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Constitutive and Inducible Expression of Invasion-related Factors in PC-3 Prostate Cancer Cells.

Hwang YS, Lindholm PF - J Cancer Prev (2015)

Bottom Line: Thrombospondin-1, interleukin-8, kallikrein 6, matrix metalloproteinase-1, and tissue factor were overexpressed in the highly invasive PC-3 variant cells and further upregulated by LPA stimulation.The results suggest that the target molecules are involved in invasiveness of prostate cancer.These molecules may have clinical value for anti-invasion therapy by serving as biomarkers for the prediction of aggressive cancers and the detection of pharmacological inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Hygiene, College of Health Science, Eulji University, Seongnam, Korea.

ABSTRACT

Background: Tumor growth and invasion are interconnected with the tumor microenvironment. Overexpression of genes that regulate cancer cell invasion by growth factors, cytokines, and lipid factors can affect cancer aggressiveness. A comparative gene expression analysis between highly invasive and low invasive cells revealed that various genes are differentially expressed in association with invasive potential. In this study, we selected variant PC-3 prostate cancer cell sublines and discovered critical molecules that contributed to their invasive potential.

Methods: The high invasive and low invasive variant PC-3 cell sublines were obtained by serial selection following Matrigel-coated Transwell invasion and were characterized by Transwell invasion, luciferase reporter assay, and Rhotekin pull-down assay. Lysophosphatidic acid (LPA) was added to the cultures to observe the response to this extracellular stimulus. The essential molecules related with cancer invasiveness were detected with Northern blotting, quantitative reverse transcription-polymerase chain reaction, and cDNA microarray.

Results: Highly invasive PC-3 cells showed higher nuclear factor kappa B (NF-κB), activator protein 1 (AP-1) and RhoA activities than of low invasive PC-3 cells. LPA promoted cancer invasion through NF-κB, AP-1, and RhoA activities. Thrombospondin-1, interleukin-8, kallikrein 6, matrix metalloproteinase-1, and tissue factor were overexpressed in the highly invasive PC-3 variant cells and further upregulated by LPA stimulation.

Conclusions: The results suggest that the target molecules are involved in invasiveness of prostate cancer. These molecules may have clinical value for anti-invasion therapy by serving as biomarkers for the prediction of aggressive cancers and the detection of pharmacological inhibitors.

No MeSH data available.


Related in: MedlinePlus

High nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities in highly invasive PC-3 cells. (A) In vitro invasion through Matrigel-coated Transwells by the variant PC-3 cell sublines in serum. Data are mean ± standard error of the mean (SEM) of five independent experiments (aP < 0.001 vs. PC-3 Low). (B) Prostate cancer cell NF-κB and AP-1 luciferase reporter activity showing increased NF-κB and AP-1 activity in PC-3 High invasive prostate cancer cells. Data are mean ± SEM of five independent experiments (aP < 0.05 vs. PC-3 Low in the same group). (C) NF-κB and AP-1 DNA binding activities in the high and low invasive PC-3 cells were determined by the electrophoretic motility shift assay. NF-κB and AP-1 DNA binding complexes are designated by arrows. ns, non-specific band. (D) RhoA activities in the variant PC-3 cell sublines were determined using the Rhotekin pull-down assay.
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f1-jcp-20-121: High nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities in highly invasive PC-3 cells. (A) In vitro invasion through Matrigel-coated Transwells by the variant PC-3 cell sublines in serum. Data are mean ± standard error of the mean (SEM) of five independent experiments (aP < 0.001 vs. PC-3 Low). (B) Prostate cancer cell NF-κB and AP-1 luciferase reporter activity showing increased NF-κB and AP-1 activity in PC-3 High invasive prostate cancer cells. Data are mean ± SEM of five independent experiments (aP < 0.05 vs. PC-3 Low in the same group). (C) NF-κB and AP-1 DNA binding activities in the high and low invasive PC-3 cells were determined by the electrophoretic motility shift assay. NF-κB and AP-1 DNA binding complexes are designated by arrows. ns, non-specific band. (D) RhoA activities in the variant PC-3 cell sublines were determined using the Rhotekin pull-down assay.

Mentions: The highly invasive PC-3 cells showed relatively higher Transwell invasion activity than that of low invasive PC-3 cells (Fig. 1A). Activation of NF-κB and AP-1 was higher in the highly invasive PC-3 High cells on the luciferase reporter assay than that in the low invasive PC-3 Low cells (Fig. 1B). The EMSA showed similar results (Fig. 1C). RhoGTPases are overexpressed in several aggressive cancers12 and RhoA stimulates expression of NF-κB-regulated, inflammation modifying genes, including TF, MMP-9, and urokinase type plasminogen activator, which are related to cancer invasion and metastasis. The Rhotekin pull-down assay was performed to determine if RhoA was also activated in the highly invasive PC-3 cells. As shown Figure 1D, significantly increased RhoA activity was observed in the highly invasive PC-3 High cells.


Constitutive and Inducible Expression of Invasion-related Factors in PC-3 Prostate Cancer Cells.

Hwang YS, Lindholm PF - J Cancer Prev (2015)

High nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities in highly invasive PC-3 cells. (A) In vitro invasion through Matrigel-coated Transwells by the variant PC-3 cell sublines in serum. Data are mean ± standard error of the mean (SEM) of five independent experiments (aP < 0.001 vs. PC-3 Low). (B) Prostate cancer cell NF-κB and AP-1 luciferase reporter activity showing increased NF-κB and AP-1 activity in PC-3 High invasive prostate cancer cells. Data are mean ± SEM of five independent experiments (aP < 0.05 vs. PC-3 Low in the same group). (C) NF-κB and AP-1 DNA binding activities in the high and low invasive PC-3 cells were determined by the electrophoretic motility shift assay. NF-κB and AP-1 DNA binding complexes are designated by arrows. ns, non-specific band. (D) RhoA activities in the variant PC-3 cell sublines were determined using the Rhotekin pull-down assay.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492356&req=5

f1-jcp-20-121: High nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities in highly invasive PC-3 cells. (A) In vitro invasion through Matrigel-coated Transwells by the variant PC-3 cell sublines in serum. Data are mean ± standard error of the mean (SEM) of five independent experiments (aP < 0.001 vs. PC-3 Low). (B) Prostate cancer cell NF-κB and AP-1 luciferase reporter activity showing increased NF-κB and AP-1 activity in PC-3 High invasive prostate cancer cells. Data are mean ± SEM of five independent experiments (aP < 0.05 vs. PC-3 Low in the same group). (C) NF-κB and AP-1 DNA binding activities in the high and low invasive PC-3 cells were determined by the electrophoretic motility shift assay. NF-κB and AP-1 DNA binding complexes are designated by arrows. ns, non-specific band. (D) RhoA activities in the variant PC-3 cell sublines were determined using the Rhotekin pull-down assay.
Mentions: The highly invasive PC-3 cells showed relatively higher Transwell invasion activity than that of low invasive PC-3 cells (Fig. 1A). Activation of NF-κB and AP-1 was higher in the highly invasive PC-3 High cells on the luciferase reporter assay than that in the low invasive PC-3 Low cells (Fig. 1B). The EMSA showed similar results (Fig. 1C). RhoGTPases are overexpressed in several aggressive cancers12 and RhoA stimulates expression of NF-κB-regulated, inflammation modifying genes, including TF, MMP-9, and urokinase type plasminogen activator, which are related to cancer invasion and metastasis. The Rhotekin pull-down assay was performed to determine if RhoA was also activated in the highly invasive PC-3 cells. As shown Figure 1D, significantly increased RhoA activity was observed in the highly invasive PC-3 High cells.

Bottom Line: Thrombospondin-1, interleukin-8, kallikrein 6, matrix metalloproteinase-1, and tissue factor were overexpressed in the highly invasive PC-3 variant cells and further upregulated by LPA stimulation.The results suggest that the target molecules are involved in invasiveness of prostate cancer.These molecules may have clinical value for anti-invasion therapy by serving as biomarkers for the prediction of aggressive cancers and the detection of pharmacological inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Dental Hygiene, College of Health Science, Eulji University, Seongnam, Korea.

ABSTRACT

Background: Tumor growth and invasion are interconnected with the tumor microenvironment. Overexpression of genes that regulate cancer cell invasion by growth factors, cytokines, and lipid factors can affect cancer aggressiveness. A comparative gene expression analysis between highly invasive and low invasive cells revealed that various genes are differentially expressed in association with invasive potential. In this study, we selected variant PC-3 prostate cancer cell sublines and discovered critical molecules that contributed to their invasive potential.

Methods: The high invasive and low invasive variant PC-3 cell sublines were obtained by serial selection following Matrigel-coated Transwell invasion and were characterized by Transwell invasion, luciferase reporter assay, and Rhotekin pull-down assay. Lysophosphatidic acid (LPA) was added to the cultures to observe the response to this extracellular stimulus. The essential molecules related with cancer invasiveness were detected with Northern blotting, quantitative reverse transcription-polymerase chain reaction, and cDNA microarray.

Results: Highly invasive PC-3 cells showed higher nuclear factor kappa B (NF-κB), activator protein 1 (AP-1) and RhoA activities than of low invasive PC-3 cells. LPA promoted cancer invasion through NF-κB, AP-1, and RhoA activities. Thrombospondin-1, interleukin-8, kallikrein 6, matrix metalloproteinase-1, and tissue factor were overexpressed in the highly invasive PC-3 variant cells and further upregulated by LPA stimulation.

Conclusions: The results suggest that the target molecules are involved in invasiveness of prostate cancer. These molecules may have clinical value for anti-invasion therapy by serving as biomarkers for the prediction of aggressive cancers and the detection of pharmacological inhibitors.

No MeSH data available.


Related in: MedlinePlus