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Cross-talk between alpha1D-adrenoceptors and transient receptor potential vanilloid type 1 triggers prostate cancer cell proliferation.

Morelli MB, Amantini C, Nabissi M, Liberati S, Cardinali C, Farfariello V, Tomassoni D, Quaglia W, Piergentili A, Bonifazi A, Del Bello F, Santoni M, Mammana G, Servi L, Filosa A, Gismondi A, Santoni G - BMC Cancer (2014)

Bottom Line: The levels of the alpha(1D)-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha(1D)-AR and TRPV1 expression levels was found.Moreover, alpha(1D)-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens.These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Camerino 62032, Italy. consuelo.amantini@unicam.it.

ABSTRACT

Background: There is evidence that calcium (Ca(2+)) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the correlation between alpha(1D)-adrenergic receptor (alpha(1D)-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha(1D)-AR to cross-talk with TRPV1 in PCa cell lines.

Methods: The expression of alpha1D-AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha(1D)-AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha(1D)-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca(2+) flux, IP3 production, western blot and MTT assay.

Results: The levels of the alpha(1D)-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha(1D)-AR and TRPV1 expression levels was found. Moreover, alpha(1D)-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha(1D)-AR- and TRPV1-dependent protons release and Ca(2+) flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha(1D)-AR/TRPV1 double knockdown.

Conclusions: We demonstrate a cross-talk between alpha1D-AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.

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Related in: MedlinePlus

PLC, PKC and MAPK inhibitors block the NA-induced PC3 cell survival. Cell growth was assessed by MTT assay in PC3 cells treated for 24 h with vehicle (control) or NA (100 μM), alone or in combination with chelerythrine (0.5 μM), U73122 (5 μM) and PD98059 (50 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells (*), NA + chelerythrine-, NA + PD98059-, NA + U73122- and NA + chelerythrine + PD98059 + U73122- with NA-treated cells (#) and NA + chelerythrine + PD98059 + U73122- with NA + chelerythrine-, NA + PD98059- and NA + U73122-treated cells (§), p ≤ 0.01.
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Fig6: PLC, PKC and MAPK inhibitors block the NA-induced PC3 cell survival. Cell growth was assessed by MTT assay in PC3 cells treated for 24 h with vehicle (control) or NA (100 μM), alone or in combination with chelerythrine (0.5 μM), U73122 (5 μM) and PD98059 (50 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells (*), NA + chelerythrine-, NA + PD98059-, NA + U73122- and NA + chelerythrine + PD98059 + U73122- with NA-treated cells (#) and NA + chelerythrine + PD98059 + U73122- with NA + chelerythrine-, NA + PD98059- and NA + U73122-treated cells (§), p ≤ 0.01.

Mentions: Then the ability of WS433 or CPZ to inhibit PLC activation, ERK1/2 phosphorylation and the production of phospho-(Ser) PKC substrates was assessed. In regard to PLC activation, when used in combination with NA CPZ, but not WS433, partially inhibited IP3 production; however, NA administered in combination with WS433 and CPZ induced a IP3 production, but at a lower rate than NA in combination with CPZ (Figure 5A). No IP3 production was found when using WS433 and CPZ alone respect to control PC3 cells (Additional file 2: Figure S1C). The NA-induced ERK1/2 phosphorylation was markedly affected by both the α1D-AR and TRPV1 antagonists or by their combination (Figure 5B), whereas the NA-induced increase of (Ser)-PKC substrate phosphorylation was reduced only by CPZ and WS433 used in combination (Figure 5C). No ERK1/2 and (Ser)-PKC substrate phosphorilation was found when using WS433 and CPZ alone respect to control PC3 cells (Additional file 2: Figure S1D, E). Next, the ability of pharmacological inhibitors of PLC, PKC and ERK to inhibit the NA-induced PC3 cell growth was evaluated. We found that U73122, chelerythrine chloride and PD98059, used at the highest doses that did not affect cell viability (Additional file 2: Figure S1F), markedly inhibited the cell growth of NA-stimulated PC3 cells; in addition, when all the three pharmacological inhibitors were used in combination, they completely reverted the NA-mediated effects (Figure 6). These results confirmed that the PLC/PKC/ERK axis plays a major role in the control of α1D-AR- and TRPV1-dependent PC3 cell growth.Figure 5


Cross-talk between alpha1D-adrenoceptors and transient receptor potential vanilloid type 1 triggers prostate cancer cell proliferation.

Morelli MB, Amantini C, Nabissi M, Liberati S, Cardinali C, Farfariello V, Tomassoni D, Quaglia W, Piergentili A, Bonifazi A, Del Bello F, Santoni M, Mammana G, Servi L, Filosa A, Gismondi A, Santoni G - BMC Cancer (2014)

PLC, PKC and MAPK inhibitors block the NA-induced PC3 cell survival. Cell growth was assessed by MTT assay in PC3 cells treated for 24 h with vehicle (control) or NA (100 μM), alone or in combination with chelerythrine (0.5 μM), U73122 (5 μM) and PD98059 (50 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells (*), NA + chelerythrine-, NA + PD98059-, NA + U73122- and NA + chelerythrine + PD98059 + U73122- with NA-treated cells (#) and NA + chelerythrine + PD98059 + U73122- with NA + chelerythrine-, NA + PD98059- and NA + U73122-treated cells (§), p ≤ 0.01.
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Related In: Results  -  Collection

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Fig6: PLC, PKC and MAPK inhibitors block the NA-induced PC3 cell survival. Cell growth was assessed by MTT assay in PC3 cells treated for 24 h with vehicle (control) or NA (100 μM), alone or in combination with chelerythrine (0.5 μM), U73122 (5 μM) and PD98059 (50 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells (*), NA + chelerythrine-, NA + PD98059-, NA + U73122- and NA + chelerythrine + PD98059 + U73122- with NA-treated cells (#) and NA + chelerythrine + PD98059 + U73122- with NA + chelerythrine-, NA + PD98059- and NA + U73122-treated cells (§), p ≤ 0.01.
Mentions: Then the ability of WS433 or CPZ to inhibit PLC activation, ERK1/2 phosphorylation and the production of phospho-(Ser) PKC substrates was assessed. In regard to PLC activation, when used in combination with NA CPZ, but not WS433, partially inhibited IP3 production; however, NA administered in combination with WS433 and CPZ induced a IP3 production, but at a lower rate than NA in combination with CPZ (Figure 5A). No IP3 production was found when using WS433 and CPZ alone respect to control PC3 cells (Additional file 2: Figure S1C). The NA-induced ERK1/2 phosphorylation was markedly affected by both the α1D-AR and TRPV1 antagonists or by their combination (Figure 5B), whereas the NA-induced increase of (Ser)-PKC substrate phosphorylation was reduced only by CPZ and WS433 used in combination (Figure 5C). No ERK1/2 and (Ser)-PKC substrate phosphorilation was found when using WS433 and CPZ alone respect to control PC3 cells (Additional file 2: Figure S1D, E). Next, the ability of pharmacological inhibitors of PLC, PKC and ERK to inhibit the NA-induced PC3 cell growth was evaluated. We found that U73122, chelerythrine chloride and PD98059, used at the highest doses that did not affect cell viability (Additional file 2: Figure S1F), markedly inhibited the cell growth of NA-stimulated PC3 cells; in addition, when all the three pharmacological inhibitors were used in combination, they completely reverted the NA-mediated effects (Figure 6). These results confirmed that the PLC/PKC/ERK axis plays a major role in the control of α1D-AR- and TRPV1-dependent PC3 cell growth.Figure 5

Bottom Line: The levels of the alpha(1D)-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha(1D)-AR and TRPV1 expression levels was found.Moreover, alpha(1D)-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens.These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Camerino 62032, Italy. consuelo.amantini@unicam.it.

ABSTRACT

Background: There is evidence that calcium (Ca(2+)) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the correlation between alpha(1D)-adrenergic receptor (alpha(1D)-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha(1D)-AR to cross-talk with TRPV1 in PCa cell lines.

Methods: The expression of alpha1D-AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha(1D)-AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha(1D)-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca(2+) flux, IP3 production, western blot and MTT assay.

Results: The levels of the alpha(1D)-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha(1D)-AR and TRPV1 expression levels was found. Moreover, alpha(1D)-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha(1D)-AR- and TRPV1-dependent protons release and Ca(2+) flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha(1D)-AR/TRPV1 double knockdown.

Conclusions: We demonstrate a cross-talk between alpha1D-AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.

Show MeSH
Related in: MedlinePlus