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Midkine is a NF-kappaB-inducible gene that supports prostate cancer cell survival.

You Z, Dong Y, Kong X, Beckett LA, Gandour-Edwards R, Melamed J - BMC Med Genomics (2008)

Bottom Line: Midkine activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in LNCaP cells.Furthermore, midkine expression was significantly increased in late stage prostate cancer, which coincides with previously reported high serum levels of TNFalpha in advanced prostate cancer.These findings provide the first demonstration that midkine expression is induced by certain growth factors and cytokines, particularly TNFalpha, which offers new insight into understanding how midkine expression is increased in the late stage prostate cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, University of California, Davis, Sacramento, CA 95817, USA. you.zongbing@gmail.com

ABSTRACT

Background: Midkine is a heparin-binding growth factor that is over-expressed in various human cancers and plays important roles in cell transformation, growth, survival, migration, and angiogenesis. However, little is known about the upstream factors and signaling mechanisms that regulate midkine gene expression.

Methods: Two prostate cancer cell lines LNCaP and PC3 were studied for their expression of midkine. Induction of midkine expression in LNCaP cells by serum, growth factors and cytokines was determined by Western blot analysis and/or real-time quantitative reverse-transcription - polymerase chain reaction (RT-PCR). The cell viability was determined by the trypan blue exclusion assay when the LNCaP cells were treated with tumor necrosis factor alpha (TNFalpha) and/or recombinant midkine. When the LNCaP cells were treated with recombinant midkine, activation of intracellular signalling pathways was determined by Western blot analysis. Prostate tissue microarray slides containing 129 cases (18 normal prostate tissues, 40 early stage cancers, and 71 late stage cancers) were assessed for midkine expression by immunohistochemical staining.

Results: We identified that fetal bovine serum, some growth factors (epidermal growth factor, androgen, insulin-like growth factor-I, and hepatocyte growth factor) and cytokines (TNFalpha and interleukin-1beta) induced midkine expression in a human prostate cancer cell line LNCaP cells. TNFalpha also induced midkine expression in PC3 cells. TNFalpha was the strongest inducer of midkine expression via nuclear factor-kappa B pathway. Midkine partially inhibited TNFalpha-induced apoptosis in LNCaP cells. Knockdown of endogenous midkine expression by small interfering RNA enhanced TNFalpha-induced apoptosis in LNCaP cells. Midkine activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in LNCaP cells. Furthermore, midkine expression was significantly increased in late stage prostate cancer, which coincides with previously reported high serum levels of TNFalpha in advanced prostate cancer.

Conclusion: These findings provide the first demonstration that midkine expression is induced by certain growth factors and cytokines, particularly TNFalpha, which offers new insight into understanding how midkine expression is increased in the late stage prostate cancer.

No MeSH data available.


Related in: MedlinePlus

Midkine partially inhibited TNFα-induced apoptosis in LNCaP cells. A. LNCaP cells in triplicate groups were not treated (as control) or treated for 4 days with 20 ng/ml TNFα, without or with 0.1 or 1 μg/ml exogenous midkine; the living cell number was counted by the trypan blue exclusion assay; the cell survival was calculated as (the living cell number of treated group ÷ the living cell number of untreated control group); the data were presented as mean ± standard deviation; *P < 0.05 compared to TNFα alone. B. LNCaP cells were transfected with the mixtures of midkine specific siRNA/Lipofectamine™ 2000, control-siRNA/Lipofectamine™ 2000, or Lipofectamine™ 2000 only (mock transfection), or no transfection as an additional control; the final concentrations used were 100 nM of siRNA or control-siRNA, and 5 μl/ml of Lipofectamine™ 2000; four h after transfection, the cells were changed into serum-free DMEM without or with 20 ng/ml TNFα; two days later, the medium supernatants were analyzed for midkine expression by Western blot. C. LNCaP cells in triplicate groups were treated as described in B; the cell survival after 2-days' treatment with 20 ng/ml TNFα was determined by the trypan blue exclusion assay as described in A; the data were presented as mean ± standard deviation; *P < 0.05 compared to the other three groups. D. LNCaP cells were treated as described in B; 16 h after treatment with 20 ng/ml TNFα, 20 μM MR-(DEVD)2 were added to the cells and incubated for 1 h, followed by addition of 1 μg/ml Hoechst 33342 for another 15 min; the red fluorescence [emitted by the cleaved MR-(DEVD)2 indicating activation of caspase-3] and blue fluorescent nuclei (stained by Hoechst 33342 to illustrate total cell number) were captured by a fluorescent microscope; original magnification: × 100.
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Figure 1: Midkine partially inhibited TNFα-induced apoptosis in LNCaP cells. A. LNCaP cells in triplicate groups were not treated (as control) or treated for 4 days with 20 ng/ml TNFα, without or with 0.1 or 1 μg/ml exogenous midkine; the living cell number was counted by the trypan blue exclusion assay; the cell survival was calculated as (the living cell number of treated group ÷ the living cell number of untreated control group); the data were presented as mean ± standard deviation; *P < 0.05 compared to TNFα alone. B. LNCaP cells were transfected with the mixtures of midkine specific siRNA/Lipofectamine™ 2000, control-siRNA/Lipofectamine™ 2000, or Lipofectamine™ 2000 only (mock transfection), or no transfection as an additional control; the final concentrations used were 100 nM of siRNA or control-siRNA, and 5 μl/ml of Lipofectamine™ 2000; four h after transfection, the cells were changed into serum-free DMEM without or with 20 ng/ml TNFα; two days later, the medium supernatants were analyzed for midkine expression by Western blot. C. LNCaP cells in triplicate groups were treated as described in B; the cell survival after 2-days' treatment with 20 ng/ml TNFα was determined by the trypan blue exclusion assay as described in A; the data were presented as mean ± standard deviation; *P < 0.05 compared to the other three groups. D. LNCaP cells were treated as described in B; 16 h after treatment with 20 ng/ml TNFα, 20 μM MR-(DEVD)2 were added to the cells and incubated for 1 h, followed by addition of 1 μg/ml Hoechst 33342 for another 15 min; the red fluorescence [emitted by the cleaved MR-(DEVD)2 indicating activation of caspase-3] and blue fluorescent nuclei (stained by Hoechst 33342 to illustrate total cell number) were captured by a fluorescent microscope; original magnification: × 100.

Mentions: 20,000 LNCaP cells in one ml 5% FBS T-medium per well in triplicate groups were plated in 12-well plates and 16 h later changed into serum-free DMEM with or without 20 ng/ml TNFα. To test the effect of exogenous MDK on cell survival, 0.1 or 1 μg/ml of recombinant human MDK (PeproTech, Inc., Rocky Hill, NJ) were added 30 min before adding TNFα. Two or four days after treatment, cell viability was determined by the trypan blue exclusion assay, in which cell survival was calculated as (the living cell number of treated group ÷ the living cell number of untreated control group) × 100. Based on our previous experience [17], we chose to determine the cell survival after four-day treatment to better evaluate the protective effects of MDK (Figure 1A), while after two-day treatment to show the combined killing effects of siRNA with TNFα (Figure 1C).


Midkine is a NF-kappaB-inducible gene that supports prostate cancer cell survival.

You Z, Dong Y, Kong X, Beckett LA, Gandour-Edwards R, Melamed J - BMC Med Genomics (2008)

Midkine partially inhibited TNFα-induced apoptosis in LNCaP cells. A. LNCaP cells in triplicate groups were not treated (as control) or treated for 4 days with 20 ng/ml TNFα, without or with 0.1 or 1 μg/ml exogenous midkine; the living cell number was counted by the trypan blue exclusion assay; the cell survival was calculated as (the living cell number of treated group ÷ the living cell number of untreated control group); the data were presented as mean ± standard deviation; *P < 0.05 compared to TNFα alone. B. LNCaP cells were transfected with the mixtures of midkine specific siRNA/Lipofectamine™ 2000, control-siRNA/Lipofectamine™ 2000, or Lipofectamine™ 2000 only (mock transfection), or no transfection as an additional control; the final concentrations used were 100 nM of siRNA or control-siRNA, and 5 μl/ml of Lipofectamine™ 2000; four h after transfection, the cells were changed into serum-free DMEM without or with 20 ng/ml TNFα; two days later, the medium supernatants were analyzed for midkine expression by Western blot. C. LNCaP cells in triplicate groups were treated as described in B; the cell survival after 2-days' treatment with 20 ng/ml TNFα was determined by the trypan blue exclusion assay as described in A; the data were presented as mean ± standard deviation; *P < 0.05 compared to the other three groups. D. LNCaP cells were treated as described in B; 16 h after treatment with 20 ng/ml TNFα, 20 μM MR-(DEVD)2 were added to the cells and incubated for 1 h, followed by addition of 1 μg/ml Hoechst 33342 for another 15 min; the red fluorescence [emitted by the cleaved MR-(DEVD)2 indicating activation of caspase-3] and blue fluorescent nuclei (stained by Hoechst 33342 to illustrate total cell number) were captured by a fluorescent microscope; original magnification: × 100.
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Figure 1: Midkine partially inhibited TNFα-induced apoptosis in LNCaP cells. A. LNCaP cells in triplicate groups were not treated (as control) or treated for 4 days with 20 ng/ml TNFα, without or with 0.1 or 1 μg/ml exogenous midkine; the living cell number was counted by the trypan blue exclusion assay; the cell survival was calculated as (the living cell number of treated group ÷ the living cell number of untreated control group); the data were presented as mean ± standard deviation; *P < 0.05 compared to TNFα alone. B. LNCaP cells were transfected with the mixtures of midkine specific siRNA/Lipofectamine™ 2000, control-siRNA/Lipofectamine™ 2000, or Lipofectamine™ 2000 only (mock transfection), or no transfection as an additional control; the final concentrations used were 100 nM of siRNA or control-siRNA, and 5 μl/ml of Lipofectamine™ 2000; four h after transfection, the cells were changed into serum-free DMEM without or with 20 ng/ml TNFα; two days later, the medium supernatants were analyzed for midkine expression by Western blot. C. LNCaP cells in triplicate groups were treated as described in B; the cell survival after 2-days' treatment with 20 ng/ml TNFα was determined by the trypan blue exclusion assay as described in A; the data were presented as mean ± standard deviation; *P < 0.05 compared to the other three groups. D. LNCaP cells were treated as described in B; 16 h after treatment with 20 ng/ml TNFα, 20 μM MR-(DEVD)2 were added to the cells and incubated for 1 h, followed by addition of 1 μg/ml Hoechst 33342 for another 15 min; the red fluorescence [emitted by the cleaved MR-(DEVD)2 indicating activation of caspase-3] and blue fluorescent nuclei (stained by Hoechst 33342 to illustrate total cell number) were captured by a fluorescent microscope; original magnification: × 100.
Mentions: 20,000 LNCaP cells in one ml 5% FBS T-medium per well in triplicate groups were plated in 12-well plates and 16 h later changed into serum-free DMEM with or without 20 ng/ml TNFα. To test the effect of exogenous MDK on cell survival, 0.1 or 1 μg/ml of recombinant human MDK (PeproTech, Inc., Rocky Hill, NJ) were added 30 min before adding TNFα. Two or four days after treatment, cell viability was determined by the trypan blue exclusion assay, in which cell survival was calculated as (the living cell number of treated group ÷ the living cell number of untreated control group) × 100. Based on our previous experience [17], we chose to determine the cell survival after four-day treatment to better evaluate the protective effects of MDK (Figure 1A), while after two-day treatment to show the combined killing effects of siRNA with TNFα (Figure 1C).

Bottom Line: Midkine activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in LNCaP cells.Furthermore, midkine expression was significantly increased in late stage prostate cancer, which coincides with previously reported high serum levels of TNFalpha in advanced prostate cancer.These findings provide the first demonstration that midkine expression is induced by certain growth factors and cytokines, particularly TNFalpha, which offers new insight into understanding how midkine expression is increased in the late stage prostate cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, University of California, Davis, Sacramento, CA 95817, USA. you.zongbing@gmail.com

ABSTRACT

Background: Midkine is a heparin-binding growth factor that is over-expressed in various human cancers and plays important roles in cell transformation, growth, survival, migration, and angiogenesis. However, little is known about the upstream factors and signaling mechanisms that regulate midkine gene expression.

Methods: Two prostate cancer cell lines LNCaP and PC3 were studied for their expression of midkine. Induction of midkine expression in LNCaP cells by serum, growth factors and cytokines was determined by Western blot analysis and/or real-time quantitative reverse-transcription - polymerase chain reaction (RT-PCR). The cell viability was determined by the trypan blue exclusion assay when the LNCaP cells were treated with tumor necrosis factor alpha (TNFalpha) and/or recombinant midkine. When the LNCaP cells were treated with recombinant midkine, activation of intracellular signalling pathways was determined by Western blot analysis. Prostate tissue microarray slides containing 129 cases (18 normal prostate tissues, 40 early stage cancers, and 71 late stage cancers) were assessed for midkine expression by immunohistochemical staining.

Results: We identified that fetal bovine serum, some growth factors (epidermal growth factor, androgen, insulin-like growth factor-I, and hepatocyte growth factor) and cytokines (TNFalpha and interleukin-1beta) induced midkine expression in a human prostate cancer cell line LNCaP cells. TNFalpha also induced midkine expression in PC3 cells. TNFalpha was the strongest inducer of midkine expression via nuclear factor-kappa B pathway. Midkine partially inhibited TNFalpha-induced apoptosis in LNCaP cells. Knockdown of endogenous midkine expression by small interfering RNA enhanced TNFalpha-induced apoptosis in LNCaP cells. Midkine activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in LNCaP cells. Furthermore, midkine expression was significantly increased in late stage prostate cancer, which coincides with previously reported high serum levels of TNFalpha in advanced prostate cancer.

Conclusion: These findings provide the first demonstration that midkine expression is induced by certain growth factors and cytokines, particularly TNFalpha, which offers new insight into understanding how midkine expression is increased in the late stage prostate cancer.

No MeSH data available.


Related in: MedlinePlus