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EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells.

Cho MC, Cho SY, Yoon CY, Lee SB, Kwak C, Kim HH, Jeong H - PLoS ONE (2015)

Bottom Line: Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines.For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control.RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Dongguk University College of Medicine, Seoul, Korea.

ABSTRACT

Objectives: To investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells.

Methods: Expression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively.

Results: In all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.

Conclusions: Our data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.

No MeSH data available.


Related in: MedlinePlus

Effect of EphA2 siRNA on cellular invasiveness in 3-dimensional (3-D) cell culture models of human RCC cell lines.3-D cell invasion was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells in each cell line. Representative images taken over a four-day period (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2). (C) Bar graph showing relative invasion in each cell line. Relative invasion was defined as a ratio: (the area obtained on day 4)/ (the area obtained on day 0). The results were presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma
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pone.0130975.g006: Effect of EphA2 siRNA on cellular invasiveness in 3-dimensional (3-D) cell culture models of human RCC cell lines.3-D cell invasion was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells in each cell line. Representative images taken over a four-day period (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2). (C) Bar graph showing relative invasion in each cell line. Relative invasion was defined as a ratio: (the area obtained on day 4)/ (the area obtained on day 0). The results were presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma

Mentions: The results of 3D culture cell invasion assays were nearly identical to the corresponding results obtained with the modified Matrigel-Boyden chamber assays. EphA2 siRNA treatment suppressed the cellular invasion in the non-metastatic RCC cell lines (Caki-2 and A498) (Fig 6B and 6C), but not in the metastatic RCC cell lines (Caki-1 or ACHN) (Fig 6A and 6C).


EphA2 Is a Potential Player of Malignant Cellular Behavior in Non-Metastatic Renal Cell Carcinoma Cells but Not in Metastatic Renal Cell Carcinoma Cells.

Cho MC, Cho SY, Yoon CY, Lee SB, Kwak C, Kim HH, Jeong H - PLoS ONE (2015)

Effect of EphA2 siRNA on cellular invasiveness in 3-dimensional (3-D) cell culture models of human RCC cell lines.3-D cell invasion was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells in each cell line. Representative images taken over a four-day period (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2). (C) Bar graph showing relative invasion in each cell line. Relative invasion was defined as a ratio: (the area obtained on day 4)/ (the area obtained on day 0). The results were presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4503437&req=5

pone.0130975.g006: Effect of EphA2 siRNA on cellular invasiveness in 3-dimensional (3-D) cell culture models of human RCC cell lines.3-D cell invasion was compared among untreated cells (control), control siRNA treated cells and EphA2 siRNA treated cells in each cell line. Representative images taken over a four-day period (A) metastatic RCC cell lines (ACHN and Caki-1) and (B) non-metastatic RCC cell lines (A498 and Caki-2). (C) Bar graph showing relative invasion in each cell line. Relative invasion was defined as a ratio: (the area obtained on day 4)/ (the area obtained on day 0). The results were presented as fold changes over controls. * p < 0.05 vs. control and control siRNA treated groups. siRNA = small interfering RNA, RCC = renal cell carcinoma
Mentions: The results of 3D culture cell invasion assays were nearly identical to the corresponding results obtained with the modified Matrigel-Boyden chamber assays. EphA2 siRNA treatment suppressed the cellular invasion in the non-metastatic RCC cell lines (Caki-2 and A498) (Fig 6B and 6C), but not in the metastatic RCC cell lines (Caki-1 or ACHN) (Fig 6A and 6C).

Bottom Line: Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines.For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control.RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Dongguk University College of Medicine, Seoul, Korea.

ABSTRACT

Objectives: To investigate the role of EphA2 in malignant cellular behavior in renal cell carcinoma (RCC) cells and whether FAK/RhoA signaling can act as downstream effectors of EphA2 on RCC cells.

Methods: Expression of EphA2 protein in non-metastatic RCC (Caki-2 and A498), metastatic RCC cells (Caki-1 and ACHN), HEK-293 cells and prostate cancer cells (PC-3 and DU-145; positive controls of EphA2 expression) was evaluated by Western blot. Changes in mRNA or protein expression of EphA2, FAK or membrane-bound RhoA following EphA2, FAK or RhoA small interfering RNA (siRNA) transfection were determined by reverse transcription polymerase chain reaction or Western blot. The effect of siRNA treatment on cellular viability, apoptosis and invasion was analyzed by cell counting kit-8, Annexin-V and modified Matrigel-Boyden assays, respectively.

Results: In all RCC cell lines, the expression of EphA2 protein was detectable at variable levels; however, in HEK-293 cells, EphA2 expression was very low. Treatment with EphA2 siRNA significantly reduced the expression of EphA2 mRNA and protein in all RCC cell lines. For non-metastatic RCC cells (Caki-2 and A498) but not metastatic RCC cells (Caki-1 and ACHN), cellular viability, invasiveness, resistance to apoptosis, expression of membrane-bound RhoA protein and FAK phosphorylation were significantly decreased in EphA2 siRNA-treated cells compared to the control. In non-metastatic RCC cells, FAK siRNA significantly attenuated the invasiveness, resistance to apoptosis, as well as expression of membrane-bound RhoA protein without changing protein expression of EphA2. RhoA siRNA significantly decreased the malignant cellular behavior and expression of membrane-bound RhoA protein without changing EphA2 protein expression or FAK phosphorylation.

Conclusions: Our data provide the first functional evidence that the EphA2/FAK/RhoA signaling pathway plays a critical role in the malignant cellular behavior of RCC and appears to be functional particularly in the early stage of malignant progression of non-metastatic RCC.

No MeSH data available.


Related in: MedlinePlus