Role of Hypoxia in Anticancer Drug-Induced Cytotoxicity for Ehrlich Ascites Cells1
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[CANCER RESEARCH 47, 2407-2412, May 1, 1987] Role of Hypoxia in Anticancer Drug-induced Cytotoxicity for Ehrlich Ascites Cells1 Vicram Gupta,2 and John J. Costanzi Division of Hematology-Oncology, Department of Internal Medicine, The University of Texas Medical Branch, Galveston, Texas 77550 tained by i.p. injection of 1 x IO7cells weekly in ICR white mice. Cells ABSTRACT from animals bearing 6-8-day old ascites tumors were used. A review of the literature on the effect of hypoxia on in vitro drug In vitro studies on the effect of oxygen on drug-induced cell survival sensitivity had suggested that there was consistently more cytotoxicity were performed as follows. EAT3 cells (1-5 x IO6)were suspended in under hypoxic conditions for the drugs misonidazole and mitomycin C 10 ml of Hams FIO media in 25-ml Erlynmyer glass flasks. The flasks while there was much conflicting data for the drugs Adriamycin and were sealed with rubber sleeve stoppers and fitted with two 21-gauge bleomycin. We have examined the effect of oxygen on the cellular needles for an inlet and outlet port. The flasks were gassed with oxygen response of Ehrlich's ascites tumor cells to the drugs mitomycin C, mixture of interest at flow rates of 1 liter/min for 1.5 h at 37*C on an misonidazole, Adriamycin, and bleomycin. Significant differences were Orbital Shaker, model 3520 (Lab-Line Instruments, Inc.) at 1800 rpm. observed when we compared the cytotoxicity of mitomycin C and mison This is similar to the method reported by Mohindra and Rauth (15). idazole as a function of oxygen concentration. For the drugs Adriamycin Gas mixtures used contained 0-20% O2, 5% CO2, and balance N2 and bleomycin no differential effects of oxygen were noted for a 1-h drug (Linde Corp., Houston, TX). Following this prééquilibraiionof oxygen, exposure with hypoxia while some differences were noted only for bleo drugs were added directly without opening the flask by using a 21- mycin for an 8-h drug exposure time. Because differences dependent on gauge needle in a 0.1 -ml volume. The effect of both short and prolonged oxygen concentration were observed for some drugs but not others at the drug exposure time during hypoxia on drug cytotoxicity was examined same experimental conditions, and as indicated by a review of the in some cases. For the 1-h drug exposures a cell density of 1 x 105/ml literature, it is suggested that some of the conflicting data in the literature was used without humidification. Measurement of volume of media with respect to some of these drugs may be cell-line dependent. Other failed to reveal any significant effect of evaporation. For the 8-h drug variables which may also be of importance were the duration of drug exposure under hypoxia, a cell density of 5 x KlVnil was used with exposure time in hypoxia and cell density. The observed oxygen concen humidification as this time period would have resulted in significant tration-dependent changes in cell survival for the Ehrlich cells with drugs evaporation. In some instances cells were exposed to a humidified examined could not be explained on the basis of changes in drug-induced preincubation atmosphere of 5% O2 (oxic) or N2 (hypoxic) for 16.5 h cellular oxygen consumption. before drug incubation for l h at the stated oxygen atmosphere in order to determine whether the duration of hypoxia before drug exposure influences cytotoxicity. After drug incubation with continuous gassing, INTRODUCTION the cells were centrifuged, washed with 10 ml of media, and resuspended Oxygen microenvironment in tumors is heterogeneous (1,2). in fresh Hams FIO media for plating. The colony assay for the EAT cells has been described previously (32). Tumor cells were suspended It is known that cell killing by radiation is dependent upon in Hams FIO media containing 15% fetal calf serum, 1% penicillin- oxygen concentration and that hypoxic cells are resistant to streptomycin, and 0.3% agar and plated in 35-mm l'etri dishes in a 2- radiation (3). Hypoxic cells may also be resistant to drugs ml volume. The dishes were then incubated in humidified Modulator because of drug delivery problems secondary to inadequate Incubator Chambers (Billup-Rothenberg Inc., Del Mar, CA) and vasculature (4) and/or intrinsic effects of hypoxia on cell re flushed with 5% O2, 5% CO2, and 90% N2. A 5% O2 atmosphere was sponse to drugs (5). There is some evidence that oxygen con used for the plating because we had previously found there was no centration may play a significant role in drug-induced cell growth of the EAT cells at 20% ( ).-in the absence of antioxidants (32). killing (5). Drugs such as mitomycin C and misonidazole are Seven to 8 days later, plates were scored for colony formation (>50 known to be selectively metabolized under hypoxic conditions cells). to active compounds (6, 7) while drugs such as Adriamycin and Oxygen measurements in solution were performed using a Diamond bleomycin may be cytotoxic through oxygen radical-mediated Electrotech, Inc. (Ann Arbor, MI) oxygen measuring system which mechanisms (8, 9). Pre-, during, and postdrug treatment influ consisted of an amplifier unit (no. 1201), calibration cell (no. 1251), and Clarke type oxygen electrode. Oxygen measurements were made ences of varying oxygen concentrations have all been described in separate experiments with and without cells similar to the drug in the literature (10,11). The effect of oxygen concentration on experiments except that phosphate buffered saline was used. For each the cytotoxicity of misonidazole has been established (12, 13) experiment a separate calibration curve using N2 and 0.1, 1, 5, 10, and and similar data with mitomycin C have been recently published 21% O2 was performed. The current was recorded as a function of time (14). However, the in vitro data are conflicting with the excep and the oxygen concentration calculated from the calibration curve. tion of misonidazole. Because of the conflicting data summa Using this system, oxygen level below 0.1 % cannot be measured accu rized in Table 1 we have examined the effect of oxygen concen rately. The data was plotted according to an equation (Equation A) tration on cell survival for the anticancer agents mitomycin C, described by Whillans and Rauth (33): misonidazole, Adriamycin, and bleomycin. (Cm - CJ = (Cw - C.)e-*" (A) MATERIALS AND METHODS where C\,\ is the oxygen tension in solution at time t. f H is the initial oxygen tension in solution, C, is the oxygen concentration at equilib Tumor line used for the experiments was a tetraploid Ehrlich's ascites rium, and *i (s ') is a physical constant independent of oxygen concen tumor (Mason Research Institute, Worcester, MA) which was main- tration and depletion rate. From the measured values of oxygen con Received4/9/86; revised 12/2/86; accepted2/4/87. sumption rate (R) and k,, one can calculate the effect of oxygen The costs of publication of this article were defrayed in part by the payment depletion on the oxygen concentration at equilibrium using the rela of page charges. This article must therefore be hereby marked advertisement in tionship C. = CM - R/ki (33) where C[girepresents oxygen concentra accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by USPHS Grant CA-32938, National Cancer Institute tion in the gas phase. (NCI), and Cancer Center Grant CA-17701, NCI. Oxygen consumption studies were performed on a Gilson Oxygraph 1To whom requests for reprints should be addressed, at Division of Hematol- located in the Division of Radiation Research, University of Texas ogy, Oncology/Internal Medicine Department, Clay Hall, H82, University of Texas Medical Branch, Galveston, TX 77550. 1The abbreviation used is: EAT, Ehrlich ascites tumor cells. 2407 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1987 American Association for Cancer Research. HYPOXIA AND DRUG CYTOTOXICITY Table 1 Summary of literature on the effect of oxygen concentration on drug cytotoxicily Preferentially toxic to Preferentially toxic Drug oxic cells to hypoxic cells No difference Misonidazole CHO"(12)EMT6(13) Metronidazole CHO and HeLa (15) Mitomycin C EMT6 (10, 16-19), CHO (14, 20), V79, CHO (16) KHT, HeLa (20), S180 (17), EAT*, WiDR and HEC-1A (19) and P3884 HTC (19) Procarbazine EMT6 (10) Actinomycin D EMT6 (10, 18) EMT6 (21) Vincristine EMT6 (10) EMT6 (21) Streptonigrin EMT6 (17) Bleomycin EMT6 (10) EMT6 (24) V79 (22) CHO (23) EAT* Adriamycin B14 FAF28 (25) EMT6(10, 17) EMT6(21) V79 (26) S180(I7) EAT* CHO (23) WHF1B(27) Bisantrene L1210, HTC (28) DTIC CHO (29) Cyclophosphamide V79 (30) CHO (23) BCNU CHO (23) EMT6(10, 21) S 1X0(17) Cisplatin EMT6 (18) EMT6 (10, 21) CHO (23) 5-Fluorouracil EMT6 (10, 21) Methotrexate EMT6(10, 21) Etoposide EMT6 (31) Alkeran EMT608) Nitrogen mustard EMT6 (18) °CHO, Chinese hamster ovary; DTIC, 5-(3,3-dimethyl-l-triereno)imidazole-4-carboxamide; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea; EMT6, mouse mammary tumor cells; V79, B14 FAF28, Chinese hamster, S180, KHT, mouse sarcoma cells; P388 and 1.1210. mouse leukemic cells; HeLa, WiDR, HEC-1 A, human epithelial cancer cell lines; HTC, human tumor biopsy cells. * This study. Medical Branch, Galveston, TX, through the courtesy of Dr. Neil 1.0 Iloud I. Oxygen consumption rates at 21% O2 were determined with and without the drugs Adriamycin, bleomycin, and mitomycin C at SO Mg/ml directly inside the oxygen consumption chamber. In addition, mitomycin C (3 /ig/ml)- and misonidazole (1000 ¿tg/ml)-treatedcells at 0.01% (>.,for l h were washed free of drug, resuspended in media, and the <>,consumption rates were determined.