Yumiko Fujita

Apoptosis-related gene transcription in human A549 lung cancer cells via A(3) adenosine receptor.

Background/Aims: Extracellular adenosine induces apoptosis in a variety of cancer cells via diverse signaling pathways. The present study investigated the mechanism underlying adenosine-induced apoptosis in A549 human lung cancer cells. Methods: MTT assay, TUNEL staining, flow cytometry using propidium iodide and annexin V-FITC, real-time RTPCR, Western blotting, monitoring of mitochondrial membrane potentials, and assay of caspase-3, -8, and -9 activities were carried out in A549 cells, and the siRNA to silence the A3 adenosine receptortargeted gene was constructed. Results: Extracellular adenosine induces A549 cell apoptosis in a concentration (0.01-10 mM)-dependent manner, and the effect was inhibited by the A3 adenosine receptor inhibitor MRS1191 or knocking-down A3 adenosine receptor. Like adenosine, the A3 adenosine receptor agonist 2-Cl-IB-MECA also induced A549 cell apoptosis. Adenosine increased expression of mRNAs for Puma, Bax, and Bad, disrupted mitochondrial membrane potentials, and activated caspase-3 and -9 in A549 cells, and those adenosine effects were also suppressed by knocking-down A3 adenosine receptor. Conclusion: Adenosine induces A549 cell apoptosis by upregulating expression of Bax, Bad, and Puma, to disrupt mitochondrial membrane potentials and to activate caspase-9 followed by the effector caspase-3, via A3 adenosine receptor.

A3 adenosine receptor-mediated p53-dependent apoptosis in Lu-65 human lung cancer cells.

Background/Aims: A3 adenosine receptor mediates apoptosis in cancer cells via diverse signaling pathways. The present study examined A3 adenosine receptor-mediated apoptosis in Lu-65 cells, a human giant cell lung carcinoma cell line. Methods: MTT assay, TUNEL staining, real-time RT-PCR, Western blotting, and assay of caspase-3, -8, and -9 activities were carried out in Lu-65 cells, and A3 adenosine receptor or p53 was knocked-down by transfecting each siRNA into cells. Results: Extracellular adenosine induces Lu-65 cell apoptosis in a concentration (0.01-10 mM)-dependent manner, and the effect was inhibited by the A3 adenosine receptor inhibitor MRS1191 or by knocking-down A3 adenosine receptor or p53. Like adenosine, the A3 adenosine receptor agonist 2-Cl-IB-MECA also induced Lu-65 cell apoptosis. Adenosine upregulated expression of p53 and Noxa mRNAs and activated caspase-3 and -9, but not caspase-8. Those adenosine effects were still inhibited by knocking-down A3 adenosine receptor or p53. Conclusion: The results of the present study show that adenosine upregulates p53 expression via A3 adenosine receptor, to promote p53-dependent Noxa gene transcription, causing activation of caspase-9 and the effector caspase-3 to induce Lu-65 cell apoptosis.

AMP Converted from Intracellularly Transported Adenosine Upregulates p53 Expression to Induce Malignant Pleural Mesothelioma Cell Apoptosis

Background/Aims: The present study investigated adenosine-induced apoptosis in human malignant pleural mesothelioma cells. Methods: MTT assay, TUNEL staining, flow cytometry using propidium iodide and annexin V-FITC, real-time RT-PCR, Western blotting, and assay of caspase-3, -8, and -9 activities were carried out using malignant pleural mesothelioma cell lines such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells, and p53 or A3 adenosine receptor was knocked-down by transfecting each siRNA into cells. Results: Adenosine induced apoptosis in all the malignant pleural mesothelioma cells used here, independently of caspase activation. The adenosine effect was prevented by the adenosine transporter inhibitor dipyridamole, the adenosine kinase inhibitor ABT-702, or the A3 adenosine receptor inhibitor MRS1191. Adenosine upregulated expression of the p53 mRNA and protein, that is abolished by ABT-702, but not by knocking-down A3 adenosine receptor. Adenosine-induced apoptosis in NCI-H28 cells was significantly inhibited by knocking-down p53 and in part by knocking-down A3 adenosine receptor. Conclusion: The results of the present study show that AMP converted from intracellularly transported adenosine upregulates p53 expression to induce caspase-independent apoptosis in malignant pleural mesothelioma cells and that A3 adenosine receptor also participates partially in the apoptosis by the different mechanism.

Mesothelioma Cell Proliferation through Autocrine Activation of PDGF- ββ Receptor

Background/Aims: Growth factors play a critical role in proliferation for a variety of cancer cells. The present study was conducted to understand the signaling cascades underlying PDGF-D/PDGF-ββ receptor-mediated proliferation of mesothelioma cells. Methods: Cell growth and cell cycle were analyzed in human non-malignant Met5A cells and malignant mesothelioma cells such as MSTO-211H, NCI-H28, NCI-H2052, and NCI-H2452 cells. Results: Growth of all the cells used here was not affected by PDGF-D, regardless of concentrations (1-30 ng/ml) or treatment time (48-72 h). Spontaneous growth of those cells was significantly inhibited by knocking-down PDGFD or PDGF-ββ receptor, without affecting cell cycling. The cell growth was significantly inhibited by the Akt inhibitor MK2206 and the ROCK inhibitor Y27632 for all the cell types, by the PDK1 inhibitor BX912 for NCI-H28 cells alone, and by the Rac1 inhibitor NSC23766 for NCI-H2052 cells alone, while the PI3 kinase inhibitor wortmannin had no effect. The cell growth, alternatively, was significantly attenuated by MAP kinase kinase inhibitor PD98059 or the ERK1/2 inhibitor FR180204 for all the cell types. Conclusion: The results of the present study show that PDGF-D promotes mesothelioma cell proliferation by targeting ROCK or MAP kinase through autocrine activation of PDGF-ββ receptor.

Adenosine induces apoptosis in SBC-3 human lung cancer cells through A(3) adenosine receptor-dependent AMID upregulation.

Background/Aims: We have shown that A3 adenosine receptor mediates apoptosis in human lung cancer cells such as A549 cells, an epithelial adenocarcinoma cell line, and Lu-65 cells, a giant cell cancer cell line, via each different signaling pathway. AMID, a pro-apoptotic protein, induces caspase-independent apoptosis by accumulating in the nucleus. The present study investigated AMID-dependent apoptosis through A3 adenosine receptor in SBC-3 cells, a human small cell lung cancer cell line. Methods: MTT assay, TUNEL staining, flow cytometry using propidium iodide and annexin V-FITC, and Western blotting were carried out in SBC-3 cells transfected with and without the siRNA to silence the A3 adenosine receptor-targeted gene or the AMID-targeted gene. Results: Adenosine induced SBC-3 cell apoptosis in a concentration (0.01-10 mM) and treatment time (24-72 h)-dependent manner, and a similar effect was obtained with the A3 adenosine receptor agonist 2-Cl-IB-MECA. Adenosine-induced SBC-3 cell death was inhibited by the A3 adenosine receptor inhibitor MRS1191, knocking-down A3 adenosine receptor, or knocking-down AMID. Adenosine upregulated expression of the AMID mRNA and protein in SBC-3 cells, that is suppressed by knocking-down A3 adenosine receptor. In addition, adenosine increased nuclear AMID localization in concert with decreased cytosolic AMID localization. Conclusion: The results of the present study show that adenosine induces SBC-3 cell apoptosis by upregulating AMID expression and promoting AMID translocation into the nucleus via A3 adenosine receptor.

A 3 Adenosine Receptor Mediates Apoptosis in 5637 Human Bladder Cancer Cells by G q Protein/PKC-Dependent AIF Upregulation

Background/Aims: A3 adenosine receptor mediates apoptosis in a variety of cancer cells via diverse signaling pathways. The present study was conducted to assess A3 adenosine receptor-mediated apoptosis in human bladder cancer cell lines and to understand the underlying mechanism. Methods: Human bladder cancer cell lines such as 253J, 5637, KK-47, TCCSUP, T24, and UMUC-3 cells were cultured. The siRNA to silence the A3 adenosine receptor-targeted gene was constructed and transfected into cells. MTT assay, TUNEL staining, Western blotting, and real-time RT-PCR were carried out. Results: For all the investigated cell types adenosine induced apoptosis in a concentration (0.01-10 mM)- and treatment time (24-48 h)-dependent manner. Adenosine-induced 5637 cell death was significantly inhibited by the A3 adenosine receptor inhibitor MRS1191 or knocking-down A3 adenosine receptor, and the A3 adenosine receptor agonist 2-Cl-IB-MECA mimicked the adenosine effect. The adenosine effect was prevented by GF109203X, an inhibitor of protein kinase C (PKC), but it was not affected by forskolin, an activator of adenylate cyclase. Adenosine-induced 5637 cell death, alternatively, was not inhibited by the pan-caspase inhibitor Z-VAD. Adenosine upregulated expression of apoptosis-inducing factor (AIF), that is suppressed by knocking-down A3 adenosine receptor, and accumulated AIF in the nucleus. Conclusion: The results of the present study show that adenosine induces 5637 cell apoptosis by upregulating AIF expression via an A3 adenosine receptor-mediated Gq protein/PKC pathway.

A2a adenosine receptor mediates HepG2 cell apoptosis by downregulating Bcl‐XL expression and upregulating bid expression

Extracellular adenosine induced apoptosis in HepG2 cells, a human hepatoma cell line, by tuning apoptosis‐mediator gene transcription. The present study aimed at identifying the responsible adenosine receptor and clarifying the signaling pathway underlying adenosine‐induced HepG2 cell apoptosis. Adenosine and CGS21680, an A2a adenosine receptor agonist, induced HepG2 cell apoptosis, and the effect was inhibited by DMPX, an A2a adenosine receptor antagonist, or by knocking‐down A2a adenosine receptors. Adenosine reduced expression of Bcl‐XL mRNA and protein but otherwise increased expression of the Bid mRNA and protein in HepG2 cells, and those effects were also prevented by knocking‐down A2a adenosine receptors. Adenosine caused disruption of mitochondrial membrane potentials and stimulated cytochrome c efflux from the mitochondria in HepG2 cells. Adenosine activated caspases‐3 and ‐9 in HepG2 cells, which was significantly inhibited by knocking‐down A2a adenosine receptors. The results of the present study indicate that extracellular adenosine downregulates Bcl‐XL expression and upregulates Bid expression, thereby disrupting mitochondrial membrane potentials to allow cytochrome c efflux from the mitochondria, and then causing activation of caspase‐9 and the effector caspase‐3, as mediated via A2a adenosine receptors. J. Cell. Biochem. 113: 1766–1775, 2012.

Sphingosine suppresses mesothelioma cell proliferation by inhibiting PKC-δ and inducing cell cycle arrest at the G(0)/G(1) phase.

Background/Aims: Sphingosine regulates cellular differentiation, cell growth, and apoptosis. The present study aimed at understanding sphingosine-regulated mesothelioma cell proliferation. Methods: Human malignant mesothelioma cells such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells were cultured. The siRNA to silence the protein kinase C (PKC)-δ-targeted gene was constructed and transfected into cells. MTT assay, cell cycle analysis using a flow cytometry, and cell-free PKC-δ assay were carried out. Results: For all the cell types sphingosine inhibited cell growth in a concentration (1-100 µM)-dependent manner. The sphingosine effect was not prevented by rottlerin, an inhibitor of protein kinase C-δ (PKC-δ); conversely, rottlerin further enhanced the sphingosine effect or rottlerin suppressed mesothelioma cell growth without sphingosine. In the cell-free PKC assay, sphingosine attenuated PKC-δ activity. Knocking-down PKC-δ induced cell cycle arrest at the G0/G1 phase and inhibited cell growth. Conclusion: The results of the present study show that sphingosine suppressed mesothelioma cell proliferation by inhibiting PKC-δ, to induce cell cycle arrest at the G0/G1 phase.

Sphingosine Induces Apoptosis in MKN-28 Human Gastric Cancer Cells in an SDK-Dependent Manner

Background/Aims: Evidence has pointed to the role of sphingosine in cellular differentiation, cell growth, and apoptosis. The present study investigated sphingosine-induced apoptosis in human gastric cancer cells. Methods: Well differentiated MKN-28 and poorly differentiated MKN-45 human gastric cancer cells were cultured. MTT assay, TUNEL staining, Western blotting, and assay of caspase-3, -8, and -9 activities were carried out in cells transfected with and without the siRNA to silence the protein kinase C (PKC)-δ-targeted gene. Results: Sphingosine induced apoptosis in MKN-28 cells, with the potential much

Inhibition of Src family kinases overcomes anoikis resistance induced by spheroid formation and facilitates cisplatin-induced apoptosis in human mesothelioma cells

Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes, and forms spheroid-like cell aggregates in pleural and peritoneal effusions. We examined the levels of anoikis, apoptosis induced by the detachment of cells from the extracellur matrix, in suspension culture in the human mesothelioma cell line NCI-H2052. NCI-H2052 cells were adherent in conventional monolayer cultures, but were found to form spheroids in suspension cultures using dishes with ultra-low cell binding capacity. NCI-H2052 cells proliferated in both cultures, but the proliferation rate was markedly lower in suspension cultures than in monolayer cultures. In addition, NCI-H2052 cells in suspension cultures showed little apoptosis, suggesting that the suspension culture induces anoikis resistance. Western blot analysis revealed that suspension cultures induced activation of Src family kinases (SFK) after spheroid formation. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, abolished anoikis resistance in suspension cultures, indicating that SFK activated by spheroid formation are responsible for anoikis resistance. Cisplatin induced apoptosis in NCI-H2052 cells, but the apoptotic rate was significantly lower in suspension cultures than in monolayer cultures, suggesting that spheroid formation is involved in cisplatin resistance. Furthermore, a combination of dasatinib and cisplatin induced apoptosis more significantly than either alone in suspension cultures. These results suggest that spheroid formation induces resistance to anoikis and to cisplatin through SFK activation and that dasatinib facilitates cisplatin-induced apoptosis in human mesothelioma cells.