Sequence-dependent, synergistic antiproliferative and proapoptotic effects of the combination of cytotoxic drugs and enzastaurin, a protein kinase Cβ inhibitor, in non-small cell lung cancer cells

Morgillo, Floriana; Martinelli, Erika; Troiani, Teresa; Laus, G.; Pepe, Stefano; Gridelli, Cesare; Ciardiello, Fortunato

doi:10.1158/1535-7163.mct-07-0547

Cited by 22 publications

(20 citation statements)

References 42 publications

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“…Erk1/2 activation has been reported to be induced by mutant GNAQ and has been found in UM independent of GNAQ, BRAF and RAS mutational status [7], [27], [34]. Akt but not Erk1/2 phosphorylation has been reported to be inhibited by enzastaurin in several types of cancer cells [18], [22], [24], [25], [35]–[39]. Interestingly, Erk1/2 phosphorylation was significantly suppressed in all three GNAQ mutant cell lines by enzastaurin but only in one GNAQ wild type cell line (Mel285) (Figure 6).…”

Section: Resultsmentioning

confidence: 99%

The Protein Kinase C Inhibitor Enzastaurin Exhibits Antitumor Activity against Uveal Melanoma

Zhu

Fletcher

et al. 2012

PLoS ONE

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GNAQ mutations at codon 209 have been recently identified in approximately 50% of uveal melanomas (UM) and are reported to be oncogenic through activating the MAPK/Erk1/2 pathway. Protein kinase C (PKC) is a component of signaling from GNAQ to Erk1/2. Inhibition of PKC might regulate GNAQ mutation-induced Erk1/2 activation, resulting in growth inhibition of UM cells carrying GNAQ mutations. UM cells carrying wild type or mutant GNAQ were treated with the PKC inhibitor enzastaurin. Effects on proliferation, apoptosis, and signaling events were evaluated. Enzastaurin downregulated the expression of several PKC isoforms including PKCβII PKCθ, PKCε and/or their phosphorylation in GNAQ mutated cells. Downregulation of these PKC isoforms in GNAQ mutated cells by shRNA resulted in reduced viability. Enzastaurin exhibited greater antiproliferative effect on GNAQ mutant cells than wild type cells through induction of G1 arrest and apoptosis. Enzastaurin-induced G1 arrest was associated with inhibition of Erk1/2 phosphorylation, downregulation of cyclin D1, and accumulation of cyclin dependent kinase inhibitor p27Kip1. Furthermore, enzastaurin reduced the expression of antiapoptotic Bcl-2 and survivin in GNAQ mutant cells. Inhibition of Erk1/2 phosphorylation with a MEK specific inhibitor enhanced the sensitivity of GNAQ wild type cells to enzastaurin, accompanied by p27Kip1 accumulation and/or inhibition of enzastaurin-induced survivin and Bcl-2 upregulation. PKC inhibitors such as enzastaurin have activity against UM cells carrying GNAQ mutations through inhibition of the PKC/Erk1/2 pathway and induction of G1 arrest and apoptosis. Inhibition of the PKC pathway provides a basis for clinical investigation in patients with UM.

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Section: Resultsmentioning

confidence: 99%

The Protein Kinase C Inhibitor Enzastaurin Exhibits Antitumor Activity against Uveal Melanoma

Zhu

Fletcher

et al. 2012

PLoS ONE

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“…Activity in a larger panel of celllines, more animal models, and autochthonous/orthotopic models may better predict for clinical activity in human subjects with TCC. Synergistic antiproliferative and proapoptotic activity was noted preclinically against non-small cell lung cancer cells when chemotherapy (gemcitabine or pemetrexed) was followed by enzastaurin, whereas concomitant treatments or enzastaurin given before chemotherapy, resulted in antagonistic effects (27). However, a determination of the sequence dependence of antitumor activity was beyond the scope of our project.…”

Section: Discussionmentioning

confidence: 97%

Enzastaurin shows preclinical antitumor activity against human transitional cell carcinoma and enhances the activity of gemcitabine

Jian

Yamashita²,

Levitt³

et al. 2009

Molecular Cancer Therapeutics

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Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signaling through protein kinase C (PKC)-β and the phosphatidylinositol 3-kinase/AKT pathways. We preclinically evaluated enzastaurin alone and in combination with gemcitabine for transitional cell cancer (TCC). Immunohistochemistry (IHC) was done on 105 human samples from a microarray to show the expression of PKC-β. The preclinical antitumor activity of enzastaurin and gemcitabine as single agents and in combination against aggressive human -lines (-SUP and 5637) and murine subcutaneous xenografts bearing 5637 cells was determined. Western Blot was done on tumor cells in vitro to detect signaling through PKC-β, GSK-3β, and AKT. The effect on cell migration was determined in vitro. Modulation of proliferation (Ki-67), apoptosis (cleaved caspase-3), and angiogenesis (CD31) in vivo was determined by IHC. IHC done on human TCC samples from a microarray showed the expression of PKC-β in 33% of tumors. Enzastaurin induced significant apoptosis and inhibited proliferation in vitro at low micromolar concentrations. The in vitro inhibitory activity of combination enzastaurin and gemcitabine by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay seemed synergistic. Western Blotting revealed down-regulation of Akt, PKC-β, and GSK-3 β phosphorylation. Enzastaurin inhibited migration at an earlier time point independent of antiproliferative activity. Combination therapy had significantly superior antitumor activity in murine xenografts compared with untreated controls, whereas single agents did not. IHC showed reduced Ki-67 and CD31 and increased cleaved caspase-3 with combination therapy compared with controls. Enzastaurin showed preclinical antitumor activity against human TCC and enhanced the activity of gemcitabine.

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“…Apoptosis was detected by flow cytometry via the examination of altered plasma membrane phospholipid packing by lipophilic dye Annexin V as described elsewhere (18). Briefly, treated cells were harvested by trypsin, washed twice with PBS, and then resuspended in binding buffer at a concentration of 1 Â 10 6 cells/mL according to the manufacturer's instruction.…”

Section: Assessment Of Apoptosismentioning

confidence: 99%

Synergistic Effects of Metformin Treatment in Combination with Gefitinib, a Selective EGFR Tyrosine Kinase Inhibitor, in LKB1 Wild-type NSCLC Cell Lines

Morgillo

Sasso

Vitagliano

et al. 2013

Clinical Cancer Research

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111

112

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Purpose: EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) have been found to be effective against lung cancer, but clinical resistance to these agents has developed as their usage has increased. Metformin is a widely used antidiabetic drug and also displays significant growth-inhibitory and proapoptotic effects in several cancer models, alone or in combination with chemotherapeutic drugs.Experimental Design: The effects of gefitinib, a selective EGFR-TKI, and metformin on a panel of nonsmall cell lung cancer (NSCLC) cell lines were assessed by using MTT, bromide assay, flow cytometry, anchorage-independent growth, coimmunoprecipitation, and Western blot analysis.Results: The combination of metformin with gefitinib induced a strong antiproliferative and proapoptotic effect in NSCLC cell lines that harbored wild-type LKB1 gene. Treatment with metformin as single agent, however, induced an activation and phosphorylation of mitogen-activated protein kinase (MAPK) through an increased C-RAF/B-RAF heterodimerization. The inhibition of EGFR phosphorylation and of downstream signaling by adding gefitinib to metformin treatment abrogated this phenomenon and induced a strong apoptotic effect in vitro and in vivo.Conclusions: Metformin and gefitinib are synergistic in LKB1 wild-type NSCLC cells. However, further studies are required to investigate better the effect of metformin action on the RAS/RAF/MAPK pathway and the best context in which to use metformin in combination with molecular targeted agents.

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Sequence-dependent, synergistic antiproliferative and proapoptotic effects of the combination of cytotoxic drugs and enzastaurin, a protein kinase Cβ inhibitor, in non-small cell lung cancer cells

Cited by 22 publications

References 42 publications

The Protein Kinase C Inhibitor Enzastaurin Exhibits Antitumor Activity against Uveal Melanoma

The Protein Kinase C Inhibitor Enzastaurin Exhibits Antitumor Activity against Uveal Melanoma

Enzastaurin shows preclinical antitumor activity against human transitional cell carcinoma and enhances the activity of gemcitabine

Synergistic Effects of Metformin Treatment in Combination with Gefitinib, a Selective EGFR Tyrosine Kinase Inhibitor, in LKB1 Wild-type NSCLC Cell Lines

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