IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apoptosis - PubMed (original) (raw)

. 2008 Oct 15;68(20):8351-60.

doi: 10.1158/0008-5472.CAN-08-0426.

Ene T Raig, Kristan Guenterberg, Lloyd Brown, Michael R Go, Nisha N Shah, Adrian Lewis, Megan Quimper, Erinn Hade, Gregory Young, Abhik Ray Chaudhury, Katherine J Ladner, Denis C Guttridge, Page Bouchard, William E Carson 3rd

Affiliations

IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apoptosis

Gregory B Lesinski et al. Cancer Res. 2008.

Abstract

We hypothesized that IFN-alpha would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-alpha induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspase-3, procaspase-7, procaspase-8, and procaspase-9 and with cleavage of Bid and poly(ADP-ribose) polymerase. Bortezomib plus IFN-alpha was effective at inducing apoptosis in melanoma cells that overexpressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The proapoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD before the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or small interfering RNA targeting Fas. These data suggest that bortezomib and IFN-alpha act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-alpha displayed statistically significant antitumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-alpha for malignant melanoma.

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Figures

Fig. 1

Fig. 1. Bortezomib and IFN-α induce apoptosis in melanoma and renal cell carcinoma cell lines

(A) The pro-apoptotic effects of IFN-α (104 U/mL), bortezomib (10 nM), or both agents combined were evaluated in human and murine melanoma cell lines (A375 and B16F1) respectively and in human RCC cell lines (RC-54) at 48 hours post-treatment. (B) Human and murine melanoma cell lines were cultured with bortezomib (B; 10 nM), IFN-α (104 U/ml) or both agents combined for 48 hours. Cells treated with PBS served as negative controls in each assay. Cell proliferation was measured as optical density (O.D.) at 570 nm using an MTT cell proliferation assay. Error bars denote the standard deviations of triplicate wells, and graphs are representative of data obtained from at least 3 individual experiments. (C) Synergistic induction of apoptosis in HT144 melanoma cells by bortezomib plus IFN-α. HT144 cells were treated for 48 hours with various doses of bortezomib alone (0.625 - 80 nM), IFN-α alone (0.625 × 103 − 80 × 103 U/mL) or in combination and examined for apoptosis via Annexin V/PI staining. This data was used to calculate the median effect plots. The fraction of cells undergoing apoptosis, or ‘effect’ is presented on the y-axis. The median effect data presented were derived from n = 3 individual experiments and have been averaged. ♦ = Bortezomib + IFN-α; ▲ = Bortezomib; ■ = IFN-α. (D) Combination indices (CI) are presented for HT144 melanoma cells following treatment with bortezomib and IFN-α combined at four doses that demonstrated synergy (5 - 80 nM bortezomib + 5 × 103 − 80 × 103 IFN-α). Data shown were derived from n = 3 individual experiments, where each data point represents an individual experiment. The combination index plots generated present CI values with their respective fraction affected levels for the four dose level combinations of IFN-α and bortezomib.

Fig. 2

Fig. 2. Treatment with bortezomib and IFN-α results in cleavage of caspases-3, -7, and PARP

(A) The HT144 human melanoma cell line was treated for 48 hours with PBS (P), IFN-α (α; 104 U/mL), bortezomib (B; 5 nM or 10 nM) or both agents combined (C) and evaluated by immunoblot analysis for caspase-3, caspase-7, cleaved caspase-7, and cleaved PARP (arrows). Membranes were probed with an anti-β-actin Ab as a loading control. Similar results were obtained in multiple human melanoma cell lines (A375, 18105 MEL, 1259 MEL). (B) Levels of cleaved caspase-3 were measured by flow cytometry with a FITC-conjugated rabbit anti-active caspase-3 mAb (Asp175; Cell Signaling Technology) in the HT144 melanoma cell line 48 hours following treatment. Shaded histograms represent staining with cleaved caspase-3 Ab. Voltage was set based on appropriate isotype control Abs (M1). The percentage of cells positive for cleaved caspase-3 is given in each histogram. (C) Inhibition of caspase activation reverses the pro-apoptotic effects of bortezomib and IFN-α treatment. HT144 cells were treated with PBS, IFN-α (104 U/mL), bortezomib (10 nM) or both agents combined in the presence of the pancaspase inhibitor Z-VAD-FMK or a negative control compound (Z-FA-FMK) at a 50 μM concentration for 48 hours.

Fig. 3

Fig. 3. Bortezomib and IFN-α induce apoptosis in melanoma cells that over-express Bcl-2 and Mcl-1

(A) Combined treatment with bortezomib (10 nM) plus IFN-α (104 U/mL) results in decreased levels of Bcl-2 and Mcl-1 and no change in the expression of Bcl-xL and Bax. A375 melanoma cells were treated with PBS (P), IFN-α (α; 104 U/mL), bortezomib (B; 20nM) or both agents combined (C) 24 hours following transient transfection with the (B) pcDNA3-Bcl-2 construct, (C) pcR3.1-Mcl-1 construct or empty vector (negative control). Lysates were evaluated for Bcl-2 expression and cleavage of PARP via immunoblot analysis. Viability of cells used to make lysates in each experiment was evaluated using trypan blue staining. Transfection efficiency in all experiments was typically > 90% as determined by transfection of A375 cells with a green fluorescent protein (GFP)-expression vector in parallel. All blots shown are representative of n = 3 experiments with similar results. Error bars are derived from n = 3 separate experiments in the A375 melanoma cell line. Comparable data were obtained using cells stably transfected with either construct following selection of transfected clones for 7 days with G418 (data not shown). (D) Decreased levels of Bcl-2 and Mcl-1 are the result of caspase-activation. A375 cells were treated PBS (P), IFN-α (α; 104 U/mL), bortezomib (B; 10nM) or both agents combined (C) in the presence of the pan-caspase inhibitor Z-VAD-FMK or a negative control compound (Z-FA-FMK) at a 50 μM concentration for 48 hours. Cell lysates were analyzed by immunoblot analysis for caspase-3 cleavage, and for Mcl-1 and Bcl-2 expression. Membranes were also probed with an anti-β-actin Ab to control for loading. Arrows denote cleaved forms of caspase-3 or PARP. Viability of cells used to make lysates for immunoblot analysis in each experiment was evaluated using trypan blue staining.

Fig. 4

Fig. 4. Bortezomib and IFN-α act via FADD-induced caspase-8 activation to initiate cell death

(A) Cell lysates were evaluated by immunoblot analysis for processing of pro-caspase-8 (55 kDa) into its cleaved 43 kDa and 41 kDa fragments, pro-caspase-9 (47 kDa) into its cleaved 37 kDa and 35 kDa fragments, and for levels of native Bid protein (22 kDa) following a 48 hour treatment with PBS, IFN-α (104 U/mL), bortezomib (10nM) or both agents combined. (B) A375 cells were treated with PBS, IFN-α, bortezomib or both agents combined in the presence of the Z-IETD-FMK caspase-8 inhibitor or Z-FA-FMK negative control compound at a 50 μM concentration for 48 hours. (C) Fas and FADD associate prior to bortezomib and IFN-α induced apoptosis. Following a 16 hour treatment with PBS, IFN-α, bortezomib or both agents combined, cell lysates were immunoprecipitated with an anti-Fas antibody, and blots were probed with an anti-FADD antibody (or anti-Fas antibody to control for loading). Comparable data were obtained in 1259 MEL, 18105 MEL and A375 cell lines. (D) Bortezomib and IFN-α induced apoptosis is inhibited by a FADD-DN construct. Twenty-four hours following transient transfection with the pcDNA3-FADD-DN or pcDNA3 vectors, A375 melanoma cells were treated for an additional 48 hours with PBS, IFN-α, bortezomib or both agents combined. Lysates were evaluated for FADD expression (or its lower molecular weight, truncated form) and cleavage of PARP as a marker of apoptosis via immunoblot analysis. Viability of cells used to make lysates for immunoblot analysis in each experiment was evaluated using trypan blue staining.

Fig. 5

Fig. 5

(A) Treatment with bortezomib plus IFN-α enhances survival in a murine model of malignant melanoma. B16F1 cells (106) were injected i.p. into C57BL/6 mice (n = 11 per treatment group). One day following tumor challenge, mice were treated intraperitoneally with either PBS (negative control), bortezomib alone (1 mg/kg, twice weekly), IFN-α alone (2 × 104 U/daily), or a combination of bortezomib and IFN-α. (B) Treatment with bortezomib plus IFN-α inhibits the growth of human melanoma xenografts in athymic mice. Human A375 cells (2 × 106) were injected s.c. into Balb/cnu/nu mice (n = 4-6 per group). Mice were treated with PBS, bortezomib, IFN-α or both agents combined. Data represents the mean tumor volume within each group (mm3) while the error bars represent the standard deviation between individual animals. (C) Mice were euthanized on Day 25 whereupon tumors were harvested and stained by TUNEL, and percentages of apoptotic cells were quantified manually. Columns, mean (n = 4-6 per group); bars, standard deviation. *, p = 0.038 versus PBS-treated controls. (D) A representative tumor section from mice treated with Bortezomib and IFN-α combined is shown with arrows depicting TUNEL+ cells used for quantitation (400X magnification).

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