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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

Lysophosphatidic acid (LPA) stimulates cancer invasion and nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities. (A) In vitro invasion assay was performed with the variant PC-3 cell sublines (PC-3 Low and High), DU145 (prostate cancer cells), MDA-MB-231 (breast cancer cells), and BT549 (breast cancer cells) under serum-free conditions. LPA (1 μM) was added to the lower Transwell chambers. Data are mean ± standard error of the mean of three independent experiments (aP < 0.001, bP < 0.05 vs. without LPA control). (B) NF-κB and AP-1 DNA binding activities in LPA-treated PC-3 cells under serum-free conditions. ns, non-specific band. Results are representative of three independent experiments. (C) RhoA activity of LPA-stimulated PC-3 cells. A western blot was performed after an LPA time course to detect maximum RhoA activity through RhoA binding to glutathione S-transferase-Rhotekin beads.
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f2-jcp-20-121: Lysophosphatidic acid (LPA) stimulates cancer invasion and nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities. (A) In vitro invasion assay was performed with the variant PC-3 cell sublines (PC-3 Low and High), DU145 (prostate cancer cells), MDA-MB-231 (breast cancer cells), and BT549 (breast cancer cells) under serum-free conditions. LPA (1 μM) was added to the lower Transwell chambers. Data are mean ± standard error of the mean of three independent experiments (aP < 0.001, bP < 0.05 vs. without LPA control). (B) NF-κB and AP-1 DNA binding activities in LPA-treated PC-3 cells under serum-free conditions. ns, non-specific band. Results are representative of three independent experiments. (C) RhoA activity of LPA-stimulated PC-3 cells. A western blot was performed after an LPA time course to detect maximum RhoA activity through RhoA binding to glutathione S-transferase-Rhotekin beads.

Mentions: Reciprocal crosstalk between tumor cells and their adjacent microenvironment is crucial during tumor growth and metastasis.2 The high and low invasive PC-3 cells were stimulated with LPA, a bioactive phospholipid present at high levels in ascites fluid and plasma of patients with cancer. LPA has been shown to stimulate cancer cell invasiveness through increasing RhoA GTPase activity.6–8 Under serum-free conditions, LPA significantly increased Transwell invasion of both the high and low invasive PC-3 cells (Fig. 2A). The LPA-stimulated increases invasion was also observed in other cancer cell lines, although there were differences in the response to LPA. NF-κB and AP-1 activities also increased in response to LPA under serum-free conditions (Fig. 2B). LPA stimulated NF-κB DNA binding activity to the maximum level at 1 hour and increased AP-1 DNA binding activity at 4 hours. LPA rapidly induced RhoA activity, and maximum Rhotekin-bound RhoA levels occurred within 4–6 min and returned to baseline by 10 minutes (Fig. 2C).


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

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

Lysophosphatidic acid (LPA) stimulates cancer invasion and nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities. (A) In vitro invasion assay was performed with the variant PC-3 cell sublines (PC-3 Low and High), DU145 (prostate cancer cells), MDA-MB-231 (breast cancer cells), and BT549 (breast cancer cells) under serum-free conditions. LPA (1 μM) was added to the lower Transwell chambers. Data are mean ± standard error of the mean of three independent experiments (aP < 0.001, bP < 0.05 vs. without LPA control). (B) NF-κB and AP-1 DNA binding activities in LPA-treated PC-3 cells under serum-free conditions. ns, non-specific band. Results are representative of three independent experiments. (C) RhoA activity of LPA-stimulated PC-3 cells. A western blot was performed after an LPA time course to detect maximum RhoA activity through RhoA binding to glutathione S-transferase-Rhotekin beads.
© Copyright Policy
Related In: Results  -  Collection

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

f2-jcp-20-121: Lysophosphatidic acid (LPA) stimulates cancer invasion and nuclear factor kappa B (NF-κB), activator protein-1 (AP-1), and RhoA activities. (A) In vitro invasion assay was performed with the variant PC-3 cell sublines (PC-3 Low and High), DU145 (prostate cancer cells), MDA-MB-231 (breast cancer cells), and BT549 (breast cancer cells) under serum-free conditions. LPA (1 μM) was added to the lower Transwell chambers. Data are mean ± standard error of the mean of three independent experiments (aP < 0.001, bP < 0.05 vs. without LPA control). (B) NF-κB and AP-1 DNA binding activities in LPA-treated PC-3 cells under serum-free conditions. ns, non-specific band. Results are representative of three independent experiments. (C) RhoA activity of LPA-stimulated PC-3 cells. A western blot was performed after an LPA time course to detect maximum RhoA activity through RhoA binding to glutathione S-transferase-Rhotekin beads.
Mentions: Reciprocal crosstalk between tumor cells and their adjacent microenvironment is crucial during tumor growth and metastasis.2 The high and low invasive PC-3 cells were stimulated with LPA, a bioactive phospholipid present at high levels in ascites fluid and plasma of patients with cancer. LPA has been shown to stimulate cancer cell invasiveness through increasing RhoA GTPase activity.6–8 Under serum-free conditions, LPA significantly increased Transwell invasion of both the high and low invasive PC-3 cells (Fig. 2A). The LPA-stimulated increases invasion was also observed in other cancer cell lines, although there were differences in the response to LPA. NF-κB and AP-1 activities also increased in response to LPA under serum-free conditions (Fig. 2B). LPA stimulated NF-κB DNA binding activity to the maximum level at 1 hour and increased AP-1 DNA binding activity at 4 hours. LPA rapidly induced RhoA activity, and maximum Rhotekin-bound RhoA levels occurred within 4–6 min and returned to baseline by 10 minutes (Fig. 2C).

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