2-Triazenoazaindoles: α novel class of triazenes inducing transcriptional down-regulation of EGFR and HER-2 in human pancreatic cancer cells

Abstract

Pancreatic cancer is a complex malignancy arising from the accumulation of genetic and epigenetic defects in the affected cells. Standard chemotherapy for patients with advanced disease shows only modest effects and is associated with considerable toxicity. Overexpression or aberrant activation of members of the epidermal growth factor receptor tyrosine kinase family, which includes EGFR and HER-2, occurs frequently and is associated with multiple drug resistance and decreased patient survival. In this study, we have investigated the therapeutic potential of AS104, a novel compound of the triazene class, with potential inhibitory effects on EGFR. We found that treatment of cells with AS104 causes significant reduction of cell growth and metabolic activity in four human pancreatic cancer cell lines. Furthermore, we show that the AS104-mediated induction of apoptotic cell death is associated with stimulation of autophagy in a dose-dependent manner. Treatment of cells with AS104 results in significant down-regulation of EGFR and HER-2 expression and activity and subsequent inhibition of downstream signaling proteins. Quantitative RT-PCR analysis and assays with proteasome inhibitors revealed that AS104 regulates the expression of EGFR and HER-2 at the transcriptional level. These findings provide for the first time experimental evidence for efficacy of AS104 in the simultaneous transcriptional repression of EGFR and HER-2 genes and suggest that AS104 may have therapeutic potential in the treatment of pancreatic cancers that express high levels of the aforementioned receptor tyrosine kinases.

Figure 1

The structural formula of AS104.

Figure 2

Effect of AS104 on viability of four human pancreatic cancer cell lines. (A) Protein expression levels of EGFR and HER-2 were analyzed by Western blotting on total cell lysates derived from the indicated cell lines. β-actin was used as loading control. (B) Cells were treated with DMSO and various concentrations of AS104 for 48 h, respectively. Proportion of viable cells measured by the WST-1 assay is indicated in arbitrary units as a difference in absorbance determined at 450 and 690 nm (reference) wavelengths, respectively. Three independent experiments were performed and data from one representative experiment [mean ± standard deviation (SD) of four replicates] are shown.

Figure 3

AS104 impairs proliferation and survival of human pancreatic cancer cells. (A) Cell proliferation of four human pancreatic cancer cell lines was determined by BrdU incorporation into genomic DNA. Results are expressed as percentage relative to the corresponding controls represented by cells incubated with DMSO. Insert to the right, shows the concentrations of AS104 inducing 50% growth inhibition (i.e., GI₅₀) of the various cell lines after 48 h incubation with the drug. Three independent experiments were performed obtaining similar results and data from one representative experiment (mean ± SD of four replicates) are shown. (B) PANC-1 cells were incubated with DMSO and increasing concentrations of AS104, respectively, for 24 h. Cells were allowed to form colonies for 14 days. Colonies were visualized by staining with crystal violet as described in Materials and methods. Representative pictures of PANC-1 cell colonies after staining are shown. (C) Bar graph shows quantification of cell colonies. Average values ± SD from six independent experiments are shown relative to DMSO-treated control. ^*Statistical significant difference in number of colonies formed after treatment with increasing concentration of AS104 as compared to the treatment with DMSO alone (Student’s t-test, P<0.05).

Figure 4

Incubation of PANC-1 cells with AS104 induces significant cell death. (A) Cells were left untreated (CT), incubated with DMSO or with various concentrations of AS104 for 48 h. Cells fixed and subsequently stained with propidium iodide were analyzed by flow cytometry. The fraction of dead cells (sub-G1) is expressed in percentage. Three independent experiments were performed obtaining similar results and data from one representative experiment are shown. (B) Representative time-course experiment performed by treating cells with either DMSO or 40 μM AS104 for the indicated time. Cells were analyzed by flow cytometry as described above. Experiments were performed three times obtaining similar results.

Figure 5

Treatment of PANC-1 cells with AS104 leads to combined induction of apoptosis and autophagy. (A) PANC-1 cells treated with the indicated concentrations of AS104 for 48 h were subjected to mitochondria isolation. U2OS cells were left untreated or incubated with doxorubicin for 24 h before harvesting and mitochondria extraction. Western blot analysis was performed using the indicated antibodies. The detection of ATP5B was carried out as a control for equal loading. (B) Cells were treated with AS104 as indicated in the figure for 48 h. Western blot analysis was performed for the detection of full-length (116 kDa) and cleaved (85 kDa) poly(ADPribose)polymerase (PARP). (C) Fluorescence-activated cell sorting analysis using the acridine orange-based assay was performed employing PANC-1 cells treated for 48 h as indicated in the bar graph. FL3-H indicates red-positive cells expressed in percentage. (D) Whole cell lysates from cells treated as indicated were subjected to Western blot analysis with antibodies directed against LC3B-I/II. All experiments were performed at least three times obtaining similar results. Data from one representative experiment are shown.

Figure 6

Cell incubation with AS104 leads to down-regulation of EGFR and HER-2 protein expression levels. (A) Total cell lysates (80 μg) from cells starved for 24 h in serum-free medium were left untreated, treated with DMSO or increasing concentrations of AS104 for 3 h. Where indicated, cells were stimulated with 100 ng/ml EGF for 10 min prior harvesting. Whole cell lysates were subjected to Western blot analysis with antibodies directed against EGFR or its phosphorylated form. (B) Experiments were performed essentially as described above except that cells were incubated with the drug for 48 h prior incubation with EGF. Whole cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. Densitometric analysis of EGFR protein was performed with ImageJ software. Values below each band were normalized to control values from cells incubated with DMSO and EGF. (C) Experiments were performed as described in (B). Cell lysates were analyzed by Western blotting employing antibodies against HER-2 protein or its phosphorylated form. Band quantitation of HER-2 protein was obtained densitometrically. Values were normalized to control values relative to cells incubated with DMSO. (D) Whole cell lysates were analyzed by Western blotting with antibodies directed against the indicated proteins. Densitometric analysis was performed on the indicated protein bands. All experiments were performed three times obtaining similar results and one representative experiment is shown. In all experiments, β-actin was detected as control for equal loading.

Figure 7

Treatment of cells with AS104 results in down-regulation of EGFR- and HER-2-mRNA expression levels. (A) PANC-1 cells were treated for 48 h with increasing concentrations of AS104. EGFR- and HER-2-mRNA was quantified (triplicate measurements) relative to β-actin by quantitative RT-PCR, respectively. Fold changes are relative to results from cells incubated with DMSO only. Error bars, SD; ^*P<0.05; ^**P<0.001; NS, not significant. (B) Cells were left untreated (CT), incubated with DMSO or various concentrations of AS104 for 24 h. Where indicated, cells were incubated with 50 μM MG132 for 6 h before harvesting. Whole cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. Two separate experiments were performed obtaining similar results. Data from one representative assay are shown.