Antibacterial activity of Dioscorea bulbifera Linn. extract and its active component flavanthrinin against skin-associated bacteria.

BACKGROUND: Dioscorea bulbifera Linn. has been used for wound care in Thailand. However, a comprehensive evaluation of its antibacterial activity is required. This study aimed to investigate the antibacterial efficacy of D. bulbifera extract against skin-associated bacteria and isolate and characterize its active antibacterial agent, flavanthrinin. METHODS: Air-dried bulbils of D. bulbifera were pulverised and extracted with hexane, dichloromethane, ethyl acetate, methanol, ethanol, and distilled water; vacuum filtered; concentrated; freeze-dried; and stored at -20 ºC. Antibacterial activity of the extracts was assessed using microdilution techniques against several skin-associated bacteria. Thin-layer chromatography (TLC) bioautography was used to identify the active compounds in the extract, which were fractionated by column chromatography and purified by preparative TLC. The chemical structures of the purified compounds were analysed using nuclear magnetic resonance (NMR). The cytotoxicity of the extract and its active compounds was evaluated in Vero cells. RESULTS: The ethyl acetate extract exhibited distinct inhibition zones against bacteria compared to other extracts. Therefore, the ethyl acetate extract of D. bulbifera in the ethyl acetate layer was used for subsequent analyses. D. bulbifera extract exhibited antibacterial activity, with minimum inhibitory concentrations (MICs) of 0.78-1.56 mg/mL. An active compound, identified through TLC-bioautography, demonstrated enhanced antibacterial activity, with MICs of 0.02-0.78 mg/mL. NMR analysis identified this bioactive compound as flavanthrinin. Both D. bulbifera extract and flavanthrinin-containing fraction demonstrated potent antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and S. epidermidis. The flavanthrinin containing fraction demonstrated low cytotoxicity against Vero cells, showing CC50 values of 0.41 ± 0.03 mg/mL. These values are lower than the MIC value, indicating that this fraction is safer than the initial ethyl acetate extract. CONCLUSIONS: Dioscorea bulbifera extract and its bioactive component flavanthrinin demonstrated significant antibacterial activity against the skin-associated bacteria Staphylococci, including MRSA. Flavanthrinin has potential as a complementary therapeutic agent for managing skin infections owing to its potent antibacterial effects and low cytotoxicity.

Distribution and Clinical Significance of Hepatitis B virus A1762T/G1764A Double Mutation in Chronic Hepatitis B Patients: A Cross-Sectional Study.

BACKGROUND: Chronic hepatitis B (CHB) is well-known as a major risk for liver cirrhosis and hepatocellular carcinoma (HCC). The A1762T/G1764A double mutation in the hepatitis B virus genome affects the production of HBe antigen and is established as a predictive marker for progression to HCC. Thus, this study aimed to investigate the prevalence and clinical significance of the mutation in Thai CHB patients. METHODS:  A cross-sectional study was conducted in 78 Thai CHB patients who were assessed for hepatitis B profiles, HBsAg, HBeAg and anti-HBeAg, transaminitis, liver fibrosis defined by FIB-4 (FIB-4) score and AST to platelet ratio index (APRI), alpha-fetoprotein (AFP) and active hepatitis B status. HBV A1762T/G1764A mutation was examined by SYBR Green I Real-time PCR. Chi-square and Mann-Whiney U tests were performed to determine the association between the mutation and variables. RESULTS: The prevalence of patients infected with the A1762T/G1764A mutation was 44.9%. The mutation was associated with HBeAg status (p=0.027) and HBsAg levels (p=0.008), transaminitis (p=0.011), and active hepatitis B (p=0.037), but not liver fibrosis markers, FIB-4 score and APRI, and AFP. Binary logistic regression identified the mutation as a predictive factor of active hepatitis B (OR 3.5, 95%CI, 1.1-11.3, p=0.037). Patients infected with the mutant exhibited significantly higher levels of HBsAg (p=0.011) and HBV viral load (p=0.047), but lower levels of HBeAg (p=0.12) than those infected with the wild-type HBV. CONCLUSION: The data indicate the high prevalence of the A1762T/G1764A mutation and its significant association with the severity of Thai CHB patients and the HBV mutation is proposed as a predictive marker of active hepatitis B status in CHB patients.

MicroRNA expression profiles associated with the metastatic ability of MDA­MB­231 breast cancer cells.

Breast cancer is an important worldwide public health concern. The incidence rate of breast cancer increases every year. The primary cause of death is metastasis, a process by which cancer cells spread from a primary site to secondary organs. MicroRNAs (miRs/miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level. Dysregulation of certain miRNAs is involved in carcinogenesis, cancer cell proliferation and metastasis. Therefore, the present study assessed miRNAs associated with breast cancer metastasis using two breast cancer cell lines, the low-metastatic MCF-7 and the highly metastatic MDA-MB-231. miRNA array analysis of both cell lines indicated that 46 miRNAs were differentially expressed when compared between the two cell lines. A total of 16 miRNAs were upregulated in MDA-MB-231 compared with MCF-7 cells, which suggested that their expression levels may be associated with the highly invasive phenotype of MDA-MB-231 cells. Among these miRNAs, miR-222-3p was selected for further study and its expression was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Under both non-adherent and adherent culture conditions, the expression levels of miR-222-3p in the MDA-MB-231 cell line were higher than those noted in the MCF-7 cell line under the same conditions. Suppression of endogenous miR-222-3p expression in MDA-MB-231 cells using a miR-222-3p inhibitor resulted in a 20-40% reduction in proliferation, and a ~30% reduction in migration, which suggested that the aggressive phenotype of MDA-MB-231 cells was partly regulated by miR-222-3p. Bioinformatic analysis of miR-222-3p using TargetScan 8.0, miRDB and PicTar identified 25 common mRNA targets, such as cyclin-dependent kinase inhibitor 1B, ADP-ribosylation factor 4, iroquois homeobox 5 and Bcl2 modifying factor. The results of the present study indicated that miR-222-3p was potentially associated with the proliferation and migratory ability of the MDA-MB-231 cell line.

Cellular signals integrate cell cycle and metabolic control in cancer.

Growth factors are the small peptides that can promote growth, differentiation, and survival of most living cells. However, aberrant activation of receptor tyrosine kinases by GFs can generate oncogenic signals, resulting in oncogenic transformation. Accumulating evidence support a link between GF/RTK signaling through the major signaling pathways, Ras/Erk and PI3K/Akt, and cell cycle progression. In response to GF signaling, the quiescent cells in the G0 stage can re-enter the cell cycle and become the proliferative stage. While in the proliferative stage, tumor cells undergo profound changes in their metabolism to support biomass production and bioenergetic requirements. Accumulating data show that the cell cycle regulators, specifically cyclin D, cyclin B, Cdk2, Cdk4, and Cdk6, and anaphase-promoting complex/cyclosome (APC/C-Cdh1) play critical roles in modulating various metabolic pathways. These cell cycle regulators can regulate metabolic enzyme activities through post-translational mechanisms or the transcriptional factors that control the expression of the metabolic genes. This fine-tune control allows only the relevant metabolic pathways to be active in a particular phase of the cell cycle, thereby providing suitable amounts of biosynthetic precursors available during the proliferative stage. The imbalance of metabolites in each cell cycle phase can induce cell cycle arrest followed by p53-induced apoptosis.

Effects of a Pedometer-Based Walking Home Program Plus Resistance Training on Inflammatory Cytokines and Depression in Thai Older People with Sarcopenia: A Three-Arm Randomized Controlled Trial.

OBJECTIVE: To examine the effects of daily walking steps plus resistive exercise on chronic inflammatory markers and depressive symptoms in older adults with sarcopenia. METHODS: Ninety men and women aged over 60 years were enrolled and divided into 60 and 30 adults with and without sarcopenia, respectively. Older individuals were screened for sarcopenia using the Asian Working Group for Sarcopenia in 2019. A simple random sample was conducted to divide the older adults with sarcopenia into two groups: control and intervention. Thirty older adults with sarcopenia were assigned to perform 12 weeks of step walking (>7500 steps) daily for 5 days/week plus resistance exercise with an elastic band twice/week; the control groups (i.e., no sarcopenia and sarcopenia) performed routine daily life Changes in depression and expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were measured before and after the 12-week intervention program. Two-way mixed ANOVA models were computed for group and interaction effects for each variable. RESULTS: Changes in depressive symptom scores (Δ2.86 ± 0.92) and TNF-α levels (Δ22.16 ± 2.30) were observed in the intervention group after the 12-week program. In addition, an interaction effect between the intervention (Δ4.04 ± 3.10) and control groups (Δ8.10 ± 4.88) was found for the symptoms of depression. CONCLUSION: Older people with sarcopenia who accumulated >7,500 steps/day, 5 days/week plus resistive elastic band twice /week show improvements in inflammation and depressive symptoms. CLINICAL IMPLICATIONS: Encourage physical activity had a positive effect on reducing inflammation and depression among older people with sarcopenia.

Beneficial effects of walking-based home program for improving cardio-respiratory performance and physical activity in sarcopenic older people: a randomized controlled trial.

BACKGROUND: Sarcopenia is an age-related loss of muscle mass, decline in physical performance, and/or loss of muscle strength. Improving physical activity (PA) is associated with a reduced risk of sarcopenia. Additionally, the World Health Organization 2020 guidelines recommend 150 minutes/week of moderate-intensity or 75 minutes/week of vigorous PA plus resistance exercise twice a week. An increase in walking steps/day also enhances PA. AIM: The study explored the benefits of a pedometer-based walking program plus a resistance exercise program in older adults with sarcopenia. DESIGN: A randomized control trial. SETTING: In community dwelling. POPULATION: Older individuals with sarcopenia. METHODS: Screening for sarcopenia was defined according to the Asian Working Group of Sarcopenia criteria in 2019, which is composed of the skeletal muscle mass index (SMI), muscle strength, and physical performance. Bioimpedance analysis, handgrip strength, and gait speed were measured for the SMI, muscle strength, and physical performance, respectively. to explore the effectiveness of a pedometer-based walking program plus TheraBand resistance exercise over 12 weeks. The intervention group was required to wear a pedometer and walk 7 500 steps/day for 5 days/week and perform the TheraBand resistance exercise twice a week, while the control group continued their routine daily activities. Respiratory muscle strength, functional capacity (i.e., 6-minute walk test), and PA were assessed before and after the 12-week program. To evaluate the benefits of the home-based walking program plus resistance exercise, two-way mixed ANOVAs were performed. RESULTS: Sixty older individuals with sarcopenia aged ≥60 years were enrolled; however, a total of 57 older adults (28 in the invention group and 29 in the control group) completed the study. Inspiratory muscle strength, functional capacity, and PA increased between and within groups after the 12-week program (all P<0.05). Furthermore, muscle strength and physical performance improved in the intervention group after the 12-week program but not in the control group. CONCLUSIONS: A pedometer-based intervention program with TheraBand resistance exercise could improve cardio-respiratory performance and PA among older Thai individuals with sarcopenia. Further study needs to consider type of exercise and dietary supplements. CLINICAL REHABILITATION IMPACT: Increasing the number of steps walked per day may reduce the risk of developing sarcopenia in older people.

Systemic Inflammation in Sarcopenia Alter Functional Capacity in Thai Community-dwelling Older People: A Preliminary Observational Study.

BACKGROUND: Sarcopenia is linked to the loss of muscle mass in older adults, leading to impaired functional capacity and quality of life. In addition, this finding was recognized as an agerelated chronic inflammatory process. We aimed to determine the relationship between sarcopenia, functional capacity, and inflammatory biomarkers and subsequent prediction of inflammatory biomarkers in older adults. METHODS: A total of 126 women and men aged ≥ 60 years were enrolled. Participants were required to complete a handgrip dynamometer, 6-meter walk test, and bioimpedance analysis. Diagnosis was based on the definition of sarcopenia from the Asian Working Group for Sarcopenia 2019. Prior to performing a 6-minute walking test (i.e., functional capacity testing), blood samples were drawn for a C-reactive protein (CRP) test. RESULTS: A total of 12.70% were categorized as having sarcopenia. Significant differences in CRP and functional capacity between the sarcopenia and non-sarcopenia groups were found (p <.05). Older people with high CRP levels had significantly reduced functional capacity and slow gait speed. CONCLUSIONS: Poor functional capacity was associated with increased CRP levels, which might be due to the development of age-related inflammation. Older patients with sarcopenia may be at higher risk for functional decline.

Prevalence and incidence of sarcopenia and low physical activity among community-dwelling older Thai people: a preliminary prospective cohort study 2-year follow-up.

Background: Sarcopenia, defined as a loss of muscle mass, has become a major health problem in older people. Few prospective studies report the incidence and risk of sarcopenia. Therefore, this study aimed to explore the prevalence of sarcopenia at the baseline and follow-up after 2 years in community-dwelling older Thai individuals. Methods: In 2019, 330 older people were recruited from a community-dwelling population, and these participants were requested to present again in 2021. Sarcopenia was diagnosed using the criteria for the Asia Working Group for Sarcopenia (AWGS). All participants were asked to perform a 6-meter walk test, handgrip strength test, and bioelectric impedance assessment, and complete the Global Physical Activity Questionnaire. Results: The study found that the prevalence of sarcopenia was 65 (19.70%) in 330 older people in 2019, and 44 of 205 participants (21.46%) were reported to have sarcopenia after 2 years. The incidence of sarcopenia was noted to be 2.44% in 2021. Analysis with ANOVA and pairwise comparisons showed that the reversibility of sarcopenia was attributed to high level of physical activity in the 2-year follow-up group (p = 0.014, 95% CI [-1753.25--195.49]). Further, participants with moderate and high physical activity had a reduced incidence of sarcopenia (odds ratio = 9.00 and 14.47, respectively). Therefore, low physical activity in older people led to the development of sarcopenia from the baseline to the 2-year follow-up, indicating that increased physical activity may be useful in reversing sarcopenia, as suggested in the 2-year follow-up study. Low physical activity could be a risk factor for the incidence of sarcopenia. Hence, the prevention of sarcopenia could promote health improvement through moderate to high physical activity.

Lack of Association between IFN-γ, CXCL10 and TGF-ß1 Gene Polymorphisms and Liver Complication in HIV-infected Thais.

OBJECTIVE: Chronic liver disease has become a leading cause of illness and death in people living with HIV and the production of the cytokines IFN-γ and TGF-ß1, and chemokine CXCL10 during chronic inflammation contributes to liver disease progression in HIV patients under long-term anti-retroviral therapy. This study aimed to examine association of IFN-γ +874T/A, CXCL10 G-201A and C-1596T, and TGF-ß1 -509C/T single nucleotide polymorphisms (SNPs) with liver complications in the HIV-infected Thais. METHODS: A cross-sectional study was conducted in 200 Thai HIV patients who were evaluated for transaminitis and significant liver fibrosis by fibrosis-4 score (FIB-4), and genotypes for IFN-γ +874T/A, CXCL10 G-201A and C-1596T, and TGF-ß1 -509C/T SNPs using PCR-based methods. RESULT: There were high rates of transaminitis (30.1%) and significant liver fibrosis assessed by FIB-4 score > 1.45 (18.8%) in this group of patients, mostly under anti-retroviral therapy (73.0%). The genotypes and alleles of IFN-γ +874T/A, CXCL10 G-201A and C-1596T, and TGF-ß1 -509C/T SNPs were not associated with either transaminitis or FIB-4 score > 1.45 (p > 005). Logistic regression analysis identified age and gender as risk factors, and CD4+ cell count higher than 350 cells/ul as a protective factor of liver fibrosis in this study group. CONCLUSION: The IFN-γ +874T/A, CXCL10 G-201A and C-1596T, and TGF-ß11 -509C/T SNPs were not significantly associated with liver complication in HIV-infected Thais, mostly under ART.

Differential growth inhibition, cell cycle arrest and apoptosis of MCF-7 and MDA-MB-231 cells to holocarboxylase synthetase suppression.

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin onto the biotin-dependent carboxylases. Recent studies have shown that HLCS is over-expressed in breast cancer patients. Here we investigated the functional roles of free biotin and HLCS in supporting growth and migration of breast cancer cell lines. Depletion of biotin from culture medium markedly reduced biotinylation of the two most abundant biotin-carboxylases, acetyl-CoA carboxylase and pyruvate carboxylase. This was accompanied by a marked decrease in cell growth. Suppression of HLCS expression in the low invasive breast cancer cell line MCF-7 resulted in an 80% reduction of biotinylated ACC, but not PC. HLCS knockdown MCF-7 cell lines showed 40-50% reduction of proliferation and 35% reduction of migration, accompanied by G1 cell cycle-arrest-induced apoptosis. In contrast, knockdown of HLCS expression in the highly invasive cell line MDA-MB-231 resulted in only marginal reduction of biotinylation of both ACC and PC, accompanied by 30% reduction of proliferation and 30% reduction of migration. Our studies provide new insights to use HLCS as a novel anti-cancer drug target.

Genotypic and allelic distribution of IFN-γ +874T/A and TGF-ß1 -509C/T single-nucleotide polymorphisms in human immunodeficiency virus-infected Thais.

Advances in antiretroviral therapy (ART) have led to a decrease of acquired immunodeficiency syndrome (AIDS)-related mortality, and an increase of non-AIDS illnesses in people living with HIV (PLWH). Risks for HIV-related chronic inflammation leading to non-AIDS illnesses in PLWH have been increasingly clarified including immunogenetic factors. This study aimed to examine distribution of genotypic and allelic frequencies of the well-characterized interferon-γ (IFN-γ) +874T/A and transforming growth factor-ß1 (TGF-ß1) -509C/T single nucleotide polymorphisms (SNPs) in Thai PLWH. The cross-sectional study was conducted in 191 Thai HIV patients. Most patients were under ART (74.3%) and maintained a relatively high median of CD4+  cell count (364.5 [5-1601] cells/µl). The frequencies of IFN-γ +874T/A  SNP genotypes were 9.0% AA, 38.3% AT, and 52.7% TT and those of the SNP alleles were 28.1% A and 71.9% T. The rates of TGF-ß1-509C/T SNP genotypes were 15.7% CC, 44.0% CT, and 40.3% TT and those of the SNP alleles were 37.7% C and 62.3% T. The more frequent TT genotypes and T allele of the IFN-γ +874T/A SNP, and relatively more prevalent TT and CT genotypes and T allele of TGF-ß1 -509C/T SNP were potentially associated with disease progression to non-AIDS complication in Thai PLWH and required further investigation. This study provides the immunogenetic data potentially supporting mechanisms for chronic immune activation in PLWH under long-term suppressive ART.

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells.

BACKGROUND: Previous studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown. METHODS: We generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics. RESULTS: PC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP. CONCLUSIONS: Suppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells. GENERAL SIGNIFICANCE: Our results highlight the possibility of the use of PC as an anti-cancer drug target.

Protein expression profiles that underpin the preventive and therapeutic potential of Moringa oleifera Lam against azoxymethane and dextran sodium sulfate-induced mouse colon carcinogenesis.

Previous studies in a mouse model have indicated the anticancer potential of boiled Moringa oleifera pod (bMO)-supplemented diets; however, its molecular mechanisms are still unclear. Therefore, the present study aimed to explore the protein expression profiles responsible for the suppressive effect of bMO supplementation on azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced mouse colon carcinogenesis. Analysis by gel electrophoresis and liquid chromatography-tandem mass spectrophotometry demonstrated that there were 125 proteins that were differentially expressed in mouse colon tissues between 14 experimental groups of mice. The differentially expressed proteins are involved in various biological processes, such as signal transduction, metabolism, transcription and translation. Venn diagram analysis of the differentially expressed proteins was performed in six selected mouse groups, including negative control, positive control mice induced by AOM/DSS, the AOM/DSS groups receiving preventive or therapeutic bMO diets and their bMO-supplemented control groups. This analysis identified 7 proteins; 60S acidic ribosomal protein P1 (Rplp1), fragile X mental retardation, cystatin 9, round spermatids protein, zinc finger protein 638, protein phosphatase 2C (Ppm1g) and unnamed protein product as being potentially associated with the preventive and therapeutic effects of bMO in AOM/DSS-induced mouse colon cancer. Analysis based on the search tool for interactions of chemicals (STITCH) database predicted that Rplp1 interacted with the apoptotic and inflammatory pathways, whereas Ppm1g was associated only with inflammatory networks. This proteomic analysis revealed candidate proteins that are responsible for the effects of bMO supplementation, potentially by regulating apoptotic and inflammatory signaling networks in colorectal cancer prevention and therapy.

Proteomic Analysis of the Anoikis-Resistant Human Breast Cancer Cell Lines.

Acquisition of anoikis resistance is a prerequisite for cancer metastasis and invasion, which are major causes of death from cancer. The molecular mechanisms underlying antianoikis properties in cancer cells are still largely unclear. Here, we describe a protocol for preparation of anoikis-resistant cultured nonmetastatic MCF-7 and metastatic MDA-MB-231 cell lines. The anoikis-resistant cultures were prepared by plating cells in the poly-2-hydroxyethyl methacrylate coated plates and cultured for 24 h. The viability of cells in the cultures was determined using trypan blue staining and annexin V cell death assay, while protein profiles associated with anoikis-resistance in both cells and conditioned media were analyzed by proteomics.

CXCL12 G801A polymorphism is associated with significant liver fibrosis in HIV-infected Thais: a cross-sectional study.

BACKGROUND: Previous studies indicate high prevalence of liver diseases in HIV-infected patients, and their genetic risk factors are still unclear. The chemokine CXCL12 plays important roles in development of chronic liver injury and a single nucleotide polymorphism (SNP) G to A change at position 801 in CXCL12 gene has been demonstrated to affect CXCL12 production levels. OBJECTIVE: This study aimed to analyze the association of CXCL12 G801A SNP with liver complication in HIV-infected Thais. METHODS: A cross-sectional study was conducted in 164 patients who were evaluated for transaminitis and significant liver fibrosis, defined by fibrosis-4 (FIB-4) score and AST to platelet ratio index (APRI), and genotyped for the SNP using tetra-primer PCR-SSP. RESULTS: There were high rates of patients with transaminits (28.0%), and significant liver fibrosis by FIB-4 score (18.9%) and by APRI (14.0%). The CXCL12 G801A AA/GA genotypes were significantly associated with transaminitis (p = 0.014) and significant fibrosis by APRI (p = 0.020). Univariate and multivariate analyses identified the AA/GA genotypes as predictive factors for significant fibrosis (OR 6.8, 95%CI 1.7-28.2, p = 0.008), together with age older than 40 years, CD4+ cell count < 350 cells/µl and hepatitis B and/or C virus coinfection. The significantly higher medians of APRI and FIB-4 score, in patients with AA/GA than those with GG genotypes (p < 0.05) were observed in the ART-naïve, but not ART-experienced groups. CONCLUSION: The CXCL12 G801A AA/GA genotypes are significant predictive factors for hepatic fibrosis potentially in the ART-naïve HIV-infected Thais.

Protein Profiles Associated with Anoikis Resistance of Metastatic MDA-MB-231 Breast Cancer Cells.

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

Comparative proteomic analysis implicates eEF2 as a novel target of PI3Kγ in the MDA-MB-231 metastatic breast cancer cell line.

BACKGROUND: Cancer cell migration is fundamentally required for breast tumour invasion and metastasis. The insulin-like growth factor 1 tyrosine kinase receptor (IGF-1R) and the chemokine G-protein coupled receptor, CXCR4 have been shown to play an important role in breast cancer metastasis. Our previous study has shown that IGF-1R can transactivate CXCR4 via a physical association in the human MDA-MB-231 metastatic breast cancer cell line and that this plays a key role in IGF-I-induced migration of these cells. In the present study we used pharmacological inhibition and RNAi to identify PI3Kγ as an important migration signalling molecule downstream of receptor transactivation in MDA-MB-231 cells. To identify PI3Kγ-regulated proteins upon transactivation of CXCR4 by IGF-I, we undertook a comparative proteomics approach using 2-D- Fluorescence Difference Gel Electrophoresis (DIGE) and identified the proteins by mass spectrometry. RESULTS: These experiments identified eukaryotic elongation factor 2 (eEF2) as a novel downstream target of PI3Kγ after activation of the IGF-1R-CXCR4 heterodimer by IGF-I. Further analysis demonstrated that eEF2 is phosphorylated in MDA-MB-231 cells in response to IGF-I and that this is dependent on PI3Kγ activity. CONCLUSIONS: Our data imply a novel role for PI3Kγ in facilitating cell migration by regulating phosphorylation of eEF2.

Differential functional activation of chemokine receptor CXCR4 is mediated by G proteins in breast cancer cells.

CXCR4 is a G protein-coupled receptor of considerable biological significance, and among its numerous functions, it is suggested to play a critical role in cancer metastasis. We have investigated the expression and function of CXCR4 in a range of breast cancer cell lines covering a spectrum of invasive phenotypes and found that, while surface levels of CXCR4 were uniform across the entire panel, only highly invasive cells that are metastatic in immunocompromised mice expressed functional receptors. CXCL12/SDF-1 induced cellular responses such as calcium mobilization, actin polymerization, and chemotaxis in metastatic cells, whereas noninvasive cells were unresponsive. Moreover, CXCL12 activated multiple signaling pathways downstream of G proteins in highly invasive cells but failed to activate any of the examined kinase cascades in noninvasive cell lines. This blockade in nonmetastatic cell lines seems to be due to the inability of G protein alpha and beta subunits to form a heterotrimeric complex with CXCR4. Galpha and Gbeta were able to bind to CXCR4 independently in all cell lines, but the association of G protein alphabetagamma heterotrimers with the receptor, a prerequisite for signal transduction downstream from G protein-coupled receptors, was only observed in the highly invasive cell lines. Our findings show, for the first time, that CXCR4 function is subject to complex and potentially tightly controlled regulation in breast cancer cells via differential G protein-receptor complex formation, and this regulation may play a role in the transition from nonmetastatic to malignant tumors.

Transactivation of CXCR4 by the insulin-like growth factor-1 receptor (IGF-1R) in human MDA-MB-231 breast cancer epithelial cells.

In the multimolecular environment in tissues and organs, cross-talk between growth factor and G protein-coupled receptors is likely to play an important role in both normal and pathological responses. In this report, we demonstrate transactivation of the chemokine receptor CXCR4 by the growth factor insulin-like growth factor (IGF)-1 is required for IGF-1-induced cell migration in metastatic MDA-MB-231 cells. The induction of chemotaxis in MDA-MB-231 cells by IGF-1 was inhibited by pretreatment of the cells with pertussis toxin (PTX) and by RNAi-mediated knockdown of CXCR4. Transactivation of the CXCR4 pathway by IGF-1 occurred independently of CXCL12, the chemokine ligand of CXCR4. Neither CXCR4 knockdown nor PTX had any effect on the ability of IGF-1 to activate IGF-1R, suggesting that CXCR4 and G proteins are activated subsequent to, or independently of, phosphorylation of IGF-1R by IGF-1. Coprecipitation studies revealed the presence of a constitutive complex containing IGF-1R, CXCR4, and the G protein subunits, G(i)alpha2 and Gbeta, and stimulation of MDA-MB-231 cells with IGF-1 led to the release of G(i)alpha2 and Gbeta from CXCR4. Based on our findings, we propose that CXCR4 constitutively forms a complex with IGF-1R in MDA-MB-231 cells, and that this interaction allows IGF-1 to activate migrational signaling pathways through CXCR4, G(i)alpha2 and Gbeta.