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Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways.

Mitsui Y, Hirata H, Arichi N, Hiraki M, Yasumoto H, Chang I, Fukuhara S, Yamamura S, Shahryari V, Deng G, Saini S, Majid S, Dahiya R, Tanaka Y, Shiina H - Oncotarget (2015)

Bottom Line: The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples.Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors.Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Shimane University Faculty of Medicine, Enya-cho, Izumo, Japan.

ABSTRACT
We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21(WAF1/CIP1) and p27(KIP1) expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2'-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

No MeSH data available.


Related in: MedlinePlus

Effects of BMP-2 overexpression on apoptosis(A) Apoptosis assays with Caki-1 and Caki-2 cells were performed at 24 hours after transfection. Representative quadrant figures of control vector BMP-2 transfectants in Caki-1 (upper) and Caki-2 (lower) cells. (B) Bar chart indicates the ratio of apoptotic cell fractions (early plus apoptotic cells) in BMP-2 transfectants as compared with the control. Data for apoptotic cell fractions are expressed as the relative value for the average expression of the control vector transfectant. *P<0.01, **P<0.001. (C) Immunoblotting analysis of apoptotic markers and GADD45β in control and BMP-2 transfected Caki-1 and Caki-2 cells. GAPDH was used as a loading control.
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Figure 6: Effects of BMP-2 overexpression on apoptosis(A) Apoptosis assays with Caki-1 and Caki-2 cells were performed at 24 hours after transfection. Representative quadrant figures of control vector BMP-2 transfectants in Caki-1 (upper) and Caki-2 (lower) cells. (B) Bar chart indicates the ratio of apoptotic cell fractions (early plus apoptotic cells) in BMP-2 transfectants as compared with the control. Data for apoptotic cell fractions are expressed as the relative value for the average expression of the control vector transfectant. *P<0.01, **P<0.001. (C) Immunoblotting analysis of apoptotic markers and GADD45β in control and BMP-2 transfected Caki-1 and Caki-2 cells. GAPDH was used as a loading control.

Mentions: Since BMP-2 restoration significantly inhibited proliferation, migration, invasion, and colony formation in RCC cell lines, we hypothesized that its expression may induce apoptosis. The results of apoptosis assays of Caki-1 and Caki-2 cells performed 24 hours post-transfection are shown in Fig. 6A and B. Apoptotic and early apoptotic fractions (upper right and lower right, respectively, in quadrant images) were significantly greater in BMP-2 transfectants as compared to the vector control. These differences were also seen in both Caki-1 and Caki-2 cells at 48 hours after transfection (data not shown). These findings indicate a pro-apoptotic role for BMP-2, as well as its effects on the apoptotic pathway and regulation of tumorigenicity. In our recent study, we found that growth arrest and DNA damage inducible gene 45α (GADD45α) may play important roles in human RCC apoptosis [26]. Therefore, we examined the expression of several apoptotic proteins and GADD45α protein using Western blot analysis. As shown in Fig. 6C, BMP-2 over-expression caused an increase in cleaved caspase-3 in both Caki-1 and Caki-2 cells, further supporting the pro-apoptotic role of BMP-2. Furthermore, GADD45α was up-regulated, indicating that overexpression of BMP-2 induced GADD45α and apoptotic effects. Fig. 7 indicates the putative BMP-2 pathway in RCC on the basis of our results and previous studies [12, 13, 25, 27].


Inactivation of bone morphogenetic protein 2 may predict clinical outcome and poor overall survival for renal cell carcinoma through epigenetic pathways.

Mitsui Y, Hirata H, Arichi N, Hiraki M, Yasumoto H, Chang I, Fukuhara S, Yamamura S, Shahryari V, Deng G, Saini S, Majid S, Dahiya R, Tanaka Y, Shiina H - Oncotarget (2015)

Effects of BMP-2 overexpression on apoptosis(A) Apoptosis assays with Caki-1 and Caki-2 cells were performed at 24 hours after transfection. Representative quadrant figures of control vector BMP-2 transfectants in Caki-1 (upper) and Caki-2 (lower) cells. (B) Bar chart indicates the ratio of apoptotic cell fractions (early plus apoptotic cells) in BMP-2 transfectants as compared with the control. Data for apoptotic cell fractions are expressed as the relative value for the average expression of the control vector transfectant. *P<0.01, **P<0.001. (C) Immunoblotting analysis of apoptotic markers and GADD45β in control and BMP-2 transfected Caki-1 and Caki-2 cells. GAPDH was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496240&req=5

Figure 6: Effects of BMP-2 overexpression on apoptosis(A) Apoptosis assays with Caki-1 and Caki-2 cells were performed at 24 hours after transfection. Representative quadrant figures of control vector BMP-2 transfectants in Caki-1 (upper) and Caki-2 (lower) cells. (B) Bar chart indicates the ratio of apoptotic cell fractions (early plus apoptotic cells) in BMP-2 transfectants as compared with the control. Data for apoptotic cell fractions are expressed as the relative value for the average expression of the control vector transfectant. *P<0.01, **P<0.001. (C) Immunoblotting analysis of apoptotic markers and GADD45β in control and BMP-2 transfected Caki-1 and Caki-2 cells. GAPDH was used as a loading control.
Mentions: Since BMP-2 restoration significantly inhibited proliferation, migration, invasion, and colony formation in RCC cell lines, we hypothesized that its expression may induce apoptosis. The results of apoptosis assays of Caki-1 and Caki-2 cells performed 24 hours post-transfection are shown in Fig. 6A and B. Apoptotic and early apoptotic fractions (upper right and lower right, respectively, in quadrant images) were significantly greater in BMP-2 transfectants as compared to the vector control. These differences were also seen in both Caki-1 and Caki-2 cells at 48 hours after transfection (data not shown). These findings indicate a pro-apoptotic role for BMP-2, as well as its effects on the apoptotic pathway and regulation of tumorigenicity. In our recent study, we found that growth arrest and DNA damage inducible gene 45α (GADD45α) may play important roles in human RCC apoptosis [26]. Therefore, we examined the expression of several apoptotic proteins and GADD45α protein using Western blot analysis. As shown in Fig. 6C, BMP-2 over-expression caused an increase in cleaved caspase-3 in both Caki-1 and Caki-2 cells, further supporting the pro-apoptotic role of BMP-2. Furthermore, GADD45α was up-regulated, indicating that overexpression of BMP-2 induced GADD45α and apoptotic effects. Fig. 7 indicates the putative BMP-2 pathway in RCC on the basis of our results and previous studies [12, 13, 25, 27].

Bottom Line: The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples.Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors.Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Shimane University Faculty of Medicine, Enya-cho, Izumo, Japan.

ABSTRACT
We investigated whether impaired regulation of bone morphogenetic protein-2 (BMP-2) via epigenetic pathways is associated with renal cell carcinoma (RCC) pathogenesis. Expression and CpG methylation of the BMP-2 gene were analyzed using RCC cell lines, and 96 matched RCC and normal renal tissues. We also performed functional analysis using BMP-2 restored RCC cells. A significant association of BMP-2 mRNA expression was also found with advanced tumor stage and lymph node involvement, while lower BMP-2 mRNA expression was significantly associated with poor overall survival after radical nephrectomy. In RCC cells, BMP-2 restoration significantly inhibited cell proliferation, migration, invasion, and colony formation. In addition, BMP-2 overexpression induced p21(WAF1/CIP1) and p27(KIP1) expression, and cellular apoptosis in RCC cells. BMP-2 mRNA expression was significantly enhanced in RCC cells by 5-aza-2'-deoxycitidine treatment. The prevalence of BMP-2 promoter methylation was significantly greater and BMP-2 mRNA expression was significantly lower in RCC samples as compared to normal kidney samples. Furthermore, a significant correlation was found between BMP-2 promoter methylation and mRNA transcription in tumors. Aberrant BMP-2 methylation and the resultant loss of BMP-2 expression may be a useful molecular marker for designing improved diagnostic and therapeutic strategies for RCC.

No MeSH data available.


Related in: MedlinePlus