Involvement of both intrinsic and extrinsic pathways in IFN-γ-induced apoptosis that are enhanced with cisplatin

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Abstract

IFN-γ has direct anti-proliferative effects on ovarian cancer cell lines and tumour cells isolated from ovarian cancer ascites. The aim of this study was to further elucidate the mechanisms involved. An IFN-γ-mediated cell cycle blockade was detectable in synchronised cell populations. Apoptosis, which was caspase dependent, was also induced. When caspase activity was blocked, the anti-proliferative effect of IFN-γ was only partially reduced indicating independent roles for both growth inhibition and apoptosis in its actions. We have demonstrated involvement of the intrinsic apoptotic pathway; IFN-γ treatment resulted in mitochondrial membrane depolarisation, cytochrome c release into the cytosol and activation of caspase 9. Cytochrome c release was blocked by the presence of a general caspase inhibitor, suggesting a role for caspases upstream of the mitochondria. One candidate is caspase 8, which was also activated in cells treated with IFN-γ. Levels of Bid, a pro-apoptotic molecule that can mediate mitochondrial membrane permeabilisation when cleaved by caspase 8, were also decreased and indicated a potential link between these two pathways in IFN-γ-induced apoptosis. Furthermore, together with cisplatin, IFN-γ exerted a more powerful anti-proliferative effect.

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.

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