The Roles of microRNAs in Cancer Multidrug Resistance.

Cancer chemotherapy may induce a multidrug resistance (MDR) phenotype. The development of MDR is based on various molecular causes, of which the following are very common: induction of ABC transporter expression; induction/activation of drug-metabolizing enzymes; alteration of the expression/function of apoptosis-related proteins; changes in cell cycle checkpoints; elevated DNA repair mechanisms. Although these mechanisms of MDR are well described, information on their molecular interaction in overall multidrug resistance is still lacking. MicroRNA (miRNA) expression and subsequent RNA interference are candidates that could be important players in the interplay of MDR mechanisms. The regulation of post-transcriptional processes in the proteosynthetic pathway is considered to be a major function of miRNAs. Due to their complementarity, they are able to bind to target mRNAs, which prevents the mRNAs from interacting effectively with the ribosome, and subsequent degradation of the mRNAs can occur. The aim of this paper is to provide an overview of the possible role of miRNAs in the molecular mechanisms that lead to MDR. The possibility of considering miRNAs as either specific effectors or interesting targets for cancer therapy is also analyzed.

Photodynamic therapy of multidrug resistant leukemic murine cells by 3,6-bis(alkylthiourea)acridine hydrochlorides.

Efforts to overcome multidrug resistance in cancer have led to the development of several novel strategies including photodynamic therapy (PDT). PDT is based on the use of photosensitizers (PSs) photoactivation, which causes the formation of reactive oxygen species that can induce cell death. In the last decade, the development of new PSs has been significantly accelerated. Recently, acridine-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been investigated as a new group of PSs and we have shown that PDT/AcrDTUs caused cell death of mouse leukemic cells L1210. In this study, we investigated the efficacy of PDT/AcrDTUs for the treatment of L1210/VCR cells as a model of chemoresistant cells (overexpressing P-glycoprotein, P-gp). The photoactivation (365 nm, 1.05 J/cm2) increased the cytotoxicity of AcrDTUs 10-15 times. Inhibition of P-gp (verapamil) has been shown to have no significant effect on the accumulation of propyl-AcrDTU (the most potent derivative) in L1210/VCR cells. The intracellular distribution of this acridine derivative has been studied. Prior to irradiation of the resistant cells, propyl-AcrDTU was sequestered mainly in the cytosol, partly in the mitochondria, and, unlike in the sensitive cells, the AcrDTU was not found in the lysosomes. PDT with 1 µM propyl-AcrDTU induced cell shrinkage and "ladder DNA" formation, and although a drastic decrease of the intracellular ATP level was observed at the same time, there was no increase in extracellular LDH activity. AIF in the nucleus can induce DNA fragmentation and we have actually observed a mitochondrio-nuclear translocation of AIF. We concluded that AcrDTUs are photocytotoxic against L1210/VCR cells and that mitochondria play an important role in cell death induced by PDT.

Insight into Bortezomib Focusing on Its Efficacy against P-gp-Positive MDR Leukemia Cells.

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.

Development of Resistance to Endoplasmic Reticulum Stress-Inducing Agents in Mouse Leukemic L1210 Cells.

Four new variants of L1210 cells resistant to endoplasmic reticulum (ER) stressors, tunicamycin (STun), thapsigargin (SThap), bortezomib (SBor), and MG-132 (SMG-132), were developed via an 18-month periodic cultivation in culture medium with a gradual increase in substance concentration. Multidrug resistance was generated for STun (to tunicamycin, bortezomib and MG-132), SThap (to tunicamycin, thapsigargin and MG-132), SBor (to bortezomib and MG-132), and SMG-132 (to bortezomib and MG-132). These cells were compared to the original L1210 cells and another two variants, which expressed P-gp due to induction with vincristine or transfection with the gene encoding P-gp, in terms of the following properties: sensitivity to either vincristine or the ER stressors listed above, proliferative activity, expression of resistance markers and proteins involved in the ER stress response, and proteasome activity. The resistance of the new cell variants to ER stressors was accompanied by a decreased proliferation rate and increased proteasome activity. The most consistent change in protein expression was the elevation of GRP78/BiP at the mRNA and protein levels in all resistant variants of L1210 cells. In conclusion, the mechanisms of resistance to these stressors have certain common features, but there are also specific differences.

Overexpression of GRP78/BiP in P-Glycoprotein-Positive L1210 Cells is Responsible for Altered Response of Cells to Tunicamycin as a Stressor of the Endoplasmic Reticulum.

P-glycoprotein (P-gp, ABCB1 member of the ABC (ATP-binding cassette) transporter family) localized in leukemia cell plasma membranes is known to reduce cell sensitivity to a large but well-defined group of chemicals known as P-gp substrates. However, we found previously that P-gp-positive sublines of L1210 murine leukemia cells (R and T) but not parental P-gp-negative parental cells (S) are resistant to the endoplasmic reticulum (ER) stressor tunicamycin (an N-glycosylation inhibitor). Here, we elucidated the mechanism of tunicamycin resistance in P-gp-positive cells. We found that tunicamycin at a sublethal concentration of 0.1 µM induced retention of the cells in the G1 phase of the cell cycle only in the P-gp negative variant of L1210 cells. P-gp-positive L1210 cell variants had higher expression of the ER stress chaperone GRP78/BiP compared to that of P-gp-negative cells, in which tunicamycin induced larger upregulation of CHOP (C/EBP homologous protein). Transfection of the sensitive P-gp-negative cells with plasmids containing GRP78/BiP antagonized tunicamycin-induced CHOP expression and reduced tunicamycin-induced arrest of cells in the G1 phase of the cell cycle. Taken together, these data suggest that the resistance of P-gp-positive cells to tunicamycin is due to increased levels of GRP78/BiP, which is overexpressed in both resistant variants of L1210 cells.

Screening of Phenanthroquinolizidine Alkaloid Derivatives for Inducing Cell Death of L1210 Leukemia Cells with Negative and Positive P-glycoprotein Expression.

We describe the screening of a set of cryptopleurine derivatives, namely thienoquinolizidine derivatives and (epi-)benzo analogs with bioactive phenanthroquinolizidine alkaloids that induce cytotoxic effects in the mouse lymphocytic leukemia cell line L1210. We used three variants of L1210 cells: i) parental cells (S) negative for P-glycoprotein (P-gp) expression; ii) P-glycoprotein positive cells (R), obtained by selection with vincristine; iii) P-glycoprotein positive cells (T), obtained by stable transfection with a human gene encoding P-glycoprotein. We identified the most effective derivative 11 with a median lethal concentration of ≈13 µM in all three L1210 cell variants. The analysis of the apoptosis/necrosis induced by derivative 11 revealed that cell death was the result of apoptosis with late apoptosis characteristics. Derivative 11 did not induce a strong alteration in the proportion of cells in the G1, S or G2/M phase of the cell cycle, but a strong increase in the number of S, R and T cells in the subG1 phase was detected. These findings indicated that we identified the most effective inducer of cell death, derivative 11, and this derivative effectively induced cell death in S, R and T cells at similar inhibitory concentrations independent of P-gp expression.

Triorganotin Derivatives Induce Cell Death Effects on L1210 Leukemia Cells at Submicromolar Concentrations Independently of P-glycoprotein Expression.

The acceleration of drug efflux activity realized by plasma membrane transporters in neoplastic cells, particularly by P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family), represents a frequently observed molecular cause of multidrug resistance (MDR). This multiple resistance represents a real obstacle in the effective chemotherapy of neoplastic diseases. Therefore, identifying cytotoxic substances that are also effective in P-gp overexpressing cells may be useful for the rational design of substances for the treatment of malignancies with developed MDR. Here, we showed that triorganotin derivatives­tributyltin-chloride (TBT-Cl), tributyltin-bromide (TBT-Br), tributyltin-iodide (TBT-I) and tributyltin-isothiocyanate (TBT-NCS) or triphenyltin-chloride (TPT-Cl) and triphenyltin-isothiocyanate (TPT-NCS)­could induce the death of L1210 mice leukemia cells at a submicromolar concentration independently of P-gp overexpression. The median lethal concentration obtained for triorganotin derivatives did not exceed 0.5 µM in the induction of cell death of either P-gp negative or P-gp positive L1210 cells. Apoptosis related to regulatory pathway of Bcl-2 family proteins seems to be the predominant mode of cell death in either P-gp negative or P-gp positive L1210 cells. TBT-Cl and TBT-Br were more efficient with L1210 cells overexpressing P-gp than with their counterpart P-gp negative cells. In contrast, TBT-I and TPT-NCS induced a more pronounced cell death effect on P-gp negative cells than on P-gp positive cells. Triorganotin derivatives did not affect P-gp efflux in native cells measured by calcein retention within the cells. Taken together, we assumed that triorganotin derivatives represent substances suitable for suppressing the viability of P-gp positive malignant cells.

Interplay between P-Glycoprotein Expression and Resistance to Endoplasmic Reticulum Stressors.

Multidrug resistance (MDR) is a phenotype of cancer cells with reduced sensitivity to a wide range of unrelated drugs. P-glycoprotein (P-gp)-a drug efflux pump (ABCB1 member of the ABC transporter gene family)-is frequently observed to be a molecular cause of MDR. The drug-efflux activity of P-gp is considered as the underlying mechanism of drug resistance against P-gp substrates and results in failure of cancer chemotherapy. Several pathological impulses such as shortages of oxygen and glucose supply, alterations of calcium storage mechanisms and/or processes of protein N-glycosylation in the endoplasmic reticulum (ER) leads to ER stress (ERS), characterized by elevation of unfolded protein cell content and activation of the unfolded protein response (UPR). UPR is responsible for modification of protein folding pathways, removal of misfolded proteins by ER associated protein degradation (ERAD) and inhibition of proteosynthesis. However, sustained ERS may result in UPR-mediated cell death. Neoplastic cells could escape from the death pathway induced by ERS by switching UPR into pro survival mechanisms instead of apoptosis. Here, we aimed to present state of the art information about consequences of P-gp expression on mechanisms associated with ERS development and regulation of the ERAD system, particularly focused on advances in ERS-associated therapy of drug resistant malignancies.

L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins.

Overexpression of P-glycoprotein (P-gp, drug transporter) in neoplastic cells is the most frequently observed molecular cause of multidrug resistance. Here, we show that the overexpression of P-gp in L1210 cells leads to resistance to tunicamycin and benzyl 2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAc-α-O-benzyl). Tunicamycin induces both glycosylation depression and ubiquitination improvement of P-gp. However, the latter is not associated with large increases in molecular mass as evidence for polyubiquitination. Therefore, P-gp continues in maturation to an active membrane efflux pump rather than proteasomal degradation. P-gp-positive L1210 cells contain a higher quantity of ubiquitin associated with cell surface proteins than their P-gp-negative counterparts. Thus, P-gp-positive cells use ubiquitin signaling for correct protein folding to a higher extent than P-gp-negative cells. Elevation of protein ubiquitination after tunicamycin treatment in these cells leads to protein folding rather than protein degradation, resulting at least in the partial lack of cell sensitivity to tunicamycin in L1210 cells after P-gp expression. In contrast to tunicamycin, to understand why P-gp-positive cells are resistant to GalNAc-α-O-benzyl, further research is needed.

The expression of P-gp in leukemia cells is associated with cross-resistance to protein N-glycosylation inhibitor tunicamycin.

In P-gp-positive cell variants obtained from L1210 cells either by selection with vincristine (L1210/R) or by transfection with the human gene encoding P-gp (L1210/T), we have previously described cross-resistance to tunicamycin (TNM), a protein N-glycosylation inhibitor. Here we studied whether this cross-resistance also underlies P-gp-positive variants of human acute myeloid leukemia cells (AML) derived from SKM-1 and MOLM-13 cells (SKM-1/VCR, SKM-1/LEN, MOLM-13/VCR) by selection with vincristine (VCR) and lenalidomide (LEN). While SKM-1/LEN cells were P-gp positive, no P-gp was detected in MOLM-13/LEN cells. P-gp-positive cells could be repeatedly passaged in medium containing TNM. In contrast, more than 90% of P-gp-negative cells were entering and progressing through cell death mechanisms after the third passage in medium containing TNM. Combined apoptosis/necrosis cell death was detected in L1210 cells after exposure to TNM. Passaging of P-gp-negative AML cells in medium containing TNM induced preferentially apoptosis. Damage to P-gp-negative cells induced with TNM was associated with arrest in the G1 phase of the cell cycle. P-gp-positive leukemia cells differed from P-gp-negative cells in the composition of plasma membrane glycoproteins, which we monitored with the aid of different lectins. The application of TNM to cells induced additional changes in membrane-linked glycosides.

A decrease in cellular microRNA-27a content is involved in azacytidine-induced P-glycoprotein expression in SKM-1 cells.

We established an azacytidine (AzaC)-resistant human acute myeloid leukemia (AML) cell line (SKM-1/AzaC) by culturing SKM-1 cells in the presence of increasing amounts of AzaC for six months. Because AzaC is not a substrate of P-glycoprotein (a product of the ABCB1 gene; ABCB1), ABCB1 was not responsible for AzaC resistance; nevertheless, it was notably upregulated in SKM-1/AzaC cells. In addition, the transcription of the Nfkb1 gene, which encodes a member of the canonical NF-kappaB regulatory pathway, was downregulated, and the transcription of the Nfkb2 gene, which encodes a member of the non-canonical NF-kappaB regulatory pathway, was upregulated in SKM-1/AzaC cells. Here, we investigate whether miRNA-27a and miRNA-138 (both of which are known to be regulators of ABCB1 expression) are involved in the regulation of ABCB1 expression in SKM-1/AzaC cells. We observed decreased levels of miRNA-27a but of not miRNA-138 in SKM-1/AzaC cells compared with SKM-1 cells. The transfection of SKM-1/AzaC cells with a miRNA-27a mimic induced the downregulation of the ABCB1 mRNA. This was associated with an increase in Nfkb1 and a decrease in Nfkb2 transcript levels in SKM-1/AzaC cells. Taken together, these data indicate that the downregulation of miRNA-27a is involved in the upregulation of ABCB1 expression in SKM-1/AzaC cells, and this effect is associated with a switch between the canonical and non-canonical NF-kappaB pathways.

Lenalidomide treatment induced the normalization of marker protein levels in blood plasma of patients with 5q-myelodysplastic syndrome.

A specific type of myelodysplastic syndrome (MDS) is associated with isolated deletion on the long arm of chromosome 5, i.e., 5q-syndrome (del(5q)). The treatment approaches for MDS del(5q) include the immunomodulating drug lenalidomide (LEN). Thirteen MDS del(5q) patients were included in this study. We found elevated activities of lactate dehydrogenase (LDH) and matrix metalloproteinase 9 (MMP-9) in the blood plasma of MDS del(5q) patients as compared with healthy controls. This was stabilized to control values after LEN treatment. Similar behavior we registered also for the thioredoxin and calnexin contents in BP. Peripheral blood mononuclear cells (PBMC) from patients with MDS del(5q) prior to and after treatment with LEN did not exhibit any detectable amount of P-glycoprotein (P-gp) gene transcript. However, we detected a measurable amount of multidrug resistance associated protein 1 (MRP1) mRNA in PBMCs from three patients prior to LEN treatment and in one patient during LEN treatment but it was not present prior to treatment. These data indicated on usefulness of applied protein markers estimation for monitoring of MDS del(5q) patient treatment effectiveness by LEN. Expression of MRP1 seems to be independent on LEN treatment and reflects probably the molecular variability in the ethiopathogenesis of MDS del(5q).

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.

Detection of glycomic alterations induced by overexpression of p-glycoprotein on the surfaces of L1210 cells using sialic acid binding lectins.

P-glycoprotein (P-gp) overexpression is the most frequently observed cause of multidrug resistance in neoplastic cells. In our experiments, P-gp was expressed in L1210 mice leukemia cells (S cells) by selection with vincristine (R cells) or transfection with the gene encoding human P-gp (T cells). Remodeling of cell surface sugars is associated with P-gp expression in L1210 cells as a secondary cellular response. In this study, we monitored the alteration of cell surface saccharides by Sambucus nigra agglutinin (SNA), wheat germ agglutinin (WGA) and Maackia amurensis agglutinin (MAA). Sialic acid is predominantly linked to the surface of S, R and T cells via α-2,6 branched sugars that tightly bind SNA. The presence of sialic acid linked to the cell surface via α-2,3 branched sugars was negligible, and the binding of MAA (recognizing this branch) was much less pronounced than SNA. WGA induced greater cell death than SNA, which was bound to the cell surface and agglutinated all three L1210 cell-variants more effectively than WGA. Thus, the ability of lectins to induce cell death did not correlate with their binding efficiency and agglutination potency. Compared to S cells, P-gp positive R and T cells contain a higher amount of N-acetyl-glucosamine on their cell surface, which is associated with improved WGA binding. Both P-gp positive variants of L1210 cells are strongly resistant to vincristine as P-gp prototypical drug. This resistance could not be altered by liberalization of terminal sialyl residues from the cell surface by sialidase.

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.

Effect of thapsigargin on P-glycoprotein-negative and P-glycoprotein-positive L1210 mouse leukaemia cells.

Expression of drug-transporting P-glycoprotein (P-gp, an integral protein of the plasma membrane) in neoplastic cells confers multidrug resistance and also involves alteration of cell sensitivity to inhibitors of the sarco/endoplasmic reticulum calcium pump thapsigargin (Th). Mouse leukaemia L1210 cell sublines that overexpress P-gp due to selection with vincristine (R) or stable transfection with a gene encoding human P-gp (T) were less sensitive to Th than the parental cell line (S). Th at a concentration of 0.1 µmol/l did not induce alterations in the amount of P-gp mRNA in R or T cells (S cells did not contain any measurable amount of this transcript as assessed by RT-PCR) or in the amount of calnexin (CNX) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in all three cell sublines. However, when using a concentration of 10 µmol/l, Th decreases the amounts of CNX, GAPDH (in S, R and T cells) and P-gp (in R and T cells) mRNAs. In contrast to R and T cells (which contain abundant P-gp), S cells did not contain any P-gp detectable by the c219 antibody on a Western blot. Th at a concentration of 0.1 µmol/l induced a reduction in the amount of P-gp present in R and T cells, particularly in isoforms with higher molecular weights (i.e., mature fully glycosylated isoforms). Similar results were observed when Th was used at a concentration of 10 µmol/l. R and T cells contained lower levels of CNX than S cells. While Th at a lower concentration did not alter the levels of CNX in S, R or T cells, a higher concentration of this substance induced a measurable decrease in the amount of CNX. S, R and T cells did not differ with respect to GAPDH content, but Th induced a reduction in the amount of this protein in all cell sublines. More pronounced results were observed when Th was applied at a concentration of 10 µmol/l comparing with a concentration of 0.1 mmol/l. These changes may be involved together with the Th efflux activity of P-gp in Th-resistance associated with the P-gp-mediated multidrug resistance of R and T cells.

The presence of P-glycoprotein in L1210 cells directly induces down-regulation of cell surface saccharide targets of concanavalin A.

Overexpression of P-glycoprotein (P-gp), a plasma membrane drug transporter (ABCB1, a member of the ABC transporter family), is the most prevalent cause of multidrug resistance in cancer tissues. Lectin concanavalin A (ConA) induces massive cell death of L1210 leukemia cells (S). Cell sublines of L1210 in which P-gp overexpression was induced by selection with vincristine (R) or by stable transfection with a plasmid encoding full-length human P-gp (T) were less sensitive to ConA. Both P-gp-positive cell lines exhibited typical P-gp-mediated multidrug resistance. Resistance of R and T cells to ConA was associated with lower binding of ConA as compared to S cells when analysed by the following methods: (i) SDS PAGE and electroblotting of proteins in the crude membrane fraction followed by detection with biotinylated ConA and avidin-peroxidase, and (ii) fluorescent cytometry or confocal microscopy of the intact cells with surfaces labeled by FITC-ConA. These data indicated that the presence of P-glycoprotein in L1210 cells independently of the mode of its expression induced down-regulation of cell surface saccharide targets of ConA. Therefore, this feature may be considered as a secondary cellular response to P-glycoprotein expression.

Does any relationship exist between P-glycoprotein-mediated multidrug resistance and intracellular calcium homeostasis.

Multidrug resistance (MDR) of neoplastic tissue represents a real obstacle to the effective chemotherapy of cancer. Several mechanisms of MDR were identified, from which the over-expression and efflux activity of P-glycoprotein (P-gp) - a plasma membrane ATPase (ABCB1 member of ABC transporter family) - represents the most commonly observed reason for neoplastic disease chemotherapy malfunction. The process of P-gp-mediated MDR seems to be related to intracellular calcium homeostasis, at least indirectly, for the following reasons: i. substances blocking calcium influx through L-type of calcium channels like verapamil were often found to antagonize P-gp-mediated MDR; ii. calcium signal abnormalities were observed in cells over-expressing P-gp; iii. cells with P-gp-mediated MDR were often resistant to thapsigargin; iv. several differences in intracellular calcium localization were observed when P-gp-negative and P-gp-positive cells were compared; and v. differences in the contents of several proteins of the endoplasmic reticulum involved in calcium homeostasis were observed to be associated with P-gp over-expression. This current study represents an attempt to summarize the knowledge about the possible relationship between P-gp-mediated MRD and intracellular calcium homeostasis.

Combined treatment of P-gp-positive L1210/VCR cells by verapamil and all-trans retinoic acid induces down-regulation of P-glycoprotein expression and transport activity.

The development of the most common multidrug resistance (MDR) phenotype associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells may result in more than one hundred fold higher resistance of these cells to several drugs. L1210/VCR is a P-gp-positive drug resistant cell line in which P-gp overexpression was achieved by repeated cultivation of parental cells with a stepwise increasing concentration of vincristine. Relatively little is known about regulation of P-gp expression. Therefore, serious efforts have been made to recognize all aspects involved in regulation of P-gp expression. Retinoic acid nuclear receptors are involved in regulating expression of a large number of different proteins. Several authors have described that all-trans retinoic acid (ATRA, ligand of retinoic acid receptors, RARs) may induce alterations in P-gp expression and/or activity in drug resistant malignant cell lines. There are also other nuclear receptors for retinoids--retinoid X receptors (RXRs)--that may be involved in the development of the P-gp-mediated MDR phenotype. The topic of the present paper is a study of the relationship, if any, between the regulatory pathways of nuclear receptors for retinoids and P-glycoprotein expression. Increased levels of mRNAs encoding the retinoic acid nuclear receptors RARalpha and gamma, as well as decreased levels of the mRNAs encoding RARbeta and the retinoid X receptor RXRgamma or slightly decreased levels of RXRbeta mRNA, were observed in L1210/VCR cells in comparison with parental L1210 cells. Neither L1210 cells nor L1210/VCR cells contained measurable amounts of mRNA encoding the RXRalpha receptor. ATRA did not influence the viability of L1210/VCR cells differently from L1210 cells. A combined treatment of L1210/VCR cells with vincristine (1.08 micromol/l) and ATRA induced slightly higher cell death than that observed with ATRA alone. When applied alone, ATRA did not influence P-gp expression (monitored by anti P-gp antibody c219 using western blot analysis) or transport activity (monitored by use of calcein/AM as a P-gp substrate by FACS) in L1210/VCR cells. In contrast, when ATRA was applied together with verapamil (an often used P-gp inhibitor), a significant decrease in P-gp expression and transport activity were observed. However, no significant differences in [11, 12-(3)H]-ATRA uptake were observed in either sensitive or resistant cells, in the latter case in the absence or presence of vincristine. Moreover, verapamil did not influence ATRA uptake under any conditions. Thus, we can conclude that the combined treatment of L1210/VCR cells with ATRA and verapamil is able to depress P-gp expression, and consequently its activity. ATRA is not a P-gp-transportable substance, and thus this effect could not be attributed to verapamil-induced inhibition of P-gp that would allow ATRA to reach retinoic acid nuclear receptors and activate them.