Reversal of intrinsic multidrug resistance in Chinese hamster ovary cells by amiloride analogs

Abstract

(Gupta, 1988). This expression of resistance, to the same group of drugs as seen in the MDR mutant, does not require prior selection with cytotoxic agents and has been termed intrinsic MDR (Gupta, 1988). In this work, for the first time, we show that several analogs of amiloride can reverse intrinsic MDR without greatly affecting the acquired MDR. Amiloride is an inhibitor of Na+/H+ antiport. The activity of this ion transporter is elevated in drug resistant cell lines (Boscoboinik et al., 1990). Analogs of amiloride vary greatly in the potency and specificity by which they inhibit this antiporter (Kleyman & Cragoe, 1988 (Keizer & Joenje, 1989;Boscoboinik et al., 1990), it seemed possible that acidification of pH,, through inhibition of the Na+/H+ antiporter, could contribute to the sensitisation of drug resistant cell lines. MaterialsAmiloride analogs were synthesised for this study by previously described methods (Cragoe et al., 1967). Minimum essential medium (a-MEM) containing L-glutamine and all four ribonucleosides and x-deoxyribonucleosides (a-MEM + nucleosides), faetal bovine serum, penicillin, streptomycin, and amphotericin were obtained from Gibco, Grand Island, NY; trypsin from Difco, Detroit, MI; methylene blue from Fisher Scientific Co., Fairlawn, NY; adriamycin, HCI, and vinblastine sulfate were purchased from Sigma Chemical Co., St. Louis, MO. Cell lines and culture conditionsThe parental Chinese hamster ovary cell line which requires proline for growth (Pro-) is referred to as wild type (WT) in our work. The cell line AUXB1 which in addition to proline requires glycine, adenosine and thymidine for growth, was derived from Pro-WT cells by a single mutational alteration (McBurney & Whitmore, 1974). The Pro-and AUXBI cell lines show similar sensitivity towards various drugs used in the present study (Gupta, R.S., unpublished results). The cell line CHRC5 was derived from AUXBI after three successive selections in presence of increasing concentrations of colchicine (Ling & Thompson, 1974). The CHRC5 cell line (Bech-Hansen et al., 1976) was kindly provided by Dr Victor Ling of the Ontario Cancer Institute, Toronto, Ontario. HeLa (clone S3) is a human cell line established from a cervical carcinoma (Puck et al., 1956). All of the above cell lines were grown as monolayer cultures in a-MEM + nucleosides supplemented with 5-10% foetal bovine serum at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO2. The cell lines were routinely grown in the absence of any selective drug without loss of resistance. Clonogenic assayThe effect of various agents on the reversal of the drugresistance was examined by determining the cloning efficiencies of the parental and resistant cell lines in the presence of different concentrations of either vinblastine, daunomycin, puromycin or colchicine, in the absence and presence of the amiloride analogs. In these experiments, which were carried out in 24-well tissue culture dishes, 0.5 ml of 11 progressive dilutions of cytotoxic drug (made at two times the fi...

Intracellular pH and the control of multidrug resistance.

“…(v) Drug influx is slower in resistant cells (4,(28)(29)(30)(31)(32)(33)(34)(35)(36), which is consistent with differences in the rate oftrapping and inconsistent with an active efflux model. (vi) Drugs which acidify the cytosol, such as amiloride, reverse MDR (37).…”

Towards an Integral Therapeutic Protocol for Breast Cancer Based upon the New H+-Centered Anticancer Paradigm of the Late Post-Warburg Era

“…Moreover, there is an important role for V-ATPases in tumor invasion and chemoresistance in several cancers, including BC [ 73 , 176 ]. In summary, pH alterations have been shown to be behind the most fundamental aspects of MDR [ 97 , 125 , 177 , 178 , 179 ]. Finally, based upon the selective H + -dynamics of cancer, an integrated mechanism to explain MDR has been developed [ 41 , 97 , 125 , 127 , 128 , 167 , 168 , 170 ].…”

Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme

“…The HeLa cell line, derived from a cervical carcinoma, is a tumour cell line commonly used in experimental cancer research. The cells do not express the P-glycoprotein that is responsible for conventional multidrug resistance in tumour cells (Epand et al, 1991;Chao et al, 1992). Chao et al (1991) established a cisplatin-resistant HeLa cell subline that had acquired crossresistance to melphalan and mitomycin C. Further study failed to demonstrate any significant change in cellular glutathione levels, but amplification of the GST P1 gene and a 6-fold increase in glutathione peroxidase activity as measured with cumene Abbreviations used: CDNB, 1-chloro-2,4-dinitrobenzene; GST, glutathione transferase; HF, HeLa cells grown in flat-bottomed flasks; HR, HeLa cells grown in roller bottles; LC50, drug concentration causing 50% -cetl death as measured by level of protein synthesis; MEM, minimum essential medium with Earle's salt; 20 x SSC, 17.35% (w/v) NaCI+ 8.82% (w/v) sodium citrate, pH 7.0.…”