Tamoxifen (TMX)/Fas induced growth inhibition of human cholangiocarcinoma (HCC) by gamma interferon (IFN-gamma) - PubMed (original) (raw)

Tamoxifen (TMX)/Fas induced growth inhibition of human cholangiocarcinoma (HCC) by gamma interferon (IFN-gamma)

Selwyn M Vickers et al. Ann Surg. 2002 Jun.

Abstract

Objectives: To evaluate the response of human cholangoicarcinoma cells to TMX treatment through the Fas pathway by pretreatment with IFN-gamma.

Summary background data: Cholangiocarcinoma remains one of the most difficult tumors to treat in clinical medicine. Currently, there are no effective chemotherapy treatments for this disease. Surgery offers the only opportunity for a cure, with the majority of patients failing to qualify for such treatment. This study seeks to evaluate a potential new modality for treatment of this disease.

Methods: Human cholangiocarcinoma cells were treated with anti Fas mab and sorted to two populations (Fas-positive and Fas-negative) by FAC analysis. In vitro individual cell populations were pretreated with IFN-gamma 250 units/mL x 18hs. The treated cells assayed for caspase 3, 7, 8, Bak, and for apoptosis with Annexin V after treatment with or without TMX. In Vivo 2 x 106 5 SK-ChA-1 Fas-negative cells were injected into nude mice for development of tumor xenografts. Mice received either no treatment or intra tumor IFN-gamma and/or intra peritoneal TMX.

Results: More than 90% (90% +/- 3.5%) of Fas-positive and 70% (71 +/- 2.3%) of Fas-negative cells underwent apoptosis after TMX treatment when pretreated with IFN-gamma. In contrast, TMX alone and IFN-gamma alone stimulated apoptosis by only 22% (22 +/- 3%) P <.00013, and 17% (17 +/- 2%) P <.0001 in Fas-ve cells respectively. In vivo human cholangiocarcinomas xenograft growth was significantly inhibited by a combination of TMX + IFN-gamma compared to controls P <.0007.

Conclusion: TMX exposure to human cholangiocarcinoma after pretreatment with IFN-gamma allows for induction of apoptosis in vitro and significant inhibition tumor xenograft growth. The combination of these two compounds may provide novel treatment regimen for cholangiocarcinoma.

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Figures

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Figure 1. IFN-γ and TMX induced apoptosis. SK-ChA-1 Fas –ve and Fas-positive a grown in all culture were pretreated for 18 hours of IFN-γ (250 units/mL) and then treated with 10 ηm of TMX. Cells were examined for apoptosis flow via cytometric analysis of Annexin V.

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Figure 2. IFN-γ up-regulates Fas, caspase-8 to 3, -7 and Bak. (A) Fas-positive and Fas-negative cells were lysed after 4 hours treatment with or without IFN-γ (250 units/mL) and total RNA was isolated. 15 μg of RNA was hybridized with 32P-labeled antisense RNA probes of Fas, caspase-8, -3, -7 and GAPDH (housekeeping gene). After RNase treatment, protected probes were resolved in 5% sequencing gel. (B) Fas-positive and Fas-negative cells were incubated with IFN-γ (250 units/mL) or medium alone, then whole cell lysates were prepared. Equal amount of proteins (20 μg) were separated by 12% SDS-PAGE and immunoblotted for Bak. (C) IFN-γ facilitates TMX-induced caspase-3 activation. Fas-positive and Fas-negative cells were incubated with IFN-γ (250 units/mL) or medium alone for 18 hours followed by additional 20 hours incubation with TMX (10 μmol/L). Whole cell lysates were prepared and separated by 15% SDS-PAGE, then immunoblotted for cleaved caspase-3 (active form of caspase-3).

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Figure 3. Effect of IFN-γ and TMX on cholangiocarcinoma tumors in nude mice. Following the protocol outlined in method, mice were inoculated subcutaneoulsy with Fas -cells and treated with PBS, IFN-γ, TMX, or IFN-γ/TMX. Tumor volumes measured were described in methods.

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