Cytotoxic effects induced by docetaxel, gefitinib, and cyclopamine on side population and nonside population cell fractions from human invasive prostate cancer cells

Abstract

The present study has been undertaken to establish the therapeutic benefit of cotargeting epidermal growth factor receptor (EGFR) and sonic hedgehog pathways by using gefitinib and cyclopamine, respectively, for improving the efficacy of the current chemotherapeutic drug docetaxel to counteract the prostate cancer progression from locally invasive to metastatic and recurrent disease stages. The data from immuofluorescence analyses revealed that EGFR/Tyr(1173)-pEGFR, sonic hedgehog ligand, smoothened coreceptor, and GLI-1 were colocalized with the CD133(+) stem cell-like marker in a small subpopulation of prostate cancer cells. These signaling molecules were also present in the bulk tumor mass of CD133(-) prostate cancer cells with a luminal phenotype detected in patient's adenocarcinoma tissues. Importantly, the results revealed that the CD133(+)/CD44(high)/AR(-/low) side population (SP) cell fraction endowed with a high self-renewal potential isolated from tumorigenic and invasive WPE1-NB26 cells by the Hoechst dye technique was insensitive to the current chemotherapeutic drug, docetaxel. In contrast, the docetaxel treatment induced significant antiproliferative and apoptotic effects on the CD133(-)/CD44(low)/AR(+) non-SP cell fraction isolated from the WPE1-NB26 cell line. Of therapeutic interest, the results have also indicated that combined docetaxel, gefitinib, and cyclopamine induced greater antiproliferative and apoptotic effects on SP and non-SP cell fractions isolated from WPE1-NB26 cells than individual drugs or two-drug combinations. Altogether, these observations suggest that EGFR and sonic hedgehog cascades may represent the potential therapeutic targets of great clinical interest to eradicate the total prostate cancer cell mass and improve the current docetaxel-based therapies against locally advanced and invasive prostate cancers, and thereby prevent metastases and disease relapse.

Figure 1

Immunohistochemical and immunofluorescence analyses of expression levels of EGFR and hedgehog signaling elements in non-malignant and malignant prostatic tissues. (A) Immunohistochemical analyses of expression levels of activated Tyr¹¹⁷³-pEGFR phosphorylated form and sonic hedgehog effector, GLI-1 transcription factor in non-malignant and malignant prostatic tissues. AccuMax array sections of non-malignant prostatic tissues and adenocarcinomas from the same PC patients were probed with antibody directed against the activated Tyr¹¹⁷³-pEGFR phosphorylated form or GLI-1 transcription factor after blocking with serum as described in Materials and Methods. All tissue sections were examined under a microscope, and the Tyr¹¹⁷³-pEGFR or GLI-1 immunoreactivity was judged by dark brown staining. Representative pictures of stained tissue samples of normal prostate and adenocarcinoma are shown at original magnifications of ×100 and ×400. (B) Immunofluorescence analyses of the co-localization of the expression of the CD133 stem cell-like marker with CK5/18, EGFR and its activated Tyr¹¹⁷³-pEGFR phosphorylated form, and SHH hedgehog ligand, SMO co-receptor or GLI-1 transcriptional effector in non-malignant and malignant prostatic tissue specimens from patients. Double-immunofluorescence staining was simultaneously performed with the phycoerythrin-labeled anti-CD133 antibody (red color) plus Alexa 340-labeled anti-CK5 antibody (blue color) or fluorescein-labeled anti-CK18, EGFR, Tyr¹¹⁷³-pEGFR, -SHH, -SMO or -GLI-1 antibody (green color) after blocking with goat serum as described in Materials and Methods. Double staining (yellow/purple color) detected by confocal analyses, which is indicative of the co-localization of these markers, are shown with an arrow. Representative pictures are shown at original magnification ×630.

Figure 2

RT-PCR and immunoconfocal analyses of expression levels of stem cell-like markers, basal/luminal markers, EGFR and sonic hedgehog signaling elements in non-malignant and malignant prostatic cell lines. The cells were maintained for 48 hours in culture medium. Then, mRNA expression levels of stem cell-like (CD133, CD44 and ABCG2), basal (CK5) and luminal (CK18) markers as well as EGF/EGFR and sonic hedgehog signaling components were estimated by RT-PCR in human non-malignant RWPE-1, tumorigenic K-ras-transformed RWPE-2, MNU-transformed tumorigenic WPE1-NA22 and invasive WPE1-NB14 and WPE1-NB26, and metastatic PC3 prostatic cell lines. Immunofluorescence staining of methanol-fixed cells was done with anti-EGFR plus Tyr¹¹⁷³p-EGFR, -SHH, -SMO or -GLI-1 primary antibody plus fluorescein (green color) and/or Texas red secondary antibody and DAPI (nuclear blue color) after blocking with goat serum. Representative pictures obtained for the overlaps of EGFR/Tyr¹¹⁷³-pEGFR (hybrid yellow), SHH (red), SMO (green) and GLI-1 (green) are shown at original magnification ×630.

Figure 3

Characterization of phenotypic features and the self-renewal ability of the SP and non-SP cell fractions isolated from parental tumorigenic and invasive WPE1-NB26 cells by Hoechst dye efflux technique and FAC sorting. (A) The WPE1-NB26 cells were stained with fluorescent Hoechst dye in the absence or presence of 50 µM verapamil and FACS analyses were performed. The Hoechst dye efflux profile shows the SP (green color), and non-SP fraction (blue color). The number of total PC cells localized in the SP fraction was significantly reduced in the presence of broad ABC transporter inhibitor, verapamil. (B) Clone formation efficacy of the SP and non-SP fractions from WPE1-NB26 corresponds to the ratio of the clone number to the plated cell numbers. (C and D) Comparative RT-PCR and immunoconfocal analyses of expression levels of prostatic stem cell-like markers (CD133, CD44 and ABCG2), AR, EGF/EGFR and sonic hedgehog signaling elements in the SP and non-SP cell fractions isolated from parental WPE1-NB26 cell line.

Figure 4

Anti-proliferative effects induced by docetaxel, gefitinib, and cyclopamine on SP and non-SP cell fractions isolated from parental tumorigenic and invasive WPE1-NB26 cells. The SP and non-SP cells were treated with the indicated concentrations of docetaxel (Doc.), gefitinib (Gef.), and cyclopamine (Cycl.), alone or in combination, for two days. (A) The effects of tested agents are expressed as the percentage of inhibition of SP and non-SP cell proliferation compared with the growth of untreated cells (control). The data are the means of three to four different experiments done in triplicate. *P < 0.05, indicates a significant difference of the anti-proliferative effects induced by docetaxel, gefitinib, and cyclopamine, alone or in combination, on the SP and non-SP cell fractions. (B) FACS analyses of the growth inhibitory effect induced by mixed docetaxel, gefitinib, and cyclopamine on SP and non-SP cell fractions. The inhibitory effect induced by drugs on the progression of PC cells in the cellular cycle was investigated by flow cytometric analyses. The SP and non-SP cell cells were untreated or treated with the indicated concentrations of 2 nM docetaxel, 0.5 µM gefitinib, and 1 µM cyclopamine, alone or in combination, for two days. At the end of incubation time, the cells were prepared as described in Materials and Methods and the cell cycle distributions were assessed by FACS analyses. Representative results obtained from three separate experiments are shown.

Figure 5

Assessment of the stimulatory effect induced by docetaxel, gefitinib, and cyclopamine on the apoptotic death, mitochondrial membrane depolarizing and cytosolic cytochrome c releasing in the SP and non-SP cell fractions isolated from parental tumorigenic and invasive WPE1-NB26 cells. The PC cells were untreated (control) or treated with the indicated concentrations of docetaxel (Doc.), gefitinib (Gef.), and cyclopamine (Cycl.), alone or in combination, for four days. (A) The cell nuclei were stained with propidium iodide and the number of apoptotic PC cells detected in the sub-G₁ phase was analyzed by flow cytometry. Plots showing the percentages of apoptotic PC cell death induced by tested drugs, alone or in combination, obtained from three separate experiments. *p < 0.05, indicates a significant difference between the apoptotic effects induced by docetaxel, gefitinib, and cyclopamine on the SP and non-SP cell fractions. After the treatments, the cells were prepared by staining with 40 nM DIOC₆(3) for analyses of MMP by flow cytometry. Moreover, the amounts of cytochrome c released into cytosol were estimated by ELISA as described in Materials and Methods. (B) Representative profiles of effects induced by tested drugs, alone or in combination, on MMP in SP and non-SP cell fractions isolated from WPE1-NB26 cells are shown. (C) Plots showing the percentages of the stimulatory effects induced by tested drugs, alone or in combination on cytochrome c release in SP and non-SP cell fractions. *p < 0.05, indicates a significant difference between the stimulatory effect induced by docetaxel, gefitinib and cyclopamine, alone or in combination, on cytochrome c release in the SP and non-SP cell fractions.