Induction of apoptosis in human colon carcinoma cells HT29 by sublethal cryo-injury: Mediation by cytochrome C release

Cryosurgery is an emerging treatment for human solid tumors, notably colorectal liver metastasis. Cryosurgical procedures generate a thermal gradient of from at least -50°C at the center of the tumor being treated to about 0°C at the periphery. Cell death occurs by necrosis in the center, while the peripheral zone of frozen tumor harbors a mix of viable and dead tissue. In order to understand the mechanisms of cell death and survival in this peripheral area at risk for tumor recurrence, we have established an in vitro freezing system that mimics in vivo conditions of sublethal injury. MT29 colon cancer cells were subjected to freezing temperatures from -6°C to -36°C, thawed at room temperature for 30 min and rewarmed at 37°C for a period of time. Post-freeze-thaw, cryolytic cells were evaluated by trypan blue exclusive assay. We also identified apoptotic cells after rewarming by cell shrinkage, nucleic condensation, TUNEL assay, DNA fragmentation and PARP degradation. The intensity of cryolysis and apoptosis was increased by lowering the freezing temperature. At -36°C, all cells were dead immediately after freeze-thaw. A kinetic analysis of cryoinduced apoptosis showed that the commitment to enter apoptosis occurred right after the freeze-thaw period and lasted less than 8 hr after rewarming. We further demonstrated that freezing triggers one of the caspase cascade involved in apoptosis: Release of cytochrome c from mitochondria to cytosol, followed by activation of caspase-9 and degradation of PARP. These results indicate the death of cancer cells under cryo-treatment at sublethal freezing temperature can be attributed 2 different modes, cryolysis as well as apoptosis. MT29 cells carrying p53 mutant have very quick response for induction of apoptosis by cryo-treatment and contain an intact pathway of caspase cascade. Further studies will address if mechanisms in cells with wild-type p53 will differ.