EGFR-mediated hyperacetylation of tubulin induced docetaxel resistance by downregulation of HDAC6 and upregulation of MCAK and PLK1 in prostate cancer cells.

Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for castration-resistant prostate cancer (PCa). However, clinical treatment with docetaxel often encounters a number of undesirable effects, including drug resistance. Tubulin isoforms have been previously examined for their resistance to docetaxel in many cancers, but their real mechanisms remained unclear. In this study, a series of docetaxel-resistant PC/DX cell sublines were established by chronically exposing PC3 to progressively increased concentrations of docetaxel. Western blotting results showed significantly higher expression of acetyl-tubulin, α-tubulin, ß-tubulin, γ-tubulin, and ßIII-tubulin in PC/DX25 than in parental PC3 cells. PC/DX25 with greater resistance to docetaxel had higher levels of acetyl-tubulin and mitotic centromere-associated kinesin (MCAK) than PC3 cells. This study found that docetaxel induced the expression of acetyl-tubulin and MCAK in PC3 cells at a dose- and time-dependent manner. Both mRNA and protein levels of histone deacetylase 6 (HDAC6) were significantly decreased in PC/DX25 compared with PC3 cells. PC3 increased the resistance to docetaxel by HDAC6 knockdown and Tubastatin A (HDAC6 inhibitor). Conversely, PC/DX25 reversed the sensitivity to docetaxel by MCAK knockdown. Notably, flow cytometry analysis revealed that MCAK knockdown induced significantly sub G1 fraction in PC/DX cells. Overexpression of polo-like kinase-1 increased the cell survival rate and resistance to docetaxel in PC3 cells. Moreover, epidermal growth factor receptor (EGFR) activation induced the upregulation of acetyl-tubulin in docetaxel-resistant PCa cells. These findings demonstrated that the EGFR-mediated upregulated expression of acetyl-tubulin played an important role in docetaxel-resistant PCa.

Circular Economy and Sustainable Recovery of Taiwanese Tilapia (Oreochromis mossambicus) Byproduct-The Large-Scale Production of Umami-Rich Seasoning Material Application.

In this study, umami-rich seasoning powder was produced from the offcuts of Taiwanese tilapia (Oreochromis mossambicus) by cooking concentration and spray drying of granules while yielding an abundance of glutamic acid (0.23 mg/100 g), glycine (0.10 mg/100 g), aspartic acid (0.11 mg/100 g), lysine (0.10 mg/100 g), and 11 other aminic acids. It exhibited water content (3.81%), water activity (0.3), powder yields (68.83%), and a good water solubility index (99.89%), while the particle microstructure was a spherical powder. Additionally, it received the highest overall preference score (7.53) in the consumer-type sensory evaluation compared to commercially available seasonings. This study proves that offcuts may be part of the human diet after proper processing and can be widely used to flavor savory food. The producers involved could increase their economic returns while meeting the environmental challenges. The practical contribution could create incremental value for products to critical stakeholders at each point in the tilapia supply chain with an operational guide for transitioning from inefficient to innovative circular practices.

Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion.

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the two metal oxides' corresponding nanoparticles-the former being as small as 22 nm and the latter 15 nm. We fed two strains of bacteria, Shewanella algae and Pandoraea sp., with various concentrations of tungstate or molybdate ions. The concentrations of tungstate and molybdate were adjusted to make microcapsules of different length-to-diameter ratios. We found that the higher the concentration the smaller the nanoparticles were. The nanoparticles came in with three length-to-diameter ratios: 10:1, 3:1 and 1:1, which were achieved by feeding the bacteria respectively with a low concentration, a medium concentration, and a high concentration. The images of the hollow microcapsules were taken via the scanning electron microsphere (SEM). Their crystal structures were verified by X-ray diffraction (XRD)-the crystal structure of molybdate microcapsules is Na2MoO4 and that of tungstate microcapsules is Na2WO4 with Na2W2O7. These syntheses all were accomplished under a near ambient condition.

Dietary flavonoids, luteolin and quercetin, inhibit invasion of cervical cancer by reduction of UBE2S through epithelial-mesenchymal transition signaling.

We previously reported that the dietary flavonoids, luteolin and quercetin, might inhibit the invasiveness of cervical cancer by reversing epithelial-mesenchymal transition (EMT) signaling. However, the regulatory mechanism exerted by luteolin and quercetin is still unclear. This study analyzed the invasiveness activation by ubiquitin E2S ligase (UBE2S) through EMT signaling and inhibition by luteolin and quercetin. We found that UBE2S expression was significantly higher in highly invasive A431 subgroup III (A431-III) than A431-parental (A431-P) cells. UBE2S small interfering (si)RNA knockdown and overexpression experiments showed that UBE2S increased the migratory and invasive abilities of cancer cells through EMT signaling. Luteolin and quercetin significantly inhibited UBE2S expression. UBE2S showed a negative correlation with von Hippel-Lindau (VHL) and a positive correlation with hypoxia-induced factor (Hif)-1α. Our findings suggest that high UBE2S in malignant cancers contributes to cell motility through EMT signaling and is reversed by luteolin and quercetin. UBE2S might contribute to Hif-1α signaling in cervical cancer. These results show the metastatic inhibition of cervical cancer by luteolin and quercetin through reducing UBE2S expression, and provide a functional role for UBE2S in the motility of cervical cancer. UBE2S could be a potential therapeutic target in cervical cancer.

Dietary Flavonoids Luteolin and Quercetin Suppressed Cancer Stem Cell Properties and Metastatic Potential of Isolated Prostate Cancer Cells.

A highly invasive Du145-III subline was isolated by three successive passages of the parental Du145 prostate tumor cell line (Du145-P) through a Boyden chamber with matrigel-coated membrane support. Du145-III cells showed great invasion potential based on their increased ability to spread/migrate and enhanced expression/secretion of the matrix metalloproteinase 9 (MMP9). Du145-III cells exerted vasculogenic mimicry (VM) properties, reminiscent of endothelial cell characteristics and expressed elevated levels of cancer stem cell (CSC) markers, including Nanog, Sox2, CD44 and ABCG2 and ability to self-renew. Of prominence, MMP9 was required for the induction of VM and for increased stemness in Du145-III cells. Using Du145-III as a model, the effects of dietary flavonoids, luteolin and quercetin, were evaluated on stemness and invasion capacity of Du145-III cells in relation to JNK signaling pathway activation. These flavonoids depressed the malignancy of highly invasive Du145-III cells, VM, anchorage-independent spheroid formation and expression of certain CSC markers. Since luteolin and quercetin were able to target CSC cells and prevent cancer cell invasiveness, may serve as potential anti-angiogenesis and anti-metastasis agents.

Dichlorido[1-(2,6-dimethyl-phenyl-imino)-1,2-diphenyl-propan-2-ol-κN,O]palladium(II) methanol monosolvate.

The title compound, [PdCl(2)(C(23)H(23)NO)]·CH(3)OH, was obtained by the reaction of 1-(2,6-dimethyl-phenyl-imino)-1,2-diphenyl-propan-2-ol and palladium chloride in methanol. The Pd atom is four-coordinated by the O atom of a tertiary alcohol, the imine N atom of the hy-droxy-limine part of the bidentate ligand and by two chloride ions, forming a nearly square-planar geometry. The complex mol-ecule and the uncoordinated methanol mol-ecule are connected via an O-H⋯O hydrogen bond.

Ling-Zhi polysaccharides potentiate cytotoxic effects of anticancer drugs against drug-resistant urothelial carcinoma cells.

The combined effects of ling-zhi polysaccharide fraction 3 (LZP-F3) and anticancer drugs (cisplatin and arsenic trioxide) were examined in three human urothelial carcinoma (UC) cells (parental, NTUB1; cisplatin-resistant, N/P(14); and arsenic-resistant, N/As(0.5)). MTT assay and median-effect analysis revealed that LZP-F3 could profoundly reverse the chemosensitivity of N/P(14) and N/As(0.5) to cisplatin and arsenic, respectively, in a dose-dependent manner, which involved activation of p38 and down-regulation of Akt and XPA. A dose of 10 mug/mL of LZP-F3 induced significant G1 arrest in N/P(14) and N/As(0.5) cells by flow cytometry, which may be mediated by the induction of p21(WAF1/CIP1). The combination of LZP-F3 and arsenic trioxide produced a significant synergistic growth inhibition of NTUB1 and N/As(0.5) cells. Similar results were also found in N/P(14) cells. These molecular events of combined effects involved significant and earlier induction of Fas, caspase 3 and 8 activation, Bax and Bad up-regulation, Bcl-2 and Bcl-x(L) down-regulatuion, and cytochrome c release.

Pyrrolidine dithiocarbamate inhibition of luteolin-induced apoptosis through up-regulated phosphorylation of Akt and caspase-9 in human leukemia HL-60 cells.

Previously, we observed that luteolin effectively inhibited cell growth and induced apoptosis in HL-60 cells. In that study, we also explored the modulatory effects and molecular mechanisms of pyrrolidine dithiocarbamate (PDTC) on the cytotoxicity of luteolin to HL-60 cells. In this study, we found that PDTC was able to inhibit luteolin-induced cell apoptosis in a dose-dependent manner. When HL-60 cells were treated with PDTC for 0.5 h before 60 microM luteolin treatment, the DNA ladder disappeared. Moreover, flow cytometry showed that PDTC had dose dependently decreased the percentage of apoptotic HL-60 cells and had not interfered with luteolin's ability to change the mitochondrial membrane potential or its ability to trigger the release of cytochrome c to cytosol. Detection by Western blotting, however, did show that PDTC had interfered with luteolin's ability to cleave poly(ADP-ribose)polymerase and DNA fragmentation of factor-45. Three hours after the PDTC-pretreated HL-60 cells were treated with 60 microM luteolin, the product cleaved from Akt started to appear. Therefore, not only was PDTC able to stop the apoptosis of HL-60 cells treated with luteolin, it was also found to increase phosphorylation of Akt and caspase-9. These results suggest that in the luteolin-induced apoptotic pathway, phosphorylation of procaspase-9 by survival signals might play an important role in the ultimate fate of HL-60 cells.

Apoptosis-inducing active components from Corbicula fluminea through activation of caspase-2 and production of reactive oxygen species in human leukemia HL-60 cells.

The anti-cancer effects and possible mechanisms of the freshwater clam (Corbicula fluminea Muller) and its active compounds (FME) on cell viability in human leukemia HL-60 cells were investigated. This study demonstrated that FME was able to inhibit cell proliferation in a concentration- and time-dependent manner. Treatment with FME caused induction of caspase-2, caspase-3, caspase-6, caspase-8, and caspase-9 activity in a time-dependent manner, but not affect caspase-1 activity; it induced the proteolysis of DNA fragmentation factor (DFF-45) and poly(ADP-ribose) polymerase (PARP). Induction of cell death by FME was completely prevented by a pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) and a caspase-2 inhibitor, Z-Val-Asp-Val-Ala-Asp-FMK (Z-VDVAD-FMK). Furthermore, treatment with FME caused a rapid loss of mitochondrial transmembrane potential, stimulation of generation of reactive oxygen species (ROS), release of mitochondrial cytochrome c into cytosol, and GSH depletion. Anti-oxidants such as N-acetylcysteine, catalase, superoxide dismutase, allopurinol, and pyrrolidine dithiocarbamate, but not diphenylene iodonium, significantly inhibited FME-induced cell death. In addition, the results showed that FME-induced apoptosis was accompanied by up-regulation of Bax and Bad, and down-regulation of Bcl-2 and Bcl-XL. Taken together, induction of apoptosis on HL-60 cells by FME was mainly associated with ROS production, GSH depletion, mitochondrial dysfunction, and caspase activation.

Targeting of focal adhesion kinase by flavonoids and small-interfering RNAs reduces tumor cell migration ability.

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells is associated with increased invasive potential. In the present study, therefore, we developed two experiments to test the hypothesis that FAK is a determinant of, and plays an important role in, regulating tumor cell migration. In the first, the biological functions of FAK were examined using flavonoid inhibition. MiaPaCa-2 cells, treated with luteolin (Lu) and quercetin (Qu), were used to dampen FAK phosphorylation and protein expression by parallel suppression of cell migration ability. The second experiment involved suppression of FAK expression using small-interfering RNAs (siRNA) specific to FAK. While not affecting cellular proliferation or apoptosis, the siRNA targeting FAK almost completely inhibited FAK protein expression in MiaPaCa-2 cells and potently blocked the cell migration mediated by FAK. Our results show that FAK functions as a key regulator of cell migration, and that FAK activity can be suppressed by specific FAK siRNA, and by luteolin and quercetin. It appears reasonable to conclude, therefore, that suppression of FAK protein has a significant impact on tumor cell invasiveness.

Transinactivation of the epidermal growth factor receptor tyrosine kinase and focal adhesion kinase phosphorylation by dietary flavonoids: effect on invasive potential of human carcinoma cells.

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells correlated with increased invasive potential. Transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase activity is proposed to stimulate cell migration and the subsequent activation of downstream signaling pathways. Quercetin (Qu) and luteolin (Lu), are potent PTK inhibitors as well as putative chemopreventive agents. The present work, we demonstrate that Qu and Lu at concentration of 20 microM transinactivated EGFR tyrosine kinase activity with marked reduction in phosphotyrosyl level of 170, 125, 65, 60 and 42 kDa cellular proteins, and induced apoptosis in MiaPaCa-2 cells. The 125 kDa protein was further identified as a FAK by immunoprecipitation and immunoblotting analyses. Tumor cells treated with Lu or Qu dampened the phosphorylation of FAK. In addition, our data clearly demonstrated that tumor cells responded to Qu and Lu by parallel reductions in the levels of phosphorylated FAK and the secreted matrix metalloproteinase (MMP) that may lead to the suppression of invasive potential and cell migration in vitro. While the molecular mechanism of FAK regulation of MMP secretion in tumor cells remains unclear, our results suggested that blockade of the EGFR-signaling pathway may contributed to the net effect. As suggested in the current study, targeting EGFR and FAK with the objective of modulating their regulatory pathways could offer prospects for the treatment of EGFR-responsive cancers in the future.

Blockade of the epidermal growth factor receptor tyrosine kinase activity by quercetin and luteolin leads to growth inhibition and apoptosis of pancreatic tumor cells.

To glean insights into the mechanism of their action, we assessed the effects of two flavonoids, quercetin (Qu) and luteolin (Lu), on the growth and epidermal growth factor receptor (EGFR) tyrosine kinase activity of MiaPaCa-2 cancer cells. Exposure of these EGFR-expressing cells to 20 microM Qu or Lu resulted in concomitant decreases in cellular protein phosphorylation and growth. On the cellular level, Qu and Lu sensitivity correlated with EGFR levels and rapid cell proliferation, indicating the possibility of targeting those cells most prone to neoplastic progression. Cell treatment with the flavonoids markedly diminished the extent of cellular protein phosphorylation, by effectively modulating protein tyrosine kinase (PTK) activities, including that of EGFR. Immunocomplex kinase assay revealed that both Qu and Lu inhibited the PTK activities responsible for the autophosphorylation of EGFR as well as for the transphosphorylation of enolase. Treatment of the cells with Qu or Lu also reduced the phosphotyrosyl levels of 170-, 125-, 110-, 65-, 60-, 44-, 30- and 25-kDa proteins. We identified the 170-kDa phosphotyrosylprotein as EGFR. Qu and Lu exhibited a specific action in hampering the levels of phosphorylation of this and the aforementioned proteins, while having no discernible effect on their synthesis. A time-dependent attenuation of the phosphorylation of the above proteins was demonstrable. Treatment of the cells with Qu or Lu for 6 hours showed little inhibition, but prolonging the cell treatment for 24 hours caused the suppression of phosphorylation. Further continuation of the cell treatment culminated in the induction of apoptosis, characteristically exhibiting shrinkage of the cell morphology, DNA fragmentation and poly(ADP-ribose)polymerase (PARP) degradation. The onset of apoptosis and associated events occurred in a time-dependent fashion. The data clearly demonstrate that MiaPaCa-2 cells respond to Qu and Lu by a parallel reduction in cellular protein phosphorylation and cellular proliferation. The flavonoid-evoked attenuation of the phosphorylation of EFGR and of other proteins appeared to be transient, since removal of the flavonoid from the cell growth medium after 24 hours of incubation followed by exposure to 10 nm EGF, restored protein phosphorylation and cellular proliferation. Such an addition of EGF was also able to reverse Qu- or Lu-induced cell growth inhibition and diminish nuclear digestion evoked by 20 microM Qu or Lu. Both Qu and Lu were able to reverse the effect of EGF biochemically as well as functionally. Based on the evidence accrued, the above proteins could be implicated in growth signal transduction and the subtle changes in their phosphorylation, as effected by flavonoids, utilized as a reliable guide to predict growth response. The antiproliferative effect of flavonoids might result, at least in part, from the modulation of the EGF-mediated signaling pathway. The results indicate that the blockade of the EGFR-signaling pathway by the PTK inhibitors Qu and Lu significantly inhibits the growth of MiaPaCa-2 cells and induces apoptosis. The modulation of EGFR kinase appears to be a critically important, intrinsic component of Qu- and Lu-induced growth suppression, even though other mechanisms could also have contributed to the net effect.

Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma A431 cells.

The purpose of this study was to investigate the effects of a potent LHRH agonist, [D-Trp(6)]LHRH on the basal and EGF-induced cell proliferation and the metastasis-associated properties in A431 human epidermoid carcinoma. [D-Trp(6)]LHRH time-dependently inhibited the basal and EGF-stimulated growth of A431 cancer cells. It is assumed that phosphorylation/dephosphorylation of cellular proteins is highly related to cell growth. This study demonstrates that [D-Trp(6)]LHRH decreased the basal and EGF-induced total cellular kinase activity, particularly the tyrosine phosphorylation of several cellular proteins including the EGFR. In contrast, [D-Trp(6)]LHRH did not cause detectable changes in basal and EGF-stimulated serine/threonine phosphorylation of A431 cellular proteins. The inhibitory effect of [D-Trp(6)]LHRH on A431 cell proliferation was associated with apoptosis as evidenced by the cell morphology and DNA integrity (ladder pattern), the expression of interleukin 1beta-converting enzyme (ICE) and activation of caspase. Furthermore, EGF could rescue the remaining attached A431 cells following [D-Trp(6)]LHRH treatment for 48 hr, which suggests that limited exposure to [D-Trp(6)]LHRH did not channel all cells to irreversible apoptotic process. We also determined the effects of [D-Trp(6)]LHRH on metastasis-associated properties in A431 cells. [D-Trp(6)]LHRH reduced both basal and EGF-stimulated secretion of MMP-9 and MMP-2. In addition, [D-Trp(6)]LHRH suppressed the basal and EGF-induced invasive activity of A431 cells based on an in vitro invasion assay. In conclusion, this study indicates that [D-Trp(6)]LHRH may act partly through activating tyrosine phosphatase activity to inhibit cell proliferation and the metastasis-associated properties of A431 cancer cells. Our work suggests that [D-Trp(6)]LHRH may be therapeutically useful in limiting the tumor growth and metastasis of some neoplasms.