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Gli1 maintains cell survival by up-regulating IGFBP6 and Bcl-2 through promoter regions in parallel manner in pancreatic cancer cells.

Xu XF, Guo CY, Liu J, Yang WJ, Xia YJ, Xu L, Yu YC, Wang XP - J Carcinog (2009)

Bottom Line: Cyclopamine inhibited cell proliferation at low concentration and induced apoptosis at high concentration.Effect of RNA interference (RNAi) for Gli1 to cell survival is mainly due to proliferation inhibition though involved in apoptosis.The transcription factor Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes and the levels of IGFBP6, proliferating cell nuclear antigen (PCNA) and Bcl-2 messenger RNA (mRNA) were decreased as well as Gli1 mRNA significantly by cyclopamine or RNAi in cultured pancreatic cancer cells (p < 0.01).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, The Tenth Hospital, Tongji University, Shanghai, China. wangxp1965@yahoo.com.cn.

ABSTRACT

Background: Aberrant activation of Hedgehog (Hh) signaling pathway has been reported to be related to malignant biological behavior of pancreatic cancer but its mechanism is unclear yet. Since IGF pathway and Bcl-2 family are involved in proliferation and apoptosis of pancreatic cancer cells, we hypothesize that they are possibly associated with Hh pathway.

Materials and methods: We studied the relationship of Shh-Gli1 signaling pathway with proliferation and apoptosis of pancreatic cancer cells and the regulation of transcription factor Gli1 to insulin-like growth factor binding protein 6 (IGFBP6) and Bcl-2 genes at the level of transcription.

Results: Sonic hedgehog (Shh), Smoothened (Smo), patched and Gli1 were expressed in pancreatic cancer cells. Cyclopamine inhibited cell proliferation at low concentration and induced apoptosis at high concentration. Effect of RNA interference (RNAi) for Gli1 to cell survival is mainly due to proliferation inhibition though involved in apoptosis. The transcription factor Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes and the levels of IGFBP6, proliferating cell nuclear antigen (PCNA) and Bcl-2 messenger RNA (mRNA) were decreased as well as Gli1 mRNA significantly by cyclopamine or RNAi in cultured pancreatic cancer cells (p < 0.01). Finally PCNA, IGFBP6 and Bcl-2 mRNA were upregulated as well as Shh or Gli1 in pancreatic cancer tissues (p < 0.01).

Conclusions: Our study reveals that Gli1 maintained cell survival by binding the promoter regions and facilitating transcription of IGFBP6 and Bcl-2 genes in a parallel manner in pancreatic cancer cells and suggests it may be one of the mechanisms of Shh-Gli1 signaling pathway in pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus

Apoptosis of pancreatic cancer cells. A: apoptosis percentage of pancreatic cancer cells. a: cyclopamine-induced apoptosis; b: purmorphamine counteract effect of cyclopamine; c: siRNA for Gli1. B: apoptotic cells exposed to 150 μM cyclopamine or 100 nM siRNA for Gli1 by FACS assay. C: apoptotic cells stained by annexin V-FITC/PI and fluorescence microscopy; D: DNA ladder of apoptotic cells. (a1, b1, c1 and d1: pancreatic cancer cells exposed to cyclopamine; a2, b2, c2 and d2: pancreatic cancer cells exposed to siRNA for Gli1; a2: BxPC3; b1, b2: AsPC-1; c1, c2: Panc-1; d1, d2: sW1990.)
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Figure 0003: Apoptosis of pancreatic cancer cells. A: apoptosis percentage of pancreatic cancer cells. a: cyclopamine-induced apoptosis; b: purmorphamine counteract effect of cyclopamine; c: siRNA for Gli1. B: apoptotic cells exposed to 150 μM cyclopamine or 100 nM siRNA for Gli1 by FACS assay. C: apoptotic cells stained by annexin V-FITC/PI and fluorescence microscopy; D: DNA ladder of apoptotic cells. (a1, b1, c1 and d1: pancreatic cancer cells exposed to cyclopamine; a2, b2, c2 and d2: pancreatic cancer cells exposed to siRNA for Gli1; a2: BxPC3; b1, b2: AsPC-1; c1, c2: Panc-1; d1, d2: sW1990.)

Mentions: Annexin V-FITC/PI assay was performed to evaluate apoptosis of cultured pancreatic cancer cells induced by cyclopamine or RNAi for Gli1. FACS assay results showed that the apoptosis percentage of pancreatic cancer cells increased significantly when the concentration of cyclopamine was raised to 100 μM (p < 0.05) and topped at 150 μM cyclopamine (p < 0.01). The percentages of early apoptosis of pancreatic cancer cells treated with cyclopamine at the concentration of < 150 μM were decreased significantly by 15 μM purmorphamine (P < 0.05). For RNAi, the results showed that apoptosis percentage began to increase significantly until the dose was increased to 50 nM (p < 0.01), and increased dose-dependently from 50 to 200 nM; however, there were no significant difference between 100 and 200 nM (p > 0.05). [Figure 3A,3B]. After being exposed to 150 μM cyclopamine or 100 nM siRNA for 24 h and treated with annexin V-FITC/PI, the pancreatic cancer was observed under fluorescence microscopy. The results showed most cells exposed to cyclopamine to have both annexin V-FITC and PI staining, and only small amount of cells were early apoptotic cells, indicated by the green fluorescence of Annexin V-FITC from cell membrane and devoid of PI staining. For RNAi there are a few cells displaying green fluorescence and several were PI stained. [Figure 3C].


Gli1 maintains cell survival by up-regulating IGFBP6 and Bcl-2 through promoter regions in parallel manner in pancreatic cancer cells.

Xu XF, Guo CY, Liu J, Yang WJ, Xia YJ, Xu L, Yu YC, Wang XP - J Carcinog (2009)

Apoptosis of pancreatic cancer cells. A: apoptosis percentage of pancreatic cancer cells. a: cyclopamine-induced apoptosis; b: purmorphamine counteract effect of cyclopamine; c: siRNA for Gli1. B: apoptotic cells exposed to 150 μM cyclopamine or 100 nM siRNA for Gli1 by FACS assay. C: apoptotic cells stained by annexin V-FITC/PI and fluorescence microscopy; D: DNA ladder of apoptotic cells. (a1, b1, c1 and d1: pancreatic cancer cells exposed to cyclopamine; a2, b2, c2 and d2: pancreatic cancer cells exposed to siRNA for Gli1; a2: BxPC3; b1, b2: AsPC-1; c1, c2: Panc-1; d1, d2: sW1990.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2746911&req=5

Figure 0003: Apoptosis of pancreatic cancer cells. A: apoptosis percentage of pancreatic cancer cells. a: cyclopamine-induced apoptosis; b: purmorphamine counteract effect of cyclopamine; c: siRNA for Gli1. B: apoptotic cells exposed to 150 μM cyclopamine or 100 nM siRNA for Gli1 by FACS assay. C: apoptotic cells stained by annexin V-FITC/PI and fluorescence microscopy; D: DNA ladder of apoptotic cells. (a1, b1, c1 and d1: pancreatic cancer cells exposed to cyclopamine; a2, b2, c2 and d2: pancreatic cancer cells exposed to siRNA for Gli1; a2: BxPC3; b1, b2: AsPC-1; c1, c2: Panc-1; d1, d2: sW1990.)
Mentions: Annexin V-FITC/PI assay was performed to evaluate apoptosis of cultured pancreatic cancer cells induced by cyclopamine or RNAi for Gli1. FACS assay results showed that the apoptosis percentage of pancreatic cancer cells increased significantly when the concentration of cyclopamine was raised to 100 μM (p < 0.05) and topped at 150 μM cyclopamine (p < 0.01). The percentages of early apoptosis of pancreatic cancer cells treated with cyclopamine at the concentration of < 150 μM were decreased significantly by 15 μM purmorphamine (P < 0.05). For RNAi, the results showed that apoptosis percentage began to increase significantly until the dose was increased to 50 nM (p < 0.01), and increased dose-dependently from 50 to 200 nM; however, there were no significant difference between 100 and 200 nM (p > 0.05). [Figure 3A,3B]. After being exposed to 150 μM cyclopamine or 100 nM siRNA for 24 h and treated with annexin V-FITC/PI, the pancreatic cancer was observed under fluorescence microscopy. The results showed most cells exposed to cyclopamine to have both annexin V-FITC and PI staining, and only small amount of cells were early apoptotic cells, indicated by the green fluorescence of Annexin V-FITC from cell membrane and devoid of PI staining. For RNAi there are a few cells displaying green fluorescence and several were PI stained. [Figure 3C].

Bottom Line: Cyclopamine inhibited cell proliferation at low concentration and induced apoptosis at high concentration.Effect of RNA interference (RNAi) for Gli1 to cell survival is mainly due to proliferation inhibition though involved in apoptosis.The transcription factor Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes and the levels of IGFBP6, proliferating cell nuclear antigen (PCNA) and Bcl-2 messenger RNA (mRNA) were decreased as well as Gli1 mRNA significantly by cyclopamine or RNAi in cultured pancreatic cancer cells (p < 0.01).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gastroenterology, The Tenth Hospital, Tongji University, Shanghai, China. wangxp1965@yahoo.com.cn.

ABSTRACT

Background: Aberrant activation of Hedgehog (Hh) signaling pathway has been reported to be related to malignant biological behavior of pancreatic cancer but its mechanism is unclear yet. Since IGF pathway and Bcl-2 family are involved in proliferation and apoptosis of pancreatic cancer cells, we hypothesize that they are possibly associated with Hh pathway.

Materials and methods: We studied the relationship of Shh-Gli1 signaling pathway with proliferation and apoptosis of pancreatic cancer cells and the regulation of transcription factor Gli1 to insulin-like growth factor binding protein 6 (IGFBP6) and Bcl-2 genes at the level of transcription.

Results: Sonic hedgehog (Shh), Smoothened (Smo), patched and Gli1 were expressed in pancreatic cancer cells. Cyclopamine inhibited cell proliferation at low concentration and induced apoptosis at high concentration. Effect of RNA interference (RNAi) for Gli1 to cell survival is mainly due to proliferation inhibition though involved in apoptosis. The transcription factor Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes and the levels of IGFBP6, proliferating cell nuclear antigen (PCNA) and Bcl-2 messenger RNA (mRNA) were decreased as well as Gli1 mRNA significantly by cyclopamine or RNAi in cultured pancreatic cancer cells (p < 0.01). Finally PCNA, IGFBP6 and Bcl-2 mRNA were upregulated as well as Shh or Gli1 in pancreatic cancer tissues (p < 0.01).

Conclusions: Our study reveals that Gli1 maintained cell survival by binding the promoter regions and facilitating transcription of IGFBP6 and Bcl-2 genes in a parallel manner in pancreatic cancer cells and suggests it may be one of the mechanisms of Shh-Gli1 signaling pathway in pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus