Research ArticleExperimental Studies

Circumvention of Inherent or Acquired Cytotoxic Drug Resistance In Vitro Using Combinations of Modulating Agents

DAVID CADAGAN and STEPHEN MERRY
Anticancer Research October 2013, 33 (10) 4381-4387;

Abstract

Background/Aim: Modulating agents are used to circumvent drug resistance in the clinical setting. However achievable serum concentrations are often lower than those which are optimal in vitro. Combination of modulating agents with non-overlapping toxicities may overcome this obstacle. We have investigated combinations of three modulating agents (quinine, verapamil, and cinnarizine) to circumvent inherent or acquired resistance to the cytotoxic drugs doxorubicin, vincristine and paclitaxel. Materials and Methods: Dose-response curves to cytotoxic drugs in the presence/absence of modulating agents were determined using colony formation and cell proliferation assays. Doxorubicin accumulation into cell monolayers was measured by fluorescence spectrophotometry. Results: Greater (1.9-fold) sensitisation to particular cytotoxic drugs was observed for certain combinations of modulating agents compared to individual effects. The most effective combination was quinine-plus-verapamil with the cytotoxic drug doxorubicin. This increase in sensitivity was associated with increased doxorubicin accumulation. Such enhanced activity was, however, not observed for all combinations of modulating agents or for all studied cytotoxic drugs. Conclusion: The findings of the present study suggest certain combinations of modulating agents to have a clinical role in circumventing drug resistance. Particular combinations of modulating agents must be carefully chosen to suit particular cytotoxic drug treatments.

The cytotoxic drugs doxorubicin hydrochloride (Adriamycin hydrochloride, DOX), vincristine sulphate (VC) and paclitaxel (taxol, PT) are used in the clinical setting to treat a variety of cancers, but it is becoming increasingly apparent that a major obstacle in their effective use is the presence or emergence of drug-resistant cells within the tumour (1).

DOX, VC and PT are all associated with the multidrug resistance phenotype (2-4), which has been observed in tumours made resistant by chronic treatment with cytotoxic drugs in vitro or in vivo (5-7). One characteristic of this phenotype is the presence of particular membrane proteins such as the P-glycoprotein (8-9) or multidrug resistance-associated protein (MRP) (10, 11) leading to altered membrane transport of a range of cytotoxic drugs. The clinical relevance of the multidrug resistance phenotype remains to be proven, but both P-glycoprotein and MRP have been shown to be expressed in a number of clinical tumour specimens (12-14).

One approach to investigate clinical relevance has been to study drug resistance in cell lines derived from clinical specimens, which have not received any prior drug treatment in vitro. While levels of resistance in this case are generally lower than those which can be derived in the laboratory, our previous studies of such inherent resistance in human glioma (15) and human non-small cell lung cancer (16) cell lines have shown similar patterns of cross-resistance between cytotoxic drugs as those observed in the multidrug resistance phenotype. The relevance of the multidrug resistance phenotype to clinical drug resistance is particularly important because such multidrug resistance can be circumvented by a number of modulating agents (17-19). The most thoroughly studied multidrug resistance-modulating agent is verapamil (VPM), which we have also shown to circumvent inherent resistance in human glioma and non-small cell lung cancer cell lines (16, 20).

Laboratory studies of multidrug resistance-modulating agents have lead to several clinical trials involving compounds such as VPM (21, 22) quinidine (23) amphotericin B (24) and cyclosporin A (25, 26). These trials have produced some encouraging results, but they have also shown that concentrations of modulating agents that can be achieved in the serum of patients are often less than those, which are optimal in reversing drug resistance in vitro. One way of effectively achieving higher serum (and hence tumour) concentrations might be to use combinations of modulating agents which have different toxicities so that each can be used at its maximum dosage in the presence of the other.

There are two additional reasons why combinations of modulating agents may be more effective than a single agent in circumventing drug resistance. Firstly, a variety of mechanisms (in an addition to changes in membrane permeability) have been shown to contribute to multidrug resistance in a variety of experimental models. Such mechanisms include changed DNA topoisomerase II activity (18, 27, 28) modified glutathione metabolism (29, 30) and altered intracellular drug binding (31). These mechanisms (and others) may contribute to inherent or acquired resistance, and they may be circumvented to different extents, by different modulating agents, such that combinations are more effective than single agents. Secondly, it has been shown that P-glycoprotein has multiple binding sites (32, 33), which have different affinities for different modulating agents. It is possible that P-glycoprotein would be completely inactivated in the presence of combinations of modulating agents, which bind to different sites rather than a single agent binding to a single site.

Previous in vitro studies have shown effective use of combinations of modulating agents including cyclosporin A, VPM and Tamoxifen in modulating the expression of the multidrug resistance phenotype (34, 35). In particular, significant synergism between tamoxifen and cyclosporine A on the sensitivity of DOX was seen at clinically administered concentrations within the hepatoma cell lines (35). However, further studies are warranted to identify which particular combinations of modulators at physiologically-achievable concentrations are most effective in overcoming the multidrug resitance phenotype.

In the present study we investigated the ability of the modulating agents quinine (QN), verapamil (VPM) and cinnarizine (CN) as single agents and in combination, in circumventing inherent resistance to DOX, VC and PT in the continuous murine cell line L929, its DOX resistant variant L929/A and the human breast cancer cell line MCF7. Each modulating agent was used at its maximum tolerated dose in vivo (ID50/10: MTD). In each case this was a similar concentration to that achieved in plasma in patients (36-38). The modulating agents used in this study were chosen with a view to future clinical studies since they fulfil the criteria of having generally non-overlapping toxicities in vivo.

Materials and Methods

Cell lines. L929 (murine fibrosarcoma) and MCF7 (human breast adenocarcinoma) cell lines were obtained from ECACC (Porton Down, Wiltshire, UK). The DOX-resistant cell line, L929/A, was established during this work by exposure of parent L929 cells to increasing concentrations of DOX over a period of 3 months. Once established, the cell line was routinely sub-cultured and grown in medium containing 0.086 μM DOX. Stock cultures were maintained in 25-cm2 tissue culture flasks and shown to be free of mycoplasma by staining with Hoechst 33258 (Sigma, Poole, Dorset, UK). Cells were grown at 37°C as monolayer cultures in RPMI 1640 medium supplemented with 10% foetal bovine serum and, when the gas phase was ambient air, 10 mM HEPES. In some cases the medium was supplemented with 50 μg/ml gentamycin sulphate (Sigma). Cell suspensions were prepared by trypsinisation and viable cells (those excluding 0.2% trypan blue) were counted using a Neubauer haemocytometer.

Chemicals. The cytotoxic drugs DOX, VC and PT, the modulating agents QN, VPM and CN, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylterazolium (MTT) were obtained from Sigma. Stock solutions of DOX, VC, PT, VPM and QN (5 mg/ml, 1mg/ml, 0.5mg/ml, 50 mg/ml and 50mg/ml respectively) were prepared in 50% ethanol. Stock solutions of CN (50 mg/ml) were prepared in dimethyl sulphoxide (DMSO). All stock solutions were stored as aliquots in the dark at −20°C, and were diluted with culture medium immediately before use. MTT (1 mg/ml) was dissolved in Dulbecco's phosphate buffered saline without calcium and magnesium (pH 7.2; PBS) (Sigma), prior to use and kept at 4°C.

Colony formation assay. Five-hundred L929 cells in 10 ml culture medium were seeded into Greiner 90-mm tissue culture petri dishes (Philip Harris, Litchfield, Staffordshire, UK) in the presence of drugs or solvent (control). The dishes were incubated for 9-11 days with ambient air as the gas phase, fixed with methanol, stained with 0.1% crystal violet and colonies ≥0.5 mm diameter were scored visually. Colony number was expressed as a percentage of control and the concentration of drug causing a 50% reduction (ID50) was determined graphically. Plating efficiency of controls was 45±3% (mean±S.E.M, n=60).

Sensitisation ratios as described by Merry et al. (1989) were calculated as: ID50 − M / ID50 + M (15). Where M is the modulating agent (or combination of modulating agents) employed in that particular assay. Sensitisation ratios were calculated using the cytotoxicity curves obtained from duplicate petri dishes at each of 5 different concentrations all within a single experiment. All experiments were carried out at least in duplicate.

Cell proliferation assay. Fifty thousand L929, L929/A or MCF7 cells in 200 μl medium were seeded per well into Nunc 96-well tissue culture plates (Fisher Scientific, Loughborough, UK) in the presence of drugs or solvent (control). Cells were incubated for 7 days with a gas phase of 5% CO2 in air before 20 μl MTT were added to each well and the plates were incubated for an additional 3 h (39). The supernatant was then aspirated from each well and 200 μl of DMSO were added to solubilize the coloured formazan product. After 20-30 min the absorbance (570 nm) of each well was determined (Anthos 2010 Microplate Reader, Anthos Labtech Instruments, Salzburg, Austria). Results were expressed as percent cytotoxicity relative to control wells.

Doxorubicin accumulation assay. One million L929 cells in 10 ml culture medium were seeded into Greiner 90-mm tissue culture petri dishes (Philip Harris), which were then incubated for 48 h with ambient air as the gas phase prior to the addition of 34 μM DOX. At 0, 30, 60, 90, 120 and 150 min the medium was poured from duplicate dishes and the cell monolayers were washed with ice cold PBS (3x5 ml). Cell suspensions were prepared from the monolayers by treatment with 2 ml 0.25% trypsin/ 0.02% EDTA in PBS and an aliquot was removed for viable cell counting. A further 1.5-ml aliquot was homogenized (4 strokes of a Potter homogeniser) and DOX was extracted into 5 ml chloroform/isopropanol (2:1) and measured by fluorescence spectrophotometry (SFM 25 Spectrofluorometer, Kontron Instruments, Watford, UK) with excitation and emission wavelengths of 480 nm and 560 nm respectively (45). All experiments were carried out at least in triplicate. Preliminary experiments showed the absence of background fluorescence from untreated cell homogenates and DOX extraction efficiencies of 101±3% (mean±s.e.m, n=14).

Figure 1.
Figure 1.

The effect of quinine on the sensitivity of L929 cells to doxorubicin. The lines join the mean of duplicate determinations at each doxorubicin concentration.

Results

In the L929 model with colony formation as end-point the ID50 values for cytotoxic drugs DOX, VC and PT in the absence of modulating agents were found to be 124±21 nM, 87±20 nM and 160±25 nM respectively (mean±S.E.M, n=25, 20 and 13 respectively). Furthermore the ID50 values of the modulating agents QN, VPM, CN -alone were found to be 35.7 μM (range: 29.7-41.6 μM), 4.4 μM (range: 3.0-5.7 μM) and 2.7 μM (range: 2.6-2.8 μM) respectively. In subsequent cytotoxicity experiments these modulating agents were used at their maximum tolerated dose (MTD) in vitro, which was determined as 1/10 ID50. For each modulator these are similar concentrations to those that have been achieved in plasma in patients (36-38) and, in all cases, the MTD inhibited colony formation in vitro by less than 6%.

Figure 1 shows the result of a typical colony formation assay. In this particular case QN is seen to produce a sensitisation ratio to DOX of 11.3 (ID50-QN=186.2 nM, ID50+QN=16.6 nM). Table I summarises the overall results of our L929 colony formation assays. It is seen that for all three cytotoxic drugs QN was the most effective modulating agent, VPM was less effective and CN showed little activity in this model. Furthermore, the size of the sensitisation ratio produced was dependent on the cytotoxic drug used. Sensitisation ratios were greatest for PT, less for DOX and smallest for VC.

View this table:
Table I.

Summary of cytotoxicity results showing the effects of maximum tolerated doses of quinine, verapamil and cinnarazine on the sensitivity of L929 cells to doxorubicin, vincristine and paclitaxel.

The combination of QN plus VPM produced greater sensitisation ratios than either modulating agent alone for DOX and for VC, but not for PT. Furthermore VPM plus CN produced greater sensitisation ratios than either modulating agent alone for DOX and PT, but not for VC, while QN plus CN produced lower sensitisation ratios than QN-alone for all three cytotoxic drugs. Toxicity (36±11%, mean±S.E.M., n=4) precluded the use of all three modulating agents together in our colony formation assay.

Figure 2 shows the effect of 3.6 μM QN (ID50/10 MTD) on DOX accumulation into L929 cells. QN is seen to produce an increase in accumulation of 63% at 150 min (DOX accumulation-QN=655 pmol/106 cells, DOX accumulation+QN=1,069 pmol/106 cells). Furthermore the increase in DOX accumulation produced by QN was statistically significant at 60, 90, 120 and 150 min (p<0.05, p<0.01, p<0.02 and p<0.05, respectively) with a statistical trend (p<0.1) at 30 min. The accumulation of DOX both in the presence and absence of QN had not reached a plateau at 150 min, but experiments were terminated at this time point because longer incubations produced reductions in cell viability.

The overall results in our DOX accumulation experiments are summarised in Table II. In all cases the shape of the accumulation curves were similar to those of Figure 2. At 150 min QN and VPM increased accumulation by 63% and 50% respectively, while CN produced a 16% reduction. The combination QN plus VPM increased DOX accumulation by 79%, whereas QN plus CN and VPM plus CN caused 3% and 8% reduction respectively. In additional experiments (data not shown) it was noted that 6.6 μM and 66 μM VPM produced increases of 74% and 71% respectively. Furthermore the combination QN plus VPM plus CN produced an increase of 53%.

Figure 2.
Figure 2.

A typical experiment showing the effect of quinine on doxorubicin accumulation into L929 cell monolayers. The lines join the mean of 3-4 determinations at each time point. Error bars indicate mean±S.E.M. and are omitted when their range is smaller than the size of the symbol used to indicate mean value.

Table III enables comparison of the effects of modulating agents on DOX resistance and DOX accumulation. It can be seen that QN and VPM enhanced accumulation both as single agents and in combination, and the increases are associated with increased DOX sensitivity with colony formation as end-point. This contrasts with the case of CN, which produced no increase in DOX accumulation both as a single agent and in combination with QN or VPM, even though CN, QN+CN and VPM+CN each produced increased sensitivity to DOX.

Figure 3 summarises results from cell proliferation assays. The effective modulator combination (VPM plus QN) identified for DOX in the colony formation assays also significantly increased DOX (100 nM) sensitivity (p<0.05, n=60) for L929 cells in cell proliferation assays. Similar sensitisation effects were additionally seen in the DOX-resistant variant L929/A (p<0.0001, n=60) and in the human breast cancer cell line MCF7 (p<0.0001, n=20).

Figure 3.
Figure 3.

Summary of the cell proliferation assay data showing the effect of maximum tolerated doses of the combined modulating agents quinine and verapamil on doxorubicin (100 nM) sensitivity. Error bars indicate mean±S.E.M. (n=20-60). *p<0.05, ***p<0.01.

Discussion

We have examined the ability of three drug resistance-modulating agents (QN, VPM and CN) both as single agents and in combination to circumvent inherent or acquired resistance to three cytotoxic drugs (DOX, VC and PT) in murine L929 and human MCF7 cells.

Initial experiments in this study utilised a cell line with inherent drug resistance mechanism (L929) since this may more truly reflect clinical drug resistance compared to multidrug resistant cell lines in which resistance has been derived by chronic treatment with cytotoxic drugs in the laboratory. Inherent drug resistance may result from a combination of mechanisms (see Introduction), which are not all equally amplified in multidrug-resistant cell lines derived in the laboratory. Subsequent experiments confirmed the relevance of observations concerning L929 to a variant cell line with acquired DOX resistance (L929/A) and to a human breast cancer cell line (MCF7).

The modulating agents were used at their MTD in this experimental model, which was determined as ID50/10. These concentrations (3.6 μM QN, 0.4 μM VPM or 0.3 μM CN) were chosen since they are equally close to the toxicity threshold of the modulating agent. Furthermore those are similar concentrations to these that have been achieved in plasma in patients (36-38). Kunnumakkara et al. (2008) have argued for the utility of conventional rather than targeted ‘smart’ drugs in cancer treatment since cancer typically involves the dysregulation of multiple pathways (44). The disruption of a single pathway by a ‘smart' drug, they argue, is unlikely to be fully effective. QN, VPM and CN are each conventional drugs, which can be regarded as multi-targeted, and the range of affected pathways is further increased by their use in combination.

View this table:
Table II.

Summary of Doxorubicin uptake data showing the effect of maximum tolerated doses of quinine, verapamil and cinnarizine on doxorubicin accumulation into L929 cells after150 min.

While the extent to which the modulating agents circumvented resistance in L929 cells varied between the three cytotoxic drugs. In all cases QN was the most effective single agent followed by VPM, and with CN showing only marginal activity. This may suggest that in this model there is a single mechanism (or group of mechanisms) that confers resistance to all three cytotoxic drugs. The use of combinations of modulating agents produced, in some cases, greater increases in sensitivity than either of the modulating agents alone with the most effective combination being QN plus VPM with the cytotoxic drug DOX. Such increased effectiveness of combinations was, however, not always observed and, when it was present, it did not apply to all cytotoxic drugs. This suggests that any combination of modulating agents used clinically would need to be first carefully chosen.

While combinations of modulating agents were sometimes more effective than single agents, with the possible exception of VPM plus CN with PT, synergistic effects were not observed. This is consistent with the presence of multiple mechanisms of inherent resistance in L929 cells since different modulating agents may well have different, but overlapping, abilities to circumvent each of the resistance mechanisms. The situations where combinations of modulating agents are less effective than one of the agents alone are more difficult to explain, but could result if a modulating agent had a mixture of activities which circumvent some resistance mechanisms, but enhance others.

View this table:
Table III.

Comparison of the effects of maximum tolerated doses of modulating agents on doxorubicin resistance and accumulation in L929 cells.

Our DOX accumulation data support the previous suggestions. One useful example is the effect of CN on the circumvention of DOX resistance by QN. The enhanced DOX accumulation produced by QN as a single agent, was reversed by the addition of CN since QN plus CN produced no increase in DOX accumulation. QN plus CN, however, still circumvented DOX resistance, but to a lesser extent than QN alone. One possible interpretation of these results is that QN circumvented DOX resistance by additional mechanisms present in L929 cells as well as by enhancing DOX accumulation. When CN is present the enhanced accumulation is prevented, but the other resistance-circumventing activities of QN remain. In this interpretation of the data CN has contradictory effects. It actually enhances one mechanism of DOX resistance (i.e. reduced DOX accumulation), but circumvents other mechanisms of DOX resistance to a sufficient extent that it overall increases DOX sensitivity when used as a single agent.

Research into combining chemotherapeutic agents to allow increases in cytotoxic efficacy is always a topic of interest. However, experiments have mainly focused on attacking different biochemical targets (40) and there have been few previous studies investigating the effect of combinations of modulating agents on drug resistance. Three studies have used cell lines in which resistance was derived in the laboratory. Lehnert et al. reported that combinations of VPM and QN can be synergistic in overcoming DOX and vinblastine resistance in a multidrug-resistant human myeloma cell line (41). Hu et al. also reported a synergistic effect for combinations of VPM and cyclosporine A in circumventing DOX resistance in human leukaemia cell lines (34) whereas VPM and trans-flupenthixol was reported to have an additive effect in circumventing DOX resistance in a human breast cancer cell line (42). A fourth study by Ishida et al. showed that VPM and cyclosporine A, at some concentrations, had a synergistic effect in overcoming inherent VC resistance in a human erythroleukaemia cell line (43). Our work also supports potential synergy in the utilisation of combinations of modulators and suggests viability in their ability to circumvent drug resistance.

Furthermore, these studies and our data on inherent and acquired resistance indicate that the complex interactions between modulating agents, could possibly be exploited to circumvent drug resistance in the clinic and that combinations of modulating agents could be more effective than single agents. However the optimum combination of modulating agents may differ for particular cytotoxic drugs. Further studies are required.

In conclusion, our data show that some combinations of modulating agents are more effective in circumventing inherent drug resistance than single agents. This increased effectiveness was not seen in all cases and the most effective combination differed for different cytotoxic drugs. The effects of modulating agents on drug resistance could not be totally explained by changes in drug accumulation. Further studies are required to identify the precise clinical applications of these findings.

  • Received August 2, 2013.
  • Revision received September 17, 2013.
  • Accepted September 18, 2013.

References

  1. ↵
    1. Endicott J
    : The Molecualr Basis of Resistance of Caner Cells to Chemotherapy. Cancer Metastases: from Mechanics to Therapies, pp. 123-144, 1995.
  2. ↵
    1. Kaye SB
    : Multidrug resistance: clinical relevance in solid tumours and strategies for circumvention. Curr Opin Oncol 10(Suppl 1): S15-19, 1998.
    OpenUrlPubMed
    1. Webster LK,
    2. Cosson EJ,
    3. Stokes KH,
    4. Millward MJ
    : Effect of the paclitaxel vehicle, Cremophor EL, on the pharmacokinetics of doxorubicin and doxorubicinol in mice. Br J Cancer 73(4): 522-524, 1996.
    OpenUrlPubMed
  4. ↵
    1. Davies GF,
    2. Juurlink BH,
    3. Harkness TA
    : Troglitazone reverses the multiple drug resistance phenotype in cancer cells. Drug Des Devel Ther 3: 79-88, 2009.
    OpenUrlPubMed
  5. ↵
    1. Blobe GC,
    2. Sachs CW,
    3. Khan WA,
    4. Fabbro D,
    5. Stabel S,
    6. Wetsel WC,
    7. Obeid LM,
    8. Fine RL,
    9. Hannun YA
    : Selective regulation of expression of protein kinase C (PKC) isoenzymes in multidrug-resistant MCF-7 cells. Functional significance of enhanced expression of PKC alpha. J Biol Chem 268(1): 658-64, 1993.
    OpenUrlAbstract/FREE Full Text
    1. Mattern J,
    2. Bak M,
    3. Hoever KH,
    4. Volm M
    : Development of drug resistance in a human epidermoid lung carcinoma xenograft line. Br J Cancer 58(1): 30-33, 1988.
    OpenUrlPubMed
  7. ↵
    1. Perez-Tomas R
    : Multidrug resistance: retrospect and prospects in anti-cancer drug treatment. Curr Med Chem 13(16): 1859-1876, 2006.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Endicott JA,
    2. Ling V
    : The biochemistry of P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 58: 137-171, 1989.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Mechetner E,
    2. Kyshtoobayeva A,
    3. Zonis S,
    4. Kim H,
    5. Stroup R,
    6. Garcia R,
    7. Parker RJ,
    8. Fruehauf JP
    : Levels of multidrug resistance (MDR1) P-glycoprotein expression by human breast cancer correlate with in vitro resistance to taxol and doxorubicin. Clin Cancer Res 4(2): 389-398, 1998.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Zaman GJ,
    2. Flens MJ,
    3. van Leusden MR,
    4. de Haas M,
    5. Mulder HS,
    6. Lankelma J,
    7. Pinedo HM,
    8. Scheper RJ,
    9. Baas F,
    10. Broxterman HJ
    : The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump. Proc Natl Acad Sci USA 91(19): 8822-8826, 1994.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Ding S,
    2. Chamberlain M,
    3. McLaren A,
    4. Goh L,
    5. Duncan I,
    6. Wolf CR
    : Cross-talk between signalling pathways and the multidrug resistant protein MDR-1. Br J Cancer 85(8): 1175-1184, 2001.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Trambas CM,
    2. Muller HK,
    3. Woods GM
    : P-glycoprotein mediated multidrug resistance and its implications for pathology. Pathology 29(2): 122-130, 1997.
    OpenUrlPubMed
    1. Roy S,
    2. Kenny E,
    3. Kennedy S,
    4. Larkin A,
    5. Ballot J,
    6. Perez De Villarreal M,
    7. Crown J,
    8. O'Driscoll L
    : MDR1/P-glycoprotein and MRP-1 mRNA and protein expression in non-small cell lung cancer. Anticancer Res 27(3A): 1325-1330, 2007.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Li L,
    2. Su LP,
    3. Ma L,
    4. Zhao J,
    5. Zhu L,
    6. Zhou YA
    : Expression and significance of P-gp/mdr1 mRNA, MRP and LRP in non-Hodgkin's lymphoma. Zhonghua Zhong Liu Za Zhi 31(3): 199-202, 2009.
    OpenUrlPubMed
  15. ↵
    1. Merry S,
    2. Kaye SB,
    3. Freshney RI
    : Cross-resistance to cytotoxic drugs in human glioma cell lines in culture. Br J Cancer 50(6): 831-835, 1984.
    OpenUrlPubMed
  16. ↵
    1. Merry S,
    2. Courtney ER,
    3. Fetherston CA,
    4. Kaye SB,
    5. Freshney RI
    : Circumvention of drug resistance in human non-small cell lung cancer in vitro by verapamil. Br J Cancer 56(4): 401-405, 1987.
    OpenUrlPubMed
  17. ↵
    1. Zamora JM,
    2. Pearce HL,
    3. Beck WT
    : Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells. Mol Pharmacol 33(4): 454-462, 1988.
    OpenUrlAbstract
  18. ↵
    1. Pauwels EK,
    2. Erba P,
    3. Mariani G,
    4. Gomes CM
    : Multidrug resistance in cancer: its mechanism and its modulation. Drug News Perspect 20(6): 371-377, 2007.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Pramanik D,
    2. Campbell NR,
    3. Das S,
    4. Gupta S,
    5. Chenna V,
    6. Bisht S,
    7. Sysa-Shah P,
    8. Bedja D,
    9. Karikari C,
    10. Steenbergen C,
    11. Gabrielson KL,
    12. Maitra A
    : A composite polymer nanoparticle overcomes multidrug resistance and ameliorates doxorubicin-associated cardiomyopathy. Oncotarget 3(6): 640-650, 2012.
    OpenUrlPubMed
  20. ↵
    1. Merry S,
    2. Fetherston CA,
    3. Kaye SB,
    4. Freshney RI,
    5. Plumb JA
    : Resistance of human glioma to adriamycin in vitro: the role of membrane transport and its circumvention with verapamil. Br J Cancer 53(1): 129-135, 1986.
    OpenUrlPubMed
  21. ↵
    1. Dalton WS,
    2. Grogan TM,
    3. Meltzer PS,
    4. Scheper RJ,
    5. Durie BG,
    6. Taylor CW,
    7. Miller TP,
    8. Salmon SE
    : Drug-resistance in multiple myeloma and non-Hodgkin's lymphoma: detection of P-glycoprotein and potential circumvention by addition of verapamil to chemotherapy. J Clin Oncol 7(4): 415-424, 1989.
    OpenUrlAbstract
  22. ↵
    1. Yu DS,
    2. Sun GH,
    3. Ma CP,
    4. Chang SY
    : Verapamil modulation of multidrug resistance in renal cell carcinoma. J Formos Med Assoc 99(4): 311-316, 2000.
    OpenUrlPubMed
  23. ↵
    1. Jones RD,
    2. Kerr DJ,
    3. Harnett AN,
    4. Rankin EM,
    5. Ray S,
    6. Kaye SB
    : A pilot study of quinidine and epirubicin in the treatment of advanced breast cancer. Br J Cancer 62(1): 133-135, 2000.
    OpenUrl
  24. ↵
    1. Presant CA,
    2. Multhauf P,
    3. Metter G
    : Reversal of cancer chemotherapeutic resistance by amphotericin B – A broad phase I-II pilot study. Eur J Cancer Clin Oncol 23(6): 683-687, 1987.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Gonzalez-Manzano R,
    2. Cid J,
    3. Brugarolas A,
    4. Piasecki CC
    : Cyclosporin A and doxorubicin-ifosfamide in resistant solid tumours: a phase I and an immunological study. Br J Cancer 72(5): 1294-1299, 1995.
    OpenUrlPubMed
  26. ↵
    1. Qadir M,
    2. O'Loughlin KL,
    3. Fricke SM,
    4. Williamson NA,
    5. Greco WR,
    6. Minderman H,
    7. Baer MR
    : Cyclosporin A is a broad-spectrum multidrug resistance modulator. Clin Cancer Res 11(6): 2320-2326, 2005.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Ferguson PJ,
    2. Fisher MH,
    3. Stephenson J,
    4. Li DH,
    5. Zhou BS,
    6. Cheng YC
    : Combined modalities of resistance in etoposide-resistant human KB cell lines. Cancer Res 48(21): 5956-5964, 1988
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Bruce AC,
    2. Longo D
    : Cancer Chemotherapy and Biotherapy: Principles and Practice. p. 406, 2011
  29. ↵
    1. Deffie AM,
    2. Alam T,
    3. Seneviratne C,
    4. Beenken SW,
    5. Batra JK,
    6. Shea TC,
    7. Henner WD,
    8. Goldenberg GJ
    : Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia. Cancer Res 48(13): 3595-3602, 1988
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Perek N,
    2. Koumanov F,
    3. Denoyer D,
    4. Boudard D,
    5. Dubois F
    : Modulation of the multidrug resistance of glioma by glutathione levels depletion--interaction with Tc-99M-Sestamibi and Tc-99M-Tetrofosmin. Cancer Biother Radiopharm 17(3): 291-302, 2002
    OpenUrlCrossRefPubMed
  31. ↵
    1. Politi PM,
    2. Sinha BK
    : Role of differential drug uptake, efflux, and binding of etoposide in sensitive and resistant human tumor cell lines: implications for the mechanisms of drug resistance. Mol Pharmacol 35(3): 271-278, 1989.
    OpenUrlAbstract
  32. ↵
    1. Ferry DR,
    2. Russell MA,
    3. Cullen MH
    : P-glycoprotein possesses a 1,4-dihydropyridine-selective drug acceptor site which is alloserically coupled to a vinca-alkaloid-selective binding site. Biochem Biophys Res Commun 188(1): 440-445, 1992
    OpenUrlCrossRefPubMed
  33. ↵
    1. Martin C,
    2. Berridge G,
    3. Higgins CF,
    4. Mistry P,
    5. Charlton P,
    6. Callaghan R
    : Communication between multiple drug binding sites on P-glycoprotein. Mol Pharmacol 58(3): 624-632, 2000.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Hu XF,
    2. Martin TJ,
    3. Bell DR,
    4. de Luise M,
    5. Zalcberg JR
    : Combined use of cyclosporin A and verapamil in modulating multidrug resistance in human leukemia cell lines. Cancer Res 50(10): 2953-2957, 1990.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Kim JH,
    2. Chung JB,
    3. Park IS,
    4. Kim BS,
    5. Yoo NC,
    6. Choi JH,
    7. Roh JK,
    8. Kim HS,
    9. Kwon OH,
    10. Lee KS
    : Combined use of tamoxifen, cyclosporin A, and verapamil for modulating multidrug resistance in human hepatocellular carcinoma cell lines. Yonsei Med J 34(1): 35-44, 1993.
    OpenUrlPubMed
  36. ↵
    1. Drewitt PN,
    2. Butterworth KR,
    3. Springall CD,
    4. Walters DG,
    5. Raglan EM
    . Toxicity threshold of quinine hydrochloride following low-level repeated dosing in healthy volunteers. Food Chem Toxicol 31(4): 235-245, 1993.
    OpenUrlPubMed
    1. Kerr DJ,
    2. Graham J,
    3. Cummings J,
    4. Morrison JG,
    5. Thompson GG,
    6. Brodie MJ,
    7. Kaye SB
    : The effect of verapamil on the pharmacokinetics of adriamycin. Cancer Chemother Pharmacol. 18(3): 239-242, 1986.
    OpenUrlPubMed
  38. ↵
    1. Morrison PJ,
    2. Bradbrook ID,
    3. Rogers HJ
    : Plasma cinnarizine levels resulting from oral administration as capsule or tablet formulation investigated by gas-liquid chromatography. Br J Clin Pharmacol 7(4): 349-352, 1979.
    OpenUrlPubMed
  39. ↵
    1. Ferrari M,
    2. Fornasiero MC,
    3. Isetta AM
    : MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 131(2): 165-172, 1990.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Lewis BJ,
    2. DeVita VT Jr..
    : Combination chemotherapy of acute leukemia and lymphoma. Pharmacol Ther 7(1): 91-121, 1979.
    OpenUrlPubMed
  41. ↵
    1. Lehnert M,
    2. Dalton WS,
    3. Roe D,
    4. Emerson S,
    5. Salmon SE
    : Synergistic inhibition by verapamil and quinine of P-glycoprotein-mediated multidrug resistance in a human myeloma cell line model. Blood 77(2): 348-354, 1991.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Ford JM,
    2. Bruggemann EP,
    3. Pastan I,
    4. Gottesman MM,
    5. Hait WN
    : Cellular and biochemical characterization of thioxanthenes for reversal of multidrug resistance in human and murine cell lines. Cancer Res 50(6): 1748-1756, 1990.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Ishida Y,
    2. Shimada Y,
    3. Shimoyama M
    : Synergistic effect of cyclosporin A and verapamil in overcoming vincristine resistance of multidrug-resistant cultured human leukemia cells. Jpn J Cancer Res 81(8): 834-841, 1990.
    OpenUrlPubMed
  44. ↵
    1. Kunnumakkara AB,
    2. Aggarwal AP
    : Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer Lett 269(2): 199-225, 2008.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Cummings J,
    2. Stuart JFB,
    3. Calman KC
    : Determination of adriamycin, adriamycinol and their 7-deoxyaglycones in human serum by high-performance liquid chromatography. Journal of Chromatography 311: 125-133, 1984.
    OpenUrlPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Circumvention of Inherent or Acquired Cytotoxic Drug Resistance In Vitro Using Combinations of Modulating Agents
DAVID CADAGAN, STEPHEN MERRY
Anticancer Research Oct 2013, 33 (10) 4381-4387;
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords