Expression Levels Of Vimentin Research Articles

Indoleamine 2,3-Dioxygenase Inhibitor Suppresses Colon Cancer Cell Migration, Invasion, and Epithelial-Mesenchymal Transition.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key enzyme in tryptophan metabolism and plays an important role in immunosuppression. The effects of IDO1 on tumor invasion and metastasis have been studied in several types of malignancies. However, the role of IDO1 in these steps in colorectal cancer (CRC) has not been elucidated. Therefore, we aimed to investigate the effects of IDO1 on invasion, migration, and epithelial-mesenchymal transition (EMT) in CRC cells. All experiments were performed using the DLD-1 colon cancer cell line that expresses IDO1. We conducted a scratch wound healing assay and Boyden chamber assay to investigate the impact of IDO1 on DLD-1 cell migration and invasion, respectively, in the presence and absence of the IDO1 inhibitor L-1-methyl-tryptophan (L-1-MT). Additionally, western blotting was performed to analyze alterations in the expression of EMT-related markers caused by L-1-MT. High expression of IDO1 was confirmed in the cytoplasm of DLD-1 by immunofluorescence staining. In the scratch wound healing assay, the invasion ability of DLD-1 cells decreased to 62% after treatment with L-1-MT at 1,000 μM for 24 h. In the Boyden chamber assay, the migration of DLD-1 cells was suppressed by 85% after treatment with L-1-MT at 2,500 μM for 24 h. L-1-MT treatment increased the expression level of E-cadherin and decreased the expression levels of vimentin, Snail, and Slug. IDO1 inhibition reduced the invasion and migration ability of IDO1-expressing DLD-1 colon cancer cells, which was accompanied by altered expression of EMT-related proteins. IDO1 could be a potential target for the treatment of advanced CRC.

Lathyrol reduces the RCC invasion and incidence of EMT via affecting the expression of AR and SPHK2 in RCC mice

ObjectiveTo investigate the effects of Lathyrol on the expression of androgen receptor (AR) and sphingosine kinase 2 (SPHK2) in renal cell carcinoma (RCC) mice and to further explore the mechanism by which Lathyrol inhibits the invasion and incidence of epithelial-mesenchymal transition (EMT).MethodsAn RCC xenograft mouse model was constructed, and the mice were randomly divided into a model group, an experiment group and a negative control group. The experiment group was intragastrically gavaged with Lathyrol solution (20 mg/kg), the model group was intragastrically gavaged with 0.9% NaCl (same volume as that used in the experiment group), and the negative control group was injected intraperitoneally with 2 mg/kg cisplatin aqueous solution. Changes in the body weight and tumor volume of the mice were recorded. Western blot (WB) was used to assess the protein expression levels of AR, p-AR, CYP17A1, PARP1, E-cadherin, N-cadherin, vimentin, α-SMA, β-catenin, and ZO-1. Protein expression levels of SPHK2, metal matrix protease 2 (MMP2), MMP9 and urokinase-type plasminogen activator (uPA) in tumor tissues were assessed by immunohistochemistry (IHC). AR expression in tumor tissues was assessed after immunofluorescence (IF) staining.ResultsAfter 14 days of drug administration, compared with that in the model group, the tumor volumes in the negative control and experiment groups were lower; the difference in tumor volume among the model, control and experiment groups was statistically significant (P < 0.05). The differences in body weight among the three groups were not statistically significant (P > 0.05). In the model group, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively increased, the protein expression levels of E-cadherin and ZO-1 were relatively reduced (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin, and α-SMA were relatively increased (P < 0.05). In the negative control and experiment groups, the protein expression levels of AR, p-AR, CYP17A1, SPHK2, and PARP1 were relatively decreased (P < 0.05), the protein expression levels of E-cadherin and ZO-1 were relatively increased (P < 0.05), and the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA were relatively decreased (P < 0.05).ConclusionLathyrol and cisplatin inhibit the proliferation of RCC xenografts, reduce the protein expression levels of AR, CYP17A1, SPHK2, PARP1, E-cadherin, and ZO-1 in tumor tissues (P < 0.05), and promote the protein expression levels of N-cadherin, β-catenin, vimentin and α-SMA (P < 0.05). Therefore, Lathyrol reduces RCC invasion and EMT by affecting the expression of AR and SPHK2 in RCC mice.

Huoxue Jiedu Huayu recipe inhibits macrophage-secreted vascular endothelial growth factor-a on angiogenesis and alleviates renal fibrosis in the contralateral kidneys of unilateral ureteral obstruction rats.

Male Wistar rats were randomly divided into 4 groups: the sham group, UUO group (180 d of left ureter ligation), UUO plus eplerenone (EPL) group, and UUO plus HJHR group. After 180 d of oral drug administration, blood and contralateral kidneys were collected for analysis. Angiogenesis- and fibrosis-related indexes were detected. HJHR and EPL improved structural damage and renal interstitial fibrosis in the contralateral kidney and reduced the protein expression levels of α-smooth muscle actin (α-SMA), vimentin and collagen I. Moreover, these treatments could reduce the expression of vascular endothelial growth factor-A (VEGFA) by inhibiting the infiltration of macrophages. Furthermore, HJHR and EPL significantly reduced the expression of CD34 and CD105 by downregulating VEGFA production, which inhibited angiogenesis. Finally, the coexpressions of CD34, CD105 and α-SMA were decreased in the HJHR and EPL groups, indicating that endothelial-to-mesenchymal transition was inhibited. These findings confirm that HJHR alleviates contralateral renal fibrosis by inhibiting VEGFA-induced angiogenesis, encourage the use of HJHR against renal interstitial fibrosis and provide a theoretical basis for the clinical management of patients with CKD.

Human amnion mesenchymal stem cells promote endometrial repair via paracrine, preferentially than transdifferentiation

BackgroundIntrauterine adhesion (IUA) is one of the most severe causes of infertility in women of childbearing age with injured endometrium secondary to uterine performance. Stem cell therapy is effective in treating damaged endometrium. The current reports mainly focus on the therapeutic effects of stem cells through paracrine or transdifferentiation, respectively. This study investigates whether paracrine or transdifferentiation occurs preferentially in treating IUA.MethodsHuman amniotic mesenchymal stem cells (hAMSCs) and transformed human endometrial stromal cells (THESCs) induced by transforming growth factor beta (TGF-β1) were co-cultured in vitro. The mRNA and protein expression levels of Fibronectin (FN), Collagen I, Cytokeratin19 (CK19), E-cadherin (E-cad) and Vimentin were detected by Quantitative real-time polymerase chain reaction (qPCR), Western blotting (WB) and Immunohistochemical staining (IHC). The Sprague-Dawley (SD) rats were used to establish the IUA model. hAMSCs, hAMSCs-conditional medium (hAMSCs-CM), and GFP-labeled hAMSCs were injected into intrauterine, respectively. The fibrotic area of the endometrium was evaluated by Masson staining. The number of endometrium glands was detected by hematoxylin and eosin (H&E). GFP-labeled hAMSCs were traced by immunofluorescence (IF). hAMSCs, combined with PPCNg (hAMSCs/PPCNg), were injected into the vagina, which was compared with intrauterine injection.ResultsqPCR and WB revealed that FN and Collagen I levels in IUA-THESCs decreased significantly after co-culturing with hAMSCs. Moreover, CK19, E-cad, and Vimentin expressions in hAMSCs showed no significant difference after co-culture for 2 days. 6 days after co-culture, CK19, E-cad and Vimentin expressions in hAMSCs were significantly changed. Histological assays showed increased endometrial glands and a remarkable decrease in the fibrotic area in the hAMSCs and hAMSCs-CM groups. However, these changes were not statistically different between the two groups. In vivo, fluorescence imaging revealed that GFP-hAMSCs were localized in the endometrial stroma and gradually underwent apoptosis. The effect of hAMSCs by vaginal injection was comparable to that by intrauterine injection assessed by H&E staining, MASSON staining and IHC.ConclusionsOur data demonstrated that hAMSCs promoted endometrial repair via paracrine, preferentially than transdifferentiation.

Bone morphogenetic protein-6 suppresses TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelium.

To evaluate the effect of bone morphogenetic protein-6 (BMP-6) on transforming growth factor (TGF)-β2-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE). Adult retinal pigment epithelial cell line (ARPE-19) were randomly divided into control, TGF-β2 (5 µg/L), and BMP-6 small interfering RNA (siRNA) group. The cell morphology was observed by microscopy, and the cell migration ability were detected by Transwell chamber. The EMT-related indexes and BMP-6 protein levels were detected by Western blotting. Furthermore, a BMP-6 overexpression plasmid was constructed and RPE cells were divided into the control group, TGF-β2+empty plasmid group, BMP-6 overexpression group, and TGF-β2+BMP-6 overexpression group. The EMT-related indexes and extracellular regulated protein kinases (ERK) protein levels were detected. Compared with the control group, the migration of RPE cells in the TGF-β2 group was significantly enhanced. TGF-β2 increased the protein expression levels of α-smooth muscle actin (α-SMA), fibronectin and vimentin but significantly decreased the protein levels of E-cadherin and BMP-6 (P<0.05) in RPE. Similarly, the migration of RPE cells in the BMP-6 siRNA group was also significantly enhanced. BMP-6 siRNA increased the protein expression levels of α-SMA, fibronectin and vimentin but significantly decreased the protein expression levels of E-cadherin (P<0.05). Overexpression of BMP-6 inhibited the migration of RPE cells induced by TGF-β2 and prevented TGF-β2 from affecting EMT-related biomarkers (P<0.05). BMP-6 prevents the EMT in RPE cells induced by TGF-β2, which may provide a theoretical basis for the prevention and treatment of proliferative vitreoretinopathy.

Neuroprotective effect of Withania somnifera leaves extract nanoemulsion against penconazole-induced neurotoxicity in albino rats via modulating TGF-β1/Smad2 signaling pathway

Penconazole (PEN) is a systemic triazole fungicide used to control various fungal diseases on grapes, stone fruits, cucurbits, and strawberries. Still, it leaves residues on treated crops after collection with many hazardous effects on population including neurotoxicity. Withania somnifera leaves extract (WSLE) is known for its memory and brain function enhancing ability. To evoke such action efficiently, WSLE bioactive metabolites are needed to cross the blood–brain barrier, that could limit the availability of such compounds to be localized within the brain. Therefore, in the present study, the association between PEN exposure and neurotoxicity was evaluated, and formulated WSLE nanoemulsion was investigated for improving the permeability of the plant extract across the blood–brain barrier. The rats were divided into five groups (n = 6). The control group was administered distilled water, group II was treated with W. somnifera leaves extract nanoemulsion (WSLE NE), group III received PEN, group IV received PEN and WSLE, and group V received PEN and WSLE NE. All rats were gavaged daily for 6 weeks. Characterization of compounds in WSLE using LC–MS/MS analysis was estimated. Neurobehavioral disorders were evaluated in all groups. Oxidative stress biomarkers, antioxidant enzyme activities, and inflammatory cytokines were measured in brain tissue. Furthermore, the gene expression patterns of GFAP, APP, vimentin, TGF-β1, Smad2 and Bax were measured. Histopathological changes and immunohistochemical expression in the peripheral sciatic nerve and cerebral cortex were evaluated. A total of 91 compounds of different chemo-types were detected and identified in WSLE in both ionization modes. Our data showed behavioral impairment in the PEN-treated group, with significant elevation of oxidative stress biomarkers, proinflammatory cytokines, neuronal damage, and apoptosis. In contrast, the PEN-treated group with WSLE NE showed marked improvement in behavioral performance and histopathological alteration with a significant increase in antioxidant enzyme activity and anti-inflammatory cytokines compared to the group administered WSLE alone. The PEN-treated group with WSLE NE in turn significantly downregulated the expression levels of GFAP, APP, vimentin, TGF-β1, Smad2 and Bax in brain tissue. In conclusion, WSLE NE markedly enhanced the permeability of plant extract constituents through the blood brain barrier to boost its neuroprotective effect against PEN-induced neurotoxicity.

Epithelial-Mesenchymal Transformation Promotes the Progression of Hepatocellular Carcinoma through NF-κB/MMP9 Axis.

Primary liver cancer (PHC) stands as one of the most prevalent malignant diseases in clinical settings. Studies have indicated that transcatheter arterial chemoembolization (TACE) treatment exhibits superior clinical outcomes, potentially increasing the complete necrosis rate in patients with PHC. A correlation exists between the clinical outcomes of TACE surgery and the process of epithelial-mesenchymal transition (EMT), yet the underlying mechanism remains a mystery. Hence, it is crucial to investigate the impact and mechanism of EMT on hepatocellular carcinoma (HCC). Retrospectively, patients with advanced liver cancer who underwent TACE were selected and categorized into two groups based on the assessment of clinical efficacy: the effective group and the ineffective group. The expression levels of nuclear factor-kappa B (NF-κB), matrix metalloproteinase 9 (MMP9), Ki-67, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), Vimentin, E-cadherin, and N-cadherin in tumor tissues were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). In vitro, Huh7 cells were cultured, and lentivirus infections were utilized to inhibit the overexpression of NF-κB and MMP9. The determination of EMT and cell viability was conducted through Cell Counting Kit-8 (CCK-8) assays, RT-PCR, and Western blot. Sixty patients diagnosed with advanced liver cancer were selected for the study. Based on their clinical outcomes, 30 patients with advanced hepatocellular carcinoma were categorized into the effective group, while the remaining 30 patients were categorized into the ineffective group. The results of the Western blot analysis indicated that, in comparison to the effective group, the expression levels of NF-κB, MMP9, Ki-67, Bcl-2, Vimentin, and N-cadherin were significantly higher in the tumor tissues of the ineffective group. Conversely, the expression of Bax and E-cadherin was notably lower in the effective group. Following the individual knockdown of NF-κB and MMP9, the cell experiments revealed a remarkable decrease in the expression levels of Ki-67, Bcl-2, Vimentin, and N-cadherin, whereas the expression of Bax and E-cadherin showed significant elevation (p < 0.05). Furthermore, there was a significant increase in cell viability and a decrease in cell apoptosis after the knockdown of NF-κB and MMP9. The NF-κB/MMP9 signaling axis serves as a pivotal regulator that fosters proliferation and impedes apoptosis in Huh7 cells by modulating the process of EMT.

Abstract 1819: Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer

Abstract Background: Esculetin is a naturally occurring dihydroxycoumarin with anti-inflammatory, antioxidant, and anti-tumor activities. Copper ion transport proteins, notably SLC31A1 and ATP7B, are essential in maintaining intracellular copper homeostasis, averting disruptions that might lead to copper-induced cell death under normal physiological conditions. However, prior research has not explored Esculetin's role in inducing copper-induced cell death. Hence, this study aims to investigate the biological process of Esculetin-induced cell death and the molecular mechanism underlying dysregulation of intracellular copper ion homeostasis. Methods: Initially, the CCK-8 assay was used to determine the IC50 values of Esculetin on colorectal cancer cell lines (HCT116, HT29, SW480) and normal colonic epithelial cell lines (NCM460). In terms of biological behaviors, the effects of Esculetin on the proliferation of colorectal cancer cell lines were assessed using CCK-8, colony formation assay, and EdU staining. Flow cytometry was used to examine the impact of Esculetin on apoptosis and cell cycle distribution in colorectal cancer cells. A subcutaneous xenograft tumor model in nude mice was employed to evaluate the in vivo anti-tumor effects of Esculetin, while IHC staining was conducted to detect Ki-67 levels, and Western blotting was performed to assess EMT-related protein expression. To investigate copper ion homeostasis, qPCR was used to measure the expression levels of copper ion transporters SLC31A1 and ATP7B before and after Esculetin treatment. Results: The CCK-8 results revealed that the IC50 of Esculetin for normal colonic epithelial cell lines was significantly higher than that for colorectal cancer cell lines. Meanwhile, a notable inhibitory effect of Esculetin on the proliferation of colorectal cancer cells was observed. Additionally, Esculetin treatment suppressed colony formation and reduced the proportion of EdU-positive cells in colorectal cancer cells. Flow cytometry demonstrated an increase in apoptosis and S-phase arrest following Esculetin treatment. The subcutaneous xenograft tumor model showed significant inhibition of tumor growth by Esculetin, as evidenced by reduced Ki-67-positive cells in the Esculetin-treated group. Western blot analysis revealed a significant decrease in the expression levels of Vimentin and N-Cadherin and an increase in E-Cadherin expression. Furthermore, qPCR analysis post-Esculetin treatment showed an elevation in the inward copper transporter SLC31A1 levels, while the outward transporter ATP7B expression decreased. Conclusion: Esculetin induces dysregulation of intracellular copper ion homeostasis by upregulating SLC31A1 expression and downregulating ATP7B expression, leading to intracellular copper accumulation and subsequent disruption of copper homeostasis, thereby inducing copper-induced cell death. Citation Format: Fangyue Guo, Mengying Liu, Kaihe Zhu, Jiapeng Ru, Pei Li, Shanfeng Zhang. Esculetin modulates SLC31A1 and ATP7B to influence copper homeostasis and trigger cell death in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1819.

Abstract 4232: Exploring chemotherapy resistance in pancreatic cancer: Insights from a 3D organoid model

Abstract Pancreatic cancer is marked by a dismal prognosis, with a 5-year survival rate at approximately 10%. A key contributor to its lethality is the development of chemotherapy resistance, followed by early metastasis. However, studies elucidating the mechanisms behind chemotherapy resistance in pancreatic cancer are limited. Previous research has relied on 2D cell line models, which present limitations, such as uniform cell types that fail to capture tumor heterogeneity and an inaccurate representation of clinical realities, wherein resistance often develops gradually rather than through continuous exposure to high drug concentrations. In this study, we developed a more sophisticated organoid model of chemotherapy resistance. Six organoids from three patients were matched to study chemotherapy resistance in a personalized context. Comprehensive whole-exome sequencing and transcriptomic analyses were performed, along with an examination of chemotherapy resistance protein expression and EMT-related markers. Notably, differential expression levels of CK19 and Vimentin were observed between samples. Our analysis also focused on the expression of the ATP-Binding Cassette Subfamily G Member 2 (ABCG2) gene in chemoresistant organoids. Western blot analysis indicated a significant increase in ABCG2 expression in these organoids compared to controls. According to the Moffitt RNA signature, the primary and corresponding chemoresistant organoids retained their molecular subtype classifications both before and after developing chemoresistance. Gene set enrichment analysis uncovered significant upregulation of pathways related to DNA repair and cell cycle regulation, particularly the G2M checkpoint, in chemoresistant organoids. Furthermore, gene ontology functional analysis suggested a notable overrepresentation of activities related to amino acid transmembrane transport and extracellular matrix constituents, underscoring their potential role in the chemoresistance phenotype. Citation Format: Hee Seung Lee. Exploring chemotherapy resistance in pancreatic cancer: Insights from a 3D organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4232.

Regulatory mechanism of the Glabrene against non-small cell lung cancer by suppressing FGFR3.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with limited effective treatment options. This study aimed to investigate the regulatory mechanism of Glabrene on NSCLC through its interaction with FGFR3. HCC827 cells were implanted into nude mice and treated with Glabrene. Tumor volume was monitored at 0, 3, 6, and 9 days after medical treatment. Tissue analysis included Hematoxylin and Eosin (HE) and Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) staining, as well as immunohistochemistry for Ki67, ERK1/2, and p-ERK1/2 expression. Cell viability was determined with the CCK8 method. We utilized immunofluorescence techniques to observe apoptosis, as well as the levels of E-cadherin and Vimentin expression. Cellular proliferation was determined via plate cloning assay and cellular mobility was determined via scratch assay. Cellular invasion ability was assessed via a transwell assay. mRNA and protein levels of FGFR3, MMP1, MMP9, vimentin, E-cadherin, ERK1/2, and p-ERK1/2 were detected via qPCR and Western blot. IGF-1, VEGF, and Estradiol (E2) levels were measured through Enzyme linked immunosorbent assay (ELISA). This study verified that Glabrene was capable of suppressing tumor growth in NSCLC mice, reversing tumor tissue's pathological morphology, attenuating the capacities of cancerous cells' proliferation, migration, and invasion, and leading to apoptosis. Besides, Glabrene could reduce the FGFR3 expression in HCC827 cells. Over-expression of FGFR3 promotes the proliferation of HCC827 cells, increase both contents of IGF-1, VEGF, and E2, and expressions of MMP1, MMP9, vimentin, and p-ERK1/2, while Glabrene inhibited FGFR3. Glabrene, and inhibition of FGFR3 expression were capable of decreasing FGFR3, MMP1, MMP9, vimentin, and p-ERK1/2 expression, as well as contents of IGF-1, VEGF, and E2 in model mice and HCC827 cells, and promoting the expression of E-cadherin. Glabrene has the potential as a therapeutic agent for NSCLC by reducing cancer invasion and migration through the inhibition of ERK1/2 phosphorylation and suppression of epithelial-mesenchymal transition (EMT).

Abstract 4208: In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes

Abstract Preclinical models of Sonic Hedgehog (SHH)-driven tumors support the therapeutic benefit of pharmacological inhibitors, yet clinical outcomes are conflicted due to biphasic tumor responses that include complete remission or faster disease progression. In such studies, the activity of the SHH pathway in the tumor-adjacent stroma supported or restrained tumor progression. Our prior studies show that altered SHH ligand levels can lead to faster tumor growth during pharmacological inhibition suggesting a role for the SHH pathway strength in the biphasic tumor responses of a triple-negative breast cancer (TNBC) model. In this study, the role of SHH pathway strength altered by tumor-fibroblast proximity was examined on the tumor growth, invasion, and transcriptional activity using in vitro and in vivo models. Tumor spheroid models composed of TNBC cell lines and fibroblasts were used to monitor SHH signal strength as a function of the culture modality and SHH ligand levels. In vitro, SHH signal strength was altered by tumor-fibroblast proximity and ligand concentration. Mixed cultures exhibited significantly higher SHH activity and reduced invasion than adjacent cultures. This effect was reversed by pharmacological inhibition of SMO, confirming the involvement of an SHH-dependent mechanism. In a tumor xenograft model, tumor metastasis outcomes were suppressed by pharmacological inhibition of the SHH pathway, but opposite results were observed in tumor xenografts composed of co-injected with fibroblasts indicating that the biphasic tumor response can be driven by the fibroblasts. Spatial transcriptional analysis identified extracellular matrix (ECM) activities in fibroblasts as the top altered bioprocesses in both in vitro and in vivo. This finding was confirmed in cultures, where fibroblast-derived decellularized matrix stimulated tumor invasion of single TNBC spheroids at levels observed in co-cultures. Further analysis of differentially expressed genes (DEGs) identified a small leucine-rich repeat proteoglycan (SLRP) as the primary ECM target altered by variations in SHH signal strength in fibroblasts. Furthermore, we confirmed the role of SLRP cues in governing tumor invasion potency within spheroid cultures embedded in a collagen hydrogel matrix. While high levels of SLRP in the matrix inhibited spheroid invasion, low SLRP matrix promoted spheroid invasion and increased secondary invasion sites. These findings agreed with the Vimentin expression levels observed in TNBC cells and confirmed the potency of SLRP in regulating tumor invasion outcomes. In summary, our study underscores the significance of ECM remodeling activities in fibroblasts and the role of SLRPs in governing tumor invasion, with implications for understanding the impact of altering SHH signaling activity in TNBC. Citation Format: Emanuel A. Carrasquillo-Dones, Heizel M. Rosado-Galindo, Ana M. Reyes-Ramos, Wandaliz Torres-Garcia, Said Cifuentes, Jan P. Rios-Grant, Gabriela Ortiz-Soto, Natalia A. Ramos-Acevedo, Miosotis Acevedo-Esquilin, Monica Colon-Vargas, Israel Almodovar-Rivera, Camilo Mora, Michelle M. Martinez-Montemayor, Maribella Domenech. In-vitro modeling of Sonic Hedgehog signaling identifies the extracellular matrix as a regulator of tumor invasion outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4208.

Expression and significance of SIRT6 in human peritoneal dialysis effluents and peritoneal mesothelial cells

Sirtuin 6 (SIRT6) can inhibit the fibrosis of many organs. However, the relationship between SIRT6 and peritoneal fibrosis (PF) in peritoneal dialysis (PD) remains unclear. We collected 110 PD patients with a duration of PD for more than 3 months and studied the influence of PD duration and history of peritonitis on SIRT6 levels in PD effluents (PDEs). We also analyzed the relationship between SIRT6 levels in PDEs and transforming growth factor beta 1 (TGF-β1), IL-6, PD duration, peritoneal function, PD ultrafiltration (UF), and glucose exposure. We extracted human peritoneal mesothelial cells (HPMCs) from PDEs and measured the protein and gene expression levels of SIRT6, E-cadherin, vimentin, and TGF-β1 in these cells. Based on the clinical results, we used human peritoneal mesothelial cells lines (HMrSV5) to observe the changes in SIRT6 levels and mesothelial-to-mesenchymal transition (MMT) after intervention with PD fluid. By overexpressing and knocking down SIRT6 expression, we investigated the effect of SIRT6 expression on E-cadherin, vimentin, and TGF-β1 expression to elucidate the role of SIRT6 in mesothelial-to-epithelial transition in PMCs. Results: (1) With the extension of PD duration, the influence of infection on SIRT6 levels in PDEs increased. Patients with the PD duration of more than 5 years and a history of peritonitis had the lowest SIRT6 levels. (2) SIRT6 levels in PDEs were negatively correlated with PD duration, total glucose exposure, TGF-β1, IL-6 levels, and the dialysate-to-plasma ratio of creatinine (Cr4hD/P), but positively correlated with UF. This indicates that SIRT6 has a protective effect on the peritoneum. (3) The short-term group (PD ≤ 1 year) had higher SIRT6 and E-cadherin gene and protein levels than the mid-term group (1 year < PD ≤ 5 years) and long-term group (PD > 5 years) in PMCs, while vimentin and TGF-β1 levels were lower in the mid-term group and long-term group. Patients with a history of peritonitis had lower SIRT6 and E-cadherin levels than those without such a history. (4) After 4.25% PD fluid intervention for HPMCs, longer intervention time resulted in lower SIRT6 levels. (5) Overexpressing SIRT6 can lead to increased E-cadherin expression and decreased vimentin and TGF-β1 expression in HPMCs. Knocking down SIRT6 expression resulted in decreased E-cadherin expression and increased vimentin and TGF-β1 expression in HPMCs. This indicates that SIRT6 expression can inhibit MMT in HPMCs, alleviate PF associated with PD, and have a protective effect on the peritoneum.

Plumbagin Regulates Snail to Inhibit Hepatocellular Carcinoma Epithelial-Mesenchymal Transition in vivo and in vitro.

Plumbagin (PL) has been shown to effe ctively inhibit autophagy, suppressing invasion and migration of hepatocellular carcinoma (HCC) cells. However, the specific mechanism remains unclear. This study aimed to investigate the effect of PL on tumor growth factor (TGF)-β-induced epithelial-mesenchymal transition (EMT) in HCC. Huh-7 cells were cultured, and in vivo models of EMT and HCC-associated lung metastasis were developed through tail vein and in situ injections of tumor cells. In vivo imaging and hematoxylin and eosin staining were used to evaluate HCC modeling and lung metastasis. After PL intervention, the expression levels of Snail, vimentin, E-cadherin, and N-cadherin in the liver were evaluated through immunohistochemistry and Western blot. An in vitro TGF-β-induced cell EMT model was used to detect Snail, vimentin, E-cadherin, and N-cadherin mRNA levels through a polymerase chain reaction. Their protein levels were detected by immunofluorescence staining and Western blot. In vivo experiments demonstrated that PL significantly reduced the expression of Snail, vimentin, and N-cadherin, while increasing the expression of E-cadherin at the protein levels, effectively inhibiting HCC and lung metastasis. In vitro experiments confirmed that PL up-regulated epithelial cell markers, down-regulated mesenchymal cell markers, and inhibited EMT levels in HCC cells. PL inhibits Snail expression, up-regulates E-cadherin expression, and down-regulates N-cadherin and vimentin expression, preventing EMT in HCC cells and reducing lung metastasis.

MiR-128-3p inhibits the proliferation, migration and invasion of human nasopharyngeal carcinoma cells via EMT

Purpose: To investigate the effect of overexpressing miR-128-3p on the migration, invasion, and proliferation of human nasopharyngeal cancer cells, and associated pathways.Methods: Cell counting kit-8 (CCK-8) and clone creation tests were used to investigate the effect of miR-128-3p inhibitor on the growth of C666-1 cells. The inhibitor control and miR-128-3p inhibitor were transfected into human nasopharyngeal cancer cells (C666-1) and allowed to incubate for 48 h. Cell migration and invasion assays were conducted using Transwell and Scratch assays. Cell cycle was assessed using propidium iodide (PI) single-staining method, while expression of miR-128-3p was evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expression levels of vimentin, E-cadherin, and N-cadherin were determined by Western blot.Results: MiR-128-3p inhibitor transfection in C666-1 cells significantly increased cell proliferation, cell viability, and clonal proliferation, and also significantly reduced miR-128-3p expression levels, and enhanced cell migration and invasion. In C666-1 cells, inhibition of miR-128-3p induced epithelial mesenchymal transition (EMT).Conclusion: MiR-128-3p inhibits nasopharyngeal cancer cells from proliferating, migrating, and invading by regulating epithelial mesenchymal transition. MiR-128-3p may therefore be a viable therapeutic target for nasopharyngeal cancer. Future studies are required to yield pertinent data in support of this hypothesis.

ZNF692 promotes cell proliferation, invasion and migration of human prostate cancer cells by targeting the EMT signaling pathway

BackgroundProstate cancer poses a considerable threat to human health. At present, the mechanism of tumor progression remains unclear. ZNF692 is overexpressed in many tumors, and the high expression of ZNF692 is correlated with tumor aggressiveness and tumor phenotype of prostate cancer, suggesting that ZNF692 may play an important role in tumor biology of prostate cancer. This paper aims to elucidate the relationship between them.MethodsThe expression level of ZNF692 was verified in normal prostate cells (RWPE-1) and prostate cancer cells (LNCaP, PC3, DU145). PC3 cells were selected to construct the ZNF692 knockout prostate cancer cell line. The changes of cell proliferation, apoptosis, invasion and metastasis were detected by CCK8, Edu staining, Transwell assay and scratch assay. The expression levels of related proteins were detected by Western blot.ResultsAt the cellular level, ZNF692 was overexpressed to varying degrees in prostate cancer cell lines, with the highest expression in PC3 cell lines. CCK8 and Edu results showed that the proliferation of prostate cancer PC3 cells that knocked down ZNF692 was slowed. Transwell assay and scratch assay showed reduced invasion and migration of prostate cancer PC3 cells that knocked out ZNF692. Flow cytometry showed that the apoptosis rate of prostate cancer PC3 cells after ZNF692 knockout was increased. In addition, after ZNF692 silencing, the expression level of epithelial phenotype E-cadherin increased in PC3 cells, while the expression level of interstitial phenotype N-cadherin, Vimentin, c-Myc, and CyclinA1 decreased. The state of prostate cancer PC3 cells that overexpressed ZNF692 was reversed from the state after ZNF692 was knocked down.ConclusionZNF692 can be used as a new prognostic marker and a potential biologic therapeutic target for PCa. By inhibiting the expression of c-myc and cyclinA1, the EMT signaling pathway is regulated to provide evidence for its potential molecular mechanism.

HMGB1 enhances the migratory and invasive abilities of A2780/DDP cells by facilitating epithelial to mesenchymal transition via GSK‑3β.

The aim of the present study was to investigate the impact and mechanism of high mobility group box 1 (HMGB1) on the regulation of cell migration and invasion in A2780/DDP cisplatin-resistant ovarian cancer cells. After transfecting small interfering (si)RNA-HMGB1 into A2780/DDP cells, Transwell migration and invasion assays were conducted to assess alterations in the cell migratory and invasive abilities. Additionally, western blotting analyses were performed to examine changes in HMGB1, phosphorylated (p)-GSK-3β, GSK-3β, E-cadherin and vimentin expression levels. The results of the present study demonstrated that the migratory and invasive abilities of A2780/DDP cells were significantly higher compared with those of A2780 cells. Additionally, the expression levels of HMGB1, p-GSK-3β and the mesenchymal phenotype marker, vimentin, in A2780/DDP cells were significantly elevated relative to the levels in A2780 cells. Conversely, the expression level of the epithelial phenotype marker, E-cadherin, was markedly decreased compared with that in A2780 cells. Following transfection of A2780/DDP cells with siRNA-HMGB1, there was a significant reduction in the rate of cell migration and invasion. Simultaneously, the expression levels of HMGB1, p-GSK-3β and vimentin were downregulated while the level of E-cadherin was upregulated. It was therefore concluded that the high expression of HMGB1 in A2780/DDP cells enhanced the cell migration and invasion abilities by facilitating epithelial to mesenchymal transition via GSK-3β.

Leech extract alleviates idiopathic pulmonary fibrosis by TGF-β1/Smad3 signaling pathway

Ethnopharmacological relevanceLeech, as a traditional Chinese medicine for the treatment of blood circulation and blood stasis, was also widely used to cure pulmonary fibrosis in China. In clinical practice, some traditional Chinese medicine preparation such as Shui Zhi Xuan Bi Hua Xian Tang and Shui Zhi Tong Luo Capsule composed of leech, could improve the clinical symptoms and pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). However, the material basis of the leech in the treatment of IPF were not yet clear. Aim of the studyScreen out the components of leech that have the anti-pulmonary fibrosis effects, and further explore the therapeutic mechanism of the active components. Materials and methodsIn this study, the different molecular weight components of leech extract samples were prepared using the semi-permeable membranes with different pore sizes. The therapeutic effects of the leech extract groups with molecular weight greater than 10 KDa (>10 KDa group), between 3 KDa and 10 KDa (3–10 KDa group), and less than 3 KDa (<3 KDa group) on pulmonary fibrosis were firstly investigated by cell proliferation and cytotoxicity assay (MTT), cell wound healing assay, immunofluorescence staining (IF) and Western blot (WB) assay through the TGF-β1-induced fibroblast cell model. Then bleomycin-induced pulmonary fibrosis (BML-induced PF) mouse model was constructed to investigate the pharmacological activities of the active component group of leech extract in vivo. Pathological changes of the mouse lung were observed by hematoxylin-eosin staining (H&E) and Masson's trichrome staining (Masson). The hydroxyproline (HYP) content of lung tissues was quantified by HYP detection kit. The levels of extracellular matrix-related fibronectin (FN) and collagen type Ⅰ (Collagen Ⅰ), pyruvate kinase M2 (PKM2) monomer and Smad7 protein were determined via WB method. PKM2 and Smad7 protein were further characterized by IF assays. ResultsUsing TGF-β1-induced HFL1 cell line as a PF cell model, the in vitro results demonstrated that the >10 KDa group could significantly inhibited the cell proliferation and migration, downregulated the expression level of cytoskeletal protein vimentin and α-smooth muscle actin (α-SMA), and reduced the deposition of FN and Collagen Ⅰ. In the BML-induced PF mouse model, the >10 KDa group significantly reduced the content of HYP, downregulated the expression levels of FN and Collagen Ⅰ in lung tissues, and delayed the pathological changes of lung tissue structure. The results of WB and IF assays further indicated that the >10 KDa group could up-regulate the expression level of PKM2 monomer and Smad7 protein in the cellular level, thereby delaying the progression of pulmonary fibrosis. ConclusionsOur study revealed that the >10 KDa group was the main material basis of the leech extract that inhibited pulmonary fibrosis through TGF-β1/Smad3 signaling pathway.

Mechanism of Hirudin-Mediated Inhibition of Proliferation in Ovarian Cancer Cells.

To investigate the inhibitory effect of hirudin on the cell proliferation of human ovarian cancer A2780 cells by preventing thrombin and its underlying molecular mechanism. Cell Counting Kit-8 (CCK-8) method was used to detect the effect of different concentrations of hirudin and thrombin on the cell proliferation of A2780 cells. PAR-1 wild-type overexpression plasmid was constructed utilizing enzyme digestion identification, and it was transferred to A2780 cells. Sequencing and Western blot were used to detect the changes in PAR-1 protein expression. Western blot detection of PKCα protein phosphorylation in A2780 cells was performed. We also implemented quantitative PCR to detect the mRNA expression levels of epithelial-mesenchymal transition (EMT)-related genes, CDH2, Snail, and Vimentin, in A2780 cells. 1μg/ml hirudin treatment maximally inhibited the promotion of A2780 cell proliferation by thrombin. Hirudin inhibited the binding of thrombin to the N-terminus of PAR-1, hindered PKCα protein phosphorylation in A2780 cells, and downregulated the mRNA expression levels of CDH2, Snail, and Vimentin. In conclusion, hirudin inhibits the cell proliferation of ovarian cancer A2780 cells, and the underlying mechanism may be through downregulating the transcription level of EMT genes, CDH2, Snail, and Vimentin. This study indicates that hirudin may have a therapeutic potential as an anti-cancer agent for ovarian cancer.

Serine/threonine kinase 36 induced epithelial-mesenchymal transition promotes docetaxel resistance in prostate cancer

To investigate the role and potential mechanism of serine/threonine kinase 36 (STK36) in docetaxel resistance-prostate cancer (PCa). The expression of STK36 in PCa and the correlation with clinicopathological characteristics of PCa patients were analyzed using the data from different databases and tissue microarrays. To investigate the role of STK36 on cell proliferation, invasion, and migration, STK36 was overexpressed and silenced in DU-145 and PC-3 cell lines. Cell counting kit-8 (CCK8) was used to test cell proliferation. Cell invasion and migration were detected by cell wound scratch assay and trans well, respectively. The expression profile of STK36, E-Cadherin, and Vimentin was analyzed by Western blot. Cell apoptosis was detected by the TUNEL assay. STK36 expression was upregulated in PCa tissue compared with adjacent benign PCa tissue; it was higher in patients with advanced stages compared with lower stages and was significantly correlated with decreased overall survival. Up-regulation of STK36 significantly promoted the proliferation, invasion, and migration of DU-145 and PC-3 cells and compensated for the suppression caused by docetaxel treatment in vitro. A striking apoptosis inhibition could be observed when dealing with docetaxel, although the apoptosis of DU-145 and PC-3 cells was not affected by the STK36 exclusive overexpression. Besides, E-Cadherin expression was restrained while the expression levels of vimentin were all enhanced. The knockdown of STK36 reversed the above process. STK36 up-regulation could accelerate the biological behavior and docetaxel resistance of PCa by epithelial-mesenchymal transition (EMT) activation. STK36 may be potentially used as a target in PCa resolvent with docetaxel.

MiR-383-5p inhibits the proliferation and migration of lung adenocarcinoma cells by targeting SHMT2.

Purpose: To explore the effects of miR-383-5p and serine hydroxymethyltransferase 2 (SHMT2) on the proliferation and migration of lung adenocarcinoma cells. Methods: SHMT2 expression in lung adenocarcinoma and normal tissues was investigated using The Cancer Genome Atlas database. Immunohistochemical analysis was performed to confirm SHMT2 expression in lung adenocarcinoma and adjacent normal lung tissues. Bioinformatics analysis and luciferase reporter assays were used to analyze the relationship between miR-383-5p and SHMT2 expression. The protein expression levels of SHMT2, vimentin, N-cadherin, E-cadherin, Bcl-2, and cyclinD1 were analyzed using western blotting. The reverse transcription-quantitative polymerase chain reaction was used to detect SHMT2 knockdown efficiency, miR-383-5p overexpression, and inhibition efficiency. The proliferative ability of cells was detected using the Cell Counting Kit-8 assay. The Transwell assay was used to detect the migration ability of cells. Results: SHMT2 expression was significantly increased in patients with lung adenocarcinoma compared to that in control patients; the higher the SHMT2 expression the worse the outcomes were in patients with lung adenocarcinoma. SHMT2 knockdown inhibited the proliferation, migration, and epithelial-mesenchymal transition of lung adenocarcinoma A549 and H1299 cells. MiR-383-5p directly targeted and downregulated SHMT2 in A549 and H1299 cells. The effects of miRNA-383-5p on the proliferation and migration of these cells differed from those of SHMT2. Exogenous overexpression of SHMT2 reversed the miR-383-5p-induced proliferation and migration inhibition in A549 and H1299 cells. Conclusion: MiR-383-5p inhibits the proliferation and migration of lung adenocarcinoma cells by targeting and downregulating SHMT2.