A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. METHODS: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. RESULTS: A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 µM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 µM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. CONCLUSION: The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Mechanistic study of dual-function inhibitors targeting topoisomerase II and Rad51-mediated DNA repair pathway against castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair. METHODS: Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis. RESULTS: A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIß but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51. CONCLUSION: The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.

Anticancer Activity and Molecular Mechanisms of an Ursodeoxycholic Acid Methyl Ester-Dihydroartemisinin Hybrid via a Triazole Linkage in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma is the third most common cause of cancer-related death according to the International Agency for Research on Cancer. Dihydroartemisinin (DHA), an antimalarial drug, has been reported to exhibit anticancer activity but with a short half-life. We synthesized a series of bile acid-dihydroartemisinin hybrids to improve its stability and anticancer activity and demonstrated that an ursodeoxycholic-DHA (UDC-DHA) hybrid was 10-fold more potent than DHA against HepG2 hepatocellular carcinoma cells. The objectives of this study were to evaluate the anticancer activity and investigate the molecular mechanisms of UDCMe-Z-DHA, a hybrid of ursodeoxycholic acid methyl ester and DHA via a triazole linkage. We found that UDCMe-Z-DHA was even more potent than UDC-DHA in HepG2 cells with IC50 of 1 µM. Time course experiments and stability in medium determined by cell viability assay as well as HPLC-MS/MS analysis revealed that UDCMe-Z-DHA was more stable than DHA, which in part accounted for the increased anticancer activity. Mechanistic studies revealed that UDCMe-Z-DHA caused G0/G1 arrest and induced reactive oxygen species (ROS), mitochondrial membrane potential loss and autophagy, which may in turn lead to apoptosis. Compared to DHA, UDCMe-Z-DHA displayed much lower cytotoxicity toward normal cells. Thus, UDCMe-Z-DHA may be a potential drug candidate for hepatocellular carcinoma.

Discovery of New Glucose Uptake Inhibitors as Potential Anticancer Agents by Non-Radioactive Cell-Based Assays.

Tumor cells rely on aerobic glycolysis to support growth and survival, thus require more glucose supply. Glucose transporters GLUTs, primarily GLUT1, are overexpressed in various cancers. Targeting GLUTs has been regarded as a promising anticancer strategy. In this study, we first evaluated 75 potential GLUT1 inhibitors obtained from virtual screening of the NCI chemical library by a high-throughput cell-based method using a fluorescent glucose analogue 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG) in COS-7 and SKOV3 cells that express high levels of GLUT1. Four compounds, #12, #16, #43 and #69, that significantly inhibited glucose uptake were further evaluated using flow cytometry directly measuring 2-NBDG uptake at the single-cell level and a Glucose Uptake-GloTM assay indirectly measuring 2-deoxy-d-glucose uptake in SKOV3, COS-7 or MCF-7 cells. The inhibitory effect on cancer cell growth was also determined in SKOV3 and MCF-7 cells, and #12 exhibited the best growth inhibitory effect equivalent to a known GLUT1 inhibitor WZB117. Although the anticancer effect of the identified potential GLUT1 inhibitors was moderate, they may enhance the activity of other anticancer drugs. Indeed, we found that #12 synergistically enhanced the anticancer activity of metformin in SKOV3 ovarian cancer cells.

Discovery of Novel Agents on Spindle Assembly Checkpoint to Sensitize Vinorelbine-Induced Mitotic Cell Death Against Human Non-Small Cell Lung Cancers.

Non-small cell lung cancer (NSCLC) accounts about 80% of all lung cancers. More than two-thirds of NSCLC patients have inoperable, locally advanced or metastatic tumors. Non-toxic agents that synergistically potentiate cancer-killing activities of chemotherapeutic drugs are in high demand. YL-9 was a novel and non-cytotoxic compound with the structure related to sildenafil but showing much less activity against phosphodiesterase type 5 (PDE5). NCI-H460, an NSCLC cell line with low PDE5 expression, was used as the cell model. YL-9 synergistically potentiated vinorelbine-induced anti-proliferative and apoptotic effects in NCI-H460 cells. Vinorelbine induced tubulin acetylation and Bub1-related kinase (BUBR1) phosphorylation, a necessary component in spindle assembly checkpoint. These effects, as well as BUBR1 cleavage, were substantially enhanced in co-treatment with YL-9. Several mitotic arrest signals were enhanced under combinatory treatment of vinorelbine and YL-9, including an increase of mitotic spindle abnormalities, increased cyclin B1 expression, B-cell lymphoma 2 (Bcl-2) phosphorylation and increased phosphoproteins. Moreover, YL-9 also displayed synergistic activity in combining with vinorelbine to induce apoptosis in A549 cells which express PDE5. In conclusion. the data suggest that YL-9 is a novel agent that synergistically amplifies vinorelbine-induced NSCLC apoptosis through activation of spindle assembly checkpoint and increased mitotic arrest of the cell cycle. YL-9 shows the potential for further development in combinatory treatment against NSCLC.

Chalcones Display Anti-NLRP3 Inflammasome Activity in Macrophages through Inhibition of Both Priming and Activation Steps-Structure-Activity-Relationship and Mechanism Studies.

Chalcones are responsible for biological activity throughout fruits, vegetables, and medicinal plants in preventing and treating a variety of inflammation-related diseases. However, their structure-activity relationship (SAR) in inhibiting inflammasome activation has not been explored. We synthesized numerous chalcones and determined their SAR on lipopolysaccharide (LPS)-primed ATP-induced NLRP3 inflammasome activation. 11Cha1 displayed good inhibitory activity on release reaction of caspase-1, IL-1ß, and IL-18. It significantly inhibited LPS-induced phosphorylation and proteolytic degradation of IĸB-α and nuclear translocation of NF-ĸB, but had little effect on mitogen-activated protein kinases (MAPKs) activities. Furthermore, 11Cha1 blocked LPS-induced up-regulation of NLRP3, pro-caspase-1, ASC, IL-18, and IL-1ß, indicating the suppression on priming step of inflammasome activation. ASC dimerization and oligomerization are considered to be direct evidence for inflammasome activation. 11Cha1 profoundly inhibited ATP-induced formation of ASC dimers, trimers, and oligomers, and the assembly of ASC, pro-caspase-1, and NLRP3 in inflammasome formation. Decrease of intracellular K+ levels is the common cellular activity elicited by all NLRP3 inflammasome activators. 11Cha1 substantially diminished ATP-mediated K+ efflux, confirming the anti-NLRP3 inflammasome activity of 11Cha1. In summary, the SAR of chalcone derivatives in anti-inflammasome activities was examined. Besides, 11Cha1 inhibited both priming and activation steps of NLRP3 inflammasome activation. It inhibited NF-ĸB activation and subsequently suppressed the up-regulation of NLRP3 inflammasome components including NLRP3, ASC, pro-caspase-1, pro-IL-18, and pro-IL-1ß. Next, 11Cha1 blocked ATP-mediated K+ efflux and suppressed the assembly and activation of NLRP3 inflammasome, leading to the inhibition of caspase-1 activation and proteolytic cleavage, maturation, and secretion of IL-1ß and IL-18.

The (+)-Brevipolide H Displays Anticancer Activity against Human Castration-Resistant Prostate Cancer: The Role of Oxidative Stress and Akt/mTOR/p70S6K-Dependent Pathways in G1 Checkpoint Arrest and Apoptosis.

Because conventional chemotherapy is not sufficiently effective against prostate cancer, various examinations have been performed to identify anticancer activity of naturally occurring components and their mechanisms of action. The (+)-brevipolide H, an α-pyrone-based natural compound, induced potent and long-term anticancer effects in human castration-resistant prostate cancer (CRPC) PC-3 cells. Flow cytofluorometric analysis with propidium iodide staining showed (+)-brevipolide H-induced G1 arrest of cell cycle and subsequent apoptosis through induction of caspase cascades. Since Akt/mTOR pathway has been well substantiated in participating in cell cycle progression in G1 phase, its signaling and downstream regulators were examined. Consequently, (+)-brevipolide H inhibited the signaling pathway of Akt/mTOR/p70S6K. The c-Myc inhibition and downregulation of G1 phase cyclins were also attributed to (+)-brevipolide H action. Overexpression of myristoylated Akt significantly rescued mTOR/p70S6K and downstream signaling under (+)-brevipolide H treatment. ROS and Ca2+, two key mediators in regulating intracellular signaling, were determined, showing that (+)-brevipolide H interactively induced ROS production and an increase of intracellular Ca2+ levels. The (+)-Brevipolide H also induced the downregulation of anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL) and loss of mitochondrial membrane potential, indicating the contribution of mitochondrial dysfunction to apoptosis. In conclusion, the data suggest that (+)-brevipolide H displays anticancer activity through crosstalk between ROS production and intracellular Ca2+ mobilization. In addition, suppression of Akt/mTOR/p70S6K pathway associated with downregulation of G1 phase cyclins contributes to (+)-brevipolide H-mediated anticancer activity, which ultimately causes mitochondrial dysfunction and cell apoptosis. The data also support the biological significance and, possibly, clinically important development of natural product-based anticancer approaches.

Effects of lipopolysaccharide on the expression of plasma membrane monoamine transporter (PMAT) at the blood-brain barrier and its implications to the transport of neurotoxins.

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter that is highly expressed in the central nervous system. This study aimed to investigate the effect of lipopolysaccharide on PMAT expression at the blood-brain barrier and the interaction between PMAT and neurotoxins. As a result, PMAT mRNA was identified in brain microvessels (BMVs), brain microvascular endothelial cells (BMECs), astrocytes, and pericytes isolated from C57BL/6 mice and/or Wistar rats using RT-qPCR. The immunofluorescence staining confirmed the expression of PMAT protein in BMVs and striatum of C57BL/6 mice. Western blotting demonstrated its localization at the luminal and abluminal sides of BMECs. In C57BL/6 mice, PMAT protein was significantly increased in BMVs 24 h after an intraperitoneal injection of 3 mg/kg lipopolysaccharide. Lipopolysaccharide treatment also significantly increased PMAT expression in cerebral cortex and the striatum in a time-dependent manner, as well as the brain-to-plasma ratio of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-benzyl-TIQ). In isolated cells, lipopolysaccharide treatment significantly increased PMAT mRNA in brain astrocytes and the BMECs co-cultured with astrocytes. In addition to 1-methyl-4-phenylpyridinium, the kinetic study indicated that both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-benzyl-TIQ are substrates of human PMAT. These findings suggest that inflammation can change PMAT expression at the blood-brain barrier, which may affect PMAT-mediated transport of neurotoxins. We demonstrated the expression of plasma membrane monoamine transporter (PMAT; mRNA or protein) at several subunits of the blood-brain barrier. Lipopolysaccharide treatment can significantly increase the expression of PMAT in vivo (in brain microvessels, cerebral cortex, and the striatum of C57BL/6 mice) and in vitro (in brain astrocytes and brain microvascular endothelial cells co-cultured with astrocytes). Lipopolysaccharide treatment also increased the brain-to-plasma ratio of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-benzyl-TIQ) in mice, where 1-benzyl-TIQ competitively inhibited 1-methyl-4-phenylpyridinium (MPP(+)) uptake in MDCK-human PMAT (hPMAT) cells and its uptake in MDCK-hPMAT is concentration dependent.

Repurposing of phentolamine as a potential anticancer agent against human castration-resistant prostate cancer: A central role on microtubule stabilization and mitochondrial apoptosis pathway.

BACKGROUND: Drug repurposing of phentolamine, an α-adrenoceptor antagonist, as an anticancer agent has been studied in human castration-resistant prostate cancer (CRPC). METHODS: Cell proliferation was examined by sulforhodamine B and CFSE staining assays. Cell cycle progression and mitochondrial membrane potential (ΔΨm) were detected by flow cytometric analysis. Protein expression was detected by Western blotting. Effect on tubulin/microtubule was determined using confocal immunofluorescence microscopic examination, microtubule assembly detection, tubulin turbidity assay, and binding assay. Several assessments were used to characterize apoptotic signaling pathways and combinatory effect. RESULTS: Phentolamine induced anti-proliferative effect in PC-3 and DU-145, two CRPC cell lines, and P-glycoprotein (P-gp) overexpressing cells. This effect was not significantly reduced in paclitaxel-resistant cells. Rhodamine 123 efflux assay showed that phentolamine was not a P-gp substrate. Phentolamine induced mitotic arrest of the cell cycle and formation of hyperdiploid cells, followed by an increase of apoptosis. Mitotic arrest was confirmed by cyclin B1 up-regulation, Cdk1 activation, and a dramatic increase of mitotic protein phosphorylation. Both in vitro and cellular identification demonstrated that phentolamine, similar to paclitaxel, induced tubulin polymerization and formation of multiple nuclei. Besides, it did not compete with paclitaxel binding on tubulin. Phentolamine induced the phosphorylation and degradation of Bcl-2 and Bcl-xL, two anti-apoptotic Bcl-2 family members, and the loss of ΔΨm indicating the induction of mitochondrial damage. It ultimately induced the activation of caspase-9, -8, and -3 and apoptotic cell death. Moreover, combination treatment with phentolamine and paclitaxel caused a synergistic apoptosis. CONCLUSIONS: The data suggest that phentolamine is a potential anticancer agent. In contrast to a wide variety of microtubule disrupting agents, phentolamine induces microtubule assembly, leading to mitotic arrest of the cell cycle which "in turn" induces subsequent mitochondrial damage and activation of related apoptotic signaling pathways in CRPC cells. Furthermore, combination between phentolamine and paclitaxel induces a synergistic apoptotic cell death. Phentolamine has a simple chemical structure and is not a P-gp substrate. Optimization of phentolamine structure may also be a potential approach for further development.

Doxazosin inhibits vasculogenic mimicry in human non­small cell lung cancer through inhibition of the VEGF­A/VE­cadherin/mTOR/MMP pathway.

Lung cancer is the leading cause of cancer-related death worldwide, and ~85% of lung cancers are non-small cell lung cancer (NSCLC), which has a low 5-year overall survival rate and high mortality. Several therapeutic strategies have been developed, such as targeted therapy, immuno-oncotherapy and combination therapy. However, the low survival rate indicates the urgent need for new NSCLC treatments. Vasculogenic mimicry (VM) is an endothelial cell-free tumor blood supply system of aggressive and metastatic tumor cells present during tumor neovascularization. VM is clinically responsible for tumor metastasis and resistance, and is correlated with poor prognosis in NSCLC, making it a potential therapeutic target. In the present study, A549 cells formed glycoprotein-rich lined tubular structures, and transcript levels of VM-related genes were markedly upregulated in VM-forming cells. Based on a drug repurposing strategy, it was demonstrated that doxazosin (an antihypertensive drug) displayed inhibitory activity on VM formation at non-cytotoxic concentrations. Doxazosin significantly reduced the levels of vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase-2 (MMP-2) in the cell media during VM formation. Further experiments revealed that the protein expression levels of VEGF-A and vascular endothelial-cadherin (VE-cadherin), which contribute to tumor aggressiveness and VM formation, were downregulated following doxazosin treatment. Moreover, the downstream signaling Ephrin type-A receptor 2 (EphA2)/AKT/mTOR/MMP/Laminin-5γ2 network was inhibited in response to doxazosin treatment. In conclusion, the present study demonstrated that doxazosin displayed anti-VM activity in an NSCLC cell model through the downregulation of VEGF-A and VE-cadherin levels, and the suppression of signaling pathways related to the receptor tyrosine kinase, EphA2, protein kinases, AKT and mTOR, and proteases, MMP-2 and MMP-9. These results support the add-on anti-VM effect of doxazosin as a potential agent against NSCLC.

Sensitization of cancer cells to paclitaxel-induced apoptosis by canagliflozin.

Cancer cells consume more glucose and usually overexpress glucose transporters which have become potential targets for the development of anticancer drugs. It has been demonstrated that selective SGLT2 inhibitors, such as canagliflozin and dapagliflozin, display anticancer activity. Here we demonstrated that canagliflozin and dapagliflozin synergistically enhanced the growth inhibitory effect of paclitaxel in cancer cells including ovarian cancer and oral squamous cell carcinoma cells. Canagliflozin also inhibited glucose uptake via GLUTs. The combination of paclitaxel and WZB117, a GLUT inhibitor, exhibited a strong synergy, supporting the notion that inhibition of GLUTs by canagliflozin may also account for the synergy between canagliflozin and paclitaxel. Mechanistic studies in ES-2 ovarian cancer cells revealed that canagliflozin potentiated paclitaxel-induced apoptosis and DNA damaging effect. Paclitaxel in the nanomolar range elevated abnormal mitotic cells as well as aneuploid cells, and canagliflozin further enhanced this effect. Furthermore, canagliflozin downregulated cyclin B1 and phospho-BUBR1 upon spindle assembly checkpoint (SAC) activation by paclitaxel, and may consequently impair SAC. Thus, paclitaxel disturbed microtubule dynamics and canagliflozin compromised SAC activity, together they may induce premature mitotic exit, accumulation of aneuploid cells with DNA damage, and ultimately apoptosis.

Cardenolide glycosides sensitize gefitinib-induced apoptosis in non-small cell lung cancer: inhibition of Na+/K+-ATPase serving as a switch-on mechanism.

The treatment of non-small cell lung cancer (NSCLC) is known as a significant level of unmet medical need in spite of the progress in targeted therapy and personalized therapy. Overexpression of the Na+/K+-ATPase contributes to NSCLC progression, suggesting its potentiality in antineoplastic approaches. Epi-reevesioside F, purified from Reevesia formosana, showed potent anti-NSCLC activity through inhibiting the Na+/K+-ATPase, leading to internalization of α1- and α3-subunits in Na+/K+-ATPase and suppression of Akt-independent mTOR-p70S6K-4EBP1 axis. Epi-reevesioside F caused a synergistic amplification of apoptosis induced by gefitinib but not cisplatin, docetaxel, etoposide, paclitaxel, or vinorelbine in both NCI-H460 and A549 cells. The synergism was validated by enhanced activation of the caspase cascade. Bax cleavage, tBid formation, and downregulation of Bcl-xL and Bcl-2 contributed to the synergistic apoptosis induced by the combination treatment of epi-reevesioside F and gefitinib. The increase of membrane DR4 and DR5 levels, intracellular Ca2+ concentrations, and active m-calpain expression were responsible for the caspase-8 activation and Bax cleavage. The increased α-tubulin acetylation and activation of MAPK (i.e., p38 MAPK, Erk, and JNK) depending on cell types contributed to the synergistic mechanism under combination treatment. These signaling pathways that converged on profound c-Myc downregulation led to synergistic apoptosis in NSCLC. In conclusion, the data suggest that epi-reevesioside F inhibits the Na+/K+-ATPase and displays potent anti-NSCLC activity. Epi-reevesioside F sensitizes gefitinib-induced apoptosis through multiple pathways that converge on c-Myc downregulation. The data support the inhibition of Na+/K+-ATPase as a switch-on mechanism to sensitize gefitinib-induced anti-NSCLC activity.

Ardisianone, a natural benzoquinone, efficiently induces apoptosis in human hormone-refractory prostate cancers through mitochondrial damage stress and survivin downregulation.

BACKGROUND: Increasing evidence suggests that mitochondria play a central role in regulating cell apoptosis. Survivin, an inhibitor of apoptosis protein (IAP) family member, mediates resistance to cancer chemotherapy particularly in prostate cancers. Therefore, development of anticancer agents targeting mitochondria and survivin is a potential strategy. METHOD: Cell proliferation was examined by sulforhodamine B, CFSE staining, and clonogenic assays. Mitochondrial membrane potential (ΔΨ(m) ) and reactive oxygen species (ROS) were detected by flow cytometric analysis. Protein expression was detected by Western blot. RNA levels were examined by reverse transcription polymerase chain reaction assay. Overexpression of constitutively active Akt was also used in this study. RESULTS: Ardisianone, a natural benzoquinone derivative, displayed anti-proliferative and apoptotic activities against human hormone-refractory prostate cancer cells (HRPC), PC-3, and DU-145. Ardisianone dramatically induced mitochondrial damage, identified by downregulation of Bcl-2 family proteins, ROS production, and loss of ΔΨ(m) . Ardisianone also inhibited Akt and mTOR/p70S6K pathways and induced a fast downregulation of survivin, leading to activation of mitochondria-involved caspase cascades. Overexpression of constitutively active Akt partly rescued ardisianone-mediated apoptotic signaling cascades. Furthermore, a long-term treatment of ardisianone caused an increase of endoplasmic reticulum (ER) stress, upregulation of cIAP1 and cIAP2, and apoptosis-inducing factor (AIF)-mediated caspase-independent apoptosis. CONCLUSIONS: The data suggest that the ardisianone induces apoptosis in human prostate cancers through mitochondrial damage stress, leading to the inhibition of mTOR/p70S6K pathway, downregulation of Bcl-2 family members, degradation of survivin, and activation of caspase cascades. The data provide evidence supporting that ardisianone is a potential anticancer agent against HRPCs.

Synthesis and biological evaluation of novel C-aryl d-glucofuranosides as sodium-dependent glucose co-transporter 2 inhibitors.

Novel C-aryl-d-glucofuranosides were synthesized and evaluated for their capacity to inhibit human sodium-dependent glucose co-transporter 2 (hSGLT2) and hSGLT1. Compound 21q demonstrated the best in vitro inhibitory activity against SGLT2 in this series (EC50=0.62µM).

Design, Synthesis, and Evaluation of Glucose Transporter Inhibitor-SN38 Conjugates for Targeting Colorectal Cancer.

Irinotecan (1), a prodrug of SN38 (2) approved by the US Food and Drug Administration for treating colorectal cancer, lacks specificity and causes many side effects. To increase the selectivity and therapeutic efficacy of this drug, we designed and synthesized conjugates of SN38 and glucose transporter inhibitors (phlorizin (5) or phloretin (6)), which could be hydrolyzed by glutathione or cathepsin to release SN38 in the tumor microenvironment, as a proof of concept. These conjugates (8, 9, and 10) displayed better antitumor efficacy with lower systemic exposure to SN38 in an orthotopic colorectal cancer mouse model compared with irinotecan at the same dosage. Further, no major adverse effects of the conjugates were observed during treatment. Biodistribution studies showed that conjugate 10 could induce higher concentrations of free SN38 in tumor tissues than irinotecan at the same dosage. Thus, the developed conjugates exhibit potential for treating colorectal cancer.

Development of hemiasterlin derivatives as potential anticancer agents that inhibit tubulin polymerization and synergize with a stilbene tubulin inhibitor.

Hemiasterlins are cytotoxic tripeptides with antimicrotubule activity originally isolated from marine sponges. We have developed new hemiasterlin derivatives BF65 and BF78 that are highly potent to induce cancer cell death in the low nanomolar range. Examination of their mechanisms of cell cycle arrest and disruption of microtubules revealed an unusual characteristic in addition to anti-tubulin effect. Immunofluorescence staining revealed that A549 lung carcinoma cells treated with BF65 or BF78 exhibited both monopolar and multipolar mitotic spindles. Centrosomes were separated with short spindle microtubules in cells with multipolar spindles. In vitro tubulin polymerization assay confirmed that both BF65 and BF78 were highly potent to inhibit tubulin polymerization. These two compounds induced the formation of monoastral spindles suggesting that they might be inhibitors of mitotic kinesins such as KSP/Eg5. However, kinetic measurement of microtubule activated kinesin ATPase activity demonstrated that unlike the positive control monastrol, neither BF65 nor BF78 suppressed KSP/Eg5 activity. Hence the effect may be a variant form of tubulin inhibition. Similar to vinca alkaloids, BF compounds synergized with a colchicine site microtubule inhibitor stilbene 5c both in vitro and in vivo, which may provide a potential drug combination in the future clinical application.

The Combination of a Novel GLUT1 Inhibitor and Cisplatin Synergistically Inhibits Breast Cancer Cell Growth By Enhancing the DNA Damaging Effect and Modulating the Akt/mTOR and MAPK Signaling Pathways.

Breast cancer is the most prevalent cancer and the second leading cause of cancer death in women. Cisplatin is a commonly used chemotherapeutic drug for breast cancer treatment. Owing to serious side effects, the combination of cisplatin with other drugs is an effective strategy to simultaneously reduce side effects and increase the anticancer efficacy. GLUT1 is an emerging target for cancer treatment since cancer cells usually consume more glucose, a phenomenon called the Warburg effect. In this study, we found that the combination of cisplatin and a novel GLUT1 inhibitor #43 identified from our previous high-throughput screening exerted a synergistic anticancer effect in MCF-7 and MDA-MB-231 breast cancer cells. Mechanism studies in MCF-7 cells revealed that combination of cisplatin and #43 significantly induced apoptosis, intracellular reactive oxygen species, and loss of mitochondrial membrane potential. Furthermore, #43 enhanced the DNA damaging effect of cisplatin. Akt/mTOR downstream signaling and the ERK signaling pathway usually involved in cell growth and survival were inhibited by the combination treatment. On the other hand, phosphorylation of p38 and JNK, which may be associated with apoptosis, was induced by the combination treatment. Altogether, our data indicate that oxidative stress, DNA damage, the Akt/mTOR and MAPK signaling pathways, and apoptosis may be involved in the synergism of cisplatin and #43 in breast cancer cells.

Characterization of a fluorescent glucose derivative 1-NBDG and its application in the identification of natural SGLT1/2 inhibitors.

Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2.

Targeting Colorectal Cancer with Conjugates of a Glucose Transporter Inhibitor and 5-Fluorouracil.

Overexpression of glucose transporters (GLUTs) in colorectal cancer cells is associated with 5-fluorouracil (1, 5-FU) resistance and poor clinical outcomes. We designed and synthesized a novel GLUT-targeting drug conjugate, triggered by glutathione in the tumor microenvironment, that releases 5-FU and GLUTs inhibitor (phlorizin (2) and phloretin (3)). Using an orthotopic colorectal cancer mice model, we showed that the conjugate exhibited better antitumor efficacy than 5-FU, with much lower exposure of 5-FU during treatment and without significant side effects. Our study establishes a GLUT-targeting theranostic incorporating a disulfide linker between the targeting module and cytotoxic payload as a potential antitumor therapy.

Phosphodiesterase Type 5 Inhibitors Synergize Vincristine in Killing Castration-Resistant Prostate Cancer Through Amplifying Mitotic Arrest Signaling.

Combination therapies that display cancer-killing activities through either coexistent targeting of several cellular factors or more efficient suppression of a specific pathway are generally used in cancer treatment. Sildenafil, a specific phosphodiesterase type 5 (PDE5) inhibitor, has been suggested to display both cardioprotective and neuroprotective activities that provide a rationale for the combination with vincristine on the treatment against castration-resistant prostate cancer (CRPC). In the present work, vincristine arrested cells in the metaphase stage of mitosis. Vincristine-induced mitotic arrest was identified by Cdk1 activation (i.e., increased Cdk1Thr161 phosphorylation and decreased Cdk1Tyr15 phosphorylation), cyclin B1 upregulation, and increased phosphorylation of multiple mitotic proteins and stathmin. Sildenafil synergistically potentiated vincristine-induced mitotic arrest and a dramatic increase of mitotic index. Furthermore, sildenafil potentiated vincristine-induced mitochondrial damage, including Mcl-1 downregulation, Bcl-2 phosphorylation and downregulation, Bak upregulation and loss of mitochondrial membrane potential, and sensitized caspase-dependent apoptotic cell death. Sildenafil-mediated synergistic effects were mimicked by other PDE5 inhibitors including vardenafil and tadalafil, and also by PDE5A knockdown in cells, suggesting PDE5-involved mechanism. Notably, sildenafil amplified vincristine-induced phosphorylation and cleavage of BUBR1, a protein kinase in spindle assembly checkpoint (SAC) function and chromosome segregation. Sildenafil also significantly decreased kinetochore tension during SAC activation. Moreover, sildenafil synergized with vincristine on suppressing tumor growth in an in vivo model. In conclusion, the data suggest that sildenafil, in a PDE5-dependent manner, potentiates vincristine-induced mitotic arrest signaling, and sensitizes mitochondria damage-involved apoptosis in CRPC. Both in vitro and in vivo data suggest the combination potential of PDE5 inhibitors and vincristine on CRPC treatment.