CoQ0-induced mitochondrial PTP opening triggers apoptosis via ROS-mediated VDAC1 upregulation in HL-60 leukemia cells and suppresses tumor growth in athymic nude mice/xenografted nude mice.

Coenzyme Q (CoQ) analogs with variable numbers of isoprenoid units have been demonstrated as anticancer and antioxidant/pro-oxidant molecules. This study examined the in vitro and in vivo antitumor and apoptosis activities of CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero isoprenoid side-chains) through upregulation of the Voltage-dependent anion channel 1 (VDAC1) signaling pathway on human promyelocytic leukemia. CoQ0 (0-40 µg/mL) treatment significantly reduced HL-60 cell viability, and up-regulated mitochondrial VDAC1 expression. CoQ0 treatment triggers intracellular ROS generation, calcium release, ΔΨm collapse and PTP opening in HL-60 cells. CoQ0 treatment induced apoptosis, which was associated with DNA fragmentation, cytochrome c release, caspase-3 and PARP activation, and Bax/Bcl-2 dysregulation. Annexin V-PI staining indicated that CoQ0 promotes late apoptosis. Furthermore, the blockade of CoQ0-induced ROS production by antioxidant NAC pretreatment substantially attenuated CoQ0-induced apoptosis. The activation of p-GSK3ß expression, cyclophilin D inhibition, and p53 activation through ROS are involved in CoQ0-induced HL-60 apoptotic cell death. Notably, ROS-independent p38 activation is involved in CoQ0-mediated apoptosis in HL-60 cells. In addition, the silencing of VDAC1 also prevented CoQ0-induced mitochondrial translocation of Bax, activation of caspase-3, and reduction in Bcl-2. Intriguingly, VDAC1 silencing did not prevent ROS production induced by CoQ0, which in turn indicates that CoQ0 induced ROS-mediated VDAC1 and then mitochondrial apoptosis in HL-60 cells. In vivo results revealed that CoQ0 is effective in delaying tumor incidence and reducing the tumor burden in HL-60-xenografted nude mice. Taken together, CoQ0 could be a promising anticancer agent for the treatment of human promyelocytic leukemia through upregulation of VDAC1 signaling pathways.

The inhibition of oleic acid induced hepatic lipogenesis and the promotion of lipolysis by caffeic acid via up-regulation of AMP-activated kinase.

BACKGROUND: Caffeic acid (CA) can inhibit toxin-induced liver injury. In this study, CA is assessed for its lipid lowering potential when oleic acid is used to induce non-alcoholic fatty liver disease in human HepG2 cells. RESULTS: The results showed that both the triglyceride and cholesterol content are decreased in the HepG2 cells by using the enzymatic colorimetric method. CA enhances the phosphorylation of AMP-activated protein kinase (AMPK) and its primary downstream targeting enzyme, acetyl-CoA carboxylase. CA down-regulates the lipogenesis gene expression of sterol regulatory element-binding protein-1 and its target genes, fatty acid synthase in the presence of oleic acid. In addition, CA significantly decreases cholesterol and triglyceride production via inhibition the expression of both 3-hydroxy-3-methyglutary coenzyme A reductase and glycerol-3-phosphate acyltransferase. These effects are eliminated by pretreatment with compound C, an AMPK inhibitor. CONCLUSIONS: These results demonstrate that CA inhibits oleic acid induced hepatic lipogenesis and the promotion of lipolysis via up-regulation of AMP-activated kinase.

Improvement of lipopolysaccharide-induced hepatic injuries and inflammation with mulberry extracts.

BACKGROUND: Mulberry water extracts (MWEs), which contain polyphenols including anthocyanins, have been used in traditional Chinese edible food. The hepatoprotective effects and molecular mechanisms of MWEs on acute liver failure induced by lipopolysaccharides (LPS) were investigated in vivo. RESULTS: Rats were administered different doses of MWEs (0.5 and 1 g kg(-1)) 1 h before injection of LPS (5 mg kg(-1)) and then sacrificed 10 h after treatment with LPS. Liver function, inflammatory factors, oxidative stress index and hepatic histopathological alteration were examined in the rats with and without MWE treatment. Pretreatment with MWEs prevented LPS-induced liver damage by preventing associated increases of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALKP), triglycerol (TG), cholesterol and low-density lipoprotein/high-density lipoprotein ratio. MWEs also suppressed oxidative stress to prevent the formation of hepatic malondialdehyde (MDA). Furthermore, the molecular mechanism involved in LPS-induced liver injury was associated with reducing the expression of COX-2, NF-κB and iNOS in liver tissues. CONCLUSION: The results support the investigation of MWEs as a therapeutic candidate for liver injuries and indicate that MWEs exhibit hepatoprotective activities via NF-κB signaling.

Afatinib triggers a Ni2+ -resistant Ca2+ influx pathway in A549 non-small cell lung cancer cells.

Afatinib is used to treat non-small cell lung cancer cells (NSCLC), and its mechanism involves irreversible inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. In this study, we examined if afatinib had cytotoxic action against NSCLC other than inhibition of tyrosine kinase. Afatinib (1-30 µM) caused apoptotic death in A549 NSCLC in a concentration-dependent manner. Afatinib triggered Ca2+ influx without causing Ca2+ release, and the Ca2+ influx was unaffected by sodium orthovanadate (SOV, an inhibitor of tyrosine phosphatase), suggesting that afatinib-triggered Ca2+ response was unrelated to its inhibition of tyrosine kinase. Addition of afatinib also promoted Mn2+ influx. Ca2+ influx triggered by afatinib was resistant to SKF96365 and ruthenium red (two general blockers of TRP channels) and, unexpectedly, Ni2+ (a non-specific Ca2+ channel blocker). Afatinib caused an increase in mitochondrial Ca2+ level, an initial mitochondrial hyperpolarization (4 h) and followed by mitochondrial potential collapse (24-48 h). Afatinib-induced cell death was slightly but significantly alleviated in low extracellular Ca2+ condition or under pharmacological block of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A. Therefore, in addition to tyrosine kinase inhibition as a major anti-cancer mechanism of afatinib, stimulation of an atypical Ca2+ influx pathway, mitochondrial Ca2+ overload, and potential collapse in part contribute to afatinib-induced cell death.

Aqueous Extract of Shi-Liu-Wei-Liu-Qi-Yin Induces G2/M Phase Arrest and Apoptosis in Human Bladder Carcinoma Cells via Fas and Mitochondrial Pathway.

Shi-Liu-Wei-Liu-Qi-Yin (SLWLQY) was traditionally used to treat cancers. However, scientific evidence of the anticancer effects still remains undefined. In this study, we aimed to clarify the possible mechanisms of SLWLQY in treating cancer. We evaluated the effects of SLWLQY on apoptosis-related experiments inducing in TSGH-8301 cells by (i) 3-(4,5-dimethylthiazol-zyl)-2,5-diphenylterazolium bromide (MTT) for cytotoxicity; (ii) cell-cycle analysis and (iii) western blot analysis of the G2/M-phase and apoptosis regulatory proteins. Human bladder carcinoma TSGH-8301 cells were transplanted into BALB/c nude mice as a tumor model for evaluating the antitumor effect of SLWLQY. Treatment of SLWLQY resulted in the G2/M phase arrest and apoptotic death in a dose-dependent manner, accompanied by a decrease in cyclin-dependent kinases (cdc2) and cyclins (cyclin B1). SLWLQY stimulated increases in the protein expression of Fas and FasL, and induced the cleavage of caspase-3, caspase-9 and caspase-8. The ratio of Bax/Bcl(2) was increased by SLWLQY treatment. SLWLQY markedly reduced tumor size in TSGH-8301 cells-xenografted tumor tissues. In the tissue specimen, SLWLQY up-regulated the expression of Fas, FasL and Bax proteins, and down-regulated Bcl(2) as well as in in vitro assay. Our results showed that SLWLQY reduced tumor growth, caused cell-cycle arrest and apoptosis in TSGH-8301 cells via the Fas and mitochondrial pathway.

Mulberry extract inhibits oleic acid-induced lipid accumulation via reduction of lipogenesis and promotion of hepatic lipid clearance.

BACKGROUND: Mulberries are a traditional edible food used to treat hepatic disease. The anti-obesity and hypolipidemic effects of mulberry water extracts (MWE) have attracted increasing interest. In the present study, MWE were assessed for their hepatic lipid-lowering potential when administered in fatty acid overload conditions in HepG2 cells. RESULTS: We found that MWE significantly reduced lipid accumulation, suppressed fatty acid synthesis, and stimulated fatty acid oxidation. Furthermore, MWE also inhibited acetyl coenzyme A carboxylase activities by stimulating adenosine monophosphate-activated protein kinase (AMPK). MWE attenuated the expression of sterol regulatory element-binding protein-1 (SREBP-1) and its target molecules, such as fatty acid synthase. Similar results were also measured in the expressions of enzymes involved in triglyceride and cholesterol biosyntheses including glycerol-3-phosphate acyltransferase, 3-hydroxy-3-methylglutaryl-CoA reductase, and SREBP-2. In contrast, the lipolytic enzyme expression of peroxisome proliferator activated receptor α and carnitine palmitoyltransferase-1 were increased. CONCLUSIONS: Our study suggests that the hypolipidemic effects of MWE occur via phosphorylation of AMPK and inhibition of lipid biosynthesis. Therefore, the mulberry extract may be active in the prevention of fatty liver.

EP4 upregulation of Ras signaling and feedback regulation of Ras in human colon tissues and cancer cells.

Previous studies indicate that COX-2 and prostaglandin E(2) (PGE(2)) receptor subtypes are involved in intestinal carcinogenesis and activation of downstream pathways. In this report, we try to understand the association of PGE(2) receptor and K-ras cellular mechanism during the development of colorectal cancer. We collected 21 colorectal cancer patients and compared the protein expression of tumor tissues and normal mucosa tissues by using immunoblot. Furthermore, we transferred empty vector and pcDNA-K-ras into Ras-HT29 colon cancer cells. Result showed that phosphorylation of Akt and EP(1)/EP(4) were over-expressed in the colorectal tumor tissue. K-ras induces HT29 cells proliferation through the expressions of COX-2, EP1/EP4, pAkt, GSK3beta and increases Tcf transcriptional factor activation. Additionally, Ras protein was suppressed when treated with EP(4) inhibitor in Ras-HT29 cell. In cell cycle assay, K-ras mutation causing cell cycle S phase was prolonged with an increase in the G2/M phase ratio. In conclusion, we suggested that Ras overexpression leads to cell proliferation through activating Ras/PI3K/GSK3beta/EP(4) PGE(2) receptor signals and caused a feedback regulation of Ras by EP4 in colorectal tumor progression.

Nelumbo nucifera Gaertn leaves extract inhibits the angiogenesis and metastasis of breast cancer cells by downregulation connective tissue growth factor (CTGF) mediated PI3K/AKT/ERK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Nelumbo nucifera Gaertn (Nymphaeaceae) has been recognized as a medicinal plant, which was distributed throughout the Asia. The aqueous extract of Nelumbo nucifera leaves extract (NLE) has various biologically active components such as polyphenols, flavonoids, oligomeric procyanidines. However, the role of NLE in breast cancer therapy is poorly understood. THE AIM OF THIS STUDY: The purpose of this study was to identify the hypothesis that NLE can suppress tumor angiogenesis and metastasis through CTGF (connective tissue growth factor), which has been implicated in tumor angiogenesis and progression in breast cancer MDA-MB-231 cells. RESULTS: We examined the effects of NLE on angiogenesis in the chicken chorioallantoic membrane (CAM) model. The data showed that NLE could reduce the chorionic plexus at day 17 in CAM and the duration of this inhibition was dose-dependent. In Xenograft model, NLE treatment significantly reduced tumor weight and CD31 (capillary density) over control, respectively. We examined the role of angiogenesis involved restructuring of endothelium using human umbilical vein endothelial cell (HUVEC) in Matrigel angiogenesis model. The results indicated that vascular-like structure formation was further blocked by NLE treatment. Moreover, knockdown of CTGF expression markedly reduced the expression of MMP2 as well as VEGF, and attenuated PI3K-AKT-ERK activation, indication that these signaling pathways are crucial in mediating CTGF function. CONCLUSION: The present results suggest that NLE might be useful for treatment in therapy-resistance triple negative breast cancer.

Protocatechuic acid inhibits oleic acid-induced vascular smooth muscle cell proliferation through activation of AMP-activated protein kinase and cell cycle arrest in G0/G1 phase.

Protocatechuic acid (PCA) has been implicated in the progression of atherosclerosis. The proliferation of vascular smooth muscle cells (VSMC) may play a crucial role in the pathogenesis of atherosclerosis. Adenosine 5'-monophosphate-activated protein kinase (AMPK) additionally exerts several beneficial effects on vascular function and improves vascular abnormalities. The current study sought to determine whether PCA has an inhibitory effect on VSMC proliferation under oleic acid (OA) treatment. A7r5 cells were treated with OA (150 µM) or cotreated with OA and PCA (150 µg/mL) for 24 and 48 h. PCA-treated cells were found to cause an increase in G0/G1 cell cycle arrest. Western blotting showed that PCA increased the expressions of p53 and p21Cip1, subsequently decreasing the expression of cyclin E1 and Cdk2. In addition, PCA induced phosphorylation of AMPK and inhibited the expression of fatty acid synthase, Akt-p, and Skp2 after stimulation with OA. After treatment with AMPK inhibitor, the effects of PCA mentioned above were reversed. Taken together, PCA inhibited OA-induced VSMC proliferation through AMPK activation and down-regulation of FAS and AKT signals, which then blocks G0/G1 phase cell cycle progression. These findings provide a new insight into the protective properties of PCA on VSMC, which may constitute a novel effective antiatherosclerosis agent.

The polyphenol extract from Sechium edule shoots inhibits lipogenesis and stimulates lipolysis via activation of AMPK signals in HepG2 cells.

Fatty liver may have implications for metabolic syndrome, such as obesity, hypertension, and diabetes. Therefore, the development of pharmacological or natural agents to reduce fat accumulation in the liver is an important effort. The Sechium edule shoots have already been verified to decrease serum lipids and cholesterol and prevent atherosclerosis. However, how Sechium edule shoots modulate hepatic lipid metabolism is unclear. This study was designed to investigate the effects and mechanisms of polyphenol extracts (SPE) of Sechium edule shoots in reducing lipid accumulation in oleic acid-treated HepG2 cells. We found that water extracts (SWE) of Sechium edule shoots could decrease serum and hepatic lipid contents (e.g., triacylglycerol and cholesterol). Furthermore, SWE and SPE through the AMPK (AMP-activating protein kinase) signaling pathway could decrease lipogenic relative enzymes, such as FAS (fatty acid synthase), HMGCoR (HMG-CoA reductase), and SREBPs (sterol regulatory element binding proteins), and increase the expression of CPT-I (carnitine palmitoyltransferase I) and PPARα (peroxisome proliferators activated receptor α), which are critical regulators of hepatic lipid metabolism. These observations suggested that Sechium edule shoots have potential for developing health foods for preventing and remedying fatty liver.