Augmented Adriamycin Sensitivity in Cells Transduced with an Antisense Tumor Necrosis Factor Gene Is Mediated by Caspase‐3 Downstream from Reactive Oxygen Species

Abstract

While transduction of an antisense tumor necrosis factor (TNF) gene sequence can augment the cytotoxicity of adriamycin (ADM) in human cancer cells, the specific effect of introducing this sequence on the signal transduction pathway leading to cell death remains unclear. In ADM-resistant pancreatic carcinoma (PANC-1) cells, both the antioxidant N-acetyl-L-cysteine (NAC) and the caspase-3 inhibitor acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspartyl-aldehyde (Ac-DMQD-CHO) prevented ADM-induced cytotoxicity. NAC additionally inhibited caspase-3 activity induced by ADM treatment, while Ac-DMQD-CHO showed no suppressive effect on reactive oxygen species (ROS). Stable antisense-TNF transfectants showed higher ADM sensitivity and greater ADMinduced ROS production and caspase-3 activity than mock transfectant or parent cells. These results indicate that increased caspase-3 activity downstream from ROS production is among the mechanisms by which transduction of the antisense TNF sequence of augments ADM sensitivity of pancreatic carcinoma cells.Key words: Tumor necrosis factor -Antisense -Reactive oxygen species -Caspase-3 Adriamycin (ADM) is widely used to treat solid tumors and malignant hematologic diseases. Numerous attempts have been made to overcome cancer cell resistance against antineoplastic drugs including ADM by inhibition of intracellular resistance factors.1, 2) We previously have reported that endogenous tumor necrosis factor (enTNF) exerts a protective effect against ADM by enhancing the scavenging of intracellular reactive oxygen species (ROS) via induction of manganous superoxide dismutase (MnSOD). 3)We also found that measurement of enTNF expression could predict ADM sensitivity in tumor cells. 4) Thus, antisense TNF gene transduction could augment ADMinduced toxicity in human cancer cells by decreasing MnSOD activity. 4,5) However, whether transduction of the antisense TNF gene actually affects the signal transduction pathway leading to ADM-induced cell death is unclear. We therefore investigated changes following antisense TNF gene transduction in expression of molecules leading to cell death in human pancreatic carcinoma (PANC-1) cells showing resistance to ADM. Our investigative group and others recently identified a signaling pathway including production of reactive oxygen species and activation of caspase-3 that leads to apoptosis in cells exposed to heat or ionizing radiation.6, 7) We therefore examined whether and how antisense TNF gene transduction augmented this signaling pathway to result in increased ADM sensitivity. As the PANC-1 cells used in this study harbor a mutant-type p53, effects from several molecules that interact with caspase-3 in cells with wild-type p53 could be excluded. MATERIALS AND METHODSCell culture and gene transduction PANC-1 cells were cultured in RPMI-1640 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS; Biological Industries, Kibbutzbeit Haemek, Israel). Transduction of antisense TNF mRNA expression vector (pLJantiTNF) to PAN...

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