The discovery of water-soluble indazole derivatives as potent microtubule polymerization inhibitors.

Taking a previously discovered indazole derivative 1 as a lead, systematic structural modifications were performed with an indazole core at the 1- and 6-positions to improve its aqueous solubility. Among the designed indazole derivatives, 6-methylpyridin-3-yl indazole derivative 8l and 1H-indol-4-yl indazole derivative 8m exhibited high potency in the low nanomolar range against A549, Huh-7, and T24 cancer cells, including Taxol-resistant variant cells (A549/Tax). As a hydrochloride salt, 8l exhibited much improved aqueous solubility, and its log P value fell into a favorable range. In mechanistic studies, 8l impeded tubulin polymerization through interacting with the colchicine site, resulting in cell cycle arrest and cellular apoptosis. In addition, compared to lead compound 1, 8l reduced cell migration and led to more potent inhibition of tumor growth in vivo without apparent toxicity. In summary, indazole derivative 8l could work as a potential anticancer agent and deserves further investigation for cancer therapy.

A trio of tumor suppressor miRNA downregulates CREB5 dependent transcription to modulate neoadjuvant hormonal therapy sensitivity.

Neoadjuvant hormonal therapy (NHT) prior to radical prostatectomy (RP) is an approach that can potentially maximize survival outcomes in prostate cancer (PCa) patients with high-risk disease. Unfortunately, subsets of patients do not respond well to such hormonal therapy. We previously identified several pathological parameters in predicting differences in response to NHT of PCa. However, little is known about the potential role and mechanism of miRNAs mediated NHT resistance (NHT-R) in PCa. Here we demonstrate that miR-l42-3p, miR-150-5p and miR-342-3p are the top downregulated miRNAs in PCa tissues with NHT-R. Functional analysis reveals that the three miRNAs inhibit cell proliferation in vitro. Transfection of miRNAs mimics strengthens the inhibitory effects of bicalutamide and enzalutamide to PCa cells. Luciferase reporter assay reveals that CREB5 is the common target of these three miRNAs. Clinically, high expression level of CREB5 correlates with high Gleason score, advanced tumor stage and NHT-R in PCa tissues. CREB5 expression promotes antiandrogen therapy resistance in LNCaP cells and IL6 signaling pathway may be involved in this process. In all, our findings highlight an important role of miR-142-3p, miR-150-5p, and miR-342-3p in contributing NHT-R by targeting CREB5 in PCa.

RUVBL1 promotes enzalutamide resistance of prostate tumors through the PLXNA1-CRAF-MAPK pathway.

Although enzalutamide improves the overall survival of patients with metastatic prostate cancers, enzalutamide resistance (ENZR) will be inevitably developed. Emerging evidence support that alternative oncogenic pathways may bypass the androgen receptor (AR) signaling to promote ENZR progression, however, the underpinning mechanisms remain poorly defined. Here, we report that the expression of RuvB like AAA ATPase 1 (RUVBL1) is upregulated in ENZR cells and xenograft models and prostate tumors in patients. Enzalutamide increases RUVBL1 accumulation in the cytoplasm, which in turn enhances the recruitment of CRAF proto-oncogene serine/threonine kinase protein to plexin A1 (PLXNA1) and the subsequent activation of the downstream MAPK pathway. Co-overexpression of RUVBL1 and PLXNA1 defines a subgroup of prostate cancer (PCa) patients with a poor prognosis. Furthermore, pharmacological inhibition of RUVBL1 by CB-6644 suppresses ENZR cell proliferation and xenograft growth and allows re-sensitization of ENZR cells and xenografts to enzalutamide, indicating that RUVBL1 may act to substitute the AR signaling to promote cancer cell survival and ENZR development. Together, these findings may lead to the identification of RUVBL1 as a potential therapeutic target for ENZR tumors.

Isotschimgine alleviates nonalcoholic steatohepatitis and fibrosis via FXR agonism in mice.

Farnesoid X receptor (FXR) agonist obeticholic acid (OCA) has emerged as a potential therapy for nonalcoholic fatty liver disease (NAFLD). However, the side effects of OCA may limit its application in clinics. We identified previously that isotschimgine (ITG) is a non-steroidal FXR selective agonist and has potent therapeutic effects on NAFLD in mice. Here, we aimed to evaluate the therapeutic effects of ITG on nonalcoholic steatohepatitis (NASH) and fibrosis in mice. We used methionine and choline deficient (MCD) diet-induced NASH mice, bile duct ligation (BDL), and carbon tetrachloride (CCl4 )-treated hepatic fibrosis mice to investigate the effects of ITG on NASH, fibrosis, and cholestatic liver injury. Our results showed that ITG improved steatosis and inflammation in the liver of MCD diet-fed mice, as well as alleviated fibrosis and inflammation in the liver of CCl4 -treated mice. Furthermore, ITG attenuated serum bile acid levels, and reduced vacuolization, inflammatory infiltration, hepatic parenchymal necrosis, and collagen accumulation in the liver of BDL mice. Mechanistically, ITG increased the expression of FXR target genes. These data suggest that ITG is an FXR agonist and may be developed as a novel therapy for NASH, hepatic fibrosis, or primary biliary cholangitis.

Suppressing NK Cells by Astragaloside IV Protects Against Acute Ischemic Stroke in Mice Via Inhibiting STAT3.

Natural killer (NK) cells, a key member of innate lymphocytes, are a promising immunotherapeutic target for ischemic stroke. Astragaloside IV (ASIV) is isolated from Astragalus mongholicus Bunge (Fabaceae), a herbal medicine possessing immunomodulatory ability. This study investigated the effect of ASIV on NK cells during the acute stage of brain ischemic injury in a mouse model of middle cerebral artery occlusion (MCAO). MCAO mice treated with ASIV had better functional outcomes, smaller brain infarction and less NK cell brain infiltration. NK cell depletion echoed the protective effect of ASIV. Notably, ASIV did not enhance the protective effect of NK cell depletion against brain ischemic injury. ASIV inhibited glial cell-derived CCL2-mediated chemotaxis to prevent post-ischemic NK cell brain recruitment. Meanwhile, ASIV also abrogated NK cell-mediated cytolytic killing of neurons subjected to oxygen-glucose deprivation and suppressed NK cell-derived IFN-γ and NKG2D expression in the ischemic brain. The inhibitory effect of ASIV on NK cell brain infiltration and activation was mimicked by cryptotanshinone, a STAT3 inhibitor. There was no additive effect when ASIV and cryptotanshinone were used together. In conclusion, ASIV inhibits post-ischemic brain infiltration and activation of NK cells through STAT3 suppression, and this inhibitory effect of ASIV on NK cells plays a key role in its protection against acute ischemic brain injury. Our findings suggest that ASIV is a promising therapeutic candidate in NK cell-based immunotherapy for the treatment of acute ischemic stroke and pave the way for potential clinical trials.

Astragaloside IV suppresses post-ischemic natural killer cell infiltration and activation in the brain: involvement of histone deacetylase inhibition.

Natural killer (NK) cells, a type of cytotoxic lymphocytes, can infiltrate into ischemic brain and exacerbate neuronal cell death. Astragaloside IV (ASIV) is the major bioactive ingredient of Astragalus membranaceus, a Chinese herbal medicine, and possesses potent immunomodulatory and neuroprotective properties. This study investigated the effects of ASIV on post-ischemic brain infiltration and activation of NK cells. ASIV reduced brain infarction and alleviated functional deficits in MCAO rats, and these beneficial effects persisted for at least 7 days. Abundant NK cells infiltrated into the ischemic hemisphere on day 1 after brain ischemia, and this infiltration was suppressed by ASIV. Strikingly, ASIV reversed NK cell deficiency in the spleen and blood after brain ischemia. ASIV inhibited astrocyte-derived CCL2 upregulation and reduced CCR2+ NK cell levels in the ischemic brain. Meanwhile, ASIV attenuated NK cell activating receptor NKG2D levels and reduced interferon-γ production. ASIV restored acetylation of histone H3 and the p65 subunit of nuclear factor-κB in the ischemic brain, suggesting inhibition of histone deacetylase (HDAC). Simultaneously, ASIV prevented p65 nuclear translocation. The effects of ASIV on reducing CCL2 production, restoring acetylated p65 levels and preventing p65 nuclear translocation were mimicked by valproate, an HDAC inhibitor, in astrocytes subjected to oxygen-glucose deprivation. Our findings suggest that ASIV inhibits post-ischemic NK cell brain infiltration and activation and reverses NK cell deficiency in the periphery, which together contribute to the beneficial effects of ASIV against brain ischemia. Furthermore, ASIV's effects on suppressing NK cell brain infiltration and activation may involve HDAC inhibition.

Cycloastragenol upregulates SIRT1 expression, attenuates apoptosis and suppresses neuroinflammation after brain ischemia.

Cycloastragenol (CAG) is the active form of astragaloside IV isolated from Astragalus Radix, which displays multiple pharmacological effects. Silent information regulator 1 (SIRT1), a class III histone deacetylase, has been shown to play an important role in neuroprotection against cerebral ischemia. In this study, we investigated whether CAG protected against ischemic brain injury and, if so, whether the beneficial effects were associated with the regulation of SIRT1 in the ischemic brain. Mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) followed by reperfusion. CAG (5, 10, 20 mg/kg) was injected intraperitoneally at the onset of reperfusion, 12 h later and then twice daily for up to three days. CAG dose-dependently reduced brain infarct volume, significantly ameliorated functional deficits, and prevented neuronal cell loss in MCAO mice. Meanwhile, CAG significantly reduced matrix metalloproteinase-9 activity, prevented tight junction degradation and subsequently ameliorated blood-brain barrier disruption. Moreover, CAG significantly upregulated SIRT1 expression in the ischemic brain but did not directly activate its enzymatic activity. Concomitant with SIRT1 upregulation, CAG reduced p53 acetylation and the ratio of Bax to Bcl-2 in the ischemic brain. CAG also inhibited NF-κB p65 nuclear translocation. As a result, CAG suppressed the mRNA expression of pro-inflammatory cytokines, including TNF-α and IL-1ß, and inhibited the activation of microglia and astrocytes in the ischemic brain. Our findings suggest that CAG is neuroprotective against ischemic brain injury in mice and that its beneficial effect may involve SIRT1 upregulation and the inhibition of apoptosis and neuroinflammation in the ischemic brain.

Buyang Huanwu Decoction Attenuates Infiltration of Natural Killer Cells and Protects Against Ischemic Brain Injury.

BACKGROUND/AIMS: Natural killer (NK) cells are among the first immune cells that respond to an ischemic insult in human brains. The infiltrated NK cells damage blood-brain barrier (BBB) and exacerbate brain infarction. Buyang Huanwu Decoction (BHD), a classic Chinese traditional herbal prescription, has long been used for the treatment of ischemic stroke. The present study investigated whether BHD can prevent brain infiltration of NK cells, attenuate BBB disruption and improve ischemic outcomes. METHODS: Transient focal cerebral ischemia was induced in rats by a 60-minute middle cerebral artery occlusion, and BHD was orally administrated at the onset of reperfusion, 12 hours later, then twice daily. Assessed parameters on Day 3 after ischemia were: neurological and motor functional deficits through neurological deficit score and rotarod test, respectively; brain infarction through TTC staining; BBB integrity through Evans blue extravasation; matrix metalloproteinase-2/9 activities through gelatin zymography; tight junction protein, nuclear factor-kB (NF-kB) p65 and phospho-p65 levels through Western blotting; NK cell brain infiltration and CXCR3 levels on NK cells through flow cytometry; interferon-γ production through ELISA; CXCL10 mRNA levels through real-time PCR; CXCL10 expression and p65 nuclear translocation through immunofluorescence staining. RESULTS: BHD markedly reduced brain infarction, improved rotarod performance, and attenuated BBB breakdown. Concurrently, BHD attenuated the upregulation of matrix metalloproteinase-2/9 activities and the degradation of tight junction proteins in the ischemic brain. Infiltration of NK cells was observed in the ischemic hemisphere, and this infiltration was blunted by treatment with BHD. BHD suppressed brain ischemia-induced interferon-γ and chemokine CXCL10 production. Furthermore, BHD significantly reduced the expression of CXCR3 on brain-infiltrated NK cells. Strikingly, BHD did not affect NK cell levels or its CXCR3 expression in the spleen or peripheral blood after brain ischemia. The nuclear translocation of NF-kB p65 and phospho-p65 in the ischemic brain was inhibited by BHD. CONCLUSION: Our findings suggest that BHD prevents brain infiltration of NK cells, preserves BBB integrity and eventually improves ischemic outcomes. The inhibitory effects of BHD on NK cell brain invasion may involve its ability of suppressing NF-kB-associated CXCL10-CXCR3-mediated chemotaxis. Notably, BHD only suppresses NK cells and their CXCR3 expression in the ischemic brain, but not those in periphery.