Thermosensitization by increasing intracellular acidity with amiloride and its analogs.
OBJECTIVE
The major mechanisms that regulate the intracellular acidity of pHi in mammalian cells are the Na+/H+ exchange and HCO3-/Cl- exchange through the plasma membrane. The purpose of this study was to investigate the feasibility of increasing the thermosensitivity of tumors by increasing intracellular acidity with the use of drugs that inhibit the pHi regulatory mechanisms.


METHODS
The pHi of SCK tumor cells in vitro was determined with the fluorescence spectroscopy method. The thermosensitizing effects of the drugs on the cells in neutral (pH 7.2-7.5) and acidic (pH 6.6) media were determined by clonogenic assay. The thermosensitization of SCK tumors in vivo by the drugs was determined with the tumor growth delay and the in vivo-in vitro assay for clonogenic cells.


RESULTS
The pHi of SCK tumor cells in pH 7.2-7.5 media was similar to the media pH, while the pHi of the cells in pH 6.6 media was about 7.0. The pHi declined and the thermosensitivity of the tumor cells increased when the Na+/H+ exchange was inhibited with amiloride (3,5 diamino-6-chloro-N-(diaminomethylene) pyrazinecarboxamide) and its analogs, HMA (3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazinecarboxamide) or EIPA (3-amino-6-chloro-5-(N-ethyl-N-isopropylamino)-N-diaminomethylene) pyrazinecarboxamide), especially in acidic medium. The potencies of HMA and EIPA to decrease the pHi and increase the thermosensitivity in vitro were more than 50 times greater than that of amiloride. DIDS (4,4-diiosothiocyanatostilbene-2,2'-disulfonic acid), an inhibitor of the Na(+)-dependent HCO3-/Cl- exchange, exerted little effect on the pHi and thermosensitivity of SCK cells in vitro, but it enhanced the effects of amiloride and its analogs. Amiloride and HMA also significantly enhanced the thermal effect on tumors in vivo, as judged by the tumor growth delay and also by the in vitro-in vivo assay for clonogenic cells. Combinations of DIDS with amiloride or HMA were more effective than either of them alone in increasing the thermal damage in vivo. As in vitro, HMA was far more potent than amiloride in increasing the thermosensitivity of tumor cells in vivo. However, EIPA was not effective in vivo, probably due to a rapid metabolic breakdown of the drug.


CONCLUSIONS
The drugs that interfere with the pHi regulatory mechanism significantly thermosensitized the tumor cells in vitro, particularly those in acidic media. The drugs were also effective in increasing the thermosensitivity of tumors. Because the interstitial environment in tumors is acidic relative to that in normal tissues, the thermosensitization by the drugs may be greater in tumors than that in normal tissues.
Song
C W
CW
University of Minnesota Medical School, Department of Therapeutic Radiology-Radiation Oncology, Minneapolis.
Kim
G E
GE
Lyons
J C
JC
Makepeace
C M
CM
Griffin
R J
RJ
Rao
G H
GH
Cragoe
E J
EJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008805:Mice, Inbred A; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
C057360:5-(N,N-hexamethylene)amiloride; D000584:Amiloride; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; C039614:ethylisopropylamiloride