Malignant progression in O6-methylguanine-DNA methyltransferase-deficient esophageal cancer cells is associated with Ezrin protein

The abnormal function of O(6)-methylguanine-DNA methyltransferase (MGMT) is reported to be associated with the occurrence of various tumors and malignant tumor progression. However, little evidence is available to describe its role in esophageal carcinogenesis. To address this issue, we constructed a stable MGMT-silenced esophageal cancer cell line by RNA interference, and exposed the cells to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) to investigate the role that MGMT plays in toxicity. During this time, we also observed the malignant behavior of cells in vitro and in vivo. In addition, two-dimensional electrophoresis and mass spectrometry were used to detect and confirm the proteins that were differentially expressed in the MGMT-deficient and MGMT-proficient cells, which might be responsible for the malignant alteration of cells. Results showed that the IC(50) of MGMT-deficient and MGMT-proficient cells exposed to MNNG was 30 μM and 65 μM, respectively, and MGMT-deficient cells had more aggressive motility and invasive abilities compared with MGMT-proficient cells. Nineteen differentially expressed proteins were detected between the MGMT-deficient and MGMT-proficient cells, 14 of which were identified, including the membrane-cytoskeleton linker protein, Ezrin, which was confirmed by both mass spectrometry and western blot analysis. The correlation between MGMT, Ezrin expression, and the malignant behavior of one normal epithelial esophageal cell line and seven esophageal cancer lines is discussed. In conclusion, loss of MGMT expression leads EC109 esophageal cancer cells to have increased malignant behavior, which may correlate with its high Ezrin protein expression.     

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.     

Effects of temozolomide (TMZ) on the expression and interaction of heat shock proteins (HSPs) and DNA repair proteins in human malignant glioma cells

We previously reported the association of HSPA1A and HSPB1 with high-grade astrocytomas, suggesting that these proteins might be involved in disease outcome and response to treatment. With the aim to better understand the resistance/susceptibility processes associated to temozolomide (TMZ) treatment, the current study was performed in three human malignant glioma cell lines by focusing on several levels: (a) apoptotic index and senescence, (b) DNA damage, and (c) interaction of HSPB1 with players of the DNA damage response. Three human glioma cell lines, Gli36, U87, and DBTRG, were treated with TMZ evaluating cell viability and survival, apoptosis, senescence, and comets (comet assay). The expression of HSPA (HSPA1A and HSPA8), HSPB1, O₆-methylguanine-DNA methyltransferase (MGMT), MLH1, and MSH2 was determined by immunocytochemistry, immunofluorescence, and Western blot. Immunoprecipitation was used to analyze protein interaction. The cell lines exhibited differences in viability, apoptosis, and senescence after TMZ administration. We then focused on Gli36 cells (relatively unstudied) which showed very low recovery capacity following TMZ treatment, and this was related to high DNA damage levels; however, the cells maintained their viability. In these cells, MGMT, MSH2, HSPA, and HSPB1 levels increased significantly after TMZ administration. In addition, MSH2 and HSPB1 proteins appeared co-localized by confocal microscopy. This co-localization increased after TMZ treatment, and in immunoprecipitation analysis, MSH2 and HSPB1 appeared interacting. In contrast, HSPB1 did not interact with MGMT. We show in glioma cells the biological effects of TMZ and how this drug affects the expression levels of heat shock proteins (HSPs), MGMT, MSH2, and MLH1. In Gli36 cells, the results suggest that interactions between HSPB1 and MSH2, including co-nuclear localization, may be important in determining cell sensitivity to TMZ.     

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.     

Differential spindle assembly checkpoint response in human lung adenocarcinoma cells

CI-980 is an antimicrotubule agent that binds the colchicine site on tubulin. We examined CI-980 cytotoxicity in two lung adenocarcinoma cell lines, A549 and A427. Depolymerization of microtubules following CI-980 treatment resulted in a mitotic arrest in the A549 population, but not in the A427 population. Similar responses were obtained following treatment with Taxol and nocodazole. Drug-treated A427 cells exited mitosis, generating a population dominated by multinucleated cells, while both multinucleated and apoptotic cells were present in the A549 population after extended drug treatment. CI-980-induced microtubule depolymerization was only partially reversible. However, regrowth of some microtubules in mitotic A549 cells following drug washout resulted in multinucleation of the population in the absence of apoptosis. These results show that A427 cells have a defective spindle assembly checkpoint. Levels of the MAD2 and BUB1 checkpoint proteins were similar in both A549 and A427 cells, suggesting that the checkpoint defect in the A427 cells is downstream of these proteins. In addition, induction of apoptosis in response to CI-980 correlates with the presence of a functional mitotic checkpoint and the extent of microtubule depolymerization.     

Investigation of antitumor potential of Ni(II) complexes with tridentate PNO acylhydrazones of 2-(diphenylphosphino)benzaldehyde and monodentate pseudohalides

Square-planar azido Ni(II) complex with condensation product of 2-(diphenylphosphino)benzaldehyde and Girard’s T reagent was synthesized and its crystal structure was determined. Cytotoxic activity of the azido complex and previously synthesized isothiocyanato, cyanato and chlorido Ni(II) complexes with this ligand was examined on six tumor cell lines (HeLa, A549, K562, MDA-MB-453, MDA-MB-361 and LS-174) and two normal cell line (MRC-5 and BEAS-2B). All the investigated nickel(II) complexes were cytotoxic against all tumor cell lines. The newly synthesized azido complex showed selectivity to HeLa and A549 tumor cell lines compared to the normal cells (for A549 IC₅₀ was similar to that of cisplatin). Azido complex interferes with cell cycle phase distribution of A549 and HeLa cells and possesses nuclease activity towards supercoiled DNA. The observed selectivity of the azido complex for some tumor cell lines can be connected with its strong DNA damaging activity.     

Focal Adhesion Kinase Inhibitors in Combination with Erlotinib Demonstrate Enhanced Anti-Tumor Activity in Non-Small Cell Lung Cancer

Blockade of epidermal growth factor receptor (EGFR) activity has been a primary therapeutic target for non-small cell lung cancers (NSCLC). As patients with wild-type EGFR have demonstrated only modest benefit from EGFR tyrosine kinase inhibitors (TKIs), there is a need for additional therapeutic approaches in patients with wild-type EGFR. As a key component of downstream integrin signalling and known receptor cross-talk with EGFR, we hypothesized that targeting focal adhesion kinase (FAK) activity, which has also been shown to correlate with aggressive stage in NSCLC, would lead to enhanced activity of EGFR TKIs. As such, EGFR TKI-resistant NSCLC cells (A549, H1299, H1975) were treated with the EGFR TKI erlotinib and FAK inhibitors (PF-573,228 or PF-562,271) both as single agents and in combination. We determined cell viability, apoptosis and 3-dimensional growth in vitro and assessed tumor growth in vivo. Treatment of EGFR TKI-resistant NSCLC cells with FAK inhibitor alone effectively inhibited cell viability in all cell lines tested; however, its use in combination with the EGFR TKI erlotinib was more effective at reducing cell viability than either treatment alone when tested in both 2- and 3-dimensional assays in vitro, with enhanced benefit seen in A549 cells. This increased efficacy may be due in part to the observed inhibition of Akt phosphorylation when the drugs were used in combination, where again A549 cells demonstrated the most inhibition following treatment with the drug combination. Combining erlotinib with FAK inhibitor was also potent in vivo as evidenced by reduced tumor growth in the A549 mouse xenograft model. We further ascertained that the enhanced sensitivity was irrespective of the LKB1 mutational status. In summary, we demonstrate the effectiveness of combining erlotinib and FAK inhibitors for use in known EGFR wild-type, EGFR TKI resistant cells, with the potential that a subset of cell types, which includes A549, could be particularly sensitive to this combination treatment. As such, further evaluation of this combination therapy is warranted and could prove to be an effective therapeutic approach for patients with inherent EGFR TKI-resistant NSCLC.     

FTIR spectra signatures reveal different cellular effects of EGFR inhibitors on nonsmall cell lung cancer cells

ATP‐analogue inhibitors, Gefitinib (Iressa) and Erlotinib (Tarceva) had been approved for advanced and metastatic nonsmall cell lung cancer (NSCLC) cells against tyrosine kinase domain of epidermal growth factor receptor (EGFR). Many techniques have been developed to better understand the drug mechanism which is multistep, time‐consuming and expensive. Herein, we performed Fourier‐transform infrared (FTIR) microscopy for evaluating the biochemical change on NSCLC (A549) cells after treatment. At levels that produced equivalent effects, Gefitinib dramatically induced cell apoptosis via impaired mitochondrial transmembrane potential. Whereas, Erlotinib had a slight effect on A549. Principal component analysis was performed to distinguish the effect of EGFR inhibitors on A549. FTIR spectra regions were divided into three regions: lipids (3000‐2800 cm^?1), proteins (1700‐1500 cm^?1) and carbohydrates and nuclei acids (1200‐1000 cm^?1). Biochemical changes can be evaluated by these spectral regions. This work may be a novel concept for utilizing FTIR spectroscopy for high‐throughput discriminative effects of a drug or compound and its derivatives on cells.     

miR‐134 inhibits non‐small cell lung cancer growth by targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently activated in a wide range of solid tumours and represents an important therapeutic target. MicroRNAs (miRNAs) have recently been recognized as a rational and potential modality for anti‐EGFR therapies. However, more EGFR‐targeting miRNAs need to be explored. In this study, we identified a novel EGFR‐targeting miRNA, miRNA‐134 (miR‐134), in non‐small‐cell lung cancer (NSCLC) cell lines. Luciferase assays confirmed that EGFR is a direct target of miR‐134. In addition, the overexpression of miR‐134 inhibited EGFR‐related signaling and suppressed NSCLC cells proliferation by inducing cell cycle arrest and/or apoptosis, suggesting that miR‐134 functions as a tumour suppressor in NSCLC. Further mechanistic investigation including RNAi and rescue experiments suggested that the down‐regulation of EGFR by miR‐134 partially contributes to the antiproliferative role of miR‐134. Last, in vivo experiments demonstrated that miR‐134 suppressed tumour growth of A549 xenograft in nude mice. Taken together, our findings suggest that miR‐134 inhibits non‐small cell lung cancer growth by targeting the EGFR.     

Nimotuzumab Enhances the Radiosensitivity of Cancer Cells In Vitro by Inhibiting Radiation-Induced DNA Damage Repair

Background

Nimotuzumab is a humanized IgG1 monoclonal antibody specifically targeting EGFR. In this study, we aimed to investigate the molecular mechanisms of nimotuzumab in its effects of enhancing cancer cell radiosensitivity.

Principal Finding

Lung cancer A549 cells and breast cancer MCF-7 cells were pretreated with or without nimotuzumab for 24 h before radiation to perform the clonogenic survival assay and to analyze the cell apoptosis by flow ctyometry. γ-H2AX foci were detected by confocal microscopy to assess the effect of nimotuzumab on radiation induced DNA repair. EGFR activation was examined and the levels of DNA damage repair related proteins in A549 cells at different time point and at varying doses exposure after nimotuzumab and radiation treatment were examined by Western blot. Pretreatment with nimotuzumab reduced clonogenic survival after radiation, inhibited radiation-induced EGFR activation and increased the radiation-induced apoptosis in both A549 cells and MCF-7 cells. The foci of γ-H2AX 24 h after radiation significantly increased in nimotuzumab pretreated cells with different doses. The phosphorylation of AKT and DNA-PKcs were remarkably inhibited in the combination group at each dose point as well as time point.

Conclusions

Our results revealed that the possible mechanism of nimotuzumab enhancing the cancer radiosensitivity is that nimotuzumab inhibited the radiation-induced activation of DNA-PKcs through blocking the PI3K/AKT pathway, which ultimately affected the DNA DSBs repair.     

The effect of tyrphostins AG494 and AG1478 on the autocrine growth regulation of A549 and DU145 cells

We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFR's inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true.     

Synthesis and biological evaluation of N-aryl salicylamides with a hydroxamic acid moiety at 5-position as novel HDAC-EGFR dual inhibitors

A novel series of N-aryl salicylamides with a hydroxamic acid moiety at 5-position were synthesized efficiently. Their activities against EGFR kinase and HDACs were evaluated. All compounds displayed inhibitory activity against EGFR and HDACs. The antiproliferative activities of synthesized compounds were evaluated by MTT method against human cancer cell lines A431, A549 and HL-60. Compound 1o showed the most potent inhibitory activity against A431 and A549. Compounds 1k and 1n exhibited higher potency against HL-60 than gefitinib and SAHA. N-Aryl salicylamides with a hydroxamic acid moiety at 5-position is another new HDAC-EGFR dual inhibitors.     

Four-membered heterocycles-containing 4-anilino-quinazoline derivatives as epidermal growth factor receptor (EGFR) kinase inhibitors

We report herein the design and synthesis of novel azaspirocycle or azetidine substituted 4-anilinoquinazoline derivatives. The EGFR inhibitory activities and in vitro antitumor potency of these newly synthesized compounds against two lung cancer cell lines HCC827 and A549 were evaluated. Most of the target compounds possess good inhibitory potency. In particular, compounds 21g with 2-oxa-6-azaspiro[3.4]octane substituent was found to possess higher EGFR inhibitory activities and similar antitumor potency comparing to the lead compound gefitinib with improved water solubility.     

Targeting of O6-MeG DNA methyltransferase (MGMT) to mitochondria protects against alkylation induced cell death

Mitochondrial DNA (mtDNA) mutations are implicated in pathogenesis of human diseases including cancer. To prevent mutations cells have developed repair systems to counteract harmful genetic changes caused by DNA damaging agents. One such DNA repair protein is the O(6)-Methylguanine-DNA methyltransferase (MGMT) that prevents certain types of alkylation damage. Yet, the role of MGMT in preventing alkylation induced DNA damage in mtDNA is unclear. We explored the idea of increasing cell survival after alkylation damage by overexpressing MGMT in mitochondria. We show that overexpression of this repair protein in mitochondria increases cell survival after treatment with the DNA damaging agent MNNG.     

The EGFR inhibitor gefitinib suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in non-small cell lung cancer cell lines

The drug gefitinib (Iressa), which is a specific inhibitor of EGFR tyrosine kinase, has been shown to suppress the activation of EGFR signaling for survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. A recent study demonstrated rapid down-regulation of ligand-induced EGFR in a gefitinib-sensitive cell line and inefficient down-regulation of EGFR in a gefitinib-resistant cell line in the exponential phase of growth; this implies that each cell type employs a different unknown down-regulation mechanism occurs. However, the mechanism of drug sensitivity to gefitinib remains unclear. In this study, to further substantiate the effect of gefitinib on the EGFR down-regulation pathway and to understand the detailed internalization mechanism of gefitinib-sensitive PC9 and gefitinib-resistant QG56 cell lines, we examined the internalization of Texas red-EGF in the absence or presence of gefitinib in both cell lines. The distribution of internalized Texas red-EGF, early endosomes, and late endosomes/lysosomes was then assessed by confocal immunofluorescence microscopy. Here, we provide novel evidence that efficient endocytosis of EGF–EGFR occurs via the endocytic pathway in the PC9 cells, because the internalized Texas red-EGF-positive small punctate vesicles were transported to the late endosomes/lysosomes and then degraded within the lysosomes after 60 min of internalization. Additionally, gefitinib exerted a strong inhibitory effect on the endocytosis of EGFR in PC9 cells, and the internalization rate of EGFR from the plasma membrane via the early endosomes to the late endosomes/lysosomes was considerably delayed. This indicates that gefitinib efficiently suppresses ligand-stimulated endocytosis of EGFR via the early/late endocytic pathway in PC9 cells. In contrast, the internalization rate of ligand-induced EGFR was not significantly changed by gefitinib in QG56 cells because even in the absence of gefitinib, internalized EGFR accumulation was noted in the early and late endosomes after 60 min of internalization instead of its delivery to the lysosomes in QG56 cells. This suggests that the endocytic machinery of EGFR might be basically impaired at the level of the early/late endosomes. Taken together, this is the first report demonstrating that the suppressive effect of gefitinib on the endocytosis of EGFR is much stronger with PC9 cells than QG56 cells. Thus, impairment in some steps of the EGF–EGFR traffic out of early endosomes toward the late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. Iressa is a trademark of the AstraZeneca group of companies.     

Rad51 and BRCA2--New molecular targets for sensitizing glioma cells to alkylating anticancer drugs

First line chemotherapeutics for brain tumors (malignant gliomas) are alkylating agents such as temozolomide and nimustine. Despite growing knowledge of how these agents work, patients suffering from this malignancy still face a dismal prognosis. Alkylating agents target DNA, forming the killing lesion O(6)-alkylguanine, which is converted into DNA double-strand breaks (DSBs) that trigger apoptosis. Here we assessed whether inhibiting repair of DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioma cells to alkylating agents. For down-regulation of HR in glioma cells, we used an interference RNA (iRNA) approach targeting Rad51 and BRCA2, and for NHEJ we employed the DNA-PK inhibitor NU7026. We also assessed whether inhibition of poly(ADP)ribosyltransferase (PARP) by olaparib would enhance the killing effect. The data show that knockdown of Rad51 or BRCA2 greatly sensitizes cells to DSBs and the induction of cell death following temozolomide and nimustine (ACNU). It did not sensitize to ionizing radiation (IR). The expression of O(6)-methylguanine-DNA methyltransferase (MGMT) abolished all these effects, indicating that O(6)-alkylguanine induced by these drugs is the primary lesion responsible for the formation of DSBs and increased sensitivity of glioma cells following knockdown of Rad51 and BRCA2. Inhibition of DNA-PK only slightly sensitized to temozolomide whereas a significant effect was observed with IR. A triple strategy including siRNA and the PARP inhibitor olaparib further improved the killing effect of temozolomide. The data provides evidence that down-regulation of Rad51 or BRCA2 is a reasonable strategy for sensitizing glioma cells to killing by O(6)-alkylating anti-cancer drugs. The data also provide proof of principle that a triple strategy involving down-regulation of HR, PARP inhibition and MGMT depletion may greatly enhance the therapeutic effect of temozolomide.     

Design,synthesis and biological activity of novel molecules designed to target PARP and DNA

In order to enhance the cytotoxic potential of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a PARP targeting scaffold. A cell-based selectivity assay involving a BRCA2-deficient Chinese hamster cell line and its corresponding BRCA2 wild type transfectant, was used to predict the PARP targeting potential of the latter agents. The results showed that adding a DNA damaging function to the PARP inhibitors decreased but did not abrogate the selective targeting of the BRCA2-deficient cells. The DNA damaging moiety augmented the potency in BRCA2 deficient cells by 2–20 fold. The most selective dual PARP–DNA targeting agent 14b was found to possess dual DNA and PARP targeting properties.     

Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM), and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors, and both EGFR and Rictor are associated with increased proliferation, invasion, metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines, including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing, tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan, temozolomide and vincristine. In LN229, co-silencing of EGFR and Rictor resulted in reduced cell migration, and increased sensitivity to vincristine and temozolomide. In U118MG, silencing of Rictor alone was sufficient to increase this line’s sensitivity to vincristine and temozolomide. In vivo, while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth, silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM.     

SR48692 inhibits non-small cell lung cancer proliferation in an EGF receptor-dependent manner

Aims

The mechanism by which SR48692 inhibits non-small cell lung cancer (NSCLC) proliferation was investigated.

Main methods

The ability of SR48692 to inhibit the proliferation of NSCLC cell lines NCI-H1299 and A549 was investigated in vitro in the presence or absence of neurotensin (NTS). The ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation was investigated by Western blot using NSCLC cells and various inhibitors. The growth effects and Western blot results were determined in cell lines treated with siRNA for NTSR1.

Key findings

Treatment of A549 or NCI-H1299 cells with siRNA for NTSR1 reduced significantly NTSR1 protein and the ability of SR48692 to inhibit the proliferation of A549 or NCI-H1299 NSCLC cells. Treatment of A549 and NCI-H1299 cells with siRNA for NTSR1 reduced the ability of NTS to cause epidermal growth factor receptor (EGFR) transactivation. SR48692 or gefitinib (EGFR tyrosine kinase inhibitor) inhibited the ability of NTS to cause EGFR and ERK tyrosine phosphorylation. NTS transactivation of the EGFR was inhibited by GM6001 (matrix metalloprotease inhibitor), Tiron (superoxide scavenger) or U73122 (phospholipase C inhibitor) but not H89 (PKA inhibitor). NTS stimulates whereas SR48692 or gefitinib inhibits the clonal growth of NSCLC cells.

Significance

These results suggest that SR48692 may inhibit NSCLC proliferation in an EGFR-dependent mechanism.     

Involvement of adenosine A2B receptor in radiation-induced translocation of epidermal growth factor receptor and DNA damage response leading to radioresistance in human lung cancer cells

BackgroundAdenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR.MethodsWe investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation.ResultsA2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression.ConclusionActivation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage.General significanceOur results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.