Possible Link Between the Intrinsic Drug Resistance of Colon Tumors and a Detoxification Mechanism of Intestinal Cells Wayne D

Home , Cytochalasin b

[CANCER RESEARCH 48, 3025-3030, June 1, 1988] Possible Link between the Intrinsic Drug Resistance of Colon Tumors and a Detoxification Mechanism of Intestinal Cells Wayne D. Klohs1 and Randall W. Steinkampf

Department of Chemotherapy, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105

ABSTRACT VER might also alter anthracycline and anthrapyrazole activity in colon tumor cells. We found that VER potentiates the The insensitiviÂ«) of colon tumors to various anticancer agents was activity of ADR and the anthrapyrazole, CI-937, in a manner studied in vitro. The activity of Adriamycin (ADR) in several colon tumor which parallels its effects on drug accumulation and efflux and cell lines was potentiated by the calcium channel blocker verapamil (VER). In the HCT-8 human colon adenocarcinoma cell Une, VER suggest that this enhanced efflux may be one factor involved in potentiation of the activities of ADR and the anthrapyrazole CI-937 the inherent resistance of certain colon tumors to chemother appeared to be related to its ability to enhance the net accumulation of apy. both drugs and inhibit their efflux. VER, which potentiated ADR activity In addition, we present data which lend support to the recent in HCT-8 cells by 4-fold, caused a 3.5-fold stimulation of ADR accumu hypotheses proposed by Beck (7) and by Sehested et al. (8) that lation and 3.5-fold inhibition of ADR efflux, when compared to non- the endosome-lysosome system is involved in this accelerated VER-treated cells. The low level of VER potentiation of CI-937 activity drug exodus process. in HCT-8 cells (1.4-fold) was also reflected in CI-937 transport studies which demonstrated a 1.5-fold enhancement of CI-937 accumulation and MATERIALS AND METHODS a 1.4-fold inhibition of its efflux. VER was also found to stimulate ADR activity and accumulation in a normal small intestinal crypt cell line Chemicals. The anthrapyrazoles CI-937, CI-941, CI-942, and [MC]- (IEC-6). The mechanism of drug efflux was examined in HCT-8 cells. CI-937 (specific activity, 83.1 ftCi/mM) were obtained from the Warner- Agents known to increase the permeability of the plasma membrane did Lambert Co., Ann Arbor, MI (9). RPMI 1640 medium, nutrient F-12 not alter ADR accumulation or its efflux in HCT-8 cells unless these medium, MEM-E, and MEM nonessential amino acids were purchased same agents were also capable of interacting with the lysosome. Tween from KC Biological, Inc., Lenexa, KS. FBS was purchased from Hy- 80 and the 1ysosomotropic detergent Triton WR-1339 as well as proton Clone Laboratories, Logan, UT. Adriamycin, daunomycin, Tween 80, ionophores and lysosomotropic amines all stimulated ADR uptake and/ Triton WR 1339, amphotericin B, filipin, deoxycholate, verapamil, or inhibited its efflux from HCT-8 cells. ADR efflux was also partially chloroquine, monensin, nigericin, cytochalasin B, monodansylcadav- blocked by cytochalasin B. Based on these observations, we suggest that erine, and CCCP were all purchased from Sigma Chemical Co., St. at least part of the inherent drug resistance of colon tumor cells results Louis, MO. from the retention of an enhanced drug efflux mechanism which is found Cell Cultures. C-38 and C-26 murine colon tumor cell lines, obtained in normal intestinal epithelium where this property may provide protec from Southern Research Institute, Birmingham, AL, were grown in tion from plant alkaloids and other xenobiotic agents ingested in the diet. MEM-E supplemented with 20% FBS, 16 mM A'-2-hydroxyethylpiper- The mechanism of this drug efflux from HCT-8 cells may involve drug azine-Ar/-2-ethanesulfonic acid, and 8 mM morpholinopropane sulfonic partitioning into acidic vesicles within the cell and their subsequent acid. HCT-8 human colon adenocarcinoma cells were grown in RPMI release from these cells by exocytosis. 1640 medium supplemented with 10% FBS. WiDr, LoVo, and LS-174 human adenocarcinoma cell lines and IEC-6 normal rat intestinal epithelial cells were obtained from the American Type Culture Collec INTRODUCTION tion, Rockville, MD. IEC-6 cells were cultured in Dulbecco's modified Eagle's medium containing 5% FBS and 0.1 Â«ig/mlof insulin. WiDr Colorectal cancer is the second leading cause of cancer death cells were grown in MEM-E with 10% FBS, nonessential amino acids, in the United States (1). It is estimated that 140,000 new cases and SO/

To determine if the effect of VER on ADR efflux was related to an Table 2 Effect ofverapamil on anthrapyrazole and anthracycline activity in effect on ADR metabolism, both IEC-6 and HCT-8 cells were incubated HCT-8 cells Verapamil(5 of for 2 h with ADR in the presence or absence of 5 /ig/ml of VER. fjg/nil) ID,,,(tiM)"210 Chloroform:met hanol (2:1) extracts of drug-loaded cells were prepared CompoundAdriamycinDaunomycinCI-937CI-941CI-942Â± potentiation4.03.31.44.41.6 Â±22.1'+ as previously described (13) and chromatographed on silica gel plates in chloroform: met hanol: water (80:20:3). Quant nation of ADR and its 53 Â±6.8129 metabolites was determined according to the method of Cradock et al. Â±4.3 (14). As expected, the extracted material was greater than 90% ADR, + 39 Â±3.5304 and this was not altered by the addition of VER. The uptake and efflux of CI-937 into HCT-8 cells were carried out Â±12.4 under similar conditions as described for ADR transport. CI-937 ac + 221 Â±29.087 cumulation (final concentration in the medium, 10.3 Â¿IM;specificactiv Â±5.4 ity, 83.1 //Ci/mmol) was measured in the presence or absence of 5 Â¿ig/ + 20Â±1.51003 ml of VER at 37'C. Cell pellets were dissolved in N NaOH at 70'C for Â±88.5 1 to 2 h, neutralized, and counted in Beckman Ready-Solv cocktail by + 626 Â±32.5Degree liquid scintillation counting. " Cells were grown as described in Table 1 in the presence or absence of the indicated compound. * Average Â±SD of two experiments, each performed in triplicate. RESULTS Verapamil Potentiation of Anthracycline and Anthrapyrazole 2000 Activities. ADR activity was determined in the presence or IEC-6 * VERAPAMIL absence of VER in 6 different colon tumor cell lines and the normal rat intestinal crypt cell line, IEC-6 (Table 1). At non- HCT-8 Â»VERAPAMIL cytotoxic concentrations of VER, ADR activity was potentiated 1500 in 5 of the 7 cell lines tested by as much as 17-fold. VER concentrations greater than 5.0 tig/m\ were toxic to all of these cell lines. VER potentiated ADR cytotoxicity in LoVo and LS- 174T cells by 3-fold, in colon-26 and HCT-8 cells by 4-fold, 2)000 and in the IEC-6 cells by 17-fold, while it had no effect on ADR activity in colon-38 or WiDr cells. In addition to ADR potentiation, VER also potentiated the activities of daunomycin and â€¢Ã•IEC-6- VERAPAMIL 3 anthrapyrazoles in HCT-8 cells (Table 2). Daunomycin activ - HCT-8 - VERAPAMIL 500 ity was increased in these cells by over 3-fold. VER potentiated the activity of the anthrapyrazole, CI-941, by over 4-fold but produced only a 1.5-fold increase in the activities of CI-937 and CI-942 in HCT-8 cells. Transport of Adriamycin and CI-937. The transport of ADR 02468 HOURS in the presence or absence of VER was examined in HCT-8 Fig. 1. Effect of VER on the net accumulation of ADR in IEC-6 normal rat human colon adenocarcinoma cells and in the IEC-6 cell line intestinal epithelial cells and HCT-8 human colon adenocarcinoma cells. Cells were incubated in 9.2 (Â¿MADRin the presence or absence of 5.0 ;ig/ml of VER (Figs. 1 and 2). After 6 h of incubation in medium containing and uptake measured at 37Â°Cas described by Skovsgaard (7). Points, average of ADR, VER stimulated the net accumulation of ADR in both two experiments, each performed in triplicate; bars, SD. HCT-8 and IEC-6 cells by greater than 3-fold (Fig. 1). The

Table 1 Effect ofverapamil on Adriamycin cytotoxicity in IEC-6 and colon tumor cell lines 100 Verapamil of CelllineIEC-6Colon-38WiDrHCT-8Colon-26LoVoLS-174TÂ±(5.0 fig/ml) IDÂ»(nM)"87.5 potentiation'17.20.950.974.04.02.9M Â±4.8C i IEC-6 Â»VERAPAMIL + 5.1Â±0.4125 80 HCT-8 * VERAPAMIL

Â±9.6 + 132Â±12.194 Seo Â±4.0 + 97 Â±10.5210

Â±22.1 40 + 53 Â±6.8237 --J IEC-6 -VERAPAMIL Â±10.1 ~1HCT-8 - VERAPAMIL + 60 Â±3.757

Â±3.3 + 20 Â±2.063 60 90 130 150 4.0+ Â± MINUTES 21 Â±1.7Degree " Cells were grown for 72 h at 37 *C in the presence of Adriamycin Â±5.0 fig/ Fig. 2. Effect of VER on the efflux of ADR from HCT-8 and IEC-6 cells. Cells were preloaded in the absence of VER for 4 h with ADR under conditions ml of verapamil. described in Fig. 1, after which cells were washed twice in ice-cold medium and ID Â»â€”verapamil Degree of potentiation = . resuspended in ADR-free medium in the presence or absence of 5.0 ftg/tnl of II).,,,+ verapamil VER. Efflux was determined as previously described (7). Points, average of 2 ' Average Â±SD of two experiments, each performed in triplicate. experiments, each performed in triplicate; bars, SD. 3026

efflux of ADR was examined in cells preloaded for 4 h with ADR. In concert with its effect on ADR accumulation, VER inhibited the high level of ADR efflux in both HCT-8 cells and 1500 IEC-6 cells by more than 3-fold (Fig. 2). The transport of the anthrapyrazole CI-937 was also deter mined in HCT-8 cells (Figs. 3 and 4). In accord with VER's potentiation of CI-937 cytotoxicity in HCT-8 cells (see Table 2), VER increased the accumulation of CI-937 (Fig. 3) and inhibited its efflux (Fig. 4) by approximately 1.5-fold. Studies on the Mechanism of Drug Efflux. Several membrane active agents were compared with VER for their effects on ADR accumulation and efflux in HCT-8 cells (Figs. 5 and 6). At concentrations that were nontoxic in growth inhibition assays (data not shown), the nonionic detergents Triton WR- 1339 and Tween 80 stimulated ADR uptake and inhibited its efflux to the same extent as observed for VER. However, at concentrations that caused an approximately 20% inhibition of Fig. 5. Comparison between the effect of VER and the effect of membrane- cell growth over 72 h (data not shown), the polyene antibiotics, active agents on ADR accumulation in HCT-8 cells. HCT-8 cells were incubated amphotericin B and filipin, and the ionic detergent, deoxycho- in ADR-uptake medium containing either 0.025% Triton WR-1339, 0.025% Tween 80, 5.0 Mg/ml of VER, 1.0 (ig/ml of amphotericin B, 2.5 jig/ml of filipin. or saline. Results are the average of 2 experiments, each performed in triplicate.

500

'VERAPAMIL

400

<->300 ^-VERAPAMIL

300

012345 HOURS 150 Fig. 3. Effect of VER on the net accumulation of the anthrapyrazole CI-937 in HCT-8 cells. Cells were incubated at 37'C in 10.3 Â»M[MC]CI-937 (specific Fig. 6. Comparison between the effect of VER and the effect of membrane- activity, 83.1 Â¿iCi/mmol)in the presence or absence of S.Ofig/ml of VER. Uptake active agents on ADR efflux from HCT-8 cells. Cells were preloaded with ADR was determined as previously described (6). Points, average of 3 experiments, as described in Fig. 1, and ADR efflux was determined in culture medium each performed in duplicate; bars, SD. containing either 0.025% Triton WR-1339,0.025% Tween 80,5.0 Â»tg/mlofVER, 1.0 Mg/ml of amphotericin B, 0.008% deoxycholate, or saline. Points, average of 2 experiments, each performed in triplicate; bars, SD. 100" late, had no effect on ADR accumulation and/or efflux (Figs. 5 and 6). Lower nontoxic concentrations of these 3 compounds were similarly without effect on ADR transport (data not shown). In view of the similarity of results between VER and the lysosomotropic detergent, Triton WR-1339, other agents known to interact with the lysosome were also tested for their effect on ADR accumulation and efflux in HCT-8 cells (Figs. 7 and 8). The proton ionophores nigericin and monensin were as effective as VER in both increasing ADR accumulation (Fig. 7) and inhibiting its efflux (Fig. 8). The other proton ionophore, CCCP, was somewhat less effective in potentiating ADR uptake (Fig. 7). The lysosomotropic amines chloroquine and mono- dansylcadaverine also potentiated cellular accumulation of ADR and/or blocked its exodus (Figs. 7 and 8). Cytochalasin 60 90 120 150 MINUTES B, which can disrupt the microfilamentous component of the Fig. 4. Effect of VER on the efflux of CI-937 from HCT-8 cells. Cells were cytoskeleton, also inhibited ADR efflux from HCT-8 cells (Fig. preloaded in the absence of VER for 4 h under conditions described in Fig. 3 and 8). Neither methylamine or ammonium chloride, however, af then washed twice prior to resuspending the cell pellet in ADR-tree medium Â± 5.0 /ig/ml of VER. Efflux was determined as previously described (6). Points, fected ADR accumulation or efflux in these cells (Figs. 7 and average of 3 experiments, each performed in duplicate; bars, SD. 8). Reducing the concentration of ADR in the incubation me- 3027

appeared to be significantly enhanced over that found for other drug-sensitive cell lines such as P388S cells (10). The degree of potentiation of ADR and CI-937 activities by VER in HCT-8 cells was the same or similar to VER's effect in blocking either drug from being transported out of these cells (Table 2; Figs. 2 and 4). While there are undoubtedly numerous factors which contribute to the overall intrinsic drug resistance of colon tumors, our data along with previous observations (4-6) suggest that this intrinsic resistance phenomenon, like acquired MDR, may, in part, be related to the ability of the tumor to transport drug out of the cell. This enhancement of drug efflux in HCT- 8 may, in fact, be a property of normal intestinal epithelial cells as well. In a carefully characterized normal IEC-6 rat intestinal epithelial cell line (IS), we demonstrated that VER potentiated ADR activity in these cells by increasing ADR uptake and inhibiting its efflux (Table 1; Figs. 1 and 2). The enhanced 246 outward transport of agents such as ADR in normal intestinal HOURS Fig. 7. Effect of lysosomal-active agents on ADR accumulation in HCT-8 cells might be one of the mechanisms involved in the protection cells. ADR uptake was determined as described in Fig. 1 in the presence of either of intestinal epithelium from plant alkaloids and other xeno- 5.0 Mg/ml of nigericin, 100 Ã•(Mchloroquine, 200 itM CCCP, 10 HIMammonium biotic agents ingested in the diet. Perhaps many colon tumors chloride, 10 mM methylamine, or saline. Results are the average of 3 experiments, each performed in duplicate. retain this property. Fojo et al. (2) observed that the mdrl gene was overexpressed in some but not all human colon tumors and adjacent normal colon tissue. Even in the limited number of colon cell lines we studied, we observed that VER did not alter ADR activity in two of six cell lines (Table 1), indirectly indicating that these two cell lines did not efflux appreciable amounts of ADR. Presumably those colon tumors which lack this efflux "pump" have some other mechanism to afford protection against xe- nobiotics. Labeling surface glycoproteins by treating cells with neuraminidase and galactose oxidase-[3H]borohydride as de scribed by Beck et al. (16), we could not detect the presence of a glycoprotein with a molecular weight of 140,000 to 170,000 in these cell lines (data not shown), but this procedure may be too insensitive to detect P-gp in these cells, and more sensitive complementary DNA probes may be necessary. Since we could not detect P-gp by surface labeling and polyacrylamide gel electrophoresis in any of the cell lines stud ied including HCT-8 cells, we cannot be certain that the drug 30 90 120 ISO efflux demonstrated in HCT-8 cells is due to the same mecha MINUTES Fig. 8. Effect of lysosomal-active agents and cytochalasin B on ADR efflux in nism as that observed in MDR cells. However, the evidence HCT-8 cells. Cells were preloaded as described in Fig. 2, and efflux was deter from other studies (4-6) and from the present study for VER mined in ADR-free medium containing either 5.0 fig/ml of VER, 5.0 /ig/ml of nigericin, 5.0 Â«Â¿g/mlofmonensin, 100 Â¿IMmonodansylcadaverine, 5.0 Â¿ig/nilof potentiation of drug activity and blocking of drug efflux in cytochalasin B, 100 HIMchloroquine, 10 mM ammonium chloride, 10 mM meth colon tumor cells as well as the finding that the mdrl mRNA ylamine, or saline. Results are the average of 3 experiments, each performed in is amplified in many colon tumors (1) suggests that P-gp is triplicate. probably responsible for the efflux of ADR from HCT-8 cells. What is still not clear, however, is the mechanism by which P- dium from 9.2 /IM to 0.46 /IM did not alter the effect of lysosomotropic amines, proton ionophores, or cytochalasin B gp removes drugs from these cells. Several groups have pro posed that the decreased ability of MDR cells to retain certain or ADR transport (data not shown). drugs was due to an active efflux "pump" (13, 17, 18), and based on its primary sequence, its apparent transmembrane DISCUSSION location, its potential nucleotide binding sites, and its homology Several recent studies have observed that VER, which poten with bacterial transport proteins (19), Chen et al. (19) also tiates drug activity in acquired or pleiotropically drug-resistant concluded that P-gp functioned as an energy-dependent drug tumors, can also increase drug sensitivity in inherently drug- efflux pump. Cornwell et al. (20) recently reported that vesicles resistant colon tumor cells (9-11). As in MDR cells, cytoflu- from MDR cells bound considerably more radiolabeled vin- orescent studies by Chauffert et al. (4) suggested that VER as blastine than did vesicles from drug-sensitive cells. Moreover, well as other pharmacological drugs could potentiate ADR they also demonstrated that VER inhibited vinblastine photo- activity in a rat colon tumor cell line by inhibiting its exodus affinity labeling of P-gp (21), and from these and other obser from these cells. In the present study, we demonstrated that vations concluded that VER and other modifiers of drug resist VER could potentiate ADR activity in a number of colon tumor ance act by inhibiting the binding of drugs such as vinblastine cell lines and that, at least for the HCT-8 human colon tumor to P-gp which is somehow involved in their efflux by an energy- line, this drug-potentiating activity was related to the ability of dependent mechanism. VER to inhibit drug efflux. ADR efflux from HCT-8 cells Our data, however, lend additional support not to the active 3028

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research. INTRINSIC DRUG RESISTANCE pump theory, but rather to a new hypothesis independently where their concentration results in alterations in lysosomal described by Sehested et al. (8) and by Beck (7). They suggest membrane phospholipids and thereby modifies lysosomal func that the lysosome or an associated acidic compartment in the tion. This possibility is supported by our observations for ADR MDR cell is involved in drug exodus and that drugs such as transport in HCT-8 cells in the presence of proton ionophores ADR or vinblastine, which are weak bases, can become en and lysosomotropic amines (Figs. 7 and 8), both of which are trapped in these acidic compartments by protonation. These known to alkalinize acidic vesicles and alter lysosomal function acidic drug-containing vesicles then migrate to the plasma (32,33). However, if the lysosomal system is somehow involved membrane where they fuse and extrude their contents to the in drug efflux from certain inherently resistant tumor cells, it outside. Beck (7) suggests that, rather than serving as an efflux is unclear why primary amines such as ammonium chloride and pump, P-gp may function at the plasma membrane to alter meth) lamine have no effect on ADR accumulation or exodus membrane turnover or vesicular trafficking, or alternatively, from HCT-8 cells (Figs. 7 and 8) or altered vincristine activity serve to transport certain drugs across the plasma membrane in MDR cell lines (34). Primary amines, like proton ionophores or into acidic vesicles. and agents such as chloroquine, have been shown to alkalinize Our data demonstrating inhibition of ADR efflux from HCT- lysosomal vesicles (Ref. 21; Footnote 3), although several recent 8 cells by lysosomotropic amines, proton ionophores, and cy- studies have noted different cellular responses to primary tochalasin B (Fig. 8) support this notion of a lysosomal involve amines versus proton ionophores or chloroquine (22-24), per ment in drug efflux. Our findings for IId -Xcells in the present haps because of the varying degrees of efficiency by which study are, indeed, virtually identical with those we have ob charged primary amines are transported into certain cells or served in our P388 MDR model.3 These findings are also in cellular compartments. agreement with those recent observations by Zamora and Beck In conclusion, our data suggest that an enhanced drug efflux (22) and by Shiraishi et al. (23) demonstrating that lysosomo may be an important factor in the overall level of inherent drug tropic amines, such as chloroquine and other agents known to resistance in certain colon tumors. This elevated efflux may be interact with the lysosome, could reverse drug resistance to a property of normal intestinal epithelium where it provides anthracyclines and Vinca alkaloids in MDR cells. In addition, some protection from toxins and carcinogens ingested in the in accord with the findings of Tsuruo and lida for MDR cells diet. Our data for modifiers of ADR exodus from HCT-8 cells (24), we observed that cytochalasin B, an agent that has several lend considerable support to the hypothesis for MDR cells that cellular effects including inhibition of microfilament function endosomal/lysosomal vesicles may be involved in drug exodus and secretion (25,26), also partially blocked the outward trans and, therefore, drug resistance. port of ADR from HCT-8 cells (Fig. 8). Beck (7) suggests that cytochalasin B might inhibit drug REFERENCES efflux by impairment of membrane flow, thereby inhibiting the movement of drug-containing vesicles to the plasma membrane. 1. American Cancer Society. Cancer statistics. Cancer J. Clin., 56:16-17,1986. It should be pointed out, however, that cytochalasin B as well 2. Fojo, A. T., Ueda, K., Siamoli, D. J., Gottesman, M. M., and Pastan, I. Expression of a multidrug-resistance gene in human tumors and tissues. as the proton ionophores and lysosomotropic agents can all Proc. Nati. Acad. Sci. USA, 84: 265-269, 1987. have multiple effects on cellular functions including alterations 3. Shen, D-W., Fojo, A., Roninson, I. B., Chin, J. E., Soffir, R., Pastan, I., and in plasma membrane functions. Several previous studies on Gottesman, M. M. Multidrug resistance Â»fDNA mediated transformants is linked to transfer of the human mdrl gene. Mol. Cell. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1988 American Association for Cancer Research. Possible Link between the Intrinsic Drug Resistance of Colon Tumors and a Detoxification Mechanism of Intestinal Cells

Wayne D. Klohs and Randall W. Steinkampf

Cancer Res 1988;48:3025-3030.

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