Involvement of both intrinsic and extrinsic pathways in IFN-γ-induced apoptosis that are enhanced with cisplatin
Introduction
Extensive experiments in a range of animal cancer models suggest that endogenous IFN-γ is involved in immune surveillance of tumours via a combination of lymphocyte mediated responses, direct actions on tumour cells and inhibition of tumour angiogenesis (reviewed in [1]). Studies looking at the direct effects of human IFN-γ on tumour cells revealed that this cytokine inhibited the growth of human ovarian tumour xenografts growing in nude mice [2]. Both DNA synthesis was inhibited and apoptosis induced [3]. Human IFN-γ can also directly inhibit the growth of human ovarian cancer cell lines and primary ovarian epithelial cancer cells derived from patients ascites in vitro. Again this was associated with the induction of apoptosis [4], [5]. These effects required 2–3 days of exposure to IFN-γ for an irreversible effect on cell survival. A pilot study, conducted to see whether exogenous IFN-γ-induced cell death in vivo, showed that 2/6 patients had a 90% reduction in the number of tumour cells in ascites after treatment; some of this response could be attributed to apoptosis [5]. Clinical benefit as assessed by intervals in paracentesis was observed in these two patients.
In addition to its ability to induce apoptosis in ovarian cancer cells, IFN-γ can also initiate apoptosis in other cell types and/or sensitise them to subsequent death signals delivered by mediators such as TNF superfamily members. This involves the induction of a number of apoptotic-related genes involved in both the extrinsic and intrinsic apoptosis pathways. IFN-γ-induced apoptosis has been associated with caspase upregulation and activation. For example, in human erythroid colony-forming cells (ECFC), IFN-γ upregulates and activates caspases 2, -6, -8 and -9 [6] and in colorectal adenocarcinoma cell lines IFN-γ activates caspase 8 and caspase 3 thus triggering apoptosis [7]. In other cell types e.g. breast and colon carcinoma cells, and lung epithelial cells, IFN-γ enhances caspase 8 expression which sensitises cells to apoptosis by CD95L/TRAIL [8], [9], [10], [11]. IFN-γ also regulates members of the Bcl-2 family, decreasing expression of anti-apoptotic molecules such as Bcl-2 and Bcl-XL and increasing expression of pro-apoptotic molecules, e.g. Bax/Bak [12].
IFN-γ can also induce growth arrest in a number of cell types. The progression of cells through all phases of the cell cycle can be slowed by IFN-γ [13], although in some cells there are more pronounced effects on G1/G0 [14]. Differences in the point of cell cycle arrest indicate that diverse growth inhibitory mechanisms are employed by IFN-γ.
Clinically, ovarian cancer remains a difficult disease to treat since most patients present with advanced disease. A regime of platinum-taxol chemotherapy following cytoreductive surgery is common, but patients often relapse. A number of clinical studies show that IFN-γ has some activity against advanced ovarian cancer. In a phase II trial, 108 patients with residual disease documented at second look laparotomy after first line cisplatin-based chemotherapy, were treated with intraperitoneal, i.p., IFN-γ twice a week for three-four months [15]. Of 98 assessable patients, 23 achieved complete and eight partial response. In a randomised phase III study, 148 women treated with cisplatin and cyclophosphamide as first line chemotherapy for ovarian cancer were randomly allocated to receive additional IFN-γ three times weekly on alternate weeks [16]. IFN-γ administration was associated with a significant increase in progression free survival but an observed increase in overall survival was not statistically significant. A large global randomised phase III trial, GRACES (Gamma Interferon and Chemotherapy Efficacy Study), is currently in progress to assess the activity of IFN-γ in combination with platinum and taxol.
In this study we sought to investigate further the mechanisms behind the direct anti-proliferative activity of IFN-γ in ovarian cancer cell lines. We also looked at the effects of IFN-γ used in combination with cisplatin, an agent commonly used in ovarian cancer. Further analysis of the molecular events involved in IFN-γ-induced apoptosis may aid identification of cancer patients that are most likely to respond to combination therapy with this cytokine.
Section snippets
Cell lines
PEO1 was derived from ascites in a patient with poorly differentiated adenocarcinoma and were obtained from Dr. S. Langdon, Cancer Research UK Edinburgh Medical Oncology Unit, UK. OVCAR-3 originated from ascites in a patient with poorly differentiated papillary adenocarcinoma (purchased from the American Type Culture Collection [ATCC]).
Tissue culture
All cell lines were grown in a humidified atmosphere at 37 °C (10% CO2) under pyrogen-free conditions. Cells were grown in RPMI 1640 (Invitrogen, UK) supplemented
Results
In two previous papers we have provided evidence that IFN-γ, at doses ranging from 10 to 5000 U/ml, has a time and dose-dependent anti-proliferative effect on the majority of ovarian cancer cell lines and freshly isolated primary tumour cells from the ascites of ovarian cancer patients in vitro [4], [5]. Here we have attempted to determine the relative contribution of cell growth arrest and apoptosis in this action. We used two cell lines that are sensitive to the anti-proliferative effects of
Discussion
We, and others, have previously determined that apoptosis (as measured, for example, by TUNEL and EM) is induced by IFN-γ in ovarian cancer cells [4], [21]. In this study we have concentrated on elucidating the mechanisms by which this apoptosis occurs and subsequently looked at the contribution apoptosis makes to the well documented anti-proliferative effect of IFN-γ.
We have shown that the intrinsic apoptosis pathway was activated in response to IFN-γ in ovarian cancer cell lines. Published
Conflict of interests statement
None declared.
Acknowledgement
This work was supported by Cancer Research UK.
References (35)
- et al.
Interferon gamma induces cell cycle arrest and apoptosis in a model of ovarian cancer: enhancement of effect by batimastat
Eur J Cancer
(1997) - et al.
Interferon gamma induces upregulation and activation of caspases 1, 3, and 8 to produce apoptosis in human erythroid progenitor cells
Blood
(1999) - et al.
Potentiation of Fas- and TRAIL-mediated apoptosis by IFN-gamma in A549 lung epithelial cells: enhancement of caspase-8 expression through IFN-response element
Cytokine
(2002) - et al.
Interferon-gamma modulates a p53-independent apoptotic pathway and apoptosis-related gene expression
J Biol Chem
(1997) - et al.
Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells
FEBS Lett
(1996) - et al.
Bid a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors
Cell
(1998) - et al.
Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis
Cell
(1998) - et al.
Non-specific effects of methyl ketone peptide inhibitors of caspases
FEBS Lett
(1999) - et al.
Modulation of cisplatin cytotoxicity by human recombinant interferon-gamma in human ovarian cancer cell lines
Eur J Cancer
(1994) - et al.
Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis
Mol Cells
(2002)
The three Es of cancer immunoediting
Annu Rev Immunol
Antitumor activity of gamma-interferon in ascitic and solid tumor models of human ovarian cancer
Cancer Res
Cytotoxic response of ovarian cancer cell lines to IFN-gamma is associated with sustained induction of IRF-1 and p21 mRNA
Brit J Cancer
IFN-gamma induces apoptosis in ovarian cancer cells in vivo and in vitro
Clin Cancer Res
Apoptosis of colorectal adenocarcinoma induced by 5-FU and/or IFN-gamma through caspase 3 and caspase 8
Int J Oncol
Interferon-gamma modulates TRAIL-mediated apoptosis in human colon carcinoma cells
Anticancer Res
Interferon-gamma treatment elevates caspase-8 expression and sensitizes human breast tumor cells to a death receptor-induced mitochondria-operated apoptotic program
Cancer Res
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