In 1970, Biedler and Riehm (3
) described an in vitro model of chemotherapeutic multidrug resistance (MDR) in which cultured cells that were selected for growth in actinomycin D developed resistance to a variety of structurally and functionally diverse cytotoxic compounds. Further studies showed that the emergence of MDR was associated with increased levels of a transmembrane glycoprotein (24
), P glycoprotein (P-gp). P-gp, the product of the MDR1
gene, is a 170-kDa protein that functions as an energy-dependent drug efflux pump whose substrates include naturally occurring, lipophilic agents with a complex ring structure such as Vinca
alkaloids, anthracyclines, epipodophyllotoxins, and certain rhodamine dyes (18
). Exposure of tumor cells to any of these substrates can generate overexpression of P-gp, resulting in the MDR phenotype.
Drug exposure is thought to cause overexpression of P-gp by both selection of resistant cells and induction of P-gp expression at the level of the MDR1
). The MDR1
promoter contains a heat-shock consensus element (46
) and a putative xenobiotic response element that responds directly to treatment with cytotoxic agents (25
), supporting the premise that induction of P-gp expression occurs in the presence of chemotherapeutic agents. Additionally, there is a correlation between specific point mutations in the MDR1
promoter and increased inducibility after treatment with chemotherapeutic agents (45
). Collectively, these studies provide supportive evidence that induction of P-gp overexpression, as a direct result of exposure to chemotherapeutic agents, does occur in vitro. Such evidence is lacking in the clinical setting, where it is unclear whether apparently increasing levels of P-gp expression are a result of selection of a P-gp-expressing subpopulation of cells or induction of P-gp expression.
However, considerable data document the importance of P-gp-mediated MDR in clinical cancer patients. Expression of P-gp has been documented elsewhere for tumor specimens derived from patients with a variety of histologic types of cancer (8
). Results from these clinical investigations were similar to in vitro results described above: P-gp expression was increased in patients with a history of exposure to chemotherapeutic drugs. Clinical studies of different malignant tumors have shown the progressive development of P-gp overexpression during chemotherapy, confirming the hypothesis that exposure to antineoplastic agents results in selection (or induction) of MDR clones of tumor cells (4
). Emergence of these resistant clones often leads to relapse of disease and therapeutic failure. For many tumor types, a relationship between P-gp expression and an adverse clinical course has been observed previously (1
). Although the role of P-gp in human cancer is not entirely defined, the consensus view is that P-gp overexpression is associated with clinical evidence of drug resistance and treatment failure for a significant number of cancer patients (22
As a result of these observations, chemotherapeutic treatment protocols are manipulated so as to prevent the development of P-gp expression. Protocols are designed to circumvent the proliferation of resistant tumor cells by judiciously combining multiple drugs and delivering them at optimal doses and intervals (34
). Considerable effort is made to avoid drugs that may have only sporadic activity against a specific tumor and yet are likely to select for MDR. In contrast, little attention is granted to the possibility that P-gp expression may be affected by other drugs that are administered to cancer patients. Many cancer patients are immunocompromised, as a direct consequence of their disease or from treatment for their disease, and are predisposed to bacterial infections. Such patients often undergo antimicrobial therapy either prophylactically or for active bacterial infections. Administration of an antimicrobial drug that enhances the development of MDR could promote therapeutic failure.
P-gp shares a high degree of homology with bacterial transport proteins (9
) and displays characteristics of bacterial multidrug efflux systems (36
). Interestingly, one of the same drugs that is used to generate overexpression of P-gp in mammalian cells, rhodamine, is capable of generating expression of a homologous bacterial transport protein. Bacillus subtilis
cells selected for rhodamine 6G resistance display amplification of the gene coding for a prokaryotic MDR transporter. This multidrug efflux system transports similar drugs (puromycin, ethidium bromide, and rhodamine) and is sensitive to the same inhibitors (verapamil and reserpine) as is the mammalian multidrug transporter P-gp (36
). These observations led to the hypothesis that exposure of cancer cells to antimicrobial drugs would result in the emergence of a P-gp-expressing MDR subline. To test this hypothesis, three agents were selected, each representing a different antimicrobial drug class. Doxycycline, a tetracycline antimicrobial agent, was selected because of its structural similarity to doxorubicin (Fig. ), and because of its lipophilicity among the tetracyclines (13
). Both doxycycline and doxorubicin are elaborated by strains of the genus Streptomyces
). Cefoperazone, a cephalosporin antimicrobial agent, was selected because of its ability to modulate P-gp function, as it has been reported previously that some P-gp modulators are actually substrates of P-gp (40
). Piperacillin, a semisynthetic penicillin, was selected because it contains a piperazine group, a chemical moiety that plays an important role in substrate binding to P-gp (21
). The well-defined human breast carcinoma cell line MCF-7 was incubated with increasing concentrations of doxycycline, cefoperazone, piperacillin, and the antineoplastic drug doxorubicin (positive control). Exposure of MCF-7 cells to the antimicrobial agent doxycycline produces a P-gp-overexpressing cell line with properties that are identical to those of well-characterized MDR cell lines generated following incubation of cells with antineoplastic agents. The fact that P-gp overexpression was generated in a previously P-gp-negative cell clone provides supportive evidence that induction of P-gp overexpression, rather than selection of a resistant cell population, is responsible for generating MDR in cell culture systems.
Chemical structures of doxorubicin and doxycycline.