Autophagy regulates Selumetinib (AZD6244) induced-apoptosis in colorectal cancer cells.

OBJECTIVE: As Selumetinib is a MEK1/2 inhibitor that has gained interest as an anti-tumor agent, the present study was designed to investigate autophagy involvement on Selumetinib-induced apoptosis in colorectal cancer (CRC) cells. METHODS: CRC cells death and cycle studies were assessed by AnnexinV-FITC and PI staining, respectively. Autophagy flux was analysed by Western Blot (LC3II and p62 protein levels) and retroviral infection of SW480 cells for siBecn1 RNA interference experiments. Confocal microscopy was used to determine mCherry-EGFP-LC3 distribution. KEY FINDINGS: The Selumetinib effects were concentration-dependent in SW480 cell line. Whereas 1 µM exerted an arrest in the cell cycle (G1 phase), higher concentrations (10 µM) induced cell death, which was accompanied by autophagy blockage in its last stages. Autophagy induction by Rapamycin (RAPA) increased cell survival, whereas pharmacology autophagy inhibition by Bafilomycin A1 (BAF), Chloroquine (CQ) or 3-Methyladenine (3-MA) increased Selumetinib-induced CRC cells death. CONCLUSIONS: Altogether, these results suggest that autophagy plays a fundamental role in CRC cells response to Selumetinib. In addition, the combination of Selumetinib with autophagy inhibitors may be a useful therapeutic strategy to enhance its activity against colorectal tumours.

HGUE-C-1 is an atypical and novel colon carcinoma cell line.

BACKGROUND: Colorectal carcinoma is a common cause of cancer. Adjuvant treatments include: 5-fluorouracil administered together with folinic acid, or more recently, oral fluoropyrimidines such as capecitabine, in combination with oxaliplatin or irinotecan. Metastatic colorectal cancer patients can benefit from other additional treatments such as cetuximab or bevacizumab. METHODS: Using cell culture techniques, we isolated clonal populations from primary cultures of ascitic effusion derived from a colon cancer patient and after several passages an established cell line, HGUE-C-1, was obtained. Genetic analysis of HGUE-C-1 cells was performed by PCR of selected exons and sequencing. Cell proliferation studies were performed by MTT assays and cell cycle analyses were performed by flow cytometry. Retinoblastoma activity was measured by luciferase assays and proteins levels and activity were analysed by Western blot or immunohistochemistry. RESULTS: We have established a new cell line from ascitic efussion of a colon cancer patient who did not respond to 5-fluorouracil or irinotecan. HGUE-C-1 cells did not show microsatellite instability and did not harbour mutations in KRAS, BRAF, PI3KCA or TP53. However, these cells showed loss of heterozygosity affecting Adenomatous Polyposis Coli and nuclear staining of ß-catenin protein. The HGUE-C-1 cell line was sensitive to erlotinib, gefitinib, NVP-BEZ235, rapamycin and trichostatin, among other drugs, but partially resistant to heat shock protein inhibitors and highly resistant to AZD-6244 and oxaliplatin, even though the patient from which this cell line was derived had not been exposed to these drugs. Molecular characterization of this cell line revealed low expression levels and activity of Retinoblastoma protein and elevated basal levels of Erk1/2 activity and p70S6K expression and activity, which may be related to chemoresistance mechanisms. CONCLUSIONS: HGUE-C-1 represents a novel and peculiar colon carcinoma model to study chemoresistance to chemotherapeutic agents and to novel anti-neoplasic drugs that interrupt signalling pathways such as the APC/ßcatenin, Ras/Raf/Mek/Erk, PI3K/mTOR/p70S6K pathways as well as histone regulation mechanisms.

Resistance to Selumetinib (AZD6244) in colorectal cancer cell lines is mediated by p70S6K and RPS6 activation.

Selumetinib (AZD6244, ARRY-142886) is a MEK1/2 inhibitor that has gained interest as an anti-tumour agent. We have determined the degree of sensitivity/resistance to Selumetinib in a panel of colorectal cancer cell lines using cell proliferation and soft agar assays. Sensitive cell lines underwent G1 arrest, whereas Selumetinib had no effect on the cell cycle of resistant cells. Some of the resistant cell lines showed high levels of ERK1/2 phosphorylation in the absence of serum. Selumetinib inhibited phosphorylation of ERK1/2 and RSK and had no effect on AKT phosphorylation in both sensitive and resistant cells. Furthermore, mutations in KRAS, BRAF, or PIK3CA were not clearly associated with Selumetinib resistance. Surprisingly, Selumetinib was able to inhibit phosphorylation of p70 S6 kinase (p70S6K) and its downstream target ribosomal protein S6 (RPS6) in sensitive cell lines. However, p70S6K and RPS6 phosphorylation remained unaffected or even increased in resistant cells. Moreover, in some of the resistant cell lines p70S6K and RPS6 were phosphorylated in the absence of serum. Interestingly, colorectal primary cultures derived from tumours excised to patients exhibited the same behaviour than established cell lines. Pharmacological inhibition of p70S6K using the PI3K/mTOR inhibitor NVP-BEZ235, the specific mTOR inhibitor Rapamycin and the specific p70S6K inhibitor PF-4708671 potentiated Selumetinib effects in resistant cells. In addition, biological inhibition of p70S6K using siRNA rendered responsiveness to Selumetinib in resistant cell lines. Furthermore, combination of p70S6K silencing and PF-47086714 was even more effective. We can conclude that p70S6K and its downstream target RPS6 are potential biomarkers of resistance to Selumetinib in colorectal cancer.

Comparative Study of 17-AAG and NVP-AUY922 in Pancreatic and Colorectal Cancer Cells: Are There Common Determinants of Sensitivity?

The use of heat shock protein 90 (Hsp90) inhibitors is an attractive antineoplastic therapy. We wanted to compare the effects of the benzoquinone 17-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) and the novel isoxazole resorcinol-based Hsp90 inhibitor NVP-AUY922 in a panel of pancreatic and colorectal carcinoma cell lines and in colorectal primary cultures derived from tumors excised to patients. PANC-1, CFPAC-1, and Caco-2 cells were intrinsically resistant to 17-AAG but sensitive to NVP-AUY922. Other cellular models were sensitive to both inhibitors. Human epidermal growth factor receptor receptors and their downstream signaling pathways were downregulated in susceptible cellular models, and concurrently, Hsp70 was induced. Intrinsic resistance to 17-AAG did not correlate with expression of ATP-binding cassette transporters involved in multidrug resistance. Some 17-AAG-resistant, NVP-AUY922-sensitive cell lines lacked NAD(P)H: quinone oxidoreductase 1 (NQO1) enzyme and activity. However, colorectal LoVo cells still responded to both drugs in spite of having undetectable levels and activity of NQO1. Pharmacological and biologic inhibition of NQO1 did not confer resistance to 17-AAG in sensitive cell lines. Therefore, even though 17-AAG sensitivity is related to NQO1 protein levels and enzymatic activity, the absence of NQO1 does not necessarily convey resistance to 17-AAG in these cellular models. Moreover, NVP-AUY922 does not require NQO1 for its action and is a more potent inhibitor than 17-AAG in these cells. More importantly, we show in this report that NVP-AUY922 potentiates the inhibitory effects of chemotherapeutic agents, such as gemcitabine or oxaliplatin, and other drugs that are currently being evaluated in clinical trials as antitumor agents.

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines.

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G(1) arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G(1) arrest. This G(1) arrest was associated with up-regulation of p27(kip1), whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G(1) arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

Roles of wound geometry, wound size, and extracellular matrix in the healing response of bovine corneal endothelial cells in culture.

It has classically been accepted that the healing of narrow wounds in epithelia occurs by the formation of a contractile actin cable, while wide wounds are resurfaced by lamellipodia-dependent migration of border cells into the denuded area. To further investigate the general validity of this idea, we performed systematic experiments of the roles of wound geometry, wound size, and extracellular matrix (ECM) in wound healing in monolayers of bovine corneal endothelial cells, a system shown here to predominantly display any of the two healing mechanisms according to the experimental conditions. We found that, in this system, it is the absence or presence of the ECM on the wound surface that determines the specific healing mode. Our observations demonstrate that, independent of their size and geometry, wounds created maintaining the ECM heal by migration of cells into the wound area, while ECM removal from the wound surface determines the predominant formation of an actin cable. While the latter mechanism is slower, the actin cable permits the maintainance of the epithelial phenotype to a larger extent during the healing process, as also confirmed by our finding of a more conserved localization of cadherin and vinculin. We also introduce a model that simulates experimental findings about the dynamics of healing mechanisms, both for the maintenance or removal of the ECM on the wound surface. The findings of this study may contribute to the understanding of physiological and pathological aspects of epithelial wound healing and to the design of therapeutic strategies.

A possible role for membrane depolarization in epithelial wound healing.

Linear narrow wounds produced on cultured bovine corneal endothelial monolayers heal by actin cable formation at the wound border and lamellar crawling of cells into the injured area. We report the novel finding that membrane potential depolarization occurs at the leading edge of wounds and gradually extends inward toward the neighboring cells. We have determined that the replacement of extracellular Na(+) by choline and the incorporation of phenamil, an inhibitor of the epithelial Na(+) channel (ENaC), provoke a decrease in the actin cable and depolarization areas and in the lamellar activity of the wound edges. To the contrary, extracellular Li(+) can successfully replace Na(+) in the determination of the depolarization and cytoskeletal responses. This finding supports the idea that membrane depolarization, not the increase in intracellular Na(+) concentration, is responsible for the formation of the actin cable, a result that is in agreement with previous evidence showing that nonspecific depolarization of the plasma membrane potential (PMP) of epithelial cells may promote characteristic cytoskeletal rearrangements per se (Chifflet S, Hernandez JA, Grasso S, and Cirillo A. Exp Cell Res 282: 1-13, 2003). We suggest that spontaneous depolarization of the PMP of the cells at the wound borders determined by a rise in the ENaC activity of these cells constitutes an additional factor in the intermediate cellular processes leading to wound healing in some epithelia.

Nonspecific depolarization of the plasma membrane potential induces cytoskeletal modifications of bovine corneal endothelial cells in culture.

Modifications in the cell membrane potential have been suggested to affect signaling mechanisms participating in diverse cellular processes, many of which involve structural cellular alterations. In order to contribute some evidence in this respect, we explored the effects of several depolarizing procedures on the structure and monolayer organization of bovine corneal endothelial cells in culture. Visually confluent cell monolayers were incubated with or without the depolarizing agent, either in a saline solution or in culture medium for up to 30 min. Membrane potential was monitored by fluorescence microscopy using oxonol V. Fluorescent probes were employed for F-actin, microtubules, and vinculin. Depolarization of the plasma membrane, achieved via the incorporation of gramicidin D into confluent endothelial cells or by modifications of the extracellular saline composition, provoked an increment of oxonol fluorescence and changes in cell morphology, consisting mainly of modifications in the cytoskeletal organization. In some areas, noticeable intercellular spaces appear. The cytoskeleton modifications mainly consist of a marked redistribution of F-actin and microtubules, with accompanying changes in vinculin localization. The results suggest that the depolarization of the plasma membrane potential may participate in mechanisms involved in cytoskeleton organization and monolayer continuity in corneal endothelial cells in culture.