Chemosensitizing Agent Research Articles

ATP binding cassette transporters and drug resistance in breast cancer.

Resistance to chemotherapy is a critical issue in the management of breast cancer patients. The nature of clinical drug resistance is likely to be multifactorial. However, in the last decade considerable attention has been dedicated to the role played by membrane transporter proteins belonging to the ATP binding cassette protein superfamily, and in particular by the MDR1 product P-glycoprotein (Pgp) and the multidrug resistance protein (MRP1). Heterogeneity of results is a common feature of studies evaluating the expression and prognostic role of these proteins, due to both methodological and biological factors. Nonetheless, Pgp and MRP1 are detected in a significant proportion of untreated breast cancers (on average 40 and 50% respectively, by immunohistochemistry), without a clear and consistent association with cancer stage. Exposure to chemotherapy increases the expression of both proteins. In vitro studies on primary cultures of breast cancer cells obtained at surgery consistently show an association between Pgp (protein) or MDR1 (mRNA) expression and resistance to chemotherapy. However, the correlation with clinical drug resistance is not as well defined. A stronger association of Pgp/MDR1 with response rates has been observed when expression or an increase in expression are detected immediately following chemotherapy. Correlations with prognosis appear more evident in studies using immunohistochemistry, in adjuvant and neoadjuvant settings. Evidence of clinical reversal of drug resistance by verapamil suggests a functional role of Pgp in drug resistance, although the significance of the evidence is generally weakened by poor trial designs. Future studies should take into account the multifactorial nature of drug resistance in breast cancer and use standardized approaches with adequate controls. Expression studies should be complemented by well-designed trials of drug-resistance reversal using target-specific chemosensitizing agents, and relating the results to the levels of expression of the target proteins.

Microplate Screening of the Differential Effects of Test Agents on Hoechst 33342, Rhodamine 123, and Rhodamine 6G Accumulation in Breast Cancer Cells that Overexpress P–Glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.

In vitro investigation of ionic polysaccharide microspheres for simultaneous delivery of chemosensitizer and antineoplastic agent to multidrug-resistant cells

Insufficient intratumoral concentration of therapeutic agents and multidrug resistance are major factors responsible for failure of treatment of solid tumors. Simultaneous delivery of chemosensitizing and antineoplastic agents by microspheres could lead to enhanced chemotherapy of multidrug-resistant (MDR) tumors. Ionic polysaccha-ride microspheres derived from dextran were used to load chemosen-sitizers (e.g., verapamil) and anticancer drugs such as vinblastine. High drug loading was achieved for both a single agent and dual agents. The equilibrium drug loading was dependent on the ratio of the microspheres (MS) to the drug, as well as the relative affinity of the agents to the MS in the case of dual agents. The drug release from drug–MS involved hydration and swelling of the MS in addition to ion exchange. The effectiveness of MS-delivered chemosensitizers in the reversal of drug resistance was evaluated by measuring the uptake of [3H]vinblastine by MDR cells (CHRC5). The concomitant delivery of verapamil with vinblastine by the MS led to a 6–7-fold increase in the uptake of vinblastine, a level similar to the uptake obtained with free drug solutions. The results suggest that the antineoplastic and chemosensitizing agents were released effectively from the MS and the bioactivity of the chemosensitizer was preserved during the process.

Linear and cyclic peptides as substrates and modulators of P-glycoprotein: peptide binding and effects on drug transport and accumulation.

One cause of multidrug resistance (MDR) in human cancers is the overexpression of the P-glycoprotein multidrug transporter, a member of the ABC superfamily of membrane proteins. Natural products and chemotherapeutic drugs are pumped out of the cell by P-glycoprotein in an ATP-dependent fashion. There is growing evidence that many hydrophobic peptides are also P-glycoprotein substrates. With the use of a fluorescence-quenching assay, we have shown that some linear and cyclic hydrophobic peptides interact with P-glycoprotein, whereas others do not. The measured values of the quenching constant, Kq, for interaction of peptides with P-glycoprotein ranged from 200 nM for cyclosporine A to 138 microM for the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Peptides that interacted with P-glycoprotein in the fluorescence assay also blocked colchicine transport into plasma membrane vesicles from MDR cells. The values of Dm, the peptide concentration causing 50% inhibition of drug uptake, were highly correlated with the values of Kq, over three orders of magnitude. The P-glycoprotein ATPase stimulation/inhibition profile of the peptides was not helpful in making a quantitative assessment of the ability of a peptide to interact with P-glycoprotein or to block drug transport. Some hydrophobic peptides were able to restore accumulation in MDR cells of the chemotherapeutic drug daunorubicin and the fluorescent dye rhodamine 123 to the levels observed in the drug-sensitive parent. Peptides that interacted with P-glycoprotein also displayed a relatively low overall toxicity to intact MDR cells, and inhibited drug transport at concentrations below the toxic range. Hydrophobic peptides should be given serious consideration for development as clinical chemosensitizing agents.

α-Tocopherol antagonizes the multidrug-resistance-reversal activity of cyclosporin A, verapamil, GF120918, clofazimine and B669

The effects of the membrane-stabilizing agent, α-tocopherol (25 μg/ml), on the chemosensitizing interactions of cyclosporin A (5 μg/ml), verapamil (2 μg/ml), clofazimine (1 μg/ml), B669 (0.5 μg/ml) and GF120918 (0.015 μg/ml) with a P-glycoprotein-expressing human lung cancer cell line (H69/LX4) have been investigated in vitro. In an assay of cell proliferation, all the chemosensitizing agents restored the sensitivity of H69/LX4 cells to doxorubicin and vinblastine. The inclusion of α-tocopherol (25 μg/ml) antagonized the multidrug-resistance (MDR)-modifying activity of all five chemosensitizing agents, effectively preventing restoration of sensitivity to both doxorubicin and vinblastine in H69/LX4 cells.

Future directions in imaging of breast diseases.

In the near future, investigation and refinement of emerging anatomic and functional breast imaging techniques will enable clinical trials that will evaluate their utility and potential for improving the survival and quality of life for patients with breast cancer. In the longer term, strategic research collaborations among investigators in the fields of functional imaging, molecular biology, and pathology are needed to merge existing science and advance the development of biomarker and genetic techniques focused on detecting and characterizing disease at the cellular and molecular levels. This research could create clinical tools for (a) detecting breast cancer earlier, (b) more accurately quantifying the extent of disease, (c) noninvasively evaluating lymph node involvement, (d) identifying micrometastases and residual microscopic disease, and (e) enhancing therapy by means of imaging-guided biomarker or tumor-specific delivery of pharmacologic, chemosensitizing, or radiosensitizing agents to tumors.

An homogeneous assay for measuring the uptake and efflux of radiolabelled drugs in adherent cells

We have developed an homogeneous assay to measure the uptake and efflux of [ 14C]adriamycin (doxorubicin hydrochloride) in human squamous lung carcinoma cells (SKMES-1), using 96 well scintillating microplates. The assay was also used to examine the effect of inhibitors of multidrug resistance in adriamycin resistant cells (SKMES-1/ADR). The effect of adriamycin on cell growth and viability was examined by continuous monitoring of the uptake of [ 14C]thymidine. The non-invasive nature of these assays, and the ease of use of the microplates, suggests a role in screens for, and characterisation of, novel chemotherapeutic or chemosensitizing agents.

Effect of quercetin on hoechst 33342 transport by purified and reconstituted p-glycoprotein

Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer. Numerous compounds are known that inhibit the drug-exporting function of P-glycoprotein. Understanding the mechanisms of action of these chemosensitizers is made difficult by the complexity of the in vivo cell systems usually employed. To examine the direct effects of chemosensitizers, we have developed a system in which purified and reconstituted P-glycoprotein transports Hoechst 33342 from the lipid membrane to the aqueous interior of proteoliposomes, requiring ATP hydrolysis (Shapiro AB and Ling V, J Biol Chem 270: 16167–16175, 1995). Here, we use this system to understand the effect on P-glycoprotein of quercetin, one of three flavonoids that have been reported to have the unique property of stimulating drug transport by P-glycoprotein in vivo (Phang et al., Cancer Res 53: 5977–5981, 1993). Since flavonoids are abundant in food, it is important to understand their effects on the function of P-glycoprotein because of the implications for cancer chemotherapy. In our hands, quercetin inhibited P-glycoprotein-mediated Hoechst 33342 efflux and enhanced accumulation, as measured by flow cytometry, by multidrug-resistant CH RC5 cells. In the purified system, quercetin strongly inhibited Hoechst 33342 transport by P-glycoprotein, at least in part by inhibiting the ATPase activity of P-glycoprotein required for transport. We conclude that the previously reported stimulatory effect of quercetin on drug efflux from multidrug-resistant cells is not a direct effect on P-glycoprotein. The ATPase domain of P-glycoprotein may be an attractive target for new chemosensitizing agents.

Cyclosporin A treatment induces overexpression of P-glycoprotein in the kidney and other tissues.

To see whether P-glycoprotein (PGP) expressed in renal brush-border membranes (BBM) could interact with compounds known as modulators of multidrug resistance (MDR), photoaffinity-labeling experiments were performed. A 145k-Da protein was photolabeled with [125I] iodoarylazidoprazosin, and this labeling was reduced in the presence of cyclosporin A (CsA) and PSC-833 (PSC). Interaction of CsA with PGP was further investigated by treating rats with daily subcutaneous injections of CsA (10 mg.kg-1.day-1). After this treatment, PGP expression levels were dramatically increased in renal BBM, intestine, liver, and many other tissues except the brain. This induction was a reversible process, since after cessation of CsA administration PGP levels declined to reach values similar to those of the control groups. The increase in PGP expression in the kidney was also detected in photolabeling experiments, suggesting the induction of a functional PGP. A higher dose of CsA (50 mg/kg) given as a bolus injection did not modify PGP expression] in renal BBM. These results demonstrate that CsA induces reversible overexpression of PGP in the rat. This may present significant relevance in the design of clinical trials using CsA as a chemosensitizing agent.

Rifampicin enhances anti-cancer drug accumulation and activity in multidrug-resistant cells

Rifampicin, a semi-synthetic antibiotic used in the treatment of tuberculosis and belonging to the chemical class of rifamycins, was examined for its effect on anti-cancer drug accumulation and activity in multidrug resistant cells overexpressing P-glycoprotein (P-gp). Rifampicin was shown to strongly enhance vinblastine accumulation in both rat hepatoma RHC1 and human leukemia K562 R7 multidrug resistant cells, but had no effect in rat SDVI drug-sensitive liver cells. By contrast, two other rifamycins, rifamycins B and SV, had no or only minor effect on vinblastine accumulation in RHC1 cells. Efflux experiments revealed that rifampicin was able, like the well-known chemosensitizer agent verapamil, to decrease export of vinblastine out of resistant cells. Rifampicin, when used at a concentration close to plasma concentrations achievable in humans (25 μM), was able to increase sensitivity of RHC1 cells to both vinblastine and doxorubicin. Rifampicin was also demonstrated to inhibit P-gp radiolabeling by the photoactivable P-gp ligand azidopine, thereby suggesting that the anti-tuberculosis compound can interfere directly with P-gp drug binding sites. These results thus indicate that rifampicin was able to down-modulate P-gp-associated resistance through inhibition of P-gp function.

A phase III randomized study of oral verapamil as a chemosensitizer to reverse drug resistance in patients with refractory myeloma. A southwest oncology group study

Multiple myeloma is considered to be a drug responsive disease; however, there is no cure for this disease and virtually all patients will develop drug resistance. One form of drug resistance that has been documented is the multidrug resistance phenotype or MDR. A randomized trial of the combination of vincristine, doxorubicin, and dexamethasone (VAD) and VAD plus oral verapamil (VAD/v) in drug refractory multiple myeloma patients was performed by the Southwestern Oncology Group. Verapamil was used as a chemosensitizing agent to attempt to overcome or prevent MDR and improve the therapeutic outcome. Response rates between the two treatment arms were similar with an overall response rate of 41% for the VAD alone arm and 36% for the VAD/v arm. Overall survival of patients was also similar with a median survival of 10 months for the VAD arm and 13 months for the VAD/v arm. The toxicity profile was also similar for both treatments, with myelosuppression being the dose-limiting toxicity. No significant correlation was observed between expression of P-glycoprotein, serum verapamil levels, and response to therapy. No beneficial effect was observed from the addition of oral verapamil to the VAD chemotherapy regimen for the treatment of drug-resistant myeloma patients. More effective and less toxic chemosensitizers are needed to study the role of chemosensitizers in reversing MDR in the clinic.

Racemic verapamil (VER) as a chemosensitizer agent of doxorubicin (DOX) in human colorectal cancer (HCC)

Racemic verapamil (VER) as a chemosensitizer agent of doxorubicin (DOX) in human colorectal cancer (HCC)

N-(p-azido-3-[125I]iodophenethyl)spiperone binds to specific regions of P-glycoprotein and another multidrug binding protein, spiperophilin, in human neuroblastoma cells

P-glycoprotein (P-gp) is an energy-dependent drug extrusion pump with broad specificity for diverse hydrophobic anticancer agents and compounds known to reverse multidrug resistance (MDR). Among MDR reversing agents, phenothiazines (PTZs) and related compounds may sensitize MDR by interacting with a specific binding site(s) on P-gp and by other mechanisms. In order (1) to identify a binding site for PTZs and related compounds on P-gp, (2) to examine whether these compounds and other MDR modulators bind to the same domains of P-gp, and (3) to identify proteins with high specificity for these neuroleptic agents and other MDR modulators, we used a butyrophenone D2-dopamine receptor photoaffinity probe, N-(p-azido-3-[125I]iodophenethyl)spiperone ([125I]NAPS). [125I]NAPS was actively effluxed from vincristine (VCR)-resistant SH-SY5Y/VCR human neuroblastoma cells, and nonradioactive I-NAPS was a potent chemosensitizing agent. After photolabeling, the probe bound specifically and with high efficiency to P-gp and to another multidrug binding 17-kDa membrane-bound protein, spiperophilin, in these cells. The efficiency of [125I]NAPS binding to P-gp was 5-6-fold more than [3H]azidopine and [125I]arylazidoprazosin ([125I]AAP), known photoaffinity analogs for P-gp. [125I]NAPS photolabeling of P-gp was preferentially competed by MDR-related drugs, with vinblastine > VCR > colchicine > doxorubicin > actinomycin D. Many drugs that are known to reverse MDR were potent inhibitors of [125I]NAPS binding to P-gp. While PTZs and related compounds were potent inhibitors of [125I]NAPS binding to P-gp, most of them enhanced the binding of [125I]AAP significantly. cis-Flupentixol increased the binding of [125I]AAP to P-gp 9-fold more than did trans-flupentixol, but both were potent inhibitors of [125I]NAPS binding, suggesting their stereoselective effect on the [125I]AAP binding site. Proteolysis of [125I]NAPS-bound P-gp with Staphylococcus aureus V8 protease revealed that this probe binds to two major peptides, 6 and 8 kDa, and a number of minor ones, while [125I]AAP binds to only an 8-kDa peptide. These results suggest that modulators of MDR may interact with separate or overlapping domains. Furthermore, most MDR modulators, dopaminergic drugs, and beta-adrenergic antagonists used also inhibited binding of [125I]-NAPS to spiperophilin, suggesting that this protein may be a target for these drugs.

Targeting chemosensitizing doses of toremifene based on protein binding.

Toremifene is currently being evaluated as a chemosensitizing agent in doxorubicin-resistant patients. Although concentrations of > 2 microM reverse resistance in vitro, target concentrations required to reverse multidrug resistance (MDR) in vivo may be highly influenced by variables such as protein binding in serum. We examined the effects of high serum concentrations on the cellular accumulation of toremifene in an MDR MDA-MB-A-1 human breast-cancer cell line. We then examined the cellular accumulation of doxorubicin at various toremifene concentrations in 5% - 100% serum. We also measured the concentrations of toremifene and its major metabolites in plasma specimens obtained from two patients receiving 360 mg/day for 5 days in a phase I study. Our results show that (1) high serum concentrations decrease toremifene accumulation, (2) toremifene concentrations of < or = 2.5 microM enhance doxorubicin accumulation, and (3) patients achieve plasma toremifene concentrations of 10-15 microM following doses of 360 mg/day x 5 days. Our findings suggest that in vivo toremifene concentrations well above those used to reverse resistance in vitro are required to overcome the effect of high serum-protein binding.

Rhodamine-123 as a new chemosensitizing versus toxic agent on human squamous carcinoma cells and fibroblast cultures.

Rhodamine-123 is a specific dye with an absorption maxima at 511 nm which was tested as a potential chemosensitizing agent for laser treatment of tumor cells. Because Rhodamine, at high doses, has direct cytotoxic effects on human cells in the absence of laser exposure, we tested the human squamous P 3 carcinoma cell line and two normal fibroblast cell lines for sensitivity to various levels of this dye. These cells were exposed to Rhodamine-123 at concentrations of 1, 3, 6, and 10 mug/ml for 1, 8, and 24 hours. The results indicate that Rhodamine-123 is nontoxic to human P 3 carcinoma cells and normal fibroblast cultures at concentrations equal or lower than 1 mug/ml. However, at concentrations equal or higher than 3 mug/ml, a significant immediate and/or delayed inhibition of cell duplication was demonstrated. The results show that Rhodamine-123 at 1 mug/ml can be used to sensitize tumor cells for targeting by monochromatic 514.5 nm Argon lasers.

Enhancement of melphalan (l-PAM) toxicity by reductive metabolites of 1-methyl-2-nitroimidazole, a model nitroimidazole chemosensitizing agent

Chemosensitization of bifunctional alkylators by misonidazole (MISO) and related nitroimidazoles in vitro has been shown to require hypoxic exposures. Presumably, reductive metabolism of the nitroimidazole under hypoxic conditions results in generation of a chemosensitizing intermediate(s) in a manner analogous to that described for the hypoxic toxicity of these compounds. In an attempt to identify these intermediates, we examined the ability of reductive metabolites of a model 2-nitroimidazole compound, 1-methyl-2-nitroimidazole (INO 2), to enhance the toxicity of melphalan ( l-PAM) in HT-29 human colon cancer cells. INO 2 was a modest chemosensitizing agent, enhancing l-PAM only under hypoxic conditions. The 2-electron reduction product, 1-methyl-2-nitrosoimidazole (INO), was a potent chemosensitizer, enhancing l-PAM toxicity at micromolar concentrations under either aerobic or hypoxic conditions. In contrast, the 4- and 6-electron reduction products, 1-methyl-2-[hydroxylamino]imidazole and 1-methyl-2-aminoimidazole, respectively, failed to modify cell kill by l-PAM even at millimolar concentrations. These results suggest that nitrosoimidazoles may be the active chemosensitizing species generated upon the reductive metabolism of nitroimidazoles.

Pharmacologic management of drug-resistant genitourinary tumors

The purpose of this article is to examine potentially useful treatment strategies for drug-resistant genitourinary (GU) tumors. To achieve the desired pharmacologic effect, adequate concentration vs time (C×T) drug kinetic profiles must be achieved at the target tissues. Inadequate C×T drug profiles can result from low vascular tissues, rapid drug metabolism, and insufficient dosing schedules. These factors may also contribute to the development of acquired drug resistance by enabling tumor cells to repair sublethal damage caused by cytotoxic drugs. Although the development of acquired drug resistance may represent a normal response to foreign agents and hence be inevitable in many tumors, dose-intensive regimens as first-line treatment may delay the clinical onset of resistance. Once acquired drug resistance does develop, several investigational treatment strategies aimed at biochemical mechanisms of resistance may further circumvent treatment failures. Finally, intrinsic drug resistance represents an additional problem in certain GU tumors, particularly renal cell carcinoma. In this case, dose-intensive treatment regimens that incorporate a chemosensitizing agent may prove to the useful as firstline therapy. Tumor-specific treatment regimens need to be developed based on many factors, including degree of intrinsic resistance, past chemotherapy, stage and site of disease, and patient tolerance to aggressive treatment regimens.

Bioinhibition of human fibroblast cultures sensitized to Q-switch II dye and treated with the Nd: YAG laser: a new technique of photodynamic therapy with lasers.

Kodak Q-switch II dye has recently proven to be an effective biostimulative agent on normal human fibroblast cultures. The potential for this dye as a new chemosensitizing agent for the treatment of connective tissue diseases and wound healing with the Nd:YAG laser was examined. Two normal fibroblast cell lines were first sensitized to a nontoxic dose of Q-switch II dye, then subjected to treatment with an Nd:YAG laser at 1,060 nm, with varying levels of energy and temperatures determined by a reproducible method of dosimetry. The results indicate that Q-switch II dye at nontoxic doses of 0.1 micrograms/ml enhances the cytotoxic effects of the Nd:YAG laser at temperatures as low as 36 degrees C. Furthermore, at physiological temperature ranges as low as 24 degrees C to 34 degrees C, cell duplication was inhibited, but cell viability was not affected. Similar results were not observed when fibroblast cultures were treated with the laser alone. These observations suggest that Q-switch II dye is an effective chemosensitizing agent for the Nd:YAG laser and could potentially be used to reduce collagen deposits in conditions such as keloids and hypertrophic scars.

The effects of argon lasers on human melanoma cells sensitized with rhodamine-123 in vitro

A human melanoma cell line, M14, was first exposed to a nontoxic dose of Rhodamine-123 (1 microgram/ml) for one hour, then subjected to a treatment with a single mode argon laser at 514.5 nm. The temperature and energy levels delivered to the target cells were determined by a reproducible method of dosimetry. Cell viability was assessed by the Trypan Blue exclusion test. Cell duplication and DNA synthesis were measured by the incorporation of 3H-thymidine at 6 and 24 hours post-treatment. At energy levels and temperatures higher or equal to 950 J/cm2 (40 degrees C), an immediate suppression of DNA synthesis was accompanied by nonviability of the M14 carcinoma cells. At energy levels between 130-900J/cm2 corresponding to temperatures between 28 to 39 degrees C, both an immediate and delayed inhibition of DNA synthesis was noted but the cells remained viable. The results indicate that Rhodamine-123 at nontoxic doses of 1 microgram/ml enhances the tumoricidal effects of the argon laser at reduced temperatures as low as 40 degrees C. Furthermore, at physiological temperature ranges as low as 28 to 30 degrees C, an immediate inhibition of cell duplication was demonstrated while cell viability was not affected. These observations suggest that Rhodamine-123 can be used effectively as a chemosensitizing agent in the treatment of human tumor cells with the argon laser at 514.5 nm.

Rhodamine 123 as a chemosensitizing agent for argon laser therapy. A new technique for treatment of superficial malignancies.

Rhodamine 123 (Rh 123), a mitochondrial-specific dye with an absorption maxima at 511 nm, was tested as a new chemosensitizing agent for argon laser treatment of P3 human squamous carcinoma cells. After exposure of P3 cells in vitro to Rh 123 at doses of 1, 3, 6, and 10 micrograms/mL for one hour, we observed significant inhibition of DNA synthesis, except at the lowest dose. Rhodamine 123 at 1 microgram/mL was selected to sensitize P3 tumor cells for treatment with the monochromatic argon laser at 514.5 nm. Exposure of P3 cells to laser energy levels of 700 to 950 J/cm2 (36 degrees C to 40 degrees C) after sensitization with Rh 123 completely inhibited tumor development of the P3 cells transplanted subcutaneously into nude mice. Control P3 cells treated with the laser alone at 36 degrees C to 40 degrees C or only with Rh 123 formed visible tumors by one week and continued to grow for the entire-week observation period. These results show that Rh 123 is a highly sensitive new fluorochrome for argon laser phototherapy of human squamous carcinoma cells.