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A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells.

ABSTRACT: Dickkopf-related protein 3 (Dkk-3) is a potential tumor suppressor reported in various cancer entities. However, we found that Dkk-3 was exceptionally upregulated in bladder cancer T24 cells. To validate the biological role of Dkk-3 other than a tumor suppressor, we examined the function of Dkk-3 in T24 cells. Gene silencing of Dkk-3 inhibited cell growth through inducing G₀/G₁ cell-cycle arrest. Furthermore, Dkk-3 knock-down caused macropinocytosis accompanied by autophagy, which were canceled in part by their inhibitors 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 3-methyladenine (3-MA). The macropinocytosis was induced by the Dkk-3 knock-down when there were sufficient extracellular nutrients. On the other hand, when the nutritional condition was poor, the autophagy was mainly induced by the Dkk-3 knock-down. These data indicated that Dkk-3 has a role in modulating macropinocytotic and autophagic pathways, a distinct function other than a Wnt antagonist.

SUBMITTER: Tsujimura N

PROVIDER: S-EPMC5133846 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells.

Tsujimura Nonoka N Yamada Nami O NO Kuranaga Yuki Y Kumazaki Minami M Shinohara Haruka H Taniguchi Kohei K Akao Yukihiro Y

International journal of molecular sciences 20161105 11

Dickkopf-related protein 3 (Dkk-3) is a potential tumor suppressor reported in various cancer entities. However, we found that Dkk-3 was exceptionally upregulated in bladder cancer T24 cells. To validate the biological role of Dkk-3 other than a tumor suppressor, we examined the function of Dkk-3 in T24 cells. Gene silencing of Dkk-3 inhibited cell growth through inducing G₀/G₁ cell-cycle arrest. Furthermore, Dkk-3 knock-down caused macropinocytosis accompanied by autophagy, which were canceled ...[more]

PMID: 27827955

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Project description:The aim of the present study was to investigate whether an increase in cyclin-dependent kinase 2 (CDK2) activity is involved in apoptosis of human bladder cancer T24 cells induced by isoliquiritigenin (ISL). The viability of T24 cells was estimated using a sulforhodamine B assay. Cell morphological changes were examined using Hoechst 33258 staining. The apoptotic rate was determined by staining cells with Annexin V-fluorescein isothiocyanate and propidium iodide labeling. The mitochondrial membrane potential (ΔΨm) was measured using 5,5,6,6-tetrachloro-1,1, 3,3-tetraethyl benzimidazole carbocyanine iodide. Alterations in the apoptosis-related regulators B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Bcl-2-interacting mediator of cell death (Bim), apoptotic protease-activating facter-1 (Apaf-1), caspase-9 and caspase-3 were determined using reverse transcription-polymerase chain reaction (PCR) and quantitative PCR methods. Western blot analysis was used to detect the expression of Bcl-2, Bax and caspase-3. CDK2 activity was measured using a spectrometric assay. Following treatment with ISL (between 30 and 70 µg/ml) for 24 h, typical apoptotic morphological changes were observed in T24 cells, exhibiting an edge set of chromosomes, nuclear condensation, nuclear fragmentation and other morphological features. Treatment with ISL increased the apoptotic ratio of T24 cells in a concentration-dependent manner and induced a decrease in the ΔΨm in a time-dependent manner. Treatment with ISL upregulated the expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, downregulated the expression of Bcl-2, and increased CDK2 activity. MK-8776 (an inhibitor of CDK2) antagonized the apoptosis induced by ISL, and, compared with treatment with ISL alone, pretreatment with MK-8776 inhibited the decrease in ΔΨm, downregulated the mRNA expression of Bax, Bim, Apaf-1, caspase-9 and caspase-3, and upregulated Bcl-2 mRNA expression. Western blot analysis demonstrated that, with increasing ISL concentration, the Bcl-2 expression level was significantly decreased (P<0.05), whereas caspase-3 and Bax expression levels were significantly increased (P<0.01). These results indicated that ISL treatment caused a significant decrease in the proliferation rate and increase in apoptosis of T24 cells. The mechanism by which ISL induces T24 cell apoptosis in vitro may be associated with an increase in CDK2 activity, downregulation of the ΔΨm and activation of caspase-3/caspase-9-mediated mitochondrial apoptotic signaling pathways.

Dataset Information

A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells.

Publications

A Novel Role of Dickkopf-Related Protein 3 in Macropinocytosis in Human Bladder Cancer T24 Cells.

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