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

Abstract

Cryosurgery is an emerging treatment for human solid tumors, notably colorectal liver metastasis. Cryosurgical procedures generate a thermal gradient of from at least -50 degrees C at the center of the tumor being treated to about 0 degrees 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. HT29 colon cancer cells were subjected to freezing temperatures from -6 degrees C to -36 degrees C, thawed at room temperature for 30 min and rewarmed at 37 degrees 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 degrees C, all cells were dead immediately after freeze-thaw. A kinetic analysis of cryo-induced 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. HT29 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.

Targeted induction of apoptosis via TRAIL and cryoablation: a novel strategy for the treatment of prostate cancer

“…19,35 Before these reports, freeze rupture and necrosis were the only forms of cell death thought to be associated with cryoablation. 12 The advent of apoptosis activation provided a novel opportunity to manipulate freezing-induced cell death in an attempt to improve procedural efficacy.…”

“…Subsequently, other studies have reported on the activation of apoptosis following the application of freezing. [17][18][19] Because of the resistant nature of CaP cells to freezing, 18,20 physicians are required to extend the freeze zone significantly beyond the diseased tissue, exposing a greater portion of the non-targeted, healthy tissue to cryoablation. 10,12 Inadequate management of the freeze zone periphery (positive freeze margin) can sometimes have a negative effect on the surrounding tissues (i.e.…”

Mesenchymal stem/stromal cells precondition lung monocytes/macrophages to produce tolerance against allo- and autoimmunity in the eye

“…However, i.v. injections of hMSCs that were made apoptotic by repeated cycles of freezing and thawing (16) did not have any effects on corneal graft survival and EAU development (Fig. S2).…”

Optimisation and molecular signalling of apoptosis in sequential cryotherapy and chemotherapy combination in human A549 lung cancer xenografts in SCID mice

“…It is established that cryosurgery, in addition to necrosis, could trigger apoptosis, and a few studies have implicated the mitochondrial pathway of apoptosis (Hanai et al, 2001;Baust et al, 2004). Clarke et al (2004) showed that freezing and chemotherapy differentially activated Bcl-2 family members in a human prostate cancer cell line.…”