The adverse and beneficial effects of polyphenols in green and black teas in vitro and in vivo.

Although numerous studies highlight the health benefits of tea, excessive consumption has been linked to toxic conditions. Thus, understanding the optimal consumption of tea is essential to minimize toxicity while maximizing its benefits. In this study, we investigated the effects of eight green tea samples (G1-G8) and eight black tea samples (R1-R8) from Camellia sinensis, the most popular teas in Asian culture, on RSC96 Schwann neural cells and embryonic cardiomyocyte H9c2 cells. The results showed that the IC50 (mg/ml, weight/volume) of both tea types were inversely proportional to their polyphenol content, suggesting a relationship between toxicity and polyphenol levels in both green and black tea. Interestingly, green teas generally have higher polyphenol content than black teas. We also assessed the protective effects of tea in vitro by pretreating cells with the teas at indicated doses of polyphenol and subsequently exposing them to H2O2. Both tea types significantly reduced the decline in cell viability for both cell lines, and there was no significant difference in protective polyphenol concentrations for green (G3 & G7) and black (R3 & R8) teas at effective concentrations (EC20 and EC40). To evaluate the preventative effects of tea in vivo, we examined the impact of two green (G3 & G7) and two black (R3 & R8) teas with varying polyphenol content on dextran sulfate sodium (DSS)-induced inflammatory colitis in mice. Tea-treated groups exhibited significantly lower inflammatory scores (DAI) than the control group. DSS treatment in the control group led to shortened colorectal lengths in mice, while tea co-treatment partially prevented this loss. Histological analysis revealed that G7 and R3 (with a moderate polyphenol content) treatment improved colorectal crypt structure, decreased the severity of inflammatory ulcerative colitis, and significantly reduced histological scores compared to the control group. However, G3 and R8 (with high and low doses of polyphenol content, respectively) did not show these effects, suggesting that a moderate polyphenol level in both tea types is optimal for preventative benefits.

Aqueous extracts of Ocimum gratissimum mitigate colitis and protect against AOM/DSS-induced colorectal cancer in mice.

In this study, we explored the in vivo effects of Ocimum gratissimum aqueous extracts (OGE) on colorectal cancer (CRC) development provoked by azoxymethane/dextran sodium sulfate (AOM/DSS). The results showed a significant reduction in the tumor load and tumor number for the OGEH group that received continued administration of OGE compared to the AOM/DSS group, with P values of <0.01, but this was not observed in the OGEHs group that received separated administration of OGE. All groups except the control group exhibited aberrant crypt foci (ACF) and adenocarcinoma of lesion pathology in colon, and both conditions were significantly reduced in the OGEH group (P < 0.01) as compared to the AOM/DSS group. Subsequent investigation into whether OGE exhibits eliminative effects on DSS-induced severe colitis (SC) in mice showed that the disease activity index score was significantly reduced in the OGE-treated groups (P < 0.01), also colon colitis histological score was reversed. These data suggest that OGE may be potentially effective in preventing CRC when administered throughout the promotional stages of carcinogenesis by inhibiting inflammatory SC.

Dietary supplementation of Ocimum gratissimum improves growth performance and immune response in broilers under high ambient temperature.

Ocimum gratissimum (OG) was found to have immunity boosting effect on Taiwan country chickens and broiler chickens raised in moderate ambient temperature in previous studies, and the current study investigates its potential on the growth performance, blood traits, intestinal traits, and immune responses in Ross 308 broilers raised in high ambient temperature which can induce mild heat-stress (26 to 33 ˚C, average 30 ˚C). Two hundred 1-d-old male/female chicks were randomly assigned to a control group, three OG (1 g/kg, 3 g/kg, and 5 g/kg)/basal diet groups, and one Amoxicillin group. Data collected during the experiment indicated that the weight gain increase of 1742 g/bird to 1815 g/bird comparing control to 5 g/kg OG supplementation was statistically significant. In addition, the production efficiency factor was also noticeably increased by OG, particularly in the 5 g/kg group, and the uric acid levels were decreased in the 3 and 5 g/kg OG groups (from 4.26 to 2.91 and 2.90 mg/dL, respectively), indicating heat-stress alleviation was observed. Several areas of the carcass saw desirable growth changes, including the increase of breast muscle ratio observed in the 5 g/kg OG group, an overall decrease in abdominal fat in all OG groups, as well as dimensional changes in several areas of the digestive system. Lastly, the hemaglutination, hemaglutination inhibition, and phytohemaglutinin tests indicated elevated immuno-response in all OG groups. In conclusion, OG has exhibited the ability to alleviate symptoms of mild heat-stress, leading to improvement of the digestive organ development and increase of carcass mass and mean weight gain for birds, and we find OG to be a potentially beneficial feed supplement for poultry raising in high ambient temperature conditions.

Ocimum gratissimum (OG) was found to have immunity boosting effect on Taiwan country chickens and broiler chickens raised in moderate ambient temperature in previous studies, and the current study investigates its potential on broilers raised in high ambient temperature. Data collected during the experiment indicated desirable changes in the physiology of the chickens, including overall increase in production efficiency factor, a significant weight gain in the 5 g/kg OG supplementation group, noticeable weight gain in several areas of the carcass especially the breast, an overall decrease in abdominal fat, dimensional changes in several areas of the digestive system, elevated immuno-response for all OG groups. In conclusion, OG has exhibited the ability to alleviate symptoms of mild heat-stress, leading to improvement of the digestive organ development and increase of carcass mass and mean weight gain for birds, and we find OG to be a potentially beneficial feed supplement for poultry raising in high ambient temperature conditions.

Involvement of matrix metalloproteinase 1 and urokinase-type plasminogen activator in the PKCα-p38 MAPK pathway-mediated progression of human liver cancer cells.

Our previous studies have shown that the plasminogen activator (PA) and matrix metalloproteinases (MMPs) proteinase systems were highly expressed in highly malignant liver cancer cells and regulated by PKCα. This study investigates whether the PKCα regulation of PA and MMPs systems is conducted through p38 mitogen-activated protein kinase (MAPK) signaling and the pathway is responsible for promoting cell progression. We found that the expressions of p38 MAPK in both highly malignant HA22T/VGH and SK-Hep-1 liver cancer cells were higher than that in other lower malignancy liver cancer cells. Since PKCα activates p38 MAPK in progression of liver cancer, we suspected the PKCα/p38 MAPK signaling pathway to be involved in the regulation of MMPs and PA systems. When SK-Hep-1 cells were treated with SB203580 or DN-p38, only MMP-1 and u-PA mRNA expressions decreased. The p38 MAPK inhibition also decreased the cell migration and invasion. In addition, the mRNA decay assays showed that the higher expressions of MMP-1 and u-PA mRNA in SK-Hep-1 cells were due to the alteration of mRNA stability by p38 MAPK inhibition. Zymography of SK-Hep-1 cells treated with siPKCα vector also showed the decrease of the activity of MMP-1 and u-PA and confirmed changes in mRNA level. Furthermore, only the transfection of MKK6 to the siPKCα-treated SK-Hep-1 stable clone cell restored the attenuation of MMP-1 and u-PA expressions. The treatment of SK-Hep-1 cells with either inhibitor of MMP-1 or u-PA reduced migration, and the reduction was enhanced with both inhibitors. In addition, tumorigenesis was also reduced with both inhibitors. These data suggest a novel finding that MMP-1 and u-PA are critical components in PKCα/MKK6/p38 MAPK signaling pathway which mediates liver cancer cell progression, and that the targeting of both genes may be a viable approach in liver cancer treatment.

Protective Effects of Ocimum gratissimumAqueous Extracts on HaCaT Cells Against UVC-Induced Inhibition of Cell Viability and Migration.

Ultraviolet C (UVC) has been applied to treatment of infections in wounds for at least the last two decades, however, cells being treated can be damaged if exposure is prolonged, which calls for protective measures, such as drug or herbal pre-treatment, to minimize damage. Ocimum gratissimum contains plant polyphenols such as isoflavones and caffeic acid, which have antioxidant effects. We hypothesize that Ocimum gratissimum aqueous extracts (OGE) can inhibit UVC-induced oxidative damage on skin cells. In this study, HaCaT skin cells are used to test the protective effects of OGE on cell proliferation and migration after exposure to UVC radiation. Pretreatment with OGE (50~150µg/mL) before 40 J/m2 UVC exposure was able to restore survival from 32.25% to between 46.77% and 68.00%, and 80 J/m2 UVC exposure from 11.49% to between 19.07% and 43.04%. Morphological observation of primarily apoptotic cell death confirms the above findings. The flow cytometry analysis revealed that UVC increased the number of cells at the sub-G1 phase in a dose dependent manner, and when pre-treated with OGE the changes were partially reversed. Moreover, the wound healing test for observing migration showed that UVC 40-80 J/m2 decreased cell migration to 47-28% activity and 100 µg/mL OGE was able to restore cell activity to81-69% at day 3. Based on the above results, we suggest that OGE has a protective effect on UVC-induced inhibition of cell proliferation and migration of skin cells and thus has potential application in wound care.

Developmental and Functional Morphology of Eulimnadia braueriana Ishikawa, 1895 (Branchiopoda: Spinicaudata) Feeding Structures: Combination of Filtering and Scraping Feeding Mechanisms.

Large branchiopods inhabit diverse continental habitats worldwide. Their feeding ecology, nevertheless, remains largely unknown. The few functional morphology studies that have been conducted have mostly focused on adults or larvae, seldom have the two been compared collectively. In this study, we examined the feeding structures in Eulimnadia braueriana Ishikawa, 1895 from nauplius to adult to clarify their feeding mechanisms and then compared them with the other two sympatric branchiopods (Branchinella kugenumaensis and Lynceus biformis) in Siangtian Pond, Taiwan. Naupliar second antennae and mandibles are similar to those of other species, suggesting filter-feeding. The naupliar feeding structures, including the mandibular palp and naupliar process, gradually degenerate during the juvenile stage. Simultaneously, the molar surface, maxillae, and second antennae continue developing, reaching their adult form in later juvenile substages. The molar surface and thoracopod setal morphology are similar to those of other filter-feeding branchiopods, but adults also have scraping setae on the first several thoracopod pairs. Nearly all naupliar primary feeding structures change through development, particularly during the early juvenile substages, whereas late juvenile substages and adult morphology are similar. Eulimnadia braueriana transforms from pelagic filtering nauplii to adults that combine benthic filtering and scraping. Comparisons of molar and thoracopod morphology between coexisting branchiopod species show some similarities and differences in filtering and scraping feeding structures, implying potential foraging resource differentiation among species.

Aqueous Ocimum gratissimum extract induces cell apoptosis in human hepatocellular carcinoma cells.

Treatment of advanced hepatocellular carcinoma (HCC) has exhibited a poor overall survival rate of only six to ten months, and the urgency of the development of more effective novel agents is ever present. In this line of research, we aimed to investigate the effects and inhibitive mechanisms of aqueous Ocimum gratissimum leaf extract (OGE), the extract of Ocimum gratissimum, which is commonly used as a therapeutic herb for its numerous pharmacological properties, on malignant HCC cells. Our results showed that OGE decreased the cell viability of HCC SK-Hep1 and HA22T cells in a dose-dependent manner (from 400 to 800 µg/mL), while there is little effect on Chang liver cells. Moreover, cell-cycle analysis shows increased Sub-G1 cell count in SK-Hep1 and HA22T cells which is not observed in Chang liver cells. These findings raise suspicion that the OGE-induced cell death may be mediated through proteins that regulate cell cycle and apoptosis in SK-Hep1 and HA22T cells, and further experimentation revealed that OGE treatment resulted in a dose-dependent decrease in caspase 3 and PARP expressions and in CDK4and p-ERK1/2expressions. Moreover, animal tests also exhibited decreased HCC tumor growth by OGE treatment. We therefore suggest that the inhibition of cell viability and tumor growth induced by OGE may be correlated to the alteration of apoptosis-related proteins.

Myeloid Zinc Finger 1 (MZF1) Maintains the Mesenchymal Phenotype by Down-regulating IGF1R/p38 MAPK/ERα Signaling Pathway in High-level MZF1-expressing TNBC cells.

BACKGROUND/AIM: Signaling regulation of myeloid zinc finger 1 (MZF1) has been implicated in the progression of many human malignancies; however, the mechanistic action of MZF1 in triple-negative breast cancer (TNBC) progression remains elusive. In this study, the aim was to investigate the molecular mechanisms of MZF1 and its functional role in TNBC cellular migration and invasion. MATERIALS AND METHODS: Hs578T and MDA-MB-231 cells were transfected to stably express the acidic domain of MZF1 (MZF160-72), or were transfected with MZF1-specific or ELK1-specific short hairpin RNA (shRNA). Changes in cell morphology and distributions of cellular proteins were observed and subsequently migration and invasion were measured by wound healing and transwell assays. Expression levels of epithelial-mesenchymal transition (EMT)-related genes were carried out using immunoblotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Data of transcriptional regulation were obtained from promoter-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. RESULTS: Herein, we found that MZF1 in high-level MZF1-expressing TNBC cells is associated with cell migration, invasion, and mesenchymal phenotype. MZF1 interacted with the promoter region of insulin-like growth factor 1 receptor (IGF1R) to drive invasion and metastasis of high-level MZF1-expressing TNBC cells. Exogenous expression of the acidic domain of MZF1 repressed the binding of endogenous MZF1 to IGF1R promoter via blocking the interaction with ETS-like gene 1 (ELK1). This blockage not only caused MZF1 protein degradation, but also restrained ELK1 nuclear localization in high-level MZF1-expressing TNBC cells. MZF1, but not ELK1, was necessary for the retention of mesenchymal phenotype by repressing IGF1R promoter activity in TNBC cells expressing high levels of MZF1. Activation of the IGF1R-driven p38MAPK-ERα-slug-E-cadherin signaling axis mediated the conversion of mesenchymal cell to epithelial phenotype, caused by MZF1 destabilization. These results suggest that MZF1 is an oncogenic inducer. CONCLUSION: Blocking of the MZF1/ELK1 interaction to reduce MZF1 protein stability by saturating the endogenous MZF1/ELK1 binding domains might be a promising therapeutic strategy for the treatment of high-level MZF1-expressing TNBC.

Cetrimonium Bromide Inhibits Cell Migration and Invasion of Human Hepatic SK-HEP-1 Cells Through Modulating the Canonical and Non-canonical TGF-ß Signaling Pathways.

BACKGROUND/AIM: Cetrimonium bromide (CTAB), a quaternary ammonium surfactant, is an antiseptic agent against bacteria and fungi. However, the mechanisms by which its pharmacological actions affect epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC) cells, such as adenocarcinoma in SK-HEP-1 cells, have not been investigated. We, thereby, investigated whether CTAB inhibits cellular mobility and invasiveness of human hepatic adenocarcinoma in SK-HEP-1 cells. MATERIALS AND METHODS: SK-HEP-1 cells were treated with CTAB, and subsequent migration and invasion were measured by wound healing and transwell assays. Protein expression was detected by immunoblotting analysis. RESULTS: Our data revealed that treatment of SK-HEP-1 cells with CTAB altered their mesenchymal spindle-like morphology. CTAB exerted inhibitory effects on the migration and invasion of SK-HEP-1 cells dose-dependently, and reduced protein levels of matrix metalloproteinase-2 (MMP-2), MMP-9, snail, slug, twist, vimentin, fibronectin, N-cadherin, Smad2, Smad3, Smad4, phosphoinositide-3-kinase (PI3K), p-PI3K, Akt, p-Akt, ß-catenin, mammalian target of rapamycin (mTOR), p-mTOR, p-p70S6K, p-extracellular signal-regulated kinases (ERK)1/2, p-p38 mitogen-activated protein kinase (MAPK) and p-c-Jun N-terminal kinase (JNK), but increased protein levels of tissue inhibitor matrix metalloproteinase-1 (TIMP-1), TIMP-2, claudin-1 and p-GSK3ß. Based on these observations, we suggest that CTAB not only inhibits the canonical transforming growth factor-ß (TGF-ß) signaling pathway though reducing SMADs (an acronym from the fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, Mothers against decapentaplegic proteins), but also restrains the non-canonical TGF-ß signaling including MAPK pathways like ERK1/2, p38 MAPK, JNK and PI3K. CONCLUSION: CTAB is involved in the suppression of TGF-ß-mediated mesenchymal phenotype and could be a potent medical agent for use in controlling the migration and invasion of hepatic adenocarcinoma.

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death.

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0-15 µg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.

Cryptotanshinone (Dsh-003) from Salvia miltiorrhiza Bunge inhibits prostaglandin E2-induced survival and invasion effects in HA22T hepatocellular carcinoma cells.

Human hepatocellular carcinoma (HCC) is currently the second most common cancer and one of the leading causes of cancer-related mortality in Taiwan. Previous reports show that the expression of (E-type prostaglandin 2) EP2 and (E-type prostaglandin 4) EP4 are elevated in HCC and further demonstrate that Prostaglandin E2 (PGE2) induces HA22T cell proliferation and metastasis through EP2 and EP4 receptor. Danshen (root of Salvia miltiorrhiza Bunge) is a very important and popular traditional Chinese herbal medicine which is widely and successfully used against breast cancer, leukemia, pancreatic cancer, and head and neck squamous carcinoma cells. In this study, we used Cryptotansinone (Dsh-003) (MW 269.14) from Danshen to investigate their effect and corresponding mechanism of action in PGE2-treated HA22T cells. Dsh-003 inhibited HA22T cell viability and further induced cell apoptosis in PGE2-treated HA22T cells. Furthermore, Dsh-003 inhibited EP2, EP4, and their downstream effector such as p-PI3K and p-Akt expression in HA22T hepatocellular carcinoma cells. We also observed that Dsh-003 blocked PGE2-induced cell migration by down-regulating PGE2-induced ß-catenin expression and by up-regulating E-cadherin and GSK3-ß expression. All these findings suggest that Dsh-003 inhibit human HCC cell lines and could potentially be used as a novel drug for HCC treatment.

Isochlorogenic Acid C Reverses Epithelial-Mesenchymal Transition via Down-regulation of EGFR Pathway in MDA-MB-231 cells.

BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) has been suggested to play an important role in survival, proliferation, migration, differentiation, and tumorigenesis of many cell types. Breast cancer patients with high EGFR expression have a poor prognosis. In this study, we investigated the molecular mechanism of the inhibitory effect of isochlorogenic acid c (ICAC) extracted from Lonicera japonica on elevated EGFR levels of the triple-negative breast cancer (TNBC) cell line, MDA-MB-231. MATERIALS AND METHODS: The cell viability and cell-cycle analysis were evaluated using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. The migration ability and invasiveness of ICAC-treated MDA-MB-231 were examined by migration and Matrigel invasion assay. The epithelial-mesenchymal-transition (EMT)-related protein expression was examined by western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: ICAC led to significant morphological changes and suppressed migration and invasion capacities of highly metastatic MDA-MB-231 cells. Western blot analysis for EGFR/EMT-associated proteins suggested that ICAC attenuated the mesenchymal traits as observed by up-regulation of epithelial markers and down-regulation of mesenchymal markers as well as decreased activities of matrix metalloproteinase-9 (MMP-9). CONCLUSION: These results suggested that the inhibitory effects of ICAC against EGFR-induced EMT and MDA-MB-231 cell invasion were dependent on the EGFR/ phospholipase Cγ (PLCγ)/extracellular regulated protein kinase ½ (ERK½)/slug signaling pathway. Therefore, the obtained results could provide us clues for the next therapeutic strategy in the treatment of TNBC.

Antitumorigenic Effects of ZAKß, an Alternative Splicing Isoform of ZAK.

Sterile alpha motif (SAM)- and leucine-zipper-containing kinase (ZAK) plays a role in the regulation of cell cycle progression and oncogenic transformation. The ZAK gene generates two transcript variants, ZAKα and ZAKß, through alternative splicing. In this study, we identified that ZAKα proteins were upregulated in tumor tissues, whereas ZAKß proteins were mostly expressed in corresponding normal tissues. The ectopically expressed ZAKß proteins in cancer cells inhibited cancer cell proliferation as well as anchorage-independent growth. The ZAKß:ZAKα protein ratio played a role in the regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway, whereas high ZAKß protein levels led to the activation of cAMP response element binding protein 1 (CREB1) and exerted antitumor properties. Overexpression of ZAKß or CREB1 cDNAs in cancer cells inhibited anchorage-independent growth and also reduced the levels of cyclooxygenase 2 (Cox2) and ß-catenin proteins. Cancer cells treated with doxorubicin (Doxo) resulted in the switching from the expression of ZAKα to ZAKß and also inhibited cancer cell growth in soft agar, demonstrating that pharmacological drugs could be used to manipulate endogenous reprogramming splicing events and resulting in the activation of endogenous antitumorigenic properties. We showed that the two ZAK transcript variants, ZAKα and ZAKß, had opposite biological functions in the regulation of tumor cell proliferation in that ZAKß had powerful antitumor properties and that ZAKα could promote tumor growth.

MZF-1/Elk-1/PKCα is Associated with Poor Prognosis in Patients with Hepatocellular Carcinoma.

Background: Protein kinase C alpha (PKCα) is a key signaling molecule in human cancer development. As a therapeutic strategy, targeting PKCα is difficult because the molecule is ubiquitously expressed in non-malignant cells. PKCα is regulated by the cooperative interaction of the transcription factors myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) in human cancer cells. Methods: By conducting tissue array analysis, herein, we determined the protein expression of MZF-1/Elk-1/PKCα in various cancers. Results: The data show that the expression of MZF-1/Elk-1 is correlated with that of PKCα in hepatocellular carcinoma (HCC), but not in bladder and lung cancers. In addition, the PKCα down-regulation by shRNA Elk-1 was only observed in the HCC SK-Hep-1 cells. Blocking the interaction between MZF-1 and Elk-1 through the transfection of their binding domain MZF-160-72 decreased PKCα expression. This step ultimately depressed the epithelial-mesenchymal transition potential of the HCC cells. Conclusion: These findings could be used to develop an alternative therapeutic strategy against patients with the PKCα-derived HCC.

Antitumor properties of Coenzyme Q0 against human ovarian carcinoma cells via induction of ROS-mediated apoptosis and cytoprotective autophagy.

Coenzyme Q0 (CoQ0, 2,3-dimethoxy-5-methyl-1,4-benzoquinone) has been reported to exert anticancer properties against human breast/lung cancer cells. This study investigated the in vitro and in vivo anticancer properties of CoQ0 on human ovarian carcinoma (SKOV-3) cells and xenografted nude mice, and revealed the underlying molecular mechanism. CoQ0 induced G2/M arrest through downregulation of cyclin B1/A and CDK1/K2 expressions. CoQ0-induced autophagy as a survival mechanism was evidenced by increased accumulation of LC3-II, GFP-LC3 puncta, AVOs formation and Beclin-1/Bcl-2 dysregulation. Increased TUNEL-positive cells and Annexin-V/PI stained cells indicated CoQ0-induced late apoptosis. Both mitochondrial (caspase-3, PARP and Bax/Bcl-2 dysregulation) and ER stress (caspase-12 and Hsp70) signals are involved in execution of apoptosis. Interestingly, CoQ0-induced apoptosis/autophagy is associated with suppression of HER-2/neu and PI3K/AKT signalling cascades. CoQ0 triggered intracellular ROS production, whereas antioxidant N-acetylcysteine prevented CoQ0-induced apoptosis, but not autophagy. Inhibition of apoptosis by Z-VAD-FMK suppressed CoQ0-induced autophagy (diminished LC3-II/AVOs), indicates CoQ0-induced apoptosis led to evoke autophagy. Contrary, inhibition of autophagy by 3-MA/CQ potentiated CoQ0-induced apoptosis (increased DNA fragmentation/PARP cleavage). Furthermore, CoQ0 treatment to SKOV-3 xenografted nude mice reduced tumor incidence and burden. Histopathological analyses confirmed that CoQ0 modulated xenografted tumor progression by apoptosis induction. Our findings emphasize that CoQ0 triggered ROS-mediated apoptosis and cytoprotective autophagy.

Attenuation of Oxidative Stress-Induced Cell Apoptosis in Schwann RSC96 Cells by Ocimum Gratissimum Aqueous Extract.

Objectives: Cell transplantation therapy of Schwann cells (SCs) is a promising therapeutic strategy after spinal cord injury. However, challenges such as oxidative stress hinder satisfactory cell viability and intervention for enhancing SCs survival is critical throughout the transplantation procedures. Ocimum gratissimum, widely used as a folk medicine in many countries, has therapeutic and anti-oxidative properties and may protect SCs survival. Methods: We examined the protective effects of aqueous O. gratissimum extract (OGE) against cell damage caused by H2O2-induced oxidative stress in RSC96 Schwann cells. Results: Our results showed that the RSC96 cells, damaged by H2O2 oxidative stress, decreased their viability up to 32% after treatment with different concentrations of up to 300 µM H2O2, but OGE pretreatment (150 or 200 µg/mL) increased cell viability by approximately 62% or 66%, respectively. Cell cycle analysis indicated a high (43%) sub-G1 cell population in the H2O2-treated RSC96 cells compared with untreated cells (1%); whereas OGE pretreatment (150 and 200 µg/mL) of RSC96 cells significantly reduced the sub-G1 cells (7% and 8%, respectively). Furthermore, Western blot analysis revealed that OGE pretreatment inhibited H2O2-induced apoptotic protein caspase-3 activation and PARP cleavage, as well as it reversed Bax up-regulation and Bcl-2 down-regulation. The amelioration of OGE of cell stress and stress-induced apoptosis was proved by the HSP70 and HSP72 decrease. Conclusion: Our data suggest that OGE may minimize the cytotoxic effects of H2O2-induced SCs apoptosis by modulating the apoptotic pathway and could potentially supplement cell transplantation therapy.

Amelioration of estrogen-deficiency-induced obesity by Ocimum gratissimum.

Objectives: Menopausal transition in women initiates with declining estrogen levels and is followed by significant changes in their physiological characteristics. These changes often lead to medical conditions, such as obesity, which is correlated with chronic low-grade/subclinical inflammation. Ocimum gratissimum L. is a food spice or traditional herb in many countries; the plant is rich in antioxidants, which possess anti-inflammation activities and multitude of other therapeutic functions. Methods: In this study, we evaluated effects of O. gratissimum extract (OGE) in preventing obesity by using ovariectomized (OVX) animal models to mimic menopausal women. Methods: OVX rats showed increase in body weight and in adipocyte size in perigonadal adipose tissue (p <0.05) and decrease in uterus weight. By contrast, OGE (0.2 mg/ml) significantly reduced body weight gain and adipocyte in OVX rats and showed insignificant changes in uterus weight. Further investigation indicated that OGE exerted no influence on levels of dorsal fat, serum total cholesterol, and serum triacylglycerol and on serum biochemical factors, calcium, phosphorus, and glucose. Conclusion: These findings suggested that OGE dietary supplements may be useful in controlling body weight of menopausal women.

Inhibition of ROS production, autophagy or apoptosis signaling reversed the anticancer properties of Antrodia salmonea in triple-negative breast cancer (MDA-MB-231) cells.

We investigated the in vitro and in vivo anticancer properties of Antrodia salmonea (AS), a well-known edible/medicinal mushroom in Taiwan, on human triple-negative breast cancer (MDA-MB-231) cells and xenografted nude mice; and revealed the underlying molecular mechanisms involved in autophagic- and apoptotic-cell death. Treatment of MDA-MB-231 cells with fermented culture broth of AS (0-200 µg/mL) inhibited cell viability/growth. AS-induced autophagy was evidenced via increased LC3-II accumulation, GFP-LC3 puncta and AVOs formation in MDA-MB-231 cells. These events are associated with increased ATG7, decreased p-mTOR, vanished SQSTM1/p62 expressions and dysregulated Beclin-1/Bcl-2 ratio. AS-induced apoptosis/necrosis through increased DNA fragmentation, Annexin-V/PI stained cells and Bax expression. Both mitochondrial (caspase-9/caspase-3/PARP) and death-receptor (caspase-8/FasL/Fas) signaling pathways are involved in execution of apoptosis. Interestingly, blockade of AS-induced ROS production by N-acetylcysteine pretreatment substantially attenuated AS-induced autophagy, mitochondrial dysfunction and autophagic/apoptotic-cell death. Inhibition of apoptosis by Z-VAD-FMK suppressed AS-induced autophagic-death (decreased LC3-II/AVOs). Similarly, inhibition of autophagy by 3-methyladenine/chloroquine diminished AS-induced apoptosis (decreased DNA fragmentation/caspase-3) in MDA-MB-231 cells. Bioluminescence imaging further confirmed that AS inhibited breast tumor growth in living MDA-MB-231-luciferase-injected nude mice. Taken together, AS crucially involved in execution/propagation of autophagic- or apoptotic-death of MDA-MB-231 cells, and decreased tumor growth in xenografted nude mice.

Antrodia salmonea induces G2 cell-cycle arrest in human triple-negative breast cancer (MDA-MB-231) cells and suppresses tumor growth in athymic nude mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Antrodia salmonea (AS), is a well-known folk medicinal mushroom in Taiwan, has been reported to exhibit anti-oxidant, anti-angiogenic, and anti-inflammatory effects. MATERIALS AND METHODS: In the present study, we examined the effects of AS on cell-cycle arrest in vitro in MDA-MB-231 cells and on tumor regression in vivo using an athymic nude mice model. RESULTS: AS (0-200µg/mL) treatment significantly induced G2 cell-cycle arrest in MDA-MB-231 cells by reducing the levels of cyclin B1, cyclin A, cyclin E, and CDC2 proteins. In addition, N-acetylcysteine (NAC) pretreatment prevented AS induced G2 cell-cycle arrest, indicating that ROS accumulation and subsequent cell cycle arrest might be a major mechanism of AS-induced cytotoxicity. Further, AS treatment decreased COX-2 expression and induced PARP cleavage was significantly reversed by NAC pretreatment in MDA-MB-231 cells. The in vivo study results revealed that AS treatment was effective in terms of delaying the tumor incidence and reducing the tumor growth in MDA-MB-231-xenografted nude mice. TUNEL assay, immunohistochemical staining and Western blotting confirmed that AS significantly modulated the xenografted tumor progression as demonstrated by induction of apoptosis, autophagy, and cell-cycle arrest. CONCLUSION: Our data strongly suggest that Antrodia salmonea could be an anti-cancer agent for human breast cancer.

Coenzyme Q0 Enhances Ultraviolet B-Induced Apoptosis in Human Estrogen Receptor-Positive Breast (MCF-7) Cancer Cells.

Coenzyme Q0 (CoQ0; 2,3-dimethoxy-5-methyl-1,4-benzoquinone), a major active constituent of Antrodia camphorata, has been shown to inhibit human triple-negative breast cancer (MDA-MB-231) cells through induction of apoptosis and cell-cycle arrest. Ecological studies have suggested a possible association between ultraviolet B (UVB) radiation and reduction in the risk of breast cancer. However, the underlying mechanism of the combination of CoQ0 and UVB in human estrogen receptor-positive breast cancer (MCF-7) remains unclear. In this study, the possible effect of CoQ0 on inducing apoptosis in MCF-7 cells under exposure to low-dose UVB (0.05 J/cm2) has been investigated. CoQ0 treatment (0-35 µM, for 24-72 hours) inhibits moderately the growth of breast cancer MCF-7 cells, and the cell viability was significantly decreased when the cells were pretreated with UVB irradiation. It was noted that there was a remarkable accumulation of subploid cells, the so-called sub-G1 peak, in CoQ0-treated cells by using flow cytometric analysis, which suggests that the viability reduction observed after treatment may result from apoptosis induction in MCF-7 cells. CoQ0 caused an elevation of reactive oxygen species, as indicated by dichlorofluorescein fluorescence, and UVB pretreatment significantly increased CoQ0-induced reactive oxygen species generation in MCF-7 cells. In addition, cells were exposed to CoQ0, and the induction of DNA damage was evaluated by single-cell gel electrophoresis (comet assay). CoQ0-induced DNA damage was remarkably enhanced by UVB pretreatment. Furthermore, CoQ0 induced apoptosis in MCF-7 cells, which was associated with PARP degradation, Bcl-2/Bax dysregulation, and p53 expression as shown by western blot. Collectively, these findings suggest that CoQ0 might be an important supplemental agent for treating patients with breast cancer.