Modulation of invasive properties of CD133(+) glioblastoma stem cells: A role for MT1‐MMP in bioactive lysophospholipid signaling

Future breakthroughs in cancer therapy must accompany targeted agents that will neutralize cancer stem cells response to circulating growth factors. Since the brain tissue microenvironmental niche is a prerequisite for expression of the stem cell marker CD133 antigen in brain tumors, we investigated the invasion mechanisms specific to CD133(+) U87 glioblastoma cells in response to lysophosphatidic acid (LPA) and sphingosine 1‐phosphate (S1P), two circulating bioactive lysophospholipids and potent inducers of cancer. A CD133(+) U87 glioma cell population was isolated from parental U87 glioblastoma cells using magnetic cell sorting technology. The CD133(+)‐enriched cell population grew as neurospheres and showed enhanced maximal response to both LPA (～5.0‐fold) and S1P (～2.5‐fold) at 1 µM when compared to parental U87 cells. The increased response to LPA in CD133(+) cells, reflected by increased levels of phosphorylated ERK, was found independent of the cooperative functions of the membrane‐type‐1 matrix metalloproteinase (MT1‐MMP), while this cooperativity was essential to the S1P response. Quantitative RT‐PCR was performed and we found higher gene expression levels of the S1P receptors S1P1 and S1P2, and of the LPA receptor LPA1 in CD133(+) cells than in their parental U87 cells. These increased levels reflected those observed from in vivo experimental U87 tumor implants. Our data suggest that the CD133(+) cell subpopulation evokes most of the lysophospholipid response within brain tumors through a combined regulation of S1P/LPA cell surface receptors signaling and by MT1‐MMP. The emergence of lead compounds targeting the stem cell niche and S1P/LPA signaling in CD133(+) cancer cells is warranted. © 2009 Wiley‐Liss, Inc.     

Expression pattern of osteopontin splice variants and its functions on cell apoptosis and invasion in glioma cells

Osteopontin (OPN) is widely overexpressed in various cancers, including gliomas, and plays an important role in tumorigenesis. However, the expression pattern and functions of OPN splice variants expressed in gliomas remain unclear. The aims of our current study were to examine the expression pattern and functions of OPN splice variants in gliomas. In present study, the mRNA levels of OPN splice variants are markedly increased in gliomas tissues, and all OPN splice variants were also found in U251 and U87 cells. Furthermore, knock-down and regain of function experiments were designed to explore the functions of OPN splice variants in U251 and U87 cells. Lentiviral vectors of OPN small interference RNA (siRNA) targeting all three endogenous mRNAs of OPN and OPN splice variants synonymous mutant that were not silenced by OPN siRNA were constructed. Our results showed that all OPN splice variants synonymous mutant-protected glioma cells from apoptosis induced by OPN siRNA through alteration of the levels of Bcl-2 family proteins and OPN-b Mu elicted a significant effect. Both OPN-a Mu and -c Mu promoted glioma cell invasion through alteration of the levels of uPA, MMP-2, and MMP-9 expressions and the activities of MMP-2 and MMP-9 via activation PI-3K/AKT/NF-κB signaling pathway. Moreover, OPN-c Mu showed the strongest effect on glioma cell invasion, while OPN-b Mu showed no effect on the invasion of U251 and U87 cells. Thus, different splice variants of OPN have divergent functions in regulating apoptosis and invasion of glioma cells, which broadens their importance in glioma biotherapy.     

Targeting notch to eradicate pancreatic cancer stem cells for cancer therapy

Pancreatic cancer is the most aggressive malignant disease once it is diagnosed and it remains the fourth leading cause of cancer-related death in the U.S.A. Recent data indicates that the Notch signaling pathway plays an important role in the development and progression of pancreatic cancer. Emerging evidence also suggests that the activation of the Notch signaling pathway is mechanistically associated with molecular characteristics of cancer stem cells (CSCs) in pancreatic cancer. Moreover, CSCs are known to be highly drug-resistant, suggesting that targeted inactivation of Notch signaling would be useful for overcoming drug resistance and the elimination of CSCs. This review describes the roles of the Notch signaling pathway in pancreatic cancer with a special emphasis on its novel functions in the regulation of pancreatic CSC. Moreover, the review also proposes that targeting the Notch signaling pathway by natural agents may represent a novel strategy for overcoming drug resistance and the elimination of CSCs, which would be useful for the successful treatment of patients diagnosed with pancreatic cancer.     

miR-205-5p/E2F1信号抑制经典Wnt/β蛋白通路调控脑胶质瘤细胞放射敏感性

目的 探索miR-205-5p/E2F1信号轴在脑胶质瘤U251、U87细胞放射耐受中的调控机制。方法 利用X射线逐步递增递间歇诱导方法照射U251、U87细胞,建立放射耐受的U251/TR、U87/TR细胞。对两种细胞进行形态学、细胞运动、侵袭及增殖能力分析。通过荧光素酶基因检测系统及点突变技术分析E2F1基因对U251/TR、U87/TR细胞的调控机制。结果 放射耐受的U251/TR、U87/TR细胞分别比251、U87细胞的增殖活力增强,运动、侵袭能力增强,X射线照射下细胞凋亡下降。miR-205-5p mimics转染能够下调U251/TR细胞E2F1因子表达,抑制细胞增殖、侵袭及运动,增加放射敏感性。miR-205-5p mimics转染协同E2F1下调是通过抑制细胞Wnt/β-catenin信号通路活性发挥抑制肿瘤作用,并降低细胞耐受。结论 逐步递增递间歇诱导方法能较好地建立U251/TR、U87/TR细胞。miR-205-5p/E2F1信号轴通过经典Wnt/β-catenin信号通路发挥抑癌作用,可以作为提高胶质瘤放射敏感性的治疗靶点。     

mTOR activation in immature cells of primary nasopharyngeal carcinoma and anti-tumor effect of rapamycin in vitro and in vivo

The mammalian target of rapamycin (mTOR) signaling is a key pathway in the progression of different cancers and in the homeostasis of stem cells. Here, we investigated the link between mTOR signaling and cancer stem cells (CSCs) in nasopharyngeal carcinoma (NPC). We found that human primary NPC expressed embryonic stem cell (ESC) markers: CD133, SOX2 and OCT4 as well as pmTOR and pS6. Primary ESC-positive NPC cells could form secondary NPC in BALB/c nude mice. Rapamycin, an mTOR inhibitor, significantly suppressed ESC-positive NPC cell growth in vitro and tumor formation in vivo. Our findings suggest that mTOR signaling is activated in CSC-like cells and plays an important role in NPC growth.     

CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway

The recent discovery of cancer stem cells (CSCs) has played a pivotal role in changing our view of carcinogenesis and chemotherapy. Based on this concept, CSCs are responsible for the formation and growth of neoplastic tissue and are naturally resistant to chemotherapy, explaining why traditional chemotherapies can initially shrink a tumor but fails to eradicate it in full, allowing eventual recurrence. Recently, we identified a CSC population in hepatocellular carcinoma (HCC) characterized by their CD133 phenotype. However, the molecular mechanism by which it escapes conventional therapies remains unknown. Here, we examined the sensitivity of these cells to chemotherapeutic agents (doxorubicin and fluorouracil) and the possible mechanistic pathway by which resistance may be regulated. Purified CD133+ HCC cells isolated from human HCC cell line and xenograft mouse models survived chemotherapy in increased proportions relative to most tumor cells which lack the CD133 phenotype; the underlying mechanism of which required the preferential expression of survival proteins involved in the Akt/PKB and Bcl-2 pathway. Treatment of CD133+ HCC cells with an AKT1 inhibitor, specific to the Akt/PKB pathway, significantly reduced the expression of the survival proteins that was normally expressed endogenously. In addition, treatment of unsorted HCC cells with both anticancer drugs in vitro significantly enriched the CD133+ subpopulation. In conclusion, our results show that CD133+ HCC cells contribute to chemoresistance through preferential activation of Akt/PKB and Bcl-2 cell survival response. Targeting of this specific survival signaling pathway in CD133+ HCC CSCs may provide a novel therapeutic model for the disease.     

Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87

A variety of malignant cancers have been found to contain a subpopulation of stem cell-like tumor cells, or cancer stem cells (CSCs). However, the existence of CSCs in U87, a most commonly used glioma cell line, is still controversial. In this study, we demonstrate that U87 cell line contained a fraction of tumor cells that could form tumor spheres and were enriched by progressively increasing the concentration of serum-free neural stem cell medium with or without low dose vincristine. These cells possessed the ability of self-renewal and multipotency, the defined characteristics of CSCs. Moreover, the tumors formed by the secondary spheres displayed typical histological features of human glioblastoma, including cellular pleomorphism, pseudopalisades surrounding necrosis, hyperchromatic nuclei, high density of microvessels and invasion to the brain parenchyma. These results indicate that gradually increasing the concentration of serum-free neural stem cell culture medium with or without vincristine is a simple and effective method for isolation of CSCs to study the initiation and progression of human glioblastoma.     

ADAM17对U87MG胶质瘤细胞增殖与侵袭的影响及作用机制

目的 探讨ADAM17在U87MG细胞增殖与侵袭中的作用及机制.方法 收集人脑胶质瘤组织65例(肿瘤组)与正常脑组织13例(对照组),采用免疫组化、RT-PCR法,检测ADAM17蛋白及mRNA的表达;利用siRNA干扰敲减或ADAM17激活剂(PMA)过表达ADAM17,并予以PI3K抑制剂抑制相关信号通路,MTT及Transwell实验检测各组U87MG细胞的增殖与侵袭变化,Western blot检测ADAM17、p-AKT及AKT蛋白变化.结果 胶质瘤组织中ADAM17mRNA及蛋白水平均高于正常脑组织;与对照组相比,siRNA干扰后,低表达组U87MG细胞的增殖与侵袭能力下降,而激活剂PMA增强ADAM17后,过表达组细胞增殖与侵袭能力增加,差异有统计学意义(P＜0.05);敲减及过表达AD-AM17可导致PI3 K/AKT信号通路下游蛋白发生变化.结果 ADAM17可通过激活PI3K/AKT信号通路促进U87MG细胞增殖与侵袭,有望为胶质瘤的治疗找到新突破点.     

PTTG1影响胶质瘤细胞侵袭力机制的研究

背景与目的：研究证实垂体肿瘤转化基因1(pituitary tumor transforming gene 1,PTTG1)的表达情况与各类肿瘤细胞的恶性程度有关,但是其在胶质瘤中的作用尚不清楚。本研究旨在探讨PTTG1在恶性胶质瘤侵袭中的作用及其临床意义。方法：应用蛋白质印迹法(Western blot)检测PTTG1蛋白在不同胶质瘤细胞系中的表达;采用小RNA质粒干扰技术抑制U87细胞中PTTG1蛋白的表达,应用Western blot技术检测转染的效率;通过体外侵袭实验检测转染后细胞侵袭力的变化;采用Western blot技术检测经过表皮细胞生长因子(epithelial growth factor,EGF)刺激后敲除PTTG1的细胞组(siPTTG1/U87)与转染空载的细胞组(Scr/U87)中Akt、ARK5磷酸化的情况。结果：PTTG1蛋白在各恶性胶质瘤细胞系中均高表达;敲除PTTG1后U87细胞的侵袭力明显下降;对Scr/U87细胞进行EGF刺激5 min后,Akt、ARK5的磷酸化情况显著增强,而无论有无EGF的刺激,siPTTG1/U87中Akt、ARK5的磷酸化情况都没有明显改变。结论：在恶性胶质瘤细胞中,PTTG1蛋白呈高表达状态并且与侵袭性显著相关,其对侵袭性的调控可能是通过Akt-ARK5通路实现的。     

MiR-21 regulates epithelial-mesenchymal transition phenotype and hypoxia-inducible factor-1α expression in third-sphere forming breast cancer stem cell-like cells

Cancer stem cells (CSCs) are predicted to be critical drivers of tumor progression due to their "stemness", but the molecular mechanism of CSCs in regulating metastasis remains to be elucidated. Epithelial-mesenchymal transition (EMT), hypoxia-inducible factor (HIF)-1α, and miR-21, all of which contribute to cell migration for metastasis, are interrelated with CSCs. In the present study, third-sphere forming (3-S) CSC-like cells, which showed elevated CSC surface markers (ALDH1(+) and CD44(+)/CD24(-/low)) and sphereforming capacity as well as migration and invasion capacities, were cultured and isolated from breast cancer MCF-7 parental cells, to evaluate the role of miR-21 in regulating the CSC-like cell biological features, especially EMT. EMT, which was assessed by overexpression of mesenchymal cell markers (N-cadherin, Vimentin, alpha-smooth muscle actin [α-SMA]) and suppression of epithelial cell marker (E-cadherin), was induced in 3-S CSC-like cells. Moreover, both of HIF-1α and miR-21 were upregulated in the CSC-like cells. Interestingly, antagonism of miR-21 by antagomir led to reversal of EMT, downexpression of HIF-1α, as well as suppression of invasion and migration, which indicates a key role of miR-21 involved in regulate CSC-associated features. In conclusion, we demonstrated that the formation of CSC-like cells undergoing process of EMT-like associated with overexpression of HIF-1α, both of which are regulated by miR-21.     

MicroRNA-653靶向调控OIP5基因介导mTOR信号通路对非小细胞肺癌细胞生物学特性的影响

目的:探讨microRNA-653（miR-653）靶向调控OIP5基因介导mTOR信号通路对非小细胞肺癌（non-small cell lung cancer,NSCLC）细胞生物学特性的影响。方法:选取人正常内皮细胞BEAS-2B和4种NSCLC细胞系（H1650、H1975、A549和H292）,分为control组、mimics组、mimics-NC组、inhibitor组和inhibitor-NC组进行瞬时转染,利用荧光定量PCR、蛋白印迹、MTT、划痕实验、Transwell侵袭实验、流式细胞术等方法分析miR-653对NSCLC细胞中OIP5、mTOR信号通路相关基因表达及增殖、迁移、侵袭、凋亡和细胞周期分布的影响。结果:与control组、mimics-NC组和inhibitor-NC组相比,mimics组mTOR信号通路被抑制,细胞增殖、迁移、侵袭能力明显降低,凋亡率明显增加,细胞多停滞于G1期（均P＜0.05）;然而,inhibitor组mTOR信号通路被激活,细胞增殖、迁移、侵袭能力明显升高,而凋亡率明显降低,G1期细胞数量较少（均P＜0.05）。结论:miR-653可通过负调控OIP5基因抑制mTOR通路激活,从而阻止NSCLC的发生与发展。     

Cancer stem cells in Epstein‐Barr virus‐associated gastric carcinoma

Cancer stem cells (CSCs) play a decisive role in the development and progression of cancer. To investigate CSCs in Epstein‐Barr virus (EBV)‐associated carcinoma (EBVaGC), we screened previously reported stem cell markers of gastric cancer in EBV‐infected gastric cancer cell lines (TMK1 and NUGC3) and identified CD44v6v9 double positive cells as candidate CSCs. CD44v6/v9^+/+ cells were sorted from EBVaGC cell line (SNU719) cells and EBV‐infected TMK1 cells and these cell populations showed high spheroid‐forming ability and tumor formation in SCID mice compared with the respective CD44v6/v9^?/? cells. Sphere‐forming ability was dependent on the nuclear factor‐κB (NF‐κB) signaling pathway, which was confirmed by decrease of sphere formation ability under BAY 11‐7082. Small interfering RNA knockdown of latent membrane protein 2A (LMP2A), one of the latent gene products of EBV infection, decreased spheroid formation in SNU719 cells. Transfection of the LMP2A gene increased the sphere‐forming ability of TMK1 cells, which was mediated through NF‐κB signaling. Together, these results indicate that CD44v6v9^+/+ cells are CSCs in EBVaGC that are maintained through the LMP2A/NF‐κB pathway. Future studies should investigate CD44v6/v9^+/+ cells in normal and neoplastic gastric epithelium to prevent and treat this specific subtype of gastric cancer infected with EBV.     

Notch signaling in CD66+ cells drives the progression of human cervical cancers

Human epithelial tumor progression and metastasis involve cellular invasion, dissemination in the vasculature, and regrowth at metastatic sites. Notch signaling has been implicated in metastatic progression but its roles have yet to be fully understood. Here we report the important role of Notch signaling in maintaining cells expressing the carcinoembryonic antigen cell adhesion molecule CEACAM (CD66), a known mediator of metastasis. CD66 and Notch1 were studied in clinical specimens and explants of human cervical cancer, including specimens grown in a pathophysiologically relevant murine model. Gene expression profiling of CD66(+) cells from primary tumors showed enhanced features of Notch signaling, metastasis, and stemness. Significant differences were also seen in invasion, colony formation, and tumor forming efficiency between CD66(+) and CD66(-) cancer cells. Notably, CD66(+) cells showed a marked sensitivity to a Notch small molecule inhibitor. In support of studies in established cell lines, we documented the emergence of a tumorigenic CD66(+) cell subset within a metastatic lesion-derived cervical-cancer cell line. Similar to primary cancers, CD66 expression in the cell line was blocked by chemical and genetic inhibitors of ligand-dependent nuclear Notch signaling. Collectively, our work on the oncogenic properties of CD66(+) cells in epithelial cancers provides insights into the nature of tumor progression and offers a mechanistic rationale to inhibit the Notch signaling pathway as a generalized therapeutic strategy to treat metastatic cancers.     

Programmed death ligand 1 regulates epithelial–mesenchymal transition and cancer stem cell phenotypes in hepatocellular carcinoma through the serum and glucocorticoid kinase 2/β-catenin signaling pathway

Programmed death ligand 1 (PD-L1) plays an important role in the occurrence of hepatocellular carcinoma (HCC). The present study indicated that epithelial–mesenchymal transition (EMT) and induction of cancer stem cell (CSC)-like properties contribute to metastasis of cancers. However, the molecular mechanisms underlying PD-L1 and EMT and CSC phenotypes in HCC remain to be elucidated. Here, we report that PD-L1 regulates not only EMT but also the stem-like transition in liver cancer cells. We observed high PD-L1 expression in CD133⁺ liver CSCs and CSC-enriched tumor spheres. Altering PD-L1 expression promoted liver CSC phenotypes by increasing the expression of stemness genes, the CD133⁺ cell population sizes, and the ability to form tumor spheres. Programmed death ligand 1 enhanced HCC cell tumorigenicity and invasion in nude mice. Additionally, PD-L1 overexpression in cells significantly increased cell motility and invasion, as well as the EMT process. Conversely, suppression of PD-L1 in cells had an opposite effect. Prolonged treatment of HCC cells with Akt inhibitor prefosine leads to activation of serum and glucocorticoid kinase 2 (SGK2) and rescued downregulation of PD-L1. Mechanistically, PD-L1 directly interacted with SGK2. Programmed death ligand 1 upregulated SGK2 and activated the SGK2/β-catenin signaling pathway, and promoted EMT and CSC expansion in liver cancer cells, highlighting the role of SGK2 in PD-L1-mediated EMT and CSC phenotypes in liver cancer cells. In conclusion, our findings suggest that PD-L1 activated the SGK2/β-catenin signaling pathway, to induce EMT and acquisition of a stem cell phenotype.     

PAR1 participates in the ability of multidrug resistance and tumorigenesis by controlling Hippo-YAP pathway

The Hippo pathway significantly correlates with organ size control and tumorigenesis. The activity of YAP/TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in cancer stem cells (CSCs). But, upstream signals that control the mammalian Hippo pathway have not been well understood. Here, we reveal a connection between the Protease-activated receptor 1 (PAR1) signaling pathway and the Hippo-YAP pathway in gastric cancer stem-like cells. The selective PAR1 agonist TFLLR-NH₂ induces an increase in the fraction of side population cells which is enriched in CSCs, and promotes tumorigenesis, multi cancer drug resistance, cell morphological change, and cell invasion which are characteristics of CSCs. In addition, PAR1 activation inhibits the Hippo-YAP pathway kinase Lats via Rho GTPase. Lats kinase inhibition in turn results in increased nuclear localization of dephosphorylated YAP. Furthermore, PAR1 activation confers CSCs related traits via the Hippo-YAP pathway, and the Hippo-YAP pathway correlates with epithelial mesenchymal transition which is induced by PAR1 activation. Our research suggests that the PAR1 signaling deeply participates in the ability of multi drug resistance and tumorigenesis through interactions with the Hippo-YAP pathway signaling in gastric cancer stem-like cells. We presume that inhibited YAP is a new therapeutic target in the treatment human gastric cancer invasion and metastasis by dysregulated PAR1 or its agonists. The Hippo pathway significantly correlates with organ size control and tumorigenesis. The activity of YAP/TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in cancer stem cells (CSCs). But, upstream signals that control the mammalian Hippo pathway have not been well understood. Here, we reveal a connection between the Protease-activated receptor 1 (PAR1) signaling pathway and the Hippo-YAP pathway in gastric cancer stem-like cells. The selective PAR1 agonist TFLLR-NH₂ induces an increase in the fraction of side population cells which is enriched in CSCs, and promotes tumorigenesis, multi cancer drug resistance, cell morphological change, and cell invasion which are characteristics of CSCs. In addition, PAR1 activation inhibits the Hippo-YAP pathway kinase Lats via Rho GTPase. Lats kinase inhibition in turn results in increased nuclear localization of Dephosphorylated YAP. Furthermore, PAR1 activation confers CSCs related traits via the Hippo-YAP pathway, and the Hippo-YAP pathway correlates with epithelial mesenchymal transition which is induced by PAR1 activation. Our research suggests that the PAR1 signaling deeply participates in the ability of multi drug resistance and tumorigenesis through interactions with the Hippo-YAP pathway signaling in gastric cancer stem-like cells. We presume that inhibited YAP is a new therapeutic target in the treatment human gastric cancer invasion and metastasis by dysregulated PAR1 or its agonists.     

Notch1 promotes glioma cell migration and invasion by stimulating β-catenin and NF-κB signaling via AKT activation

The Notch signaling pathway has been implicated in both developmental processes and tumorigenesis. Aberrant Notch signaling has been repeatedly demonstrated to facilitate the proliferation and survival of glioma cells by regulating downstream effectors or other signaling pathways. In glioblastoma multiforme specimens from 59 patients, Notch1 was highly expressed in tumor tissues compared with normal brain tissues, and this expression was correlated with elevated AKT phosphorylation and Snail expression. Increased nuclear localization of β-catenin and p50 as well as enhanced IKKα/AKT interaction were also observed in glioma tissues. In U87MG cells, the activation of Notch1 by DLL4 stimulation or by the overexpression of Notch intracellular domain (NICD) resulted in AKT activation and thereby promoted β-catenin activity and NF-κB signaling. Inhibition of EGFR partially blocked the β-catenin and NF-κB signaling stimulated by Notch1 activation. Furthermore, NICD overexpression in U87MG cells led to the upregulated expression of several metastasis-associated molecules, which could be abrogated by the knockdown of either β-catenin or p50. In U87MG and U251 cells, DLL4-induced cellular migration and invasion could be inhibited by either β-catenin or a p50 inhibitor. Collectively, these results indicate that Notch activation could stimulate β-catenin and NF-κB signaling through AKT activation in glioma cells. Thus, Notch activation-stimulated β-catenin and NF-κB signaling synergistically promote the migratory and invasive properties of glioma cells.     

Glutamate Promotes Cell Growth by EGFR Signaling on U-87MG Human Glioblastoma Cell Line

Accumulating evidences suggest that glutamate plays a key role in the proliferation and invasion of malignant glioblastoma (GBM) tumors. It has been shown that GBM cells release and exploit glutamate for proliferation and invasion through AMPA glutamate receptors. Additionally, amplification of the epidermal growth factor receptor (EGFR) gene occurs in 40–50% of GBM. Since, PI3K/Akt is considered one of the main intracellular pathways involved in EGFR activation, AKT functions could trigger EGFR signaling. Thus, we investigated whether EGFR-phospho-Akt pathway is involved on the glutamate inducing U-87MG human GBM cell line proliferation. For these purpose, we treated the U-87MG cell line with 5 to 200 mM of glutamate and assessed the number of viable cells by trypan blue dye exclusion test. An increase in cell number (50%) was found at 5 mM glutamate, while the addition of DNQX (500 μM), an antagonist of AMPA receptor, inhibited the effect of glutamate on the U87-MG cells proliferation. Also, at 5 mM glutamate we observed an increase on the EGFR and phospho-Akt contents evaluated by immunohistochemistry. Moreover, U-87MG cells treated with glutamate exhibited an increase about 2 times in the EGFR mRNA expression. While, in the presence of the anti-EGFR gefitinib (50 μM) or the PI3K inhibitor wortmannin (5 μM), the U-87MG proliferation was restored to control levels. Together, our data suggest that glutamate signaling mediated by AMPA receptor induces U-87MG human GBM cell line proliferation via EGFR-phospho-Akt pathway.     

NO donor and MEK inhibitor synergistically inhibit proliferation and invasion of cancer cells

Nitric oxide (NO) shows tumoricidal activity. We had previously reported that NO downregulates the phosphatidylinositol-3-kinase/Akt pathway, but upregulates the MEK/ERK pathway downstream of growth factor signaling. We hypothesized that NO donor and MEK inhibitor in combination synergistically inhibit the viability of cancer cells compared to either NO donor or MEK inhibitor alone. We determined the effects of S-nitrosoglutathione (GSNO, NO-donor) and U0126 (MEK inhibitor) on insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) signaling, proliferation and invasion in cancer cell lines. GSNO inhibits phosphorylation of IGF-I receptor (IGF-IR), EGF receptor (EGFR) and Akt, but upregulates ERK1/2 phosphorylation in MIAPaCa-2 and HCT-116 cells after stimulation by IGF-I and EGF. On the other hand, U0126 inhibits phosphorylation of ERK1/2, but upregulates phosphorylation of IGF-IR and EGFR in MIAPaCa-2 and HCT-116 cells. The combination of GSNO and U0126 downregulates phosphorylation of IGF-IR, EGFR, Akt and ERK1/2 after stimulation by IGF-I and EGF. GSNO as well as U0126, inhibits the proliferation of MIAPaCa-2, HCT-116, Panc-1, MCF-7, HT-29 and AGS cells in a dose-dependent manner. GSNO and U0126 in combination synergistically inhibit proliferation and invasion of cancer cells. These results indicate that the combined treatment of NO donor and MEK inhibitor may be promising in cancer therapy.