α7 nicotinic acetylcholine receptor agonist attenuates allergen-induced immediate nasal response in murine model of allergic rhinitis.

The expression of nicotinic acetylcholine receptor (nAChR) subunits on various immune cells suggests their involvement in allergic rhinitis. However, how exactly they contribute to this pathogenesis is not yet confirmed. Our present study examined the therapeutic potential of GTS-21, an α7 nAChR agonist, for treating allergic rhinitis by employing its mouse models. GTS-21 treatment reduced allergen-induced immediate nasal response in ovalbumin (OVA)-sensitized model. However, nasal hyperresponsiveness or eosinophil infiltration elicited in either the OVA-sensitized or T helper 2 cell-transplanted model was not affected by GTS-21. GTS-21 did not alter allergen-induced passive cutaneous anaphylaxis response in anti-dinitrophenyl IgE-sensitized mice. This evidence implies GTS-21's potential to alleviate allergic rhinitis without perturbing T cells or mast cells.

α7 nicotinic acetylcholine receptor agonist GTS-21 attenuates DSS-induced intestinal colitis by improving intestinal mucosal barrier function.

BACKGROUND AND AIMS: Cholinergic output, which could modulate innate immune responses through stimulation of α7 nicotinic acetylcholine receptor (α7nAChR), might be a target to minimize tissue damage in autoimmune disease. GTS-21, a selective α7nAChR agonist, has previously demonstrated to inhibit synovium inflammation in rheumatoid arthritis. In this study, we investigated the effect of GTS-21 on dextran sulfate sodium (DSS)-induced colitis model and its potential mechanism. METHODS: Male BABL/c mice (n = 32) were randomly divided into four groups: normal control group, DSS-induced colitis group, GTS-21 treatment with or without α7nAChR antagonist α-BGT treatment group. Disease activity index (DAI), histological activity index (HAI) and colonic macroscopic damage were evaluated. Fluorescein isothiocyanate (FITC)-dextran assay was applied to measure intestinal permeability. The expressions of tight junction (TJ) proteins and NF-κB associated proteins were detected by Western blot. RESULTS: GTS-21 could decrease DAI scores, HAI scores, intestinal permeability and reduce the intestinal bacterial translocation in DSS-induced colitis group, whereas α7nAChR antagonist α-BGT could impair this protective influence. The expressions of TJ proteins were increased with administration of GTS-21 both in vivo and in vitro. Furthermore, GTS-21 also inhibited the NF-қB activation in intestinal epithelial cells and colitis model, while α-BGT reversed the inhibitory effect. CONCLUSION: The α7nAChR agonist GTS-21 attenuated DSS-induced colitis through increasing expressions of TJ proteins in colon tissues and improved intestinal barrier function, which might be due to  modulating NF-қB activation in intestinal epithelial cells.

EGPI-1, a novel eIF4E/eIF4G interaction inhibitor, inhibits lung cancer cell growth and angiogenesis through Ras/MNK/ERK/eIF4E signaling pathway.

eIF4E plays an important role in regulating tumor growth and angiogenesis, and eIF4E is highly expressed in a variety of lung cancer cell lines. siRNA eIF4E can significantly inhibit the proliferation of lung cancer cells, indicating that inhibition of eIF4E may become a novel anti-tumor target. In the previous study, we synthesized a series of small molecule compounds with the potential to inhibit eIF4E. Among them, the compound EGPI-1 significantly inhibited the proliferation of a variety of lung cancer cells such as A549, NCI-H460, NCI-H1650 and 95D without inhibiting the proliferation of HUVEC cells. Further studies found that EGPI-1 interfered with the eIF4E/eIF4G interaction and inhibited the phosphorylation of eIF4E in NCI-H460 cells. The results of flow cytometry showed that EGPI-1 induced apoptosis and G0/G1 cycle arrest in NCI-H460 cell. Interestingly, we also found that EGPI-1 induced autophagy and DNA damage in NCI-H460 cells. The mechanism results showed that EGPI-1 inhibited the Ras/MNK/ERK/eIF4E signaling pathway. Moreover, EGPI-1 inhibited tube formation of HUVECs, as well as inhibited the neovascularization of CAM, proving the anti-angiogenesis activity of EGPI-1. The NCI-H460 xenograft studies showed that EGPI-1 inhibited tumor growth and angiogenesis in vivo by regulating Ras/MNK/ERK/eIF4E pathway. Our studies proved that eIF4E was a novel target for regulating tumor growth, and the eIF4E/eIF4G interaction inhibitor EGPI-1 was promising to develop into a novel anti-lung cancer drug.

A nanounit strategy reverses immune suppression of exosomal PD-L1 and is associated with enhanced ferroptosis.

In addition to increasing the expression of programmed death-ligand 1 (PD-L1), tumor cells can also secrete exosomal PD-L1 to suppress T cell activity. Emerging evidence has revealed that exosomal PD-L1 resists immune checkpoint blockade, and may contribute to resistance to therapy. In this scenario, suppressing the secretion of tumor-derived exosomes may aid therapy. Here, we develop an assembly of exosome inhibitor (GW4869) and ferroptosis inducer (Fe3+) via amphiphilic hyaluronic acid. Cooperation between the two active components in the constructed nanounit induces an anti-tumor immunoresponse to B16F10 melanoma cells and stimulates cytotoxic T lymphocytes and immunological memory. The nanounit enhances the response to PD-L1 checkpoint blockade and may represent a therapeutic strategy for enhancing the response to this therapy.

Nonopioid GTS-21 Mitigates Burn Injury Pain in Rats by Decreasing Spinal Cord Inflammatory Responses.

BACKGROUND: Burn injury (BI) pain consists of inflammatory and neuropathic components and activates microglia. Nicotinic alpha 7 acetylcholine receptors (α7AChRs) expressed in microglia exhibit immunomodulatory activity during agonist stimulation. Efficacy of selective α7AChR agonist GTS-21 to mitigate BI pain and spinal pain-mediators was tested. METHODS: Anesthetized rats after hind-paw BI received intraperitoneal GTS-21 or saline daily. Allodynia and hyperalgesia were tested on BI and contralateral paw for 21 days. Another group after BI receiving GTS-21 or saline had lumbar spinal cord segments harvested (day 7 or 14) to quantify spinal inflammatory-pain transducers or microglia activation using fluorescent marker, ionized calcium-binding adaptor protein (Iba1). RESULTS: BI significantly decreased allodynia withdrawal threshold from baseline of ~9-10 to ~0.5-1 g, and hyperalgesia latency from ~16-17 to ~5-6 seconds by day 1. Both doses of GTS-21 (4 or 8 mg/kg) mitigated burn-induced allodynia from ~0.5-1 to ~2-3 g threshold (P = .089 and P = .010), and hyperalgesia from ~5-6 to 8-9 seconds (P < .001 and P < .001) by day 1. The GTS-21 group recovered to baseline pain threshold by day 15-17 compared to saline-treated, where the exaggerated nociception persisted beyond 15-17 days. BI significantly (P < .01) increased spinal cord microgliosis (identified by fluorescent Iba1 staining), microglia activation (evidenced by the increased inflammatory cytokine), and pain-transducer (protein and/or messenger RNA [mRNA]) expression (tumor necrosis factor-α [TNF-α], interleukin-1ß [IL-1ß], nuclear factor-kappa B [NF-κB], interleukin-6 [IL-6], Janus-associated kinase signal transducer and activator of transcription 3 [JAK-STAT3], and/or N-methyl-D-aspartate receptor [NMDAR]). GTS-21 mitigated pain-transducer changes. The α7AChR antagonist methyllycaconitine nullified the beneficial effects of GTS-21 on both increased nociception and pain-biomarker expression. CONCLUSIONS: Nonopioid, α7AChR agonist GTS-21 elicits antinociceptive effects at least in part by decreased activation spinal-cord pain-inducers. The α7AChR agonist GTS-21 holds promise as potential therapeutic adjunct to decrease BI pain by attenuating both microglia changes and expression of exaggerated pain transducers.

Novel poly (ADP-ribose) polymerases inhibitor DHC-1 exhibits in vitro and in vivo anticancer activity on BRCA-deficient pancreatic cancer cells.

Poly (ADP-ribose) polymerases (PARPs) play a key role in DNA repair. In this study we designed a novel small-molecular compound, (E)-2-(2,3-dibromo-4,5-dimethoxybenzylidene)hydrazine-1-carbothioamide (DHC-1), which was a potent and selective PARP-1 inhibitor. DHC-1 selectively inhibited PARP-1 activity with an IC50 value of 41.12 ± 13.28 nM. Cytotoxicity results showed that DHC-1 selectively inhibited the proliferation of BRCA1-deficient breast cancer HCC-1937 and BRCA2-deficient pancreatic cancer Capan-1 cells. Mechanism studies found that DHC-1 stabilized PARP-1-DNA complexes and inhibited PAR formation in BRCA2-/- Capan-1 cells. Further experiments found that DHC-1 induced DNA double-strand damage in BRCA2-/- Capan-1 cells, which was demonstrated by accumulation of γ-H2AX foci. Flow cytometry experiments revealed that DHC-1 induced G2/M phase arrest and activate mitochondrial-induced apoptotic pathways. Interestingly, we also found that DHC-1 enhanced cell proliferation inhibitory effect of oxaliplatin (OXA). The further in vivo nude mouse studies showed that DHC-1 inhibited the growth of Capan-1 xenografts and showed a similar mechanism to that in vitro. Collectively, our results demonstrate that DHC-1 may be an excellent candidate for treatment of BRCA-deficient pancreatic cancers.

Cardioprotective role of GTS-21 by attenuating the TLR4/NF-κB pathway in streptozotocin-induced diabetic cardiomyopathy in rats.

The cholinergic anti-inflammatory pathway (CAP) was investigated in a variety of inflammatory conditions and constitutes a valuable line in their treatment. In the current study, we investigated the anti-inflammatory effect of GTS-21 (GTS) as a partial selective α7 nicotinic acetylcholine receptor (α7-nAchR) agonist in diabetic cardiomyopathy model in rats. This mechanism was elaborated to study whether it could alleviate the electrocardiographic, histopathological, and molecular levels of Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway proteins. Diabetes was induced by the injection of streptozotocin (STZ) (50 mg/kg). Diabetic rats were treated with GTS (1 or 2 mg/kg/day), methyllycaconitine (MLA), a selective α7-nAchR antagonist (2 mg/kg/day) plus GTS (2 mg/kg/day), or the vehicle. All treatments were given by the intraperitoneal route. Ventricular rate and different electrocardiograph (ECG) anomalies were detected. Plasma levels of cardiac troponin T (cTnT) and creatine kinase MB (CK-MB) were measured by ELISA. Additionally, we elucidated the levels of several proteins involved in the TLR4/NF-κB pathway. Cardiac levels of TLR4 and phosphorylated protein kinase B (p-Akt) were detected by ELISA. The cardiac expression of myeloid differentiation primary response 88 (Myd88), tumor necrosis factor receptor-associated factor 6 (TRAF6), NF-κB, interleukin 1ß (IL-1ß), and active caspase-1 were evaluated by immunohistochemical staining. Finally, the cardiac levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by ELISA. Diabetic rats showed (i) ECG signs of cardiomyopathy such as significant ST segment elevations, prolonged QRS, QT intervals, and ventricular tachycardia; (ii) increased plasma levels of cTnT and CK-MB; (iii) increased expression of cardiac TLR4; (iv) elevated immunohistochemical expression of cardiac, Myd88, TRAF6, and NF-κB; (v) diminution in the cardiac expression of p-Akt; and (vi) adaptive increases in cardiac expression of TNF-α and IL-6. These effects were ameliorated in diabetic rats treated with both doses of GTS. Pretreatment with MLA did not completely reverse the ameliorative effect of GTS on cTnT, TRAF6, TNF-α, and IL-6, thereby reinforcing the presence of possible α7-nAchR-independent mechanisms. The activation of α7-nAchR with GTS offers a promising prophylactic strategy for diabetic cardiomyopathy by attenuating the TLR4/NF-κB pathway.

Benzylidene thiazolidinediones: Synthesis, in vitro investigations of antiproliferative mechanisms and in vivo efficacy determination in combination with Imatinib.

Thiazolidinedione (TZD) has been an interesting scaffold due to its proven antidiabetic activity and encouraging findings in anticancer drug discovery. We synthesised benzylidene thiazolidinedione derivatives which exhibited excellent antiproliferative effects in chronic myeloid leukemic cells K562 and the most active compounds 3t and 3x had GI50 value of 0.9 and 0.23 µM respectively. Both the compound was found to arrest the growth of K562 cells in G0/G1 phase in a time and dose dependent manner. Further, western blot analysis revealed that 3t and 3x could also inhibit the expression of cell proliferation markers, PCNA and Cyclin D1 and compound 3x up-regulated apoptosis markers, cleaved PARP1 and activated caspase 3, which could be a possible mechanism for the excellent antiproliferative effects exhibited by these compounds. In vitro combination studies of 3t and 3x with Imatinib found to potentiate the antitumor effects of Imatinib. Further in vivo efficacy in K562 xenografts, of 3t and 3x alone and in combination with Imatinib was found to be promising and far better than control group and combination treatment was found to be more effective as compared to only Imatinib treated or test compound treated animals. Thus, our findings suggest that these compounds are promising antitumor agents and could help to enhance the anticancer effects of Imatinib and other tyrosine kinase inhibitors, when used in combination.

Antidepressant-Like Activities of Hispidol and Decursin in Mice and Analysis of Neurotransmitter Monoamines.

The antidepressant activities of hispidol and decursin (both potent monoamine oxidase A (MAO-A) inhibitors) were evaluated using the forced swimming test (FST) and the tail suspension test (TST) in mice, and thereafter, levels of neurotransmitter monoamines and metabolites in brain tissues were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hispidol (15 mg/kg) caused less or comparable immobility than fluoxetine (15 mg/kg; the positive control) in immobility time, as determined by FST (9.6 vs 32.0 s) and TST (53.1 vs 48.7 s), respectively, and its effects were dose-dependent and significant. Decursin (15 mg/kg) also produced immobility comparable to that of fluoxetine as determined by FST (47.0 vs 43.4 s) and TST (55.6 vs 63.4 s), and its effects were also dose-dependent and significant. LC-MS/MS analysis after FST showed that hispidol (15 mg/kg) greatly increased dopamine (DA) and serotonin levels dose-dependently in brain tissues as compared with the positive control. Decursin (15 mg/kg) dose-dependently increased DA level after TST. Slight changes in norepinephrine and 3,4-dihydroxyphenylacetic acid levels were observed after FST and TST in hispidol- or decursin-treated animals. It was observed that hispidol and decursin were effective and comparable to fluoxetine in immobility tests. These immobility and monoamine level results suggest that hispidol and decursin are potential antidepressant agents for the treatment of depression, and that they act mainly through serotonergic and/or dopaminergic systems.

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties.

We sought to design ubiquitin-proteasome system inhibitors active against solid cancers by targeting ubiquitin receptor RPN13 within the proteasome's 19S regulatory particle. The prototypic bis-benzylidine piperidone-based inhibitor RA190 is a michael acceptor that adducts Cysteine 88 of RPN13. In probing the pharmacophore, we showed the benefit of the central nitrogen-bearing piperidone ring moiety compared to a cyclohexanone, the importance of the span of the aromatic wings from the central enone-piperidone ring, the contribution of both wings, and that substituents with stronger electron withdrawing groups were more cytotoxic. Potency was further enhanced by coupling of a second warhead to the central nitrogen-bearing piperidone as RA375 exhibited ten-fold greater activity against cancer lines than RA190, reflecting its nitro ring substituents and the addition of a chloroacetamide warhead. Treatment with RA375 caused a rapid and profound accumulation of high molecular weight polyubiquitinated proteins and reduced intracellular glutathione levels, which produce endoplasmic reticulum and oxidative stress, and trigger apoptosis. RA375 was highly active against cell lines of multiple myeloma and diverse solid cancers, and demonstrated a wide therapeutic window against normal cells. For cervical and head and neck cancer cell lines, those associated with human papillomavirus were significantly more sensitive to RA375. While ARID1A-deficiency also enhanced sensitivity 4-fold, RA375 was active against all ovarian cancer cell lines tested. RA375 inhibited proteasome function in muscle for >72h after single i.p. administration to mice, and treatment reduced tumor burden and extended survival in mice carrying an orthotopic human xenograft derived from a clear cell ovarian carcinoma.

Anti-Cancer and Ototoxicity Characteristics of the Curcuminoids, CLEFMA and EF24, in Combination with Cisplatin.

In this study, we investigated whether the curcuminoids, CLEFMA and EF24, improved cisplatin efficacy and reduced cisplatin ototoxicity. We used the lung cancer cell line, A549, to determine the effects of the curcuminoids and cisplatin on cell viability and several apoptotic signaling mechanisms. Cellular viability was measured using the MTT assay. A scratch assay was used to measure cell migration and fluorescent spectrophotometry to measure reactive oxygen species (ROS) production. Western blots and luminescence assays were used to measure the expression and activity of apoptosis-inducing factor (AIF), caspases-3/7, -8, -9, and -12, c-Jun N-terminal kinases (JNK), mitogen-activated protein kinase (MAPK), and proto-oncogene tyrosine-protein kinase (Src). A zebrafish model was used to evaluate auditory effects. Cisplatin, the curcuminoids, and their combinations had similar effects on cell viability (IC50 values: 2-16 µM) and AIF, caspase-12, JNK, MAPK, and Src expression, while caspase-3/7, -8, and -9 activity was unchanged or decreased. Cisplatin increased ROS yield (1.2-fold), and curcuminoid and combination treatments reduced ROS (0.75-0.85-fold). Combination treatments reduced A549 migration (0.51-0.53-fold). Both curcuminoids reduced auditory threshold shifts induced by cisplatin. In summary, cisplatin and the curcuminoids might cause cell death through AIF and caspase-12. The curcuminoids may potentiate cisplatin's effect against A549 migration, but may counteract cisplatin's effect to increase ROS production. The curcuminoids might also prevent cisplatin ototoxicity.

Exosomes isolated from CAPS1­overexpressing colorectal cancer cells promote cell migration.

Calcium­dependent activator protein for secretion 1 (CAPS1) has been reported to promote metastasis in colorectal cancer (CRC), however, the underlying mechanisms have not yet been elucidated. The present study revealed that exosomes derived from CAPS1­overexpressing CRC cells could enhance the migration of normal colonic epithelial FHC cells. GW4869, an inhibitor of exosomes, could attenuate the migration of FHC cells. Furthermore, liquid chromatography­mass spectrometry (LC­MS) and bioinformatics analysis demonstrated that overexpression of CAPS1 could alter the expression pattern of exosomal proteins involved in cell migration. Bone morphogenetic protein 4, which may serve vital roles in the process of CAPS1­induced cell migration, was downregulated in the exosomes. In summary, the present results demonstrated that CAPS1 promotes cell migration by regulating exosomes. Inhibiting the secretion of exosomes may be helpful for the treatment of patients with metastatic CRC.

Phosphorylation of HSF1 by PIM2 Induces PD-L1 Expression and Promotes Tumor Growth in Breast Cancer.

Heat shock transcription factor 1 (HSF1) is the master regulator of the proteotoxic stress response, which plays a key role in breast cancer tumorigenesis. However, the mechanisms underlying regulation of HSF1 protein stability are still unclear. Here, we show that HSF1 protein stability is regulated by PIM2-mediated phosphorylation of HSF1 at Thr120, which disrupts the binding of HSF1 to the E3 ubiquitin ligase FBXW7. In addition, HSF1 Thr120 phosphorylation promoted proteostasis and carboplatin-induced autophagy. Interestingly, HSF1 Thr120 phosphorylation induced HSF1 binding to the PD-L1 promoter and enhanced PD-L1 expression. Furthermore, HSF1 Thr120 phosphorylation promoted breast cancer tumorigenesis in vitro and in vivo. PIM2, pThr120-HSF1, and PD-L1 expression positively correlated with each other in breast cancer tissues. Collectively, these findings identify PIM2-mediated HSF1 phosphorylation at Thr120 as an essential mechanism that regulates breast tumor growth and potential therapeutic target for breast cancer. SIGNIFICANCE: These findings identify heat shock transcription factor 1 as a new substrate for PIM2 kinase and establish its role in breast tumor progression.

Benzylideneacetone Derivatives Inhibit Osteoclastogenesis and Activate Osteoblastogenesis Independently Based on Specific Structure-Activity Relationship.

(E)-3,4-Dihydroxybenzylideneacetone (compound 1) inhibited receptor activator of NF-κB ligand-induced osteoclastogenesis of C57BL/6 bone marrow monocyte/macrophages with IC50 of 7.8 µM (IC50 of alendronate, 3.7 µM) while stimulating the differentiation of MC3T3-E1 osteoblastic cells, accompanied by the induction of Runt-related transcription factor 2, alkaline phosphatase, and osteocalcin. (E)-4-(3-Hydroxy-4-methoxyphenyl)-3-buten-2-one (compound 2c) showed a dramatically increased osteoclast-inhibitory potency with IC50 of 0.11 µM while sustaining osteoblast-stimulatory activity. (E)-4-(4-Hydroxy-3-methoxyphenyl)-3-buten-2-one (compound 2g) stimulated alkaline phosphatase production 2-fold at 50 µM without changing osteoclast-inhibitory activity, compared with compound 1. Oral administration of compounds 1, 2c, and 2g prevented ovariectomy-induced osteoporosis in ddY mice to a degree proportional to their osteoclastogenesis-inhibitory potencies. The administration of 1 (mg/kg)/d compound 2c ameliorated histomorphometry of osteoporotic bone to a degree comparable with 10 (mg/kg)/d alendronate. Conclusively, the in vitro capacity of a few benzylideneacetone derivatives to inhibit osteoclastogenesis supported by independent osteoblastogenesis activation was convincingly reflected in in vivo management of osteoporosis, suggesting a potential novel therapeutics for osteopenic diseases.

PIM1 inhibitor SMI-4a attenuated lipopolysaccharide-induced acute lung injury through suppressing macrophage inflammatory responses via modulating p65 phosphorylation.

PIM kinase is involved in the cellular processes of growth, differentiation and apoptosis. However, the role of PIM1 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains largely unknown. A trend of PIM1 in the lung tissue of LPS-induced ALI at different time points was detected. Histology, wet/dry (W/D) ratio, inflammatory cells in the bronchoalveolar lavage fluid (BALF) and survival rate analyses were performed when mice received the PIM1 inhibitor SMI-4a intratracheally 3 h before LPS administration. Cytokine production in vivo and in vitro was measured after SMI-4a pretreatment. NF-κB subunit p65 expression in nuclei and phosphorylation at Ser276 in lung tissues or cells were detected by Western blot analysis. The results showed that PIM1 mRNA and protein were upregulated in the lung tissue of LPS-induced ALI. The PIM1 inhibitor SMI-4a markedly improved the survival rate after lethal LPS administration, reduced the severity of lung edema, attenuated the histologic injuries of the lung tissue and reduced the counts of infiltrated inflammatory cells in the BALF. The PIM1 inhibitor SMI-4a suppressed the production of cytokines in LPS-treated RAW264.7 cell supernatants and BALF. Furthermore, LPS administration upregulated the levels of nuclear p65 and phosphorylated p65 (p-p65) at Ser276, whereas pretreatment with the PIM1 inhibitor SMI-4a reduced p65 upregulation in the nucleus and p-p65 at Ser276. Taken together, these data indicate that the PIM1 inhibitor SMI-4a may serve as a promising therapeutic strategy for LPS-induced ALI by suppressing macrophage production of cytokines via a reduction of p65 activities.

Design and optimize N-substituted EF24 as effective and low toxicity NF-κB inhibitor for lung cancer therapy via apoptosis-to-pyroptosis switch.

As NF-κB signaling pathway is constitutively activated in lung cancer, targeting NF-κB has a potential for the treatment. EF24 has been proved to be a NF-κB inhibitor with good antitumor activity, while whose toxicity possibly became one of the obstacles to enter into clinical application. In order to find high efficiency and low toxicity NF-κB inhibitors, EF24 was modified and 13d was screened out. It was proved that 13d possessed an effective combination of inhibiting NF-κB pathway and showing lower cytotoxicity on normal cells as well as less toxicity in acute toxicity experiment compared with the lead compound of EF24. In addition, 13d was found to inhibit cell vitality, arrest cell cycle in G2/M phase, promote cell apoptosis, and suppress the xenograft tumor growth. Furthermore, 13d was elucidated to induce pyroptosis developing from apoptosis, which was associated with the inhibition of NF-κB. Taken together, it was suggested that 13d was a potent antitumor agent.

GrgA as a potential target of selective antichlamydials.

Chlamydia is a common pathogen that can causes serious complications in the reproductive system and eyes. Lack of vaccine and other effective prophylactic measures coupled with the largely asymptomatic nature and unrare clinical treatment failure calls for development of new antichlamydials, particularly selective antichlamydials without adverse effects on humans and the beneficial microbiota. We previously reported that benzal-N-acylhydrazones (BAH) can inhibit chlamydiae without detectable adverse effects on host cells and beneficial lactobacilli that dominate the human vaginal microbiota among reproductive-age women. However, the antichlamydial mechanism of BAH is not known. Whereas 4 single nucleotide polymorphisms (i.e., SNP1-4) were identified in a rare Chlamydia variant with a low level of BAH resistance, termed MCR, previous studies failed to establish a causal effect of any particular SNP(s). In the present work, we performed recombination to segregate the four SNPs. Susceptibility tests indicate that the R51G GrgA allele is both necessary and sufficient for the low level of BAH resistance. Thus, the Chlamydia-specific transcription factor GrgA either is a direct target of BAH or regulates BAH susceptibility. We further confirm an extremely low rate of BAH resistance in Chlamydia. Our findings warrant exploration of GrgA as a therapeutic and prophylactic target for chlamydial infections.

PIM inhibitor SMI-4a induces cell apoptosis in B-cell acute lymphocytic leukemia cells via the HO-1-mediated JAK2/STAT3 pathway.

OBJECTIVES: The serine/threonine PIM protein kinases are critical regulators of tumorigenesis in multiple cancers. However, whether PIMs are potential therapeutic targets for treating B-cell acute lymphocytic leukemia (B-ALL) remains unclear. Therefore, here, PIM expression was detected in B-ALL patients and the effects of SMI-4a, a pan-PIM small molecule inhibitor, were investigated in B-ALL cells. METHODS: PIM1 and PIM2 expression in 26 newly diagnosed B-ALL cases was detected by real-time PCR and Western blot. B-ALL cells were treated with varied SMI-4a doses and the viability of treated cells was investigated using a cell-counting kit-8 (CCK-8) assay. Apoptosis and cell cycles were analyzed by flow cytometry. Western blot analysis was then used to explore the expression of apoptosis-related proteins and the JAK2/STAT3 pathway. RESULTS: PIM1 and 2 were overexpressed in B-ALL patients with high HO-1 level. SMI-4a induced decreases in PIMs and HO-1 expressions and inhibited B-ALL cell viability. Treatment with SMI-4a induced apoptosis by downregulating Bcl-2, upregulating Bax and other antiapoptotic proteins, and decreasing protein levels of p-JAK2 and p-STAT3. In addition, upregulation of HO-1 alleviated decrease in p-JAK2 and p-STAT3 expression, reduced SMI-4a-induced apoptosis of B-ALL cells, and influenced B-ALL cell survival. CONCLUSIONS: PIMs were highly expressed in B-ALL patients. SMI-4a inhibited B-ALL proliferation and induced apoptosis via the HO-1-mediated JAK2/STAT3 pathway. SMI-4a might be applicable for treatment of B-ALL cells.

2-Benzylidene-1-Indanone Analogues as Dual Adenosine A1/A2a Receptor Antagonists for the Potential Treatment of Neurological Conditions.

Previous studies explored 2-benzylidine-1-tetralone derivatives as innovative adenosine A1 and A2A receptor antagonists for alternative non-dopaminergic treatment of Parkinson's disease. This study's aim is to investigate structurally related 2-benzylidene-1-indanones with substitutions on ring A and B as novel, potent and selective adenosine A1 and A2A receptor blockers. 2-Benzylidene-1-indanone derivatives were synthesised via acid catalysed aldol condensation reactions and evaluated via radioligand binding assays to ascertain structure activity relationships to govern A1 and A2A AR affinity. The results indicated that hydroxy substitution at C4 of ring A and meta (3'), or para (4') substitution on ring B of the 2-benzylidene-1-indanone scaffold (2C: ) is preferred over substitution at C5 (2D: ) or C6 (2E: ) of ring A for adenosine A1 receptor activity and selectivity in the micromolar range. Furthermore, substitution at the meta (3') position of ring B with chlorine lead to the highly potent and selective adenosine A2A receptor antagonist, compound 2 H: . Compound 2C: and the 2Q: behaved as adenosine A1 receptor antagonists in the performed GTP shift assays. In view of these findings, compounds 2C: , 2 H: , 2Q: and 2P: are potent and selective adenosine A1 and A2A receptor antagonists for the potential treatment of neurological conditions.

Inhibition of Carrageenan/Kaolin-Induced Arthritis in Rats and of Inflammatory Cytokine Expressions in Human IL-1ß-Stimulated Fibroblast-like Synoviocytes by a Benzylideneacetophenone Derivative.

The benzylideneacetophenone derivative JC3 [(2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one] (JC3) was synthesized by modifying yakuchinone B obtained from the seeds of Alpinia oxyphylla, a member of the ginger family (Zingiberaceae), which are widely used as a folk remedy and as an anti-inflammatory. The aim of this study was to investigate the anti-arthritic effects of JC3 in rat models of carrageenan-induced paw pain and carrageenan/kaolin-induced knee arthritis. The anti-nociceptive effect of JC3 was assessed by measuring paw withdrawal pressure thresholds using an analgesy-meter. Arthritic symptoms in our monoarthritic rat model were evaluated using weight distribution ratios (WDR), paw thicknesses, and serum prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and vascular endothelial growth factor (VEGF) levels (determined by ELISA). Histological analyses of knee joints were performed after injecting JC3 intraperitoneally into rats before carrageenan treatment at 5 or 10 mg/kg/day for 6 days. The anti-inflammatory effects of JC3 were investigated in vitro using interleukin-1beta (IL-1ß)-stimulated fibroblast-like synoviocytes (FLS) derived from arthritis patients. PGE2, IL-6, and IL-8 levels were measured after treating FLS with JC3. In arthritis-induced rats, JC3 treatment significantly decreased nociceptive and arthritic symptoms at days 5 to 6 after carrageenan/kaolin injection. Histological staining of knee tissue showed that JC3 significantly reduced inflammatory areas in the knee joints. Furthermore, JC3 inhibited the expressions of IL-6 and IL-8 in FLS cells at concentrations of 5-10 µg/ml and decreased PGE2 levels in FLS cells. These findings suggest JC3 has anti-arthritic effects in in vivo and in vitro, and that it might be useful for the treatment of arthritis.