Hisao Nagaya

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.

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to α-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.

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.

Regulated motion of glycoproteins revealed by direct visualization of a single cargo in the endoplasmic reticulum

The quality of cargo proteins in the endoplasmic reticulum (ER) is affected by their motion during folding. To understand how the diffusion of secretory cargo proteins is regulated in the ER, we directly analyze the motion of a single cargo molecule using fluorescence imaging/fluctuation analyses. We find that the addition of two N-glycans onto the cargo dramatically alters their diffusion by transient binding to membrane components that are confined by hyperosmolarity. Via simultaneous observation of a single cargo and ER exit sites (ERESs), we could exclude ERESs as the binding sites. Remarkably, actin cytoskeleton was required for the transient binding. These results provide a molecular basis for hypertonicity-induced immobilization of cargo, which is dependent on glycosylation at multiple sites but not the completion of proper folding. We propose that diffusion of secretory glycoproteins in the ER lumen is controlled from the cytoplasm to reduce the chances of aggregation.

Antitumor Action of α1-Adrenoceptor Blockers on Human Bladder, Prostate and Renal Cancer Cells

The present study investigated the antitumor action of α1-adrenoceptor blockers on human bladder, prostate and renal cancer cells. For bladder cancer cell lines used here such as 253J, 5637, KK-47, T24 and UM-UC-3 cells, prazosin, a selective α1-adrenoceptor blocker, reduced cell viability at concentrations more than 30 µmol/l. Likewise, naftopidil, a blocker of α1A- and α1D-adrenoceptors, reduced cell viability for all the bladder cancer cells used here in a concentration (10–100 µmol/l)-dependent manner, with a much greater advantage than prazosin. Naftopidil also reduced cell viability for human prostate cancer cell lines such as DU145, LNCap and PC-3 cells and ACHN human renal cancer cells, with a much higher potential than prazosin. Thus, the results of the present study suggest that naftopidil could be a beneficial antitumor drug for the treatment of urological cancers.

PTP1B Inhibition Causes Rac1 Activation by Enhancing Receptor Tyrosine Kinase Signaling

Background/Aims: The present study investigated the signaling pathway underlying Rac1 activation induced by the linoleic acid derivative 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). Methods: Activity of protein tyrosine phosphatase 1B (PTP1B) was assayed under cell-free conditions. Western blot was carried out to quantify phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt in PC-12 cells. Rac1 activity was monitored in the föerster resonance energy transfer (FRET) analysis using living and fixed PC-12 cells. Results: DCP-LA markedly suppressed PTP1B activity in a concentration (100 pM-100 µM)-dependent manner. In the DCP-LA binding assay, fluorescein-conjugated DCP-LA produced a single fluorescent signal band at 60 kDa, corresponding to the molecule of PTP1B, and the signal was attenuated or abolished by co-treatment or pretreatment with non-conjugated DCP-LA. DCP-LA significantly enhanced nerve growth factor (NGF)-stimulated phosphorylation of IRS-1 at Tyr1222 and Akt1/2 at Thr308/309 and Ser473/474 in PC-12 cells. In the FRET analysis, DCP-LA significantly enhanced NGF-stimulated Rac1 activation, which is abrogated by the phosphatidylinositol 3 kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1) inhibitor BX912, or the Akt inhibitor MK2206. Conclusion: The results of the present study show that DCP-LA-induced PTP1B inhibition, possibly through its direct binding, causes Rac1 activation by enhancing a pathway along a receptor tyrosine kinase (RTK)/IRS-1/PI3K/Akt/Rac1 axis.

Newly synthesized anticancer drug HUHS1015 is effective on malignant pleural mesothelioma

The newly synthesized naftopidil analogue HUHS1015 reduced cell viability in malignant pleural mesothelioma cell lines MSTO‐211H, NCI‐H28, NCI‐H2052, and NCI‐H2452, with the potential greater than that for the anticancer drugs paclitaxel or cisplatin at concentrations higher than 30 μM. HUHS1015 induced both necrosis and apoptosis of MSTO‐211H and NCI‐H2052 cells. HUHS1015 upregulated expression of mRNAs for Puma, Hrk, and Noxa in MSTO‐211H and NCI‐H2052 cells, suggesting HUHS1015‐induced mitochondrial apoptosis. HUHS1015 clearly suppressed tumor growth in mice inoculated with NCI‐H2052 cells. Taken together, the results of the present study indicate that HUHS1015 could be developed as an effective anticancer drug for treatment of malignant pleural mesothelioma.

Oleic Acid Stimulates Glucose Uptake Into Adipocytes by Enhancing Insulin Receptor Signaling

The present study investigated cis-unsaturated free fatty acid (FFA)-regulated glucose uptake. In the cell-free assay of protein tyrosine phosphatase 1B (PTP1B), cis-unsaturated FFAs such as linoleic, linolenic, and oleic acid significantly suppressed PTP1B activity in a concentration (1 - 100 μM)-dependent manner, with the highest potential for oleic acid. Oleic acid (1 μM) stimulated insulin (0.1 nM)-induced phosphorylation of the insulin receptor at Tyr1185 and increased insulin (0.1 nM)-induced phosphorylation of Akt at Thr308 and Ser473 in differentiated 3T3-L1-GLUT4myc adipocytes. In the föerster resonance energy transfer analysis, oleic acid activated Rac1 in PC-12 cells, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, the 3-phosphoinositide-dependent protein kinase-1 (PDK1) inhibitor BX912, or the Akt inhibitor MK2206. Oleic acid (1 μM) significantly increased insulin (0.1 nM)-stimulated glucose uptake in 3T3-L1-GLUT4myc adipocytes, although oleic acid by itself had no effect on the glucose uptake. Taken together, the results of the present study show that oleic acid enhances insulin receptor signaling through a pathway along an insulin receptor/PI3K/PDK1/Akt/Rac1 axis in association with PTP1B inhibition and facilitates insulin-induced glucose uptake into adipocytes.

Naftopidil Is Useful for the Treatment of Malignant Pleural Mesothelioma

Naftopidil, an α1-adrenoceptor blocker, induced apoptosis of human malignant pleural mesothelioma NCI-H2052 cells. Naftopidil upregulated the expression of tumor necrosis factor-α (TNF-α) mRNA in these cells. Naftopidil, alternatively, increased FasL secretion from NCI-H2052 cells, without affecting the expression of FasL mRNA and protein, and activated caspase-3 and -8 in NCI-H2052 cells. Naftopidil drastically suppressed tumor growth in mice inoculated with these cells. The results of the present study indicate that naftopidil induces apoptosis of NCI-H2052 cells by upregulating the expression of TNF-α and stimulating the secretion of FasL, a ligand for the death receptor Fas, both to activate caspase-8 and the effector caspase-3, leading to the suppression of NCI-H2052 cell proliferation in vivo. This raises the possibility that naftopidil could be developed as an effective drug for the treatment of malignant pleural mesothelioma.

Abstract 3353: Cancer stem cell markers (ALDH1 and CD133) expression could be associated with a poor prognosis in the patients with lung adenocarcinoma

[Introduction and Purpose] Cancer stem cells (CSCs) may have abilities of self-renewal and multi-potent differentiation and be responsible for tumor initiation, progression, metastasis and highly resistant to radiation or chemotherapy. Aldehyde dehydrogenase 1 (ALDH1) enzymes are a family of intracellular enzymes that participate in cellular detoxification, differentiation and drug resistance through the oxidation of cellular aldehydes. The biochemical function of CD133 currently remains unclear, but its expression on the cell surface has been demonstrated to be a specific marker for CSCs in many malignant tumors. ALDH1 and CD133 have been identified as putative CSCs marker in patients with lung adenocarcinoma (ad patients) (Miyata et al, 2015). In this study, we investigated the relationship CSCs markers (ALDH1 and CD133 expression) and various clinical parameters in ad patients. We also showed that the expression of CSCs markers (ALDH1 and CD133 expression) related with prognostic potential in ad patients. [Materials and Methods] We examined 92 of 154 (59.7%) in Japanese ad patients, who underwent surgical resection in Fukuoka-Wajiro Hosp. Those 92 ad sections were performed immunohistochemical (IHC) staining for ALDH1 and CD133 using a standard immunoperoxidase technique. The staining intensity of cytoplasmic staining of ALDH1 was scored as 0, 1, 2, or 3, corresponding to negative, weak, intermediate, or strong immunoreactivity, respectively. Percentage of cells with positive ALDH1 was graded as 0 to 100%. The ALDH1-score was assigned to each case by multiplying the intensity score by the each percentage of cells staining. The ALDH1-score was calculated as follows: H = (% of cells that stained at intensity category 1 × 1) + (% of cells that stained at intensity category 2 × 2) + (% of cells that stained at intensity category 3 × 3). We defined as positive cases when more than 100 of the ALDH1-score were calculated. We also defined as CD133 positive cases when more than 10% of tumor was stained (negative cases; > 10% positivity, positive cases; Citation Format: Takeaki Miyata, Takashi Yoshimatsu, Atsushi Sekimura, Tetsuya So, Naohiro Nose, Tsunehiro Oyama, Hisao Nagaya, Akinobu Gotoh, Takeaki Miyata, Tsunehiro Oyama. Cancer stem cell markers (ALDH1 and CD133) expression could be associated with a poor prognosis in the patients with lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3353.