Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies.

MicroRNAs (miRs) serve either as oncogenes or tumor-suppressor genes in tumor progression. MicroRNA-20b (miR‑20b) is known to be involved with the oncomirs of several types of cancers. However, in the present study we describe how miR-20b inhibits the proliferation, migration and invasion of bladder cancer EJ cells. In the present study, miR-20b was downregulated in bladder cancer cell lines, and its overexpression resulted in a significant reduction in the proliferation of EJ cells. In addition, via a bioinformatics approach, we identified cell cycle-regulated genes that are the putative targets of miR-20b. The transfection of miR-20b into EJ cells induced G1 phase cell cycle arrest via the decreased expression of cyclin D1, CDK2 and CDK6 without affecting another G1 phase cell cycle regulator, cyclin E. The cell cycle inhibitor p21WAF1 was upregulated in the miR-20b transfected cells. Moreover, the enforced expression of miR-20b resulted in impaired wound-healing migration and invasion in the EJ cells. Based on our target prediction analysis of miRs, we confirmed that miR-20b overexpression strongly impedes MMP-2 expression via suppressive activation of the Sp-1 binding motif, an important transcription factor present in the MMP-2 promoter. Herein, we report the novel concept that miR-20b exerts a suppressive effect on both cell cycle-modulated proliferation and MMP-2-mediated migration and invasion in bladder cancer EJ cells.

Interleukin-7 (IL-7) plays a pivotal role in the development and survival of lymphocytes, but its role in cancer cell responses remains unexplained. In this study, IL-7 treatment resulted in a significant induction in the wound-healing migration and Matrigel invasion of the 5637 bladder cancer cells, but it did not result in cell proliferation. In addition, IL-7 treatment strongly induced MMP-9 expression, and increased the binding activation of NF-κB and AP-1 motifs, the important transcription factors that regulate MMP-9 expression. Moreover, the treatment of 5637 cells with IL-7 stimulated the phosphorylation of ERK1/2. U0126, an ERK1/2-specific inhibitor, blocked IL-7-induced cell migration and invasion, and also suppressed the expression of MMP-9 in the presence of IL-7. Inhibition of the ERK1/2 function consistently reversed the binding activity of NF-κB without altering AP-1 activation in IL-7-stimulated cells. Among the cell cycle regulators examined, only the expression of the cell cycle inhibitor p27KIP1 was induced by IL-7. Moreover, the inhibition of p27KIP1 by small interfering RNA (siRNA) abolished the migration, invasion and phosphorylation of ERK1/2, the expression of MMP-9, and the binding activity of the NF-κB motif in IL-7-stimulated 5637 cells. These results demonstrated that the cell cycle inhibitor p27KIP1 is involved in ERK1/2-mediated MMP-9 expression via activation of the NF-κB binding motif, which leads to the migration and invasion of bladder cancer cells induced by IL-7. These novel results could help explain the migration and invasion of bladder tumor cells.

The pharmacological effects of Rosa hybrida are well known in the cosmetics industry. However, the role of Rosa hybrida in cardiovascular biology had not previously been investigated, to the best of our knowledge. The aim of the present study was to elucidate the effect of water extract of Rosa hybrida (WERH) on platelet‑derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs). VSMC proliferation, which was stimulated by PDGF, was inhibited in a non-toxic manner by WERH treatment, which also diminished the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT. Treatment with WERH also induced G1-phase cell cycle arrest, which was due to the decreased expression of cyclins and cyclin-dependent kinases (CDKs), and induced p21WAF1 expression in PDGF-stimulated VSMCs. Moreover, WERH treatment suppressed the migration and invasion of VSMCs stimulated with PDGF. Treatment with WERH abolished the expression of matrix metalloproteinase-9 (MMP-9) and decreased the binding activity of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and specificity protein 1 (Sp1) motifs in PDGF-stimulated VSMCs. WERH treatment inhibited the proliferation of PDGF‑stimulated VSMCs through p21WAF1‑mediated G1-phase cell cycle arrest, by decreasing the kinase activity of cyclin/CDK complexes. Furthermore, WERH suppressed the PDGF-induced phosphorylation of ERK1/2 and AKT in VSMCs. Finally, treatment with WERH impeded the migration and invasion of VSMCs stimulated by PDGF by downregulating MMP-9 expression and a reduction in NF-κB, AP-1 and Sp1 activity. These results provide new insights into the effects of WERH on PDGF-stimulated VSMCs, and we suggest that WERH has the potential to act as a novel agent for the prevention and/or treatment of vascular diseases.

We report a pivotal role for IL-5 as an angiogenic activator. IL-5 increased proliferation, migration and colony tube formation in HUVECs associated with the phosphorylation of ERK and AKT/eNOS, and promoted microvessel sprouting from an angiogenesis animal model. The angiogenic effects were confirmed in IL-5-deficient mice and addition of IL-5 antibody. HSP70-1 was identified via expression profiling following IL-5 stimulation. A siRNA knockdown of HSP70-1 suppressed angiogenic responses and eNOS phosphorylation induced by IL-5. HSP70-1 overexpression enhanced IL-5-induced angiogenic responses. In addition, IL-5-induced neo-vascular formation was verified in both HSP70-1 knockout and HSP70-1 transgenic mice. Furthermore, transcription factor AP-1 was a main factor in IL-5-induced HSP70-1 in response to ERK and AKT signaling pathway. Angiogenic responses induced by VEGF had no effect in either HSP70-1 siRNA in vitro or HSP70-1 knockout mice. IL-5-induced angiogenic responses depended on the binding of IL-5Rα. Our data demonstrate that binding of IL-5 to IL-5Rα receptors enhances angiogenic responses by stimulating the expression of HSP70-1 via the eNOS signaling pathway.

Here, we explored the anti-tumor efficacy of a cyclic pentadepsipeptide, N-methylsansalvamide (MSSV), in bladder cancer. MSSV inhibited the proliferation of both bladder cancer 5637 and T24 cells, which was attributed to the G1-phase cell cycle arrest, apoptosis induction, and alteration of mitogen-activated protein kinases (MAPKs) and protein kinase b (AKT) signaling pathways. Additionally, the treatment of bladder cancer cells with MSSV suppressed migratory and invasive potential via the transcription factor-mediated expression of matrix metalloproteinase 9 (MMP-9). MSSV abrogated vascular endothelial growth factor (VEGF)-induced angiogenic responses in vitro and in vivo. Furthermore, our result showed the potent anti-tumor efficacy of MSSV in a xenograft mouse model implanted with bladder cancer 5637 cells. Finally, acute toxicity test data obtained from blood biochemical test and liver staining indicated that the oral administration of MSSV at 2000 mg/kg caused no adverse cytotoxic effects. Our preclinical data described the potent anti-angiogenic and anti-tumor efficacy of MSSV and showed no signs of acute toxicity, thereby suggesting the putative potential of oral MSSV as a novel anti-tumor agent in bladder cancer treatment.

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Interleukin-16 (IL-16) is a lymphocyte chemoattractant factor well known for its role in immune responses, but its role in vascular disease is unknown. Here, we explored the novel physiological function of IL-16 in vascular smooth muscle cells (VSMCs). The expression of IL-16 and its receptor CD4 was observed in VSMCs. Treatment with IL-16 enhanced the migration and invasion by VSMCs without altering the proliferative potential. IL-16 induced MMP-9 expression via the binding activity of transcription factors NF-κB, AP-1, and Sp-1 motifs in VSMCs. Among the relevant signaling pathways examined, only p38MAPK phosphorylation was significantly stimulated in IL-16-treated VSMCs. Treatment with p38MAPK inhibitor SB203580 prevented the IL-16-induced migration and invasion of VSMCs. SB203580 treatment inhibited the MMP-9 expression and activation of Sp-1 binding in IL-16-treated VSMCs, and siRNA knockdown of CD4 expression blocked the induction of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation stimulated by IL-16. The IL-16 induced cell-cycle-inhibitor p21WAF1 expression in VSMCs, but had no effect on the expression levels of other cell-cycle negative regulators. Finally, blockage of p21WAF1 function with specific siRNA abolished the IL-16-induced elevation of migration, invasion, p38MAPK phosphorylation, MMP-9 expression, and Sp-1 binding activation in VSMCs. Taken together, p21WAF1 was required for the induction of p38MAPK-mediated MMP-9 expression via activation of the Sp-1 binding motif, which led to migration and invasion of VSMCs interacting with IL-16/CD4. These results could provide that IL-16 is a new target in the treatment of vascular diseases such as atherosclerosis and re-stenosis.