Twist-related Protein Research Articles

Transcription factor 4 is a key mediator of oncogenesis in neuroblastoma by promoting MYC activity.

Super-enhancer-associated transcription factor networks define cell identity in neuroblastoma (NB). Dysregulation of these transcription factors contributes to the initiation and maintenance of NB by enforcing early developmental identity states. We report that the class I basic helix-loop-helix (bHLH) transcription factor 4 (TCF4; also known as E2-2) is a critical NB dependency gene that significantly contributes to these identity states through heterodimerization with cell-identity-specific bHLH transcription factors. Knockdown of TCF4 significantly induces apoptosis in vitro and inhibits tumorigenicity in vivo. We used genome-wide expression profiling, TCF4 chromatin immunoprecipitation sequencing (ChIP-seq) and TCF4 immunoprecipitation-mass spectrometry to determine the role of TCF4 in NB cells. Our results, along with recent findings in NB for the transcription factors T-box transcription factor TBX2, heart- and neural crest derivatives-expressed protein 2 (HAND2) and twist-related protein 1 (TWIST1), propose a role for TCF4 in regulating forkhead box protein M1 (FOXM1)/transcription factor E2F-driven gene regulatory networks that control cell cycle progression in cooperation with N-myc proto-oncogene protein (MYCN), TBX2, and the TCF4 dimerization partners HAND2 and TWIST1. Collectively, we showed that TCF4 promotes cell proliferation through direct transcriptional regulation of the c-MYC/MYCN oncogenic program that drives high-risk NB. Mechanistically, our data suggest the novel finding that TCF4 acts to support MYC activity by recruiting multiple factors known to regulate MYC function to sites of colocalization between critical NB transcription factors, TCF4 and MYC oncoproteins. Many of the TCF4-recruited factors are druggable, giving insight into potential therapies for high-risk NB. This study identifies a new function for class I bHLH transcription factors (e.g., TCF3, TCF4, and TCF12) that are important in cancer and development.

Twist-related protein 1 promotes transforming growth factor β receptor 1 in keloid fibroblasts via regulating the stability of myocyte enhancer factor 2A.

Keloid scarring is caused by a fibroproliferative disorder due to abnormal activation of genes, the underlying mechanism of which is still unclear. The basic helix-loop-helix transcription factor Twist-related protein 1 (TWIST1) controls cell proliferation and differentiation in tissue development and disease processes. In this study, we aimed to clarify the essential role of TWIST1 in the pathogenesis of keloids. Immunohistochemistry, cell counting kit-8 assays, western blotting, PCR, matrigel invasion assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of TWIST1 in fibroblasts derived from normal skin and keloids. Mass spectrometry, ubiquitination assays, chromatin immunoprecipitation and dual luciferase reporter assay were applied to explore the interaction of TWIST1 with downstream molecules. In the present study, we confirmed that TWIST1 was upregulated in keloid tissue of patients and in keloid-derived fibroblasts (KFBs). In vitro, TWIST1 inhibition prevented KFB proliferation, invasion and activation. We also discovered a link between TWIST1 and the transforming growth factor β (TGF-β) signaling related molecules TGF-β receptor 1 (TΒR1), SMAD family member 2 (Smad2) and Smad3, and the fibrosis markers α-smooth muscle actin, collagen type I and collagen type III in KFBs. Mechanistically, we uncovered a brand-new mechanism by which TWIST1 interacts with myocyte enhancer factor 2A (MEF2A) and suppresses its ubiquitination and degradation. Using chromatin immunoprecipitation and dual-luciferase reporter assay, TΒR1 was identified as a novel downstream target of MEF2A, which directly binds to its promoter. Overexpression of TWIST1 promoted the recruitment of MEF2A to the TΒR1 promoter and restored TΒR1 functional expression. Our research highlights a significant function of TWIST1 in the development of keloid and its related fibroblasts, partially facilitated by elevated MEF2A-dependent TΒR1 expression. Blocking the expression of TWIST1 in KFBs could potentially pave a novel therapeutic avenue for keloid treatment.

Multifaceted Characterization of Human Embryonic Stem Cell-Derived Mesenchymal Stem/Stromal Cells Revealed Amelioration of Acute Liver Injury in NOD-SCID Mice.

Human embryonic stem cells (hESCs) are advantaged sources for large-scale and homogeneous mesenchymal stem/stromal cells (MSCs) generation. However, due to the limitations in high-efficiency procedures for hESC-MSCs induction, the systematic and detailed information of mesengenesis and early MSC development are largely obscure. In this study, we took advantage of the well-established twist-related protein 1 (TWIST1)-overexpressing hESCs and two small molecular cocktails (CHIR99021, decitabine) for high-efficient MSC induction. To assess the multidimensional biological and transcriptomic characteristics, we turned to cellular and molecular methods, such as flow cytometry (FCM), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in vitro tri-lineage differentiation, cytokine secretion analysis, in vivo transplantation for acute liver injury (ALI) management, and bioinformatics analyses (eg, gene ontology-biological processes [GO-BP], Kyoto Encyclopedia of Genes and Genomes [KEGG], HeatMap, and principal component analysis [PCA]). By combining TWIST1 overexpression (denoted as T) and the indicated small molecular cocktails (denoted as S), hESCs high-efficiently differentiated into MSCs (denoted as TS-MSCs, induced by T and S combination) within 2 weeks. TS-MSCs satisfied the criteria for MSC definition and revealed comparable tri-lineage differentiation potential and ameliorative efficacy upon ALI mice. According to RNA-sequencing (SEQ) analysis, we originally illuminated the gradual variations in gene expression pattern and the concomitant biofunctions of the programmed hESC-MSCs. Overall, our data indicated the feasibility of high-efficient generation of hESC-MSCs by TWIST1 and cocktail-based programming. The generated hESC-MSCs revealed multifaceted in vivo and in vitro biofunctions as adult BM-MSCs, which collectively suggested promising prospects in ALI management in future.

Twist-1 Stimulates the Malignant Behaviors of Hydatidiform Mole via the PI3K/AKT Pathway.

Hydatidiform mole (HM) is a common pregnancy disease among women of gestational age. Twist-related protein 1 (Twist-1) is involved in the development of various tumors, but its role in HM is poorly defined. This study aimed to explore Twist-1 expression and its biological function in HM cells. Twist-1 expression in HM was detected by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR). The effects of silencing Twist-1 on choriocarcinoma (CCA) cell proliferation were detected by cell counting kit-8 (CCK-8) and clone formation assays. CCA cell migration and invasion were detected through transwell assay. Western blot was used to detect epithelial-mesenchymal transition (EMT) and the expression of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway-related proteins. Twist-1 expression was upregulated in HM tissues (p < 0.001) and CCA cells (p < 0.01). Twist-1 silencing inhibited proliferation of BeWo and JAR cells (p < 0.01, p < 0.05) as shown by CCK-8 assay (p < 0.01) and clone formation assays (p < 0.01, p < 0.05). Twist-1 silencing inhibited the migration (p < 0.01) and invasion activity (p < 0.01, p < 0.05) of BeWo and JAR cells. Western blot results showed that Twist-1 silencing promoted E-cadherin (p < 0.01) expression, and inhibited N-cadherin (p < 0.01, p < 0.05) and vimentin (p < 0.01, p < 0.05) expression in BeWo and JAR cells. Twist-1 downregulation decreased protein levels of p-PI3K (p < 0.01) and p-AKT (p < 0.01, p < 0.05) in BeWo and JAR cells. Silencing Twist-1 inhibits the malignant behavior of CCA cells, which may play a part by inhibiting the EMT process and the PI3K/AKT pathway.

Promyelocytic leukemia protein regulates angiogenesis and epithelial-mesenchymal transition to limit metastasis in MDA-MB-231 breast cancer cells.

Promyelocytic leukemia protein (PML) modulates diverse cell functions that contribute to both tumor-suppressor and pro-oncogenic effects, depending on the cellular context. We show here that PML knockdown (KD) in MDA-MB-231, but not MCF7, breast cancer cells, prolonged stem-cell-like survival, and increased cell proliferation and migration, which is in line with gene-enrichment results from their RNA sequencing analysis. Of note, increased migration was accompanied by higher levels of the epithelial-mesenchymal transition (EMT) regulator Twist-related protein 2 (TWIST2). We showed here that PML binds to TWIST2 via its basic helix-loop-helix (bHLH) region and functionally interferes with suppression of the epithelial target of TWIST2, CD24. In addition, PML ablation in MDA-MB-231 cells led to higher protein levels of hypoxia-inducible factor 1-alpha (HIF1a), resulting in a higher cell hypoxic response. Functionally, PML directly suppressed induction of the HIF1a target gene vascular endothelial growth factor A (VEGFa). In line with these results, tumor xenografts of MDA-MB-231 PML-KD cells had enhanced aggressive properties, including higher microvessel density, faster local growth and higher metastatic ability, with a preference for lung. Collectively, PML suppresses the cancer aggressive behavior by multiple mechanisms that impede both the HIF-hypoxia-angiogenic and EMT pathways.

Probing into the Network of Transcription Factor Twist-Related Protein 1 (TWIST1) in Breast Cancer Metastasis

Probing into the Network of Transcription Factor Twist-Related Protein 1 (TWIST1) in Breast Cancer Metastasis

Prognostic impact of tumor microenvironment-related markers in patients with adenocarcinoma of the lung

Cancer-associated fibroblasts (CAFs) are a prominent component in the tumor microenvironment (TME), which plays an important role in lung carcinogenesis. Here, we investigated microenvironmental markers expressed by CAFs, including α-smooth muscle actin, CD10, podoplanin, fibroblast-specific protein 1, platelet-derived growth factor α and β, fibroblast-associated protein, tenascin-C, zinc finger E-box binding homeobox 1 (ZEB1), and twist-related protein 1 expression levels. We evaluated samples from 257 patients with lung adenocarcinoma (LAD) to assess the associations of CAF-related protein expression patterns with prognosis. LAD cases were stratified using cluster analysis. To determine the utility of prognostic markers in LAD, univariate and multivariate analyses were performed. LAD cases were classified into subgroups 1 and 2. Subgroup 2 was shown to be significantly correlated with disease-free and overall survival using univariate and multivariate analyses in this group. Upregulation of podoplanin was identified as a single prognostic marker in this study by univariate and multivariate analyses. In addition, ZEB1 overexpression was correlated with disease-free survival. Our current results suggested that the specific CAF phenotype (e.g., the expression pattern of CAF-related proteins) could predict outcomes in patients with LAD. In addition, podoplanin upregulation may predict outcomes in these patients.

Twist1 as a target for prevention of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) represents an important clinical problem requiring novel approaches for both prevention and treatment. The transcription factor, Twist-related protein 1 (Twist1), has been identified as having a key mechanistic role in the development and progression of cSCC. Studies in relevant mouse models of cSCC have shown that Twist1 regulates epithelial-mesenchymal transition (EMT) and stemness driving progression and metastasis of cSCC. In addition, further research has shown that Twist1 regulates the balance between keratinocyte proliferation and differentiation and therefore impacts earlier stages of cSCC development. Through use of keratinocyte specific Twist1 knockout models, a role for this gene in keratinocyte stem cell homeostasis has been revealed. As a transcription factor, Twist1 regulates a large number of genes both in a positive, as well as a negative manner across several interdependent pathways. Studies in keratinocyte specific knockout models have shown that Twist1 upregulates the expression of genes involved in proliferation, stemness, and EMT while downregulating the expression of genes associated with differentiation. Furthermore, a number of compounds, including naturally occurring compounds, have been identified that target Twist1 and can block its effects in cancer cells and in keratinocytes in vivo. Collectively, the current understanding of Twist1 function in cSCC development and progression suggests that it represents a potential target for prevention and treatment of cSCC.

Suppression of the malignancy of mammary tumor in mice model by inactivated preparation of Mycobacterium obuense.

Breast cancer (BC) is a significant cause of global mortality in women. This study was aimed to evaluate the immune-activation of malignant BC via the administration of attenuated Mycobacterium obuense. For this purpose, an in vivo model was developed with BALB/c mice. Mice were injected with 2.00 × 106 4T1 cells with breast tumor cell line. Forty-two mice were equally divided into control as well as low dose (0.20 mg 100 µL-1) and high dose (0.50 mg 100 µL-1) groups of M. obuense to investigate gene expression in the antitumor effects of M. obuense. In one group, paclitaxel was administrated as a choice drug in BC treatment. Antitumor manners were characterized by cytotoxicity against tumor target cells, size of the tumor and the expression of some BC metastatic genes together with pathology. The MTT assay demonstrated that different concentrations of both low and a high dose of bacteria did present no cytotoxicity effect on 4T1 cells. According to our findings, M. obuense significantly repressed tumor growth. M. obuense downregulated the expression of collagen type I alpha 1 (COLIA1), cFos, alkaline phosphatase (ALP), claudin 3 (cldn3), and conversely, activated transcription factor 4 (ATF4) and Twist related protein-1 (Twist1). All these alternations induced a decrease in the migratory and invasive capabilities of BC. The result of pathology was indicative of tumor regression in the paclitaxel and HK- M. obuense -recipient group. Thus, it seems most likely that M. obuense might impinge upon cell growth and metastatic behavior of malignant cells exerting anti-tumor activity in BC.

Twist-related protein 1 induces epithelial-mesenchymal transition and renal fibrosis through the upregulation of complement 3.

We have demonstrated that complement 3 (C3) is upregulated and induces epithelial-mesenchymal transition (EMT) phenomenon and renal fibrosis in unilateral ureteral obstruction (UUO) kidney. We investigated roles of twist-related protein 1 (TWIST1) in EMT phenomenon and renal fibrosis through C3 upregulation in a mouse UUO model with gene silencer pyrrole-imidazole (PI) polyamides targeting TWIST1. We designed and synthesized PI polyamides targeting TWIST1 binding site on mouse pre-pro C3 promoter. Increased expression C3 mRNA with interferon-γ was significantly inhibited with PI polyamide in nephrotubular epithelial cells. Immunofluorescence showed suppression of E-cadherin and enhancement of α-smooth muscle actin (α-SMA) stainings as EMT phenomena in UUO kidney. TWIST1 and C3 expression was significantly increased in UUO kidney versus contralateral unobstructed kidney (CUK). Expression of transforming growth factor-β1 (TGF-β1), α-SMA and renin mRNAs was increased in UUO kidney versus CUK. Systemic administration of TWIST1 PI polyamide significantly suppressed increased C3 expression in UUO kidney versus CUK. PI polyamide administration also suppressed the increased expression of TGF-β1, α-SMA and renin mRNAs and histologically improved renal fibrosis in UUO kidney. These findings indicate that TWIST1 induces EMT phenomenon and renal fibrosis by TGF-β1 upregulation of C3 in mouse UUO model and that TWIST1 PI polyamide may be a novel medicine for renal fibrosis.

Analyses of selected tumour-associated factors expression in normotensive and preeclamptic placenta.

Human placenta is often considered a controlled-tumour because of shared properties such as invasion and angiogenesis. We assessed the status of a few selected tumour-associated factors (TAFs) in late onset pre-eclamptic (PE) and normotensive (NT) placentae, to understand their involvement in trophoblast invasion. These molecules include aldehyde dehydrogenase (ALDH3A1), aurora kinases (AURK-A/C), platelet derived growth factor receptor-α (PDGFRα), jagged-1 (JAG1) and twist related protein-1 (TWIST1). The expression of TAF was compared in 13 NT and 11 PE (late onset) placentae using immunoblotting/immunohistochemistry. We then used a novel spheroidal cell model developed from transformed human first trimester trophoblast cell lines HTR8/SVneo and TEV-1 to determine the expression and localization of these six factors during invasion. We also compared the expression of these TAFs during migration and invasion. Our results suggest that expressions of ALDH3A1, AURK-A, PDGFRα, and TWIST1 are significantly upregulated in PE placentae (p<0.05) when compared to NT placentae, whereas AURK-C and JAG1 are down-regulated (p<0.05). The protein expression pattern of all the six factors were found to be similar in spheroids in comparison to their parental counterparts. The invasive potential of the spheroids was also enhanced when compared with the parental cells. Collectively, data from our present study suggests that these TAFs are involved in placental invasion and their altered expressions may be regarded as a compensatory mechanism against reduced invasion.

Twist1 Promoter Methylation Regulates the Proliferation and Apoptosis of Acute Myeloid Leukemia Cells via PI3K/AKT Pathway.

Twist-related protein 1 (Twist1) is a widely recognized oncogene in acute myeloid leukemia (AML), and its promoter methylation is related with the progression of solid tumors. However, the association between Twist1 promoter methylation and AML has not been well studied. Twist1 mRNA expression was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The protein levels of Twist1 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signal were measured via western blotting. Methylation-specific PCR was performed to detect the methylation status of Twist1 promoter. CCK-8 assay and flow cytometry were used to reveal cellular biological effects. Twist1 expression and promoter methylation level were significantly upregulated in AML tissues and cell lines and were further downregulated in demethylating agent 5'-azacitidine (5-Aza)-treated cells. Ectopic expression of Twist1 increased AML cell viability, while reducing apoptosis, and attenuated the effects of 5-Aza on the proliferation and apoptosis. We also found that the PI3K/AKT signaling pathway was positively regulated by Twist1. Our findings revealed that Twist1 accelerates the tumorigenesis of AML cells by promoting its promoter methylation via the activation of PI3K/AKT signaling pathway.

Antiproliferative efficacy of the antioxidant bioactive compounds of defatted seeds of Azadirachta indica and Momordica charantia against the regulatory function of tumor suppressor gene inducing oral carcinoma

The present study focuses on the antiproliferative activity of polyphenolic flavonoids found in defatted seeds of Azadirachta indica and Momordica charantia with the regulatory function of tumor suppressor genes inducing Oral Squamous Cell Carcinoma. Polyphenolic flavonoid in extracts was characterized using chromatographic analysis and has confirmed the presence of quercetin, rutin and tannic acid in the extracts of A. indica and M. charantia. According to DPPH assay and reducing power assays, free radical scavenging was found to be high in ethanolic extract of defatted seeds. Antiproliferative efficacies of defatted seed extracts against KB cell line (mouth) were studied by MTT assay and revealed that aqueous extract of defatted seeds of M. charantia has exhibited maximum antiproliferative activity against KB cells. Antioxidant activity of defatted seed extracts were observed on treated KB cells by determining enzymatic activity (SOD, Cat, and GST) and nonenzyme content (GSH and MDA Content). Using the AutoDock tool, quercetin, rutin and tannin acid revealed that mutant p53, TWIST related protein, TGF-β and Snail I have the best binging energy results. MD simulation was observed on best docking results between the molecule and identified flavonoid by Desmond V 5.9 package . This leads to the conclusion that bioactive extracts with antiproliferative activity, antioxidant capacity and polyphenols with binding efficacy against tumor suppressor gene regulatory function could be used as a herbal remedy. Communicated by Ramaswamy H. Sarma

Overexpression of Twist1 in vascular endothelial cells promotes pathological retinal angiogenesis in mice.

Retinal angiogenesis is a critical process for normal retinal function. However, uncontrolled angiogenesis can lead to pathological neovascularization (NV), which is closely related to most irreversible blindness-causing retinal diseases. Understanding the molecular basis behind pathological NV is important for the treatment of related diseases. Twist-related protein 1 (TWIST1) is a well-known transcription factor and principal inducer of epithelial-mesenchymal transition (EMT) in many human cancers. Our previous study showed that Twist1 expression is elevated in pathological retinal NV. To date, however, the role of TWIST1 in retinal pathological angiogenesis remains to be elucidated. To study the role of TWIST1 in pathological retinal NV and identify specific molecular targets for antagonizing pathological NV, we generated an inducible vascular endothelial cell (EC)-specific Twist1 transgenic mouse model (Tg-Twist1iEC+). Whole-mount retinas from Tg-Twist1iEC+ mice showed retarded vascular progression and increased vascular density in the front end of the growing retinal vasculature, as well as aneurysm-like pathological retinal NV. Furthermore, overexpression of Twist1 in the ECs promoted cell proliferation but disturbed cell polarity, thus leading to uncontrolled retinal angiogenesis. TWIST1 promoted pathological NV by activating the Wnt/β-catenin signaling pathway and inducing the expression of NV formation-related genes, thereby acting as a ‘valve’ in the regulation of pathological angiogenesis. This study identified the critical role of TWIST1 in retinal pathological NV, thus providing a potential therapeutic target for pathological NV.

CEACAM6 promotes cholangiocarcinoma migration and invasion by inducing epithelial-mesenchymal transition through inhibition of the SRC/PI3K/AKT signaling pathway.

The immunoglobulin superfamily member carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is overexpressed in a wide variety of human cancer types, and is associated with tumor invasion and migration. The aim of the present study was to determine the role of CEACAM6 in cholangiocarcinoma (CCA) invasion and migration in vitro. The results showed that CEACAM6 was highly expressed in CCA tissues, and that the expression level of CEACAM6 was negatively associated with the degree of differentiation of CCA. Silencing CEACAM6 inhibited cell viability, invasion and migration but promoted cell apoptosis in a human CCA cell line (RBE). In addition, CEACAM6 knockdown decreased the expression of an antiapoptotic protein (Bcl-2), an interstitial cell marker (N-cadherin), extracellular matrix proteins (MMP-2 and MMP-9), a transcription factor helix protein (Twist-related protein 1), an intermediate tumor cell scaffold marker (vimentin), a protein involved in tumor nutrient vascular formation (VEGFA) and a tumorigenesis factor (intercellular cell adhesion molecule-1), but increased the expression of pro-apoptotic proteins (Bax, and cleaved caspases-3, −8 and −9) and an epithelial cell marker protein (E-cadherin). Furthermore, CEACAM6-small interfering RNA reduced the expression of the SRC/PI3K/AKT signaling transduction pathway. Taken together, these results suggested that CEACAM6 may be an epithelial-mesenchymal transition biomarker and a potential therapeutic target in human CCA.

Bisdemethoxycurcumin Promotes Apoptosis and Inhibits the Epithelial-Mesenchymal Transition through the Inhibition of the G-Protein-Coupled Receptor 161/Mammalian Target of Rapamycin Signaling Pathway in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is one of the leading causes of cancer death in the world and lacks an effective targeted therapy. G-protein-coupled receptor 161 (GPR161) has been demonstrated to perform the functional regulations on TNBC progression and might be a potential new target for TNBC therapy. This study showed the effects of bisdemethoxycurcumin (BDMC) on GPR161 regulation, indicating that BDMC effectively inhibited GPR161 expression and downregulated GPR161-driven signaling. BDMC showed the potent inhibitory effects on TNBC proliferation through suppressing GPR161-mediated mammalian target of rapamycin (mTOR)/70 kDa ribosomal protein S6 kinase (p70S6K) activation. Besides, in this study, we discover the mechanism of GPR161-driven TNBC metastasis, linking to GPR161-mediated twist-related protein 1 (Twist1)/matrix metallopeptidase 9 (MMP9) contributing to the epithelial-mesenchymal transition (EMT). BDMC effectively repressed GPR161-mediated TNBC metastasis via inhibiting Twist1/MMP9-induced EMT. The three-dimensional invasion assay also showed that BDMC significantly inhibited TNBC invasion. The combination treatment of BDMC and rapamycin enhanced the inhibition of TNBC proliferation and metastasis through increasing the blockage of mTOR activation. Furthermore, this study also observed that BDMC activated the caspase 3/9 signaling pathway to induce TNBC apoptosis. Therefore, BDMC could be applicable to anticancer therapy, especially targeting on the GPR161-driven cancer type.

Retraction notice to MicroRNA-539 Inhibits the Epithelial-Mesenchymal Transition of Esophageal Cancer Cells by Twist-Related Protein 1-Mediated Modulation of Melanoma-Associated Antigen A4 [Oncology Research 26(4) (2018) 529-536

Retraction notice to MicroRNA-539 Inhibits the Epithelial-Mesenchymal Transition of Esophageal Cancer Cells by Twist-Related Protein 1-Mediated Modulation of Melanoma-Associated Antigen A4 [Oncology Research 26(4) (2018) 529-536

SLC17A3 rs9379800 and Ischemic Stroke Susceptibility at the Northern Region of Malaysia

ObjectivesThe relationships of Paired Like Homeodomain 2 (PITX2), Ninjurin 2 (NINJ2), TWIST-Related Protein 1 (TWIST1), Ras Interacting Protein 1 (Rasip1), Solute Carrier Family 17 Member 3 (SLC17A3), Methylmalonyl Co-A Mutase (MUT) and Fer3 Like BHLH Transcription Factor (FERD3L) polymorphisms and gene expression with ischemic stroke have yet to be determined in Malaysia. Hence, this study aimed to explore the associations of single nucleotide polymorphisms (SNPs) and gene expression with ischemic stroke risk among population who resided at the Northern region of Malaysia. Materials and methodsStudy subjects including 216 ischemic stroke patients and 203 healthy controls were recruited upon obtaining ethical clearance. SNP genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism assays. Gene expression levels were quantified by real-time polymerase chain reaction assays. Statistical and genetic analyses were conducted with SPSS version 22.2, PLINK version 1.07 and multifactor dimensionality reduction software. ResultsStudy subjects with G allele, CG or GG genotypes of SLC17A3 rs9379800 demonstrated increased risk of ischemic stroke with the odds ratios ranging from 1.76-fold to 3.14-fold (p<0.05). When stratified study subjects according to the ethnicity, SLC17A3 rs9379800 G allele and CG genotype contributed to 2.14- and 2.96-fold of ischemic stroke risk among Malay population significantly, in the multivariate analysis (p<0.05). However, no significant associations were observed for PITX2, NINJ2, TWIST1, Rasip1, and MUT polymorphisms with ischemic stroke risk in the multivariate analysis for the pooled cases and controls as well as when stratified them according to the ethnicity. Lower mRNA expression levels of Rasip1, SLC17A3, MUT and FERD3L were observed among cases (p<0.05). After FDR adjustment, the mRNA level of SLC17A3 remained significantly associated with ischemic stroke among Malay population (q=0.034). ConclusionIn conclusion, this study suggests that SLC17A3 rs9379800 polymorphism and its gene expression contribute to significant ischemic stroke risk among Malaysian population, particularly the Malay who resided at the Northern Region of the country. Our findings can provide useful information for the future diagnosis, management and treatment of ischemic stroke patients.

Microenvironmental markers are correlated with lymph node metastasis in invasive submucosal colorectal cancer.

AimsRecent studies have shown that the microenvironment can include cancer cells and cancer‐associated fibroblasts (CAFs), and that both play important roles in the progression and metastasis of CRC. Here, we aimed to analyse the expression patterns of cancer cell‐ and CAF‐related proteins in submucosal invasive colorectal cancer (SiCRC) and whether such markers are correlated with lymph node metastasis (LNM).Methods and resultsQuantitative analysis was conducted for Ki‐67, p53, β‐catenin and matrix metalloproteinase‐7 (MMP7) to assess cancer cell markers. In addition, we examined CAF markers, including smooth muscle alpha‐actin (α‐SMA), CD10, podoplanin, fibroblast‐specific protein 1 (FSP‐1), platelet‐derived growth factor receptor (PDGFR)‐α, PDGFR‐β, adipocyte enhancer‐binding protein 1 (AEBP1), fibroblast‐associated protein 1 (FAP‐1), zinc finger E‐box binding homeobox 1 (ZEB1) and TWIST‐related protein 1 (TWIST1). In both cases, we conducted digital pathology with Aperio software. We also examined the expression patterns of biomarkers using hierarchical cluster analysis. Two subgroups were established based on the expression patterns of cancer cell‐ and CAF‐ related markers, and the associations of these subgroups with clinicopathological variables. In multivariate analysis, subgroup 2, which was characterised by high expression of Ki‐67, p53, FAP‐1, platelet‐derived growth factor receptor (PDGFR)‐α, PDGFR‐β and TWIST1, was correlated with LNM (P < 0.01). Next, we examined the associations of individual biomarkers with LNM. Multivariate analysis showed that high expression levels of Ki‐67 and FAP‐1 were significantly associated with LNM (P < 0.05).ConclusionsOur findings showed that expression patterns of cancer cell‐ and CAF‐related proteins may allow for stratification of patients into risk categories for LNM in SiCRC. In addition, Ki‐67‐ and FAP‐1‐expressing microenvironmental cells might be helpful for identification of correlations with LNM in SiCRC.

The Dual Effects of Silibinin on Human Pancreatic Cells

Objective: Silibinin is a flavonoid with antihepatotoxic properties, and exhibits pleiotropic anticancer effects. However, the molecular mechanisms responsible for its anticancer actions in pancreatic cancer cells, and the effects on such cells and normal pancreatic cells, remain unclear. The objective of this study was to determine the effect of silibinin on human pancreatic cancer cells and normal ductal cells. Methods: Human pancreatic cancer cells (MIA PaCa-2 and PANC-1) and normal ductal cells (hTERT-HPNE) were cultured with 0-400 μM silibinin for 48 h. Thereafter, the proliferation, invasion, apoptosis, and signaling pathways of the pancreatic cells were evaluated. Results: Silibinin significantly inhibited the proliferation, invasion, and spheroid formation of human pancreatic cancer cells in vitro in a dosedependent manner (p&lt;0.05). It also induced apoptosis in a dose-dependent manner. Western blot analysis showed that silibinin downregulated extracellular signaling-regulated kinase (ERK) and serine/threonine protein kinase (AKT) in human pancreatic cancer cells. It also upregulated microtubule associated protein 1 light chain 3 β (LC3B) and cleaved caspase-3 via c-Jun N-terminal kinases (JNK) signaling. On the other hand, silibinin increased the mRNA and protein levels of c-Jun, Twist-related protein 1, and Snail. It also decreased exogenous p53 levels, but increased endogenous c-Jun protein levels in human pancreatic cancer cells. However, silibinin did not affect cell viability and endogenous c-Jun levels in pancreatic normal ductal cells. It increased exogenous p53 levels, but decreased stemness-related gene expression in pancreatic normal ductal cells. Silibinin increased Ki-67 levels in pancreatic cancer cells, but decreased them in pancreatic normal ductal cells. Conclusion: Silibinin not only exerted anticancer effects by inhibiting AKTERK and JNK signaling, but also upregulated cancer stemness-related genes in human pancreatic cancer cells. These results suggest that silibinin should be used as a therapeutic agent for human pancreatic cancer with caution.