Zdenka Sulová

P-glycoprotein--implications of metabolism of neoplastic cells and cancer therapy.

Multidrug resistance (MDR) of neoplastic tissues is a major obstacle in cancer chemotherapy. The predominant cause of MDR is the overexpression and drug transport activity of P-glycoprotein (P-gp, a product of the MDR gene). P-gp is a member of the ATP binding cassette (ABC) transporters family, with broad substrate specificity for several substances including anticancer drugs, linear and cyclic peptides, inhibitors of HIV protease, and several other substances. The development of P-gp-mediated MDR is often associated with several changes in cell structure and metabolism of resistant cells. In the present review are discussed the relations between glucosylceramide synthase activity, Pregnane X receptor and development of P-gp mediated MDR phenotype. Attention is also focused on the changes in protein kinase systems (mitogen-activated protein kinases, protein kinase C, Akt kinase) that are associated with the development of MDR phenotype and to the possible role of these kinase cascades in modulation of P-gp expression and function. The overexpression of P-gp may be associated with changes in metabolism of sugars as well as energy production. Structural and ultrastructural characteristics of multidrug resistant cells expressing P-gp are typical for cells engaged in a metabolically demanding process of protein synthesis and transport. P-gp mediated MDR phenotype is often also associated with alterations in cytoskeletal elements, microtubule and mitochondria distribution, Golgi apparatus, chromatin texture, vacuoles and caveolae formation. The current review also aims at bringing some state-of-the-art information on interactions of P-glycoprotein with various substances. To capture and transport the numerous unrelated substances, P-gp should contain site(s) able to bind compounds with a molecular weight of several hundreds and comprising hydrophobic and/or base regions that are protonated under physiological conditions. Drug binding sites that are able to recognize substances with different chemical structures may have a complex architecture in which different parts are responsible for binding of different drugs. For P-gp substrates and inhibitors, a pharmacophore-based model has been described. The pharmacophores have to contain parts with hydrophobic and aromatic characteristics and functional groups that can act as hydrogen-bond donors and/or acceptors. Several drugs are known to be P-glycoprotein antagonizing agents. They represent a large group of structurally unrelated substances that can act via direct interaction with P-gp and inhibition of its transport activity, or via possible modulation of processes (such as phosphorylation) regulating P-gp transport activity. Effects of MDR reversal agents on the P-gp expression have also been reported. Function and expression of P-gp can be affected indirectly as well, e.g. through cyclooxygenase-2 or carbonic anhydrase-IX expression and effects.

New insight into p-glycoprotein as a drug target.

Multidrug resistance (MDR) of cancer tissue is a phenomenon in which cancer cells exhibit reduced sensitivity to a large group of unrelated drugs with different mechanisms of pharmacological activity. Mechanisms that reduce cell sensitivity to damage induced by a variety of chemicals were found to be caused by diverse, albeit well-defined, phenotypic alterations. The molecular basis of MDR commonly involves overexpression of the plasma membrane drug efflux pump - P-glycoprotein (P-gp). This glycoprotein is an ABCB1 member of the ABC transporter family. Cells that develop MDR of this type express massive amounts of P-gp that can induce a drug resistance of more than 100 times higher than normal cells to several drugs, which are substrates of P-gp. Expression of P-gp could be inherent to cancer cells with regard to the specialized tissues from which the cells originated. This is often designated as intrinsic Pgp- mediated MDR. However, overexpression of P-gp may be induced by selection and/or adaptation of cells during exposure to anticancer drugs; this particular example is known as acquired P-gp-mediated MDR. Drugs that are potential inducers of P-gp are often substrates of this transporter. However, several substances that have been proven to not be transportable by P-gp (such as cisplatin or alltrans retinoic acid) could induce minor improvements in P-gp overexpression. It is generally accepted that the drug efflux activity of Pgp is a major cause of reduced cell sensitivity to several compounds. However, P-gp may have side effects that are independent of its drug efflux activity. Several authors have described a direct influence of P-gp on the function of proteins involved in regulatory pathways, including apoptotic progression (such as p53, caspase-3 and Pokemon). Moreover, alterations of cell regulatory pathways, including protein expression, glycosylation and phosphorylation, have been demonstrated in cells overexpressing P-gp, which may consequently induce changes in cell sensitivity to substances that are not P-gp substrates or modulators. We recently reported that P-gppositive L1210 cells exhibit reduced sensitivity to cisplatin, concanavalin A, thapsigargin and tunicamycin. Thus, P-gp-mediated MDR represents a more complex process than was expected, and the unintended effects of P-gp overexpression should be considered when describing this phenotype. The present review aims to provide the most current informations about P-gp-mediated MDR while paying particular attention to the possible dual function of this protein as a drug efflux pump and a regulatory protein that influences diverse cell processes. From a clinical standpoint, overexpression of P-gp in cancer cells represents a real obstacle to effective chemotherapy for malignant diseases. Therefore, this protein should be considered as a viable target for pharmaceutical design.

P-glycoprotein depresses cisplatin sensitivity in L1210 cells by inhibiting cisplatin-induced caspase-3 activation.

Multidrug resistance (MDR) is a phenomenon in which cells become resistant to cytostatic drugs and other substances with diverse chemical structures and cytotoxicity mechanisms. The most often observed molecular mechanism for MDR includes high levels of P-glycoprotein (P-gp)--an ABCB1 member of the ABC drug transporter family. Overexpression of P-gp in neoplastic tissue is an obstacle to chemotherapeutic treatment. Herein, we were focused on differences in apoptosis induced by cisplatin (no substrate for P-gp) between P-gp-positive and P-gp-negative L1210 cells. P-gp-positive cells were obtained by either L1210 cell adaptation to vincristine (R) or L1210 cell transfection with the human gene for P-gp (T) and compared with parental L1210 cells (S). R and T cells were more resistant to CisPt than S cells. R and T cell resistance to CisPt-induced apoptosis could not be reversed by verapamil (a well-known P-gp inhibitor), which excludes P-gp transport activity as a cause of CisPt resistance. CisPt induced a more pronounced entry into apoptosis in S than R and T cells, which was measured using the annexin-V/propidium iodide apoptosis kit. CisPt induced more pronounced caspase-3 activation in S than R and T cells. CisPt did not induce changes in the P-gp protein level for R and T cells. While similar levels of Bax and Bcl-2 proteins were observed in P-gp-negative and P-gp-positive cells, CisPt induced a more significant decrease in Bcl-2 levels for S cells than P-gp-positive cells. Expression of p53 and its molecular chaperone Hsp90 were more pronounced in R and T than S cells. Moreover, CisPt enhanced the upregulation of p53 and Hsp90 in R and T cells to a higher degree than S cells. Apoptosis was shown to be the prevalent mode of cell death in S, R and T cells by the typical DNA fragmentation and cell ultrastructure changes. All of the above findings indicate that P-gp, independent of its drug efflux activity, induced changes in cell regulatory pathways that confer a partial loss of cisplatin sensitivity.

Tunicamycin Depresses P-Glycoprotein Glycosylation Without an Effect on Its Membrane Localization and Drug Efflux Activity in L1210 Cells

P-glycoprotein (P-gp), also known as ABCB1, is a member of the ABC transporter family of proteins. P-gp is an ATP-dependent drug efflux pump that is localized to the plasma membrane of mammalian cells and confers multidrug resistance in neoplastic cells. P-gp is a 140-kDa polypeptide that is glycosylated to a final molecular weight of 170 kDa. Our experimental model used two variants of L1210 cells in which overexpression of P-gp was achieved: either by adaptation of parental cells (S) to vincristine (R) or by transfection with the human gene encoding P-gp (T). R and T cells were found to differ from S cells in transglycosylation reactions in our recent studies. The effects of tunicamycin on glycosylation, drug efflux activity and cellular localization of P-gp in R and T cells were examined in the present study. Treatment with tunicamycin caused less concentration-dependent cellular damage to R and T cells compared with S cells. Tunicamycin inhibited P-gp N-glycosylation in both of the P-gp-positive cells. However, tunicamycin treatment did not alter either the P-gp cellular localization to the plasma membrane or the P-gp transport activity. The present paper brings evidence that independently on the mode of P-gp expression (selection with drugs or transfection with a gene encoding P-gp) in L1210 cells, tunicamycin induces inhibition of N-glycosylation of this protein, without altering its function as plasma membrane drug efflux pump.

Vincristine-Induced Overexpression of P-Glycoprotein in L1210 Cells Is Associated with Remodeling of Cell Surface Saccharides

Multidrug resistance of murine leukemic cell line L1210/VCR (R), obtained by adaptation of parental L1210 cells (S) on vincristine, is associated with overexpression of P glycoprotein (P-gp, the ATP-dependent drug efflux pump). Previously, we found that cytochemical staining of negatively charged cell surface binding sites (probably sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of S cells. This is in contrast to R cells and L1210/VCR cells cultured in the presence of vincristine during the last cultivation prior to the experiment (V cells), where the RR layer was either reduced or absent. In the current paper, we observed differences in the interactions of S, R and V cells with Concanavalin A (ConA) and tomato lectin (lycopersicum esculentum agglutinin, LEA). ConA bound and induced cell damage more effectively in S cells than in R or V cells. Both of these effects could be prevented by methyl-manopyranose, but not by N-acetylglucosamine. In contrast, LEA lectin preferentially bound to R and V cells. While LEA agglutinated cells more effectively than ConA, it did not cause cell damage comparable to ConA. Binding of LEA to the cell surface could be prevented by chitooligosaccharides. Both LEA and ConA failed to identify P-gp in lectin blots. Thus, changes in ConA and LEA interactions are not caused by massive expression of P-gp in the plasma membrane and the consequent exposure of the inner saccharides to the external side of the plasma membrane.Taken together, the above facts suggest that S cells differ from R and V cells in the composition of cell surface glycosides not directly linked to P-gp.

Selection of resistant acute myeloid leukemia SKM-1 and MOLM-13 cells by vincristine-, mitoxantrone- and lenalidomide-induced upregulation of P-glycoprotein activity and downregulation of CD33 cell surface exposure.

Bone marrow cells and peripheral blood mononuclear cells obtained from both acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients contain upregulated levels of cell surface antigen CD33 compared with healthy controls. This difference enables the use of humanized anti-CD33 antibody conjugated to cytotoxic agents for CD33 targeted immunotherapy. However, the expression of the membrane-bound drug transporter P-glycoprotein (P-gp) has been shown to be critical for resistance against the cytotoxicity of a humanized anti-CD33 antibody conjugated to maytansine-derivative DM4. The aim of the present study was to examine whether the expression of P-gp in AML cell lines is associated with changes in CD33 expression. For this purpose, we established drug resistant variants of SKM-1 and MOLM-13 AML cell lines via the selection of parental cells for resistance to vincristine, mitoxantrone and lenalidomide. All three substances induced a multidrug resistance (MDR) phenotype in SKM-1 cells associated with strong upregulation of P-gp and downregulation of CD33. However, in MOLM-13 cells, the upregulation of P-gp and downregulation of CD33 were present only in cells selected for resistance to vincristine and mitoxantrone but not lenalidomide. Inverse expression of P-gp and CD33 were observed in all resistant variants of SKM-1 and MOLM-13 cells. The MDR phenotype of resistant variants of SKM-1 and MOLM-13 cells was associated with alterations in apoptotic regulatory proteins and downregulation of the multidrug resistance associated protein 1 and breast cancer resistance protein.