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The Ron receptor tyrosine kinase positively regulates angiogenic chemokine production in prostate cancer cells.

Thobe MN, Gurusamy D, Pathrose P, Waltz SE - Oncogene (2009)

Bottom Line: The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB).Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling.Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Overexpression of the Ron receptor tyrosine kinase has recently been shown in a wide variety of human cancers. However, no studies have examined Ron receptor expression or function during prostate tumorigenesis. In this study we report that Ron is highly expressed in human prostate adenocarcinoma and metastatic lymph nodes when compared with normal prostate or benign prostate hyperplasia. Furthermore, we show that Ron is overexpressed in PC-3 and DU145 prostate cancer cell lines, and that the levels of angiogenic chemokines produced by prostate cancer cells positively correlate with Ron expression. The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling. Given that the function of angiogenic chemokines is important in the development of new blood vessels, we also examined the ability of Ron to modulate endothelial cell migration. Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo. In total, our data suggest that the Ron receptor is important in modulating prostate tumor growth by modulating angiogenic chemokine production and subsequent endothelial cell recruitment.

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DU145 and PC-3 prostate cancer cells with high Ron expression produce high amounts of angiogenic chemokinesA, B, and C, Levels of CXCL8, CXCL1, and CXCL5 in prostate cancer cells. LNCaP, 22RV1, DU145 and PC-3 cells were placed in serum free media for 72 hours and supernatant was used to determine levels of CXCL8 (Figure 3A), CXCL1 (Figure 3B), and CXCL5 (Figure 3C) by ELISA. Only DU145 and PC-3, which overexpress Ron, produce CXCL8 and CXCL1 while limited to no expression of these chemokines is observed in LNCaP and 22RV1 cells. Only PC-3 cells had detectable levels of CXCL5. Similar levels of all three chemokines were also observed at 96 hours (data not shown). D, DU145 cells grow at a greater rate than LNCaP, 22RV1, and PC-3 cells. 72 hours after plating cells, crystal violet assays were performed to determine changes in cell number over time. LNCaP, 22RV1 and PC-3 cells all had similar rates of cell growth over 72 hours, while DU145 had a higher growth rate. The relative levels of chemokines in A–C reflects a normalization to cell number for the DU145 cells (see Materials and Methods). E, F and G, RNA levels of CXCL8, CXCL5 and VEGF in prostate cancer cells. Similar to results as determined by ELISA, quantitative real-time PCR analyses confirmed that both DU145 and PC-3 cells have high levels of CXCL8 (Figure 3E), while PC-3 cells are the only cell line that had detectable levels of CXCL5 (Figure 3F). Conversely, DU145 and PC-3 have low levels of VEGF compared to LNCaP and 22RV1 cells (Figure 3G).
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Figure 3: DU145 and PC-3 prostate cancer cells with high Ron expression produce high amounts of angiogenic chemokinesA, B, and C, Levels of CXCL8, CXCL1, and CXCL5 in prostate cancer cells. LNCaP, 22RV1, DU145 and PC-3 cells were placed in serum free media for 72 hours and supernatant was used to determine levels of CXCL8 (Figure 3A), CXCL1 (Figure 3B), and CXCL5 (Figure 3C) by ELISA. Only DU145 and PC-3, which overexpress Ron, produce CXCL8 and CXCL1 while limited to no expression of these chemokines is observed in LNCaP and 22RV1 cells. Only PC-3 cells had detectable levels of CXCL5. Similar levels of all three chemokines were also observed at 96 hours (data not shown). D, DU145 cells grow at a greater rate than LNCaP, 22RV1, and PC-3 cells. 72 hours after plating cells, crystal violet assays were performed to determine changes in cell number over time. LNCaP, 22RV1 and PC-3 cells all had similar rates of cell growth over 72 hours, while DU145 had a higher growth rate. The relative levels of chemokines in A–C reflects a normalization to cell number for the DU145 cells (see Materials and Methods). E, F and G, RNA levels of CXCL8, CXCL5 and VEGF in prostate cancer cells. Similar to results as determined by ELISA, quantitative real-time PCR analyses confirmed that both DU145 and PC-3 cells have high levels of CXCL8 (Figure 3E), while PC-3 cells are the only cell line that had detectable levels of CXCL5 (Figure 3F). Conversely, DU145 and PC-3 have low levels of VEGF compared to LNCaP and 22RV1 cells (Figure 3G).

Mentions: Cells were plated in a 24-well tissue culture dish in complete media. After reaching ~80% confluency, serum-free media was added (Defined Keratinocyte Media, Gibco, Carlsbad, CA). Supernatants were collected over time and chemokine levels were determined by Enzyme-Linked ImmunoSorbent Assays (ELISAs) according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Crystal violet assays were performed by fixing cells in 10% neutral buffered formalin for 15 minutes, and then incubating cells with 0.1% crystal violet in 25% methanol for 30 minutes. The cells were washed, air-dried, and 500µl of DMSO was added. After shaking 30 minutes, the absorbance was read at 570nm. Relative cell number was plotted by calculating the fold change in cell growth from 0 hours to 72 hours and normalizing the values to one cell line set at 1.0. The relative level of chemokine production between cell lines was calculated with the amount of chemokines produced normalized to the relative cell number observed during the time course of the experiment (Figure 3A and B). LNCaP, 22RV1 and PC-3 cells all grew to similar extents and the chemokine values were not adjusted. However, DU145 cells grew significantly faster during the experimental observation period (Figure 3D) and the relative chemokine values for these cells were decreased accordingly by the change in cell number.


The Ron receptor tyrosine kinase positively regulates angiogenic chemokine production in prostate cancer cells.

Thobe MN, Gurusamy D, Pathrose P, Waltz SE - Oncogene (2009)

DU145 and PC-3 prostate cancer cells with high Ron expression produce high amounts of angiogenic chemokinesA, B, and C, Levels of CXCL8, CXCL1, and CXCL5 in prostate cancer cells. LNCaP, 22RV1, DU145 and PC-3 cells were placed in serum free media for 72 hours and supernatant was used to determine levels of CXCL8 (Figure 3A), CXCL1 (Figure 3B), and CXCL5 (Figure 3C) by ELISA. Only DU145 and PC-3, which overexpress Ron, produce CXCL8 and CXCL1 while limited to no expression of these chemokines is observed in LNCaP and 22RV1 cells. Only PC-3 cells had detectable levels of CXCL5. Similar levels of all three chemokines were also observed at 96 hours (data not shown). D, DU145 cells grow at a greater rate than LNCaP, 22RV1, and PC-3 cells. 72 hours after plating cells, crystal violet assays were performed to determine changes in cell number over time. LNCaP, 22RV1 and PC-3 cells all had similar rates of cell growth over 72 hours, while DU145 had a higher growth rate. The relative levels of chemokines in A–C reflects a normalization to cell number for the DU145 cells (see Materials and Methods). E, F and G, RNA levels of CXCL8, CXCL5 and VEGF in prostate cancer cells. Similar to results as determined by ELISA, quantitative real-time PCR analyses confirmed that both DU145 and PC-3 cells have high levels of CXCL8 (Figure 3E), while PC-3 cells are the only cell line that had detectable levels of CXCL5 (Figure 3F). Conversely, DU145 and PC-3 have low levels of VEGF compared to LNCaP and 22RV1 cells (Figure 3G).
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Figure 3: DU145 and PC-3 prostate cancer cells with high Ron expression produce high amounts of angiogenic chemokinesA, B, and C, Levels of CXCL8, CXCL1, and CXCL5 in prostate cancer cells. LNCaP, 22RV1, DU145 and PC-3 cells were placed in serum free media for 72 hours and supernatant was used to determine levels of CXCL8 (Figure 3A), CXCL1 (Figure 3B), and CXCL5 (Figure 3C) by ELISA. Only DU145 and PC-3, which overexpress Ron, produce CXCL8 and CXCL1 while limited to no expression of these chemokines is observed in LNCaP and 22RV1 cells. Only PC-3 cells had detectable levels of CXCL5. Similar levels of all three chemokines were also observed at 96 hours (data not shown). D, DU145 cells grow at a greater rate than LNCaP, 22RV1, and PC-3 cells. 72 hours after plating cells, crystal violet assays were performed to determine changes in cell number over time. LNCaP, 22RV1 and PC-3 cells all had similar rates of cell growth over 72 hours, while DU145 had a higher growth rate. The relative levels of chemokines in A–C reflects a normalization to cell number for the DU145 cells (see Materials and Methods). E, F and G, RNA levels of CXCL8, CXCL5 and VEGF in prostate cancer cells. Similar to results as determined by ELISA, quantitative real-time PCR analyses confirmed that both DU145 and PC-3 cells have high levels of CXCL8 (Figure 3E), while PC-3 cells are the only cell line that had detectable levels of CXCL5 (Figure 3F). Conversely, DU145 and PC-3 have low levels of VEGF compared to LNCaP and 22RV1 cells (Figure 3G).
Mentions: Cells were plated in a 24-well tissue culture dish in complete media. After reaching ~80% confluency, serum-free media was added (Defined Keratinocyte Media, Gibco, Carlsbad, CA). Supernatants were collected over time and chemokine levels were determined by Enzyme-Linked ImmunoSorbent Assays (ELISAs) according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Crystal violet assays were performed by fixing cells in 10% neutral buffered formalin for 15 minutes, and then incubating cells with 0.1% crystal violet in 25% methanol for 30 minutes. The cells were washed, air-dried, and 500µl of DMSO was added. After shaking 30 minutes, the absorbance was read at 570nm. Relative cell number was plotted by calculating the fold change in cell growth from 0 hours to 72 hours and normalizing the values to one cell line set at 1.0. The relative level of chemokine production between cell lines was calculated with the amount of chemokines produced normalized to the relative cell number observed during the time course of the experiment (Figure 3A and B). LNCaP, 22RV1 and PC-3 cells all grew to similar extents and the chemokine values were not adjusted. However, DU145 cells grew significantly faster during the experimental observation period (Figure 3D) and the relative chemokine values for these cells were decreased accordingly by the change in cell number.

Bottom Line: The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB).Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling.Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

ABSTRACT
Overexpression of the Ron receptor tyrosine kinase has recently been shown in a wide variety of human cancers. However, no studies have examined Ron receptor expression or function during prostate tumorigenesis. In this study we report that Ron is highly expressed in human prostate adenocarcinoma and metastatic lymph nodes when compared with normal prostate or benign prostate hyperplasia. Furthermore, we show that Ron is overexpressed in PC-3 and DU145 prostate cancer cell lines, and that the levels of angiogenic chemokines produced by prostate cancer cells positively correlate with Ron expression. The knockdown of Ron in PC-3 or DU145 cells results in a significant decrease in angiogenic chemokine production and is associated with a decreased activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Moreover, exogenous overexpression of Ron in LNCaP cells is sufficient to induce a significant increase in angiogenic chemokines that can be abrogated by inhibition of NF-kappaB signaling. Given that the function of angiogenic chemokines is important in the development of new blood vessels, we also examined the ability of Ron to modulate endothelial cell migration. Our data show that knockdown of Ron in prostate cancer cells results in significantly less endothelial cell chemotaxis when compared with Ron-expressing cells in vitro as well as in reduced tumor growth and decreased microvessel density after orthotopic transplantation into the prostate in vivo. In total, our data suggest that the Ron receptor is important in modulating prostate tumor growth by modulating angiogenic chemokine production and subsequent endothelial cell recruitment.

Show MeSH
Related in: MedlinePlus