Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Mar-Apr;11(2):349-61.
doi: 10.1111/j.1582-4934.2007.00013.x. Epub 2007 Mar 22.

bcl-2-specific siRNAs restore gemcitabine sensitivity in human pancreatic cancer cells

Kinya Okamoto  1 , Matthias Ocker, Daniel Neureiter, Otto Dietze, Steffen Zopf, Eckhart G Hahn, Christoph Herold
Affiliations

bcl-2-specific siRNAs restore gemcitabine sensitivity in human pancreatic cancer cells

Kinya Okamoto et al. J Cell Mol Med. 2007 Mar-Apr.

Abstract

Gemcitabine has been shown to ameliorate disease related symptoms and to prolong overall survival in pancreatic cancer.Yet, resistance to Gemcitabine is commonly observed in this tumour entity and has been linked to increased expression of anti-apoptotic bcl-2. We therefore investigated if and to what extend silencing of bcl-2 by specific siRNAs (siBCL2) might enhance Gemcitabine effects in human pancreatic carcinoma cells. siBCL2 was transfected into the pancreatic cancer cell line YAP C alone and 72 hrs before co-incubation with different concentrations of Gemcitabine. Total protein and RNA were extracted for Western-blot analysis and quantitative polymerase chain reaction. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siBCL2 alone, Gemcitabine and control siRNA or Gemcitabine and siBCL2 for 21 days. Combination of both methods lead to a synergistic induction of apoptosis at otherwise ineffective concentrations of Gemcitabine. Tumour growth suppression was also potentiated by the combined treatment with siBCL2 and Gemcitabine in vivo and lead to increased TUNEL positivity. In contrast, non-transformed human foreskin fibroblasts showed only minor responses to this treatment. Our results demonstrate that siRNA-mediated silencing of anti-apoptotic bcl-2 enhances chemotherapy sensitivity in human pancreatic cancer cells in vitro and might lead to improved therapy responses in advanced stages of this disease.

PubMed Disclaimer

Figures

1
1
Sensitivity of YAP C pancreatic carcinoma cells and non-malignant human foreskin fibroblasts (HF) to Gemcitabine treatment. (A) Induction of apoptosis after treatment with different concentrations of Gemcitabine. (B) Decrease in cell numbers relative to untreated control (= 100%) after incubation with different concentrations of Gemcitabine. Values are mean ± S.E.M. of three independent experiments. *P < 0.05 versus untreated controls.
2
2
bcl-2 mRNA levels (normalized to GAPDH on log tables). (A) siRNA transfection: Bcl-2 mRNA level was down-regulated by bcl-2 specific siRNA (siBCL2) in YAP C, but not by control siRNA, with maximum effect 96 hrs after transfection. (B) Gemcitabine treatment: 1 μM but not 0.01 μM Gemcitabine treatment up-regulated bcl-2 mRNA levels in YAP C, but downregulated it in HF. (C) Combination treatment: Gemcitabine treatment 72 hrs after siBCL2 transfection leads to pronounced down regulation of bcl-2 mRNA in YAP C compared to control siRNA and Gemcitabine treatment as well as HF controls. Bars represent means of two independent experiments with pooled RNA samples from three independent experimental settings.
3
3
Expression of the bcl-2 protein determined by Western blotting. (A) siRNA transfection inhibited bcl-2 protein expression of YAP C but not in HF. (B) 1 μM Gemcitabine treatment tended to down-regulate bcl-2 protein expression in YAP C in comparison to 0.01 μM Gemcitabine treated YAPC cell lines or controls. (C) The combination treatments of siBCL2 and Gemcitabine lead to a pronounced down-regulation of bcl-2 protein in YAP C but not in HF cells.
4
4
Apoptosis rate and numbers of viable cells after siRNA transfection. (A) 10 nM siBCL2 lead to a moderate increase in apoptosis in pancreatic cancer cells and in HF while the control siRNA was ineffective in both cell lines. (B) siBCL2 lead to a significant reduction in of viable cells in YAP C, while in HF only a moderate but not significant reduction of cell number was observed. Mock or control siRNA transfections did not induce apoptosis or reduce the number of viable cells in both cell lines.Values are mean ± S.E.M. of three independent experiments. *P < 0.05 versus mock transfected cells and untreated controls.
5
5
Sensitivity of YAP C pancreatic carcinoma cells and HF to combined siRNA and Gemcitabine treatment. (A) Apoptosis rate of YAP C, but not of HF, significantly increased after addition of 1 or 0.01 μM Gemcitabine 72 hrs after transfection with 10 nM siBCL2. In combination with control siRNA, only the high concentration of Gemcitabine leads to significant levels of apoptosis, indicating the predominant cytotoxic effect of Gemcitabine at this concentration. (B) Addition of 0.01 μM Gemcitabine to siBCL2 reduced the number of viable YAP C cells by more than 50%, while HF showed only a minor response to 0.01 μM Gemcitabine and siBCL2 combination therapy. Combinations with control siRNA and 1 μM Gemcitabine show only the Gemcitabine effect but no further reduction in both cell lines. Results are mean ± S.E.M. of three independent experiments. *P < 0.05 versus untreated controls and 0.01 μM Gemcitabine with pre-treatment of mock transfection.
6
6
Results of pancreatic cancer xenografts in nude mice. (A) Growth rate of pancreatic cancer xenografts: After 21 days, single agents (siBCL2, control siRNA or Gemcitabine at 8 mg/kg) as well as the combination of Gemcitabine with control siRNA only lead to a minor decrease in relative tumour size compared to saline controls (1.6, 1.7, 1.45 and 1.46, respectively), while tumour size relative to day 1 reached 1.11 in animals receiving Gemcitabine and siBCL2. *P < 0.05 versus untreated control or control experiments. (B) Western-blot analysis of pancreatic cancer xenografts revealed a down regulation of bcl- 2 protein in animals receiving siBCL2 and Gemcitabine, while no change of bcl-2 expression was observed in the other groups. (C) Immunohistochemistry staining of pancreatic cancer xenografts. A strong cytoplasmic staining for bcl-2 was detectable in saline controls in contrast to intermediate or little staining in sections from xenografts of Gemcitabine and siBCL2 treated animals. Lowest proliferation index (percentage of PCNA-positive nuclei) was detected in xenografts treated with siBCL2/Gemciatbine (25.6%). The maximum of TUNEL-positive areas were seen in xenografts with siBCL2/Gemcitabine (3.6%). 200 × magnification of bcl-2 and PCNA immunohistochemistry and 100 × for TUNEL staining.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Keighley MR. Gastrointestinal cancers in Europe. Aliment Pharmacol Ther. 2003;18:7–30. - PubMed
    1. DiMagno EP, Reber HA, Tempero MA. AGA technical review on the epidemiology, diagnosis, and treatment of pancreatic ductal adenocarcinoma. American Gastroenterological Association. Gastroenterology. 1999;117:1464–84. - PubMed
    1. Simon B, Printz H. Epidemiological trends in pancreatic neoplasias. Dig Dis. 2001;19:6–14. - PubMed
    1. Raraty MG, Magee CJ, Ghaneh P, Neoptolemos JP. New techniques and agents in the adjuvant therapy of pancreatic cancer. Acta Oncol. 2002;41:582–95. - PubMed
    1. Nitecki SS, Sarr MG, Colby TV, Van Heerden JA. Long-term survival after resection for ductal adenocarcinoma of the pancreas. Is it really improving? Ann Surg. 1995;221:59–66. - PMC - PubMed

Publication types