Noninvasive detection of multidrug resistance in patients with hematological malignancies: are we there yet?

The success of chemotherapy in the treatment of malignancies may be limited by cellular mechanisms leading to drug resistance. In hematological malignancies, mechanisms leading to the development of multidrug resistance (MDR) include overexpression of the membrane-based export pump P-glycoprotein (Pgp) and the MDR-associated protein (MRP). Recently, the overexpression of the lung-resistance protein (LRP) has also been associated with reduced intracellular drug accumulation. A major problem in assessing the significance of the expression of these resistance proteins in clinical MDR has been the variability of detection techniques either at the mRNA or protein level. Currently, the detection of resistance proteins relies heavily on antibody and cDNA probes, and these methods may not be informative about the in vivo function of Pgp, MRP, or LRP. Nuclear medicine imaging techniques such as single-photon emission tomography (SPECT) and positron emission tomography (PET) have been evaluated for noninvasive determination of the presence and the function of Pgp- and MRP-mediated transport systems. Technetium 99m ((99m)Tc)-sestamibi, an agent in clinical use for myocardial perfusion and tumor imaging, is recognized as a substrate for Pgp and MRP, and has been used to visualize Pgp expression. (99m)Tc-tetrofosmin is also a substrate for the Pgp efflux pump mechanism and is used to evaluate Pgp function in in vitro and in vivo studies. Recently, radiopharmaceuticals including carbon 11-labeled colchicine, verapamil, and daunorubicin have been used in cell line and animal studies for the evaluation of Pgp-mediated transport functions using PET technology. Preliminary results suggest that the potential to detect MDR in tumors prior to or after exposure to chemotherapeutic agents exists in imaging using either (99m)Tc-labeled compounds and SPECT or positron emitting compounds and PET.     

Overexpression of P-glycoprotein in mammalian tumor cell lines after fractionated X irradiation in vitro

We observed that in vitro exposure of mammalian tumor cells to fractionated x irradiation results in the expression of drug resistance. The cause of this resistance was investigated in a series of Chinese hamster ovary cell lines that had survived exposure to multiple lethal doses of radiation. These cell lines had increased levels of P-glycoprotein (Pgp), the multidrug-resistance-associated membrane glycoprotein. Consistent with the classic multidrug resistance phenotype, they exhibited cross-resistance to multiple drugs, as well as sensitivity to reversal of vincristine resistance by verapamil. However, the cell lines showed no change in their sensitivity to x rays. Pgp overexpression occurred in these cells, despite a lack of Pgp gene amplification or of significant alteration in Pgp messenger RNA levels. Although the cause of increased Pgp levels is not yet known, these data suggest a biological basis for the clinical problem of drug resistance that can occur in previously irradiated tumors.     

P-glycoprotein expression during tumor progression in the rat liver.

P-Glycoprotein (Pgp) has been shown to mediate multidrug resistance in tumor cell lines. Overexpression of Pgp has been detected in clinical cancer samples of many histological types. The basis and biological significance of such increases in Pgp expression are not well understood. In this study, the expression of Pgp during stepwise progression to rat liver cancer was examined to investigate the possible role of Pgp in carcinogenesis. An immunohistochemical technique was used to detect Pgp at the single-cell level, in a large number of liver nodules, hepatocellular carcinoma, and in distant metastases of the carcinomas. The results showed that distinct changes in Pgp expression occurred during stepwise liver carcinogenesis and that these changes were closely associated with the microscopic anatomy of the lesions. In contrast to gamma-glutamyl transpeptidase and glutathione S-transferase-7.7, whose expression appeared to correlate with the early steps of liver carcinogenesis, Pgp expression was higher in the large hyperplastic nodules and in hepatocellular carcinomas than in the early microscopic lesions. A particularly striking finding was the consistent expression of Pgp in the lung metastases. These findings suggested that Pgp was associated with a more progressed malignant phenotype in liver carcinogenesis.     

Immunization with liposome-anchored pegylated peptides modulates doxorubicin sensitivity in P-glycoprotein-expressing P388 cells

The clinical use of chemotherapy in cancer treatment is limited by the occurrence of multidrug resistance (MDR) associated with the overexpression of membrane transporters, one of the best known is P-glycoprotein (Pgp), that actively expels drugs out of tumor cells. To overcome Pgp-mediated MDR, synthetic peptides corresponding to fragments from extracellular loops 1, 2 and 4 of the murine Pgp were coupled to polyethylene glycol-distearoylphosphatidylethanolamine and inserted into empty or monophosphoryl lipid A-containing liposomes. This formulation elicited specific antibodies which blocked Pgp-mediated efflux of doxorubicin, resulting in increased intracellular drug accumulation and subsequent potentiation of the cytotoxic effect of doxorubicin on multidrug-resistant P388 (P388R) cells. Previous immunizations with MDR1 peptides improved the efficiency of chemotherapy against P388R cells in vivo, with an increase of 83% of mice survival time. Overall, these results suggest that this approach can modulate Pgp activity by blocking drug efflux and may have clinical relevance as an alternative strategy to toxic chemosensitizers in drug-resistant cancer therapy.     

Correlation between functional and molecular analysis of mdr1 P-glycoprotein in human solid-tumor xenografts

The contribution of P-glycoprotein (Pgp) to multidrug resistance in human solid tumors is generally estimated from bulk mRNA measurements or immunohistochemistry, while direct measurement of the effect of Pgp on intracellular drug concentrations has not been pursued. We investigated the feasibility and sensitivity of a method for probing Pgp-mediated drug transport in cells isolated from solid tumors, using xenograft models. Human tumor xenografts (XG) were grown by s.c. injection of Pgp-expressing cell lines 2780^AD, BRO/mdrl and KB8-5. Tumor uptake of doxorubicin (DOX) after administration of DOX to the mice was determined. XG from untreated mice were enzymatically dissociated. The effect of the Pgp modulator bepridil on steady-state cellular daunorubicin (DNR) and vincristine (VCR) accumulation and chemosensitivity of these XG cells was compared with its effects in the cell lines (CL). mdrl mRNA and Pgp (by flow cytometry) were measured. Also, the dependence on intraceilular ATP concentration, [ATP]_i, of the modulator effect was determined in intact KB8-5 cells. The results showed that i.v. administration of DOX to the mice led to lower DOX levels in the Pgp-expressing XG than in the “sensitive” XG, suggesting the presence of an in vivo functional Pgp in these XG tumor models. Dissociated, viable XG cells appeared to have ATP levels sufficient to sustain Pgp-ATPase-coupled drug transport. This was inferred from experiments using KB8- 5 CL, which showed half-maximal inhibition of DNR transport at an [ATP]_i of 1 to 2 mM. The effect of bepridii on DNR and VCR accumulation and chemosensitivity in the XG cells was in accordance with the XG expression of mdr1/Pgp, In KB8-5 XG cells, Pgp function was hardly detectable, in accordance with decreased mdrl/Pgp expression in vivo. In conclusion, Pgp activity can be determined in freshly dissociated XG human tumor cells. The results obtained with the more necrotic KB8-5 XG may represent some of the interpretation problems arising when low levels of Pgp expression occur within a heterogeneous cell population, such as may be expected in clinical human tumors. Also our results indicate that Pgp activity may be impaired in vivo at [ATP]_i below 2 mM, which are realistic values for human solid tumors. © 1995 Wiley-Liss, Inc.     

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.     

P-glycoprotein in human sarcoma: evidence for multidrug resistance

Overexpression of an immunologically conserved, cell-surface glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines in vitro. A preliminary survey of specimens from 12 solid tumor types in our laboratories indicates significant overexpression of P-glycoprotein in some sarcomas. When tested by immunoblotting with monoclonal antibodies directed against P-glycoprotein; tumors from six of 25 sarcoma patients displayed elevated levels of P-glycoprotein. Three of the sarcoma patients exhibiting P-glycoprotein had not previously been exposed to chemotherapy, implying that overexpression of this marker and possible concomitant multidrug resistance may not depend only on selection during prior drug treatments. The P-glycoprotein overexpression in the sarcoma specimens is evidence for the presence of multidrug resistant cells in these tumors; thus, our data suggest that this mode of resistance may have clinical significance in sarcoma patients.     

P-glycoprotein and survivin simultaneously regulate vincristine-induced apoptosis in chronic myeloid leukemia cells

Overexpression of P-glycoprotein (Pgp/ABCB1) in tumor cells is associated with a classic phenotype of multidrug resistance (MDR). Moreover, some members of the inhibitor of apoptosis protein (IAP) family, such as survivin, contribute to an apoptosis-resistant phenotype, by inhibiting chemotherapy-induced cell death and promoting MDR. By using Western blotting, qRT-PCR, Annexin V and immunofluorescence assays we have demonstrated a relationship between Pgp and survivin in a prior sensitive chronic myeloid leukemia (CML) cell line (K562). A high dose of vincristine induced a concomitant overexpression of Pgp and survivin, which was associated with a low apoptotic index in the K562 cell line. In addition, we observed a cytoplasmic co-localization of Pgp and survivin, suggesting a functional association between these two proteins in apoptosis control by a common mechanism. In summary, our data suggest that Pgp and survivin should be analyzed in aggregate because they may have significant impact on drug resistance in CML cells.     

Immunohistochemical detection of P-glycoprotein in breast cancer and its significance as a prognostic factor

Overexpression of P-glycoprotein (Pgp) in tumors is one of the major mechanisms which mediates the multidrug resistance (MDR) phenotype. To evaluate the prognostic significance of Pgp in breast cancer, Pgp expression was examined in paraffin-embedded tissue sections of 94 breast cancer specimens by immunohistochemistry. Tissue specimens were obtained by mastectomy without preoperative chemotherapy. UIC2 monoclonal antibody which recognizes an extracellular epitope of human Pgp was employed. Of the 94 breast cancer specimens, 35 (37.2%) were positive for Pgp expression. Pgp expression had no correlation with menopausal or hormone receptor status, axillary lymph node involvement or tumor size. However, a significant correlation was observed between Pgp expression and disease relapse (p = 0.0322). Pgp-positive patients showed a significantly shorter disease-free survival period than Pgp-negative patients by the Kaplan-Meier method (p = 0.0433). These results suggest that immunohistochemical detection of Pgp in breast cancer tissue may have prognostic value after radical operation.     

Overexpression of P-glycoprotein is associated with a decreased mitochondrial transmembrane potential in doxorubicin-selected K562 human leukemia cells

We tested the hypothesis of whether overexpression of P-glycoprotein (Pgp) could be coupled with changes in specific mechanisms of antioxidant defense (in particular, transition of mitochondrial transmembrane potential, MTP) in tumor cells chronically exposed to anticancer drugs known to exert their cytotoxicity via oxidative stress. We show elevation of Pgp associated with decreased MTP in doxorubicin-selected K562Dox subline as compared with parental K562 cells. The low MTP was not due to a fewer number of mitochondria in K562Dox cells, nor was it associated with altered content of Bcl-XL protein. We discuss a model for coordinated up-regulation of Pgp and MTP transition in cells that survived chemotherapy-induced oxidative stress.     

Characterization of a new antibody raised against the NH2 terminus of P-glycoprotein.

PURPOSE: Cancers exposed to chemotherapy develop multidrug resistance, a major cause for chemotherapy failure. One mechanism of multidrug resistance development is due to overexpression of P-glycoprotein (Pgp) in these cancer cells. Thus, a prechemotherapy evaluation of Pgp in cancer cells aids in the design of alternative regimens that can circumvent such failure. As few Pgp-specific antibodies are available in detecting low levels of Pgp, there is a need for preparing an antibody that allows the detection of Pgp by various immunologic methods. EXPERIMENTAL DESIGN: We selected the amino acid stretch 11 to 34 in the cytoplasmically located NH2 terminus of Pgp as antigen, which was chemically synthesized and used to raise an antibody in a rabbit, termed NH2 11 antibody. We compared the properties of NH2 11 antibody with that of the well-characterized Pgp-specific antibody, C219, by Western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. RESULTS: Immunoblotting analysis suggested that NH2 11 antibody efficiently interacts with both recombinant and constitutively expressed Pgp in cancerous and noncancerous human cells. Immunoprecipitation reactions indicated that the NH2 11 antibody selectively immunoprecipitates Pgp. Immunocytochemical analyses indicated that the NH2 11 antibody detects Pgp in drug-resistant breast cancer cells as well as in human prostate and breast adenocarcinoma tissue sections. CONCLUSION: As the NH2 11 antibody detects Pgp present in cells and tissues, we conclude that the amino acid sequence to which this antibody was raised is highly antigenic and the antibody is useful in the detection of Pgp by a variety of immunologic methods.     

De novo expression of P-glycoprotein associated with poor prognosis in high grade non-Hodgkin's lymphoma

Chemotherapy has proven to be effective in the management of high grade non-Hodgkin's lymphoma (NHL). However drug resistance remains a major clinical obstacle to effective disease control. One such well documented system involves multidrug resistance (MDR), characterized by the overexpression of a 170 kDa membrane protein termed P-glycoprotein (Pgp). Analysis of P-glycoprotein was done on lymph node biopsies obtained from 109 patients with high grade NHL. Patients were followed up for time periods ranging from 7 to 26 months after chemotherapy or until recurrent/residual disease was diagnosed. Analysis of Pgp in tissue samples was carried out by immunocytochemistry and Western blot. Of the 109 patients, 36 had either residual or recurrent disease. All these patients had a second lymph node biopsy done which was also analyzed for Pgp. P-glycoprotein was detected by immunocytochemistry in only 5 of the 73 patients who remained disease-free, while it was expressed in 26 of the 36 patients with residual/recurrent disease. All the 36 tissue samples of the latter group and 42 of the 73 biopsies of the disease-free group were re-analyzed by Western blot. Only one of the 42 samples from the disease-free group showed a positive Western blot reaction while 30 of the 36 samples from patients with recurrent/residual disease gave a positive reaction. Thirty-four of the 36 repeat biopsy samples from patients with recurrent/residual disease were positive for Pgp. Correlation analysis, thus showed significant relationship between prognosis and detection of Pgp by immunocytochemistry (r=0.85, p<0.001) and Western blot (r=0.90, p<0.001). Moreover, the odds ratio of a tumor positive for Pgp not responding to chemotherapy was 35.36 (CI 11.03, 113.37). Thus evaluation for Pgp may prove to be a useful prognostic marker for high grade NHL, and may also prove useful in designing or altering chemotherapy protocols in such patients.     

Targeting multidrug resistant tumor cells with a recombinant single-chain FV fragment directed to P-glycoprotein.

The MDR1 gene product P-glycoprotein (Pgp) plays a key role in multidrug resistance of cancer cells. Pgp is an ATP-driven efflux pump that extrudes a variety of dissimilar hydrophobic cytotoxic compounds. P-glycoprotein overexpression results in multidrug resistance (MDR) of tumor cell lines in vitro as well as in cancer patients. To selectively target and eliminate MDR tumor cells, we have isolated a monoclonal antibody that specifically reacts with the first extracellular loop of the human Pgp. We have cloned the variable domain genes of this antibody and assembled a functional single-chain Fv fragment capable of specifically targeting various Pgp-expressing MDR carcinoma cells lines. Targeting and specific elimination of Pgp-dependent MDR human cancer cells was achieved by constructing a single-chain immunotoxin in which the scFv fragment was fused to a truncated form of Pseudomonas exotoxin (PE38). We conclude that recombinant Fv-immunotoxins or other Fv-based molecules armed with potent cytotoxins represent an effective tool in targeted cancer therapy aimed at specific elimination of MDR tumor cell sub-populations. Recombinant antibody fragments targeting MDR proteins such as Pgp may be also used for intracellular expression and consequent phenotypic knockout of MDR.     

The farnesyl protein transferase inhibitor SCH66336 is a potent inhibitor of MDR1 product P-glycoprotein

P-glycoprotein (Pgp)-mediated drug efflux is a major factor contributing to the variance of absorption and distribution of many drugs, particularly cancer chemotherapeutics. Multidrug resistance (MDR) is caused largely by the efflux of therapeutics out of the tumor cell by Pgp, resulting in reduced efficacy of chemotherapy. SCH66336, a farnesyl transferase inhibitor in development for cancer therapy, was examined in the present study for its ability to inhibit Pgp. In a test system consisting of a NIH-G185 cell line presenting an overexpressed amount of the human transporter Pgp, known Pgp inhibitors, such as cyclosporin A, paclitaxel, verapamil, tamoxifen, and vinblastine, were demonstrated to inhibit the Pgp-mediated efflux of daunorubicin. SCH66336 significantly inhibited daunorubicin transport with an IC50 of about 3 microM and similarly affected the transport of rhodamine 123 with a potency similar to cyclosporin A. Additionally, by an ATP-hydrolysis assay, SCH66336 was shown to decrease Pgp-mediated ATP hydrolysis by >70% with a Km of 3 microM. This observation indicates that SCH66336 directly interacts with the substrate binding site of Pgp, a quality unique to SCH66336 and its analogues, although not inherent to farnesyl transferase inhibitors in general. Moreover, low concentrations of SCH66336 exhibit synergy with the Pgp substrate/inhibitors paclitaxel, tamoxifen, and vinblastine respectively by significantly potentiating their inhibition of Pgp. Treatment with SCH66336 would be predicted to be synergistic with coadministered cancer therapeutics that are substrates of Pgp. A further benefit of coadministration of SCH66336 could be reduced chemotherapy dosage, hence, lower exposure to normal cells and, therefore, less undesired toxicity.     

A pilot study of amiodarone with infusional doxorubicin or vinblastine in refractory breast cancer

Increasing evidence suggests that P-glycoprotein (Pgp) expression can mediate drug resistance in refractory breast cancer. We studied 33 patients with refractory breast cancer enrolled in a pilot study of oral amiodarone as a Pgp antagonist given in combination with infusional doxorubicin or vinblastine. Whenever possible, tumors were biopsied and Pgp expression was assayed. Patients received either 60 mg/m² doxorubicin over 96 h or 8.5 mg/m² vinblastine over 120 h by continuous intravenous infusion. Beginning with the second cycle of chemotherapy, 600–800 mg amiodarone was given orally each day. Patients who experienced toxicity due to amiodarone but were responding to chemotherapy were placed on quinidine. Partial responses were observed in 9 of 33 patients on study and were sometimes observed after the first cycle of chemotherapy, before amiodarone was given, suggesting that some patients may have responded to treatment because of the infusional schedule. Toxicities were primarily the known side effects of the antineoplastic agents and of amiodarone. The major amiodarone toxicity was gastrointestinal, with nausea, vomiting, anorexia, or diarrhea being noted in 21 patients. Biopsy samples were obtained from 29 patients and in 21 cases, viable tumor tissue was present and the results were interpretable. Of the 21 samples, 9 had Pgp expression as determined by immunohistochemical staining; 12 were considered negative. The presence of Pgp expression was associated with an acceleration of the time to treatment failure. Whereas normal-tissue toxicities related to the combination of a Pgp antagonist with chemotherapy were not observed, amiodarone was associated with too many untoward effects to be utilized as a drug resistance-reversing agent.     

Spontaneous apoptosis in the intra-ductal component's stroma of breast invasive carcinoma

Spontaneous apoptosis by in situ detection of DNA fragmentation (DNAf) was investigated in breast invasive ductal carcinoma (IDC) frozen samples removed from 61 untreated patients. The incidence of DNAf was low in carcinoma cells and was mainly detected in the stroma. In the stroma at a distance from carcinoma cells, DNAf was inversely related to estradiol plasma level variations (p=0.01), indicating that it probably remained under physiological hormonal regulation. In the stroma adjacent to carcinoma cells, DNAf was correlated to tumor progression parameters such as the presence of a comedo intra ductal carcinoma (DCIS) component (p=0.001) and axillary lymph node metastasis (p=0.002), suggesting that this stromal compartment more probably represented a tumoral component closely associated to epithelial tumor cells. Therefore, the detection of DNAf in the adjacent stroma of breast carcinoma could help to predict progression in non invasive tumors and also in invasive tumors in those patients without lymph node invasion.     

Strategies to circumvent multidrug resistance due to P-glycoprotein or to altered DNA topoisomerase II

Strategies to circumvent different forms of multidrug resistance (MDR) in tumor cells will be discussed. The form of MDR associated with overexpression of P-glycoprotein. Pgp-MDR, is well-understood, and its features are briefly described. Many clinically useful lipophilic organic bases have been shown to interfere with drug efflux mediated by Pgp, consequently circumventing or overcoming this form of MDR. Based on these empiric observations, screening and molecular modeling efforts are being employed to develop new modulators of Pgp-MDR. However, because inhibition of normal tissue Pgp can cause unacceptable toxicities, new strategies to circumvent Pgp-MDR in tumors must be sought. Possibilities range from pharmacokinetic modeling to the development of tissue-specific inhibitory antibodies or antisense oligonucleotides. Tumor cells expressing altered DNA topoisomerase II express a more restricted form of MDR, termed at-MDR, that will be discussed briefly and compared with Pgp-MDR. Modulators of Pgp-MDR are without effect in cells expressing only at-MDR. However, some analogs of anthracyclines appear to act via a topo II-independent pathway and can circumvent this form of resistance. Also, alterations in topoisomerase II may have consequences for other cellular functions, as at-MDR cells appear to have defects in DNA repair pathways, suggesting other areas for therapeutic exploitation.     

Modulation of tumor cell response to chemotherapy by the organ environment

The outcome of cancer metastasis depends on the interaction of metastatic cells with various host factors. The implantation of human cancer cells into anatomically correct (orthotopic) sites in nude mice can be used to ascertain their metastatic potential. While it is clear that vascularity and local immunity can retard or facilitate tumor growth, we have found that the organ environment also influences tumor cell functions such as production of degradative enzymes. The organ microenvironment can also influence the response of metastases to chemotherapy. It is not uncommon to observe the regression of cancer metastases in one organ and their continued growth in other sites after systemic chemotherapy. We demonstrated this effect in a series of experiments using a murine fibrosarcoma, a murine colon carcinoma, and a human colon carcinoma. The tumor cells were implanted subcutaneously or into different visceral organs. Subcutaneous tumors were sensitive to doxorubicin (DXR), whereas lung or liver metastases were not. In contrast, sensitivity to 5-FU did not differ between these sites of growth. The differences in response to DXR between s.c. tumors (sensitive) and lung or liver tumors (resistant) were not due to variations in DXR potency or DXR distribution. The expression of the multidrug resistance-associated P-glycoprotein as determined by flow cytometric analysis of tumor cells harvested from lesions in different organs correlated inversely with their sensitivity to DXR: increased P-glycoprotein was associated with overexpression ofmdr1 mRNA. However, the organ-specific mechanism for upregulatingmdr1 and P-glycoprotein has yet to be elucidated.     

Effect of PSC 833, an inhibitor of P-glycoprotein on N-methyl-N-nitrosourea induced mammary carcinogenesis in rats

Studies in our laboratory on the role of P-glycoprotein (Pgp, coded by mdr1 gene) have led to the hypothesis that over-expression of Pgp is closely associated with the development of cancer. It was conceived therefore that inhibitors of Pgp should inhibit the development of cancer. We have reported that PSC833 (PSC), a potent inhibitor of Pgp, inhibits the development of liver cancer in rats. Similarly, based on the intrinsic over-expression of Pgp in experimental mammary carcinogenesis, we studied the effect of PSC on N-methyl-N-nitrosourea induced mammary cancer in female Sprague-Dawley rats. The study indicates that PSC at daily dietary doses of 15 (PSC15) and 30 mg/kg (PSC30) body wt resulted in dose-dependent inhibition of the incidence as well as the growth of mammary tumors. Compared with controls, PSC15 and PSC30 inhibited: (i) mean tumor multiplicity by 32 and 67%, (ii) median tumor burden by 46 and 93% and (iii) incidence of ulcerated tumors by 40 and 82%, respectively. Most remarkably, PSC delayed median tumor incidence by 8 weeks, and exerted a 100% inhibitory effect on the incidence of large tumors, 4 cm(3) and greater. In all the cases, although the inhibitory effect of PSC was evident at both doses, only PSC30 exhibited statistical significance. A possible compounding effect that was also observed in PSC30-treated rats was a decrease in body weight gain not attributed to diminished food consumption. All in all, consistent with recent reports, which have demonstrated inhibition of cancer development by compromising Pgp function, this study introduces a novel role for Pgp in breast cancer and potentially an unexplored therapeutic approach in treating the disease.