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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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Effects of mesothelin on pancreatic cancer growth in the xenograft nude mouse model.A. Subcutaneous tumor volume of HPAC- mesothelin,Capan-2- mesothelin and MIA PaCa-2- mesothelin and their mock cells(2 × 106)were subcutaneously inoculated into nude mice (8 mice per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05,*p>0.05. B. Subcutaneous tumor volume of AsPC-1-shRNA mesothelin, Capan-2-shRNA mesothelin and Capan-1-shRNA mesothelin (2 × 106) were injected into the flank of nude mice (eight per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05. C, Ki-67-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group. D, Mesothelin,P53,PUMA,bax and bcl-2 protein was detected by Western blot in tumor samples. E, Mesothelin regulates cell apoptosis in orthotopic pancreatic cancer tumors. TUNEL-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean±SE of 8 tumor samples from individual mouse in each group. F, Cleaved capase-3-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group.
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Figure 6: Effects of mesothelin on pancreatic cancer growth in the xenograft nude mouse model.A. Subcutaneous tumor volume of HPAC- mesothelin,Capan-2- mesothelin and MIA PaCa-2- mesothelin and their mock cells(2 × 106)were subcutaneously inoculated into nude mice (8 mice per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05,*p>0.05. B. Subcutaneous tumor volume of AsPC-1-shRNA mesothelin, Capan-2-shRNA mesothelin and Capan-1-shRNA mesothelin (2 × 106) were injected into the flank of nude mice (eight per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05. C, Ki-67-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group. D, Mesothelin,P53,PUMA,bax and bcl-2 protein was detected by Western blot in tumor samples. E, Mesothelin regulates cell apoptosis in orthotopic pancreatic cancer tumors. TUNEL-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean±SE of 8 tumor samples from individual mouse in each group. F, Cleaved capase-3-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group.

Mentions: In ASPC-1 cells with mt-p53, mesothelin sliencing significantly increased PUMA and bax levels (Figure5F) and caspase-3 activity (Figure5B), but decreased bcl-2 levels (Figure5F). PUMA re-inhibition by PUMA siRNA transfection in mesothelin-sliencing ASPC-1 cells lead to increased survival (Figure6C), decreased apoptosis (Figures5D and E) and caspase-3 activity (Figure5B). This data shown mesothelin sliencing promotes apoptosis and inhibits survival was by p53-independent pathway in ASPC-1 cells with mt-p53. Similar results was shown in CaPan-1 cells(data not shown).


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)

Effects of mesothelin on pancreatic cancer growth in the xenograft nude mouse model.A. Subcutaneous tumor volume of HPAC- mesothelin,Capan-2- mesothelin and MIA PaCa-2- mesothelin and their mock cells(2 × 106)were subcutaneously inoculated into nude mice (8 mice per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05,*p>0.05. B. Subcutaneous tumor volume of AsPC-1-shRNA mesothelin, Capan-2-shRNA mesothelin and Capan-1-shRNA mesothelin (2 × 106) were injected into the flank of nude mice (eight per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05. C, Ki-67-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group. D, Mesothelin,P53,PUMA,bax and bcl-2 protein was detected by Western blot in tumor samples. E, Mesothelin regulates cell apoptosis in orthotopic pancreatic cancer tumors. TUNEL-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean±SE of 8 tumor samples from individual mouse in each group. F, Cleaved capase-3-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Effects of mesothelin on pancreatic cancer growth in the xenograft nude mouse model.A. Subcutaneous tumor volume of HPAC- mesothelin,Capan-2- mesothelin and MIA PaCa-2- mesothelin and their mock cells(2 × 106)were subcutaneously inoculated into nude mice (8 mice per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05,*p>0.05. B. Subcutaneous tumor volume of AsPC-1-shRNA mesothelin, Capan-2-shRNA mesothelin and Capan-1-shRNA mesothelin (2 × 106) were injected into the flank of nude mice (eight per treatment group). Tumor size was measured weekly for 4 weeks. **p < 0.05. C, Ki-67-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group. D, Mesothelin,P53,PUMA,bax and bcl-2 protein was detected by Western blot in tumor samples. E, Mesothelin regulates cell apoptosis in orthotopic pancreatic cancer tumors. TUNEL-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean±SE of 8 tumor samples from individual mouse in each group. F, Cleaved capase-3-positive cells were counted under ×400 magnifications in five randomly selected areas in each tumor sample. Mean ± SE of 8 tumor samples from individual mouse in each group.
Mentions: In ASPC-1 cells with mt-p53, mesothelin sliencing significantly increased PUMA and bax levels (Figure5F) and caspase-3 activity (Figure5B), but decreased bcl-2 levels (Figure5F). PUMA re-inhibition by PUMA siRNA transfection in mesothelin-sliencing ASPC-1 cells lead to increased survival (Figure6C), decreased apoptosis (Figures5D and E) and caspase-3 activity (Figure5B). This data shown mesothelin sliencing promotes apoptosis and inhibits survival was by p53-independent pathway in ASPC-1 cells with mt-p53. Similar results was shown in CaPan-1 cells(data not shown).

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