Drug-induced caspase 8 upregulation sensitises cisplatin-resistant ovarian carcinoma cells to rhTRAIL-induced apoptosis

Abstract

Background: Drug resistance is a major problem in ovarian cancer. Triggering apoptosis using death ligands such as tumour necrosis factor-related apoptosis inducing ligand (TRAIL) might overcome chemoresistance.

Methods: We investigated whether acquired cisplatin resistance affects sensitivity to recombinant human (rh) TRAIL alone or in combination with cisplatin in an ovarian cancer cell line model consisting of A2780 and its cisplatin-resistant subline CP70.

Results: Combining cisplatin and rhTRAIL strongly enhanced apoptosis in both cell lines. CP70 expressed less caspase 8 protein, whereas mRNA levels were similar compared with A2780. Pre-exposure of particularly CP70 to cisplatin resulted in strongly elevated caspase 8 protein and mRNA levels. Caspase 8 mRNA turnover and protein stability in the presence or absence of cisplatin did not differ between both cell lines. Cisplatin-induced caspase 8 protein levels were essential for the rhTRAIL-sensitising effect as demonstrated using caspase 8 small-interfering RNA (siRNA) and caspase-8 overexpressing constructs. Cellular FLICE-inhibitory protein (c-FLIP) and p53 siRNA experiments showed that neither an altered caspase 8/c-FLIP ratio nor a p53-dependent increase in DR5 membrane expression following cisplatin were involved in rhTRAIL sensitisation.

Conclusion: Cisplatin enhances rhTRAIL-induced apoptosis in cisplatin-resistant ovarian cancer cells, and induction of caspase 8 protein expression is the key factor of rhTRAIL sensitisation.

Figure 1

Resistance to cisplatin causes cross-resistance to rhTRAIL. Combination therapy overcomes resistance. (A) Survival after 96 h exposure to 0–25 μM (A2780) or 0–100 μM (CP70) cisplatin and (B) survival after 96 h exposure to 0 μg ml⁻¹–0.25 μg ml⁻¹ rhTRAIL as measured by cytotoxicity assays. (C) To determine apoptosis induction, cells were treated for 4 h with cisplatin (2.5, 10 and 30 μM) after which cisplatin was washed away. Twenty hours later, the cells were treated for 4 h with 0.25 μg ml⁻¹ rhTRAIL. Apoptosis was determined with acridine orange staining. Apoptosis in the combinations marked with ^* was significantly enhanced (P<0.05) over apoptosis after single-agent treatment. Data represent the mean±s.d. of at least three independent experiments.

Figure 2

Cellular characteristics involved in sensitivity to rhTRAIL. Cisplatin can induce caspase 8 protein in the low caspase 8-expressing CP70. (A) Basic levels of TRAIL receptor membrane expression in A2780 and CP70 as determined by FACS analysis. Receptor expression is expressed as MFI. (B) Western blot analysis of basic protein expression levels of key determinants of the TRAIL pathway in A2780 and CP70. (C) The effect of cisplatin on DR5 expression in both cell lines. Flow cytometry was performed at 24 h after 4 h incubation with cisplatin. (D) Western blot analysis of caspases 8 and 9 after exposure to cisplatin and rhTRAIL. A2780 and CP70 were incubated with 2.5 μM or 30 μM cisplatin during 4 h, after which the cells were washed. Twenty hours later, unexposed or cisplatin-exposed cells were treated during 4 h with 0.25 μg ml⁻¹ rhTRAIL. β-Actin serves as a loading control. The blots are representative for at least three independent experiments.

Figure 3

Caspase 8 downregulation inhibits cisplatin and rhTRAIL-induced apoptosis, whereas caspase 8 upregulation augmented apoptosis induction. A2780 and CP70 were transfected with siRNA against caspase 8 or negative control siRNA (A and B). The CP70 cells were transiently transfected with a caspase 8 construct (C and D). At 48 h after transfection, cells were treated with 30 μM cisplatin or medium control for 4 h, after which all cells were washed with PBS. Following 16 h of recovery, cells were treated with 0.25 μg ml⁻¹ rhTRAIL or medium control for 4 h. (A) Apoptosis induction was determined by acridine orange staining. Caspase 8 siRNA strongly reduced apoptosis induction by rhTRAIL and cisplatin in both cell lines. (B) Caspase 8 and 3 expression was determined using western blot analysis. The exposure time of the caspase 8 blot of CP70 was increased compared with the blot of A2780. β-Actin serves as a loading control. The blots are representative for at least three independent experiments. (C) Cytospins of CP70 cells (1,2) were generated and stained for caspase 8 and show increased caspase 8 levels following transfection (2a+b) compared with untransfected cells (1a+b). (D) Apoptosis induction following cisplatin and rhTRAIL was determined by acridine orange staining. Caspase 8 upregulation increased apoptosis induction by rhTRAIL alone and by rhTRAIL in combination with cisplatin.

Figure 4

Stability of caspase 8 mRNA or protein does not differ between A2780 and CP70. Differences in caspase 8 protein levels in CP70 are due to changes in protein translation. (A) Expression of caspase 8 mRNA was determined with quantitative RT–PCR after exposure to 30 μM cisplatin, 0.25 μg ml⁻¹ rhTRAIL or the combination in both cell lines. Cells were incubated for 4 h with cisplatin, after which the cells were washed and total RNA was isolated 20 h after cisplatin exposure. Total RNA was isolated 4 h after exposure to rhTRAIL. Quantitative RT–PCR was carried out on cDNA with the SYBR Green method. Quantification was performed with the standard curve method with GAPDH as reference. Basic mRNA levels between A2780 and CP70 were not different. Cisplatin induced caspase 8 mRNA by 1.5 fold. (B) To assess caspase 8 mRNA stability, cells were exposed to 30 μM cisplatin for 4 h or left untreated. Hereafter, all conditions were washed with PBS and received new medium, followed by addition of 5 μg ml⁻¹ actinomycin D (Act D). Total RNA was extracted at the time of Act D addition (t=0 h) and at the indicated time points. Quantitative RT–PCR was performed as in (A). Data represent the mean±s.d. of at least three independent experiments. (C) A2780 and CP70 were exposed to 0.5 μM of the proteasome inhibitor MG132 for 24 h. The following day either rhTRAIL was added the last 4 h of incubation, or the cells were left untreated. Then cells were lysed and subjected to western blot analysis with caspase 8 and 3 antibodies. (D and E) The A2780 and CP70 cell lines were treated with 0.5 μM MG132 for the indicated time points in the presence of 50 μM of z-VAD and lysed or treated with 20 μg ml⁻¹ cycloheximide (CHX) for the indicated time points and lysed. Following SDS–PAGE, immunoblotting was carried out with caspase 8 antibodies. β-Actin serves as a loading control. All immunoblots are representative for at least three independent experiments.

Figure 5

The caspase 8/c-FLIP ratio is not involved in resistance to rhTRAIL. Cells were transfected with siRNA against c-FLIP or luciferase. The next day, cells were exposed to cisplatin for 4 h, after which all cells including those conditions left unexposed were washed. The following day after 4 h incubation with or without 0.25 μg ml⁻¹ rhTRAIL cell lysates were made (A) Cellular FLICE-inhibitory protein (c-FLIP) and caspase 8 cleavage was determined with western blot analysis. The exposure time of the caspase 8 blot of CP70 was increased compared with the blot of A2780. The blots are representative for at least three independent experiments. (B) A small fraction of the same cell suspension used for western blot was plated in a 96-wells plate and apoptosis levels were determined with acridine orange apoptosis assays. Data represent the mean±s.d. of at least three independent experiments. After siRNA against c-FLIP a significant decrease in apoptosis occurred in CP70 after treatment with cisplatin and rhTRAIL (P<0.05).

Figure 6

The tumor suppressor protein p53 causes cisplatin-induced DR5 expression but is not required for rhTRAIL sensitisation of A2780 and CP70. The A2780 and CP70 cell lines were transfected with siRNA against p53 or luciferase and were 24 h later exposed to 30 μM of cisplatin during 4 h, after which all cells including those not exposed to cisplatin, were washed. The next day the cells were treated with 0.25 μg ml⁻¹ rhTRAIL for 4 h. (A) Subsequently, western blotting of p53 and p21 levels was performed on the lysates. β-Actin serves as a loading control. (B) A small fraction of the same cell suspension used for western blot was plated in a 96-wells plate and apoptosis levels were determined with acridine orange apoptosis assays. After downregulation of p53, a significant increase in apoptosis occurred in CP70 after treatment with rhTRAIL. (C) Cleavage patterns of caspase 8, caspase 9 and caspase 3 were determined on lysates after transfection with siRNA against luciferase and p53 as indicated above. The exposure time of the caspase 8 blot of CP70 was increased compared with the blot of A2780. β-Actin serves as a loading control. All blots are representative for at least three independent experiments. (D) At 24 h after siRNA transfection, the cells were exposed for 4 h to 2.5, 10 and 30 μM cisplatin, after which all cells were washed. The next day cells were collected and analysed for DR5 expression by flow FACS cytometry. DR5 expression is represented as MFI. Data represent the mean±s.d. of at least three independent experiments.