Migration Of T24 Cells Research Articles

Genetic deletion and pharmacological inhibition of Akt1 isoform attenuates bladder cancer cell proliferation, motility and invasion

Isoform specific expression, intracellular localization and function of Akt in bladder cancer are not known. In the current study, we identified Akt1, followed by Akt2 and Akt3 as the predominant Akt isoform in human T24 and UM-UC-3 metastatic bladder cancer cells. Whereas Akt1 is localized at the membrane, cytoplasm and nucleus, Akt2 is solely cytoplasmic and Akt3 is mostly localized in the nucleus in T24 cells. ShRNA-mediated Akt1 knockdown resulted in impaired T24 cell survival, proliferation, colony formation, migration and microinvasion. Whereas pharmacological inhibition of Akt1 resulted in impaired T24 and UM-UC-3 cell motility, viability and proliferation, effect of pharmacological inhibition by Akt2 inhibitor was limited to proliferation in T24, but not UM-UC-3 cells. Our data provide important clues on the therapeutic benefits of targeting Akt1 for bladder cancer therapy.

The combined use of Camellia sinensis and metronomic zoledronate in 4T1 mouse carcinoma against tumor growth and metastasis.

In previous studies, we demonstrated that the green tea Camellia sinensis (CS) water extract had potent antitumor and antimetastatic effects on 4T1 breast cancer. The metronomic regimen (0.0125 mg/kg twice a week for 4 weeks) of zoledronate (ZOL) was found to be effective in decreasing tumor burden and metastasis as compared with conventional regimen. The aim of the present study was to investigate the antitumor, antimetastatic and anti-osteolytic effects of the combined use of CS water extract and metronomic ZOL against 4T1 breast carcinoma in vitro and in vivo. The results demonstrated that the combination of CS+ZOL exerted a more potent effect on lung and liver by decreasing tumor burden and metastasis, when compared to CS or metronomic ZOL as monotherapies. The combination of CS+ZOL demonstrated optimal bone protection against breast cancer-induced osteolysis for the three groups of CS, ZOL and CS+ZOL. The in vitro results further demonstrated that ZOL enhanced CS-induced apoptosis in 4T1 cells as assessed by the Annexin V-FITC/PI staining and caspase-3 activity assays. In addition, the combined use of CS+ZOL significantly inhibited 4T1 cell migration. Mechanistic studies showed that the enzyme levels of matrix metalloproteinases (MMP)-2 and MMP-9 were suppressed significantly by CS+ZOL. In conclusion, to the best of our knowledge, this is the first study to investigate the novel combined application of herbal extract CS and chemotherapy ZOL in 4T1 breast cancer. The combination of CS plus metronomic ZOL demonstrated significant antitumor, antimetastatic and anti-osteolytic effects against breast cancer, and suggested potential clinical application for breast cancer patients.

MP36-07 ROLE OF ADIPOSE TISSUE IN BLADDER CANCER PROGRESSION

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-07 ROLE OF ADIPOSE TISSUE IN BLADDER CANCER PROGRESSION Nisha Hariharan, Teresa Johnson-Pais, Robert Svatek, Keith Ashcraft, and Robin Leach Nisha HariharanNisha Hariharan More articles by this author , Teresa Johnson-PaisTeresa Johnson-Pais More articles by this author , Robert SvatekRobert Svatek More articles by this author , Keith AshcraftKeith Ashcraft More articles by this author , and Robin LeachRobin Leach More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.735AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Obesity is associated with an increased risk of disease recurrence in patients with bladder cancer. One potential explanation is that adipose tissue surrounding the bladder may secrete tumor-promoting factors as this has been shown in other solid malignancies. The objective of our study is to elucidate the paracrine interactions between adipose tissue and surrounding bladder tumor as a potential contributor to disease progression. Hence, we characterized potential secreted pro-tumor factors in adipose tissue of obese and non-obese muscle-invasive bladder cancer (MIBC) patients. METHODS Adipose tissue in the abdominal subcutaneous space and surrounding the urinary bladder, urachus, and prostate was collected from normal weight [Body Mass Index (BMI) under 25 kg/m2, n=5], overweight (BMI 25 to 29.9 kg/m2, n=8) and obese (BMI>=30 kg/m2, n=7) patients undergoing radical cystectomy for MIBC. To isolate adipose stromal cells (ASCs), bladder and subcutaneous fat was digested and the stromal vascular fraction was placed into culture-specific media. The adherent ASCs were expanded, plated in 0.5% serum media and the conditioned (CM) was harvested. In addition, whole fat explants were incubated in serum-free media to harvest as whole fat CM. CM was characterized using the Millipore multi-analyte profiling. We evaluated the effect of ASC CM and whole fat CM on T24 cells migration, proliferation, and invasion. RESULTS We found several secreted factors in fat CM including Interleukin-8 and Growth Regulated Oncogene-alpha (GRO-alpha), which were highly elevated across all subjects, irrespective of BMI and fat source. Depot-specific whole fat CM from all the tested subjects increased T24 cell migration, but did not affect T24 cell proliferation or invasion. Treatment of T24 cells with GRO-alpha enhanced their migration and neutralizing antibody against GRO-alpha significantly inhibited migration. Secreted factors in the ASC CM were sufficient to induce T24 cells migration. ASC CM from an obese subject's subcutaneous fat significantly elevated T24 cell migration compared to ASC CM from normal and overweight subject's subcutaneous fat. Notably, we detected high levels of Plasminogen Inhibitor Activator-1 in ASC CM from multiple depots. Ongoing studies are focused on understanding the mechanism by which these factors promote bladder cancer progression in vivo. CONCLUSIONS Secreted factors from adipose cells collected from bladder cancer patient influence bladder tumor cells in vitro. These studies provide the first step in identifying adipose tissue-derived factors that promote bladder tumor progression. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e431 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nisha Hariharan More articles by this author Teresa Johnson-Pais More articles by this author Robert Svatek More articles by this author Keith Ashcraft More articles by this author Robin Leach More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

CCL21/CCR7 enhances the proliferation, migration, and invasion of human bladder cancer T24 cells.

ObjectiveTo investigate the effects of CCL21/CCR7 on the proliferation, migration, and invasion of T24 cells and the possible associated mechanisms: expression of MMP-2 and MMP-9, and regulation of BCL-2 and BAX proteins.MethodsT24 cells received corresponding treatments including vehicle control, antibody (20ng/mL CCR7 antibody and 50 ng/ml CCL21), and 50, 100, and 200 ng/ml CCL21. Proliferation was evaluated by MTT assay; cell migration and invasion were assayed using a transwell chamber. Cell apoptosis was induced by Adriamycin (ADM). The rate of cell apoptosis was examined by flow cytometry using annexin V-FITC/PI staining. Western-blot was used to analyze MMP-2 and MMP-9 and BCL-2 and BAX proteins.ResultsCCL21 promoted T24 cell proliferation in concentration-dependent manner with that 200 ng/mL induced the largest amount of proliferation. Significant differences of cell migration were found between CCL21treatment groups and the control group in both the migration and invasion studies (P < 0.001 for all). The expressions of MMP-2 and MMP-9 proteins were significantly increased after CCL21 treatment (p < 0.05 for all). Protein expression of Bcl-21 follows an ascending trend while the expression of Bax follows a descending trend as the concentration of CCL21 increases. No difference was found between the control group and antibody group for all assessments.ConclusionCCL21/CCR7 promoted T24 cell proliferation and enhanced its migration and invasion via the increased expression of MMP-2 and MMP-9. CCL21/CCR7 had antiapoptotic activities on T24 cells via regulation of Bcl-2 and Bax proteins. CCL21/CCR7 may promote bladder cancer development and metastasis.

Sarsaparilla (Smilax Glabra Rhizome) extract inhibits migration and invasion of cancer cells by suppressing TGF-β1 pathway.

Sarsaparilla, also known as Smilax Glabra Rhizome (SGR), was shown to modulate immunity, protect against liver injury, lower blood glucose and suppress cancer. However, its effects on cancer cell adhesion, migration and invasion were unclear. In the present study, we found that the supernatant of water-soluble extract from SGR (SW) could promote adhesion, inhibit migration and invasion of HepG2, MDA-MB-231 and T24 cells in vitro, as well as suppress metastasis of MDA-MB-231 cells in vivo. Results of F-actin and vinculin dual staining showed the enhanced focal adhesion in SW-treated cells. Microarray analysis indicated a repression of TGF-β1 signaling by SW treatment, which was verified by real-time RT-PCR of TGF-β1-related genes and immunoblotting of TGFBR1 protein. SW was also shown to antagonize TGF-β1-promoted cell migration. Collectively, our study revealed a new antitumor function of Sarsaparilla in counteracting invasiveness of a subset of cancer cells by inhibiting TGF-β1 signaling.

Human adipose derived stem cells induced cell apoptosis and s phase arrest in bladder tumor.

The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs) on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM), respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy.

Analysis of the in vivo and in vitro effects of photodynamic therapy on breast cancer by using a sensitizer, sinoporphyrin sodium.

Photodynamic therapy (PDT) is an emerging theranostic modality for various cancers and diseases. Photosensitizers are critical components for PDT. Sinoporphyrin sodium, referred to as DVDMS, is a newly identified photosensitizer that was isolated from Photofrin. Here, we evaluated the effects of DVDMS-mediated PDT (DVDMS-PDT) on tumor cell proliferation and metastasis in the highly metastatic 4T1 cell line and a mouse xenograft model. DVDMS-PDT elicited a potent phototoxic effect in vitro, which was abolished using the reactive oxygen species (ROS) scavenger N-acetylcysteine. In addition, DVDMS-PDT effectively inhibited the migration of 4T1 cells in scratch wound-healing and transwell assays. Using an in vivo mouse model, DVDMS-PDT greatly prolonged the survival time of tumor-bearing mice and inhibited tumor growth and lung metastasis, consistent with in vitro findings. PDT with DVDMS had a greater anti-tumor efficacy than clinically used Photofrin. Moreover, preliminary toxicological results indicate that DVDMS is relatively safe. These results suggest that DVDMS is a promising sensitizer that warrants further development for use in cancer treatment with PDT or other sensitizing agent-based therapies.

Simultaneous delivery of therapeutic antagomirs with paclitaxel for the management of metastatic tumors by a pH-responsive anti-microbial peptide-mediated liposomal delivery system

The roles of microRNAs (miRNAs) in the regulation of metastasis have been widely recognized in the recent years. Mir-10b antagomir (antagomir-10b) was shown to impede metastasis through the down-regulation of mir-10b; however, it could not stunt the growth of primary tumors. In this study we showed that the co-delivery of antagomir-10b with paclitaxel (PTX) by a novel liposomal delivery system modified with an anti-microbial peptide [D]-H6L9 (D-Lip) could significantly both hinder the migration of 4T1 cells and induce evident cellular apoptosis and cell death in the meantime. The histidines in the sequence of [D]-H6L9 allowed the peptide to get protonated under pH5.0 (mimicking the lysosome/endosome environment), and strong membrane lytic effect could thus be activated, leading to the escape of liposomes from the lysosomes and the decrease of of mir-10b expression. The in vivo and ex vivo fluorescence imaging showed that D-Lip could reach 4T1 tumors efficaciously. Incorporation of PTX did not influence the antagomir-10b delivery effect of D-Lip; for the in vivo tumor inhibition assay, compared with all the other groups, the combination of antagomir-10b and PTX delivered by D-Lip could prominently delay the growth of 4T1 tumors and reduce the lung metastases at the same time, and the expression of Hoxd10 in tumors was also significantly up-regulated. Taken together, these results demonstrated that D-Lip could act as a sufficient tool in co-delivering antagomir-10b and PTX.

Divergent behaviors and underlying mechanisms of cell migration and invasion in non-metastatic T24 and its metastatic derivative T24T bladder cancer cell lines

Previous studies on cancer cell invasion were primarily focused on its migration because these two events were often considered biologically equivalent. Here we found that T24T cells exhibited higher invasion but lower migration abilities than T24 cells. Expression of Rho-GDPases was much lower and expression of SOD2 was much higher in T24T cells than those in T24 cells. Indeed, knockdown of SOD2 in T24T cells can reverse the cell migration but without affecting cell invasion. We also found that SOD2 inhibited the JNK/c-Jun cascade, and the inhibition of c-Jun activation by ectopic expression of TAM67 impaired Rho-GDPases expression and cell migration in T24T shSOD2 cells. Further, we found that Sp1 can upregulate SOD2 transcription in T24T cells. Importantly, matrix metalloproteinase-2 (MMP-2) was overexpressed in T24T and participated in increasing its invasion, and MMP-2 overexpression was mediated by increasing nuclear transport of nucleolin, which enhanced mmp-2 mRNA stability. Taken together, our study unravels an inverse relationship between cell migration and invasion in human bladder cancer T24T cells and suggests a novel mechanism underlying the divergent roles of SOD2 and MMP-2 in regulating metastatic behaviors of human bladder T24T in cell migration and invasion.

M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells

The role of muscarinic receptors in several diseases including cancer has recently emerged. To evaluate the hypothesis that muscarinic acetylcholine receptors may play a role in bladder cancer as well as in other tumor types, we investigated their expression in bladder tumor specimens. All examined samples expressed the M1, M2 and M3 receptor subtypes. We also found that the level of M2 transcripts, but not those of M1 or M3, significantly increased with the tumor histologic grade. In view of these results, we proceeded to investigate whether the M2 agonist Arecaidine had any effect on in vitro cell growth and migration of T24 cells, a bladder tumor cell line expressing the muscarinic receptors, including the M2 subtype. We observed that Arecaidine significantly reduced T24 and 5637 cell proliferation and migration in a concentration dependent manner. The silencing of M2 receptor by siRNA in T24 and 5637 cell lines showed the inability of Arecaidine (100 μM) to inhibit cell proliferation after 48 hours, whereas the use of M1 and M3 antagonists in T24 appeared not to counteract the Arecaidine effect, suggesting that the inhibition of cell proliferation was directly dependent on M2 receptor activation. These data suggest that M2 muscarinic receptors may play a relevant role in bladder cancer and represent a new attractive therapeutic target.

Antimetastatic Effects of Licochalcone B on Human Bladder Carcinoma T24 by Inhibition of Matrix Metalloproteinases‐9 and NF‐кB Activity

This study investigated the mechanisms by which licochalcone B (LCB) inhibits the adhesion,invasion and metastasis of human malignant bladder cancer T24 cells. Cell viability was evaluated using a sulforhodamine B (SRB) assay. Cell migration and invasion ability were conducted using wound-healing assay and matrigel transwell invasion assay. The activities of matrix metalloproteinases (MMP)-2 and MMP-9 were measured by gelatin zymography protease assays. The expression in protein level of NF-κBP65 and AP-1 was determined using the ELISA method; the protein levels of MMP-9, NF-κBP65, IκBα and P-IκBα were detected by Western blot. The expression in mRNA level of MMP-9 was assessed using quantitative real-time polymerase chain reaction (PCR) and reverse transcription PCR. The results indicated that LCB attenuated T24 cell migration, adhesion and invasion in a concentration-dependent manner. LCB treatment down-regulated the mRNA expression, protein expression and activity of MMP-9 but had no effect on MMP-2. In addition, LCB treatment decreased the protein level of NF-кBP65 and nuclear translocation of NF-кB. These findings suggested that LCB attenuated migration of bladder cancer T24 cells and adhesion and invasion accompanied with down-regulated protein expression of MMP-9 and the nuclear translocation of NF-кB. Our results provide support that LCB may be a potent adjuvant therapeutic agent in the prevention and therapy of bladder cancer.

Effect of Numb overexpression on invasion and migration in human bladder cancer cell line T24

Objective To study the effect of Numb overexpression on invasion and migration in human bladder cancer cell line T24,and its related mechanism.Methods In June 2013,the plasmids including Numb-ORF plasmid and its blank vector pCMV6-Entry were transfected into T24 cells,and set the blank control as usual.The expressions of Numb and MMP-9 (Matrix metalloproteinase-9) were detected by Real-Time PCR and Western Blot.Then T24 cell invasion and migration were measured respectively by Transwell assay and Scratch Wound Healing Assay.Results Compared to negative control group,which transfected with blank-vector plasmid and untransfected group,Numb expression level in Numb-ORF group was significantly higher (P＜0.01).Meanwhile,MMP-9 level in Numb-ORF group was decreased,△ CT-value as follows:Numb-ORF group was 8.423±0.202,negative control group was 7.294±0.138,untransfected group was 7.220±0.118 (P＜0.01).In Scratch Wound Healing Assay,the repair ratio of Numb-ORF group was less than negative control group and untransfected group.The repair ratio in 12 hours as follow:Numb-ORF group was 0.525±0.037,negative control group was 0.693±0.034,untransfected group was 0.701 ±0.038(P=0.004).In Transwell assay,the number of invasion cells in Numb-ORF group was 12.6±3.2,less than in control group (130.8±9.2) and untransfected group (132.2± 10.6) (P＜0.05).Conclusions Overexpression of Numb by transfection significantly decreases the capability of T24 cell invasion and migration.This suppressed effect may be due to the down-regulation of MMP-9 expression in human bladder cancer. Key words: Bladder neoplasms; Numb Gene; Matrix metalloproteinase-9; Invasion; Migration

MicroRNA-99a inhibiting cell proliferation, migration and invasion by targeting fibroblast growth factor receptor 3 in bladder cancer.

The expression of microRNA-99a (miRNA-99a) has been investigated in a number of human cancers. It has been reported to be downregulated in several types of cancer, including ovarian carcinoma, squamous cell carcinoma of the tongue, squamous cell lung carcinoma, hepatocellular carcinoma, bladder cancer, prostate cancer and childhood adrenocortical tumors. In the present study, the effects of miRNA-99a on bladder cancer cell proliferation, migration and invasion were examined. Following transfection of miRNA-99a, cell viability, cell migration assay, cell invasion, western blot analysis and luciferase assays were conducted in bladder cancer cell lines. It was found that miRNA-99a inhibits cell proliferation, migration and invasion in T24 and EJ cells. Additionally, this study provided the first evidence that miRNA-99a is likely to directly target fibroblast growth factor receptor 3 in bladder cancer. The study provided evidence that miRNA-99a suppresses cell proliferation, migration and invasion by targeting growth factor receptor 3 in bladder cancer cell lines. These results indicated that it could be investigated as a target for therapeutic drugs designed to treat bladder cancer.

Inhibition of FGFR signaling by PD173074 improves antitumor immunity and impairs breast cancer metastasis

Aberrant fibroblast growth factor (FGF) and FGF receptor (FGFR) system have been associated with breast cancer. The objectives of our study were to investigate the effects and mechanisms of FGFR inhibition on tumor growth and metastasis on breast cancer. Our studies showed that the FGFR inhibitor PD173074 decreased the viability of several human breast cancer cells, as well as 4T1 murine mammary tumor cells. Therefore, we chose 4T1 cells to study PD173074's antitumor mechanism. Flow cytometry showed that PD173074 induced 4T1 cell apoptosis in a concentration-dependent manner. Western blot demonstrated that PD173074-induced apoptosis was correlated with the inhibition of Mcl-1 and survivin. Moreover, PD173074 also significantly increased the ratio of Bax/Bcl-2. PD173074 could also block 4T1 cell migration and invasion in vitro. In 4T1 tumor-bearing mice, PD173074 significantly inhibited tumor growth without obvious side effects. Meanwhile, PD173074 functionally reduced microvessel density and proliferation index and induced tumor apoptosis. Importantly, we found that FGFR inhibition by PD173074 reduced myeloid-derived suppressor cells (MDSCs) in the blood, spleens and tumors, accompanied by the increased infiltration of CD4(+) and CD8(+) T cells in the spleens and tumors. Furthermore, PD173074 significantly inhibited breast tumor metastasis to the lung of inoculated 4T1 breast cancer cells, which was accompanied by a reduction in MDSCs. Our findings suggested that FGFR inhibition could delay breast tumor progression, impair lung metastasis and break immunosuppression by effecting on tumor microenvironment, which may provide a promising therapeutic approach for breast cancer patient.

Effect of siRNA targeting EZH2 on cell viability and apoptosis of bladder cancer T24 cells.

We investigated the effect of siRNA targeting enhancer of EZH2 on cell proliferation, invasion, migration, and apoptosis of human bladder cancer T24 cells. An siRNA-expressing plasmid targeting the EZH2 gene was transfected into T24 cells. Quantitative polymerase chain reaction and Western blot analysis were used to detect EZH2 expression at the mRNA and protein levels, respectively. Proliferation, invasion, and migration of T24 cells were examined in vivo using MTT, wound healing, and transwell chamber migration assays, respectively. Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometric analysis was performed to determine cell apoptosis levels. Expression of EZH2 in T24 cells was suppressed at the mRNA and protein levels. Following transfection for 48 h, growth was inhibited by 37.9%, which was markedly lower than that in the negative control group (P < 0.05). Following a wound-healing assay for 24 h, transfected cell migration distance was 1.37 ± 0.12, which was markedly less than the horizontal migration distance of negative control group cells (P < 0.01). In addition, the cell invasion ability of EZH2- siRNA group cells decreased by 67% compared with negative control group cells (P < 0.01). Following transfection for 48 h, early- and late-stage apoptosis rates for T24 cells were 22.8 and 3.60%, respectively, which were higher than in the negative control group (P < 0.01). EZH2 gene silencing effectively suppressed the proliferation, invasion, and migration abilities of human bladder cancer cells, promoting apoptosis.

Green tea (Camellia sinensis) extract inhibits both the metastasis and osteolytic components of mammary cancer 4T1 lesions in mice

Green tea (Camellia sinensis, CS), a kind of Chinese tea commonly consumed as a healthy beverage, has been demonstrated to have various biological activities, including antioxidation, antiobesity and anticancer. Our study aims to investigate the antitumor, antimetastasis and antiosteolytic effects of CS aqueous extract both in vitro and in vivo using metastasis-specific mouse mammary carcinoma 4T1 cells. Our results showed that treatment of 4T1 cells with CS aqueous extract resulted in significant inhibition of 4T1 cell proliferation. CS extract induced 4T1 apoptosis in a dose-dependent manner as assessed by annexin-V and propidium iodide staining and caspase-3 activity. Western blot analysis showed that CS increased the expression of Bax-to-Bcl-2 ratio and activated caspase-8 and caspase-3 to induce apoptosis. CS also inhibited 4T1 cell migration and invasion at 0.06–0.125 mg/ml. In addition, CS extract (0.6 g/kg, orally fed daily for 4 weeks) was effective in decreasing the tumor weight by 34.8% in female BALB/c mice against water treatment control (100%). Apart from the antitumor effect, CS extract significantly decreased lung and liver metastasis in BALB/c mice bearing 4T1 tumors by 54.5% and 72.6%, respectively. Furthermore, micro-computed tomography and in vitro osteoclast staining analysis suggested that CS extract was effective in bone protection against breast cancer-induced bone destruction. In conclusion, the present study demonstrated that the CS aqueous extract, which closely mimics green tea beverage, has potent antitumor and antimetastasis effects in breast cancer and could protect the bone from breast cancer-induced bone destruction.

α-Lipoic acid suppresses migration and invasion via downregulation of cell surface β1-integrin expression in bladder cancer cells

Our previous study showed α-lipoic acid (LA) downregulated cell surface β1-integrin expression of v-H-ras-transformed derivative of rat fibroblast with amelioration of their malignant phenotype. Here, we evaluated the ameliorating effect of LA on the malignant characters in H-ras-transformed bladder cancer cells. H-ras mutated bladder cancer line, T24 cells were incubated with LA to evaluate the inhibitory effect on proliferation, migration, invasion and β1-integrin expression. Fluorescence staining of F-actin and western blotting analyses of the related signaling pathways were also performed. LA inhibited the proliferation of T24 cells. Cell adhesion to collagen IV and fibronectin was strikingly inhibited by LA treatment accompanied by downregulation of cell surface but not whole cell β1-integrin expression. LA clearly inhibited cell migration and invasion of T24 cells, which were mimicked by extracellular signal-regulated kinase (ERK) and Akt pathway inhibition. Actually, LA significantly downregulated the phosphorylated ERK and Akt levels. Moreover, LA downregulated phosphorylated focal adhesion kinase level with disappearance of stress fiber formation. Finally, although LA induced the internalization of cell surface β1-integrin, disruption of the raft did not affect the action of LA. Taken together, LA is a promising agent to improve malignant character of bladder cancer cells through regulation of cellular β1-integrin localization.

Hydrazinocurcumin Encapsuled Nanoparticles “Re-Educate” Tumor-Associated Macrophages and Exhibit Anti-Tumor Effects on Breast Cancer Following STAT3 Suppression

Tumor-associated macrophages (TAMs) are essential cellular components within tumor microenvironment (TME). TAMs are educated by TME to transform to M2 polarized population, showing a M2-like phenotype, IL-10high, IL-12low, TGF-βhigh. STAT3 signaling triggers crosstalk between tumor cells and TAMs, and is crucial for the regulation of malignant progression. In our study, legumain-targeting liposomal nanoparticles (NPs) encapsulating HC were employed to suppress STAT3 activity and “re-educate” TAMs, and to investigate the effects of suppression of tumor progression in vivo. The results showed that TAMs treated by HC encapsuled NPs could switch to M1-like phenotype, IL-10low, IL-12high, TGF-βlow, and the “re-educated” macrophages (M1-like macrophages) considerably demonstrated opposite effect of M2-like macrophages, especially the induction of 4T1 cells migration and invasion in vitro, and suppression of tumor growth, angiogenesis and metastasis in vivo. These data indicated that inhibition of STAT3 activity of TAMs by HC-NPs was able to reverse their phenotype and could regulate their crosstalk between tumor cells and TAMs in order to suppress tumor progression.

MicroRNA-101 suppresses motility of bladder cancer cells by targeting c-Met

MicroRNAs (miRNAs) are small non-coding RNAs that play regulatory roles by repressing translation or cleaving RNA transcripts. Here, we report that the expression of microRNA-101 (miR-101) is down-regulated in human bladder cancer tissue versus normal adjacent tissue. To better characterize the role of miR-101 in bladder cancer, we conducted a gain-of-function analysis by transfecting the bladder cancer cell line T24 with chemically synthesized miR-101 mimics. We found that miR-101 directly targets c-Met via its 3′-UTR. Specifically, forced expression of miR-101 decreased c-Met expression at both mRNA and protein levels, consequently inhibiting T24 cell migration and invasion in a c-Met-dependent manner. In conclusion, we have shown miR-101 to be a novel suppressor of T24 cell migration and invasion through its negative regulation of c-Met.

5-Aza-2′-Deoxycytidine Enhances Maspin Expression and Inhibits Proliferation, Migration, and Invasion of the Bladder Cancer T24 Cell Line

Downregulation of maspin expression has been linked to bladder cancer development, and that DNA methylation may be important for regulating maspin gene activation in bladder cancer cells. Thus, we attempted to explore the effects of the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR), on the maspin expression and the biological behaviors in bladder cancer T24 cells. The methylation status of maspin in T24 cells was investigated by methylation-specific polymerase chain reaction (PCR). After treated with different concentrations of 5-Aza-CdR (0, 0.25, 0.5, 1, and 2 μM), the maspin gene mRNA expression and protein expression were examined by real-time PCR and western blotting analysis. Cell proliferations were evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Flow cytometry was used to identify the apoptosis rates. Migration and invasive ability were determined by the transwell assay. Using the western blotting analysis, the changes of Cyclin D1, VEGF-C, VEGFR-3, MMP-2, MMP-9, caspase-3 p17, Bax, and Bcl-2 expression were measured. Promoter DNA methylation of maspin was observed in T24 cells. The expression levels of maspin mRNA and protein in T24 cells were increased in a dose manner after treatment with increasing 5-Aza-CdR (p<0.05). The proliferation, migration, and invasion of cells were significantly inhibited with increasing 5-Aza-CdR, whereas the apoptosis was greatly increased (p<0.05). These were associated with the decreased ratio of Bcl-2/Bax, activation of caspase-3, and decreased expression of Cyclin D1, VEGF-C, VEGFR-3, MMP-2 and MMP-9. The present study demonstrates that maspin is silenced by DNA methylation in bladder T24 cells, and its expression can be reactivated by treatment with 5-Aza-CdR. 5-Aza-CdR could result in obvious inhibitions of the proliferation, migration, and invasion of T24 cells, which may serve as a potential strategy for the treatment of bladder cancer.