Exome Sequencing Reveals Novel Germline Variants in Breast Cancer Patients in the Southernmost Region of Thailand.

Germline carriers of pathogenic variants in cancer susceptibility genes are at an increased risk of breast cancer (BC). We characterized germline variants in a cohort of 151 patients diagnosed with epithelial BC in the southernmost region of Thailand, where the predominant ethnicity differs from that of the rest of the country. Whole exome sequencing was used to identify and subsequently filter variants present in 26 genes known to be associated with cancer predisposition. Of the 151 individuals assessed, 23, corresponding to 15.2% of the sample, exhibited the presence of one or more pathogenic or likely pathogenic variants associated with BC susceptibility. We identified novel germline truncating variants in BRIP1, CHEK2, MSH6, PALB2, and PTEN and annotated variants of uncertain significance (VUSs), both novel and previously documented. Therefore, it is advisable to use genetic testing as an additional risk screening method for BC in this area.

Immunohistochemistry-based molecular subtyping of triple-negative breast cancer and its prognostic significance.

Background: Immunohistochemistry (IHC)-based protein markers representing molecular subtypes are of great value for routine use. This study aimed to evaluate the frequency distributions of the molecular subtypes of triple-negative breast cancer (TNBC) using IHC-based surrogate markers and examined their prognostic value. Methods: Patients with TNBC treated at a university hospital in Southern Thailand were included in this study. Expression levels of androgen receptor, CD8, Forkhead box transcription factor C1, and Doublecortin-like kinase 1 were detected in tumor tissue to classify them into luminal androgen receptor (LAR), immunomodulatory (IM), basal-like immunosuppressed (BLIS), mesenchymal-like (MES), and unclassifiable (UC) subtypes. The association between variables and disease-free survival (DFS) and overall survival (OS) was analyzed using Cox proportional hazards regression. Results: Among the 195 cases of TNBC, the frequency distribution of the IHC-based subtype was as follows: BLIS, 52.8%; LAR, 19.0%; IM, 17.4%; MES, 0.5%; and un-classifiable, 10.3%. BLIS subtype was significantly found in younger ages (mean: 49.6 years) than other subtypes (mean: 51-57.7 years). LAR and BLIS subtypes were significantly associated with poorer OS compared to the IM subtype in univariate analysis, however, only BLIS was significant in multivariate analysis (HR: 3.29, 95% CI: 1.01-10.72). IHC-based subtype was not found to be associated with DFS. Conclusion: This study revealed the differences in the proportion frequency of IHC-based TNBC subtypes in Thai patients compared to other populations. IHC-based molecular subtyping may be beneficial for prognosis. However further refinement of the molecular classification of TNBC is needed for better clinical relevance.

Prevalence of Pathogenic Germline Mutations in 13 Hereditary Cancer-Related Genes in Breast Cancer Patients in Narathiwat Province, Thailand.

BACKGROUND: BRCA1 and BRCA2 genes are known to increase breast cancer's lifetime risk. Early identification of women with this inherited risk can potentially reduce the risk of breast and/or ovarian cancer and, together with early screening, decrease the mortality rate. OBJECTIVE: This study explored the frequency and distribution of genetic variants in consecutive cases of breast cancer in Narathiwat province, one of the three provinces in the southernmost Thai border. MATERIAL & METHOD: A series of 64 consecutive breast cancer patients who underwent treatment in two general hospitals in the province during the period from the year 2021 to 2022. Genotyping studies were performed using a whole exome sequencing platform. Moderate to high penetrance variants recommended by the National Comprehensive Cancer Network (NCCN) guidelines 2022 (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, NF1, PALB2, PTEN, RAD51C, RAD51D, STK11, TP53) were annotated and filtered for pathogenic, likely pathogenic, or high-impact variants. RESULTS: Pathogenic germline variants were found in 8/64 cases (12.5%), namely BRCA1 in 3 (4.7%), BRCA2 in 4 (6.3%), ATM in 1 (1.6%), and PALB2 in 1 (1.6%). One patient had two concomitant germline mutations in BRCA2 and ATM. CONCLUSION: This is the first study on the frequency of germline mutations in BRCA1/2 and other breast cancer-predisposing genes in the southernmost provinces of Thailand. At least one pathogenic germline mutation was identified in 12.5% of the study patients, which suggests that genetic testing in this population has a high potential to provide benefits.

Potential target identification for osteosarcoma treatment: Gene expression re-analysis and drug repurposing.

Survival rate of osteosarcoma has remained plateaued for the past three decades. New treatment is needed to improve survival rate. Drug repurposing, a method to identify new indications of previous drugs, which saves time and cost compared to the de novo drug discovery. Data mining from gene expression profile was carried out and new potential targets were identified by using drug repurposing strategy. Selected data were newly categorized as pathophysiology and metastasis groups. Data were normalized and calculated the differential gene expression. Genes with log fold change ≥ 2 and adjusted p-value ≤ 0.05 were selected as primary candidate genes (PCGs). PCGs were further enriched to determine the secondary candidate genes (SCGs) by protein interaction analysis, upstream transcription factor and related-protein kinase identification. PCGs and SCGs were further matched with gene targeted of corresponding drugs from the Drug Repurposing Hub. A total of 778 targets were identified (360 from PCGs, and 418 from SCGs). This newly identified KLHL13 is a new candidate target based on its molecular function. KLHL13 was upregulated in clinical samples. We found 256 drugs from matching processes (50anti-cancerand206non-anticancerdrugs). Clinical trials of anti-cancer drugs from 5 targets (CDK4, BCL-2, JUN, SRC, PIK3CA) are being performed for osteosarcoma treatment. Niclosamide and synthetic PPARÉ£ ligands are candidates for repurposing due to the possibility based on their mechanism and pharmacology properties. Re-analysis of gene expression profile could identify new potential targets, confirm a current implication, and expand the chance of repurposing drugs for osteosarcoma treatment.

Integrated bioinformatic analysis of potential biomarkers of poor prognosis in triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease associated with late-stage diagnosis and high metastatic rates. However, a gene signature for reliable TNBC biomarkers is not available yet. We aimed to identify potential key genes and their association with poor prognosis in TNBC through integrated bioinformatics. Methods: Microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) in TNBC vs. non-TNBC and TNBC vs. normal tissues were analyzed. Overlapping upregulated and downregulated DEGs were selected as inputs for Gene Ontology and pathway enrichment analyses using Metascape. Then, UALCAN and Kaplan-Meier plotter were employed to analyze the prognostic values of all overlapping DEGs. Results: We identified 21 upregulated and 24 downregulated overlapping DEGs in TNBC vs. non-TNBC and TNBC vs. normal breast tissue. The upregulated overlapping DEGs were mainly enriched in various pathways including chromosome segregation, cell cycle phase transition, and cell division, whereas overlapping DEGs were significantly downregulated in pathways, such as multicellular organismal homeostasis, tissue homeostasis, and negative regulation of cell population proliferation. Key genes were identified by association with poor overall survival (OS). Our results showed that high expression of CENPW and HORMAD1 was associated with poor OS of TNBC patients. Conversely, the low expression of PIP, APOD, and ZNF703 indicated worse OS. Conclusions: We identified key genes (CENPW, HORMAD1, APOD, PIP, and ZNF703) associated with poor OS. Thus, these genes might serve as candidate prognostic markers for TNBC.

Anticancer mechanism of 7-α-hydroxyfrullanolide on microtubules and computational prediction of its target binding in triple-negative breast cancer cells.

Background: Triple-negative breast cancer (TNBC) responds poorly to the available drugs; thus, the mortality rate associated with TNBC remains high. 7-α-Hydroxyfrullanolide (7HF) possesses anticancer properties and arrests cells in the G2/M-phase via modulation of several proteins involved in the G2/M-phase transition, as well as the mitotic checkpoint in MDA-MB-468 (TNBC) cells. Microtubules (MTs) dynamically regulate cell division in the G2/M phase and are related to cancer cell stress response. However, antimitotic drug cytotoxicity to multiple cancer resistance developed in response to drugs are obstacles faced to date. Here, the activity and mechanism via which 7HF controls MTs dynamics was investigated in MDA-MB-468 cells. Methods: 7HF uptake by MDA-MB-468 cells was assessed using spectrophotometry. The drug-like properties of 7HF were predicted using the Swiss-absorption, distribution, metabolism, and excretion (ADME) webtool. Then, the effect of 7HF treatment (6, 12, and 24 µM) on the dynamic arrangement of MTs was assessed for 1, 12, and 24 h using indirect immunofluorescence. Polymerization of α- and ß-tubulin was assessed using different 7HF concentrations in a cell-free system for 1 h. Cell proliferation assay with bromodeoxyuridine plus propidium iodide staining and flow cytometry was performed at different 7HF concentrations and time points. The mechanism of action was assessed by detecting the expression of proteins, including Bub3, cyclin B1, p-Cdk1 (Tyr15), Rb, p-Rb (Ser780), Chk1, p-Chk1 (Ser345), Chk2, p-Chk2 (Ser516), and p-H2AX (Ser139), using western blotting. Molecular docking was used to predict the molecular interactions between 7HF and tubulins in MTs. Results: We observed that 7HF was able to enter the MDA-MB-468 cells. The ADME webtool analysis predicted that it possesses the high passive permeation and gastrointestinal absorption properties of drugs. Various concentrations of 7HF disrupted the dynamic arrangement of spindle MTs by causing radial spindle array shrinkage and expansion of fibrous spindle density and radial array lengths in a time-dependent manner. 7HF reduced polymerization of α-, ß-tubulin in dose-dependent manner. 7HF also triggered DNA damage response by inducing G2/M and G1 phase arrests in a concentration and time-dependent manner, which occurred due to the upregulation of Bub3, Chk1, p-Chk1 (Ser345), p-Cdk1 (Tyr15), and cyclin B1. According to molecular docking analysis, 7HF preferred to bind to ß-tubulin over α-tubulin. The lactone, ketone, and hydroxyl groups of 7HF supported the 7HF-tubulin interactions. Hydrogen bonding with a hydrocarbon ring and salt bridge attractive forces were responsible for the binding versatility of 7HF. Conclusions: This is the first study to investigate the molecular mechanism, MTs interacting sites, and the internalization and drug-like properties of 7HF in TNBC cells. The findings will be useful for developing 7HF-based treatment for patients with TNBC.

Interruptin C, a Radioprotective Agent, Derived from Cyclosorus terminans Protect Normal Breast MCF-10A and Human Keratinocyte HaCaT Cells against Radiation-Induced Damage.

Radiotherapy is a common method to treat cancers, with the goal of maximizing the dose to tumors while minimizing the dose to normal tissues. Radioprotectors can reduce the toxicity to normal tissues during radiotherapy. Several plant-derived compounds can function as radioprotectors by scavenging free radicals. We investigated the radioprotective activity of interruptin C from the fern Cyclosorus terminans. The molecular mechanism of interruptin C's activity in X-ray-irradiated cells was evaluated. Superoxide dismutase activity was examined to investigate the antioxidant enzyme activity. Clonogenic cell survival was also investigated following radiation exposure. DNA damage and cell cycle progression were detected using micronuclei formation assays. DNA repair after irradiation was analyzed in a γH2AX assay. The levels of the proteins related to the radioprotective responses were analyzed by Western blotting. Interruptin C increased the antioxidant enzyme activity and significantly decreased the DNA damage by reducing the γH2AX foci and micronucleus formation in irradiated MCF-10A normal breast and HaCaT human keratinocyte cells. The apoptotic protein levels decreased, whereas the antiapoptotic protein levels increased. Interruptin C pretreatment increased the survival rate of irradiated MCF-10A and HaCaT cells. Moreover, the compound did not promote the survival of MDA-MB-231 and Hs578T breast cancer cells. Therefore, interruptin C may exert radioprotective activity without enhancing cancer cell proliferation.

Anticancer Effects and Molecular Action of 7-α-Hydroxyfrullanolide in G2/M-Phase Arrest and Apoptosis in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 expression. TNBC cells respond poorly to targeted chemotherapies currently in use and the mortality rate of TNBC remains high. Therefore, it is necessary to identify new chemotherapeutic agents for TNBC. In this study, the anti-cancer effects of 7-α-hydroxyfrullanolide (7HF), derived from Grangea maderaspatana, on MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells were assessed using MTT assay. The mode of action of 7HF in TNBC cells treated with 6, 12 and 24 µM of 7HF was determined by flow cytometry and propidium iodide (PI) staining for cell cycle analysis and annexin V/fluorescein isothiocyanate + PI staining for detecting apoptosis. The molecular mechanism of action of 7HF in TNBC cells was investigated by evaluating protein expression using proteomic techniques and western blotting. Subsequently, 7HF exhibited the strongest anti-TNBC activity toward MDA-MB-468 cells and a concomitantly weak toxicity toward normal breast cells. The molecular mechanism of action of low-dose 7HF in TNBC cells primarily involved G2/M-phase arrest through upregulation of the expression of Bub3, cyclin B1, phosphorylated Cdk1 (Tyr 15) and p53-independent p21. Contrastingly, the upregulation of PP2A-A subunit expression may have modulated the suppression of various cell survival proteins such as p-Akt (Ser 473), FoxO3a and ß-catenin. The concurrent apoptotic effect of 7HF on the treated cells was mediated via both intrinsic and extrinsic modes through the upregulation of Bax and active cleaved caspase-7-9 expression and downregulation of Bcl-2 and full-length caspase-7-9 expression. Notably, the proteomic approach revealed the upregulation of the expression of pivotal protein clusters associated with G1/S-phase arrest, G2/M-phase transition and apoptosis. Thus, 7HF exhibits promising anti-TNBC activity and at a low dose, it modulates signal transduction associated with G2/M-phase arrest and apoptosis.

Decreased body-fat accumulation and increased vasorelaxation to glyceryl trinitrate in middle-aged male rats following six-weeks consumption of coconut milk protein

Abstract We investigated whether coconut milk protein (CMP) contributes to the beneficial effects of coconut milk consumption on cardiovascular health markers previously found in middle-aged rats. CMP was isolated and precipitated from dried fresh coconut milk, then gavaged (1 g/kg) to middle-aged male rats for six weeks; control rats received distilled water. Compared to controls, CMP caused decreased body fat and lipid accumulation in liver cells and the platelet count. CMP did not affect basal blood pressure or heart rate in anesthetized rats. Vascular responsiveness to phenylephrine, DL-propargylglycine (PAG), acetylcholine or sodium nitroprusside was unaffected, but vasorelaxation to glyceryl trinitrate (GTN) increased. Effects of ODQ on vasorelaxation to GTN were similar in both groups. Expression of blood vessel eNOS, CSE and sGC was normal. The cyclic guanosine monophosphate (cGMP) level of CMP-treated rats was normal but addition of GTN increased cGMP and NO concentration more in CMP-treated rats than in controls, an effect unaltered by addition of diadzin. Taken together, CMP appears partially responsible for the improvement in cardiovascular health markers caused by coconut milk in middle-aged male rats

Molecular Classification Models for Triple Negative Breast Cancer Subtype Using Machine Learning.

Triple negative breast cancer (TNBC) lacks well-defined molecular targets and is highly heterogenous, making treatment challenging. Using gene expression analysis, TNBC has been classified into four different subtypes: basal-like immune-activated (BLIA), basal-like immune-suppressed (BLIS), mesenchymal (MES), and luminal androgen receptor (LAR). However, there is currently no standardized method for classifying TNBC subtypes. We attempted to define a gene signature for each subtype, and to develop a classification method based on machine learning (ML) for TNBC subtyping. In these experiments, gene expression microarray data for TNBC patients were downloaded from the Gene Expression Omnibus database. Differentially expressed genes unique to 198 known TNBC cases were identified and selected as a training gene set to train in seven different classification models. We produced a training set consisting of 719 DEGs selected from uniquely expressed genes of all four subtypes. The highest average accuracy of classification of the BLIA, BLIS, MES, and LAR subtypes was achieved by the SVM algorithm (accuracy 95-98.8%; AUC 0.99-1.00). For model validation, we used 334 samples of unknown TNBC subtypes, of which 97 (29.04%), 73 (21.86%), 39 (11.68%) and 59 (17.66%) were predicted to be BLIA, BLIS, MES, and LAR, respectively. However, 66 TNBC samples (19.76%) could not be assigned to any subtype. These samples contained only three upregulated genes (EN1, PROM1, and CCL2). Each TNBC subtype had a unique gene expression pattern, which was confirmed by identification of DEGs and pathway analysis. These results indicated that our training gene set was suitable for development of classification models, and that the SVM algorithm could classify TNBC into four unique subtypes. Accurate and consistent classification of the TNBC subtypes is essential for personalized treatment and prognosis of TNBC.

The Anticancer Effects of FDI-6, a FOXM1 Inhibitor, on Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) presents an important clinical challenge, as it does not respond to endocrine therapies or other available targeting agents. FOXM1, an oncogenic transcriptional factor, has reported to be upregulated and associated with poor clinical outcomes in TNBC patients. In this study, we investigated the anti-cancer effects of FDI-6, a FOXM1 inhibitor, as well as its molecular mechanisms, in TNBC cells. Two TNBC cell lines, MDA-MB-231 and HS578T, were used in this study. The anti-cancer activities of FDI-6 were evaluated using various 2D cell culture assays, including Sulforhodamine B (SRB), wound healing, and transwell invasion assays together with 3D spheroid assays, mimicking real tumour structural properties. After treatment with FDI-6, the TNBC cells displayed a significant inhibition in cell proliferation, migration, and invasion. Increased apoptosis was also observed in the treated cells. In addition, we found that FDI-6 lead to the downregulation of FOXM1 and its key oncogenic targets, including CyclinB1, Snail, and Slug. Interestingly, we also found that the FDI-6/Doxorubicin combination significantly enhanced the cytotoxicity and apoptotic properties, suggesting that FDI-6 might improve chemotherapy treatment efficacy and reduce unwanted side effects. Altogether, FDI-6 exhibited promising anti-tumour activities and could be developed as a newly effective treatment for TNBC.

Anti-breast cancer potential of frullanolide from Grangea maderaspatana plant by inducing apoptosis.

Breast cancer is the leading cause of female mortality worldwide. Although there are several modern treatments for breast cancer, there is a high rate of recurrence for the majority of treatments; therefore, the search for effective anticancer agents continues. The present study aimed to investigate the anti-breast cancer potential of frullanolide, a compound which is isolated and purified from the Grangea maderaspatana plant, for selected human breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231). The MTT assay was used to assess cytotoxic activity in breast cancer cell lines of treatment with frullanolide at 1.25, 2.5, 5.0, 10.0 and 20.0 µg/ml. Additionally, the apoptotic induction ability of frullanolide at various concentrations [0.5×, 1× and 2× half maximal inhibitory concentration (IC50)] was investigated by flow cytometry and western blot analysis. Frullanolide exhibited strong anti-breast cancer activity against MDA-MB-468 (IC50, 8.04±2.69 µg/ml) and weak cytotoxicity against the MCF-7 (IC50, 10.74±0.86 µg/ml) and MDA-MB-231 (IC50, 12.36±0.31 µg/ml) cell lines. The IC50 of frullanolide was high in the human normal epithelial breast cell line (MCF-12A) and mouse fibroblast cell line (L-929). Density plot diagrams revealed that frullanolide induced apoptosis in MCF-7, MDA-MB-468 and MDA-MB-231 cells. Notably, a plausible anticancer mechanism was elucidated via cellular apoptosis by p53-independence in the treated MCF-7 cell line and p53-dependence in the treated MDA-MB-468 and MDA-MB-231 cell lines. In conclusion, the present study demonstrated that frullanolide may exert anticancer activity on breast cancer cell lines by inducing apoptosis. Frullanolide offers a possible novel approach to breast cancer therapy.

Anticancer Potential of Fruit Extracts from Vatica diospyroides Symington Type SS and Their Effect on Program Cell Death of Cervical Cancer Cell Lines.

Vatica diospyroides Symington is locally known as Chan-Ka-Pho in Thailand. Ancient people have used it as therapeutic plant for cardiac and blood tonic cure. The purpose of this study was to investigate the potential cytotoxicity and selectivity of the extracts from V. diospyroides type SS fruit on cervical cancer HeLa and SiHa cell lines and to examine its underlying mechanism of action. MTT assay revealed that the extracts showed inhibition of cell survival in a dose-dependent manner and exhibited highly cytotoxic activity against both HeLa and SiHa cells with IC50 value less than 20 µg/mL along with less toxicity against L929 cells. Acetone cotyledon extract (ACE) showed the best selectivity index value of 4.47 (HeLa) and 3.51 (SiHa). Distinctive morphological changes were observed in ACE-treated cervical cancer cells contributing to apoptosis action. Flow cytometry analysis with Annexin V-FITC and PI staining precisely indicated that ACE induced apoptosis in HeLa and SiHa cell lines in a dose-dependent manner. Treatment of ACE with half IC50 caused DNA fragmentation and also activated increasing of bax and cleaved caspase-8 protein in HeLa cells after 48 h exposure. The results suggest that ACE has potent and selective cytotoxic effect against cervical cancer cells and the potential to induce bax and caspase-8-dependent apoptosis. Hence, the ACE could be further exploited as a potential lead in cancer treatment.

6 weeks consumption of pure fresh coconut milk caused up-regulation of eNOS and CSE protein expression in middle-aged male rats

Coconut milk (CCM) has been an important cooking ingredient in the Asia-Pacific region since ancient time. Due to its high content of saturated fatty acids, it has been considered atherogenic. We have tested if chronic consumption of fresh coconut milk by middle-aged male rat affects vascular function, plasma glucose and lipid profiles. Compared to control, CCM caused lower maximal contraction to phenylephrine of thoracic aortic rings and increased relaxation to acetylcholine that was abolished by N G-nitro-L-arginine (L-NA) or disruption of the endothelium. DL-propargylglycine caused slight increase in baseline tension of L-NA treated aortic rings of CCM-treated rats and produced higher contractile response of the aortic rings to low concentrations of phenylephrine. The aortic eNOS- and cystathionine-γ-lyase(CSE) proteins expression of the CCM-treated rats were also higher than in controls. Except for lower fasting plasma glucose there were no changes in blood chemistry for the CCM treated rats. CCM consumption caused up-regulation of eNOS and CSE protein expression which resulted in increased production of NO and H2S from the blood vessels with attenuation of vasocontraction to phenylephrine and increased relaxation to acetylcholine. These novel benefits may be expected to reduce the development of cardiovascular risk factors in the aging rat

The role of WT1 isoforms in vasculogenic mimicry and metastatic potential of human triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is highly aggressive and has a few therapeutic treatments, so new targeted therapy and biomarkers are required to provide alternative choices for treating TNBC patients. Recent studies showed that vasculogenic mimicry (VM), the formation of blood channels by aggressive cancer cells that mimic endothelial cells, is a factor contributing to poor prognosis in TNBC. Wilms' tumor 1 (WT1) gene has been found to be highly expressed in TNBC, and has 4 major distinct isoforms; isoform A (-17AA/-KTS; -/-), isoform B (+17AA/-KTS; +/-), isoform C (-17AA/+KTS; -/+) and isoform D (+17AA/+KTS; +/+). The involvement of each WT1 isoform in TNBC progression remains largely unclear. In this study, WT1 isoform-overexpressing cell sublines were established from a TNBC cell line, MDA-MB-231, by stable transfection, and the aggressive behavior of the cell sublines were evaluated. Only the WT1 isoform B- and isoform C-overexpressing cell sublines showed the significant increase in VM forming capability compared to the parental cell line and other isoform cell sublines. qRT-PCR was used to explore the change in expression level of two VM-related genes, EphA2 and VE-cadherin. All WT1 isoform cell sublines showed up-regulation of EphA2 but the levels detected in the isoform B- and isoform C-cell sublines were higher than those observed in other cell sublines. In contrast, significant up-regulation of VE-cadherin was found only in isoform A- and isoform D-cell sublines. Isoform B- and isoform C-cell sublines showed higher rates of cell migration compared to those of other cell sublines, as determined by both wound healing and Transwell assays. Gelatin zymography revealed increased MMP-9 enzyme production in isoform D-cell subline compared to the parental cell line, but this change was not observed in other cell sublines. Western blot analysis showed significantly increased expression of ß-catenin in isoform B- and isoform C-cell sublines, compared to parental cell line and other isoform cell sublines. In conclusion, our findings demonstrate that WT1 isoforms play different roles in modulating the VM-forming capacity and metastatic potential of TNBC cells.

Increased vascular eNOS and cystathionine-γ-lyase protein after 6 weeks oral administration of 3, 5, 7, 3', 4'-pentamethoxyflavone to middle-aged male rats.

Effects of treatment of middle-aged male rats with 3, 5, 7, 3', 4'-pentamethoxyflavone (PMF) on vascular and perivascular adipose tissue (PVAT) functions and blood chemistry were investigated. Rats received PMF (22 mg/kg), orally or vehicle, twice a day for 6 weeks. The PMF-treated rats had lower serum glucose, higher HDL-C levels, but no change in other parameters. Thoracic aortic and mesenteric rings of PMF treated rats produced lower maximal contraction to phenylephrine that was normalized by NG-nitro-L-arginine (L-NA) or endothelial removal. The aortic- and mesenteric rings of the PMF treated rats showed improved relaxation to acetylcholine, but not to glyceryl trinitrate, and had higher eNOS protein. DL-propargylglycine (PAG) caused greater increase in the baseline tension of the PMF-treated aortic ring and higher contraction to low concentrations of phenylephrine. PVAT lowered the contractile response of the L-NA pretreated aortic rings to phenylephrine for both groups, but PAG had no effect. The cystathionine-γ-lyase (CSE) protein of the thoracic rings, but not of the PVAT, shows increased expression after PMF treatment. Overall, PMF treatment of middle aged rats appeared to increase production of NO and H2S from the blood vessels by upregulating the expression of eNOS and CSE. PMF also decreased fasting serum glucose and increased HDL-C levels, with no toxicity to liver and kidney functions. Thus, PMF is a novel compound for possible use as a health product to prevent and/or to reduce the development of diabetes type II and/or cardiovascular disease.

Effects of 6 weeks oral administration of Phyllanthus acidus leaf water extract on the vascular functions of middle-aged male rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves of Phyllanthus acidus (PA) have been used in Thai traditional medicine for the treatment of hypertension. We have previously shown that chronic treatment of a PA water extract to middle-aged male rats caused a lowering of the body and serum lipids, two of the parameters that are implicated in cardiovascular disease. AIM OF THE STUDY: To investigate if chronic treatment of middle-aged male rats with a PA water extract affected the perivascular (aortic) adipose tissue (PVAT) and/or their vascular functions MATERIALS AND METHODS: Fresh leaves of PA were extracted with water and orally gavaged to the middle-aged male rats for 6 weeks. Vascular functions were studied in vitro using isolated thoracic aorta with and without PVAT, and mesenteric rings in Krebs Heinseleit solution with results recorded with a Polygraph or a Myograph system. The amount of blood vessel eNOS and CSE (cystathionine-γ-lyase) expression was measured by Western blotting. RESULTS: PA treatment caused a lower maximal contractile response to phenylephrine (Phe) of the endothelium-intact aortic ring than that of the control group. This effect was abolished by N(G)-nitro-l-arginine (l-NA) or by denudation of the endothelium. dl-propargylglycine (PAG, H2S inhibitor) and TEA (Ca(2+)-activated K(+) channel blocker), but not glybenclamide (ATP-activated K(+) channel blocker), caused a similar increase in the baseline of the endothelium-intact aortic ring in the presence of l-NA in both the PA-treated and control aortic rings. This effect sequentially resulted in a greater contractile response of the aortic rings of both groups to Phe. Glybenclamide also caused a similar increase in the maximal contraction of the endothelium-intact blood vessels with l-NA to both groups. PAG, TEA or glybenclamide did not modify the phenylephrine C-R curves for either group of the PVAT-endothelium-intact aortic rings preincubated with l-NA. The CSE levels of the thoracic aorta and at the PVAT were not different between the PA-treated and the control group. Relaxation of the Phe-precontracted thoracic aortic ring to acetylcholine, but not to glyceryl trinitrate, was higher for the PA-treated than for the control aortic rings and this effect was abolished by l-NA. The mesenteric rings of the PA treated group showed a lower sensitivity on the contractile response to Phe than that of the control group, and this effect was abolished by l-NA. Vasodilatation to acetylcholine, but not to glyceryl trinitrate, of the PA treated-mesenteric ring was more sensitive than that of the control group and this effect was abolished by l-NA. The expression of eNOS by the PA treated thoracic aorta and the mesenteric arteries was higher than the control group. These results demonstrated that chronic treatment with a PA water extract to middle-aged rats affected their vascular functions by increasing the nitric oxide production from the endothelial cells and also modulated the responsiveness of the thoracic aortic- and mesenteric rings to phenylephrine and acetylcholine.

The dose dependent in vitro responses of MCF-7 and MDA-MB-231 cell lines to extracts of Vatica diospyroides symington type SS fruit include effects on mode of cell death.

BACKGROUND: Vatica diospyroides type LS is a known source of valuable compounds for cancer treatment, however, in contrast little is known about therapeutic efficacy of type SS. OBJECTIVE: This study focused on in vitro cytotoxicity of these fruit extracts, and the cell death mode they induce in breast cancer cells. MATERIALS AND METHODS: Acetone extracts of fruit were tested for cytotoxicity against MCF-7 and MDA-MB-231 cell lines. The apoptosis and necrosis of these cells were quantified by fluorescence activated cell sorter (FACS) and western blot analyses. RESULTS: After 72 h of treatment, the 50% growth inhibition concentrations (IC50) levels were 16.21 ± 0.13 µg/mL against MCF-7 and 30.0 ± 4.30 µg/mL against MDA-MB-231, indicating high and moderate cytotoxicity, respectively. From the FACS results, we estimate that the cotyledon extract at half IC50 produced 11.7% dead MCF-7 cells via apoptosis, whereas another concentrations both apoptosis and necrosis modes co-existed in a dose-dependent manner. In MDA-MB-231 cell line, only the apoptosis was induced by the pericarp extract in a dose-dependent manner. With the extracts at half IC50 concentration, in both cells, the expression of p21 decreased while that of Bax increased within 12-48 h of dosing, confirming apoptosis induced by time-dependent responses. Apoptosis dependent on p53 was found in MCF-7, whereas the mutant p53 of MDA-MB-231 cells was expressed. CONCLUSION: The results indicate that fruit extracts of V. diospyroides have cytotoxic effects against MCF-7 and MDA-MB-231 cells via apoptosis pathway in a dose-dependent manner. This suggests that the extracts could provide active ingredients for the development, targeting breast cancer therapy.

Extracts from Vatica diospyroides type SS fruit show low dose activity against MDA-MB-468 breast cancer cell-line via apoptotic action.

Very strong antiproliferative action of V. diospyroides type SS fruit extracts (IC50 range of 1.60-17.45 µg/mL) in MDA-MB-468 cell-line was observed in an MTT assay. After dosing of an extract concentration at half IC50 to cell line for 24 to 72 hours, treated cells were subjected to Annexin V-FITC/PI binding assay, followed by FACS and western blot analyses. Significant apoptotic death was observed with all extract treatments and both exposure times. Dosing with acetone extract of pericarp and cotyledon induced the highest apoptotic populations (33 and 32%, resp.), with the lowest populations of viable cells (65 and 67%, resp.). During 24 to 72 hours of dosing with methanolic extract of pericarp, the populations of viable and early apoptotic cells decreased significantly from 72.40 to 71.32% and from 12.00 to 6.36%, respectively, while the late apoptotic and nonviable cell populations continuously increased from 15.30 to 19.18% and from 0.30 to 3.14%, respectively. The expression of Bax increased within 12-48 hours of dosing, confirming apoptosis induced by time-dependent responses. The mutant p53 of MDA-MB-468 cells was expressed. Our results indicate that apoptosis and time-dependent therapeutic actions contribute to the cytotoxic effects of V. diospyroides type SS fruit on MDA-MB-468 cell.

Response of cardiac endothelial nitric oxide synthase to plasma viscosity modulation in acute isovolemic hemodilution.

BACKGROUND: Endothelial nitric oxide synthase (eNOS) is generally expressed in endocardial cells, vascular endothelial cells and ventricular myocytes. However, there is no experimental study elucidating the relationship between cardiac eNOS expression and elevated plasma viscosity in low oxygen delivery pathological conditions such as hemorrhagic shock-resuscitation and hemodilution. This study tested the hypothesis that elevated plasma viscosity increases cardiac eNOS expression in a hemodilution model, leading to positive effects on cardiac performance. MATERIALS AND METHODS: Two groups of golden Syrian hamster underwent an acute isovolemic hemodilution where 40% of blood volume was exchanged with 2% (low-viscogenic plasma expander [LVPE]) or 6% (high-viscogenic plasma expander [HVPE]) of dextran 2000 kDa. In control group, experiment was performed without hemodilution. All groups were performed in awake condition. Experimental parameters, i.e., mean arterial blood pressure (MAP), heart rate, hematocrit, blood gas content and viscosity, were measured. The eNOS expression was evaluated by eNOS Western blot analysis. RESULTS: After hemodilution, MAP decreased to 72% and 93% of baseline in the LVPE and HVPE, respectively. Furthermore, pO2 in the LVPE group increased highest among the groups. Plasma viscosity in the HVPE group was significantly higher than that in control and LVPE groups. The expression of eNOS in the HVPE group showed higher intensity compared to other groups, especially compared with the control group. CONCLUSION: Our results demonstrated that cardiac eNOS has responded to plasma viscosity modulation with HVPE and LVPE. This particularly supports the previous studies that revealed the positive effects on cardiac function in animals hemodiluted with HVPE.