Ultrafine particles of Ulmus davidiana var. japonica induce apoptosis of gastric cancer cells via activation of caspase and endoplasmic reticulum stress

Small-sized particles are more suitable for targeted delivery and are therapeutically more effective than large-sized particles. In this study, we investigated the anticancer effects of ultrafine particles of Ulmus davidiana var. japonica (ufUJ) on human gastric cancer cell lines SNU-1, SNU-216, and SNU-484. ufUJ induced apoptosis by the proteolytic activation of caspase-9, caspase-6, and caspase-3 and cleavage of poly (ADP-ribose) polymerase. The expression levels of the endoplasmic reticulum stress-related protein BiP markedly increased after ufUJ treatment. BiP knockdown decreased ufUJ-induced cell death. ufUJ-induced apoptosis was inhibited by the caspase-3 inhibitor z-DEVD-fmk, caspase-6 inhibitor z-VEID-fmk, and caspase-9 inhibitor z-LEHD-fmk, and by siRNAs against caspases 3, 6, and 9. Gastric cancer cells did not show anchorage-independent growth in the presence of ufUJ. However, cells treated with caspase inhibitors showed an enhanced colony-forming ability. These findings may be helpful in the prevention of gastric cancer and in the development of functional foods.ope

Protein kinase CK2-dependent aerobic glycolysis-induced lactate dehydrogenase A enhances the migration and invasion of cancer cells

We investigated the intracellular metabolic fluxes of protein kinase CK2-activating (C alpha OE) cells and role of lactate dehydrogenase A (LDHA) as a contributor of tumorigenesis after reprogrammed glucose metabolism. Facilitated aerobic glycolysis was confirmed via isotope tracer analysis, in which C-13(6)-Glc or C-13(5)-Gln was added to the media, following which metabolites converted from C alpha OE cells were identified. We found a greater decrease in cell survival, colony-forming ability, migration, and C alpha OE cell invasion under glucose (Glc)-depletion conditions than under glutamine (Gln)-depletion conditions. Cancer cell migration and invasion increased due to LDHA elevation of the altered metabolic axis driven by activated CK2. FX11 treatment and LDHA knockdown suppressed migration and invasion through ROS generation, but this was partially reversed by the antioxidant N-acetylcysteine (NAC). Moreover, LDHA inhibition decreased tumor growth in a mouse xenograft model transplanted with C alpha OE cells. Finally, we concluded that LDHA is an excellent metabolic target for tumor therapy, based on CK2 alpha derived aerobic glycolysis.ope

Phosphorylation-dependent stabilization of MZF1 upregulates N-cadherin expression during protein kinase CK2-mediated epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a critical process in invasion and metastasis of cancer cells. E-cadherin to N-cadherin switching is considered a molecular hallmark of EMT. Recently, we reported that increased CK2 activity fully induces E-cadherin to N-cadherin switching, but the molecular mechanisms of N-cadherin upregulation are unknown. In this study, we examined how N-cadherin is upregulated by CK2. N-cadherin promoter analysis and ChIP analysis identified and confirmed myeloid zinc finger 1 (MZF1) as an N-cadherin transcription factor. Molecular analysis showed that MZF1 directly interacts with CK2 and is phosphorylated at serine 27. Phosphorylation stabilizes MZF1 and induces transcription of N-cadherin. MZF1 knockdown (MKD) in N-cadherin-expressing cancer cells downregulates N-cadherin expression and reverts the morphology from spindle and fibroblast-like to a rounded, epithelial shape. In addition, we showed that that MKD reduced the motility and invasiveness of N-cadherin-expressing cancer cells. Collectively, these data indicate that N-cadherin upregulation in CK2-mediated E-cadherin to N-cadherin switching is dependent on phosphorylation-mediated MZF1 stabilization. CK2 could be a good therapeutic target for the prevention of metastasis.ope

Protein kinase CK2 activation is required for transforming growth factor b-induced epithelial–mesenchymal transition

Transforming growth factor β (TGFβ) is overexpressed in advanced cancers and promotes tumorigenesis by inducing epithelial-mesenchymal transition (EMT), which enhances invasiveness and metastasis. Although we previously reported that EMT could be induced by increasing CK2 activity alone, it is not known whether CK2 also plays an essential role in TGFβ-induced EMT. Therefore, in the present study, we investigated whether TGFβ signaling could activate CK2 and, if so, whether such activation is required for TGFβ-induced EMT. We found that CK2 is activated by TGFβ treatment, and that activity peaks at 48 h after treatment. CK2 activation is dependent on TGFβ receptor (TGFBR) I kinase activity, but independent of SMAD4. Inhibition of CK2 activation through the use of either a CK2 inhibitor or shRNA against CSNK2A1 inhibited TGFβ-induced EMT. TGFβ signaling decreased CK2β but did not affect CK2α protein levels, resulting in a quantitative imbalance between the catalytic α and regulatory β subunits, thereby increasing CK2 activity. The decrease in CK2β expression was dependent on TGFBRI kinase activity and the ubiquitin-proteasome pathway. The E3 ubiquitin ligases responsible for TGFβ-induced CK2β degradation were found to be CHIP and WWP1. Okadaic acid (OA) pretreatment protected CK2β from TGFβ-induced degradation, suggesting that dephosphorylation of CK2β by an OA-sensitive phosphatase might be required for CK2 activation in TGFβ-induced EMT. Collectively, our results suggest CK2 as a therapeutic target for the prevention of EMT and metastasis of cancers.ope

High temperature stimulates acetic acid accumulation and enhances the growth inhibition and ethanol production by Saccharomyces cerevisiae under fermenting conditions

Cellular responses of Saccharomyces cerevisiae to high temperatures of up to 42 °C during ethanol fermentation at a high glucose concentration (i.e., 100 g/L) were investigated. Increased temperature correlated with stimulated glucose uptake to produce not only the thermal protectant glycerol but also ethanol and acetic acid. Carbon flux into the tricarboxylic acid (TCA) cycle correlated positively with cultivation temperature. These results indicate that the increased demand for energy (in the form of ATP), most likely caused by multiple stressors, including heat, acetic acid, and ethanol, was matched by both the fermentation and respiration pathways. Notably, acetic acid production was substantially stimulated compared to that of other metabolites during growth at increased temperature. The acetic acid produced in addition to ethanol seemed to subsequently result in adverse effects, leading to increased production of reactive oxygen species. This, in turn, appeared to cause the specific growth rate, and glucose uptake rate reduced leading to a decrease of the specific ethanol production rate far before glucose depletion. These results suggest that adverse effects from heat, acetic acid, ethanol, and oxidative stressors are synergistic, resulting in a decrease of the specific growth rate and ethanol production rate and, hence, are major determinants of cell stability and ethanol fermentation performance of S. cerevisiae at high temperatures. The results are discussed in the context of possible applications.ope

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.restrictio

Nanoparticulation improves bioavailability of Erlotinib

OBJECTIVES: Nanoparticulation using fat and supercritical fluid (NUFSTM) is a drug delivery platform technology enabling efficient and effective formulation of poorly soluble drugs. We performed experiments to examine whether NUFS™ could improve poor bioavailability and reduce fed-fasted bioavailability variances of erlotinib (Ert). METHODS: NUFS-Ert was prepared using NUFS™ technology; its physical properties were characterized, and drug release was measured. Furthermore, in vitro and in vivo efficacy tests and pharmacokinetic analysis were performed. RESULTS: NUFS-Ert nanoparticles had an average size of 250 nm and were stable for 2 months at 40 °C, 4 °C, and room temperature. The dissolution rate of NUFS-Ert increased in bio-relevant dissolution media. NUFS-Ert was more potent in inhibiting EGF signaling and in suppressing the proliferation of A549, a human non-small cell lung cancer cell line. Furthermore, A549 xenografts in BALB/c nude mice treated with NUFS-Ert regressed more efficiently than those in the mice treated with vehicle or Tarceva®. In addition, experimental lung metastasis was more efficiently inhibited by NUFS-Ert than by Tarceva®. The relative bioavailability of NUFS-Ert compared with that of Tarceva® was 550% and the ratio of the area under the concentration-time curve (AUC) of fed state to the AUC of fasted state was 1.8 for NUFS-Ert and 5.8 for Tarceva®. CONCLUSIONS: NUFS-Ert could improve poor bioavailability and reduce fed-fasted bioavailability variances of Ert. NUFS-Ert was more efficacious than Tarceva®.restrictio

Protein kinase CK2 modulation of pyruvate kinase M isoforms augments the Warburg effect in cancer cells.

Protein kinase CK2 is active in cancer cells. Previously, we reported that increased CK2 activity could induce epithelial mesenchymal transition of cancer cells. CK2 also induced epithelial mesenchymal transition in colon cancer cell lines such as HT29 and SW620, and the transitioned cells (CK2α cells) became more proliferative than the controls. We assumed that CK2 could affect cancer cell growth by modulating their energy metabolism. Here, we examined the molecular effects of CK2 on the glucose metabolism of cancer cells. We found that CK2α cells consumed more glucose and produced more lactate than control cells did. An XF glycolysis stress test showed that aerobic glycolysis was augmented up to the cancer cell's maximal glycolytic capacity in CK2α cells. Molecular analysis revealed that pyruvate kinase M1 was downregulated and pyruvate kinase M2 was nuclear localized in CK2α cells. Consequently, the expression and activity of lactate dehydrogenase A (LDHA) were upregulated. Treatment with FX11-a specific LDHA inhibitor-or clustered regularly interspaced short palindromic repeats (CRISPR)-mediated knockout of LDHA inhibited the CK2-driven proliferation of cancer cells. We conclude that CK2 augments the Warburg effect, resulting in increased proliferation of cancer cells.restrictio

Catechin-7-O-xyloside induces apoptosis via endoplasmic reticulum stress and mitochondrial dysfunction in human non-small cell lung carcinoma H1299 cells

The medicinal plant Ulmus davidiana var. japonica has significant potential as a cancer chemoprevention agent. Catechin-7-O-xyloside (C7Ox) was purified from ultrafine U. davidiana var. japonica ethanol extract. In the present study, we investigated the apoptotic effect of C7Ox in the non-small cell lung cancer (NSCLC) cell line H1299. C7Ox treatment induced cell death and decreased plasma membrane integrity, an event typical of apoptosis. C7Ox-induced apoptosis was associated with the proteolytic activation of caspase-6, cleavage of poly(ADP-ribose) polymerase (PARP) and loss of mitochondrial membrane potential. C7Ox also induced the endoplasmic reticulum (ER) stress-regulated pro-apoptotic transcription factor CHOP. The suppression of CHOP expression significantly decreased C7Ox-induced cell death, LDH leakage and caspase-6 activation. Antitumor effects, evaluated based on protracted tumor regression, were observed when nude-mice bearing H1299 xenografts were treated with C7Ox. C7Ox-induced tumor regression was accompanied by enhanced expression of CHOP mRNA. Our data suggest that C7Ox can trigger mitochondrial-mediated apoptosis, and that ER stress is critical for C7Ox-induced apoptosis in H1299 NSCLC cells.ope

ω-Hydroxyundec-9-enoic acid induces apoptosis through ROS-mediated endoplasmic reticulum stress in non-small cell lung cancer cells

ω-Hydroxyundec-9-enoic acid (ω-HUA), a hydroxyl unsaturated fatty acid derivative, is involved in the antifungal activity of wild rice (Oryza officinalis). Here, we investigated the anti-cancer activity of ω-HUA on a non-small cell lung cancer (NSCLC) cell line. ω-HUA increased apoptosis and induced cleavages of caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP). ω-HUA treatment significantly induced endoplasmic reticulum (ER) stress response. Suppression of CHOP expression and inhibiting ER stress by 4-phenylbutyrate (4-PBA) significantly attenuated the ω-HUA treatment-induced activation of caspase-6, caspase-9, and PARP, and subsequent apoptotic cell death, indicating a role for ER stress in ω-HUA-induced apoptosis. In addition, cells subjected to ω-HUA exhibited significantly increased quantity of reactive oxygen species (ROS), and the ROS scavenger N-acetyl-l-cysteine (NAC) inhibited ω-HUA-induced apoptotic cell death and ER stress signals, indicating a role for ROS in ER stress-mediated apoptosis in ω-HUA-treated cells. Taken together, these results suggest that sequential ROS generation and ER stress activation are critical in ω-HUA treatment-induced apoptosis and that ω-HUA represents a promising candidate for NSCLC treatment.ope