Akinobu Gotoh

Honokiol, a natural plant product, inhibits the bone metastatic growth of human prostate cancer cells

Honokiol, a soluble nontoxic natural product derived from Magnolia spp., has been shown to induce apoptosis in malignant cells. The effect of honokiol and the combined therapy with docetaxel on prostate cancer (PCa) growth and bone metastasis was investigated in experimental models.

Isolation and characterization of neural stem/progenitor cells from post-stroke cerebral cortex in mice

The CNS has the potential to marshal strong reparative mechanisms, including activation of endogenous neurogenesis, after a brain injury such as stroke. However, the response of neural stem/progenitor cells to stroke is poorly understood. Recently, neural stem/progenitor cells have been identified in the cerebral cortex, as well as previously recognized regions such as the subventricular or subgranular zones of the hippocampus, suggesting that a contribution of cortex‐derived neural stem/progenitor cells may repair ischemic lesions of the cerebral cortex. In the present study, using a highly reproducible murine model of cortical infarction, we have found nestin‐positive cells in the post‐stroke cerebral cortex, but not in the non‐ischemic cortex. Cells obtained from the ischemic core of the post‐stroke cerebral cortex formed neurosphere‐like cell clusters expressing nestin; such cells had the capacity for self‐renewal and differentiated into electrophysiologically functional neurons, astrocytes and myelin‐producing oligodendrocytes. Nestin‐positive cells from the stroke‐affected cortex migrated into the peri‐infarct area and differentiated into glial cells in vivo. Although we could not detect differentiation of nestin‐positive cells into neurons in vivo, our current observations indicate that endogenous neural stem/progenitors with the potential to become neurons can develop within post‐stroke cerebral cortex.

Etodolac, a Selective Cyclooxygenase-2 Inhibitor, Induces Upregulation of E-Cadherin and Has Antitumor Effect on Human Bladder Cancer Cells In Vitro and In Vivo

OBJECTIVES Cyclooxygenase-2 (COX-2) is highly expressed in several human cancers, including bladder cancer. Thus, a selective COX-2 inhibitor could be useful as an antitumor agent for a range of cancers. In the present study, we investigated the antitumor effect and E-cadherin induction of etodolac, a highly selective COX-2 inhibitor, on human bladder cancer cells in vitro and in vivo. METHODS We examined the cytotoxicity of etodolac against three human bladder cancer cell lines, T24, 5637, and KK47, and performed quantitative reverse transcriptase-polymerase chain reaction to measure the mRNA expression of COX-2, and E-cadherin. RESULTS Etodolac showed significant cytotoxicity only to T24 cells, which expressed the greatest level of COX-2 mRNA and the lowest level of E-cadherin mRNA among the three cell lines. Etodolac also increased the E-cadherin mRNA expression in T24 cells in vitro. We also found that etodolac suppressed tumor growth and induced E-cadherin expression and cell apoptosis in a T24 tumor xenograft mouse model. CONCLUSIONS Etodolac exhibited antitumor activity and induced E-cadherin expression in bladder cancer cells and might be useful for the clinical treatment and prevention of bladder cancer, especially in poorly differentiated bladder cancer with high COX-2 and low E-cadherin expression.

Crosstalk between PI3 Kinase/PDK1/Akt/ Rac1 and Ras/Raf/MEK/ERK Pathways Downstream PDGF Receptor

Background/Aims: Our earlier studies suggested crosstalk between IRS/PI3 kinase/PDK1/Akt/Rac1/ROCK and (Shc2/Grb2/SOS)/Ras/Raf/MEK/ERK pathways downstream PDGF-ββ receptor responsible for chemotaxis and proliferation of malignant mesothelioma cells. The present study was conducted to obtain evidence for this. Methods: To assess activation of Akt, MEK, and ERK, Western blotting was carried out on MSTO-211H malignant mesothelioma cells using antibodies against phospho-Thr308-Akt, phopho-Ser473-Akt, Akt, phospho-MEK, MEK, phopho-ERK1/2, and ERK1/2. To knock-down Akt, PI3 kinase, PDK1, and Rac1, siRNAs silencing each-targeted gene were constructed and transfected into cells. To monitor Rac1 activity, FRET monitoring was carried out on living and fixed cells. Results: ERK was activated under the basal conditions in MSTO-211H cells, and the activation was prevented by inhibitors for PI3 kinase, PDK1, Akt, and Rac1 or by knocking-down PI3 kinase, PDK1, Akt, and Rac1. Akt was also activated under the basal conditions, and the activation was suppressed by a MEK inhibitor and an ERK1/2 inhibitor. In the FRET analysis, Rac1 was activated under the basal conditions, and the activation was inhibited by a MEK inhibitor and an ERK1/2 inhibitor. Conclusion: The results of the present study show that ERK could be activated by PI3 kinase, PDK1, Akt, and Rac1 and that alternatively, Akt and Rac1 could be activated by MEK and ERK in MSTO-211H cells.

Quantitative proteomic analysis to discover potential diagnostic markers and therapeutic targets in human renal cell carcinoma.

Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2‐nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six 12C or 13C. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI‐TOF MS. NBS‐labeled peptides with a 6 Da difference were identified by MS/MS. Thirty‐four proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT‐PCR. The results suggest that NBS‐based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC.

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.

A3 Adenosine Receptor Mediates Apoptosis in In Vitro RCC4-VHL Human Renal Cancer Cells by Up-Regulating AMID Expression

PURPOSE Accumulating studies have shown that extracellular adenosine induces apoptosis in various cancer cells via diverse signaling pathways. We sought to understand adenosine induced apoptosis in human renal cancer cells and the underlying pathway. MATERIALS AND METHODS RCC4-VHL (European Collection of Animal Cell Cultures, Salisbury, United Kingdom), ACHN (Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohuku University, Aoba-ku, Sendai, Japan) and 786-O (ATCC®) human renal cancer cells were cultured. MTT assay, TUNEL staining, reverse transcriptase-polymerase chain reaction and Western blot were done in cells untransfected and transfected with siRNA silencing the A(3) adenosine receptor targeted gene or the AMID targeted gene. RESULTS Adenosine induced apoptosis in all cell types used in a concentration (1 to 10 mM) dependent manner. A similar effect was obtained with the A(3) adenosine receptor agonist 2-Cl-IB-MECA. Adenosine induced RCC4-VHL cell death was inhibited by the A(3) adenosine receptor inhibitor MRS1191 or by knocking down A(3) adenosine receptor or AMID. Adenosine up-regulated the expression of AMID mRNA and protein in RCC4-VHL cells, which was suppressed by A(3) adenosine receptor knockdown. Moreover, adenosine promoted AMID translocation from cytosol to nucleus. CONCLUSIONS Adenosine induces RCC4-VHL cell apoptosis by up-regulating AMID expression and accumulating AMID in the nucleus via A(3) adenosine receptor.

Potential tumor markers of renal cell carcinoma: α-Enolase for postoperative follow up, and galectin-1 and galectin-3 for primary detection

The diagnosis of renal cell carcinoma is currently based on imaging techniques, mainly because there is no blood marker available for its detection. Thus, there is still the need for the development of novel tumor markers. We examined plasma levels of eight proteins in 15 renal cell carcinoma patients before and after surgery, and in 51 healthy controls using enzyme‐linked immunosorbent assay. Plasma levels of α‐enolase, calnexin, galectin‐1, galectin‐3 and lectin mannose‐binding 2 were significantly higher in renal cell carcinoma patients than in controls (P < 0.05). Among these proteins, the sensitivities for galectin‐1 and galectin‐3 were higher than those for calnexin and lectin mannose‐binding 2 in the specificity range from 80% to 100%. A combined use of galectin‐1 and galectin‐3 showed 98% specificity and 47% sensitivity. In addition, the assays showed that plasma α‐enolase levels decreased significantly 4 weeks after nephrectomy (P = 0.0034), and this tendency continued until 12 weeks after nephrectomy (P = 0.0156). These findings suggest that α‐enolase could be used in the postoperative follow up of renal cell carcinoma patients, whereas the combined use of galectin‐1 and galectin‐3 might represent a useful tool for primary detection.

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.