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Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

Li J, Ng EK, Ng YP, Wong CY, Yu J, Jin H, Cheng VY, Go MY, Cheung PK, Ebert MP, Tong J, To KF, Chan FK, Sung JJ, Ip NY, Leung WK - Br. J. Cancer (2009)

Bottom Line: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001).In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia.Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest.

View Article: PubMed Central - PubMed

Affiliation: Institute of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis.

Methods: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro.

Results: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar.

Conclusion: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

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Related in: MedlinePlus

mRNA expression levels of ramp in (A) human gastric cancer cell lines and (B) primary gastric cancer and their adjacent non-cancerous tissues were determined by quantitative real-time PCR. The results were expressed as the ratio of copies of ramp relevant to β-actin from at least three independent experiments. Data are expressed as mean±s.d.; *P<0.05, **P<0.01, ***P<0.0001. (C) Representative immunohistochemical staining of RAMP protein expression in gastric cancer (C1) and intestinal metaplasia (C2). (C3) Subcellular localisation of RAMP protein in human cancer cells. Black arrowheads indicate some examples of cancer cells with RAMP expression.
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fig1: mRNA expression levels of ramp in (A) human gastric cancer cell lines and (B) primary gastric cancer and their adjacent non-cancerous tissues were determined by quantitative real-time PCR. The results were expressed as the ratio of copies of ramp relevant to β-actin from at least three independent experiments. Data are expressed as mean±s.d.; *P<0.05, **P<0.01, ***P<0.0001. (C) Representative immunohistochemical staining of RAMP protein expression in gastric cancer (C1) and intestinal metaplasia (C2). (C3) Subcellular localisation of RAMP protein in human cancer cells. Black arrowheads indicate some examples of cancer cells with RAMP expression.

Mentions: To investigate whether RAMP might be involved in gastric carcinogenesis, the mRNA expression of ramp was first examined in 7 human gastric cancer cell lines (MKN45, AGS, SNU1, MKN28, NCI-N87, SNU16 and KATO III) and 47 gastric cancer tissues. Ramp mRNA was highly expressed in all seven cell lines (P<0.05; Figure 1A). Of the 47 paired gastric tumour tissues analysed, 38 cases (81%) showed increased ramp mRNA expression compared with their adjacent non-cancerous tissues (P<0.001, Wilcoxon test; Figure 1B). Consistent with the mRNA expression, strong and dense RAMP immunoreactivity was presented in gastric tumour tissues, whereas RAMP immunoactivity was only noted occasionally in adjacent non-cancerous tissues (Figure 1C1). Ramp protein expression was identified in both nuclear and cytoplasmic regions of gastric cancer cells (Figure 1C3). Furthermore, moderate level of RAMP expression was detected in precancerous gastric lesion, intestinal metaplasia (Figure 1C2).


Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

Li J, Ng EK, Ng YP, Wong CY, Yu J, Jin H, Cheng VY, Go MY, Cheung PK, Ebert MP, Tong J, To KF, Chan FK, Sung JJ, Ip NY, Leung WK - Br. J. Cancer (2009)

mRNA expression levels of ramp in (A) human gastric cancer cell lines and (B) primary gastric cancer and their adjacent non-cancerous tissues were determined by quantitative real-time PCR. The results were expressed as the ratio of copies of ramp relevant to β-actin from at least three independent experiments. Data are expressed as mean±s.d.; *P<0.05, **P<0.01, ***P<0.0001. (C) Representative immunohistochemical staining of RAMP protein expression in gastric cancer (C1) and intestinal metaplasia (C2). (C3) Subcellular localisation of RAMP protein in human cancer cells. Black arrowheads indicate some examples of cancer cells with RAMP expression.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2736823&req=5

fig1: mRNA expression levels of ramp in (A) human gastric cancer cell lines and (B) primary gastric cancer and their adjacent non-cancerous tissues were determined by quantitative real-time PCR. The results were expressed as the ratio of copies of ramp relevant to β-actin from at least three independent experiments. Data are expressed as mean±s.d.; *P<0.05, **P<0.01, ***P<0.0001. (C) Representative immunohistochemical staining of RAMP protein expression in gastric cancer (C1) and intestinal metaplasia (C2). (C3) Subcellular localisation of RAMP protein in human cancer cells. Black arrowheads indicate some examples of cancer cells with RAMP expression.
Mentions: To investigate whether RAMP might be involved in gastric carcinogenesis, the mRNA expression of ramp was first examined in 7 human gastric cancer cell lines (MKN45, AGS, SNU1, MKN28, NCI-N87, SNU16 and KATO III) and 47 gastric cancer tissues. Ramp mRNA was highly expressed in all seven cell lines (P<0.05; Figure 1A). Of the 47 paired gastric tumour tissues analysed, 38 cases (81%) showed increased ramp mRNA expression compared with their adjacent non-cancerous tissues (P<0.001, Wilcoxon test; Figure 1B). Consistent with the mRNA expression, strong and dense RAMP immunoreactivity was presented in gastric tumour tissues, whereas RAMP immunoactivity was only noted occasionally in adjacent non-cancerous tissues (Figure 1C1). Ramp protein expression was identified in both nuclear and cytoplasmic regions of gastric cancer cells (Figure 1C3). Furthermore, moderate level of RAMP expression was detected in precancerous gastric lesion, intestinal metaplasia (Figure 1C2).

Bottom Line: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001).In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia.Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest.

View Article: PubMed Central - PubMed

Affiliation: Institute of Digestive Disease, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis.

Methods: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro.

Results: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar.

Conclusion: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

Show MeSH
Related in: MedlinePlus