Migration Of T24 Cells Research Articles

The effects of modified RNA-binding proteins HuR on the biological behavior of the bladder cancer T24 cell line.

BackgroundIn tumors, the role of human antigen R (HuR) includes regulating tumor cell proliferation, differentiation, apoptosis, angiogenesis, and lymphangiogenesis. Previous studies have revealed that the expression of HuR can be detected in bladder cancer, and is related to the biological behavior of malignancy.MethodsT24 cells were transfected by HuR overexpression and HuR knockdown vectors, and divided into the control group, the overexpression-HuR group, and the cas9-HuR group. Cell viability was detected after 48 h by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, apoptosis was detected by Annexin V-allophycocyanin (APC)/7-aminoactinomycin D (7-AAD) double staining, cell migration was detected by Transwell assays, and the expression levels of HuR, cyclin D1, and apoptosis-related factors [i.e., B-cell lymphoma 2 (Bcl-2)] were detected by fluorescence quantitative polymerase chain reaction (PCR) and Western blot.ResultsCompared to the control group, cell viability after 48 h increased significantly in the overexpression-HuR group, and decreased significantly in the cas9-HuR group (P<0.05). The number of migrating cells increased significantly in the overexpression-HuR group, and decreased significantly in the cas9-HuR group (P<0.05). The apoptosis rate was significantly decreased in the overexpression-HuR group, and significantly increased in the cas9-HuR group (P<0.05). The messenger ribonucleic acid and protein expression levels of HuR, cyclin D1, and Bcl-2 were significantly increased in the overexpression-HuR group, and significantly decreased in the cas9-HuR group (P<0.05).ConclusionsHuR promotes the proliferation and migration of T24 cells, and inhibits cell apoptosis. The mechanism may be related to the expression of cyclin D and the apoptosis-related protein, Bcl-2.

Synthesis and biological evaluation of indazole derivatives as anti-cancer agents.

Several FDA approved small molecule anti-cancer drugs contain indazole scaffolds. Here, we report the design, synthesis and biological evaluation of a series of indazole derivatives. In vitro antiproliferative activity screening showed that compound 2f had potent growth inhibitory activity against several cancer cell lines (IC50 = 0.23–1.15 μM). Treatment of the breast cancer cell line 4T1 with 2f inhibited cell proliferation and colony formation. 2f dose-dependently promoted the apoptosis of 4T1 cells, which was connected with the upregulation of cleaved caspase-3 and Bax, and downregulation of Bcl-2. 2f also decreased the mitochondrial membrane potential and increased the levels of reactive oxygen species (ROS) in 4T1 cells. Additionally, treatment with 2f disrupted 4T1 cells migration and invasion, and the reduction of matrix metalloproteinase metalloproteinase-9 (MMP9) and increase of tissue inhibitor matrix metalloproteinase 2 (TIMP2) were also observed. Moreover, 2f could suppress the growth of the 4T1 tumor model without obvious side effects in vivo. Taken together, these results identified 2f as a potential small molecule anti-cancer agent.

Anti-tumor effects of isoliquiritigenin in Bcl-2/Bax and PCNA expression of T24 human bladder cancer cells

IntroductionThe aim of this study was to investigate the proliferation and apoptosis of T24 after treatment with isoliquiritigenin (ISL) and to explore the underlying mechanism.Material and methodsT24 cells were divided into 6 groups and cultured. The five experimental groups were seeded into medium with 10, 20, 40, 80, and 160 µM ISL and the control group was administered with 0.01% DMSO. The cell morphology was observed using an optical microscope. The proliferation of cells and half maximal inhibitory concentration (IC50) were determined and calculated by MTS. Cell cycle and apoptosis were evaluated by flow cytometry. The migration ability of cells was detected by wound healing assay. The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and proliferating cell nuclear antigen (PCNA) was determined by RT-qPCR and western blot.ResultsAt 24 h after treatment with ISL, morphology revealed that cell number diminished and the cell volume shrank with increasing concentration. According to the results of MTS, ISL significantly inhibited the T24 cell proliferation in a dose- and time-dependent manner. Flow cytometry showed that ISL could block the replication of T24 cell DNA from S phase to G2, therefore promoting cell apoptosis. Wound healing assay showed that at 48 h after treatment with ISL, the migration of T24 cells was remarkably inhibited. The results of RT-qPCR and Western blot revealed that after 24 h of treatment, the expression levels of Bcl-2 and PCNA were down-regulated, and the expression level of Bax was increased in the different dose of the ISL treated group.ConclusionsISL possesses detrimental effects on the viability of BC cell T24 in a dose-dependent manner via blocking the cell cycle and inducing apoptosis by down-regulation of PCNA and Bcl-2 expression and up-regulation of Bax expression.

Fingerroot (Boesenbergia pandurata) Extract Inhibits Proliferation and Migration of 4T1 Metastatic Breast Cancer Cells

Fingerroot (Boesenbergia pandurata) is an Indonesian herb, with anti-proliferation and anti-migratory effects against several cancer cells. This study aims to investigate the anticancer property of Fingerroot Extract (FE) in combination with doxorubicin (Dox) against 4T1, a metastatic breast cancer cell lines. FE was prepared by 96% ethanol maceration and characterized by thin-layer chromatography analysis. FE was subjected to a cytotoxicity test with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay alone or in combination with 10 nM Dox against 4T1 cells. Cytotoxic effect was then confirmed by measure reactive oxygen species (ROS) intracellular level using 2’,7’-dichloroflourescin diacetate (DCFDA)-staining flow cytometry-based assay. The anti-migratory effect was observed using scratch wound healing assay and gelatin zymography to investigate matrix metalloproteinase (MMP)-9 expression. FE showed a cytotoxic effect with an inhibitory concentration 50 (IC50) value of 25.5±3.9 μg/mL and performed an improved effect in combination with 10 nM Dox. A single treatment of FE decreased ROS intracellular level, while in combination with Dox, FE increased the ROS intracellular level. Further, at 42 h observation, FE and its combination with Dox inhibited the migration of 4T1 cells with % closure of 82.6 and 82.5, respectively, correlates with a significant decrease of MMP-9 expression. Overall, FE performs a cytotoxic activity and anti-migration activity on 4T1 breast cancer cells.Keywords: Boesenbergia pandurata, cytotoxic, ROS, anti-migration, 4T1

Notch3 Knockout Suppresses Mouse Mammary Gland Development and Inhibits the Proliferation of 4T1 Murine Mammary Carcinoma Cells via CCL2/CCR4 Axis.

Our previous study found that Notch3 knockout mice exhibit defects in mammary gland development. To elucidate the underlying mechanism, tissue samples were subjected to RNA-seq, GO, and KEGG enrichment analyses and qRT-PCR validation. Of enriched pathways, chemokine signaling pathway and cytokine–cytokine receptor interaction were noticed in both Notch3wt/wt/Notch3wt/– and Notch3wt/wt/Notch3–/– mice, in which the expression of chemokine ligand 2 (CCL2) was sharply reduced in Notch3wt/– and Notch3–/– mammary gland tissues. The Mouse ENCODE transcriptome data reveal that the mammary gland fat pad exhibits a high CCL2, CCR2, and CCR4 expression, indicating that these molecules play important roles during mammary gland development. Specifically, defective mammary glands in Notch3 knockout mice could be partially rescued by CCL2 overexpression lentivirus through intraductal injection. An in vitro study showed that CCL2 overexpression promoted the proliferation, migration, and cancerous acinar formation of 4T1 cells, which could rescue the defective migration of 4T1 cells caused by Notch3 knockdown. We also found that Notch3 transcriptionally regulated the expression of CCL2 in a classical pattern. Our findings illustrated that Notch3-regulating CCL2/CCR4 axis should be an important signaling pathway for mammary gland development and should be a candidate target for breast cancer therapy.

Evaluating the biological functions of the prognostic genes identified by the Pathology Atlas in bladder cancer.

The prognosis-associated genes of urinary bladder cancer have been systematically investigated in the Pathology Atlas project based on The Cancer Genome Atlas data. However, the biological functions of most genes in bladder cancer remain unknown. The present study investigated the biological function of 12 of the most significant survival-associated genes (ABRACL, MITD1, ZNF524, EMP1, HSPB6, CXorf38, TRIM38, ZNF182, ZNF195, SPRN, PTPN6 and LIPT1) in urothelial cancer reported by the Pathology Atlas project, with respect to cell proliferation and migration. In vitro, proliferation and migration analyses of T24 cells were performed following the transfection of the 12 prognostic genes. The results were validated with a small interfering (si)RNA library. Immunohistochemistry (IHC) analysis of clinical samples was performed to determine the association between gene expression and tumor metastasis. Furthermore, RNA sequencing was used to investigate the downstream signals. Among the 12 prognostic genes, MIT-domain containing protein 1 (MITD1) transfection was demonstrated to inhibit T24 cell migration to a certain degree. Experiments performed with a 7-gene siRNA library demonstrated that MITD1 knockdown markedly upregulated cell migratory abilities. Mechanistically, the influence of MITD1 on cell signal transduction was assessed via RNA sequencing. Cell migration-associated genes, including KISS1, SPANXB1, SPINT1, PIWIL2, SNAI1, APLN and CTHRC1 were dysregulated. IHC analysis demonstrated that MITD1 protein expression was notably lower in metastatic lymph nodes compared with the primary tumors. Taken together, the results of the present study suggest that the prognostic gene, MITD1 may serve as a migration inhibitor, and be developed as a potential therapeutic target for improving the prognosis of bladder cancer.

Mir-29c-3p targeting TUG1 affects migration and invasion of bladder cancer cells by regulating CAPN7 expression

To investigate the mechanism by which long non-coding RNA TUG1 affects bladder cancer cell migration and invasion. The expressions of TUG1 and miR-29c-3p were examined by quantitative RT-PCR (qRT-PCR) in 10 bladder cancer tissues and 5 bladder cancer cell lines. Trans-well assay was used to detect the changes in migration and invasion abilities of bladder cancer T24 cells after TUG1 knockdown using RNA interference technique, and the alteration in the expression of CAPN7 was also detected. The expression of CAPN7 was examined in T24 cells overexpressing mir-29c-3p by Western blotting, and luciferase reporter assay was performed to confirm the targeting of miR-29c-3p to TUG1 and CAPN7. The effects of CAPN7 overexpression and sh-TUG1 on the migration and invasion of T24 cells were investigated. The expression of TUG1 was up-regulated and mir-29c-3p was down-regulated significantly in bladder cancer tissue with a negative correlation between their expressions. TUG1 knockdown significantly inhibited the migration and invasion of T24 cells (P < 0.01). Overexpression of mir-29c-3p in T24 cells obviously down-regulated the expression of CAPN7 protein, whose expression was positively correlated with TUG1 expression (r=0.4081, P=0.0139). The results of luciferase reporter assay confirmed both TUG1 and CAPN7 as the targets of mir-29c-3p. CAPN7 overexpression could partially reverse the tumor suppressing effect of sh-TUG1 in T24 cells. Mir-29c-3p targeting TUG1 affects the migration and invasion of bladder cancer cells by regulating the expression of CAPN7.

Knockdown of TRPM7 prevents tumor growth, migration, and invasion through the Src, Akt, and JNK pathway in bladder cancer

BackgroundBladder cancer (BC) is one of the most common malignancies of the urinary tract. The role of transient receptor potential melastatin 7 (TRPM7) in BC remains unclear. The aim of this study was to investigate the function and signal transduction pathway of TRPM7 in BC.MethodsT24 and UMUC3 cells were used to evaluate the molecular mechanism of TRPM7 by immunoblot analysis. Small interfering RNA was used to knockdown TRPM7, and the effect of silencing TRPM7 was studied by wound healing, migration, and invasion assays in T24 and UMUC3 cells. Xenograft model study was obtained to analyze the effect of TRPM7 inhibition in vivo.ResultsSilencing of TRPM7 decreased the migration and invasion ability of T24 and UMUC3 cells. The phosphorylation of Src, Akt, and JNK (c-Jun N-terminal kinase) was also suppressed by TRPM7 silencing. Src, Akt, and JNK inhibitors effectively inhibited the migration and invasion of T24 and UMUC3 cells. In addition, the TRPM7 inhibitor, carvacrol, limited the tumor size in a xenograft model.ConclusionOur data reveal that TRPM7 regulates the migration and invasion of T24 and UMUC3 cells via the Src, Akt, and JNK signaling pathway. Therefore, TRPM7 suppression could be a potential treatment for BC patients.

Serum LUCAT1 implicates the pathogenesis of muscle-invasive bladder cancer via targeting miR-199a-5p and miR-199b-5p.

Muscle-invasive bladder cancer (MIBC) is a common malignancy of urinary system cancers, accounting for about 1/3 of all newly diagnosed bladder cancer cases. Due to its strong metastasis, the 5-year survival of MIBC is less than 50%, and in serious cases, the overall survival of metastatic bladder cancer patients is about 1.3years. LncRNAs, a type of non-coding RNAs defined as the transcripts exceeding 200 nucleotides in length, are frequently aberrant in multiple cancers including cervical, ovarian, breast and bladder cancers. Recently, LUCAT1 (short for lung cancer-associated transcript 1), a lncRNA first reported to be involved in smoking-related lung cancer, has been observed to exhibit crucial roles in the epithelial-to-mesenchymal transition (EMT), migration and invasion processes of clear cell renal cell carcinoma (ccRCC) and colorectal cancer. However, whether it involves in the pathogenesis of MIBC remains underexplored. In the present study, LUCAT1 was up-regulated in the serum samples of MIBC patients and bladder cancer cell lines, as assessed using real-time PCR. Our in vitro data (including wound healing and trans-well assays) showed that LUCAT1 was required for the proliferation, EMT, migration and invasion processes of T24 cells. Moreover, LUCAT1 directly targeted miR-199a-5p and miR-199b-5p, as affirmed using the luciferase reporter assay, and manipulation of LUCAT1 significantly suppressed miR-199a-5p and miR-199b-5p. Collectively, our findings highlight an axis of LUCAT1/miR-199a/b-5p in MIBC pathogenesis. Therefore, LUCAT1 may possibly be a promising candidate for diagnostic biomarker and therapeutic target of MIBC.

Abstract 2578: An essential role for Argonaute 2 in HRAS and NRAS driven oncogenesis

Abstract The RAS gene family is among the most commonly mutated genes in cancer, and targeting the potential binding partners of mutated RAS may present a promising alternative therapeutic strategy. We recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of KRAS through its Switch II domain. Further, we demonstrated a role for AGO2 through the development of a KRASG12D driven mouse model of pancreatic cancer. Specifically, our findings revealed that AGO2 ablation drove early precursor pancreatic intraepithelial (PanIN) lesions to enter oncogene-induced senescence in lieu of progressing to late stage PanINs and pancreatic ductal adenocarcinoma. These studies suggested that AGO2 plays a key role in the development of mutant KRAS driven cancers. In order to assess the role of AGO2 in other mutant RAS driven cancers, we performed co-IP of purified AGO2 and purified NRAS/HRAS proteins using isoform specific antibodies. Both HRAS and NRAS bound AGO2, regardless of GDP or GTP nucleotide loading. Using a panel of cell lines (including T24 cells (HRASG12V) and Mel-Juso cells (NRASQ61L)), we observed that AGO2 interacted with both wild-type (WT) and mutant forms of HRAS and NRAS. Additionally, using a variety of cell line models, we observed that EGFR-mediated phosphorylation of AGO2Y393 disrupted the interaction between WT RAS and AGO2. However, the mutant NRAS-, HRAS-, and KRAS-AGO2 interactions were resistant to EGFR regulation. Considering the strong role of AGO2 in mutant KRAS driven cancer, we next asked if loss of AGO2 could induce a similar inhibition of RAS driven disease in human cell line models. In a panel of mutant NRAS and HRAS driven cell lines, AGO2 knockdown led to a substantial decrease in cell proliferation; however, WT RAS expressing cells were unperturbed following loss of AGO2. Similar to the loss of AGO2 in the KRASG12D driven mouse model, knockdown of AGO2 led to increased beta-galactosidase staining in a panel of oncogenic NRAS/HRAS driven cells. Furthermore, knockdown of AGO2 was associated with an induction of multiple senescence pathways, including increased p53, p21, and p16 expression. These results suggest that decreased AGO2 expression is sufficient to induce senescence in mutant but not WT RAS driven cells. In these cell line models, AGO2 loss reduced not only the mutant RAS isoform expression but also the WT RAS isoforms. Finally, knockdown of AGO2 led to an inhibition of T24 and Mel-Juso cell migration and metastasis in a zebrafish xenograft model. Taken together, these results suggest that 1) AGO2 interactions with mutant HRAS and NRAS play a key role in mutant RAS driven oncogenesis and 2) both mutant HRAS and NRAS depend on AGO2 to overcome senescence. Citation Format: Ronald F. Siebenaler, Jessica Waninger, Sunita Shankar, Seema Chugh, Shih-Chun Chu, Jean Tien, Vijaya L. Dommeti, Malay Mody, Anudeeta Gautam, Carson Kenum, Chandan Kumar-Sinha, Arul M. Chinnaiyan. An essential role for Argonaute 2 in HRAS and NRAS driven oncogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2578.

Protein-Bound Polysaccharides from Coriolus Versicolor Fungus Disrupt the Crosstalk Between Breast Cancer Cells and Macrophages through Inhibition of Angiogenic Cytokines Production and Shifting Tumour-Associated Macrophages from the M2 to M1 Subtype.

The tumour microenvironment is rich in multiple cells that influence cancer development. Among them, macrophages are the most abundant immune cells, which secrete factors involved in carcinogenesis. Since protein-bound polysaccharides (PBP) from the Coriolus versicolor fungus are believed to inhibit the growth of cancers, in the present study, we investigated whether these PBP influence crosstalk between triple-negative 4T1 breast cancer cells and RAW 264.7 macrophages. 4T1 cells were cultured in conditioned media (CM) collected after: stimulation of the macrophages with PBP (CM-PBP) or incubation of non-treated macrophages (CM-NT). A co-cultured model of both cell lines was also employed to investigate the crosstalk between the cells. Cell viability was measured using the MTT assay. The levels of cytokines and chemokines were determined by ELISA methods. Commercial assay kits were used to assess the activity of both arginase 1 and inducible nitric oxide synthase (iNOS) and the level of cell migration. The results revealed that CM-NT promotes proliferation and migration of 4T1 cells, and increases the secretion of pro-angiogenic factors (VEGF, MCP-1) by cancer cells. In contrast, CM-PBP inhibits 4T1 cell growth and migration, decreases the secretion of pro-angiogenic factors (VEGF, MCP-1) and upregulates the production of pro-inflammatory mediators (IL-6, TNF-α) with certain anti-tumoral properties Moreover, PBP-treated CM significantly decreases the level of M2 macrophage markers (arginase 1 activity, IL-10 and TGF-β concentrations), but upregulates iNOS activity and IL-6 and TNF-α production, which are M1 cell markers. The results suggest that PBP suppress the favourable tumour microenvironment by inhibiting the crosstalk between 4T1 cells and macrophages through the regulation of production of angiogenic and inflammatory mediators, and modulating the M1/M2 macrophage subtype.

Hsa_circ_0068307 mediates bladder cancer stem cell-like properties via miR-147/c-Myc axis regulation

BackgroundCircular RNAs (circRNAs) play an essential role in the regulation of gene expression. However, the underlying mechanisms remain unknown. This study aimed to evaluate the role of hsa_circ_0068307 in bladder cancer (BCa).MethodsRt-qPCR was used to detect hsa_circ_0068307 expression in BCa cell lines. The CCK8, colony formation, and Transwell assays were used to evaluate the effect of hsa_circ_0068307 on BCa cell migration and proliferation. Bioinformatics and luciferase reporter experiments were used to study the regulatory mechanism. Nude mouse xenografts were generated to examine the effect of hsa_circ_0068307 on tumor growth.ResultsThe results showed that hsa_circ_0068307 was upregulated in BCa cell lines. Downregulation of hsa_circ_0068307 suppressed cell migration and proliferation in T24 and UMUC3 cells. Hsa_circ_0068307 silencing suppressed cancer stem cell differentiation by upregulating miR-147 expression. Upregulation of miR-147 suppressed c-Myc expression, which is involved in cancer stem cell differentiation. Luciferase reporter assays confirmed that hsa_circ_0068307 upregulated c-Myc expression by targeting miR-147. In vivo studies showed that hsa_circ_0068307 knockdown suppressed T24 tumor growth.ConclusionsThese data indicate that downregulation of hsa_circ_0068307 reversed the stem cell-like properties of human bladder cancer through the regulation of the miR-147/c-Myc axis.

Erdafitinib exerts the anticancer effect on urothelial carcinoma via induction of authophagy.

Erdafitinib has recently been approved for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma (UC). The current study aimed to evaluate whether erdafitinib inhibits the proliferation in UC via inducing authophagy. Our data showed that erdafitinib demonstrated strong toxicity for the T24 and UMUC6 cell lines. Flow cytometry analysis showed that erdafitinib increased the proportion of G0/G1 phase in both T24 and UMUC6 cells. Additionally, Annexin V staining indicated that erdafitinib enhanced the apoptosis of UC cells. Meanwhile, the expression of apoptosis-related proteins was significantly elevated after treatment with erdafitinib. Transwell assay showed that erdafitinib significantly reduced T24 and UMUC6 cell migration and invasion. More importantly, western blot assay showed that erdafitinib induced the expression of autophagy-related proteins. Besides, GFP-LC3 transfection assay and electron microscopy examination showed that erdafitinib could partially diminish 3-methyladenine (3-MA) induced impairment of autophagy in UC cells. In summary, for the first time we showed novel data that erdafitinib inhibited UC cell malignant proliferation via inducing cell autophagy.

Antitumor mechanism of MAP30 in bladder cancer T24 cells, and its potential toxic effects in mice

To investigate the antitumor mechanism of MAP30 in human bladder cell line (T24) and its potential toxic effects in mice. In this study, the biological behavior of MAP30's influence on bladder cell was investigated to reveal the antitumor mechanism and role of MAP30 in bladder cancer. MAP30 gene sequence optimized by gene synthesis codon was inserted into the prokaryotic expression vector pET-28a to produce a large amount of target protein in Escherichia coli. The protein product was obtained after purification. Membrane hydration method was used to prepare MAP30 liposome in order to enhance its membrane permeability. The effects of MAP30 on the viability, apoptosis and migration of T24 cell were assessed using 3‑(4,5‑dimethyl‑thiazol‑2‑yl)‑2,5‑diphenyl‑2H‑tetrazolium bromide (MTT), flow cytometric and TUNEL assays, respectively. Mice were transfected with bladder cancer cells for 48 h. The expressions of apoptotic and non-apoptotic proteins were determined using Western blotting. Changes in tumor volume and occurrence of metastasis were assessed using luciferase assay. After 7 days, liver and kidney were excised for histological examination. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH), and activities of catalase and glutathione peroxidase (GPx) were determined in serum or homogenate using enzyme-linked immunosorbent assay (ELISA). The yield of MAP30 after purification was significantly increased. The results of MTT assay showed that MAP30 significantly and concentration-dependently inhibited the proliferation and migration of T24 cells (p &lt; 0.05). The prepared liposomes had uniform hydrated particle size of 132.6 nm, with encapsulation efficiency of 78 %. The inhibitory effect of MAP30 liposome on T24 cells was significantly higher than that of MAP30, and MAP30 significantly increased the number of apoptotic cells (p &lt; 0.05). Western blotting showed that MAP30 significantly promoted the expression of caspase 3 (p &lt; 0.05), but did not significantly affect the expressions of bcl-2 and bax (p &gt; 0.05). It also significantly down-regulated the expressions of NF-kB, JNK and MMP2 (p &lt; 0.05). Tumor formation was significantly inhibited, and tumor volume reduced in bladder cancer-bearing mice after treatment with MAP30 (p &lt; 0.05). Histological examination showed that MAP30 induced mild histological changes in the liver and kidney of mice, and significantly increased the level of MDA at day 1 (p &lt; 0.05). It also significantly and time-dependently increased ROS, but reduced GSH levels and activities of catalase and GPx (p &lt; 0.05). However, MAP30 had no significant effect on DNA (p &gt; 0.05). The apoptotic effect of MAP30 in T24 cells is mediated via activation of caspase-3 signaling pathway. The protein produces mild histological changes in the liver and kidney of mice, but has no significant effect on DNA.

MiR-20a acted as a ceRNA of lncRNA PTENPL and promoted bladder cancer cell proliferation and migration by regulating PDCD4.

Bladder cancer is the most frequent tumor of the urinary system. Despite variety of new treatment options, bladder cancer remains a main global medical problem. Our purpose was to explore the potential molecular and therapeutic targets of bladder cancer diagnosis. The qRT-PCR was used to assess the expression of miR-20a in tissues and cell lines. Counting Cell Kit-8 (CCK-8) assay was carried out to evaluate cell proliferation. Cell migration was calculated using the transwell assay. The expression of miR-20a increased and PDCD4 decreased in bladder cancer tissues compared with normal tissues. Overexpression of miR-20a promoted T24 cell proliferation and migration, while miR-20a inhibitor suppressed cell proliferation and migration. MiR-20a targeted PDCD4 to regulate its expression in T24 cells. MiR-20a is inversely related to PDCD4 and PTENPL in bladder cancer tissues. Upregulation of PDCD4 suppressed T24 cell proliferation and migration. The PTENP1/miR-20a/PTEN axis was involved in the progression of bladder cancer. Our study investigated the function of miR-20a in bladder cancer and provided new insights into the treatment of bladder cancer.

Bladder Cancer Chemosensitivity is Affected by Paraoxonase-2 Expression.

The goal of the current study was to identify potential roles of paraoxonase-2 in bladder carcinogenesis. T24 bladder cancer cells were transfected with plasmids inducing paraoxonase-2 silencing or overexpression. Upon the selection of clones stably down- or upregulating paraoxonase-2, cell proliferation, migration, and the production of reactive oxygen species were evaluated, before and after treatment with cisplatin and gemcitabine, used alone or in combination. The activity levels of both caspase-3 and caspase-8 were also analyzed. shRNA-mediated gene silencing and the overexpression of paraoxonase-2 revealed that the enzyme was able to promote both the proliferation and migration of T24 cells. Moreover, the knockdown of paraoxonase-2 was significantly associated with a reduced cell viability of T24 cells treated with chemotherapeutic drugs and led to both an increase of reactive oxygen species production and caspase-3 and caspase-8 activation. Conversely, under treatment with anti-neoplastic compounds, a higher proliferative capacity was found in T24 cells overexpressing paraoxonase-2 compared with controls. In addition, upon enzyme upregulation, both the production of reactive oxygen species and activation of caspase-3 and caspase-8 were reduced. Although further analyses will be required to fully understand the involvement of paraoxonase-2 in bladder tumorigenesis and in mechanisms leading to the development of chemoresistance, the data reported in this study seem to demonstrate that the enzyme could exert a great impact on tumor progression and susceptibility to chemotherapy, thus suggesting paraoxonase-2 as a novel and interesting molecular target for effective bladder cancer treatment.

MiR‑21 inhibits autophagy and promotes malignant development in the bladder cancer T24cell line.

MicroRNA‑21 (miR‑21) is reported to exhibit cancer‑promoting activity in various types of cancer. It has been previously demonstrated that miR‑21 is overexpressed in bladder tumor tissue compared with normal mucosa. However, the functional mechanism of miR‑21 in bladder cancer remains largely unknown. Thus, the current study aimed to determine the roles of miR‑21 in autophagy and the malignant development of bladder cancer in T24cells. Upregulation or downregulation of miR‑21 was achieved following the transfection of miR‑21 mimic or miR‑21 inhibitor. An MTT assay was additionally performed to measure cell growth. Wound healing and transwell invasion assays were used to detect cell migration and invasion. The apoptotic potential and cell cycle were examined via flow cytometry and reverse transcription‑quantitative PCR was performed to evaluate the expression of phosphatase and tensin homolog (PTEN), beclin1, microtubule‑associated proteinl light chain3B (LC3‑II), cyclinD1, caspase‑3, E‑cadherin, matrix metallopeptidase‑9 (MMP‑9) and vimentin. The results revealed that the proliferation, migration and invasion of T24cells was greatly increased in the miR‑21 mimic group, while apoptosis was greatly inhibited. Additionally, T24cells treated with miR‑21 mimic exhibited G1‑phase arrest. In the miR‑21 mimic group, the expression of PTEN, beclin1, LC3‑II, caspase‑3 and E‑cadherin were decreased, while the expression of cyclinD1, MMP‑9 and vimentin were increased. Opposite effects were observed in the miR‑21 inhibitor group. The data of the current study may indicate that miR‑21 overexpression inhibited autophagy and promoted the proliferation, migration, invasion and epithelial to mesenchymal transition of bladder cancer T24cells. The results may further elucidate the molecular mechanism of miR‑21 in the development of bladder cancer.

Revealing the Reversal Effect of Galangal (Alpinia galanga L.) Extract Against Oxidative Stress in Metastatic Breast Cancer Cells and Normal Fibroblast Cells Intended as a Co- Chemotherapeutic and Anti-Ageing Agent.

Objective:This study intends to explore the potential of galangal extract as a co-chemotherapeutic agent through the analysis of its cytotoxic and migratory effects on metastatic breast cancer cells and as an anti-ageing agent through its senescence inhibitory effect on normal fibroblast cells. Methods:Galangal ethanolic extract (GE) was subjected to a cytotoxicity test with the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay alone or in combination with doxorubicin (Dox) against 4T1 cells but not in NIH-3T3 cells. Evidence of senescent cells was detected using a SA-β galactosidase based assay. In addition, the level of reactive oxygen species (ROS), apoptosis, and cell cycle were measured with a flow cytometry-based assay. Meanwhile, cell migration and matrix metalloproteinase (MMP)-9 expression after GE treatment on 4T1 cells were measured using the scratch wound healing assay and gelatin zymography assay, respectively. The metabolomic profiles of GE were traced using gas chromatography-mass spectrometry (GC-MS) analysis. Results:GE effectively inhibited the growth of 4T1 cells with an IC50 value of 135 µg/mL and increased the cytotoxic effect of Dox at concentrations of 50 and 100 µg/mL. GE increased the number of senescent cells arrested in the G2/M phase but did not cause apoptosis. This effect is compounded by increasing intracellular levels of ROS. However, GE reduced senescence to normal in fibroblast cells (NIH 3T3 cells) under oxidative stress by Dox without any changes in the ROS level. Moreover, GE also inhibited the migration of 4T1 cells and suppressed the expression of MMP-9 induced by Dox. Conclusion:Galangal has the potential for use as a co-chemotherapeutic agent by inducing senescence in correlation with increasing intracellular ROS toward metastatic breast cancer. However, the effect of GE in decreasing the senescence phenomena toward normal fibroblast cells illustrates its potential as a promising anti-ageing agent.

The mechanism of Annexin A1 regulating the proliferation, apoptosis and migration of bladder cancer cells

Objective Explore the function and regulatory mechanism of Annexin A1 (ANXA1) in bladder cancer cell proliferation, apoptosis and migration. Methods From February 2018 to June 2019, we use T24 cells as the model and divide it into over-expression control group (ctrl), ANXA1 over-expression group (ANXA1), knockdown control group (shctrl), ANXA1 knockdown group 1 (shANXA1-1), ANXA1 knockdown group 2 (shANXA1-2) and ANXA1 knockdown group 3 (shANXA1-3). 24 hours after the culture, the cells were collected and the mRNA expression level of ANXA1 was detected by Real-Time quantitative PCR. The cell activity was detected by CCK-8; the cell apoptosis and cycle were detected by flow cytometry. The cell migration was detected by Transwell assay. Results The Real-Time quantitative PCR showed that the expression of ANXA1 in the over expression group was significantly higher than that in the over expression control group (15 369.00±874.20 and 1.00±0.07, P 0.05), while the number of apoptosis in the knockdown group 2 and 3 were significantly higher than that in the knockdown control group (13.04%, 14.58% and 7.76%, P<0.001). Cell function analysis showed that the number of cells passing through the membrane of the over expression group was significantly higher than that of the over expression group (525.00±9.30 and 385.70±13.40, P<0.01), while that of the knockdown group 2 and 3 were significantly lower than that of the knockdown control group (214.70±6.40, 226.00±5.30 and 398.70±10.00, P<0.001). Conclusions Over-expression of ANXA1 significantly promoted the proliferation, cycle and migration of T24 cells and inhibited apoptosis. On the contrary, ANXA1 knockdown inhibited the proliferation, cycle and migration of T24 cells and promoted apoptosis. Key words: Urinary bladder neoplasms; Annexin A1; Cell proliferation; Cell apoptosis; Tumor invasion and metastasis

TLR5: A prognostic and monitoring indicator for triple-negative breast cancer

A novel, highly selective biomarker is urgently needed to predict and monitor triple-negative breast cancer (TNBC) because targeting molecules are not currently available. Although associated with various malignant tumors, the role of toll-like receptor 5 (TLR5) in TNBC remains uncertain. We aimed to define the effects of TLR5 in TNBC to determine whether it could serve as a prognostic and monitoring indicator for TNBC. We established TNBC cell line 4T1 with low TLR5 expression (GFP tag; TLR5− 4T1) and with normal TLR5 expression (GFP tag; TLR5+ 4T1) using lentivirus-shRNA-TLR5 knockdown transfection and negative lentivirus transfection, respectively. Detected by western blot and qPCR, we found knockdown of TLR5 resulted in decreased expression of TLR5 and E-cadherin and increased expression of N-cadherin, vimentin, fibronectin, TRAF6, SOX2, and Twist1, which were related to EMT (epithelial–mesenchymal transition). In addition, downregulation of TLR5 increased the invasion and migration of 4T1 cells in vitro, which were investigated by CCK-8 and wound healing, as well as transwell assay and colony formation. Furthermore, the metastatic ability of TLR5− 4T1 cells to the lungs was also increased compared to TLR5+ 4T1 cells in vivo. To verify the effect of TLR5 as a monitor indicator, mice bearing TLR5+ and TLR5− 4T1 tumors injected with 125I-anti-TLR5 mAb or isotype 125I-IgG were assessed by whole body phosphor-autoradiography and fluorescence imaging in vivo. Phosphor-autoradiography of model mice revealed early tumors at 6 days after inoculation with TLR5+ 4T1, but not TLR5− 4T1 cells. Intratumoral accumulation of radioactivity positively correlated with TLR5 expression, and fluorescence imaging in vivo revealed both TLR5+ and TLR5− 4T1 tumors. Our results suggested that downregulation of TLR5 in TNBC increased tumor invasiveness and EMT expression via TRAF6 and SOX2 pathway and TLR5 could serve as a prognostic and monitoring indicator for TLR5-positive tumors.