Morin overcomes doxorubicin resistance in human breast cancer by inducing DNA damage and modulating the LKB1/AMPK/mTORC1 signaling pathway.

Breast cancer chemoresistance hampers chemotherapy efficacy; researchers investigate the pharmacological activities of natural products for potential solutions. This study aimed to determine the effect of morin, a bioflavonoid isolated from Maclura pomifera, on two Dox-resistant human breast cancer cell lines MDA-MB-231 (MDA-DR) and MCF-7 (MCF-DR). Sulforhodamine B and colony-forming assays demonstrated the cytotoxic effect of morin on both cell lines. Morin induced DNA damage and reduced the DNA repair mechanism, a feature of chemoresistance. In addition, morin reduced the protein expressions of cell cycle regulators, such as cyclin D1, CDK4, cyclin E1, cyclin B1, and p-Rb, thereby halting cell cycle progression. Moreover, morin slightly reduced PARP and Bcl-xL expressions but left LC3-II and RIPK3 expressions unchanged. Annexin-V/7-AAD analysis showed morin increased 7-AAD positive cells and annexin-V positive cells among MDA-DR and MCF-DR cells, respectively. In addition, morin increased p-AMPK and p-LKB1 levels; and, thus, inhibited phosphorylation of the mTOR pathway, but decreased t-AMPK levels by inducing lysosomal degradation, and AICAR, an AMPK activator, reduced Raptor, cyclin D1, CDK4, cyclin E1 and phosphorylated, and total mTOR levels, indicating AMPK is a key player in inducing cell death. Also, morin modulated MAPK phosphorylation and attenuated p-Akt and p-GSK3αß levels; and thus, inhibited cell survival. In addition, morin suppressed tumor growth in our MDA-DR xenografted mouse model. These findings indicate that morin is a potential treatment for Dox-resistant breast cancer and that it does so by inducing DNA damage and modulating the LKB1/AMPK/mTORC1 pathway, along with regulating the MAPK, and Akt/GSK3αß signaling pathways.

Resource Utilization of Residual Organic Sludge Generated from Bioenergy Facilities Using Hermetia illucens Larvae.

Residual organic sludge generated from bioenergy facilities (BF-rOS) is often disposed instead of recycled, thus contributing to further environmental pollution. This study explored the resource utilization of BF-rOS using Hermetia illucens larvae (BSFL). When BF-rOS was fed to BSFL for two weeks, the dry weight per individual BSFL was approximately 15% of that of BSFL that were fed food waste (FW). However, the dry weight increased by approximately two-fold in BSFL that were fed effective microorganism (EM)-supplemented BF-rOS containing 60% moisture. However, under both conditions, the BSFL did not mature into pupae. In contrast, the highest dry weight per BSFL was observed with the BF-rOS/FW (50%:50%) mixture, regardless of EM supplementation. Furthermore, the highest bioconversion rate was observed when the BSFL were fed the BF-rOS/FW (50%:50%) mixture, and the frass produced by the BSFL contained fertilizer-appropriate components. In addition, the nutritional components of the BSFL exhibited a nutrient profile suitable for animal feed, except for those fed BF-rOS only. In conclusion, this investigation demonstrates that BF-rOS should be recycled for fertilizer production by mixing it with FW as a BSFL feed, which generates the valuable insect biomass as potential nutrition for animal feeding.

Combined doxorubicin and arctigenin treatment induce cell cycle arrest-associated cell death by promoting doxorubicin uptake in doxorubicin-resistant breast cancer cells.

Chemotherapy failure is often caused by drug resistance, for which no effective treatment strategy has been established. Many studies have been undertaken with the aim of overcoming drug resistance using natural products. Arctigenin (ATG), a natural product, has been investigated for its anti-cancer effects in HER2-overexpressing, ER-positive, and triple-negative breast cancer cells. We investigated the efficacy of ATG against self-established doxorubicin (DOX)-resistant breast cancer cells (MCF-DR and MDA-DR cells) derived from MCF-7 and MDA-MB-231 cells, respectively. ATG was found to increase DOX intracellular levels by downregulating multidrug Resistance 1 (MDR1) mRNA expression in DOX-resistant cells. In addition, combined treatment with DOX and ATG (DOX/ATG) reduced the viability of and colony formation by DOX-resistant cells. DOX/ATG also significantly induced G2/M cell cycle arrest by suppressing the Cyclin D1/CDK4/RB pathways and suppressed the expressions of MDR1 and Cyclin D1 by inhibiting the Mitogen-activated protein kinase (MAPK)/Activating protein-1 (AP-1) signaling pathways. Furthermore, DOX/ATG induced DNA damage and attenuated the expressions of RAD51 and Ku80. However, PARP1 (Poly [ADP-ribose] polymerase1) cleavage and AIF (Apoptosis-inducing factor) induced apoptosis did not occur despite DNA damage-induced cell death. Rather, flow cytometry showed that DOX/ATG caused necrosis. In summary, DOX/ATG increased intracellular DOX levels by inhibiting MDR1 and inducing G2/M arrest by inhibiting the Cyclin D1/CDK4/RB pathways and causing necrosis by damaging DNA. Our results suggest that ATG might be used as an adjuvant to enhance the efficacy of DOX in DOX-resistant breast cancer.

Development of Optimized Feed for Lipid Gain in Zophobas morio (Coleoptera: Tenebrionidae) Larvae.

Super mealworm Zophobas morio (Coleoptera: Tenbrionidea) larvae (ZML) are being investigated as potential candidates for biodiesel production. Several studies have revealed that the crude fat content of ZML can be enhanced by increasing the feed consumed. We aimed to develop an optimized ZML feed that enhances the lipid gain using 10 different ingredients. The results revealed that the highest lipid content was observed in ZML fed food waste (FW). Furthermore, we found that the weight gain of ZML improved when fed fermented FW using three selected microorganisms (3M), Lactobacillus fermentum, Lactobacillus acidophilus, and Pediococcus acidilactici. We also analyzed the effects of preservatives on the weight gain of ZML, and the results revealed that ZML fed 5-day 3M-fermented FW (FFW) containing 0.05% sorbic acid exhibited the highest weight gain. Based on these findings, we produced solid FFW containing 0.05% sorbic acid using 5% agar and established a manufacturing process. Body composition analysis revealed that the lipid content of the ZML fed manufactured feed was higher than that of the ZML fed wheat bran. Therefore, this study suggests that solid FFW containing 0.05% sorbic acid should be used as a commercial feed for ZML breeding to enhance lipid gain, making it an economical substrate for raw biodiesel production.

Morin Sensitizes MDA-MB-231 Triple-Negative Breast Cancer Cells to Doxorubicin Cytotoxicity by Suppressing FOXM1 and Attenuating EGFR/STAT3 Signaling Pathways.

Considerable emphasis is being placed on combinatorial chemotherapeutic/natural treatments for breast cancer. This study reveals the synergistic anti-tumor activity of morin and Doxorubicin (Dox) co-treatment on MDA-MB-231 triple-negative breast cancer (TNBC) cell proliferation. Morin/Dox treatment promoted Dox uptake and induced DNA damage and formation of nuclear foci of p-H2A.X. Furthermore, DNA repair proteins, RAD51 and survivin, and cell cycle proteins, cyclin B1 and forkhead Box M1 (FOXM1), were induced by Dox alone but attenuated by morin/Dox co-treatment. In addition, Annexin V/7-AAD analysis revealed that necrotic cell death after co-treatment and apoptotic cell death by Dox alone were associated with the induction of cleaved PARP and caspase-7 without Bcl-2 family involvement. FOXM1 inhibition by thiostrepton showed that co-treatment caused FOXM1-mediated cell death. Furthermore, co-treatment downregulated the phosphorylation of EGFR and STAT3. Flow cytometry showed that the accumulation of cells in the G2/M and S phases might be linked to cellular Dox uptake, p21 upregulation, and cyclin D1 downregulation. Taken together, our study shows that the anti-tumor effect of morin/Dox co-treatment is due to the suppression of FOXM1 and attenuation of EGFR/STAT3 signaling pathways in MDA-MB-231 TNBC cells, which suggests that morin offers a means of improving therapeutic efficacy in TNBC patients.

Evaluation of Antimicrobial Activity in the Extract of Defatted Hermetia illucens Fed Organic Waste Feed Containing Fermented Effective Microorganisms.

Hermetia illucens (black soldier fly) larvae (HIL) are considered useful industrial insects for the production of feed for livestock, eco-friendly fertilizer from organic wastes, and biodiesel. Therefore, we evaluated the antimicrobial activity in the extract of crude-oil-extracted crushed HIL powder prepared from HIL fed organic waste containing fermented effective micro-organisms for biodiesel production. The result showed that antimicrobial activity was not fully induced in HIL fed L. casei-containing feed. In contrast, increased antimicrobial activity was observed in defatted HIL extract prepared from crude-oil-extracted crushed HIL powder. We found that the extract effectively inhibited the growth of pathogens and antimicrobial-peptide-resistant bacteria, such as three kinds of Salmonella species, and Enterococcus faecalis, Streptococcus mutans, Candida albicans, Serratia marcescens, and Pseudomonas tolaasii, with a minimum inhibitory concentration of 200-1000 µg/100 µL. Furthermore, no cytotoxicity to CaCO-2 human intestinal cells was observed in the extract. We also found that the production fee of extract equivalent to the antimicrobial activity of melittin was approximately 25-fold less than the production fee of melittin. Therefore, the results demonstrate that crude-oil-extracted crushed HIL powder prepared from HIL fed organic waste containing fermented effective micro-organisms for biodiesel production should be used as the feedstock for synthetic, preservative-free livestock feed and food additives. Taken together, the present study supports the usefulness of HIL as an eco-friendly feedstock in the biodiesel, agricultural, food, and feed industries.

Optimization of Feed Components to Improve Hermetia illucens Growth and Development of Oil Extractor to Produce Biodiesel.

HIL are useful in agriculture because they can be used as feed for livestock or fertilizer and can bioconvert organic wastes, such as food waste and human and animal manure to usable fertilizer. In addition, HIL are being studied as a source of biodiesel because of their high-fat content. However, their use for biodiesel production has not been fully adopted. Here, the results showed that survival, weight gains, and total dried weight were significantly enhanced when HIL were fed dried-food waste (DFW)/chicken manure (CM). Furthermore, increased weight gain was observed in HIL fed DFW containing 5 mL waste cooking oil (WCO) per 100 g and 1.2% (v/w) fermented effective microorganism (F-EM). Based on these results, we prepared experimental feeds containing DFW, CM, WCO, and F-EM to establish an optimal feed for biodiesel production. We found that FT-1-2, a feed prepared with 60 g DFW, 40 g CM, 2 mL WCO, and 0.8% F-EM (v/w), significantly enhanced fat content, weight gain, and total dried weight of HIL. Our results indicate FT-1-2 is a suitable feed to breed HIL for biodiesel production. We then developed an automatic oil extractor for biodiesel production. The yield of the oil extractor was higher than that of solvent extraction. The study shows FT-1-2 is an optimal HIL feed for biodiesel production and that the developed oil extractor is useful for the extraction of crude oil from HIL and for the harvesting of defatted HIL frass for livestock feed and fertilizer. Taken together, we established an optimized low-cost feed for HIL breeding and developed an automatic oil extractor for the production of biodiesel from HIL.

Cell cycle arrest-mediated cell death by morin in MDA-MB-231 triple-negative breast cancer cells.

BACKGROUND: Morin, a flavonoid extracted from Moraceace family and exhibits several pharmacological activities including anti-cancer activity. Although the anticancer activity of morin in breast cancer was estimated in some investigations, the pharmaceutical mechanism has not been fully elucidated. Therefore, we investigated to unveil the detail signaling pathway in morin-treated in MDA-MB-231 triple-negative breast cancer cells. METHODS: The cytotoxicity of morin in MDA-MB-231 cells was confirmed by sulforhodamine B (SRB) assay and colony formation assay. Flow cytometry was performed to examine the cell cycle and cell death patterns and the protein expression and phosphorylation were detected by western blotting. RESULTS: Our results showed that morin inhibited MDA-MB-231 cells proliferation in time and concentration-dependent manner. Morphological changes were observed when treated with various concentration of morin in MDA-MB-231 cells. In regard to protein expression, morin induced the phosphorylation of ERK and p-H2A.X and decreased the level of DNA repair markers, RAD51 and survivin. In addition, flow cytometry showed S and G2/M arrest by morin that was associated with the decrease in the protein expression of cyclin A2 and cyclin B1 and upregulation of p21. Interestingly, annexin V/PI staining result clearly showed that morin induced cell death without apoptosis. Furthermore, attenuated FoxM1 by morin was co-related with cell cycle regulators including p21, cyclin A2 and cyclin B1. CONCLUSION: Taken together, our study indicates that morin-induced cell death of MDA-MB-231 is caused by sustained cell cycle arrest via the induction of p21 expression by activation of ERK and repression of FOXM1 signaling pathways.

Evaluation of the antimetastatic and anticancer activities of morin in HER2­overexpressing breast cancer SK­BR­3 cells.

Morin (2',3,4',5,7­pentahydroxyflavone), a flavonoid isolated from members of the Moraceae family and the leaves of Cudranaia tricuspidata Buread, is a well­known natural substance with anti­inflammatory, antioxidative, antimetastasis, and anticancer effects. However, its anticancer activity has not been comprehensively investigated in human epidermal growth factor receptor 2 (HER2)­overexpressing breast cancer cells. Here, we evaluated the effects of morin on metastasis and cell viability in HER­2­overexpressing human breast cancer SK­BR­3 cells. Our results revealed that morin (150­200 µM) prevented endothelial growth factor (EGF)­induced metastatic potential and suppressed cell migration and MMP­9 activity by inhibiting the EGFR signaling pathway in SK­BR­3 cells by gelatin zymography, wound healing assay and western blotting. Interestingly, morin­induced reductions in cell viability were found to be associated with inhibition of the HER2/EGFR signaling pathway by sulforhodamine B assay and western blotting. Morin also induced the phosphorylation of H2A.X and downregulated the expression levels of RAD51 and survivin, which implied morin­induced DNA damage and that this damage accumulated in HER­2­overexpressing SK­BR­3 cells. Western blot analysis and fluorescent immunocytochemisty showed that morin also activated autophagy after 24 h of treatment and this was maintained at 48 h when activation of apoptosis via PARP cleavage resulted in the activation of caspase­3 and ­7, which was associated with the release of cytochrome c to the cytosol from mitochondria. In addition, the phosphorylation of p38 and JNK was enhanced in the HER­2­overexpressing SK­BR­3 cells by morin after 24 and 48 h of treatment, which suggested p38 and JNK participate in morin­induced cell death. Taken together, the present investigation indicates that morin is a powerful therapeutic candidate for the treatment of HER2­overexpressing breast cancer because it suppresses the EGFR signaling pathway, induces cell death by inhibiting the HER2/EGFR signaling pathway, and suppresses metastatic potential.

Arctigenin-mediated cell death of SK-BR-3 cells is caused by HER2 inhibition and autophagy-linked apoptosis.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is well-known as the therapeutic marker in breast cancer. Therefore, we evaluated anti-cancer activity of arctigenin (ATG) on in SK-BR-3 HER2-overexpressing human breast cancer cells. METHODS: Cell viability and cytotoxicity were analyzed with MTT and colony-forming assay and cell cycle analysis was performed by flow cytometry. The expression and/or phosphorylation of proteins in whole cell lysate and mitochondrial fraction were analyzed by Western blotting. Cellular levels of LC3 and sequestosome 1 (SQSTM1/P62) were observed by immunofluorescence analysis. RESULTS: The result showed that ATG decreased cell viability of SK-BR-3 cells in a concentration-dependent manner. Moreover, ATG increased the sub G1 population linked to the suppression of HER2/EGFR1 signaling pathway. Furthermore, ATG increased the phosphorylation of H2AX and down-regulated RAD51 and survivin expressions, indicating that ATG induced DNA damage and inhibited the DNA repair system. We also found that cleavages of caspase-7 and PARP by releasing mitochondrial cytochrome c into the cytoplasm were induced by ATG treatment for 72 h through the reduction of Bcl-2 and Bcl-xL levels in mitochondria. In contrast, the levels of LC-3 and SQSTM1/P62 were increased by ATG for 24 h through the Akt/mTOR and AMPK signaling pathway. CONCLUSIONS: Taken together, this study indicates that autophagy-linked apoptosis is responsible for the anti-cancer activity of ATG in SK-BR-3 cells, and suggests that ATG is considered a potential therapeutic for the treatment of HER2-overexpressing breast cancer.

Evaluation of the Antimicrobial Activity of an Extract of Lactobacillus casei-Infected Hermetia illucens Larvae Produced Using an Automatic Injection System.

In the present study, we developed an automatic mass-injection system (AMIS) to produce an extract of infected H. illucens larvae (iHIL-E) and then evaluated antimicrobial peptide (AMP) expressions and assessed the antimicrobial activity of iHIL-E against various pathogens and Lactobacillus species. AMP gene expressions were assessed by real-time quantitative polymerase chain reaction (PCR) and the antimicrobial activities of iHIL-E were estimated using a radial diffusion assay and by determining minimal inhibitory concentrations. Results showed that the antimicrobial activity of HIL extract was effectively enhanced by L. casei infection and that the gene expressions of cecropin 3 and defensin 3 (antimicrobial peptides) were up-regulated. iHIL-E also prevented the growths of Enterococcus faecalis, Streptococcus mutans, and Candida vaginitis (MICs 200, 500, and 1000 µg/100 µL, respectively) and demonstrated high protease resistance. Moreover, the growths of methicillin-resistant Staphylococcus aureus, antibiotic-resistant Pseudomonas aeruginosa and AMP-resistant bacteria, Serratia marcescens, and Pseudomons tolaasii were significantly suppressed by iHIL-E. In addition, although iHIL completely cleared Salmonella species at concentrations of >200 µg/100 µL, Lactobacillus species were unaffected by iHIL at concentrations of <1000 µg/100 µL. The present investigation shows that the devised automatic mass injection system is effective for the mass production of the extract of infected HIL and that this extract is a novel, natural, protease-resistant, antibiotic candidate with broad-spectrum antibiotic activity.

Antimicrobial Activity of an Extract of Hermetia illucens Larvae Immunized with Lactobacillus casei against Salmonella Species.

The expressions of antimicrobial peptides (AMPs) in the larvae of the black soldier fly, Hermetia illucens, were significantly increased by pathogen or stimulant induced innate immunity activation. We immunized H. illucens fifth instar larvae with five different Lactobacillus species, that is, Lactobacillus acidophilus, L. brevis, L. casei, L. fermentum, or L. delbrueckii, to induce the mass production of AMPs and selected optimal immune inducers. Antimicrobial activities in hemolymph and H. illucens larvae (HIL) extract were evaluated against three salmonella species (Salmonella pullorum, Salmonella typhimurium, and Salmonella enteritidis). Highest antimicrobial activity was shown by the hemolymph of HIL immunized by L. casei and its activity was closely linked with the inductions of cecropin 1 (HiCec1) and defensin 1 (HiDef1) gene expressions. Furthermore, antimicrobial activity in hemolymph was stable to heat and pH and the growth of three Salmonella species were dramatically suppressed by HIL hemolymph and extract after immunization with L. casei. The minimal inhibitory concentration (MICs) of L. casei-immunized HIL extract against Staphylococcus aureus, Escherichia coli, and Salmonella species ranged from 100~200 µg/100 µL and no cytotoxicity to CaCo-2 and L929 cells were observed in the concentration range 100~40,000 µg/100 µL. Taken together, the present investigation demonstrates that L. casei-immunized HIL extract is a powerful natural antibiotic and preservative that can prevent contamination by Salmonella species.

Refined Deep Seawater Improves Serum Lipid Profile in Hypercholesterolemia: A Randomized, Double-Blind, Placebo-Controlled, Clinical Trial.

Deep seawater (DSW) has been investigated for its lipid-lowering effects, but clinical evidence is still far from conclusive. Therefore, this study was conducted to examine the effects of refined DSW (RDSW) on hypercholesterolemia. In this randomized, double-blind, placebo-controlled trial, 78 Korean participants were randomized to either an RDSW group that drank RDSW for 8 weeks or a placebo group. Clinical laboratory information was collected from all subjects at 0, 4, and 8 weeks. Both groups showed a significant reduction in total cholesterol (TC), whereas only the RDSW group demonstrated a significant decrease in low-density lipoprotein cholesterol (LDL-c) during the study. Stratified analysis of both groups revealed a significant reduction of TC in the moderately high TC subgroup. However, only the RDSW exhibited a significant decline of LDL-c in the high LDL-c subgroup. In addition, lipoprotein(a) decreased significantly in the RDSW group, but not in the placebo. RDSW did not affect other lipid profiles, including high-density lipoprotein cholesterol (HDL-c), triglyceride, free fatty acid, apolipoproteins, and other markers including inflammation marker, hematological parameters, blood and urine chemistry, and vital signs. RDSW improved lipid profiles by decreasing TC and LDL-c while maintaining HDL-c levels in people with hypercholesterolemia.

Arctigenin Enhances the Cytotoxic Effect of Doxorubicin in MDA-MB-231 Breast Cancer Cells.

Several reports have described the anti-cancer activity of arctigenin, a lignan extracted from Arctium lappa L. Here, we investigated the effect of arctigenin (ATG) on doxorubicin (DOX)-induced cell death using MDA-MB-231 human breast cancer cells. The results showed that DOX-induced cell death was enhanced by ATG/DOX co-treatment in a concentration-dependent manner and that this was associated with increased DOX uptake and the suppression of multidrug resistance-associated protein 1 (MRP1) gene expression in MDA-MB-231 cells. ATG enhanced DOX-induced DNA damage and decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the expressions of RAD51 and survivin. Cell death caused by ATG/DOX co-treatment was mediated by the nuclear translocation of apoptosis inducing factor (AIF), reductions in cellular and mitochondrial Bcl-2 and Bcl-xL, and increases in mitochondrial BAX levels. However, caspase-3 and -7 did not participate in DOX/ATG-induced cell death. We also found that DOX/ATG-induced cell death was linked with activation of the p38 signaling pathway and suppressions of the phosphorylations and expressions of Akt and c-Jun N-terminal kinase. Taken together, these results show that ATG enhances the cytotoxic activity of DOX in MDA-MB-231 human breast cancer cells by inducing prolonged p21 expression and p38-mediated AIF-dependent cell death. In conclusion, our findings suggest that ATG might alleviate the side effects and improve the therapeutic efficacy of DOX.

Anti­metastatic effects of arctigenin are regulated by MAPK/AP­1 signaling in 4T­1 mouse breast cancer cells.

Arctigenin is a natural lignan that is found in burdock with anti­viral, ­oxidative, ­inflammatory and anti­tumor activities. In the current study, the effect of arctigenin on metastatic potential was examined in 4T­1 mouse triple­negative breast cancer cells. The results indicated that arctigenin inhibited cell motility and invasiveness, which was determined using wound healing and transwell invasion assays. Arctigenin suppressed matrix metalloprotease­9 (MMP­9) activity via gelatin zymography, and protein expression of cyclooxygenase­2 (COX­2) and MMP­3. Furthermore, arctigenin attenuated the mRNA expression of metastatic factors, including MMP­9, MMP­3 and COX­2. Based on these results, the effect of arctigenin on the mitogen­activated protein kinase (MAPK)/activating protein­1 (AP­1) signaling pathway was assessed in an attempt to identify the regulatory mechanism responsible for its anti­metastatic effects. Arctigenin was demonstrated to inhibit the phosphorylation of extracellular signal­regulated protein kinase (ERK) and c­Jun N­terminal kinase (JNK), and the nuclear translocations of the AP­1 subunits, c­Jun and c­Fos. In summary, the present study demonstrated that in 4T­1 mouse triple­negative breast cancer cells the anti­metastatic effect of arctigenin is mediated by the inhibition of MMP­9 activity and by the inhibition of the metastasis­enhancing factors MMP­9, MMP­3 and COX­2, due to the suppression of the MAPK/AP­1 signaling pathway. The results of the current study demonstrated that arctigenin exhibits a potential for preventing cell migration and invasion in triple negative breast cancer.

Inhibition of TPA­induced metastatic potential by morin hydrate in MCF­7 human breast cancer cells via the Akt/GSK­3ß/c­Fos signaling pathway.

Plant flavonoid 2',3,4',5,7­pentahydroxyflavone (morin hydrate), isolated from the family Moraceae (Morus alba L.), is known to have anti­inflammatory and anticancer effects. However, its pharmaceutical effects on metastasis have not been fully elucidated to date. Therefore, the current study investigated the effects of morin hydrate on cancer metastasis in MCF­7 human breast cancer cells. The results showed that morin hydrate suppressed 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced cell migration and invasion via the inhibition of matrix metalloproteinase (MMP)­9 activity. Furthermore, gene expression level of MMP­9, MMP­7, urokinase plasminogen activator (uPA), uPA receptor (uPAR) and fibronectin were significantly decreased by morin hydrate treatment. Morin hydrate inhibited the phosphorylation of Akt and glycogen synthase kinase (GSK)­3ß, and downregulated the expression of an activator protein­1 subunit c­Fos. In addition, the GSK­3ß phosphorylation and c­Fos expression were suppressed by PI3K/Akt pathway inhibitors, LY294002 and wortmannin. Taken together, these results demonstrated that morin hydrate reduced the metastatic potential in TPA­treated MCF­7 human breast cancer cells via the inhibition of MMPs, uPA and uPAR, and the underlying Akt/GSK­3ß/c­Fos pathway. Therefore, the present investigation suggested that morin hydrate may be a natural substance with a preventive potential for metastasis in breast cancer cells.

Anti­platelet activity of mineral­balanced deep sea water is mediated via the regulation of Akt and ERK pathway crosstalk.

Mineral­balanced deep sea water (MBDSW), an unlimited natural sea source, has been demonstrated to minimize the risk of developing cardiovascular diseases, such as obesity, hypertension, inflammation and hyperlipidemia. This study investigated the effects of MBDSW [magnesium (Mg):calcium (Ca) ratio, 3:1] on platelet activation. MBDSW significantly inhibited the collagen­ and thrombin­induced platelet aggregation of human platelets. In collagen­induced platelets, MBDSW inhibited intracellular calcium mobilization, granule secretion [serotonin, adenosine triphosphate (ATP) and P­selectin expression] and thromboxane A2 (TXA2) production. Moreover, MBDSW markedly inhibited Akt and extracellular signal­regulated kinase (ERK) phosphorylation, but not that of c­Jun N­terminal kinase (JNK) and p38. Moreover, MBDSW phosphorylated inositol 1,4,5­triphosphate receptor (IP3R) and vasodilator­stimulated phosphoprotein (VASP), and it increased the cyclic adenosine monophosphate (cAMP) level in collagen­induced human platelets. Dipyridamole, a phosphodiesterase (PDE) inhibitor, significantly increased the cAMP level and regulated the Akt, ERK and VASP (Ser157) levels in a manner similar to that of MBDSW. In addition, LY294002, an Akt inhibitor, inhibited the phosphorylation of ERK, and U0126, an ERK inhibitor, inhibited the phosphorylation of Akt. Taken together, the results of the present investigation suggest that the inhibitory effects of MBDSW on platelet aggregation may be associated with the cross­inhibition of Akt and ERK phosphorylation. These results strongly indicate that MBDSW may have preventive or therapeutic potential for platelet aggregation­mediated diseases, such as thrombosis, atherosclerosis and myocardial infarction.

Morin hydrate inhibits platelet activation and clot retraction by regulating integrin αIIbß3, TXA2, and cAMP levels.

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbß3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule. In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbß3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25-100 µM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbß3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbß3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.

The association of changes in RAD51 and survivin expression levels with the proton beam sensitivity of Capan­1 and Panc­1 human pancreatic cancer cells.

Fewer than 20% of patients diagnosed with pancreatic cancer can be treated with surgical resection. The effects of proton beam irradiation were evaluated on the cell viabilities in Panc­1 and Capan­1 pancreatic cancer cells. The cells were irradiated with proton beams at the center of Bragg peaks with a 6­cm width using a proton accelerator. Cell proliferation was assessed with the MTT assay, gene expression was analyzed with semi­quantitative or quantitative reverse transcription­polymerase chain reaction analyses and protein expression was evaluated by western blotting. The results demonstrated that Capan­1 cells had lower cell viability than Panc­1 cells at 72 h after proton beam irradiation. Furthermore, the cleaved poly (ADP­ribose) polymerase protein level was increased by irradiation in Capan­1 cells, but not in Panc­1 cells. Additionally, it was determined that histone H2AX phosphorylation in the two cell lines was increased by irradiation. Although a 16 Gy proton beam was only slightly up­regulated cyclin­dependent kinase inhibitor 1 (p21) protein expression in Capan­1 cells, p21 expression levels in Capan­1 and Panc­1 cells were significantly increased at 72 h after irradiation. Furthermore, it was observed that the expression of DNA repair protein RAD51 homolog 1 (RAD51), a homogenous repair enzyme, was decreased in what appeared to be a dose­dependent manner by irradiation in Capan­1 cells. Contrastingly, the transcription of survivin in Panc­1 was significantly enhanced. The results suggest that RAD51 and survivin are potent markers that determine the therapeutic efficacy of proton beam therapy in patients with pancreatic cancer.

The preventive effect of deep sea water on the development of cancerous skin cells through the induction of autophagic cell death in UVB-damaged HaCaT keratinocyte.

Ultraviolet light (UV) is a major inducer of skin cancer. Therefore, recovery and removal of UV-damaged skin cells are important in the prevention of skin carcinogenesis. Here, we investigated the effect of deep sea water (DSW) in HaCaT keratinocyte exposed by UVB (λ = 290∼320 nm). The result showed that UVB-induced cell death was reinforced by DSW treatment in a hardness-dependent manner. Furthermore, the increase of cell death by DSW was associated with the down-regulation of survivin and RAD51 expressions induced by UVB. Moreover, we confirmed the inhibition of H2 A.X phosphorylation, a marker for double-stranded DNA damage, and the enhancement of LC3-II and SQSTM1/p62 expressions by DSW administration in UVB-radiated HaCaT keratinocyte. The results imply that the enhancement of UVB-induced cell death by DSW is associated with autophagy. Therefore, we further explored the regulation of autophagy-regulating proteins and apoptosis-related factors expression. Phosphorylation of mammalian target of rapamycin (mTOR), ribosomal protein S6, and S6 kinase by UVB radiation were regressed via DSW treatment, underlying the increase of AMP-activated protein kinase (AMPK) phosphorylation. Furthermore, UVB-enhanced nuclear factor κB (NF-κB) and c-Jun N-terminal kinase (JNK) phosphorylations were increased with DSW treatment. Contrastingly, DSW lessened the Ser15 phosphorylation of p53 and cleavage of poly (ADP-ribose) polymerase induced by UVB radiation. Consequently, the results demonstrate that DSW enhances UVB-damaged skin cell clearance through the activation of autophagic cell death underlying the regulation of AMP-activated protein kinase (AMPK)/mTOR signaling as well as NF-κB and JNK phosphorylations. In conclusion, this investigation suggests that DSW is a potent candidate for the prevention of UV-induced skin cancer development.