Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer.

Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.

TW-01, a piperazinedione-derived compound, inhibits Ras-mediated cell proliferation and angioplasty-induced vascular restenosis.

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the pathogenesis of atherosclerosis and restenosis. This study investigated piperazinedione derived compound TW-01-mediated inhibitory effects on VSMC proliferation and intimal hyperplasia. METHODS: Cell proliferation was determined using [(3)H]-thymidine incorporation and MTT assay; cell cycle distribution was measured using flow cytometry; proteins and mRNA expression were determined using western blotting and RT-PCR analyses; DNA binding activity of nuclear factor-κB (NF-κB), as measured using enzyme-linked immunosorbent assays (ELISA); in vivo effects of TW-01 were determined using balloon angioplasty in the rat. RESULTS: TW-01 significantly inhibited cell proliferation. At the concentrations used, no cytotoxic effects were observed. Three predominant signaling pathways were inhibited by TW-01: (a) extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activation and its downstream effectors of c-fos, c-jun, and c-myc; (b) DNA binding activity of nuclear factor-κB (NF-κB); and, (c) Akt/protein kinase B (PKB) and cell cycle progression. Furthermore, TW-01 also inhibited Ras activation, a shared upstream event of each of these signaling cascades. In vascular injury studies, oral administration of TW-01 significantly suppressed intimal hyperplasia induced by balloon angioplasty. CONCLUSION: The present study suggests that TW-01 might be a potential candidate for atherosclerosis treatment.

Activated PAR-2 regulates pancreatic cancer progression through ILK/HIF-α-induced TGF-α expression and MEK/VEGF-A-mediated angiogenesis.

Tissue factor initiates the process of thrombosis and activates cell signaling through protease-activated receptor-2 (PAR-2). The aim of this study was to investigate the pathological role of PAR-2 signaling in pancreatic cancer. We first demonstrated that activated PAR-2 up-regulated the protein expression of both hypoxia-inducible factor-1α (HIF-1α) and HIF-2α, resulting in enhanced transcription of transforming growth factor-α (TGF-α). Down-regulation of HIFs-α by siRNA or YC-1, an HIF inhibitor, resulted in depleted levels of TGF-α protein. Furthermore, PAR-2, through integrin-linked kinase (ILK) signaling, including the p-AKT, promoted HIF protein expression. Diminishing ILK by siRNA decreased the levels of PAR-2-induced p-AKT, HIFs-α, and TGF-α; our results suggest that ILK is involved in the PAR-2-mediated TGF-α via an HIF-α-dependent pathway. Furthermore, the culture medium from PAR-2-treated pancreatic cancer cells enhanced human umbilical vein endothelial cell proliferation and tube formation, which was blocked by the MEK inhibitor, PD98059. We also found that activated PAR-2 enhanced tumor angiogenesis through the release of vascular endothelial growth factor-A (VEGF-A) from cancer cells, independent of the ILK/HIFs-α pathways. Consistent with microarray analysis, activated PAR-2 induced TGF-A and VEGF-A gene expression. In conclusion, the activation of PAR-2 signaling induced human pancreatic cancer progression through the induction of TGF-α expression by ILK/HIFs-α, as well as through MEK/VEGF-A-mediated angiogenesis, and it plays a role in the interaction between cancer progression and cancer-related thrombosis.

Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer.

BACKGROUND: The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. MATERIALS AND METHODS: Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. RESULTS: Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. CONCLUSIONS: Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation.

Effect of C7-substitution of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines on the selectivity towards a subclass of histone deacetylases.

This study focused on the substitution effect at position C7 of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines. Compound 9, (E)-3-(7-amino-1-(4-methoxyphenylsulfonyl)indolin-5-yl)-N-hydroxyacrylamide, displayed 4- to 14-fold more potent antiproliferative activity than vorinostat (SAHA, 1). Notably, 9 possessed specific histone deacetylase (HDAC) inhibitory activity toward HDAC1 and HDAC2, but had no effect on HDAC6, indicating that 9 has the potential to be developed as a class I HDAC inhibitor. In a xenograft tumor model, 9 suppressed the growth of HCT116 cells at 100 mg kg(−1), which led to a TGI (tumor growth inhibition) of 40.3%. Taken together, the C7 substitutions have a crucial effect on class I HDACs, which is beneficial for synthesizing efficient anticancer agents.

Synthesis of analogues of gingerol and shogaol, the active pungent principles from the rhizomes of Zingiber officinale and evaluation of their anti-platelet aggregation effects.

The present study was aimed at discovering novel biologically active compounds based on the skeletons of gingerol and shogaol, the pungent principles from the rhizomes of Zingiber officinale. Therefore, eight groups of analogues were synthesized and examined for their inhibitory activities of platelet aggregation induced by arachidonic acid, collagen, platelet activating factor, and thrombin. Among the tested compounds, [6]-paradol (5b) exhibited the most significant anti-platelet aggregation activity. It was the most potent candidate, which could be used in further investigation to explore new drug leads.

Depletion of 4E-BP1 and regulation of autophagy lead to YXM110-induced anticancer effects.

Natural products have always been a profuse database for developing new chemotherapeutics. YXM110 is a newly synthesized phenanthroquinolizidines that exhibits excellent anticancer activity in numerous cancer cells. In this study, we examined the anticancer mechanisms of YXM110 both in vitro and in vivo. Protein level of 4E-binding protein 1, which is crucial in cap-independent translation, was decreased significantly after YXM110 treatment via c-Jun N-terminal kinases-mediated proteasomal degradation. Moreover, the effects of YXM110 were associated with several characteristics of autophagy, including accumulation of autophagic vacuoles, elevation of Atg12-Atg5 and light chain 3 (LC3)-II, and levels of GFP-LC3 puncta. The results suggested that depletion of Mcl-1 contributes to YXM110-triggered autophagy, whereas downregulation of lysosomal-related genes could cause autophagy impairment. Furthermore, YXM110-induced cell death was prevented by autophagy inhibitor 3-methyladenine and Atg5 silencing, indicating that YXM110-mediated autophagy impairment leads to cancer cell death. These data reveal key mechanisms that support the further development of YXM110 as a promising anticancer agent.

1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines histone deacetylase inhibitors are potent cytokine release suppressors.

A series of 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines (7-15) has been developed; the compounds exhibited potent histone deacetylase (HDAC) inhibitory activities. Notably, almost all of this series exhibited better HDAC-inhibitory and antiproliferative activities than 3-(1-benzenesulfonyl-1H-indol-5-yl)-N-hydroxyacrylamide (6), as reported in a previous study. Among these compounds, 3-[1-(4-methoxybenzenesulfonyl)-2,3-dihydro-1H-indol-5-yl]-N-hydroxyacrylamide (9) showed a two- to tenfold increase in activity compared to SAHA (1) in the suppression of lipopolysaccharide-induced cytokine production. Compound 9 also caused a marked reduction in carrageenan-induced acute inflammation in a rat model. Taken together, these data indicated that 1-arylsulfonyl-5-(N-hydroxyacrylamide)indolines HDAC inhibitors exhibit potent anti-inflammatory activity.

Energy restriction-mimetic agents induce apoptosis in prostate cancer cells in part through epigenetic activation of KLF6 tumor suppressor gene expression.

Although energy restriction has been recognized as an important target for cancer prevention, the mechanism by which energy restriction-mimetic agents (ERMAs) mediate apoptosis remains unclear. By using a novel thiazolidinedione-derived ERMA, CG-12 (Wei, S., Kulp, S. K., and Chen, C. S. (2010) J. Biol. Chem. 285, 9780-9791), vis-à-vis 2-deoxyglucose and glucose deprivation, we obtain evidence that epigenetic activation of the tumor suppressor gene Kruppel-like factor 6 (KLF6) plays a role in ERMA-induced apoptosis in LNCaP prostate cancer cells. KLF6 regulates the expression of many proapoptotic genes, and shRNA-mediated KLF6 knockdown abrogated the ability of ERMAs to induce apoptosis. Chromatin immunoprecipitation analysis indicates that this KLF6 transcriptional activation was associated with increased histone H3 acetylation and histone H3 lysine 4 trimethylation occupancy at the promoter region. Several lines of evidence demonstrate that the enhancing effect of ERMAs on these active histone marks was mediated through transcriptional repression of histone deacetylases and H3 lysine 4 demethylases by down-regulating Sp1 expression. First, putative Sp1-binding elements are present in the promoters of the affected histone-modifying enzymes, and luciferase reporter assays indicate that site-directed mutagenesis of these Sp1 binding sites significantly diminished the promoter activities. Second, shRNA-mediated knockdown of Sp1 mimicked the repressive effect of energy restriction on these histone-modifying enzymes. Third, ectopic Sp1 expression protected cells from the repressive effect of CG-12 on these histone-modifying enzymes, thereby abolishing the activation of KLF6 expression. Together, these findings underscore the intricate relationship between energy restriction and epigenetic regulation of tumor suppressor gene expression, which has therapeutic relevance to foster novel strategies for prostate cancer therapy.

QS-ZYX-1-61 induces apoptosis through topoisomerase II in human non-small-cell lung cancer A549 cells.

Agents that cause DNA damage have been widely used as anticancer drugs because DNA lesions can initiate DNA checkpoints that induce cell death. The results presented here indicate that QS-ZYX-1-61, a derivative of VP-16, was significantly more potent than VP-16 in suppressing the viability of A549 cells. Treatment of cells with QS-ZYX-1-61 led to a DNA damage response and a dramatic increase of apoptosis. Our results also suggest that QS-ZYX-1-61 may be a topoisomerase (topo) II targeting agent, as evidenced by relaxation assay and induction of reversible cleavable complexes. Moreover, blocking of p53, topo IIα, and topo IIß greatly protected against caspase-3 activation, poly-ADP-ribose polymerase cleavage, and cell growth inhibition, indicating that QS-ZYX-1-61 acts through these proteins. These results support our conclusion that QS-ZYX-1-61 has potential as an anticancer agent because it causes accumulation of DNA cleavable complexes, with downstream consequences that include double-strand breaks and DNA damage response signaling for apoptosis. Taken together, our results indicate that QS-ZYX-1-61 is a novel DNA damaging agent and displays an outstanding activity that could be worthy of further investigation.

DYZ-2-90, a novel neo-tanshinlactone ring-opened compound, induces ERK-mediated mitotic arrest and subsequent apoptosis by activating JNK in human colorectal cancer cells.

Over the past several decades, there has been a considerable and still growing interest in discovering natural products with anticancer potential from traditional Chinese medicine and increasing their anticancer selectivity by chemical modification. In addition, total synthesis of active compounds from natural products can overcome problems related to poor resource availability. DYZ-2-90 is a novel ring-opened compound modified from neo-tanshinlactone, which is isolated from Chinese medicinal herb tanshen. Both in vitro and in vivo tubulin polymerization assays showed that DYZ-2-90 directly bound to microtubules and rapidly induced tubulin depolymerization, inducing ERK-mediated mitotic arrest and subsequent apoptosis by JNK activation in cancer cells, respectively. These results suggest that the fate of cells that undergo mitotic arrest is dictated by two competing networks activated by DYZ-2-90: the cytoprotective ERK pathway and the stress-related JNK pathway. DYZ-2-90 is therefore a novel microtubule-destabilizing agent and a new drug candidate for cancer therapy. This paper provides a new insight into the model of mitotic cell death, which was proposed in order to elucidate how cancer cells respond to microtubule-interfering agents and prolonged cell cycle delay.

Novel mechanism by which histone deacetylase inhibitors facilitate topoisomerase IIα degradation in hepatocellular carcinoma cells.

UNLABELLED: Histone deacetylase (HDAC) inhibitors exhibit a unique ability to degrade topoisomerase (topo)IIα in hepatocellular carcinoma (HCC) cells, which contrasts with the effect of topoII-targeted drugs on topoIIß degradation. This selective degradation might foster novel strategies for HCC treatment in light of the correlation of topoIIα overexpression with the aggressive tumor phenotype and chemoresistance. Here we report a novel pathway by which HDAC inhibitors mediate topoIIα proteolysis in HCC cells. Our data indicate that HDAC inhibitors transcriptionally activated casein kinase (CK)2α expression through increased association of acetylated histone H3 with the CK2α gene promoter. In turn, CK2 facilitated the binding of topoIIα to COP9 signalosome subunit (Csn)5 by way of topoIIα phosphorylation. Furthermore, we identified Fbw7, a Csn5-interacting F-box protein, as the E3 ligase that targeted topoIIα for degradation. Moreover, knockdown of CK2α, Csn5, or Fbw7 reversed HDAC inhibitor-induced topoIIα degradation. Mutational analysis indicates that the (1361) SPKLSNKE(1368) motif plays a crucial role in regulating topoIIα protein stability. This motif contains the consensus recognition sites for CK2 (SXXE), glycogen synthase kinase (GSK)3ß (SXXXS), and Fbw7 (SPXXS). This study also reports the novel finding that topoIIα may be a target of GSK3ß phosphorylation. Evidence suggests that CK2 serves as a priming kinase, through phosphorylation at Ser1365, for GSK3ß-mediated phosphorylation at Ser1361. This double phosphorylation facilitated the recruitment of Fbw7 to the phospho-degron (1361) pSPKLpS(1365) of topoIIα, leading to its ubiquitin-dependent degradation. CONCLUSION: This study shows a novel pathway by which HDAC inhibitors facilitate the selective degradation of topoIIα, which underlies the complexity of the functional role of HDAC in regulating tumorigenesis and aggressive phenotype in HCC cells.

Cytotoxic apigenin derivatives from Chrysopogon aciculatis.

Four new apigenin derivatives, 7-de-O-methylaciculatin, 8-C-ß-D-boivinopyranosylapigenin, aciculatinone, and 4'-O-glucosylaciculatin, along with eight known compounds, apigenin-8-carbaldehyde, kaempferol, tricin, taxifolin, 6,7,4'-trihydroxyflavone, trans-oxyresveratrol, aciculatin, and luteolin-7-sulfate, were isolated from an ethanolic extract of Chrysopogon aciculatis. Their chemical structures were elucidated by spectroscopic methods. Among the known compounds, the natural occurrence of apigenin-8-carbaldehyde and luteolin-7-sulfate is demonstrated for the first time. Some of the isolates were evaluated for cytotoxic activity against human cancer cell lines including MCF-7, H460, HT-29, and CEM.

Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases.

This study investigates the mechanism by which histone deacetylase (HDAC) inhibitors up-regulate histone H3 lysine 4 (H3K4) methylation. Exposure of LNCaP prostate cancer cells and the prostate tissue of transgenic adenocarcinoma of the mouse prostate mice to the pan- and class I HDAC inhibitors (S)-(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (AR42), N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-aminomethyl]-benzamide (MS-275), and vorinostat led to differential increases in H3K4 methylation. Chromatin immunoprecipitation shows that this accumulation of methylated H3K4 occurred in conjunction with decreases in the amount of the H3K4 demethylase RBP2 at the promoter of genes associated with tumor suppression and differentiation, including KLF4 and E-cadherin. This finding, together with the HDAC inhibitor-induced up-regulation of KLF4 and E-cadherin, suggests that HDAC inhibitors could activate the expression of these genes through changes in histone methylation status. Evidence indicates that this up-regulation of H3K4 methylation was attributable to the suppressive effect of these HDAC inhibitors on the expression of RBP2 and other JARID1 family histone demethylases, including PLU-1, SMCX, and LSD1, via the down-regulation of Sp1 expression. Moreover, shRNA-mediated silencing of the class I HDAC isozymes 1, 2, 3, and 8, but not that of the class II isozyme HDAC6, mimicked the drug effects on H3K4 methylation and H3K4 demethylases, which could be reversed by ectopic Sp1 expression. These data suggest a cross-talk mechanism between HDACs and H3K4 demethylases via Sp1-mediated transcriptional regulation, which underlies the complexity of the functional role of HDACs in the regulation of histone modifications.

CHM-1, a new vascular targeting agent, induces apoptosis of human umbilical vein endothelial cells via p53-mediated death receptor 5 up-regulation.

CHM-1 (2'-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone) has been identified as a potent antitumor agent in human hepatocellular carcinoma; however, its role in tumor angiogenesis is unclear. This study investigated the effects of CHM-1 and the mechanisms by which it exerts its antiangiogenic and vascular disrupting properties. Using a xenograft model antitumor assay, we found that CHM-1 significantly inhibits tumor growth and microvessel formation. Flow cytometry, immunofluorescence microscopy, and cell death enzyme-linked immunosorbent assay kit revealed that CHM-1 inhibits growth of human umbilical vein endothelial cells (HUVEC) by induction of apoptotic cell death in a concentration-dependent manner. CHM-1 also suppresses HUVEC migration and capillary-like tube formation. We were able to correlate CHM-1-induced apoptosis in HUVEC with the cleavage of procaspase-3, -7, and -8, as well as with the cleavage of poly(ADP-ribose) polymerase by Western blotting assay. Such sensitization was achieved through up-regulation of death receptor 5 (DR5) but not DR4 or Fas. CHM-1 was also capable of increasing the expression level of p53, and most importantly, the induction of DR5 by CHM-1 was abolished by p53 small interfering RNA. Taken together, the results of this study indicate that CHM-1 exhibits vascular targeting activity associated with the induction of DR5-mediated endothelial cell apoptosis through p53 up-regulation, which suggests its potential as an antivascular and antitumor therapeutic agent.

Thrombin induces expression of twist and cell motility via the hypoxia-inducible factor-1α translational pathway in colorectal cancer cells.

Deep vein thrombosis associated with advanced cancer is known as Trousseau's syndrome. We hypothesized that thrombin, an activator of protease-activated receptor (PAR)-1 and PAR-4 contributes to tumor metastasis. In this study, we demonstrated that thrombin and the PAR-1 activating peptide (AP) SFLLRN, but not the PAR-4 AP GYPGKF, induced HIF-1α activities, protein expression, and cell motility in colorectal cancer cells, and these actions were significantly inhibited by the PAR-1 antagonist SCH79797. Moreover, thrombin-induced HIF-1α activity and cell motility were blocked by inhibiting important mediators of signaling transduction, including the ERK, PI3K, and mTOR pathways. These results showed that thrombin induced HIF-1α protein expression through PAR-1 and HIF-1α translational de novo protein synthesis. Twist can regulate epithelial-mesenchymal transition (EMT) and increase tumor metastasis. However, we observed that thrombin-induced HIF-1α increased Twist mRNA and its protein level was mediated by the modulation of PAR-1 activation and the HIF-1α translational pathway. In addition, Twist could increase N-cadherin but not E-cadherin to promote tumor metastasis. Overexpression of dominant-negative HIF-1α reversed thrombin-mediated Twist and Twist-induced N-cadherin expression. Moreover, siTwist inhibited Twist-induced N-cadherin and Thrombin-induced cell motility. In conclusion, our study showed that thrombin-induced HIF-1α upregulated Twist at the transcriptional level to enhance cell motility. These findings show that thrombin upregulates Twist via HIF-1α to make tumor cells malignant and also establish a link between the coagulation disorder and cancer metastasis.

Aciculatin inhibits lipopolysaccharide-mediated inducible nitric oxide synthase and cyclooxygenase-2 expression via suppressing NF-κB and JNK/p38 MAPK activation pathways.

OBJECTIVES: Natural products have played a significant role in drug discovery and development. Inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) have been suggested to connect with various inflammatory diseases. In this study, we explored the anti-inflammatory potential of aciculatin (8-((2R,4S,5S,6R)-tetrahydro-4,5-dihydroxy-6-methyl-2H-pyran-2-yl)-5-hydroxy-2-(4-hydroxyphenyl)-7-methoxy-4H-chromen-4-one), one of main components of Chrysopogon aciculatis, by examining its effects on the expression and activity of iNOS and COX-2 in lipopolysaccharide (LPS)-activated macrophages. METHODS: We used nitrate and prostaglandin E2 (PGE2) assays to examine inhibitory effect of aciculatin on nitric oxide (NO) and PGE2 levels in LPS-activated mouse RAW264.7 macrophages and further investigated the mechanisms of aciculatin suppressed LPS-mediated iNOS/COX-2 expression by western blot, RT-PCR, reporter gene assay and confocal microscope analysis. RESULTS: Aciculatin remarkably decreased the LPS (1 µg/mL)-induced mRNA and protein expression of iNOS and COX-2 as well as their downstream products, NO and PGE2 respectively, in a concentration-dependent manner (1-10 µM). Such inhibition was found, via immunoblot analyses, reporter gene assays, and confocal microscope observations that aciculatin not only acts through significant suppression of LPS-induced NF-κB activation, an effect highly correlated with its inhibitory effect on LPS-induced IκB kinase (IKK) activation, IκB degradation, NF-κB phosphorylation, nuclear translocation and binding of NF-κB to the κB motif of the iNOS and COX-2 promoters, but also suppressed phosphorylation of JNK/p38 mitogen-activated protein kinases (MAPKs). CONCLUSION: Our results demonstrated that aciculatin exerts potent anti-inflammatory activity through its dual inhibitory effects on iNOS and COX-2 by regulating NF-κB and JNK/p38 MAPK pathways.

Ring-opening of five-membered heterocycles conjugated 4-isopropylresorcinol scaffold-based benzamides as HSP90 inhibitors suppressing tumor growth in vitro and in vivo.

A series of ring-opened dihydroxybenzamides have been designed and synthesized as heat shock protein 90 inhibitors. One of derivatives, compound 6b ((N-ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)benzamide)) demonstrated remarkable antiproliferative activity against in human KRAS mutant A549 and EGFR T790 M mutant H1975 lung cancer cell lines with GI50 values of 0.07 and 0.05 µM, respectively. It is also active against in other cancer cell lines, such as colorectal HCT116 (GI50 = 0.09 µM), liver Hep3B (GI50 = 0.20 µM) and breast MDA-MB-231 (GI50 = 0.09 µM), and shows no evidence of toxicity in normal cell line. Compound 6b has an IC50 of 110.18 nM in HSP90α inhibitory activity, slightly better than reference compound 1 (17-AAG, IC50 = 141.62 nM) and achieves the degradation of multiple HSP90 client proteins in a dose- and time-dependent manner and downstream signaling of Akt in a concentration- and time-dependent manner in the human A549 lung cancer cell line. In the Boyden chamber assay, compound 6b can efficiently inhibit the migration of A549 cells when compared to the reference compound 1. It also induce significant activity through the apoptotic pathway. Treatment with 6b showed no vision toxicity (IC50 > 10 µM) on 661w photoreceptor cells as compared to AUY922 (3a) with a 0.04 µM values of IC50 and has no effect in hERG test. In a bidirectional Caco-2 permeability assay, compound 6b was classified as a highly permeable compound which is not a substrate of efflux transporters. In a pharmacokinetic study in rats, 6b showed an F = 17.8% of oral bioavailability. The effect of metabolic stability of compound 6b in human hepatocytes showed a T1/2 of 67.59 min. Compound 6b (50 mg/kg, po, daily) exhibits antitumor activity with a 72% TGD (tumor growth delay) in human A549 lung xenograft. The combination of 6b and afatinib, orally administered, showed tumor growth suppression with 67.5% of TGI in lung H1975 xenograft model. Thus compound 6b is a lead compound for further development of potential agents to treat lung cancer.

Retraction: Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

[This retracts the article DOI: 10.18632/oncotarget.6427.].