Estrogen Receptor Is Required for Metformin-Induced Apoptosis in Breast Cancer Cells Under Hyperglycemic Conditions.

Backgrounds: About 25% to 30% of estrogen receptor (ER)-positive breast cancer patients develop resistance to endocrine therapy. Human epidermal growth factor receptor 2 (HER2) has been shown to cooperate with several growth factors that regulate cellular energy metabolism, including the insulin-like growth factor 1 receptor (IGF-1R). Objective: As the first-line therapy for type 2 diabetes mellitus (T2DM) patients, metformin is widely known to inhibit the metabolic reprogramming of cancer cells. This study aims to investigate metformin's efficacy in inhibiting endocrine resistance related to genes regulating energy metabolism in both ER-positive and ER-negative breast cancer cell lines under hyperglycemic conditions. Design and methods: MDA-MB-361 (ER-positive, HER2-positive) and SKBR3 (ER-negative, HER2-positive) cancer cell lines were used to represent ER status. Cell viability and cell survival rate were measured using the colorimetric assay of Cell Counting Kit-8. All mRNA levels were quantified using real-time quantitative polymerase chain reaction preceded by reverse transcription. A P value of <.05 was considered statistically significant. Results: Unlike MDA-MB-361, SKBR3 were found to acquire resistance upon metformin treatment in hyperglycemic conditions. Moreover, the mRNA expression of IGF-1R and its downstream signaling, such as the mammalian target of rapamycin (mTOR), was not affected by metformin. Meanwhile, the mRNA expression level of ribosomal S6 kinase 1 (S6K1) was upregulated, whereas forkhead box O1 (FOXO1) was downregulated after metformin treatment in hyperglycemic conditions. Conclusions: This preliminary study suggests that an alternative pathway of metformin resistance may exist in the absence of ERα. Therefore, relying solely on metformin may be inadequate to inhibit the aggressiveness of breast cancer cells.

Navigating metformin's impact on breast cancer: insights into resistance, alternative pathways, and the crucial role of estrogen receptor under high-glucose conditions Around 25% to 30% of breast cancer patients with estrogen receptor (ER)-positive tumors become resistant to hormone therapy. This study explores whether metformin, a drug commonly used for type 2 diabetes, can counteract this resistance by affecting genes linked to energy metabolism. The research focused on both ER-positive (MDA-MB-361) and ER-negative (SKBR3) breast cancer cell lines under high-glucose conditions. Results showed that although metformin inhibited the growth of ER-positive cells, it surprisingly promoted resistance in ER-negative cells. The expression of insulin-like growth factor 1 receptor (IGF-1R) and its downstream signals like mammalian target of rapamycin (mTOR) remained unaffected by metformin. However, metformin did alter the expression of other genes related to energy metabolism, suggesting that a different resistance pathway might exist in ER-negative cases. In conclusion, this early study implies that relying solely on metformin might not be sufficient to combat the aggressiveness of breast cancer cells, particularly in cases lacking ERα. More research is needed to understand alternative pathways and develop more effective strategies against resistance.

Role of urinary H2O2, 8-iso-PGF2α, and serum oxLDL/ß2GP1 complex in the diabetic kidney disease.

Oxidant species is reported as a major determinant in the pathophysiology of diabetic kidney disease. However, reactive oxygen species (ROS) formation in the initial phase and progressing phase of diabetic kidney disease remains unclear. Therefore, we conducted this study to find out what ROS and their modified product are associated with eGFR in type 2 diabetes mellitus (T2DM) patients. A cross-sectional study was performed on 227 T2DM patients. The study subjects were divided into three groups based on their eGFR stage (Group 1, eGFR > 89 ml/min/1.73 m2; Group 2, eGFR = 60-89 ml/min/1.73 m2; and Group 3, eGFR < 60 ml/min/1.73 m2). Enzyme-linked immunosorbent assay (ELISA) was used to measure serum oxLDL/ß2GPI complex and urinary 8-iso-PGF2α, while ferrous ion oxidation xylenol orange method 1 (FOX-1) was used to measure urinary hydrogen peroxide (H2O2). H2O2 significantly decreased across the groups, whereas OxLDL/ß2GPI complex increased, but not significant, and there was no trend for 8-iso-PGF2α. Consistently, in the total study population, only H2O2 showed correlation with eGFR (r = 0.161, p = 0.015). Multiple linear regression analysis showed that significant factors for increased eGFR were H2O2, diastolic blood pressure, and female. Whereas increased systolic blood pressure and age were significant factors affecting the decrease of eGFR. We also found that urinary H2O2 had correlation with serum oxLDL/ß2GPI complex in total population. This finding could lead to further research on urinary H2O2 for early detection and research on novel therapies of diabetic kidney disease.

Antioxidative attributes of rice bran extracts in ameliorative effects of atherosclerosis-associated risk factors.

Oxidative stress, chronic inflammation, dyslipidemia, hyperglycemia, and shear stress (physical effect) are risk factors associated with the pathogenesis of atherosclerosis. Rice bran, a by-product of rice milling process, is known to house polyphenols and vitamins which exhibit potent antioxidant and anti-inflammatory properties. Through recent emerging knowledge of rice bran in health and wellness, the present study was aimed to assess the ameliorative effects of rice bran extracts (RBE) derived from Japanese colored rice varieties in modulating risk factors of atherosclerosis via in vitro and in vivo study models. Pre-treatment of lipopolysaccharide (LPS)-stimulated murine J774A.1 macrophage-like cells with RBE alleviated nitric oxide (NO) overproduction and downregulated gene expressions of pro-inflammatory modulators: tumor necrosis factor-α (TNF-α), interleukin (IL)-α (IL-1α), IL-1ß, IL-6, and inducible nitric oxide synthase (iNOS). In addition, RBE also significantly attenuated LPS-stimulated protein expressions of iNOS, TNF-α, IL-1α, and IL-6 in J774A.1 macrophage-like cells as compared to non-treated LPS control group. In in vivo, 12 weeks of RBE dietary supplementations significantly reduced (p < 0.05) total cholesterol, triglycerides, and pro-atherogenic oxidized LDL/ß2-glycoprotein I (oxLDL/ß2GPI) complexes at plasma levels, in high fat diet (HFD) induced low density lipoprotein receptor knockout (Ldlr -/-) mice. En face pathological assessments of murine aortas also revealed significant reductions by 38% (p < 0.05) in plaque sizes of RBE-supplemented HFD mice groups as compared to non RBE-supplemented HFD control mice group. Moreover, gene expressions of aortic (iNOS, TNF-α, IL-1ß) and hepatic (TNF-α, IL-1α, IL-1ß) pro-inflammatory modulators were also downregulated in RBE-supplemented mice groups. Present study has revealed the potent health attributes and application of RBE as a dietary supplement to attenuate risks of inadvertent oxidative damage and chronic inflammation underlying the pathogenesis of atherosclerosis. Intrinsically, present preliminary findings may provide global health prospects for future dietary implementation of RBE in management of atherosclerosis.

Identification and visualization of oxidized lipids in atherosclerotic plaques by microscopic imaging mass spectrometry-based metabolomics.

BACKGROUND AND AIMS: Dysregulated lipid metabolism has emerged as one of the major risk factors of atherosclerosis. Presently, there is a consensus that oxidized LDL (oxLDL) promotes development of atherosclerosis and downstream chronic inflammatory responses. Due to the dynamic metabolic disposition of lipoprotein, conventional approach to purify bioactive lipids for subsequent comprehensive analysis has proven to be inadequate for elucidation of the oxidized lipids species accountable for pathophysiology of atherosclerotic lesions. Herein, we aimed to utilize a novel mass microscopic imaging technology, coupled with mass spectrometry (MS) to characterize oxidized lipids in atherosclerotic lesions. METHODS: We attempted to use MALDI-TOF-MS and iMScope to identify selected oxidized lipid targets and visualize their respective localizations in study models of atherosclerosis. RESULTS: Based on the MS analysis, detection of 7-K under positive ionization through product ion peak at m/z 383 [M + H-H2O] indicated the distinctive presence of targeted lipid within Cu2+-oxLDL and Cu2+-oxLDL loaded macrophage-like J774A.1 cells, along with other cholesterol oxidation products. Moreover, the application of two-dimensional iMScope has successfully visualized the localization of lipids in aortic atherosclerotic plaques of the Watanabe heritable hyperlipidemic (WHHL) rabbit. Distinctive lipid distribution profiles were observed in atherosclerotic lesions of different sizes, especially the localizations of lysoPCs in atherosclerotic plaques. CONCLUSIONS: Taken together, we believe that both MALDI-TOF-MS and iMScope metabolomics technology may offer a novel proposition for future pathophysiological studies of lipid metabolism in atherosclerosis.

Cytotoxic Effects of Alcohol Extracts from a Plastic Wrap (Polyvinylidene Chloride) on Human Cultured Liver Cells and Mouse Primary Cultured Liver Cells.

An increasing accumulation of microplastics and further degraded nanoplastics in our environment is suspected to have harmful effects on humans and animals. To clarify this problem, we tested the cytotoxicity of two types of plastic wrap on human cultured liver cells and mouse primary cultured liver cells. Alcohol extracts from plastic wrap, i.e., polyvinylidene chloride (PVDC), showed cytotoxic effects on the cells. Alcohol extracts of polyethylene (PE) wrap were not toxic. The commercially available PVDC wrap consists of vinylidene chloride, epoxidized soybean oil, epoxidized linseed oil as a stiffener and stabilizer; we sought to identify which component(s) are toxic. The epoxidized soybean oil and epoxidized linseed oil exerted strong cytotoxicity, but the plastic raw material itself, vinylidene chloride, did not. Our findings indicate that plastic wraps should be used with caution in order to prevent health risks.

Rapid and specific detection of oxidized LDL/ß2GPI complexes via facile lateral flow immunoassay.

ß2-Glycoprotein I (ß2GPI) forms indissociable complex with oxidized LDL (oxLDL) into proatherogenic oxLDL/ß2GPI complex through a specific ligand known as 7-ketocholesteryl-9-carboxynonanoate (oxLig-1). Recent discoveries have demonstrated the atherogenicity of these complexes in patients of both systemic and non-systemic autoimmune diseases. Hence, serological level of oxLDL/ß2GPI complexes may represent one crucial clinical parameter for disease prognosis of atherosclerosis-related diseases. Herein, we established a simple, specific and rapid gold nanoparticle (GNP) based lateral flow immunoassay (LFIA) to quantify oxLDL/ß2GPI complexes from test samples. Specificities of hybridoma cell-derived monoclonal antibodies against antigen, optimal conditions for conjugation of antibody with GNP, and sensitivity of oxLDL/ß2GPI LFIA in comparison to an ELISA-based detection method were assessed accordingly. The established oxLDL/ß2GPI LFIA was capable of detecting oxLDL/ß2GPI specifically without interference from autoantibodies and solitary components of oxLDL/ß2GPI present in test samples. A significant correlation (R2 > 0.8) was also obtained with the oxLDL/ß2GPI LFIA when compared to the ELISA-based detection. On the whole, the oxLDL/ß2GPI LFIA remains advantageous over the oxLDL/ß2GPI ELISA. The unnecessary washing step, short developmental and analytical time support facile and rapid detection of oxLDL/ß2GPI as opposed to the laborious ELISA system.

Urinary Bile Acid Profile of Newborns Born by Cesarean Section Is Characterized by Oxidative Metabolism of Primary Bile Acids: Limited Roles of Fetal-Specific CYP3A7 in Cholate Oxidations.

This work aims to investigate how the bile acid metabolism of newborns differs from that of adults along the axis of primary, secondary, and tertiary bile acids (BAs). The total unconjugated BA profiles were quantitatively determined by enzyme digestion techniques in urine of 21 newborns born by cesarean section, 29 healthy parturient women, 30 healthy males, and 28 healthy nonpregnant females. As expected, because of a lack of developed gut microbiota, newborns exhibited poor metabolism of secondary BAs. Accordingly, the tertiary BAs contributed limitedly to the urinary excretion of BAs in newborns despite their tertiary-to-secondary ratios significantly increasing. As a result, the primary BAs of newborns underwent extensive oxidative metabolism, resulting in elevated urinary levels of some fetal-specific BAs, including 3-dehydroCA, 3ß,7α,12α-trihydroxy-5ß-cholan-24-oic acid, 3α,12-oxo-hydroxy-5ß-cholan-24-oic acid, and nine tetrahydroxy-cholan-24-oic acids (Tetra-BAs). Parturient women had significantly elevated urinary levels of tertiary BAs and fetal-specific BAs compared with female control, indicating that they may be excreted into amniotic fluid for maternal disposition. An in vitro metabolism assay in infant liver microsomes showed that four Tetra-BAs and 3-dehydroCA were hydroxylated metabolites of cholate, glycocholate, and particularly taurocholate. However, the recombinant cytochrome P450 enzyme assay found that the fetal-specific CYP3A7 did not contribute to these oxidation metabolisms as much as expected compared with CYP3A4. In conclusion, newborns show a BA metabolism pattern predominated by primary BA oxidations due to immaturity of secondary BA metabolism. Translational studies following this finding may bring new ideas and strategies for both pediatric pharmacology and diagnosis and treatment of perinatal cholestasis-associated diseases. SIGNIFICANCE STATEMENT: The prenatal BA disposition is different from adults because of a lack of gut microbiota. However, how the BA metabolism of newborns differs from that of adults along the axis of primary, secondary, and tertiary BAs remains poorly defined. This work demonstrated that the urinary BA profiles of newborns born by cesarean section are characterized by oxidative metabolism of primary BAs, in which the fetal-specific CYP3A7 plays a limited role in the downstream oxidation metabolism of cholate.

Stereoselective Oxidation Kinetics of Deoxycholate in Recombinant and Microsomal CYP3A Enzymes: Deoxycholate 19-Hydroxylation Is an In Vitro Marker of CYP3A7 Activity.

The primary bile acids (BAs) synthesized from cholesterol in the liver are converted to secondary BAs by gut microbiota. It was recently disclosed that the major secondary BA, deoxycholate (DCA) species, is stereoselectively oxidized to tertiary BAs exclusively by CYP3A enzymes. This work subsequently investigated the in vitro oxidation kinetics of DCA at C-1ß, C-3ß, C-4ß, C-5ß, C-6α, C-6ß, and C-19 in recombinant CYP3A enzymes and naive enzymes in human liver microsomes (HLMs). The stereoselective oxidation of DCA fit well with Hill kinetics at 1-300 µM in both recombinant CYP3A enzymes and pooled HLMs. With no contributions or trace contributions from CYP3A5, CYP3A7 favors oxidation at C-19, C-4ß, C-6α, C-3ß, and C-1ß, whereas CYP3A4 favors the oxidation at C-5ß and C-6ß compared with each other. Correlation between DCA oxidation and testosterone 6ß-hydroxylation in 14 adult single-donor HLMs provided proof-of-concept evidence that DCA 19-hydroxylation is an in vitro marker reaction for CYP3A7 activity, whereas oxidation at other sites represents mixed indicators for CYP3A4 and CYP3A7 activities. Deactivation caused by DCA-induced cytochrome P450-cytochrome P420 conversion, as shown by the spectral titrations of isolated CYP3A proteins, was observed when DCA levels were near or higher than the critical micelle concentration (about 1500 µM). Unlike CYP3A4, CYP3A7 showed abnormally elevated activities at 500 and 750 µM, which might be associated with an altered affinity for DCA multimers. The disclosed kinetic and functional roles of CYP3A isoforms in disposing of the gut bacteria-derived DCA may help in understanding the structural and functional mechanisms of CYP3A.

Corrigendum to "Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes".

[This corrects the article DOI: 10.1155/2016/6943053.].

Mutants of ß2-glycoprotein I: Their features and potent applications.

ß2-Glycoprotein I (ß2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of ß2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K287C288 was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact ß2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18-30% as compared to control. Only DI and nicked ß2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.

Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes.

This study was aimed at preliminarily assessing the cytoprotective and antioxidative effects of rice bran extracts (RBEs) from a Sarawak local rice variety (local name: "BJLN") and a commercial rice variety, "MR219," on oxidative stress in rat H9c2(2-1) cardiomyocytes. The cardiomyocytes were incubated with different concentrations of RBE and hydrogen peroxide (H2O2), respectively, to identify their respective IC50 values and safe dose ranges. Two nonlethal and close-to-IC50 doses of RBE were selected to evaluate their respective effects on H2O2 induced oxidative stress in cardiomyocytes. Both RBEs showed dose-dependent cytotoxicity effects on cardiomyocytes. H2O2 induction of cardiomyocytes pretreated with RBE further revealed the dose-dependent cytoprotective and antioxidative effects of RBE via an increase in IC50 values of H2O2. Preliminary analyses of induction effects of RBE and H2O2 on cellular antioxidant enzyme, catalase (CAT), also revealed their potential in regulating these activities and expression profile of related gene on oxidative stress in cardiomyocytes. Pretreated cardiomyocytes significantly upregulated the enzymatic activity and expression level of CAT under the exposure of H2O2 induced oxidative stress. This preliminary study has demonstrated the potential antioxidant effects of RBE in alleviating H2O2-mediated oxidative injuries via upregulation in enzymatic activities and expression levels of CAT.