C.M. Kuiper

Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.

Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.

Early multidrug resistance, defined by changes in intracellular doxorubicin distribution, independent of P-glycoprotein.

Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance.

Molecular downstream events and induction of thymidylate synthase in mutant and wild-type p53 colon cancer cell lines after treatment with 5-fluorouracil and the thymidylate synthase inhibitor raltitrexed

Inhibition of the key enzyme in DNA synthesis, thymidylate synthase (TS), by 5-fluorouracil (5-FU) and the novel antifolate raltitrexed (Tomudex; ZD1694), induces dTTP depletion, resulting in DNA strand breaks, which can initiate pathways leading to an apoptotic mode of cell death. We studied 5-FU- and ZD1694-induced TS inhibition in relation to the expression of p53, p21, Bcl-2 and Bax in six colon carcinoma cell lines, two with a wild-type (wt) p53 (Lovo, LS174T) and four with a mutant (mt) p53 (WiDr, WiDr/F, HT29 and SW948) phenotype. In untreated cells, a reciprocal correlation between p53 and Bcl-2 was found: in cells with a low wt p53, Bcl-2 expression was present; whilst in cells with mt p53, Bcl-2 expression was not detectable. Exposure to 5-FU (50 and 100 microM) and ZD1694 (50 and 100 nM) for 24 and 48 h induced p53 and p21 expression in wt p53 cells, but not in mt p53 cells. TS was induced approximately 2-10-fold in all cell lines. TS induction was highest after ZD1694 exposure in the mt p53 cells HT29 and WiDr/F (6-10-fold). After 5-FU treatment, TS was present both as the free enzyme and in the ternary complex; however, predominantly as the ternary complex between TS, FdUMP and 5,10-methylenetetrahydrofolate. In wt p53 cells, both drugs increased Bax expression up to 5-fold, whereas in mt p53 cells, only a very slight induction was found. In wt p53 cells, Bcl-2 expression hardly changed after drug treatment. These results indicate a p53-independent induction of TS but a regulatory role of wt p53 in the synthesis of Bax in the colon carcinoma cell lines after TS inhibition.

Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein

An overexpression of plasma membrane 170-180 kDa P-glycoproteins is consistently found in multidrug-resistant (MDR) cell lines. In this study MRK-16, a monoclonal antibody (mAb) reacting with P-glycoprotein is used to study the putative functional role of this protein in vincristine (VCR) and daunorubicin (DNR) cellular accumulation in the MDR human ovarian carcinoma cell line 2780AD. We established that this cell line is highly cross-resistant to vincristine and daunomycin, related to a greatly reduced drug accumulation. Verapamil (Vp) (8 microM) caused a 3.6-fold increase in DNR as well as VCR accumulation. Exposition of 2780AD cells to MRK-16 led to an increase of 30% in cellular accumulation of VCR, both in normal growth medium as well as in medium without added glucose and with sodium azide, which largely depleted cellular ATP levels. No increase in DNR accumulation was found under these conditions. However, in the presence of 8 microM Vp, MRK-16 increased not only VCR but also DNR accumulation with about 30%. The relative increase of DNR accumulation was constant in a concentration range of 0.2-4 microM DNR. These data indicate that mAbs against P-glycoprotein might potentiate the action of calcium antagonists like Vp to increase cellular anthracycline accumulation.

Glycolysis in P-glycoprotein-overexpressing human tumor cell lines. Effects of resistance-modifying agents.

We show that drugs, such as verapamil, which reverse multidrug resistance (MDR), in P‐glycoprotein‐overexpressing tumor cells, increased the rate of lactate production in four human MDR cell lines, but not in the parent, sensitive cell lines. The effect on glycolytic rate was maximal at a medium concentration of 2 μM verapamil. The glycolytic rate in sensitive (A2780) and MDR (2780AD) cells showed the same pH dependence, but the effect of verapamil was seen only in 2780AD cells at all pH values investigated (6.6, 7.4 and 8.2). A series of drugs such as nigericin, oligomycin, amiloride and monensin had similar effects in the two cells. Phorbol myristate acetate increased lactate formation in neither cell line. Verapamil induced an extra amount of ATP consumption in P‐glycoprotein‐expressing 2780AD cells of approx. 25 pmol/s per 106 cells, which was estimated to be about 10% of cellular energy turnover.

Differences in the induction of DNA damage, cell cycle arrest, and cell death by 5-fluorouracil and antifolates.

Thymidylate synthase (TS) is an important target for chemotherapy and can be inhibited by 5-fluorouracil (5-FU) and the antifolates, AG337 (Nolatrexed) and multitargeted antifolate (MTA or Pemetrexed). In addition, 5-FU can be incorporated into RNA and DNA, and MTA can inhibit two other enzymes. It is, however, unclear to what extent these differences in drug action will influence activation of downstream mechanisms mediated via TS inhibition. Therefore, two human colon cancer cell lines, WiDr and Lovo, with a different clonogenic origin, were treated with equitoxic concentrations of 5-FU, AG337, and MTA to determine the induction of DNA damage, cell cycle arrest, downstream protein expression, and cell death. At these concentrations, the specific TS inhibitor AG337 induced more DNA damage (up to 20%) than MTA and 5-FU. FACS analysis showed that all drugs induced S phase arrest in Lovo and WiDr that was most pronounced after 5-FU and AG337 exposure (50-70%). Western blotting showed that p53 induction was not detectable in mutant (mt) p53 WiDr and increased much earlier in wild-type (wt) Lovo cells after 5-FU and MTA (24 h) than after AG337 exposure (72 h). In contrast to 5-FU-treated Lovo cells, the bcl-2/bax ratio decreased after antifolate exposure. Nevertheless, both 5-FU and antifolates induced similar amounts of cell death (up to 60%). These results demonstrate that in human colon cancer cells differences in downstream events between AG337 and 5-FU or MTA are related to the additional effects of 5-FU and MTA, which are not associated with TS inhibition.

Daunomycin accumulation in resistant tumor cells as a screening model for resistance modifying drugs: role of protein binding.

A potential screening system for drugs that modify anthracycline resistance was investigated. The effect of the calcium antagonistic agents verapamil, diltiazem, bepridil and Ro 11-2933/001 on daunorubicin (DNR) accumulation in the doxorubicin resistant cell line 2780AD was measured. For this purpose cells were suspended in medium with varying concentrations of bovine serum albumin (BSA) and in human plasma to establish the influence of protein binding on DNR accumulation. Increase of protein concentration did not affect DNR accumulation when no calcium antagonists were present but caused an increase in concentrations of calcium antagonists needed to promote DNR accumulation. It appeared that DNR accumulation in suspended cells correlated well with cytotoxicity assays, performed on cells growing in monolayer in 96-well microtiter plates, when accumulation was measured in medium with the same protein constitution as in growth medium (10% fetal calf serum). It is concluded that protein binding effects should be taken into account in the screening of compounds for the circumvention of multidrug resistance.

Synergistic interaction between cisplatin and gemcitabine in ovarian and colon cancer cell lines.

Cisplatin (cis-diammine dichloroplatinum, CDDP) is an established square planar coordination compound, which is effective against ovarian cancer (1). The antitumour activity of CDDP is the result of formation of adducts within the DNA (2). Resistance against CDDP treatment may be related to an increased DNA repair. Like CDDP, Gemcitabine (2’,2’-difluorodeoxycytidine, dFdC) is active against human ovarian carcinoma (3, 4). After entering the cell, dFdC requires activation catalyzed by deoxycytidine kinase (dCK), and can be inactivated by the action of deoxycytidine deaminase (dCDA)(5). The active metabolite dFdCTP can be incorporated into both DNA and RNA (6). For both 1-s-D-arabinofuranosylcytosine (ara-C) and 2’-deoxy-5-azacytidine (DAC) two other deoxycytidine analogues, synergy with CDDP has already been described (7, 8). The next logical step was to combine dFdC with CDDP. Both agents have a different mechanism of action. A combination is also attractive from a clinical point of view, since both drugs have different side effects. We tested this combination in different concentrations and schedules in the human ovarian cancer cell line A2780, its 50 fold CDDP resistant variant ADDP (9), its 150,000 fold dFdC resistant variant AG6000 (10), and in the murine colon cancer cell line C26-10, which has an inherent lower sensitivity to both drugs compared to A2780 (11). These results were related to both dCK and dCDA activities and the effect of CDDP on dFdCTP accumulation.

Tumor size and origin determine the antitumor activity of cisplatin or 5-fluorouracil and its modulation by leucovorin in murine colon carcinomas

Abstract 5-Fluorouracil (FUra) is one of the few effective agents in the treatment of patients with colorectal cancer. Its effects on the target enzyme thymidylate synthase (TS) can be modulated by leucovorin (LV) or cisplatin (CDDP). Tumor size and differentiation of tumor characteristics can influence therapeutic efficacy. We therefore studied the relationship between tumor size (cutoff point 200 mm3) and the antitumor activity of FUra and its modulation by LV in murine Colon 26 and Colon 38 tumors. The doubling time of tumors measuring >200 mm3 was about 160% longer. The antitumor effect of FUra in these large tumors was decreased and could not be modulated by LV. In addition, three subtypes of Colon 26 (Colon 26-A, Colon 26-B, and Colon 26-10) were identified and characterized for tumor-induced weight loss, TS activity, response to chemotherapy, and histological features. Mice bearing Colon 38 and Colon 26-10 did not lose weight as a result of tumor growth. Colon 26-A caused a weight loss of up to 19%, whereas mice with Colon 26-B tumors remained within 10% of their initial weight and tolerated at least 2.5 times more tumor load than did mice bearing Colon 26-A, which induces cachexia. Among untreated tumors, TS catalytic activity was highest in Colon 26-B (5536 pmol mg protein-1 h-1) and lowest in Colon 38 (799 pmol mg protein-1 h-1); Colon 26-A and Colon 26-10 had intermediate activities (about 2500 pmol mg protein-1 h-1). 5-Fluoro-2′-deoxyuridine monophosphate (FdUMP) binding was comparable in the three Colon 26 subtypes but was lower in Colon 38. The antitumor activity of FUra could be modulated by LV in Colon 38, Colon 26-10, and Colon 26-A but could not in Colon 26-B, with complete responses (CR) being obtained in Colon 26-10 and Colon 38. The latter two were highly sensitive to CDDP, followed by Colon 26-A and Colon 26-B (CRs: 50%, 40%, 25%, and 0, respectively). Furthermore, necrosis was noted in Colon 26-B and Colon 38 but not in Colon 26-A. In conclusion, (1) the antitumor activity of FUra in large tumors is decreased and cannot be modulated by LV and (2) characteristics and sensitivity to chemotherapeutics can vary substantially in closely related tumors of the same origin.