Cryotherapy, a method of in situ ablation, is used in the treatment of colorectal liver metastases with variable results. During the treatment, the central area of treated tumor undergoes necrotic destruction by lethal cryo-injury; however, the cellular response of tumor exposed to sublethal cryo-injury at the peripheral zone is unclear. In our study, we have identified the induction of apoptosis by cryo-injury at -10 degrees C in 4 colorectal cancer cell lines (HT29, HCT116, KM12C and KM12SM). The apoptosis was characterized by chromatin condensation, transferase-mediated dUTP nick end-labeling (TUNEL) staining, proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and cytokeratin 18, and activation of caspase-3. The occurrence and intensity of cryo-induced apoptosis did not correlate with the functional status of p53 in the cell lines studied. The expression of anti-apoptotic proteins (Bcl-2, Bcl-X(L)) and pro-apoptotic proteins (Bax, Bcl-X(S), Bad, and Bak) in response to cryo-injury varied in this cell line panel. The basal level of Bcl-2/Bax protein ratio correlated inversely to the apoptotic rate. We further demonstrated that Bax level decreased in cytosol and increased in mitochondria, followed by a loss of mitochondrial membrane potential after cryo-injury in HT29 cells. These findings indicate that cryo-injury induces apoptosis in colorectal cancer cells via disruption of mitochondrial integrity. The cryo-induced apoptosis was also identified in a nude mouse tumor xenograft model. Our elucidation of the apoptosis pathway induced by cryo-injury implies that synergistic combination of cryosurgery with pharmacological agents that augment of apoptosis induction may have clinical relevance in treating colorectal liver metastasis.
Copyright 2002 Wiley-Liss, Inc.