miR-4487 Enhances Gefitinib-Mediated Ubiquitination and Autophagic Degradation of EGFR in Non-Small Cell Lung Cancer Cells by Targeting USP37.

PURPOSE: With the identification of epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) cells, EGFR-tyrosine kinase inhibitors (TKIs) are being used widely as the first-line of treatment in NSCLC. These inhibitors block auto-phosphorylation of activated EGFR by competing with ATP binding and mediate EGFR degradation independent of exogenous epidermal growth factor, which is associated with the mutation variants of EGFR. However, the precise mechanisms underlying the TKI-mediated EGFR degradation are still unclear. MATERIALS AND METHODS: To examine the physiological roles of miR-4487 and ubiquitin-specific peptidase 37 (USP37) in gefitinib-mediated EGFR degradation in NSCLC cells, multiple NSCLC cell lines were applied. The level of EGFR expression, apoptosis marker and autophagic flux were determined by western blot. Expression level of miR-4487 and cell cycle arrest was analyzed by TaqMan assay and flow cytometry respectively. RESULTS: We found that gefitinib mediates EGFR degradation under normal culture conditions, and is dependent on autophagic flux and the mutation variants of EGFR. Gefitinib reduced expression levels of USP37, which mediated EGFR degradation similar to gefitinib. Our results also showed a gefitinib-mediated increase in endogenous miR-4487 level and presented evidence for the direct targeting of USP37 by miR-4487, resulting in the sequential enhancement of ubiquitination, autophagy, and EGFR degradation. Thus, the depletion of USP37 and overexpression of miR-4487 led to an increase in gefitinib-mediated apoptotic cell death. CONCLUSION: These data suggest that miR-4487 is a potential target for treating NSCLC, and miR-4487/USP37-regulated EGFR degradation is a determinant for developing gefitinib resistance.

Afatinib Mediates Autophagic Degradation of ORAI1, STIM1, and SERCA2, Which Inhibits Proliferation of Non-Small Cell Lung Cancer Cells.

BACKGROUND: The expression of calcium signaling pathway molecules is altered in various carcinomas, which are related to the proliferation and altered characteristics of cancer cells. However, changes in calcium signaling in anti-cancer drugresistant cells (bearing a T790M mutation in epidermal growth factor receptor [EGFR]) remain unclear. METHODS: Afatinib-mediated changes in the level of store-operated Ca2+ entry (SOCE)-related proteins and intracellular Ca2+ level in non-small cell lung cancer cells with T790M mutation in the EGFR gene were analyzed using western blot and ratiometric assays, respectively. Afatinib-mediated autophagic flux was evaluated by measuring the cleavage of LC3B-II. Flow cytometry and cell proliferation assays were conducted to assess cell apoptosis and proliferation. RESULTS: The levels of SOCE-mediating proteins (ORAI calcium release-activated calcium modulator 1 [ORAI1], stromal interaction molecule 1 [STIM1], and sarco/endoplasmic reticulum Ca2+ ATPase [SERCA2]) decreased after afatinib treatment in non-small cell lung cancer cells, whereas the levels of SOCE-related proteins did not change in gefitinibresistant non-small cell lung cancer cells (PC-9/GR; bearing a T790M mutation in EGFR ). Notably, the expression level of SOCE-related proteins in PC-9/GR cells was reduced also responding to afatinib in the absence of extracellular Ca2+. Moreover, extracellular Ca2+ influx through the SOCE was significantly reduced in PC-9 cells pre-treated with afatinib than in the control group. Additionally, afatinib was found to decrease the level of SOCE-related proteins through autophagic degradation, and the proliferation of PC-9GR cells was significantly inhibited by a lack of extracellular Ca2+. CONCLUSION: Extracellular Ca2+ plays important role in afatinib-mediated autophagic degradation of SOCE-related proteins in cells with T790M mutation in the EGFR gene and extracellular Ca2+ is essential for determining anti-cancer drug efficacy.

Restricting extracellular Ca2+ on gefitinib-resistant non-small cell lung cancer cells reverses altered epidermal growth factor-mediated Ca2+ response, which consequently enhances gefitinib sensitivity.

Non-small cell lung cancer (NSCLC), one of the leading causes of cancer-related death, has a low 5-year survival rate owing to the inevitable acquired resistance toward antitumor drugs, platinum-based chemotherapy, and targeted therapy. Epidermal growth factor (EGF)-EGF receptor (EGFR) signaling activates downstream events leading to phospholipase C/inositol trisphosphate (IP3)/Ca2+ release from IP3-sensitive Ca2+ stores to modulate cell proliferation, motility, and invasion. However, the role of EGFR-mediated Ca2+ signaling in acquired drug resistance is not fully understood. Here, we analyzed alterations of intracellular Ca2+ ([Ca2+]i) responses between gefitinib-sensitive NSCLC PC-9 cells and gefitinib-resistant NSCLC PC-9/GR cells, and we found that acute EGF treatment elicited intracellular Ca2+ ([Ca2+]i) oscillations in PC-9 cells but not in PC-9/GR cells. PC-9/GR cells presented a more sustained basal [Ca2+]i level, lower endoplasmic reticulum Ca2+ level, and higher spontaneous extracellular Ca2+ ([Ca2+]e) influx than PC-9 cells. Notably, restricting [Ca2+]e in both cell types induced identical [Ca2+]i oscillations, dependent on phospholipase C and EGFR activation. Consequently, restricting [Ca2+]e in PC-9/GR cells upregulated gefitinib-mediated poly (ADP-ribose) polymerase cleavage, an increase in Bax/Bcl-2 ratio, cytotoxicity, and apoptosis. In addition, nuclear factor of activated T cell (NFAT1) induction in response to EGF was inhibited by gefitinib in PC-9 cells, whereas EGF-mediated NFAT1 induction in PC-9/GR cells was sustained regardless of gefitinib treatment. Restricting [Ca2+]e in PC-9/GR cells significantly reduced EGF-mediated NFAT1 induction. These findings indicate that spontaneous [Ca2+]e influx in NSCLC cells plays a pivotal role in developing acquired drug resistance and suggest that restricting [Ca2+]e may be a potential strategy for modulating drug-sensitivity.

Construction of late transition metal complex on amine-functionalized SiO2 and SBA-15 for L-lactide polymerization.

We report the in-situ synthesis of a late transition metal catalyst on amine-functionalized SBA-15 and amorphous silica and their ring opening polymerizations of L-lactide. Funtionalizations of SBA-15 and amorphous silica with N-[3-(trimethoxysilyl)propyl]-ethylenediamine (2NS) were carried out to in-situ synthesize late transition metal catalysts on the surface of nanoporous materials. FT-IR spectra indicates that the 1,2-dimethoxyethane ligand did not participate in the reaction when (DME)NiBr2 was immobilized on the amine-functionalized silica. In addition, the XPS spectrum confirmed the presence of Ni compound on the 2NS-functionalized SiO2. SBA-15/2NS/Ni and SBA-15/ 2NS/Pd catalysts showed higher conversion of PLA polymerization in comparison to SiO2/2NS/Ni and SiO2/2NS/Pd, respectively. The molecular weights of PLA produced by SBA-15/2NS/Ni or Pd were larger than those of SiO2/2NS/Ni or Pd, respectively. The effects of polymerization conditions such as the polymerization temperature, time, the catalyst amount in the feed, and the polymerization medium in L-lactide polymerization were also investigated.

Influenza vaccination among adults 65 years or older: a 2009-2010 community health survey in the Honam region of Korea.

The present study examined the rates and related factors for influenza vaccination among the elderly Korean population during the 2008/09 influenza seasons. We obtained data for 6,391 adults aged 65 years or older from Community Health Surveys conducted in 2009 and 2010 in 13 communities in the Honam region of Korea. A multiple logistic regression analysis was used to identify the factors associated with self-reported influenza vaccinations. In this elderly population, 81.7% reported to having received an influenza vaccination in the past year. The main contributing factors were older age, lower economic status, lower educational level, married, non-smoking, regular alcohol consumption, regular walking exercise, receiving a health check-up during the past two years, not stressed, and having comorbid conditions. The influenza vaccination coverage rate among elderly Koreans was relatively high, but improvements in vaccination rates are required.

Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes.

It has recently been known that berberine, an alkaloid of medicinal plants, has anti-hyperglycemic effects. To explore the mechanism underlying this effect, we used 3T3-L1 adipocytes for analyzing the signaling pathways that contribute to glucose transport. Treatment of berberine to 3T3-L1 adipocytes for 6 h enhanced basal glucose uptake both in normal and in insulin-resistant state, but the insulin-stimulated glucose uptake was not augmented significantly. Inhibition of phosphatidylinositol 3-kinase (PI 3-K) by wortmannin did not affect the berberine effect on basal glucose uptake. Berberine did not augment tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate (IRS)-1. Further, berberine had no effect on the activity of the insulin-sensitive downstream kinase, atypical protein kinase C (PKCzeta/lambda). However, interestingly, extracellular signal-regulated kinases (ERKs), which have been known to be responsible for the expression of glucose transporter (GLUT)1, were significantly activated in berberine-treated 3T3-L1 cells. As expected, the level of GLUT1 protein was increased both in normal and insulin-resistant cells in response to berberine. But berberine affected the expression of GLUT4 neither in normal nor in insulin-resistant cells. In addition, berberine treatment increased AMP-activated protein kinase (AMPK) activity in 3T3-L1 cells, which has been reported to be associated with GLUT1-mediated glucose uptake. Together, we concluded that berberine increases glucose transport activity of 3T3-L1 adipocytes by enhancing GLUT1 expression and also stimulates the GLUT1-mediated glucose uptake by activating GLUT1, a result of AMPK stimulation.

Insulin sensitization of MAP kinase signaling by fibroin in insulin-resistant Hirc-B cells.

Fibroin has been shown to enhance insulin-stimulated glucose uptake in 3T3-L1 adipocytes, and the mechanism underlying the fibroin effect focused on phosphatidylinositol 3-kinase (PI 3-K) pathway has been reported. In the present study, for defining the insulin-sensitizing effects of fibroin synthetically, we have used the Hirc-B cells which are rat fibroblasts over-expressing wild-type human insulin receptors to investigate the insulin-stimulation of mitogen-activated protein (MAP) kinase signaling cascades. Cultivation of Hirc-B cells in high-glucose medium for 6 days led to an insulin-resistant state in which insulin-stimulated DNA synthesis was blocked completely. Chronic exposure to fibroin for 16 h markedly recovered DNA synthesis in insulin-resistant cells. Development of insulin resistance caused a reduction of c-Jun N-terminal kinase (JNK) phosphorylation, which was also recovered by fibroin exposure. Fibroin sensitized the insulin-stimulated c-Jun accumulation and phosphorylation in insulin-resistant cells. In the time course for c-Jun accumulation, fibroin had a vanadate-like effect. Further, fibroin was shown to delay the degradation of c-Jun. It is suggested that fibroin may sensitize insulin action by blocking JNK dephosphorylation caused by MAP kinase phosphatase-1 (MKP-1).