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Pathobiological implications of the expression of EGFR, pAkt, NF-κB and MIC-1 in prostate cancer stem cells and their progenies.

Mimeault M, Johansson SL, Batra SK - PLoS ONE (2012)

Bottom Line: The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse.Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells.Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse. The present study was undertaken to establish the possibility of using a combination of specific oncogenic products, including epidermal growth factor receptor (EGFR), pAkt, nuclear factor-kappaB (NF-κB) and macrophage inhibitory cytokine-1 (MIC-1) as biomarkers and therapeutic targets for optimizing the management of patients with localized PC at earlier disease stages. The immunohistochemical and immunofluorescence data have revealed that the expression levels of EGFR, Ser(473)-pAkt, NF-κB p65 and MIC-1 proteins were significantly enhanced in the same subset of 76 cases of prostatic adenocarcinoma specimens during the disease progression and these biomarkers were expressed in a small subpopulation of CD133(+) PC cells and the bulk tumor mass of CD133(-) PC cells. Importantly, all of these biomarkers were also overexpressed in 80-100% of 30 PC metastasis bone tissue specimens. Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells. Of therapeutic interest, the targeting of EGFR, pAkt, NF-κB or MIC-1 was also effective at suppressing the basal and EGF-promoted prostasphere formation by SP WPE1-NB26 cells, inducing disintegration of SP cell-derived prostaspheres and decreasing the viability of SP and non-SP WPE1-NB26 cell fractions. Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel. These findings suggest that the combined use of EGFR, pAkt, NF-κB and/or MIC-1 may represent promising strategies for improving the accuracy of current diagnostic and prognostic methods and efficacy of treatments of PC patients in considering the disease heterogeneity, thereby preventing PC progression to metastatic and lethal disease states.

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Related in: MedlinePlus

Estimation of the disintegration effects induced by different tested drugs on prostaspheres derived from SP WPE1-NB26 cell-and implication of the mitochondrial and caspase pathways.a) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P<0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.
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pone-0031919-g008: Estimation of the disintegration effects induced by different tested drugs on prostaspheres derived from SP WPE1-NB26 cell-and implication of the mitochondrial and caspase pathways.a) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P<0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.

Mentions: The results from MTT analyses have indicated that a treatment with increasing concentrations of gefitinib, Akt inhibitor VIII, partenolide or anti-MIC-1 antibody for 72 hours significantly reduced the number of viable SP and non-SP WPE1-NB26 cells (Figure 7). We have also observed that 10 µl/ml of the pre-immune rabbit serum used as control has not significant effect on the viability of cells as compared to 10 µl/ml anti-MIC-1 antibody (data not shown). Moreover, a treatment with the current chemotherapeutic drug docetaxel induced only a small cytotoxic effect on the SP WPE1-NB26 cell fraction while it markedly reduced the number of viable non-SP WPE1-NB26 cells (Figure 7). As shown in Figure 8a, a treatment with gefitinib, LY294002, Akt inhibitor VIII, partenolide or anti-MIC-1 antibody for 4 days was also effective at inducing the disintegration of the dense prostaspheres generated by SP WPE1-NB26 cells after 7 days of cultures under ultra-low attachment plate while docetaxel had no significant effect (Figure 8a).


Pathobiological implications of the expression of EGFR, pAkt, NF-κB and MIC-1 in prostate cancer stem cells and their progenies.

Mimeault M, Johansson SL, Batra SK - PLoS ONE (2012)

Estimation of the disintegration effects induced by different tested drugs on prostaspheres derived from SP WPE1-NB26 cell-and implication of the mitochondrial and caspase pathways.a) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P<0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285632&req=5

pone-0031919-g008: Estimation of the disintegration effects induced by different tested drugs on prostaspheres derived from SP WPE1-NB26 cell-and implication of the mitochondrial and caspase pathways.a) Representative pictures of the disintegration effect induced by a treatment with tested drugs during 4 days on the prostaspheres derived from SP WPE1-NB26 cells. b) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with anti-cytochrome c primary antibody plus fluorescein (green) secondary antibody and the mitochondria and nuclei stained with MitoTracker Red CMXRos (red) and DAPI (blue), respectively. Representative pictures showing the expression level and cellular localization of mitochondria (red), cytochrome c in mitochondria (green/red; hybrid yellow) or cytoplasm (diffuse green staining) are shown at the original magnification of ×630. c) Immunofluorescence staining of SP WPE1-NB26 cells after a treatment with indicated cytotoxic agents was done with a primary antibody directed against the cleaved caspase-9 fragment plus Texas red secondary antibody and TUNEL reactive mixture (green) and the nuclei counterstained with DAPI (blue). Representative pictures showing the expression level and cellular localization of the cleaved caspase-9 fragment (red) and TUNEL and DAPI (green/blue; hybrid cyan) in SP WPE1-NB26 cells are shown at original magnification ×630. The overlaps of double nuclear staining with TUNEL and DAPI (green/blue; hybrid cyan) associated with DNA fragmentation which is indicative of the apoptotic nuclei in SP cells are indicated by arrows. d) The SP WPE1-NB26 cells were untreated or treated with the indicated concentrations of a specific inhibitory agent including EGFR (gefitinib), PI3K (LY294002), pAkt (pAkt inhibitor VIII), NF-κB (partenolide) and MIC-1 (anti-MIC-1 antibody), alone or in combination with 5 nM docetaxel for 4 days, and the apoptotic cell death was analyzed by FACS. The panel shows the apoptotic effect induced by the tested agents that are expressed as the percentage of apoptotic SP WPE1-NB26 cells compared to non-treated SP cells (control). *, P<0.05, indicates a significant difference between the apoptotic effect induced by tested drugs plus 5 nM docetaxel versus individual drugs on the SP WPE1-NB26 cell fraction.
Mentions: The results from MTT analyses have indicated that a treatment with increasing concentrations of gefitinib, Akt inhibitor VIII, partenolide or anti-MIC-1 antibody for 72 hours significantly reduced the number of viable SP and non-SP WPE1-NB26 cells (Figure 7). We have also observed that 10 µl/ml of the pre-immune rabbit serum used as control has not significant effect on the viability of cells as compared to 10 µl/ml anti-MIC-1 antibody (data not shown). Moreover, a treatment with the current chemotherapeutic drug docetaxel induced only a small cytotoxic effect on the SP WPE1-NB26 cell fraction while it markedly reduced the number of viable non-SP WPE1-NB26 cells (Figure 7). As shown in Figure 8a, a treatment with gefitinib, LY294002, Akt inhibitor VIII, partenolide or anti-MIC-1 antibody for 4 days was also effective at inducing the disintegration of the dense prostaspheres generated by SP WPE1-NB26 cells after 7 days of cultures under ultra-low attachment plate while docetaxel had no significant effect (Figure 8a).

Bottom Line: The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse.Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells.Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT
The progression of prostate cancers (PCs) to locally invasive, androgen-independent and metastatic disease states is generally associated with treatment resistance and disease relapse. The present study was undertaken to establish the possibility of using a combination of specific oncogenic products, including epidermal growth factor receptor (EGFR), pAkt, nuclear factor-kappaB (NF-κB) and macrophage inhibitory cytokine-1 (MIC-1) as biomarkers and therapeutic targets for optimizing the management of patients with localized PC at earlier disease stages. The immunohistochemical and immunofluorescence data have revealed that the expression levels of EGFR, Ser(473)-pAkt, NF-κB p65 and MIC-1 proteins were significantly enhanced in the same subset of 76 cases of prostatic adenocarcinoma specimens during the disease progression and these biomarkers were expressed in a small subpopulation of CD133(+) PC cells and the bulk tumor mass of CD133(-) PC cells. Importantly, all of these biomarkers were also overexpressed in 80-100% of 30 PC metastasis bone tissue specimens. Moreover, the results have indicated that the EGF-EGFR signaling pathway can provide critical functions for the self-renewal of side population (SP) cells endowed with stem cell-like features from highly invasive WPE1-NB26 cells. Of therapeutic interest, the targeting of EGFR, pAkt, NF-κB or MIC-1 was also effective at suppressing the basal and EGF-promoted prostasphere formation by SP WPE1-NB26 cells, inducing disintegration of SP cell-derived prostaspheres and decreasing the viability of SP and non-SP WPE1-NB26 cell fractions. Also, the targeting of these oncogenic products induced the caspase-dependent apoptosis in chemoresistant SP WPE1-NB26 cells and enhanced their sensibility to the cytotoxic effects induced by docetaxel. These findings suggest that the combined use of EGFR, pAkt, NF-κB and/or MIC-1 may represent promising strategies for improving the accuracy of current diagnostic and prognostic methods and efficacy of treatments of PC patients in considering the disease heterogeneity, thereby preventing PC progression to metastatic and lethal disease states.

Show MeSH
Related in: MedlinePlus