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Does valproic acid induce neuroendocrine differentiation in prostate cancer?

Sidana A, Wang M, Chowdhury WH, Toubaji A, Shabbeer S, Netto G, Carducci M, Lupold SE, Rodriguez R - J. Biomed. Biotechnol. (2010)

Bottom Line: Western blot analysis for CgA was performed to confirm the results of the TMA.In the case of CgA, VPA treatment decreased its expression in vitro in a dose-dependent manner, as determined by western blot analysis.Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevant in vivo setting.

View Article: PubMed Central - PubMed

Affiliation: James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, Marburg 205A, 600 North Wolfe Street, Baltimore, MD 21287, USA.

ABSTRACT
Valproic Acid (VPA) is a histone deacetylase inhibitor that holds promise for cancer therapy. Here, we investigate whether VPA treatment induces neuroendocrine differentiation of Prostate Cancer (PCa). A tissue microarray of VPA-treated and untreated tumor xenografts and cell lines of human PCa (LNCaP, C4-2, DU145, and PC-3) were generated and were analyzed by immunohistochemical analysis (IHC) for NE markers chromogranin A (CgA), synaptophysin, and NCAM (neural cell adhesion molecule). Western blot analysis for CgA was performed to confirm the results of the TMA. IHC analysis did not reveal any induction of CgA, synaptophysin, or NCAM in any xenograft after VPA treatment in vivo. In vitro, VPA treatment induced little synaptophysin expression in C4-2 and PC-3 cells and NCAM expression in LNCaP and PC-3 cells. In the case of CgA, VPA treatment decreased its expression in vitro in a dose-dependent manner, as determined by western blot analysis. Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevant in vivo setting.

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Chromogranin A staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CgA staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and human prostate were positive internal controls for CGA staining, while another human prostate section served as a negative control. (b) Synaptophysin staining in prostate xenografts animals treated with or without VPA. A: Representative images of synaptophysin staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and mouse colon were positive internal controls for synaptophysin staining, while mouse bladder as negative internal controls. (c) NCAM staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CD56 staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human colon carcinoid and normal human prostate were positive internal controls for NCAM staining, while mouse bladder was a negative control.
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fig4: Chromogranin A staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CgA staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and human prostate were positive internal controls for CGA staining, while another human prostate section served as a negative control. (b) Synaptophysin staining in prostate xenografts animals treated with or without VPA. A: Representative images of synaptophysin staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and mouse colon were positive internal controls for synaptophysin staining, while mouse bladder as negative internal controls. (c) NCAM staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CD56 staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human colon carcinoid and normal human prostate were positive internal controls for NCAM staining, while mouse bladder was a negative control.

Mentions: Unilateral tumor xenografts were established in 20 animals each for every cell line. Half of the animals were randomized to receive 0.4% VPA in drinking water. We have shown previously that administration of 0.4% VPA in mouse drinking water can achieve plasma VPA levels similar to the levels obtained in human patients [4]. VPA treatment at these levels was shown to induce acetyl H3K9, p21, and reduce tumor volume, thus confirming the pharmacologic activity of VPA [4]. Animals were sacrificed, and tumors were harvested on day 35. To investigate the effects of VPA on NE markers of PCa tumors in vivo, we evaluated expression of NE markers by IHC on the excised tumors. IHC did not reveal any CgA staining in either treatment or control arms in all cell lines (Figure 4(a)). TMAs from C4-2 tumors revealed decreased expression of both synaptophysin (mean weighted score 47(±10) versus 15(±5), P  < .001) and NCAM (44(9) versus 5(6), P  =  .002) in treatment arms (Figure 4(b)). None of the other arms revealed any significant staining (weighted scores less than 30) for NCAM or synaptophysin (Figures 4(b) and 4(c)). Thus VPA does not induce any NE markers in the physiologically relevant in vivo setting.


Does valproic acid induce neuroendocrine differentiation in prostate cancer?

Sidana A, Wang M, Chowdhury WH, Toubaji A, Shabbeer S, Netto G, Carducci M, Lupold SE, Rodriguez R - J. Biomed. Biotechnol. (2010)

Chromogranin A staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CgA staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and human prostate were positive internal controls for CGA staining, while another human prostate section served as a negative control. (b) Synaptophysin staining in prostate xenografts animals treated with or without VPA. A: Representative images of synaptophysin staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and mouse colon were positive internal controls for synaptophysin staining, while mouse bladder as negative internal controls. (c) NCAM staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CD56 staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human colon carcinoid and normal human prostate were positive internal controls for NCAM staining, while mouse bladder was a negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: Chromogranin A staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CgA staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and human prostate were positive internal controls for CGA staining, while another human prostate section served as a negative control. (b) Synaptophysin staining in prostate xenografts animals treated with or without VPA. A: Representative images of synaptophysin staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (Scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human pancreas and mouse colon were positive internal controls for synaptophysin staining, while mouse bladder as negative internal controls. (c) NCAM staining in prostate xenografts animals treated with or without VPA. (a): Representative images of CD56 staining for xenograft sections from control and VPA-treated groups: LNCaP, C4-2, PC-3, and DU-145 (scanned at 20X magnification using the APERIO imaging system). The tissue specimens of human colon carcinoid and normal human prostate were positive internal controls for NCAM staining, while mouse bladder was a negative control.
Mentions: Unilateral tumor xenografts were established in 20 animals each for every cell line. Half of the animals were randomized to receive 0.4% VPA in drinking water. We have shown previously that administration of 0.4% VPA in mouse drinking water can achieve plasma VPA levels similar to the levels obtained in human patients [4]. VPA treatment at these levels was shown to induce acetyl H3K9, p21, and reduce tumor volume, thus confirming the pharmacologic activity of VPA [4]. Animals were sacrificed, and tumors were harvested on day 35. To investigate the effects of VPA on NE markers of PCa tumors in vivo, we evaluated expression of NE markers by IHC on the excised tumors. IHC did not reveal any CgA staining in either treatment or control arms in all cell lines (Figure 4(a)). TMAs from C4-2 tumors revealed decreased expression of both synaptophysin (mean weighted score 47(±10) versus 15(±5), P  < .001) and NCAM (44(9) versus 5(6), P  =  .002) in treatment arms (Figure 4(b)). None of the other arms revealed any significant staining (weighted scores less than 30) for NCAM or synaptophysin (Figures 4(b) and 4(c)). Thus VPA does not induce any NE markers in the physiologically relevant in vivo setting.

Bottom Line: Western blot analysis for CgA was performed to confirm the results of the TMA.In the case of CgA, VPA treatment decreased its expression in vitro in a dose-dependent manner, as determined by western blot analysis.Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevant in vivo setting.

View Article: PubMed Central - PubMed

Affiliation: James Buchanan Brady Urological Institute, Johns Hopkins University School of Medicine, Marburg 205A, 600 North Wolfe Street, Baltimore, MD 21287, USA.

ABSTRACT
Valproic Acid (VPA) is a histone deacetylase inhibitor that holds promise for cancer therapy. Here, we investigate whether VPA treatment induces neuroendocrine differentiation of Prostate Cancer (PCa). A tissue microarray of VPA-treated and untreated tumor xenografts and cell lines of human PCa (LNCaP, C4-2, DU145, and PC-3) were generated and were analyzed by immunohistochemical analysis (IHC) for NE markers chromogranin A (CgA), synaptophysin, and NCAM (neural cell adhesion molecule). Western blot analysis for CgA was performed to confirm the results of the TMA. IHC analysis did not reveal any induction of CgA, synaptophysin, or NCAM in any xenograft after VPA treatment in vivo. In vitro, VPA treatment induced little synaptophysin expression in C4-2 and PC-3 cells and NCAM expression in LNCaP and PC-3 cells. In the case of CgA, VPA treatment decreased its expression in vitro in a dose-dependent manner, as determined by western blot analysis. Thus our data demonstrates that VPA does not induce NE differentiation of PCa cells in the physiologically relevant in vivo setting.

Show MeSH
Related in: MedlinePlus