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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

NA induces PLC, PKC and ERK activation in PC3 cells. (A) The measurement of IP3 levels using the IP3 [3H] Radioreceptor assay kit was performed in PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells, *p ≤ 0.01. (B-C) Lysates from PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM) were separated on SDS-polyacrylamide gels, transferred and then blotted with anti-pERK, anti-ERK, anti-p38, anti-pp38 and anti-phospho-(Ser) PKC substrate Abs. The GAPDH protein level was evaluated as a loading control. Data shown are representative of one out of three separate experiments. Densitometric analysis was performed evaluating three different experiments and statistical analysis was carried out comparing NA-treated with control cells, *p ≤ 0.01.
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Fig4: NA induces PLC, PKC and ERK activation in PC3 cells. (A) The measurement of IP3 levels using the IP3 [3H] Radioreceptor assay kit was performed in PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells, *p ≤ 0.01. (B-C) Lysates from PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM) were separated on SDS-polyacrylamide gels, transferred and then blotted with anti-pERK, anti-ERK, anti-p38, anti-pp38 and anti-phospho-(Ser) PKC substrate Abs. The GAPDH protein level was evaluated as a loading control. Data shown are representative of one out of three separate experiments. Densitometric analysis was performed evaluating three different experiments and statistical analysis was carried out comparing NA-treated with control cells, *p ≤ 0.01.

Mentions: Several reports have examined the role of the PLC-PKC-MAPK signalling pathways in the catecholamine-induced α1D-AR-mediated proliferation of prostate cancer cells [22, 23]. Thus, we studied the effect of α1D-AR and TRPV1 cross-talk in the NA-induced activation of PLC, PKC and ERK signalling pathways. Time-course analysis of NA-induced PLC activation, evaluated as IP3 production, evidenced that NA stimulates IP3 production 5 min after NA treatment, remaining sustained at 30 min and declining thereafter (Figure 4A). Moreover, we found that ERK1/2 is phosphorylated at basal level and NA induces a rapid and transient increase of its phosphorylation at 3 min after treatment (Figure 4B); NA also increased the (Ser)-PKC substrate phosphorylation at 3–5 min and then return to basal level (Figure 4C); p38 was basally phosphorylated, and NA stimulation did not affect its phosphorylation state (Figure 4B).Figure 4


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)

NA induces PLC, PKC and ERK activation in PC3 cells. (A) The measurement of IP3 levels using the IP3 [3H] Radioreceptor assay kit was performed in PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells, *p ≤ 0.01. (B-C) Lysates from PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM) were separated on SDS-polyacrylamide gels, transferred and then blotted with anti-pERK, anti-ERK, anti-p38, anti-pp38 and anti-phospho-(Ser) PKC substrate Abs. The GAPDH protein level was evaluated as a loading control. Data shown are representative of one out of three separate experiments. Densitometric analysis was performed evaluating three different experiments and statistical analysis was carried out comparing NA-treated with control cells, *p ≤ 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4306515&req=5

Fig4: NA induces PLC, PKC and ERK activation in PC3 cells. (A) The measurement of IP3 levels using the IP3 [3H] Radioreceptor assay kit was performed in PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM). Data shown are the mean ± SD of three separate experiments. Statistical analysis was performed by comparing NA-treated with control cells, *p ≤ 0.01. (B-C) Lysates from PC3 cells vehicle-treated (control) or treated for different times with NA (100 μM) were separated on SDS-polyacrylamide gels, transferred and then blotted with anti-pERK, anti-ERK, anti-p38, anti-pp38 and anti-phospho-(Ser) PKC substrate Abs. The GAPDH protein level was evaluated as a loading control. Data shown are representative of one out of three separate experiments. Densitometric analysis was performed evaluating three different experiments and statistical analysis was carried out comparing NA-treated with control cells, *p ≤ 0.01.
Mentions: Several reports have examined the role of the PLC-PKC-MAPK signalling pathways in the catecholamine-induced α1D-AR-mediated proliferation of prostate cancer cells [22, 23]. Thus, we studied the effect of α1D-AR and TRPV1 cross-talk in the NA-induced activation of PLC, PKC and ERK signalling pathways. Time-course analysis of NA-induced PLC activation, evaluated as IP3 production, evidenced that NA stimulates IP3 production 5 min after NA treatment, remaining sustained at 30 min and declining thereafter (Figure 4A). Moreover, we found that ERK1/2 is phosphorylated at basal level and NA induces a rapid and transient increase of its phosphorylation at 3 min after treatment (Figure 4B); NA also increased the (Ser)-PKC substrate phosphorylation at 3–5 min and then return to basal level (Figure 4C); p38 was basally phosphorylated, and NA stimulation did not affect its phosphorylation state (Figure 4B).Figure 4

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