stage, invasion depth and lymph-node metastasis (P<0.05). VEGF expression level was also positively associated with MVD. MVD in GC was significantly larger than that in normal tissue (P<0.01), and it was significantly associated with TNM stage (P<0.05), invasion depth (P<0.01) and lymph-node metastasis (P<0.01). EPCs in the peripheral blood have an important role in GC development, and may be a promising indicator of GC diagnosis and prognosis.

Effects of celastrol on Tau hyperphosphorylation and expression of HSF-1 and HSP70 in SH-SY5Y neuroblastoma cells induced by amyloid-ß peptides.

To observe the effects of celastrol on Tau hyperphosphorylation induced by amyloid-ß peptides (Aß) in SH-SY5Y neuroblastoma cells, the changes of Tau hyperphosphorylation and the expression of heat shock protein 90 (HSP90), HSP70, and heat shock factor 1 (HSF-1) in SH-SY5Y cells treated with Aß1-42 and celastrol were measured. Tau hyperphosphorylation and HSP90 expression induced by Aß1-42 was also measured by Western blotting after HSP70 or HSF-1 knockdown by siRNA. The interaction between HSP70 and Tau or HSP70 and carboxyl terminus of HSP70 interacting protein (CHIP) was measured by co-immunoprecipitation. Compared with the control group, the expressions of HSP70 and HSF-1 were markedly decreased after the induction of Aß1-42 , whereas the expressions of HSP90, Tau phospho S199/202, and Tau phospho S396 were markedly increased. Meanwhile, both celastrol treatment and knockdown of HSP70 or HSF-1 in SH-SY5Y cells significantly inhibited the Tau hyperphosphorylation and HSP90 expression induced by Aß1-42 . Moreover, celastrol treatment had no effects on Aß1-42 -induced decreased expression of HSP70 and HSF-1, Tau ubiquitination, and the interaction of HSP70/Tau and HSP70/CHIP. These results suggest that celastrol- inhibited Tau hyperphosphorylation may not be dependent on the cause of HSF-1/HSP70/CHIP-mediated ubiquitination of Tau.

Mutations Y493G and K546D in human HSP90 disrupt binding of celastrol and reduce interaction with Cdc37.

Celastrol, a natural compound derived from the Chinese herb Tripterygium wilfordii Hook F, has been proven to inhibit heat shock protein 90 (HSP90) activity and has attracted much attention because of its promising effects in cancer treatment and in ameliorating degenerative neuron diseases. However, the HSP90 structure involved in celastrol interaction is not known. Here, we report a novel celastrol-binding pocket in the HSP90 dimer, predicted by molecular docking. Mutation of the two key binding pocket amino acids (Lys546 and Tyr493) disrupted the binding of celastrol to HSP90 dimers, as detected by isothermal titration calorimetry (ITC). Interestingly, such mutations also reduced binding between HSP90 and the cochaperone Cdc37, thus providing a new explanation for reported findings that celastrol shows more obvious effects in disrupting binding between HSP90 and Cdc37 than between HSP90 and other cochaperones. In short, our work discloses a novel binding pocket in HSP90 dimer for celastrol and provides an explanation as to why celastrol has a strong effect on HSP90 and Cdc37 binding.

Celastrol targets IRAKs to block Toll-like receptor 4-mediated nuclear factor-κB activation.

OBJECTIVE: Celastrol has been established as a nuclear factor-κB (NF-κB) activation inhibitor; however, the exact mechanism behind this action is still unknown. Using text-mining technology, the authors predicted that interleukin-1 receptor-associated kinases (IRAKs) are potential celastrol targets, and hypothesized that targeting IRAKs might be one way that celastrol inhibits NF-κB. This is because IRAKs are key molecules for some crucial pathways to activate NF-κB (e.g., the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily). METHODS: The human hepatocellular cell line (HepG2) treated with palmitic acid (PA) was used as a model for stimulating TLR4/NF-κB activation, in order to observe the potential effects of celastrol in IRAK regulation and NF-κB inhibition. The transfection of small interfering RNA was used for down-regulating TLR4, IRAK1 and IRAK4, and the Western blot method was used to detect changes in the protein expressions. RESULTS: The results showed that celastrol could effectively inhibit PA-caused TLR4-dependent NF-κB activation in the HepG2 cells; PA also activated IRAKs, which were inhibited by celastrol. Knocking down IRAKs abolished PA-caused NF-κB activation. CONCLUSION: The results for the first time show that targeting IRAKs is one way in which celastrol inhibits NF-κB activation.

Inhibiting inducible miR-223 further reduces viable cells in human cancer cell lines MCF-7 and PC3 treated by celastrol.

BACKGROUND: Celastrol is a novel anti-tumor agent. Ways to further enhance this effect of celastrol has attracted much research attention. METHODS AND RESULTS: Here, we report that celastrol treatment can elevate miR-223 in human breast cancer cell line MCF-7 and prostate cancer PC3. Down-regulating miR-223 could increase the number of viable cells, yet it further reduced viable cells in samples that were treated by celastrol; up-regulation of miR-223 displayed opposite effects. Celastrol's miR-223 induction might be due to NF-κB inhibition and transient mTOR activation: these two events occurred prior to miR-223 elevation in celastrol-treated cells. NF-κB inhibitor, like celastrol, could induce miR-223; the induction of miR-223 by NF-κB inhibitor or celastrol was reduced by the use of mTOR inhibitor. Finally and interestingly, miR-223 also could affect NF-κB and mTOR and the effects were different between cells treated or not treated with celastrol, thus providing an explanation for differing effects of miR-223 alteration on cellular viability in the presence of celastrol or not. CONCLUSIONS: For the first time, we disclose that celastrol could induce miR-223 in breast and prostate cancer cells, and that inhibiting miR-223 could further reduce the living cells in celastrol-treated cancer cell lines. We thus provide a novel way to increase celastrol's anti-cancer effects.

Celastrol Ameliorates EAE Induction by Suppressing Pathogenic T Cell Responses in the Peripheral and Central Nervous Systems.

Multiple sclerosis (MS) is the prototypical inflammatory demyelinating disease of the central nervous system (CNS), and MS results in physical and cognitive impairments, such as fatigue, pain, depression and bladder dysfunction. Though many therapies for MS have been developed, the safety profile and effectiveness of these therapies still need to be defined. Thus, new therapies for MS must be explored. Celastrol, a quinonemethide triterpene, is a pharmacologically active compound present in Thunder God Vine root extracts used to treat inflammatory and autoimmune diseases. Molecular studies have identified several molecular targets, which are mostly centered on the inhibition of IKK-NF-κB signaling. The animal model of experimental autoimmune encephalomyelitis (EAE) has been widely used in MS studies; thus, we tried to explore the role of celastrol in EAE development in this study. We demonstrated that the intraperitoneal injection of celastrol significantly attenuated EAE disease. Th17 cell responses in the peripheral lymph nodes in EAE mice were also inhibited by celastrol. We determined that celastroldownregulated cytokine production in bone-marrow derived dendritic cells (BMDCs). Accordingly, T cells that were co-cultured with either BMDCs pre-treated with celastrolor splenic DCs and then collected on day 7 after EAE immunizationshowed that Th17 cell polarization is suppressed in the above two situations. Moreover, celastrol was required for tissue-infiltrating DCs to sustain Th17 responses in the central nervous system (CNS). Taken together, the results of our study demonstrate that celastrol ameliorates EAE development by suppressing pathogenic Th17 responses; this finding offers a better understanding of the role of celastrol in EAE development as well as new proposals for clinical interventions.

Celastrol inhibits lung infiltration in differential syndrome animal models by reducing TNF-α and ICAM-1 levels while preserving differentiation in ATRA-induced acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) is a revolutionary agent for acute promyelocytic leukemia (APL) treatment via differentiation induction. However, ATRA treatment also increases cytokine, chemokine, and adhesive molecule (mainly ICAM-1) expression, which can cause clinical complications, including a severe situation known as differentiation syndrome (DS) which can cause death. Therefore, it is of clinical significance to find a strategy to specifically blunt inflammatory effects while preserving differentiation. Here we report that the natural compound, celastrol, could effectively block lung infiltrations in DS animal models created by loading ATRA-induced APL cell line NB4. In ATRA-treated NB4 cells, celastrol could potently inhibit ICAM-1 elevation and partially reduce TNF-α and IL-1ß secretion, though treatment showed no effects on IL-8 and MCP-1 levels. Celastrol's effect on ICAM-1 in ATRA-treated NB4 was related to reducing MEK1/ERK1 activation. Strikingly and encouragingly, celastrol showed no obvious effects on ATRA-induced NB4 differentiation, as determined by morphology, enzymes, and surface markers. Our results show that celastrol is a promising and unique agent for managing the side effects of ATRA application on APL, and suggest that hyper-inflammatory ability is accompanied by, but not necessary for, APL differentiation. Thus we offered an encouraging novel strategy to further improve differentiation therapy.

Peptide deformylase inhibitor actinonin reduces celastrol's HSP70 induction while synergizing proliferation inhibition in tumor cells.

BACKGROUND: Celastrol is a promising anti-tumor agent, yet it also elevates heat shock proteins (HSPs), especially HSP70, this effect believed to reduce its anti-tumor effects. Concurrent use of siRNA to increase celastrol's anti-tumor effects through HSP70 interference has been reported, but because siRNA technology is difficult to clinically apply, an alternative way to curb unwanted HSP70 elevation caused by celastrol treatment is worth exploring. METHODS: In this work, we explore three alternative strategies to control HSP70 elevation: (1) Searching for cancer cell types that show no HSP70 elevation in the presence of celastrol (thus recommending themselves as suitable targets); (2) Modifying HSP70-inducing chemical groups, i.e.: the carboxyl group in celastrol; and (3) Using signaling molecule inhibitors to specifically block HSP70 elevation while protecting and/or enhancing anti-tumor effects. RESULTS: The first strategy was unsuccessful since celastrol treatment increased HSP70 in all 7 of the cancer cell types tested, this result related to HSF1 activation. The ubiquity of HSF1 expression in different cancer cells might explain why celastrol has no cell-type limitation for HSP70 induction. The second strategy revealed that modification of celastrol's carboxyl group abolished its ability to elevate HSP70, but also abolished celastrol's tumor inhibition effects. In the third strategy, 11 inhibitors for 10 signaling proteins reportedly related to celastrol action were tested, and five of these could reduce celastrol-caused HSP70 elevation. Among these, the peptide deformylase (PDF) inhibitor, actinonin, could synergize celastrol's proliferation inhibition. CONCLUSIONS: Concurrent use of the chemical agent actinonin could reduce celastrol's HSP70 elevation and also enhance proliferation inhibition by celastrol. This combination presents a novel alternative to siRNA technology and is worth further investigation for its potentially effective anti-tumor action.

IP-FCM platform detects the existence and regulator-caused dissociation of components in naturally assembled HSP90 complex.

Flow cytometry, in conjunction with immunoprecipitation (IP-FCM), is suggested to have some advantages to conventional IP-western blot technology in analyzing protein complexes. In this paper, to further examine its practicability, we test the use of IP-FCM in detecting the HSP90 complex, which has gained importance in drug research and development and involves more than a dozen components. We found that IP-FCM could effectively detect HSP70, p23, Cdc37, and Cdk6 components in the HSP90 complex naturally formed in U937 cells when this complex was captured by anti-HSP90 antibody-coated polystyrene microspheres. IP-FCM could also detect alteration in components caused by treating cells with HSP90 inhibitors. In a cell-free environment, IP-FCM could detect the direct effects of ATP and/or HSP90 inhibitors (17-N-allylamino-17-demethoxygeldanamycin or celastrol) in causing component dissociation and the time- and dose-effects of inhibitor-caused dissociation. IP-FCM is a practical and powerful platform for analyzing HSP90 complex components, and is thus a useful tool in studying HSP90 complex function and screening inhibitors.

Celastrol regulates multiple nuclear transcription factors belonging to HSP90's clients in a dose- and cell type-dependent way.

Celastrol, a novel HSP90 inhibitor, has recently attracted much attention due to its potential in multiple applications, such as anti-inflammation use, degenerative neuron disease relief, and tumor management. At present, the studies in celastrol's effects on HSP90's clients have focused on the kinase sub-population, while another key sub-population, nuclear transcription factors (TFs), is not being well-explored. In this study, we observe the effects of celastrol on 18 TFs (belonging to HSP90 clients) in three human cell lines: MCF-7 (breast cancer), HepG2 (hepatoma), and THP-1 (monocytic leukemia). The results show that at least half of the detectable TFs were affected by celastrol, though the effect patterns varied with cell type and dosage. Bi-directional regulations of some TFs were identified, a phenomenon not yet seen with other HSP90 inhibitors. Celastrol's capability to affect multiple TFs was consistent with its altering HSP90/TFs interactions and disrupting HSP90/Hop interaction, in addition to the reported damaging HSP90/Cdc37 interaction. This work confirms, for the first time, that celastrol has broad effects on TFs belonging to HSP90's clients, casts new light on understanding these reported actions, and suggests new possible applications for celastrol, such as diabetes management.