Difeng Zhu

Insulin sensitizing activity of ethyl acetate fraction of Acorus calamus L. in vitro and in vivo

UNLABELLED Acorus calamus L. (AC), family Araceae, have been used in the Indian and Chinese systems of medicine for hundreds of years. The radix of AC is widely used in the therapy of diabetes in traditional folk medicine of America and Indonesia. AIM OF THE STUDY To investigate the insulin sensitizing activity and antidiabetic effects of the ethyl acetate fraction of AC (ACE). MATERIALS AND METHODS Glucose consumption mediated by insulin was detected in L6 rat skeletal muscle cells. Diabetes and its complications related indexes were monitored after orally administrating to genetically obese diabetic C57BL/Ks db/db mice daily for 3 weeks. RESULTS ACE (12.5 and 25 microg/ml) increased glucose consumption mediated by insulin in L6 cells (p<0.05 and p<0.01). In db/db mice, ACE (100 mg/kg) significantly reduced serum glucose, triglyceride, reinforce the decrease of total cholesterol caused by rosiglitazone (at least p<0.05), and markedly reduced free fatty acid (FFA) levels and increased adiponectin levels (p<0.01 and p<0.05) as rosiglitazone did (p<0.05 and p<0.001). Serum insulin was decreased but not significantly. In addition, ACE decreased the intake of food and water, and did not increase body weight gain whereas rosiglitazone did. CONCLUSIONS Owing to the ability of insulin sensitizing, ACE has the potential to be useful for the treatment of diabetes and cardiovascular complications without body weight gain.

BZYX, a novel acetylcholinesterase inhibitor, significantly improved chemicals-induced learning and memory impairments on rodents and protected PC12 cells from apoptosis induced by hydrogen peroxide.

BZYX was designed as a dual-binding-site acetylcholinesterase (AChE) inhibitor and selected from series of indanone derivatives. The present study was designed to examine the cognition-enhanced, anti-cholinesterase, and neuroprotective effects of BZYX. In the passive avoidance performance and radial arm maze, BZYX showed a comparable effect to donepezil and rivastigmine on memory deficits in different stages induced by scopolamine, NaNO(2) and ethanol, respectively. Ellmans assay indicated BZYX exhibited high inhibition on AChE activity. IC(50) values for BZYX: 0.058+/-0.022 microM; donepezil: 0.019+/-0.004 microM; rivastigmine: 3.81+/-2.81 microM; glantamine: 3.01+/-1.85 microM and huperzine A: 0.053+/-0.016 microM. BZYX also presented great neuroprotecive function from apoptosis induced by hydrogen peroxide(H(2)O(2)) in PC12 cells. MTT assay and Annexin V-FITC Apoptosis Detection showed the viability of PC12 cells remarkably decreased with 400 microM H(2)O(2), while it significantly increased when the cells were pretreated with 0.1-1.0 microM BZYX. BZYX pretreatment remarkably reversed the loss of mitochondria membrane potential (DeltaPsim), scavenged reactive oxygen species formation induced by H(2)O(2) and resulted in up-regulation of procaspase3 and xIAP protein level and down-regulation of phosphorylated JNK protein, p53 protein level and cleavage of caspase 3. It is speculated that the mitochondrial pathway, mediated by Bcl-2 family and Mitogen-Activated Protein Kinases (MAPKs), might involved in the neuroprotection of BZYX. These results first demonstrated that BZYX had neuroprotective effects as well as cognition enhancement and acetylcholinesterase inhibition. It is hopeful that BZYX becomes a potential candidate for use in the intervention for neurodegenerative diseases.

Inhibition of all‐Trans‐retinoic acid‐induced proteasome activation potentiates the differentiating effect of retinoid in acute myeloid leukemia cells

All‐trans retinoic acid (ATRA) is nowadays considered to be the sole efficient agent for differentiation‐based therapy in leukemia; however, the mechanisms of ATRAs biological effects remain largely unknown. Here we first reported that ATRA‐induced myeloid leukemia differentiation was accompanied with the increased level of ubiquitin–protein conjugates and the upregulation of proteasome activity. To explore the functional role of the activated proteasome in retinoic acid (RA) signaling, the effects of proteasome inhibitors on RA‐induced cell differentiation were determined. Our results demonstrated that inhibition of ATRA‐elevated proteasome activity obviously promoted the myeloid maturation program triggered by ATRA, suggesting that the overactivated proteasome is not beneficial for ATRAs effects. Further studies demonstrated that the synergistic differentiating effects of ATRA and proteasome inhibitors might be associated with the protection of retinoic acid receptor alpha (RARα) from degradation by the ubiquitin–proteasome pathway (UPP). Moreover, the accumulated RARα was able to enhance the transcription of its target gene, which might also contribute to the enhanced differentiation of leukemia cells. Together, by linking the UPP to ATRA‐dependent signaling, our data provide a novel insight into studying the mechanisms of ATRA‐elicited cellular effects and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy. Mol. Carcinog.

Nuclear translocation and activation of YAP by hypoxia contributes to the chemoresistance of SN38 in hepatocellular carcinoma cells

Although hypoxia is a prominent feature contributing to the therapeutic resistance of hepatocellular carcinoma cells (HCC) against chemotherapeutic agents, including the Topoisomerase I inhibitor SN38, the underlying mechanism is not fully understood and its understanding remains a major clinical challenge. In the present study, we found that hypoxia-induced nuclear translocation and accumulation of YAP acted as a survival input to promote resistance to SN38 in HCC. The induction of YAP by hypoxia was not mediated by HIF-1α because manipulating the abundance of HIF-1α with CoCl2, exogenous expression, and RNA interference had no effect on the phosphorylation or total levels of YAP. The mevalonate-HMG-CoA reductase (HMGCR) pathway may modulate the YAP activation under hypoxia. Combined YAP inhibition using either siRNA or the HMGCR inhibitor statins together with SN38 treatment produced improved anti-cancer effects in HCC cells. The increased anti-cancer effect of the combined treatment with statins and irinotecan (the prodrug of SN-38) was further validated in a human HepG2 xenograft model of HCC in nude mice. Taken together, our findings identify YAP as a novel mediator of hypoxic-resistance to SN38. These results suggest that the administration of SN28 together with the suppression of YAP using statins is a promising strategy for enhancing the treatment response in HCC patients, particularly in advanced stage HCC cases presenting hypoxic resistance.

Q39, a quinoxaline 1,4-Di-N-oxide derivative, inhibits hypoxia-inducible factor-1α expression and the Akt/mTOR/4E-BP1 signaling pathway in human hepatoma cells

SummaryCumulative evidence has established that hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in hepatocellular carcinoma angiogenesis, invasiveness and metastasis. 3-(4-bromophenyl)-2-(ethylsulfonyl)-6-methylquinoxaline 1,4-dioxide (Q39) has recently shown great antiproliferative activity in extensive cell lines in normoxia and hypoxia. In this study, Q39 exhibited high antiproliferative activity against hepatoma both in vitro and in vivo, mainly by inducing apoptosis. In addition, suppression of HIF-1α by Q39 resulted in a drastic decrease in VEGF expression. These results indicate that Q39 is an effective inhibitor of HIF-1α and provide new perspectives into the mechanism of its anticancer activity. Interestingly, neither the HIF-1α degradation rate nor the HIF-1α steady-state mRNA level was affected by Q39. Instead, suppression of HIF-1α accumulation by Q39 correlated with prominent dephosphorylation of mTOR and 4E-BP1, a pathway known to regulate protein expression at the translational level.

The Selectivity and Potency of the New PDE5 Inhibitor TPN729MA

INTRODUCTION TPN729MA is a newly developed phosphodiesterase type 5 inhibitor (PDE5i) for the treatment of erectile dysfunction, which offers potential for greater selectivity and longer duration of action than PDE5i in current clinical use. AIM We investigated the in vitro inhibitory potency and selectivity of TPN729MA on PDE isozymes, and its efficacy in animal models. METHODS The inhibition of 11 human recombinant PDEs by TPN729MA, sildenafil, and tadalafil were determined using radioimmunoassay. The effect of TPN729MA and sildenafil on intracavernous pressure (ICP), blood pressure (BP), and ICP/BP ratio were determined in a rat model of erection induced by electric stimulation and in a dog model of erection induced by sodium nitroprusside injection. MAIN OUTCOME MEASURES The main outcome measures were IC50 of TPN729MA, sildenafil, and tadalafil for PDE1-PDE11; maximum ICP; BP and ICP/BP ratio. RESULTS The IC50 of TPN729MA, sildenafil, and tadalafil for PDE5 was 2.28, 5.22, and 2.35 nM, respectively. TPN729MA showed 248, 366, 20, and 2671-fold selectivity against PDE1, PDE4, PDE6, and PDE11, respectively. TPN729MA showed excellent selectivity against PDE2, 3, 7, 8, 9, and 10 (>10,000-fold). In the rat model of erection, TPN729MA (5.0 and 2.5 mg/kg), but not sildenafil, significantly increased the maximum ICP compared with vehicle. Significantly increased ICP/BP was observed in the TPN729MA (5.0 mg/kg) group at all time points, in the TPN729MA (2.5 mg/kg) group at 75, 90, 105, and 120 minutes time points, and in sildenafil group at 75 and 90 minutes time points compared with vehicle. In the dog model of erection, TPN729MA and sildenafil significantly increased ICP and ICP/BP but showed no significant effect on BP compared with vehicle. CONCLUSIONS TPN729MA is a potent PDE5i with a balanced selectivity profile. TPN729MA shows excellent in vitro and in vivo potency, and a longer effect on erectile function than sildenafil in animal model.

Kinetics of aristolochic acid I after oral administration of Radix Aristolochiae or Guanxinsuhe preparation in canines

ETHNOPHARMACOLOGICAL RELEVANCE Aristolochic acid I (AAI), a major component derived from Aristolochia species, which have been known for a long time and remain in use today, particularly in Asia and Central America. It has been confirmed to induce a type of so-called aristolochic acid nephropathy (AAN) and involved in the development of Balkan endemic nephropathy (BEN). AIM OF THE STUDY To investigate the kinetic of AAI in beagle dogs after single-dose oral administration of Radix Aristolochiae or its preparation, Guanxinsuhe, as well as the effects of compound compatibility in traditional Chinese medicine on the pathologic processes of AAN. MATERIALS AND METHODS Beagle dogs were orally administrated Radix Aristolochiae (0.3 g/kg/day), Guanxinsuhe preparation (0.9 g/kg/day) (with an identical dosage of AAI), and empty capsules respectively for 180 days. Canines (n=2) were euthanized on day 90, 180, 210, HPLC was established to determine the AAI level in plasma and the kinetic behaviors of AAI in dogs were elucidated after single dosing. Hematoxylin and eosin (H&E)-staining was applied for histopathologic examination to evaluate the pathological status of kidneys. RESULTS Compared to canines with Radix Aristolochiae treatment, the Cmax, AUC, Tmax, and t(1/2β) of AAI in Guanxinsuhe preparation group were elevated, while t(1/2α) of AAI was decreased. The results indicated the co-existing components in Guanxinsuhe preparation could increase the absorption, accelerate the distribution, but delay the absorption and elimination of AAI. After long-term dosing, animals treated with Radix Aristolochiae were found with more severe renal impairment and higher AAI level in plasma. CONCLUSIONS It was demonstrated that the compound compatibility in Guanxinsuhe preparation can affect the kinetic process of AAI and attenuate the toxic effect on kidney when the duration of treatment was prolonged.

Bortezomib sensitizes human osteosarcoma cells to adriamycin-induced apoptosis through ROS-dependent activation of p-eIF2α/ATF4/CHOP axis

Osteosarcoma is the most common bone cancer, and chemotherapy is currently indispensable for its treatment. Adriamycin has been claimed to be the most effective agent for osteosarcoma, however, the outcome of adriamycin chemotherapy remains unsatisfactory. Here, we reported a potent combination therapy that bortezomib, a proteasome inhibitor, enhances adriamycin‐induced apoptosis to eliminate osteosarcoma cells and we revealed that the activation of p‐eIF2α/ATF4/CHOP axis is the underlying associated mechanisms. First, we observed that bortezomib enhances adriamycin‐mediated inhibition of cell proliferation and enhances the apoptosis in osteosarcoma cell lines. Moreover, this drug combination produced more potent tumor‐growth inhibitory effects in human osteosarcoma cell line KHOS/NP xenografts. Our study showed that reactive oxygen species (ROS) plays an important role in apoptosis induced by adriamycin plus bortezomib, whereas ROS scavenger NAC could almost completely block the apoptosis induced by the combination treatment. Meanwhile, p‐eIF2α is remarkably elevated in the combination group. As a result, ATF4 exhibits strong activation which consequently induces the activation of CHOP and leads to the cell death. Finally, 13 primary osteosarcoma cells demonstrated potent response to the combination treatment. In a human osteosarcoma patient‐derived xenograft (PDX) model, our finding suggests that when combined with bortezomib, a relatively low dose of adriamycin produced more potent tumor‐growth inhibitory effects without increased toxicity. Thus, our findings not only provide a promising combination strategy to overcome osteosarcoma but also shed new light on the strategy of combining increased ROS and inhibited proteasome to open up new opportunities for the clinical development of chemotherapy regimens.

Efficacy of all-trans retinoid acid in preventing nickel induced cardiotoxicity in myocardial cells of rats.

Nickel, a metal commonly found in battery plants and welding factories, has potential cardiotoxicity, while all-trans retinoid acid (atRA) can promote cardiovascular repair and myocardial recovery. The purpose of this study was to investigate whether atRA could prevent cardiotoxicity induced by nickel both in vitro and in vivo. In the study, a rat myocardial cell line (H9c2) exposed to different concentrations of nickel chloride (NiCl(2)) displayed apoptotic features accompanied by reactive oxygen species generation. In addition, NiCl(2) also caused obvious apoptosis and systolic dysfunction in primary myocardial cells. Treatment with atRA efficiently attenuated the cytotoxicities triggered by NiCl(2) as it significantly mitigated ROS generation and decreased MAP kinases activity in NiCl(2)-treated cardiomyocytes. Additionally, NiCl(2) exposure caused obvious arrhythmia in Sprague-Dawley rats with the maximum tolerance dose of NiCl(2) between 2 and 3mg/kg. A combinational intragastric administration of 40mg/kg atRA can partially reverse NiCl(2)-induced arrhythmia in rats. Our results suggested that atRA might have therapeutic potential in alleviating the adverse effects of nickel on the cardiovascular system.

Nutlin-1 strengthened anti-proliferation and differentiation-inducing activity of ATRA in ATRA-treated p-glycoprotein deregulated human myelocytic leukemia cells

Unlike its cytotoxicity in p53-functional cell lines, Nutlin-1, the small-molecule inhibitor of murine double minute (MDM2), significantly enhanced the differentiation-inducing activity of all-trans retinoic acid (ATRA) in HL60 and NB4 cells (p53-nonfunctional) but not in U937 cells (p53 wild-type). Moreover, we demonstrated that the synergistic differentiation-inducing activity of Nutlin-1 combined with ATRA appeared in a p53-independent manner. In the present study, we found that ATRA could selectively induce expression of p-glycoprotein (p-gp) in HL60 and NB4 cells but not in U937 cells. Investigation of p-gp-ATPase activity showed that Nutlin-1 and ATRA were likely to act as p-gp transport substrates. Furthermore, Nutlin-1 enhanced the ability of ATRA to induce expression of the myeloid differentiation-related transcription factor C/EBPβ and to reduce expression of c-myc. Additionally, the expression of retinoic acid receptor α (RARα) was further reduced in cells treated with ATRA in combination with Nutlin-1. Taken together, the mechanisms of synergistic differentiation-inducing activity of Nutlin-1 combined with ATRA could be attributed to Nutlin-1 competitive binding to p-gp, leading to ATRA efflux inhibition, and then the differentiation pathways involved were therefore further activated. Nutlin-1 might be a useful adjuvant with ATRA for patients with retinoid-resistant leukemia induced by overexpression of p-gp.