PERK-dependent regulation of ceramide synthase 6 and thioredoxin play a key role in mda-7/IL-24-induced killing of primary human glioblastoma multiforme cells

Abstract

Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors. In this study, we defined the proximal mechanisms by which Ad-mda-7 kills glioma cells. Key factors implicated included activation of the endoplasmic reticulum stress kinase protein kinase R-like endoplasmic reticulum kinase (PERK), Ca(++) elevation, ceramide generation and reactive oxygen species (ROS) production. PERK inhibition blocked ceramide or dihydroceramide generation, which were critical for Ca(++) induction and subsequent ROS formation. Activation of autophagy and cell death relied upon ROS formation, the inhibition of which ablated Ad.mda-7-killing activity. In contrast, inhibiting TRX induced by Ad.MDA-7 enhanced tumor cytotoxicity and improved animal survival in an orthotopic tumor model. Our findings indicate that mda-7/IL-24 induces an endoplasmic reticulum stress response that triggers production of ceramide, Ca(2+), and ROS, which in turn promote glioma cell autophagy and cell death.

Figure 1

MDA-7/IL-24 promotes mitochondrial ROS generation. A, GBM6 cells transfected with empty vector plasmid (CMV) or plasmids to express wild-type or mutant TRX. Twelve hours later, cells were infected with Ad.cmv or with Ad.mda-7. The levels of ROS were measured 6, 12, and 24 h after virus infection (n = 12, three experiments ± SEM). *, P < 0.05 greater than parallel CMV transfection; #, P < 0.05 less than parallel CMV transfection. B, GBM6 cells transfected with expression plasmids: CMV, mutant TRX, or SOD2; or with siRNA molecules: scrambled (siSCR) or siSOD2. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. The levels of ROS were measured 24 h after virus infection (n = 8, three experiments ± SEM). *, P < 0.05 greater than parallel CMV transfection; #, P < 0.05 less than parallel CMV transfection. C, GBM6 cells infected with Ad.cmv or Ad.mda-7. Twelve hours after infection, cells were treated as indicated with vehicle (DMSO), apocynin (Apo., 30 μmol/L), diphenyleneiodonium (DPI., 10 μmol/L), cyclosporine A (Csa, 100 nmol/L), and bongkrekic (Bong., 10 μmol/L), and 30 min after drug treatment, the levels of ROS were measured (n = 6, three experiments ± SEM). *, P < 0.05 greater than parallel vehicle control; #, P < 0.05 less than parallel Ad.mda-7 infection. D, GBM6 cells transfected with expression plasmids: CMV, mutant TRX, or SOD2; or with siRNA molecules: scrambled (siSCR) or siSOD2. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Forty-eight hours after infection, viability was determined (± SEM, n = 3). *, P < 0.05 greater than corresponding Ad.cmv value; #, P < 0.05 less than parallel CMV transfection.

Figure 2

MDA-7/IL-24 promotes autophagy via ROS signaling. A, GBM6 cells transfected with vector plasmid (CMV) or to express wild-type or mutant TRX. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Twenty-four hours after infection, cells were isolated and subjected to SDS-PAGE and immunoblotting to determine the expression and/or the phosphorylation of the indicated proteins (n = 3). B, GBM6 cells transfected with vector plasmid (CMV) or plasmids to express wild-type or mutant TRX; all cells were transfected with a plasmid to express LC3-GFP. Following transfection, cells were treated with vehicle (PBS) or with N-acetyl cysteine (10 mmol/L). Twelve to 48 h after infection, slides were examined for punctate GFP vesicles (±SEM, n = 3). *, P < 0.05 greater than parallel CMV transfection; #, P < 0.05 less than parallel CMV transfection. C, GBM6 cells transfected with CMV plasmid or a plasmid to express mutant TRX and in parallel transfected with siRNA molecules: scrambled (siSCR) or to knock down ATG5 or Beclin1. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Forty-eight hours after infection, cells were isolated and viability determined (±SEM, n = 3). #, P < 0.05 less than parallel siSCR transfection/vehicle treatment. D, GBM6 cells transfected with CMV plasmid or a plasmid to express BiP/GRP78. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7, and in parallel adenoviruses to activate or inhibit MEK1 or AKT or p38 MAPK. Where indicated, cells were treated with the JNK inhibitory peptide (10 μmol/L). Forty-eight hours after infection, cells were isolated and viability determined (±SEM, n = 3). *, P < 0.05 greater than parallel CMV transfection/infection; #, P < 0.05 less than parallel CMV transfection/infection.

Figure 3

MDA-7/IL-24 increases cytosolic Ca²⁺, quenching of Ca²⁺ blocks ROS, and inhibition of de novo ceramide synthesis blocks enhanced Ca²⁺. A, GBM6 cells transfected with plasmids: CMV, or to express TRX, mutant TRX, dominant negative PERK or calbindin. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Twelve hours after infection, levels of cytosolic Ca²⁺ were measured (n = 12, two experiments ± SEM). #, P < 0.05 less than parallel CMV transfection. B, GBM6 cells transfected with plasmids: CMV, or to express mutant TRX or calbindin. Twelve hours later, cells infected with Ad.cmv or Ad.mda-7. Twelve hours after infection, levels of ROS were measured (n = 12, two experiments ± SEM). #, P < 0.05 less than parallel CMV transfection. C, GBM6 cells were transfected with CMV or a plasmid to express mutant TRX and in parallel transfected with siRNA molecules: scrambled (siSCR) or to knockdown ASMase. Twelve hours later, cells were infected with Ad.cmv or with Ad.mda-7 and in parallel treated with vehicle (DMSO) or with myriocin (Myr, 1 μmol/L). Twelve hours after infection, levels of ROS were measured (n = 12, two experiments ± SEM). *, P < 0.05 greater than parallel Myr treatment; #, P < 0.05 less than corresponding vehicle-treated values; ##, P < 0.05 less than corresponding value in Myr + siSCR cells. D, GBM6 cells transfected with plasmids: CMV, or to express siLASS6 or calbindin. Twelve hours later, cells infected with Ad.cmv or Ad.mda-7, and in parallel treated with vehicle (DMSO) or with myriocin (Myr, 1 μmol/L). Twelve hours after infection, levels of cytosolic Ca²⁺ were measured (n = 12, two experiments ± SEM). *, P < 0.05 greater than parallel Ad.cmv infection.

Figure 4

Loss of PERK function blocks MDA-7/IL-24–induced increases in Ca²⁺ and ROS, and cell killing. A, left, GBM6 cells transfected with plasmids: CMV; to express mutant TRX or dominant negative PERK. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Twelve hours after infection, levels of ROS were measured (n = 12, two experiments ± SEM). Right, SV40-transformed MEFs were infected with Ad.cmv or Ad.mda-7. Twelve hours after infection, levels of ROS were measured (n = 12, two experiments ± SEM). #, P < 0.05 less than corresponding CMV transfected value. B, SV40-transformed MEFs were infected with Ad.cmv or Ad.mda-7. Twelve hours after infection, levels of cytosolic Ca²⁺ were measured (n = 12, two experiments ± SEM). #, P < 0.05 less than corresponding wild-type MEF value. C, left, GBM6 cells were transfected with siRNA molecules: scrambled (siSCR) or to knock down ASMase. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7 and in parallel treated with vehicle (DMSO) or with myriocin (Myr, 1 μmol/L). Forty-eight hours after infection, cells were isolated and viability determined (±SEM, n = 3). ##, P < 0.05 less than corresponding vehicle value; #, P < 0.05 less than corresponding siSCR value. Right, GBM6 cells were transfected with plasmids: CMV; to express calbindin D28 or dominant negative PERK. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Forty-eight hours after infection, cells were isolated and viability determined (±SEM, n = 3). #, P < 0.05 less than corresponding CMV transfected value. D, GBM6 cells were transfected with plasmids: CMV, or to express calbindin D28 or dominant negative PERK. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Twelve hours after infection, cells were scraped into PBS, the cells lysed and lipids extracted prior to analyses by tandem mass spectrometry (n = 4; three experiments ± SEM). *, P < 0.05 greater than Ad.cmv control.

Figure 5

MDA-7/IL-24 radiosensitizes GBM cells through a ceramide/Ca²⁺/ROS–dependent mechanism that is partially dependent on altered levels of autophagy. A, GBM6 cells infected with Ad.cmv or with Ad.mda-7 and 12 h later irradiated (0–6 Gy). Cells were isolated 48 h after infection and viability determined (±SEM, n = 3). *, P < 0.05 greater differential between Ad.cmv and Ad.mda-7 values in unirradiated cells. B, GBM6 cells transfected with plasmids: to express LC3-GFP and to express nothing, TRX, calbindin D28, or dominant negative PERK. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Twenty-four hours after infection, slides were examined for punctate GFP vesicles (±SEM, n = 3). #, P < 0.05 less than corresponding value in CMV-transfected irradiated cells. C, GBM6 cells transfected: scrambled (siSCR) or to knock down ATG5 or Beclin1. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7. Forty-eight hours after infection, cells viability determined (±SEM, n = 3). #, P < 0.05 less than corresponding value in siSCR transfected cells. D, GBM6 cells were transfected with siRNA molecules: scrambled (siSCR) or to knock down ATG5 or Beclin1. Twelve hours later, cells were infected with Ad.cmv or Ad.mda-7 and 12 h later were irradiated (4 Gy) and the plates incubated for a further ~20 d to permit colonies of >50 cells to form (±SEM, n = 3). *, P < 0.05 greater reduction in colony formation than Ad.mda-7 or irradiation; #, P < 0.05 less than corresponding value in siSCR cells; ##, P < 0.05 greater reduction in colony formation than Ad.mda-7 or irradiation alone and less than corresponding value in siSCR cells.