Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells.

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H+ ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC.

Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death.

Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction.

TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

Blockage of Stat3 enhances the sensitivity of NSCLC cells to PI3K/mTOR inhibition.

The PI3K/Akt/mTOR axis in lung cancer is frequently activated and implicated in tumorigenesis. Specific targeting of this pathway is therefore an attractive therapeutic approach for lung cancer. However, non-small cell lung cancer cells are resistant to BEZ235, a dual inhibitor of PI3K and mTOR. Interestingly, blockage of Stat3 with a selective inhibitor, S3I-201, or siRNA dramatically sensitized the BEZ235-induced cell death, as evident from increased PARP cleavage. Furthermore, inhibition of Stat3 led to enhancement of cell death induced by LY294002, a PI3K inhibitor. Treatment of cells with a combination of BEZ235 and S3I-201 significantly induced the proapoptotic transcription factor, CHOP, and its targets, Bim and DR4. Knockdown of CHOP or Bim suppressed cell death stimulated by the combination treatment, implicating the involvement of these BEZ235/S3I-201-induced factors in pronounced cell death. Moreover, the BEZ235/S3I-201 combination enhanced TRAIL-induced cell death. Our results collectively suggest that blockage of Stat3 presents an effective strategy to overcome resistance to PI3K/Akt/mTOR inhibition.

Inhibition of vacuolar H+ ATPase enhances sensitivity to tamoxifen via up-regulation of CHOP in breast cancer cells.

Resistance of estrogen receptor-positive breast cancer cells to tamoxifen represents a major barrier to the successful treatment of breast cancer. In the present study, we found that vacuolar H+ ATPase (vATPase) inhibitors, bafilomycin A1 and concanamycin A, sensitize tamoxifen-induced cell death. siRNA targeting ATP6V0C, a 16-kDa hydrophobic proteolipid subunit of vATPase that plays a central role in H+ transport, markedly increased cell death induced by tamoxifen. Interestingly, bafilomycin A1 induced up-regulation of DR4/DR5 and CHOP. Knock-down of CHOP by siRNA suppressed the cell death induced by bafilomycin A1 and tamoxifen, suggesting that bafilomycin A1-mediated CHOP activation sensitizes to tamoxifen. In addition, we found that bafilomycin A1 enhances TRAIL-induced cell death in breast cancer cells. Furthermore, we showed that combination of vATPase inhibitors with tamoxifen also effectively induced cell death in HER2- and ERα-overexpressing breast cancer cells. Overall, our results demonstrate that inhibition of vATPase can potentiate the apoptotic effects of tamoxifen through up-regulation of CHOP.

Learning to Forget for Meta-Learning via Task-and-Layer-Wise Attenuation.

Few-shot learning is an emerging yet challenging problem in which the goal is to achieve generalization from only few examples. Meta-learning tackles few-shot learning via the learning of prior knowledge shared across tasks and using it to learn new tasks. One of the most representative meta-learning algorithms is the model-agnostic meta-learning (MAML), which formulates prior knowledge as a common initialization, a shared starting point from where a learner can quickly adapt to unseen tasks. However, forcibly sharing an initialization can lead to conflicts among tasks and the compromised (undesired by tasks) location on optimization landscape, thereby hindering task adaptation. Furthermore, the degree of conflict is observed to vary not only among the tasks but also among the layers of a neural network. Thus, we propose task-and-layer-wise attenuation on the compromised initialization to reduce its adverse influence on task adaptation. As attenuation dynamically controls (or selectively forgets) the influence of the compromised prior knowledge for a given task and each layer, we name our method Learn to Forget (L2F). Experimental results demonstrate that the proposed method greatly improves the performance of the state-of-the-art MAML-based frameworks across diverse domains: few-shot classification, cross-domain few-shot classification, regression, reinforcement learning, and visual tracking.

Redd1 inhibits the invasiveness of non-small cell lung cancer cells.

Redd1 acts as a negative regulator of mTOR in response to various stress conditions, but its specific physiological role is currently unclear. In the present study, we showed that Redd1 inhibits the invasive activity of non-small cell lung cancer (NSCLC) cells. Interestingly, expression of Redd1 was extremely low in H1299 cells displaying high invasiveness, compared with that in H460 cells with lower invasive activity. Overexpression of Redd1 inhibited the invasive activity of H1299 cells, while suppression with specific siRNAs enhanced the invasiveness of H460 cells. Knockdown of the mTOR downstream substrate, S6K, resulted in a decrease in the invasive property of H1299 cells. Our results provide preliminary evidence that Redd1 inhibits the invasive activity of NSCLC cells via suppression of the mTOR downstream pathway.

A truncated form of p23 down-regulates telomerase activity via disruption of Hsp90 function.

The Hsp90-associated protein p23 modulates Hsp90 activity during the final stages of the chaperone pathway to facilitate maturation of client proteins. Previous reports indicate that p23 cleavage induced by caspases during cell death triggers destabilization of client proteins. However, the specific role of truncated p23 (Delta p23) in this process and the underlying mechanisms remain to be determined. One such client protein, hTERT, is a telomerase catalytic subunit regulated by several chaperone proteins, including Hsp90 and p23. In the present study, we examined the effects of p23 cleavage on hTERT stability and telomerase activity. Our data showed that overexpression of Delta p23 resulted in a decrease in hTERT levels, and a down-regulation in telomerase activity. Serine phosphorylation of Hsp90 was significantly reduced in cells expressing high levels of Delta p23 compared with those expressing full-length p23. Mutation analyses revealed that two serine residues (Ser-231 and Ser-263) in Hsp90 are important for activation of telomerase, and down-regulation of telomerase activity by Delta p23 was associated with inhibition of cell growth and sensitization of cells to cisplatin. Our data aid in determining the mechanism underlying the regulation of telomerase activity by the chaperone complex during caspase-dependent cell death.

Activation of epidermal growth factor receptor and its downstream signaling pathway by nitric oxide in response to ionizing radiation.

Epidermal growth factor receptor (EGFR) is activated by ionizing radiation (IR), but the molecular mechanism for this effect is unknown. We have found that intracellular generation of nitric oxide (NO) by NO synthase (NOS) is required for the rapid activation of EGFR phosphorylation by IR. Treatment of A549 lung cancer cells with IR increased NOS activity within minutes, accompanied by an increase of NO. 2-Phenyl-4,4,5,5,-tetramethylimidazolline-1-oxyl-3-oxide, an NO scavenger, and NG-monomethyl-l-arginine, an NOS inhibitor, abolished the increase in intracellular NO and activation of EGFR by IR. In addition, an NO donor alone induced EGFR phosphorylation. Transient transfection with small interfering RNA for endothelial NOS reduced IR-induced NO production and suppressed IR-induced EGFR activation. Overexpression of endothelial NOS increased IR-induced NO generation and EGFR activation. These results indicate a novel molecular mechanism for EGFR activation by IR-induced NO production via NOS.

Nuclear protein 1 induced by ATF4 in response to various stressors acts as a positive regulator on the transcriptional activation of ATF4.

Nuclear protein 1 (NUPR1) was originally identified as p8, a member of the family of HMG-I/Y transcription factors induced in response to various cellular stressors. However, the signaling pathway underlying NUPR1 induction by cellular stresses remains to be established. In this study, we found that the expression of NUPR1 by various stresses induced by activating transcription factor 4 (ATF4). Loss of ATF4 using siRNA significantly diminished NUPR1 expression. Overexpression of ATF4 caused NUPR1 levels to rise. NUPR1 expression was associated with enhanced transcriptional activation of genes of ATF4 downstream, suggesting that the protein promoted the transcription of stress-regulated genes via positive feedback on the ATF4 pathway.

HSP27 regulates cell adhesion and invasion via modulation of focal adhesion kinase and MMP-2 expression.

Heat-shock protein 27 (HSP27), a member of the small heat-shock protein family, is a molecule involved in cellular protection in response to a variety of stresses such as heat shock, toxicants, and oxidative stress. HSP27 is also known to modulate cell functions via interaction with the actin cytoskeleton. To elucidate the functions of HSP27 in adhesion and invasion in more detail, we examined NIH3T3 cells overexpressing HSP27. HSP27 overexpression affected FAK phosphorylation and focal adhesion formation, depending on integrin-mediated actin cytoskeleton polymerization. In addition, the HSP27-overexpressing cells showed a retarded cell migration and invasion in wound-healing assays. Such HSP27-mediated retarded wound healing was correlated with reduced matrix metalloproteinase-2 (MMP-2) expression. The transcription factor for MMP-2 expression, signal transducer and activator or transcription 3 (STAT3), was correspondingly less phosphorylated. When a phosphomimetic form of HSP27 was transiently transfected, migration and invasion were similarly decreased via the regulation of the FAK/STAT3/MMP-2 signaling pathway, whereas a non-phosphorylatable form of HSP27 blocked HSP27-mediated phenotypes probably due to a dominant-negative effect on phosphorylation of endogenous HSP27. Altogether, our results suggest that HSP27 can enhance cell adhesion and modulate cell migration and invasion via the coordination of FAK-dependent actin organization and STAT3-dependent MMP-2 expression, and that phosphorylation of HSP27 is indispensable to regulate this signal pathway.

Combined effects of sulindac and suberoylanilide hydroxamic acid on apoptosis induction in human lung cancer cells.

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. Treatment of cancer cells with HDAC blockers, such as suberoylanilide hydroxamic acid (SAHA), leads to the activation of apoptosis-promoting genes. To enhance proapoptotic efficiency, SAHA has been used in conjunction with radiation, kinase inhibitors, and cytotoxic drugs. In the present study, we show that at the suboptimal dose of 250 muM, sulindac [2-[6-fluoro-2-methyl-3-[(4-methylsulfinylphenyl)methylidene]inden-1-yl]-acetic acid] significantly enhances SAHA-induced growth suppression and apoptosis of A549 human non-small cell lung cancer cells, primarily via enhanced collapse of the mitochondrial membrane potential, release of cytochrome c, and caspase activation. Furthermore, sulindac/SAHA cotreatment induced marked down-regulation of survivin at both the mRNA and protein levels and stimulated the production of reactive oxygen species (ROS), which were blocked by the antioxidant N-acetyl-l-cysteine. Overexpression of survivin was associated with reduced sulindac/SAHA-induced apoptosis of A549 cells, whereas suppression of survivin levels with antisense oligonucleotides or small interfering RNA further sensitized cells to sulindac/SAHA-induced cell death. Our results collectively demonstrate that sulindac/SAHA-induced apoptosis is mediated by ROS-dependent down-regulation of survivin in lung cancer cells.

A combination of sulindac and arsenic trioxide synergistically induces apoptosis in human lung cancer H1299 cells via c-Jun NH2-terminal kinase-dependent Bcl-xL phosphorylation.

SUMMARY: In the present study, we show that a combination of sulindac and arsenic trioxide (ATO) induces more extensive apoptosis than either drug alone in H1299 human non-small cell lung carcinoma (NSCLC) cells. Treatment with sulindac/ATO triggered three major apoptotic signaling events, namely, collapse of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases. Furthermore, the sulindac/ATO combination induced reactive oxygen species (ROS) generation, and the antioxidant, N-acetyl-L-cysteine, blocked this apoptotic signaling. The c-Jun NH(2)-terminal kinase (JNK) was activated downstream of ROS production in H1299 cells. Blockage of JNK by pretreatment with SP600125, a pharmacological inhibitor, or transfection with dominant-negative (DN) JNK1 vectors abrogated sulindac/ATO-induced apoptosis, as evident from the disruption of caspase activation. Interestingly, a slower migrating Bcl-xL band was observed on immunoblots after treatment of cells with sulindac/ATO. The band was absent upon the treatment of cell lysates with lambda protein phosphatase. Moreover, confocal microscopy findings disclose that active JNK translocates to mitochondria. Treatment with SP600125 and transfection with DN-JNK blocked Bcl-xL phosphorylation, suggesting that JNK plays an important role in sulindac/ATO-induced Bcl-xL phosphorylation. In conclusion, in H1299 human NSCLC cells, sulindac and ATO synergistically induce a high degree of apoptosis, which is mediated by the ROS-dependent JNK activation pathway via Bcl-xL phosphorylation.

Hypoxic condition- and high cell density-induced expression of Redd1 is regulated by activation of hypoxia-inducible factor-1alpha and Sp1 through the phosphatidylinositol 3-kinase/Akt signaling pathway.

Redd1, a recently discovered stress-response gene, is regulated by hypoxia via hypoxia-inducible factor 1 (HIF-1) and by DNA damage via p53/p63; however, the signaling pathway by which its expression is induced by hypoxia has not been elucidated. In the present study, we demonstrated that the expression of Redd1 in response to hypoxia (1% O(2)), hypoxia-mimetic agent, cobalt chloride (CoCl(2)) and high cell density (HCD) requires coactivation of HIF-1alpha and Sp1. CoCl(2) and HCD induced the activation of HIF-1alpha and Sp1 in HeLa cells, and siRNAs targeting HIF-1alpha and Sp1 abrogated Redd1 expression. Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 and by a dominant-negative PI3K mutant reduced the expression of Redd1 and activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Also, suppression of Akt activation blocked the expression of Redd1 and the activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Furthermore, we found that the induction of Redd1 expression by CoCl(2) can be mediated by activation of Sp1 in HIF-1alpha-deficient cells but that a higher level of Redd1 expression is achieved when these cells are transfected with HIF-1alpha. These results demonstrate that hypoxic condition-and HCD-induced expression of Redd1 is mediated by coactivation of Sp1 and HIF-1alpha downstream of the PI3K/Akt signaling pathway.

Acute radio frequency irradiation does not affect cell cycle, cellular migration, and invasion.

Although in vitro studies have been previously conducted to determine the biological effects of radio frequency (RF) radiation, it has not yet been determined whether or not RF radiation poses a potential hazard. This study was conducted to determine whether RF radiation exposure exerts detectable effects on cell cycle distribution, cellular invasion, and migration. NIH3T3 mouse fibroblasts were exposed to 849 MHz of RF radiation at average SAR values of 2 or 10 W/kg for either 1 h, or for 1 h per day for 3 days. During the exposure period, the temperature in the exposure chamber was maintained isothermally by circulating water throughout the cavity. Cell cycle distribution was analyzed at 24 and 48 h after exposure, by flow cytometry. We detected no statistically significant differences between the sham-exposed and RF radiation-exposed cells. Cellular invasion and migration were assessed by in vitro Matrigel invasion and Transwell migration assays. The RF radiation-exposed groups evidenced no significant changes in motility and invasiveness compared to the sham-exposed group. However, the ionizing radiation-exposed cells, used as a positive control group, manifested dramatic alterations in their cell cycle distribution, cellular invasiveness, and migration characteristics. Our results show that 849 MHz RF radiation exposure exerts no detectable effects on cell cycle distribution, cellular migration, or invasion at average SAR values of 2 or 10 W/kg.

Transforming growth factor-beta1 induces tissue inhibitor of metalloproteinase-1 expression via activation of extracellular signal-regulated kinase and Sp1 in human fibrosarcoma cells.

The net balance of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) system has been known to be a key factor in tumor cell invasion. In the present study, we investigated the molecular mechanisms of anti-invasive and antimigrative activity of transforming growth factor (TGF)-beta1 on HT1080 human fibrosarcoma cells. In in vitro Matrigel invasion and Transwell migration assays, TGF-beta1 dose-dependently inhibited the invasion and migration of HT1080 cells, respectively. Gelatin zymography, Western blot, and real-time PCR analysis showed that TGF-beta1 enhanced the expression and secretion of MMP-2, TIMP-1, and, to a lesser degree, MMP-9 but not membrane type 1-MMP and TIMP-2. The addition of recombinant TIMP-1 protein reduced the Matrigel invasion and Transwell migration of HT1080 cells, similar to TGF-beta1. Because augmentation of TIMP-1 might be the major factor for the anti-invasive and antimigrative activity of TGF-beta1, we investigated possible molecular mechanisms responsible for the expression of TIMP-1 induced by TGF-beta1. Treatment of HT1080 cells with TGF-beta1 rapidly phosphorylated three mitogen-activated protein kinases [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun NH2-terminal kinase] and Akt. Among these kinases, the inhibition of only ERK1/2 pathway by PD98059, a specific inhibitor of MAPK/ERK kinase(MEK)-1, and transfection of dominant-negative MEK 1 effectively blocked the TIMP-1 induction by TGF-beta1. Mithramycin, a specific inhibitor of Sp1 transcription factor, but not curcumin, an inhibitor of activator protein-1, and transfection of Sp1 small interfering RNA significantly inhibited the TGF-beta1-induced expression of TIMP-1. In addition, electrophoretic mobility shift assay showed that TGF-beta1 up-regulated Sp1 DNA-binding activity, and PD98059 and mithramycin effectively inhibited these events. Finally, pretreatment of HT1080 cells with PD98059 and mithramycin, but not curcumin, restored the invasive activity of these cells. Taken together, these data suggest that TGF-beta1 modulates the net balance of the MMPs/TIMPs the systems in HT1080 cells for anti-invasion and antimigration by augmenting TIMP-1 through ERK1/2 pathway and Sp1 transcription factor.

Synergistic induction of apoptosis by sulindac and arsenic trioxide in human lung cancer A549 cells via reactive oxygen species-dependent down-regulation of survivin.

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, may be a good target for cancer therapy because it is expressed in a variety of human tumors but not in differentiated adult tissues. In the present study, we show that a combination of sulindac and arsenic trioxide (ATO) induces more extensive apoptosis than either drug alone in A549 human non-small cell lung carcinoma (NSCLC) cells. Treatment with sulindac/ATO reduced the expression of survivin and promoted major apoptotic signaling events, namely, collapse of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases. Combined sulindac/ATO treatment did not significantly affect the levels of other members of the IAP family (XIAP, cIAP1 and cIAP2), indicating that the effects were specific to survivin. In addition, sulindac/ATO treatment induced the production of reactive oxygen species and the antioxidant N-acetyl-l-cysteine blocked the down-regulation of survivin and induction of apoptotic signaling by the combination of sulindac and ATO. Combined sulindac/ATO treatment also activated p53 expression, and inhibition of p53 expression by small interfering RNA (siRNA) prevented sulindac/ATO-induced down-regulation of survivin, suggesting that survivin expression is negatively regulated by p53. Overexpression of survivin reduced sulindac/ATO-induced apoptosis in A549 cells and reduction of survivin levels by siRNA sensitized the cells to sulindac/ATO-induced cell death. These results demonstrate that, in A549 human NSCLC cells, sulindac/ATO-induced apoptosis is mediated by the reactive oxygen species-dependent down-regulation of survivin.

Induction of p53-mediated apoptosis and recovery of chemosensitivity through p53 transduction in human glioblastoma cells by cisplatin.

Cisplatin is a DNA-damaging chemotherapeutic drug that may have a role in the adjuvant chemotherapy of several solid tumors, such as malignant glioblastoma, and the status of p53 tumor suppressor protein is a critical determinant of cisplatin chemosensitivity. In the present study, we showed the relationship of p53 status and chemosensitivity of cisplatin between two human malignant glioblastoma cell lines, A172 and T98G, harboring wild-type and mutant-type p53, respectively. Cisplatin was found to be more cytotoxic to A172 than T98G cells in a time- and concentration-dependent manner. Cisplatin-induced cytotoxicity manifested as apoptosis, characterized by genomic DNA fragmentation, nuclear condensation and an increase in sub-G1 population. Cisplatin induced the accumulation of p53 and p21 proteins in A172 cells, but not in T98G cells. The introduction of the adenovirus-mediated wild-type p53 gene into T98G cells resulted in the decrease of viability as well as the increase in sub-G1 population with p53 accumulation, activation of caspase-3 protease and release of cytochrome c from the mitochondria. These data strongly suggest that the expression of p53 is essential for the cytotoxic effect of cisplatin in human malignant glioblastoma cells, A172 and T98G, and the introduction of apoptotic signal molecules, such as p53, will be beneficial to achieve chemosensitivity in malignant glioma.

Heterogeneity of capillary endothelial cells for basic fibroblast growth factor-induced paracrine signaling.

In this study, the authors isolated morphologically different capillary endothelial cells, designated as BCE-1 and BCE-2 cells, from bovine adrenal cortex. By a series of experiments involving proliferation, migration, and tubular-like structure formation assays, the authors found that the two BCE clones showed a clearly different response to basic fibroblast growth factor (bFGF). Similar to these results, the ERK-1/2 in the BCE-1 cells was phosphorylated by bFGF or vascular endothelial growth factor (VEGF), whereas that of the BCE-2 cells was phosphorylated only by VEGF. However, when the BCE-2 cells were transfected with FGF receptor 1 cDNA, the ERK-1/2 of these cells was phosphorylated by exogenous bFGF. Receptor binding experiments revealed that BCE-2 cells expressed high-affinity tyrosine-kinase FGF receptors approximately twofold less than BCE-1 cells. Transfection and receptor binding studies suggest a possibility that the poor response of the BCE-2 cells to exogenous bFGF is derived from the limitation of functional availability of high affinity FGF receptors. On the other hand, when both BCE clones were treated with anti-bFGF antibodies, basal formation of tubular-like structure in both clones were strongly inhibited, indicating that endogenous bFGF plays a role in in vitro angiogenesis of both BCE clones. Taken together, these data show that the isolated capillary endothelial cells are heterogeneous for paracrine but not autocrine bFGF signaling, and suggest that the diversity of capillary endothelial cells can occur by angiogenic factors, such as bFGF.

PTEN suppresses hyaluronic acid-induced matrix metalloproteinase-9 expression in U87MG glioblastoma cells through focal adhesion kinase dephosphorylation.

Glioblastoma is a severe type of primary brain tumor and its invasion is strongly correlated with the secretion of matrix metalloproteinases (MMPs). To investigate a role of PTEN, a tumor suppressor gene, in the regulation of hyaluronic acid (HA)-induced invasion of glioma cells, we examined the secretion of MMP-9 in various glioma cells with or without a functional PTEN gene. The secretion of MMP-9 in glioma cells lacking functional PTEN (U87MG, U251MG, and U373MG) was induced by HA, although not in wildtype (wt)-PTEN-harboring cells (LN229, LN18, and LN428). In addition, stable expression of wt-PTEN into U87MG cells significantly decreased the secretion of HA-induced MMP-9 and basal levels of MMP-2, inhibiting the activation of focal adhesion kinase and extracellular signal-regulated kinase 1/2, whereas the secretion levels of the tissue inhibitor of metalloproteinase-1 and -2 were increased, finally resulting in the inhibition of invasion by HA in vitro. Ectopic expressions of adenoviral (Ad)-wt-PTEN and -lipid phosphatase-deficient (G129E)-PTEN, but not both protein and -lipid phosphatase-deficient (C124S)-PTEN, reduced MMP-9 secretion and invasion by HA. These results were also confirmed by expressions of Ad-wt-PTEN and Ad-G129E-PTEN in other glioblastoma cells lacking functional PTEN, U251MG, and U373MG. These findings strongly suggest the possibility that PTEN may block HA-induced MMP-9 secretion and invasion through its protein phosphatase activity.