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Background/Aims: This study aims to identify whether Urothelial Cancer Associated 1 (UCA1) regulates mitochondrial metabolic reprogramming in bladder cancer, and to explore how UCA1 participates in mitochondrial metabolism by the UCA1/miR-195/ARL2 signaling pathway; these findings may be aid in the development of tumor diagnostic and therapeutic strategies. Methods: Bladder tissues were obtained from patients. Stable cell lines were constructed, with ectopic expression of UCA1 in UMUC2 cells and knockdown of UCA1 in 5637 cells. The expression levels of UCA1, miR-195, and ARL2 were detected by real-time PCR, western blotting, and immunohistochemistry. Cell viability was detected by Cell Counting Kit-8 (CCK8) assay; mitochondrial DNA copy numbers were tested by realtime PCR; ATP level was evaluated by ATP assay kit; mitochondrial membrane potential was analyzed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescent probe. miRNAs between UCA1 and ARL2 were predicted by TargetScan and RNAHybrid, and then determined by real-time PCR. Dual-luciferase activity assay and RNA immunoprecipitation (RIP) assay were used to verify the relationship between UCA1 and miR-195. The expression level of ARL2 was silenced by small interfering RNA(siRNA). For in vivo experiments, UCA1-silencing 5637 cells were subcutaneously injected into BALB/C nude mice to evaluate the effects of UCA1 on tumor progression by the regulation of miR-195 and ARL2. Results: We demonstrate here that UCA1 enhances mitochondrial function in bladder cancer cells. UCA1 contributes to ARL2-induced mitochondrial activity, which plays an important role in mitochondrial function. UCA1, as a competing endogenous RNA (ceRNA), regulates mitochondrial function through upregulating ARL2. In this way, it inhibited the miR-195 signaling pathway to enhance mitochondrial function in bladder cancer. Additionally, ARL2 is a direct target of miR-195 and can be repressed by either miR-195 overexpression or UCA1 inhibition. Knockdown of ARL2 was analogous to the inhibition of UCA1 and the upregulation of miR-195. Animal experiments further indicated that UCA1 promoted bladder tumor growth by regulating miR-195/ARL2. Conclusion: These data suggest that UCA1 enhanced mitochondrial function and cell viability through the UCA1/miR-195/ARL2 axis in vitro and in vivo. The elucidation of this signaling network provides a more adequate theoretical basis for understanding the molecular pathology of bladder cancer, and also UCA1 as a potential diagnosis and treatment target for bladder cancer. [ABSTRACT FROM AUTHOR]

Aim:The efficacy of the Akt inhibitor perifosine against chronic myeloid leukemia (CML) cells and its mechanisms of action are unknown. In this study, the cytotoxic effects of perifosine on CML and acute myeloid leukemia (AML) cell lines were compared to elucidate the mechanisms underlying the differences.Methods:Human AML cell lines Kasumi-1 and HL-60, and the CML cell line K562 were used. Cell viability was quantitated using MTT assay. Apoptosis was determined using Annexin V-FITC/propidium iodide and Hoechst staining, which were followed by flow cytometry and fluorescence microscopy analysis, respectively. Caspase pathway activation and the expression of autophagy-related genes were examined using Western blot. Autophagy was studied using electron microscopy, the acridine orange staining method, and GFP-LC3 was examined with fluorescence microscopy.Results:In contrast to AML cell lines, the CML cell lines K562 and K562/G (an imatinib-insensitive CML cell line) were resistant to perifosine (2.5-20 μmol/L) in respect to inhibiting cell growth and inducing apoptosis. Perifosine (2.5, 5, and 10 μmol/L) inhibited Akt and its phosphorylation in AML cells, but not in CML cells. Treatment with perifosine (20 μmol/L) resulted in autophagy in CML cells as shown by the increased formation of acidic vesicular organelles and the accumulation of LC3-II. Treatment of CML cells with perifosine (5, 10, and 20 μmol/L) dose-dependently upregulated AGT5, but not Beclin 1 at the protein level. Furthermore, inhibition of autophagy by chloroquine (40 nmol/L) significantly suppressed the cell growth and induced apoptosis in CML cells treated with perifosine (20 μmol/L).Conclusion:Our results show that CML cell lines were resistant to the Akt inhibitor perifosine in vitro, which is due to perifosine-induced protective autophagy and upregulation of ATG5. [ABSTRACT FROM AUTHOR]

Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer (NK) cells enter target tumor cells, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internalization and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we carried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Importantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells. [ABSTRACT FROM AUTHOR]