Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24

Abstract

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

Figure 1

A, Purification of MDA7/IL-24 by immunoaffinity chromatography. All samples were resolved by 12% SDS-PAGE. Lane 1, Heat-denatured, reduced-starting supernatant. Lane 2, Heat-denaturation and reduced-affinity purification of MDA7/IL-24 performed with resolving gel (pH 8.9) 0.5 M Tris-HCl. An analysis of MDA7/IL-24 by ELISA showed a 3000-fold increase in purity over the supernatant. BSA = bovine serum albumin. Lane 3, Western blot with anti–N-terminal monoclonal of the elution fraction. B, Further purification of MDA7/IL-24 by immunoaffinity and cation exchange purification. All samples were resolved by 12% SDS-PAGE (pH 8.32) 1.5 M Tris-HCl. Affinity-purified MDA7/IL-24 was further purified by cation exchange chromatography. Lane 1, MDA7/IL-24 affinity purified (starting material loaded onto column). Lane 2, Wash from the column. Lane 3, Elution at 1 M NaCl. Lane 4, Western blot with anti–N-terminal monoclonal of the elution fraction.

Figure 2

A, Affinity-purified MDA7/IL-24 induces cell death in melanoma cells. A panel of melanoma cells (A375, A375.S2, MeWo, SK-Mel-1, WM35, WM793) was exposed to a dose titration of affinity-purified MDA7/IL-24 and assessed for cell death by trypan blue exclusion after 5 days. Cells were analyzed for apoptosis via Annexin V–FITC staining followed by fluorescence-activated cell sorter analysis. Black bars = percentage of dead cells (trypan blue positive) and gray bars = percentage of apoptotic cells based on total cells. B, MDA7/IL-24 kills melanoma cells via ligand binding to type 1 IL-20R. MeWo melanoma cells were exposed to a dose increase of affinity-purified MDA7/IL-24 and assessed for cell death (cytotoxicity and apoptosis) after 5 days. Black bars = percentage of dead cells (trypan blue positive) and gray bars = percentage of apoptotic cells (by Annexin-V-FITC staining) based on total cells. .Cells were also treated with antibodies to MDA7/IL-24, IL-20R1 antibody, or IL-22R1 antibody. MDA7/IL-24 protein was added to antibodies, and cell killing was assessed. Data were plotted as mean + SD. Error bars represent one standard deviation of the mean. C, MDA7/IL-24–transfected HEK-293 cells (293M) kill melanoma cells. MeWo melanoma cells were cocultured with MDA7/IL-24–transfected 293 cells and assessed for cell death after 5 days. Black bars = percentage of dead cells (trypan blue positive) and gray bars = percentage of living cells based on total cells. Cells were also cocultured with nontransfected HEK-293 cells and MeWo cells. Data were plotted as mean + SD. Error bars represent one standard deviation of the mean.

Figure 3

A and B, Purified MDA7/IL-24 induces accumulation of cells in the S phase. A, MeWo melanoma cells were exposed to a dose titration of affinity-purified MDA7/IL-24 and assessed by propidium iodide staining for distribution of cells in each cell cycle. B, MeWo melanoma cells were exposed to a dose titration of affinity/ion exchange–purified MDA7/IL-24 and assessed by propidium iodide for distribution of cells in each cell cycle. Each arrow indicates S phase population bars. C and D, Affinity-purified and affinity/ion exchange–purified MDA7/IL-24 is cytotoxic. C, MeWo melanoma cells were exposed to a dose titration of affinity-purified MDA7/IL-24 and assessed for cell death by trypan blue exclusion after 5 days. Cells were analyzed for apoptosis via Annexin V–FITC staining followed by fluorescence-activated cell sorter analysis. For (C) and (D), black bars = percentage of dead cells (trypan blue positive) and gray bars = percentage of apoptotic cells based on total cells. Error bars represent one standard deviation of the mean. D, MeWo melanoma cells were exposed to a dose increase of affinity/ion-purified MDA7/IL-24 and assessed for cell death after 5 days. Error bars represent one standard deviation of the mean.

Figure 4

MDA7/IL-24-induced interferon (IFN) beta secretion from melanoma cells. A, MeWo cells exposed to affinity; B, affinity/ion-purified MDA7/IL-24 and supernatants from melanoma cells treated with affinity-purified and affinity/ion exchange–purified MDA7/IL-24, respectively, were tested by ELISA after 5 days for IFN beta. Error bars represent one standard deviation of the mean.

Figure 5

Interferon (IFN) beta blockade partially diminishes MDA7/IL-24-induced cell death. MeWo melanoma cells were exposed to a dose titration of affinity-purified MDA7/IL-24 in the presence of neutralizing antibodies to IFN beta and assessed for cell death after 5 days. Gray bars = percentage of living cells, and black bars = percentage of dead cells (trypan blue positive) based on total cells. Anti-IFN beta–neutralizing antibody was used at 5× molar excess for predicting the level of IFN beta in supernatant with the IgG control added at the same concentration. Error bars represent one standard deviation of the mean.

Figure 6

Class I (alfa [a] and beta [b]) interferons (IFNs) are cytotoxic to melanoma cells. A, A375 melanoma cells were exposed to a dose titration of both class I and class II (gamma [g]) IFNs and assessed for cell death after 24 h. For (A) and (B), gray bars = total number of live cells and black bars = total number of dead cells, trypan blue positive. Error bars represent one standard deviation of the mean. B, MeWo melanoma cells were exposed to a dose titration of both class I and II IFN sand assessed for cell death after 24 h. Error bars represent one standard deviation of the mean.

Figure 7

MDA7/IL-24 differentially regulates genes on signaling and apoptosis. A, MeWo melanoma cells were exposed to either a dose titration of affinity-purified MDA7/IL-24 or cocultured with MDA7/IL-24–transfected HEK-293 cells. After 48 h of treatment, cells were assessed for their apoptotic and signaling pathways in gene regulation with commercially available gene array analysis. B, MeWo melanoma cells were exposed to either a dose titration of affinity-purified MDA7/IL-24 or cocultured with MDA7/IL-24–transfected HEK-293 cells. After 48 h of treatment, lysates were taken and proteins were precipitated and washed in 1× PBS, resolved by SDS-PAGE, transferred to nitrocellulose, and Western blotted for apoptotic and signaling pathway markers

Figure 7

MDA7/IL-24 differentially regulates genes on signaling and apoptosis. A, MeWo melanoma cells were exposed to either a dose titration of affinity-purified MDA7/IL-24 or cocultured with MDA7/IL-24–transfected HEK-293 cells. After 48 h of treatment, cells were assessed for their apoptotic and signaling pathways in gene regulation with commercially available gene array analysis. B, MeWo melanoma cells were exposed to either a dose titration of affinity-purified MDA7/IL-24 or cocultured with MDA7/IL-24–transfected HEK-293 cells. After 48 h of treatment, lysates were taken and proteins were precipitated and washed in 1× PBS, resolved by SDS-PAGE, transferred to nitrocellulose, and Western blotted for apoptotic and signaling pathway markers

Figure 8

MDA7/IL-24 induces tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–receptor 2 (TRAIL-R2 or DR5) in human melanoma cells. A, MeWo melanoma cells were exposed to a dose titration of affinity-purified MDA7/IL-24. After 48 h of treatment, cells were collected and cytospins prepared for cytochemical assessment of their TRAIL receptor (R1 and R2) expression. (anti-DR4 or anti-DR5, AEC, hematoxylin. Original magnification X400). B, A second set of identical component were reserved in culture for 48 h of treatment, lysates were taken, and proteins were precipitated and washed in 1× PBS, resolved by SDS-PAGE, transferred to nitrocellulose and Western blotted for TRAIL-R1 and R2. MeWo melanoma cells were exposed to media only (MeWo I), protein preparation media only (MeWo II), and a dose titration of affinity-purified MDA7/IL-24. After 48 h of treatment, lysates were taken, and proteins were precipitated and washed in 1× PBS, resolved by SDS-PAGE, transferred to nitrocellulose and Western blotted for TRAIL-R2.