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Down-regulation of PKCζ in renal cell carcinoma and its clinicopathological implications.

Pu YS, Huang CY, Chen JY, Kang WY, Lin YC, Shiu YS, Chuang SJ, Yu HJ, Lai MK, Tsai YC, Wu WJ, Hour TC - J. Biomed. Sci. (2012)

Bottom Line: Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC.Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P=0.04), but no such association was found in TNM stage (P=0.13).PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT

Background: Metastatic renal cell carcinoma (RCC) is highly resistant to systemic chemotherapy. Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC. Recent studies have shown the atypical PKCζ is an important regulator of tumorigenesis. However, the correlation between PKCζ expression and the clinical outcome in RCC patients is unclear. We examined the level of PKCζ expression in human RCC.

Methods: PKCζ mRNA and protein expressions were examined by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) respectively in RCC tissues of 144 patients. Cellular cytotoxicity and proliferation were assessed by MTT.

Results: PKCζ expression was significantly higher in normal than in cancerous tissues (P<0.0001) by real-time PCR and IHC. Similarly, PKCζ expression was down-regulated in four renal cancer cell lines compared to immortalized benign renal tubular cells. Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P=0.04), but no such association was found in TNM stage (P=0.13). Tumors with higher PKCζ expression were associated with tumor size (P=0.048). Expression of higher PKCζ found a poor survival in patients with high tumor grade. Down-regulation of PKCζ showed the significant chemoresistance in RCC cell lines. Inactivation of PKCζ expression enhanced cellular resistance to cisplatin and paclitaxel, and proliferation in HK-2 cells by specific PKCζ siRNA and inhibitor.

Conclusions: PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.

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Inhibition of PKCζ expression led to increased chemoresistance and cell proliferation in HK-2 cells. (A) siRNA-mediated suppression of PKCζ gene expression in HK-2 cells. PKCζ siRNA (100 nM) were transfected into parental HK-2 cells. Note, the level of targeted PKCζ gene was significantly reduced by > 50% using RT-PCR analysis. (B) Similarly, the level of PKCζ protein was significantly inhibited by 57% at 100 nm siRNA using Western blotting analysis. C: control group, lipofectamine only, and α-tubulin was used as an internal control. (C) Parental HK-2 cells were treated 100 nm PKCζ siRNA (HK-2-siRNA) or lipofectamine only (HK-2-C), respectively for 24 hours. Chemosensivity of HK-2-C (IC50 = 2.05 μM) and HK-2-siRNA (IC50 = 4.02 μM) cells to cisplatin were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Similar results, chemosensivity of HK-2-C (IC50 = 0.09 μM) and HK-2-siRNA (IC50 = 0.17 μM) cells to paclitaxel were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. P* < 0.05, P** < 0.005 and P*** < 0.001. (D) Parental HK-2 cells were treated with 0.16 μM PKCζ pseudosubstrate. After 60 h, cell proliferation was assessed by MTT assay. Points, means of two experiments plated in replicates of six; Data are presented as the mean ± standard error of the means (SEM). P* < 0.05, compared with untreated cells.
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Figure 5: Inhibition of PKCζ expression led to increased chemoresistance and cell proliferation in HK-2 cells. (A) siRNA-mediated suppression of PKCζ gene expression in HK-2 cells. PKCζ siRNA (100 nM) were transfected into parental HK-2 cells. Note, the level of targeted PKCζ gene was significantly reduced by > 50% using RT-PCR analysis. (B) Similarly, the level of PKCζ protein was significantly inhibited by 57% at 100 nm siRNA using Western blotting analysis. C: control group, lipofectamine only, and α-tubulin was used as an internal control. (C) Parental HK-2 cells were treated 100 nm PKCζ siRNA (HK-2-siRNA) or lipofectamine only (HK-2-C), respectively for 24 hours. Chemosensivity of HK-2-C (IC50 = 2.05 μM) and HK-2-siRNA (IC50 = 4.02 μM) cells to cisplatin were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Similar results, chemosensivity of HK-2-C (IC50 = 0.09 μM) and HK-2-siRNA (IC50 = 0.17 μM) cells to paclitaxel were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. P* < 0.05, P** < 0.005 and P*** < 0.001. (D) Parental HK-2 cells were treated with 0.16 μM PKCζ pseudosubstrate. After 60 h, cell proliferation was assessed by MTT assay. Points, means of two experiments plated in replicates of six; Data are presented as the mean ± standard error of the means (SEM). P* < 0.05, compared with untreated cells.

Mentions: Knockdown of PKCζ expression in HK-2 by specific PKCζ siRNA (Figure 5A and 5B), indicated down-regulated PKCζ expression enhanced HK-2 cells the resistance to cisplatin at the concentrations of 1, 3 and 10 μM, respectively (P* < 0.05, P** < 0.005 and P*** < 0.001) (Figure 5C). Chemosensivity of HK-2-C (IC50 = 2.05 μM) and HK-2-siRNA (IC50 = 4.02 μM) cells to cisplatin were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Similar results, chemosensivity of HK-2-C (IC50 = 0.09 μM) and HK-2-siRNA (IC50 = 0.17 μM) cells to paclitaxel were examined by MTT assay (Figure 5C). The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Herein, knockdown of PKCζ expression enhanced HK-2 cells the resistance to paclitaxel at the concentrations of 0.1 and 3 μM, respectively (P* < 0.05 and P** < 0.005). Cell permeable protein kinase Cζ pseudosubstrate inhibitor was used to inhibit the PKCζ activity of HK-2 cells. Interestingly, we also found inactivation of PKCζ by specific inhibitor of 0.16 μM PKCζ pseudosubstrate could promote significantly cell growth in HK-2 cells (P < 0.05) (Figure 5D).


Down-regulation of PKCζ in renal cell carcinoma and its clinicopathological implications.

Pu YS, Huang CY, Chen JY, Kang WY, Lin YC, Shiu YS, Chuang SJ, Yu HJ, Lai MK, Tsai YC, Wu WJ, Hour TC - J. Biomed. Sci. (2012)

Inhibition of PKCζ expression led to increased chemoresistance and cell proliferation in HK-2 cells. (A) siRNA-mediated suppression of PKCζ gene expression in HK-2 cells. PKCζ siRNA (100 nM) were transfected into parental HK-2 cells. Note, the level of targeted PKCζ gene was significantly reduced by > 50% using RT-PCR analysis. (B) Similarly, the level of PKCζ protein was significantly inhibited by 57% at 100 nm siRNA using Western blotting analysis. C: control group, lipofectamine only, and α-tubulin was used as an internal control. (C) Parental HK-2 cells were treated 100 nm PKCζ siRNA (HK-2-siRNA) or lipofectamine only (HK-2-C), respectively for 24 hours. Chemosensivity of HK-2-C (IC50 = 2.05 μM) and HK-2-siRNA (IC50 = 4.02 μM) cells to cisplatin were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Similar results, chemosensivity of HK-2-C (IC50 = 0.09 μM) and HK-2-siRNA (IC50 = 0.17 μM) cells to paclitaxel were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. P* < 0.05, P** < 0.005 and P*** < 0.001. (D) Parental HK-2 cells were treated with 0.16 μM PKCζ pseudosubstrate. After 60 h, cell proliferation was assessed by MTT assay. Points, means of two experiments plated in replicates of six; Data are presented as the mean ± standard error of the means (SEM). P* < 0.05, compared with untreated cells.
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Related In: Results  -  Collection

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Figure 5: Inhibition of PKCζ expression led to increased chemoresistance and cell proliferation in HK-2 cells. (A) siRNA-mediated suppression of PKCζ gene expression in HK-2 cells. PKCζ siRNA (100 nM) were transfected into parental HK-2 cells. Note, the level of targeted PKCζ gene was significantly reduced by > 50% using RT-PCR analysis. (B) Similarly, the level of PKCζ protein was significantly inhibited by 57% at 100 nm siRNA using Western blotting analysis. C: control group, lipofectamine only, and α-tubulin was used as an internal control. (C) Parental HK-2 cells were treated 100 nm PKCζ siRNA (HK-2-siRNA) or lipofectamine only (HK-2-C), respectively for 24 hours. Chemosensivity of HK-2-C (IC50 = 2.05 μM) and HK-2-siRNA (IC50 = 4.02 μM) cells to cisplatin were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Similar results, chemosensivity of HK-2-C (IC50 = 0.09 μM) and HK-2-siRNA (IC50 = 0.17 μM) cells to paclitaxel were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. P* < 0.05, P** < 0.005 and P*** < 0.001. (D) Parental HK-2 cells were treated with 0.16 μM PKCζ pseudosubstrate. After 60 h, cell proliferation was assessed by MTT assay. Points, means of two experiments plated in replicates of six; Data are presented as the mean ± standard error of the means (SEM). P* < 0.05, compared with untreated cells.
Mentions: Knockdown of PKCζ expression in HK-2 by specific PKCζ siRNA (Figure 5A and 5B), indicated down-regulated PKCζ expression enhanced HK-2 cells the resistance to cisplatin at the concentrations of 1, 3 and 10 μM, respectively (P* < 0.05, P** < 0.005 and P*** < 0.001) (Figure 5C). Chemosensivity of HK-2-C (IC50 = 2.05 μM) and HK-2-siRNA (IC50 = 4.02 μM) cells to cisplatin were examined by MTT assay. The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Similar results, chemosensivity of HK-2-C (IC50 = 0.09 μM) and HK-2-siRNA (IC50 = 0.17 μM) cells to paclitaxel were examined by MTT assay (Figure 5C). The IC50 value was a near 2-fold increase in HK-2-siRNA compared to HK-2-C. Herein, knockdown of PKCζ expression enhanced HK-2 cells the resistance to paclitaxel at the concentrations of 0.1 and 3 μM, respectively (P* < 0.05 and P** < 0.005). Cell permeable protein kinase Cζ pseudosubstrate inhibitor was used to inhibit the PKCζ activity of HK-2 cells. Interestingly, we also found inactivation of PKCζ by specific inhibitor of 0.16 μM PKCζ pseudosubstrate could promote significantly cell growth in HK-2 cells (P < 0.05) (Figure 5D).

Bottom Line: Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC.Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P=0.04), but no such association was found in TNM stage (P=0.13).PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT

Background: Metastatic renal cell carcinoma (RCC) is highly resistant to systemic chemotherapy. Unfortunately, nearly all patients die of the metastatic and chemoresistant RCC. Recent studies have shown the atypical PKCζ is an important regulator of tumorigenesis. However, the correlation between PKCζ expression and the clinical outcome in RCC patients is unclear. We examined the level of PKCζ expression in human RCC.

Methods: PKCζ mRNA and protein expressions were examined by real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC) respectively in RCC tissues of 144 patients. Cellular cytotoxicity and proliferation were assessed by MTT.

Results: PKCζ expression was significantly higher in normal than in cancerous tissues (P<0.0001) by real-time PCR and IHC. Similarly, PKCζ expression was down-regulated in four renal cancer cell lines compared to immortalized benign renal tubular cells. Interestingly, an increase of PKCζ expression was associated with the elevated tumor grade (P=0.04), but no such association was found in TNM stage (P=0.13). Tumors with higher PKCζ expression were associated with tumor size (P=0.048). Expression of higher PKCζ found a poor survival in patients with high tumor grade. Down-regulation of PKCζ showed the significant chemoresistance in RCC cell lines. Inactivation of PKCζ expression enhanced cellular resistance to cisplatin and paclitaxel, and proliferation in HK-2 cells by specific PKCζ siRNA and inhibitor.

Conclusions: PKCζ expression was associated with tumorigenesis and chemoresistance in RCC.

Show MeSH
Related in: MedlinePlus