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Mesothelin regulates growth and apoptosis in pancreatic cancer cells through p53-dependent and -independent signal pathway.

Zheng C, Jia W, Tang Y, Zhao H, Jiang Y, Sun S - J. Exp. Clin. Cancer Res. (2012)

Bottom Line: We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis.When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased.Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: General surgery, the affiliated Jinan central hospital of Shandong university, No105, Jiefang Road, District Lixia, Jinan, 250013, R.P China.

ABSTRACT
Mesothelin, a secreted protein, is overexpressed in some cancers, including pancreatic cancer. Rescent studies have shown that overexpression of mesothelin significantly increased tumor cell proliferation, and downregulation of mesothelin inhibited cell proliferation in pancreatic cancer cells, but its exact function and mechanism remains unclear. The aim of the present study was to evaluate the effects of mesothelin on proliferation and apoptosis in pancreatic cancer cells with different p53 status and to explore its signal pathway. Mesothelin levels were detected by western blot and RT-PCR assay in human pancreatic cancer AsPC-1, HPAC and Capan-2, Capan-1 and MIA PaCa-2 cell lines. Mesothelin was slienced by shRNA in AsPC-1, Capan-2 and Capan-1 cells with rich mesothelin level, and mesothelin was overexpressed in the HPAC and Capan-2 cells with less mesothelin level. We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis. When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased. However, in the Capan-2 cells with wt-p53, suppression of the mesothelin significantly increased wt-p53 levels. When p53 was blocked by siRNA in the stable mesothelin shRNA transfected Capan-2 cells, PUMA was inhibited, followed by increased proliferative capability and decreased cell apoptosis. In the HPAC and Capan-2 cells with wt-p53 and in the MIA PaCa-2 cells with mt-p53, overexpression of the mesothelin significantly decreased bax levels and increased bcl-2 levels, followed by increased proliferative and colony forming capability. Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. However, mesothelin-transfected cells exhibited a increased rate of tumor growth under in vivo conditions. Our data demonstrated that mesothelin promotes proliferation and inhibited apoptosis through p53-dependent pathway in pancreatic cancer cells with wt-p53, and p53-independent pathway in pancreatic cancer cells with mt-p53. Targeting mesothelin by shRNA is the important method for pancreatic cancer therapy.

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Mesothelin sliencing suppresses cell survival,proliferation and promotes apoptosis by p53-dependent and -independent pathway in pancreatic cancer cells.A, Western blot assay for p53, PUMA,bax and bcl-2 in Capan-2 cells with wt-p53. Mesothelin sliencing significantly increased the P53,PUMA and bax levels and decreased bcl-2 level. Knockdown of p53 by shRNA(3 days transfection) decreased the PUMA and bax level and increased the bcl-2 level in stable mesothelin silenced CaPan-2 cells. B, Determination of caspase-3 activity. Caspase-3 activity was determined by fluorogenic substrates. Caspase-3 activity was measured fluorometrically at 510 nm on a microplate fluorescence reader. Mesothelin sliencing significantly increased the caspase-3 activity. The activity in mock shRNA transfected cells was defined 1.* denote p < 0.05, compared with mock shRNA controls, t test. C, Cytotoxicity assay was by MTT. .* denote p < 0.05,**p<0.01, compared with mesothelin shRNA groups, t test. D, Cell apoptosis was determined by FCM assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA. E, Cell apoptosis was determined by TUNEL assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA.* denote p < 0.05, compared with combined shRNA treatment groups, t test. F, Western blot assay for p53, PUMA,bax and bcl-2 in ASPC-1 cells with mt-p53. Mesothelin sliencing significantly increased the PUMA and bax levels and decreased the bcl-2 level.
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Figure 5: Mesothelin sliencing suppresses cell survival,proliferation and promotes apoptosis by p53-dependent and -independent pathway in pancreatic cancer cells.A, Western blot assay for p53, PUMA,bax and bcl-2 in Capan-2 cells with wt-p53. Mesothelin sliencing significantly increased the P53,PUMA and bax levels and decreased bcl-2 level. Knockdown of p53 by shRNA(3 days transfection) decreased the PUMA and bax level and increased the bcl-2 level in stable mesothelin silenced CaPan-2 cells. B, Determination of caspase-3 activity. Caspase-3 activity was determined by fluorogenic substrates. Caspase-3 activity was measured fluorometrically at 510 nm on a microplate fluorescence reader. Mesothelin sliencing significantly increased the caspase-3 activity. The activity in mock shRNA transfected cells was defined 1.* denote p < 0.05, compared with mock shRNA controls, t test. C, Cytotoxicity assay was by MTT. .* denote p < 0.05,**p<0.01, compared with mesothelin shRNA groups, t test. D, Cell apoptosis was determined by FCM assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA. E, Cell apoptosis was determined by TUNEL assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA.* denote p < 0.05, compared with combined shRNA treatment groups, t test. F, Western blot assay for p53, PUMA,bax and bcl-2 in ASPC-1 cells with mt-p53. Mesothelin sliencing significantly increased the PUMA and bax levels and decreased the bcl-2 level.

Mentions: It has shown above mesothelin sliencing suppresses cell survival and proliferation.We next investigated the signal transduction mechanism of cell survival and proliferation in mesothelin-sliencing Capan-1, Capan-2 and ASPC-1 cells with wt- and mt- p53 status. To identify signals activated by mesothelin sliencing, we examined transcription factors p53, PUMA, bax and bcl-2. In the Capan-2 cell with wt-p53 cells, mesothelin sliencing significantly increased the p53, PUMA and bax levels (Figure5A), caspase-3 activity (Figure5B) and decreased bcl-2 levels (Figure5A). When p53 was knockdown by p53 siRNA transfection (3 days after transfection) in stable mesothelin-sliencing cells, PUMA and bax levels (Figure5B) and caspase-3 activity (Figure5B) was significantly decreased. But the bcl-2 level was increased (Figure5B). This data shown mesothelin sliencing decreased PUMA, caspase-3, bax and increased bcl-2 levels was by p53-dependent pathway in Capan-1 cells with wt-p53.


Mesothelin regulates growth and apoptosis in pancreatic cancer cells through p53-dependent and -independent signal pathway.

Zheng C, Jia W, Tang Y, Zhao H, Jiang Y, Sun S - J. Exp. Clin. Cancer Res. (2012)

Mesothelin sliencing suppresses cell survival,proliferation and promotes apoptosis by p53-dependent and -independent pathway in pancreatic cancer cells.A, Western blot assay for p53, PUMA,bax and bcl-2 in Capan-2 cells with wt-p53. Mesothelin sliencing significantly increased the P53,PUMA and bax levels and decreased bcl-2 level. Knockdown of p53 by shRNA(3 days transfection) decreased the PUMA and bax level and increased the bcl-2 level in stable mesothelin silenced CaPan-2 cells. B, Determination of caspase-3 activity. Caspase-3 activity was determined by fluorogenic substrates. Caspase-3 activity was measured fluorometrically at 510 nm on a microplate fluorescence reader. Mesothelin sliencing significantly increased the caspase-3 activity. The activity in mock shRNA transfected cells was defined 1.* denote p < 0.05, compared with mock shRNA controls, t test. C, Cytotoxicity assay was by MTT. .* denote p < 0.05,**p<0.01, compared with mesothelin shRNA groups, t test. D, Cell apoptosis was determined by FCM assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA. E, Cell apoptosis was determined by TUNEL assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA.* denote p < 0.05, compared with combined shRNA treatment groups, t test. F, Western blot assay for p53, PUMA,bax and bcl-2 in ASPC-1 cells with mt-p53. Mesothelin sliencing significantly increased the PUMA and bax levels and decreased the bcl-2 level.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585882&req=5

Figure 5: Mesothelin sliencing suppresses cell survival,proliferation and promotes apoptosis by p53-dependent and -independent pathway in pancreatic cancer cells.A, Western blot assay for p53, PUMA,bax and bcl-2 in Capan-2 cells with wt-p53. Mesothelin sliencing significantly increased the P53,PUMA and bax levels and decreased bcl-2 level. Knockdown of p53 by shRNA(3 days transfection) decreased the PUMA and bax level and increased the bcl-2 level in stable mesothelin silenced CaPan-2 cells. B, Determination of caspase-3 activity. Caspase-3 activity was determined by fluorogenic substrates. Caspase-3 activity was measured fluorometrically at 510 nm on a microplate fluorescence reader. Mesothelin sliencing significantly increased the caspase-3 activity. The activity in mock shRNA transfected cells was defined 1.* denote p < 0.05, compared with mock shRNA controls, t test. C, Cytotoxicity assay was by MTT. .* denote p < 0.05,**p<0.01, compared with mesothelin shRNA groups, t test. D, Cell apoptosis was determined by FCM assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA. E, Cell apoptosis was determined by TUNEL assay in samples treated with mesothelin shRNA or mesothelin shRNA plus PUMA shRNA.* denote p < 0.05, compared with combined shRNA treatment groups, t test. F, Western blot assay for p53, PUMA,bax and bcl-2 in ASPC-1 cells with mt-p53. Mesothelin sliencing significantly increased the PUMA and bax levels and decreased the bcl-2 level.
Mentions: It has shown above mesothelin sliencing suppresses cell survival and proliferation.We next investigated the signal transduction mechanism of cell survival and proliferation in mesothelin-sliencing Capan-1, Capan-2 and ASPC-1 cells with wt- and mt- p53 status. To identify signals activated by mesothelin sliencing, we examined transcription factors p53, PUMA, bax and bcl-2. In the Capan-2 cell with wt-p53 cells, mesothelin sliencing significantly increased the p53, PUMA and bax levels (Figure5A), caspase-3 activity (Figure5B) and decreased bcl-2 levels (Figure5A). When p53 was knockdown by p53 siRNA transfection (3 days after transfection) in stable mesothelin-sliencing cells, PUMA and bax levels (Figure5B) and caspase-3 activity (Figure5B) was significantly decreased. But the bcl-2 level was increased (Figure5B). This data shown mesothelin sliencing decreased PUMA, caspase-3, bax and increased bcl-2 levels was by p53-dependent pathway in Capan-1 cells with wt-p53.

Bottom Line: We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis.When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased.Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions.

View Article: PubMed Central - HTML - PubMed

Affiliation: General surgery, the affiliated Jinan central hospital of Shandong university, No105, Jiefang Road, District Lixia, Jinan, 250013, R.P China.

ABSTRACT
Mesothelin, a secreted protein, is overexpressed in some cancers, including pancreatic cancer. Rescent studies have shown that overexpression of mesothelin significantly increased tumor cell proliferation, and downregulation of mesothelin inhibited cell proliferation in pancreatic cancer cells, but its exact function and mechanism remains unclear. The aim of the present study was to evaluate the effects of mesothelin on proliferation and apoptosis in pancreatic cancer cells with different p53 status and to explore its signal pathway. Mesothelin levels were detected by western blot and RT-PCR assay in human pancreatic cancer AsPC-1, HPAC and Capan-2, Capan-1 and MIA PaCa-2 cell lines. Mesothelin was slienced by shRNA in AsPC-1, Capan-2 and Capan-1 cells with rich mesothelin level, and mesothelin was overexpressed in the HPAC and Capan-2 cells with less mesothelin level. We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis. When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased. However, in the Capan-2 cells with wt-p53, suppression of the mesothelin significantly increased wt-p53 levels. When p53 was blocked by siRNA in the stable mesothelin shRNA transfected Capan-2 cells, PUMA was inhibited, followed by increased proliferative capability and decreased cell apoptosis. In the HPAC and Capan-2 cells with wt-p53 and in the MIA PaCa-2 cells with mt-p53, overexpression of the mesothelin significantly decreased bax levels and increased bcl-2 levels, followed by increased proliferative and colony forming capability. Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. However, mesothelin-transfected cells exhibited a increased rate of tumor growth under in vivo conditions. Our data demonstrated that mesothelin promotes proliferation and inhibited apoptosis through p53-dependent pathway in pancreatic cancer cells with wt-p53, and p53-independent pathway in pancreatic cancer cells with mt-p53. Targeting mesothelin by shRNA is the important method for pancreatic cancer therapy.

Show MeSH
Related in: MedlinePlus