Liposomal doxorubicin increases radiofrequency ablation-induced tumor destruction by increasing cellular oxidative and nitrative stress and accelerating apoptotic pathways.
OBJECTIVE
To determine if oxidative and nitrative stress and/or apoptosis contribute to increased coagulation when combining radiofrequency (RF) ablation with liposomal doxorubicin.


METHODS
Animal care committee approval was obtained. R3230 mammary adenocarcinomas in Fischer rats were treated with either RF ablation (n = 43), 1 mg of intravenously injected liposomal doxorubicin (n = 26), or combined therapy (n = 30) and were compared with control subjects (n = 11). A subset of animals receiving combination therapy (n = 24) were treated in the presence or absence of N-acetylcysteine (NAC) administered 24 hours and 1 hour before RF ablation. Tumors were analyzed 2 minutes to 72 hours after treatment to determine the temporal range of response by using immunohistochemical staining of the apoptosis marker cleaved caspase-3, phosphorylated gammaH2AX, and HSP70 and of markers of oxidative and nitrative stress (8-hydroxydeoxyguanosine [8-OHdG], 4-hydroxynonenal [4-HNE]-modified proteins, and nitrotyrosine [NT]). Statistical analyses, including t tests and analysis of variance for comparisons where appropriate, were performed.


RESULTS
By 4 hours after RF ablation alone, a 0.48-mm +/- 0.13 (standard deviation) peripheral band with 57.0% +/- 7.3 cleaved caspase-3 positive cells was noted at the ablation margin, whereas a 0.73-mm +/- 0.18 band with 77.7% +/- 6.3 positivity was seen for combination therapy (P < .03 for both comparisons). Combination therapy caused increased and earlier staining for 4-HNE-modified proteins, 8-OHdG, NT, and gammaH2AX with colocalization to cleaved caspase-3 staining. A rim of increased HSP70 was identified peripheral to the area of cleaved caspase-3. Parameters of oxidative and nitrative stress were significantly inhibited by NAC 1 hour following RF ablation, resulting in decreased cleaved caspase-3 positivity (0.28-mm +/- 0.09 band of 25.9% +/- 7.4 positivity vs 0.59-mm +/- 0.11 band of 62.9% +/- 6.0 positivity, P < .001 for both comparisons).


CONCLUSIONS
Combining RF ablation with liposomal doxorubicin increases cell injury and apoptosis in the zone of increased coagulation by using a mechanism that involves oxidative and nitrative stress that leads to accelerated apoptosis.
Laboratory for Minimally Invasive Tumor Therapies, Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215, USA.
Stephanie A
SA
Solazzo
Muneeb
M
Ahmed
Rachel
R
Schor-Bardach
Wei
W
Yang
Geoffrey D
GD
Girnun
Syed
S
Rahmanuddin
Tatyana
T
Levchenko
Sabina
S
Signoretti
Douglas R
DR
Spitz
Vladimir
V
Torchilin
S Nahum
SN
Goldberg
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000111:Acetylcysteine / Q000494:pharmacology; D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D017115:Catheter Ablation; D003131:Combined Modality Therapy; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy* / Q000378:metabolism / Q000601:surgery*; D018384:Oxidative Stress; D051381:Rats; D011916:Rats, Inbred F344; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000447:Aldehydes; D018840:HSP70 Heat-Shock Proteins; D006657:Histones; C002744:3-nitrotyrosine; D014443:Tyrosine; D004317:Doxorubicin; D000080242:8-Hydroxy-2'-Deoxyguanosine; D053148:Caspase 3; D003849:Deoxyguanosine; C027576:4-hydroxy-2-nonenal; D000111:Acetylcysteine