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 co-targeting 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 (PC) progression from locally invasive to metastatic and recurrent disease stages. The data from immuofluorescence analyses revealed that EGFR/Tyr1173-pEGFR, sonic hedgehog ligand (SHH), smoothened co-receptor (SMO) and GLI-1 were co-localized with the CD133+ stem cell-like marker in a small subpopulation of PC cells. These signaling molecules were also present in the bulk tumor mass of CD133− PC cells with a luminal phenotype detected in patient’s adenocarcinoma tissues. Importantly, the results revealed that the CD133+/CD44high/AR−/low side population (SP) cell fraction endowed with a high self-renewal potential isolated from tumorigenic and invasive WPE1-NB26 cells by Hoechst dye technique was insensitive to current chemotherapeutic drug, docetaxel. In contrast, the docetaxel treatment induced significant anti-proliferative and apoptotic effects on the CD133−/CD44low/AR+ non-SP cell fraction isolated from WPE1-NB26 cell line. Of therapeutic interest, the results have also indicated that combined docetaxel, gefitinib and cyclopamine induced greater anti-proliferative 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 PC cell mass and improve the current docetaxel-based therapies against locally advanced and invasive PCs, and thereby prevent metastases and disease relapse.

Frequent Gene Products and Molecular Pathways Altered in Prostate Cancer- and Metastasis-Initiating Cells and Their Progenies and Novel Promising Multitargeted Therapies

“…For instance, it was reported that the cotargeting of EGFR and hedgehog pathways by using gefitinib and cyclopamine improved the cytotoxic effects induced by mitoxantrone on parental AI PC3 and DU145 cells and CD44 high cell fractions enriched from these metastatic PC cell lines (203). Furthermore, a combination of low concentrations of gefitinib and cyclopamine plus docetaxel also induced greater antiproliferative and apoptotic effects on parental PC3 cells as well as on the CD133 + SP subpopulation and CD133 -non-SP cell fractions from highly invasive WPE1-NB26 PC cells than individual drugs or two drug combinations (15,69). In addition, since the chemoattractant gradient mediated by SDF-1 can regulate the proliferation, migration and metastatic spread of CXCR4 + PC cells, the inhibition of the SDF-1/CXCR4 axis also may constitute a potential therapeutic approach to prevent their invasion and metastases at distant sites, including bones and disease relapse (Figures 1-3) (51,(167)(168)(169).…”

“…Similarly, the highly tumorigenic CD133 + / CD117 high / ABCG2 high /nestin + PC cell subpopulation coexpressing Oct3/4, Nanog and Sox2 from the PC cell line 22RV1 was also more resistant to treatment with a variety of chemotherapeutic drugs such as cisplatin, paclitaxel, adriamycin, and methotrexate than the CD133 -/ABCG2 low 22RV1 cell fraction (67). Moreover, the CD133 + SP cells endowed with stem cell-like properties isolated from the highly invasive WPE1-NB26 PC cell line were less sensitive to the antiproliferative and apoptotic effects induced by docetaxel treatment than the CD133 -non-SP cell fraction (69). In addition, it has also been observed that an enrichment of Sca + PC stem/progenitor cells occurred after androgen-deprivation or docetaxel treatment in transgenic adenocarcinoma of the mouse prostate (TRAMP) and PTEN knockdown transgenic mouse models of PC and led to tumor regrowth and metastases (63,98,(181)(182)(183)(184).…”

“…Recent lines of experimental evidence have revealed that the PC stem/progenitor cells, including side population (SP) of PC cells isolated by using the Hoechst dye efflux technique, exhibiting an AI phenotype and expressing high levels of ATP-binding cassette (ABC) multidrug transporters such as ABCG2, may be more resistant than their differentiated progenies and non-SP cells to the antihormonal and chemotherapeutic treatments (67)(68)(69)180). For example, it was observed that the nonadherent suspension culture of metastatic and AI PC3 cells under the form of prostaspheres resulted in an enrichment of CD133 + / CD44 + PC cells that are more resistance to cisplatin than adherent cells (68).…”

“…These signaling elements include telomerase, ALDH, CD44, EGFR, hedgehog, Wnt/β-catenin, Notch, integrins, IL-6/IL-6R and SDF-1/CXCR4 and downstream signaling elements such as miRNAs, Myc, PI3K/Akt, NF-κB, HIFs, MIC-1, Nrf2 and ABC multidrug transporters such as P-gp and ABCG2 (Figures 2 and 3) (22,47,51,57,58,60,69,72,73,76,108,129,195,196,(201)(202)(203)(204)(205)(206)(207). The data from several preclinical studies have indicated that the targeting of these deregulated gene products and oncogenic pathways by RNA silencing or using specific inhibitory agents induced the antiproliferative, antiinvasive, antimetastatic and cytotoxic effects on PC stem/ progenitor cells and their progenies in vitro and in vivo and/or reversed chemoresistance (Figure 3) (22,47,51,57,58,60,69,72,73,76,108,129,195,196,(201)(202)(203)(204)(205)(206)(207)<...>…”

“…These observations, which are consistent with a reduction of the bulk mass of proliferative androgen sensitive PC cells, while AI PC cells with stem cell-like features can persist after AR inhibition, underline the importance of using casodex in combination with other cytotoxic drugs for eradicating the total PC cell mass (108). Of particular interest, the blockade of EGFR and/or hedgehog cascades by using a specific inhibitor of EGFR tyrosine kinase activity (such as gefitinib, erlotinib or lapitinib), smoothened (SMO) hedgehog coreceptor inhibitor, cyclopamine or anti-SHH antibody has also been shown to induce the antiproliferative, antiinvasive and cytotoxic effects on PC stem/ progenitor cells with stem cell-like properties and their progenies in vitro and in vivo (Figure 3) (14,15,21,22,69,203,209,210). For instance, it was reported that the cotargeting of EGFR and hedgehog pathways by using gefitinib and cyclopamine improved the cytotoxic effects induced by mitoxantrone on parental AI PC3 and DU145 cells and CD44 high cell fractions enriched from these metastatic PC cell lines (203).…”