Month: July 2019

It has previously been shown that treatment with BEZ235 or ZSTK474 results in cell cycle arrest at G1. Our study demonstrates that cells were more likely to arrest in G1 if they had been treated with either BEZ235 or ZSTK474 with ICI 182780 temsirolimus compared to controls or single agent treatment. This may be attributed to the ability of BEZ235 to promote increased expression of the CDKI p27. Accordingly, we also detected elevated p27 expression when endometrial cancer cells were treated with BEZ235 alone or in combination with temsirolimus. While inhibition of prosurvival Akt signaling is cytotoxic, the mechanism of cell death involves autophagy and apoptosis. We observed a decrease in the autophagy marker, LC-3I, in response to dual mTOR/PI3K inhibition, implicating autophagy. Others have shown that depletion of all three Akt isoforms promoted tumor regression through initiation of autophagy, and inhibition of mTOR with the alkylphospholipid perifosine induces autophagic cell death. BEZ235 has also been shown to Niltubacin supply induce caspase-independent apoptosis in a mechanism that includes PARP cleavage, which we also observed in our study. Taken together, these data suggest that the mechanism of cell death is through autophagy and caspase-independent apoptosis. Molecular profiling of the endometrial cancer cell lines revealed that sensitivity toward drug treatment correlates with loss of PTEN expression and hyper-activation of Akt. In endometrial tumors, loss of PTEN has previously been shown to correlate with elevated Akt phosphorylation and results in poor outcomes. Our findings are consistent with earlier studies showing low expression of PTEN in RL95-2, AN3CA, ECC-1, and Ishikawa H cells and high expression of PTEN in Hec50, Hec1A, and KLE cells. Furthermore, RL95-2, AN3CA, ECC-1, and Ishikawa H cells harbor mutant PTEN, whereas KLE, Hec50, Hec1A, and Hec1Bcells express wildtype PTEN. The fact that Hec50 cells contain both wildtype PTEN and high Akt phosphorylation can be explained by recent data demonstrating that PI3KR1, a regulatory subunit of PI3K, is mutated in Hec50 cells and thus may phenocopy loss of PTEN. Additional investigation is necessary to understand why Ishikawa H cells, which have high Akt phosphorylation and a loss of active PTEN, are relatively resistant to temsirolimus. These data highlight the fact that molecular profiling does not always predict for response due to the complexity of pathways governing tumor initiation and progression. Furthermore, basal Akt phosphorylation correlates with response to another rapalog, RAD001, in a panel of various cancer cell lines. Studies involving PTEN +/2 mice or cell lines devoid of PTEN show that PTEN-deficient tumors are sensitive to mTOR inhibition. A phase I clinical trial demonstrated that 63% of patients with PTEN-negative tumors displayed tumor regression when treated with drugs targeting the PI3K/Akt/ mTOR signaling pathway. These results are consistent with our data that cell lines with little or no PTEN are more sensitive to temsirolimus alone and with the combination of temsirolimus and BEZ235. In accord with our findings, single-agent BEZ235 has been shown to inhibit proliferation of endometrial cancer cells harboring PIK3CA and/or PTEN mutations by other investigators. These studies reported herein promote a further understanding of the potential underlying mechanisms of both primary cell resistance and the development of acquired resistance after therapy, both of which can be overcome with the combination of mTOR and PI3K inhibitors. Our data underscore the need to inhibit PI3K/Akt/mTOR signaling at multiple levels to achieve sustained cellular responses.

The NuRD complex, which contains both the CHD4 ATPase and the HDAC2 deacetylase, is required for cells to repair and survive IR-induced DNA damage. Importantly, expression of the MTA1 sub-unit of NuRD is induced under hypoxic conditions, and both MTA1 and histone deacetylases contribute to Hif1a SCH727965 stability. This indicates that components of the NuRD complex may be upregulated by Hif1a. Therefore, we examined if components of the NuRD complex were increased by hypoxia mimics and if this induction contributed to the observed radioprotection by DMOG. To examine the transcriptional activity of Hif1a, we first reduced expression of Hif1a using shRNA. shRNA targeting Hif1a blocked the accumulation of Hif1a after treatment with either DMOG or CoCl2and reduced the levels of Hif1a mRNA. Silencing of Hif1a also inhibited the accumulation of VEGF mRNA, a key target of Hif1a, demonstrating that Hif1a function is abolished in the shRNAHif1a cells. Next, we determined if Hif1a regulates expression of the CHD4 and MTA3 genes. Figure 2D and 2E demonstrate that activation of Hif1a by CoCl2 increased the levels of CHD4 and MTA3 mRNA, and this increase was abolished when Hif1a was silenced with shRNA. This demonstrates that Hif1a is required for the accumulation of CHD4 and MTA3 mRNA after exposure to CoCl2. Further, CoCl2 increased the levels of Hif1a, CHD4 and MTA3 protein with similar kinetics, consistent with the Hif1adependent increase in their mRNA levels. Significantly, suppression of Hif1a with shRNA also reduced the basal levels of CHD4 protein, whereas basal levels of MTA3 were unaffected by loss of Hif1a. Further, loss of Hif1a greatly attenuated the accumulation of CHD4 after exposure to CoCl2, but had only a small impact on the accumulation of MTA3 protein. Figure 2 therefore demonstrates that increased levels of Hif1a lead to increased levels of MTA3 and CHD4 mRNA, potentially identifying CHD4 and MTA3 as transcriptional targets for Hif1a. Significantly, loss of Hif1a decreased both the basal and stimulated levels of CHD4 protein, indicating that Hif1a plays a critical role in maintaining basal and stimulated levels of CHD4. Previous work indicates that CHD4 can participate in the cells response to IR-induced DNA damage. This suggests that, because cells expressing shRNA to Hif1a have decreased levels of CHD4, they should be more sensitive to IR. Figure 3A demonstrates that cells lacking Hif1a exhibit a small but significant increase in radiosensitivity, consistent with a key role for Hif1a in regulating radiosensitivity. However, when shRNA was used to deplete CHD4 protein levels to a level similar to those detected in Hif1a depleted cellsno significant impact on radiosensitivity was seen. Similarly, silencing MTA3 expression with shRNA did not alter cellular radiosensitivity. We interpret this to mean that, while Hif1a contributes to cell survival after exposure to IR, this regulation of radiosensitivity is not mediated Adriamycin through the ability of Hif1a to regulate the expression of either CHD4 or MTA3. Therefore, although we have identified CHD4 and MTA3 as potential transcriptional targets for Hif1a, the ability of Hif1a to protect cells from radiation damage does not require either CHD4 or MTA3. It is more likely that upregulating CHD4 and MTA3, which are components of NuRD deacetylase complex, plays a key role in other processes, such as transcriptional repression, which are a feature of the hypoxia response. To further explore how stabilization of Hif1a regulates radiosensitivity, we examined a second group of proteins, the histone demethylases, which are transcriptionally activated by Hif1a.

While it has been generally assumed that all PIs possess the same degree of isoform selectivity as indinavir, direct comparisons of glucose transport blockade in GLUT1 versus GLUT4 expressing cells have been lacking. The binding affinityof indinavir for GLUT4 in the oocyte systemdiffers from that observed in primary adipocytes. While the basis for this difference is unknown, contributing factors may include subtle structural differences in the expressed transporter due to lipid composition, assay temperature, the presence of additional proteins, or other factors. It was therefore necessary to directly compare the ability of both first generation and newer PIs to alter GLUT1 versus GLUT4 activity. These data provide a more comprehensive assessment of similarities and differences in the behavior of these PIs on facilitative glucose transport. Several observations related to the ability of PIs examined in this study to compete for endofacial ATB BMPA binding have direct relevance to understanding the metabolic toxicities of these drugs in antiretroviral treatment regimens. Importantly, few studies to date have directly assessed the relationship between EX 527 intracellular PI concentrations and impaired glucose uptake. Whether PI import occurs via simple diffusion or through mediated transport, sufficient drug levels may be present within the cytosol even when serum levels are low. In addition, while it has been assumed that all PIs possess the same degree of GLUT isoform selectivity as indinavir, several PIs including ritonavir do not appear to distinguish among these transporters. Thus, the effects of some PIs on glucose homeostasis in tissues that do not express GLUT4may still be mediated by changes in glucose transport. Comparison of the effects of various PIs in these tissues may provide further insight into the mechanistic basis for altered glucose homeostasis. More comprehensive assessment of the ability of individual PIs to block each of the other known GLUTs may provide insight into glucotoxicities. While atazanavir has a more favorable metabolic profile relative to first generation PIs, the current studies demonstrate that at drug levels above those typically CUDC-907 achieved during clinical use, the potential for significantly altering glucose transport exists. The inability of tipranavir to alter either ATB BMPA binding or 2DG transport further supports the role of peptidomimetic structure in mediating binding to GLUTs. Understanding of the molecular basis for the development of insulin resistance in HIV infected patients treated with PIs has already contributed to success in developing drugs within this class that do not directly alter glucose homeostasis. Nevertheless, many of these newer agents including tipranavir are associated with dyslipidemiaand may therefore indirectly contribute to impaired insulin signaling. Furthermore, with the potential for development of viral resistance over time, the need for continued drug development remains. Characterization of the molecular interactions between candidate drugs and GLUTs will assist ongoing efforts for rationale drug design, not only for antiviral efficacy, but also for metabolic toxicity. Beyond further understanding of the mechanisms for PImediated insulin resistance, the ability to distinguish compounds that selectively interact with GLUT4 from those that bind to both GLUT1 and GLUT4 suggests that it may be possible to identify small molecule inhibitors of each of the other known GLUTs. The availability of specific pharmacologic inhibitors of these transporters would provide a means to further characterize the functional role of these isoforms prior to the induction of potential compensatory changes in gene knockout models.

ABHD12 has remained a challenging target for inhibitor development as there are no crystal structures available, number of known inhibitors is low and the existing MLN4924 side effects activity data are GDC-0879 limited. In order to find novel lead structures for selective inhibitors of recently discovered serine hydrolases, exploring the activity of natural compounds may offer valuable information for this developing process. For instance, plant-derived pentacyclic triterpenes such as betulinic, oleanolic and ursolic acid are interesting molecules as they all are bioactive and widespread in nature and their therapeutic potential is well documented see also reviews and references cited therein. In addition, their multi-targeting biological activity, low toxicity, easy availability, and core structure offering good starting point for chemical modifications, make triterpenoids appealing source for the drug discovery. Along this line, recent studies have revealed that triterpenes may include potential candidates for novel inhibitors of e.g. endocannabinoid hydrolases. Indeed, pristimerin has been shown to inhibit MAGL activity in in vitro studies. In another study, a mixture of a/b-amyrin was shown to reduce inflammatory and neuropathic hyperalgesia in mice through activation of the cannabinoid CB1 and CB2 receptors. Interestingly, despite their high affinity towards CB1R, the compounds failed to show any cannabimimetic effects in the tetrad test. In addition, a- and b-amyrin were reported to inhibit 2-AG-hydrolysis in pig brain homogenates. The molecular target of this action was not identified. Our preliminary screening efforts to identify novel serine hydrolase inhibitors among various chemical compounds revealed unexpectedly that ursolic acid was able to selectively inhibit ABHD12 with negligible effect on ABHD6 or MAGL activity. Inspired by this finding, we selected various commercial triterpenes/triterpenoids as well as recently reported betulin-based triterpenes for further evaluation. In this paper, we report the inhibitory activity of these compounds towards human ABHD12. Based on the activity data we have established preliminary structure-activity relationships and constructed the first pharmacophore model for betulin-based triterpenes. This model should prove useful in the discovery of novel lead structures for ABHD12 selective inhibitors. Although the triterpenoids typically interact with multiple protein targets, we witnessed unprecedented selectivity towards ABHD12 among the metabolic serine hydrolases, as activity-based protein profiling of mouse brain membrane proteome indicated that the representative ABHD12 inhibitors did not inhibit other serine hydrolases, nor did they target cannabinoid receptors. Pentacyclic triterpenes can be classified into three different groups: lupanes, oleananes and ursanes. Derivatives of triterpenes are called triterpenoids. In this study, commercially available triterpenes 1�C11 and triterpenoids 12�C15 were purchased from different chemical vendors and tested for their ability to inhibit hydrolase activity in lysates of HEK293 cells transiently overexpressing human ABHD12. The inhibition data are presented in Table 1. In the lupane series, an importance of a carboxyl group at position 17 was shown as betulinic acid had the highest inhibitory activity. However, lipophilicity differences should also be taken into consideration as the compound with the lowest logD also had the highest inhibitory activity. In the ursane series, similar effect of the carboxyl group at position 17 was observed as ursolic acid showed higher inhibition activity compared to a-amyrin that has a methyl group at this position. Asiatic acid, which has a primary hydroxyl group at the position 4, was completely devoid of activity, demonstrating the importance of this position for hABHD12 inhibition.

Although the effects of eNOS-derived NO on 26S proteasome functionality are not completely elucidated, the effect of NO on proteasome has drawn increased attention. NO has been found to inhibit the 26S proteasome, resulting in diminished p53 degradation or loss of cell viability. The suppressive effect is PF-4217903 956905-27-4 mediated by S-nitrosylation and reduced proteasomal subunit expression in vascular smooth muscle cells. However, others report that NO enhances proteasome activity and that the activation promoted by NO donor is thought to reduce neutral ceramidase or to protect the endothelial cell from damage induced by H2O2. It is unclear how to reconcile these discrepancies. One plausible solution could be testing the NO-exerted effects in an appropriate 26S proteasome reporter system in intact cells. The 26S proteasome functionality can be regulated through mechanisms dependent and/or independent of proteasomal protease-like activities. Until the first report of imaging 26S proteasome in living cells with a reporter system expressing UbG76V-GFP, assessments of 26S proteasome functionality have predominantly relied on the assay of protease-like activities in whole cell lysates or purified 26S proteasomes. UbG76V-GFP was engineered by expressing a surrogate protein substrate fused with a GDC-0879 Raf inhibitor ubiquitin mutant. The UbG76V mutation is crucial because it makes this GFP-bound ubiquitin resistant to removal by deubiquitinase, which would otherwise prevent the GFP from recognition and degradation by 26S proteasomes. Thus, the UbG76V mutation makes the UbG76V-GFP protein a perfect 26S proteasome substrate. As such, protein levels of GFP represent 26S proteasome functionality in cells. As a surrogate proteasome substrate, UbG76V-GFP was initially designed for the assessment of 26S proteasome inhibition in intact cells or mice. This is because when the 26S proteasome is sufficiently suppressed, the otherwise degraded poly Ub-GFP would accumulate to a significant level for quantification of the GFP fluorescence. Accordingly, UbG76V-GFP mice have been used to monitor proteasome inhibition in models of amyotrophic lateral sclerosis, Alzheimer’s disease, and polyglutamine diseases. By taking the advantage of the UbG76V-GFP reporter, together with an additional modification to its detection, we have been able to monitor the enhancement of 26S proteasome functionality in early diabetes and in glucosetreated cultured cells. This has been accomplished by employing a more sensitive approach that has combined ubiquitin enrichment through ubiquitin affinity binding purification followed by Western blotting of the GFP proteins. Physiological regulation of 26S proteasomes are complex which mechanisms remain incompletely understood; however, it is believed that the mechanisms are multifaceted and include posttranslational modifications. O-GlcNAc modification was the first endogenous inhibitor of the 26S proteasome identified in cells, although the physiological relevance has yet to be established. By utilizing the 26S proteasome reporter system both in cultured cells and mice, the present study sought to identify NO, particularly the eNOS-derived, as an endogenous regulator of the 26S proteasome in vascular endothelial cells and the involvement of proteasome O-GlcNAcylation. With a 26S proteasome reporter system, this study has identified a new mechanism by which NO affects 26S proteasome functionality in vascular endothelial cells. The presented evidence supports an alternative pathway where eNOS-derived NO blocks 26S proteasome functionality through OGT, the essential enzyme that upregulates protein O-GlcNAc modification. Mechanistically, like NO donors, the eNOS-generated NO increased an OGT-dependent O-GlcNAc modification, likely of Rpt2, one of the subunit of the proteasome regulatory.