Resistance of Leukemia Cells to 5-Azacytidine: Different Responses to the Same Induction Protocol.

Three AML cell variants (M/A, M/A* from MOLM-13 and S/A from SKM-1) were established for resistance by the same protocol using 5-azacytidine (AZA) as a selection agent. These AZA-resistant variants differ in their responses to other cytosine nucleoside analogs, including 5-aza-2'-deoxycytidine (DAC), as well as in some molecular features. Differences in global DNA methylation, protein levels of DNA methyltransferases, and phosphorylation of histone H2AX were observed in response to AZA and DAC treatment in these cell variants. This could be due to changes in the expression of uridine-cytidine kinases 1 and 2 (UCK1 and UCK2) demonstrated in our cell variants. In the M/A variant that retained sensitivity to DAC, we detected a homozygous point mutation in UCK2 resulting in an amino acid substitution (L220R) that is likely responsible for AZA resistance. Cells administered AZA treatment can switch to de novo synthesis of pyrimidine nucleotides, which could be blocked by inhibition of dihydroorotate dehydrogenase by teriflunomide (TFN). This is shown by the synergistic effect of AZA and TFN in those variants that were cross-resistant to DAC and did not have a mutation in UCK2.

Different mechanisms of drug resistance to hypomethylating agents in the treatment of myelodysplastic syndromes and acute myeloid leukemia.

Resistance to the hypomethylating agents (HMAs) 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) represents a major obstacle in the treatment of elderly patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) which are not suitable for hematopoietic stem cells transplantation. Approximately 50 % of patients do not respond to HMA treatment because of intrinsic (primary) resistance, while others could acquire drug resistance during the repeated cycles of the treatment. To prevent, delay or surmount resistance development, the molecular mechanisms underlying drug resistance must be first identified. This is crucial as no further standard therapeutic opportunities are available for these patients who failed hypomethylating agents-based treatment. The current review provides an updated information about the different mechanisms that may contribute to the development of resistance to HMAs. Despite the similar structure and mechanism of action of HMA, several studies did not report the expected development of cross-resistance. It is clear that in addition to the common modalities of chemoresistance, there must be some specific mechanisms of drug resistance. Changes in transport and metabolism of HMAs are among the most studied mechanisms of resistance. Drug uptake provided by two solute carrier (SLC) families: SLC28 and SLC29 (also known as the concentrative and equilibrative nucleoside transporter families, respectively), could represent one of the mechanisms of cross-resistance. Changes in the metabolism of these drugs are the most likely mechanism responsible for the unique mode of resistance to AZA and DAC. Deoxycytidine kinase and uridine-cytidine kinase due to their necessity for drug activation, each could represent one of the response markers to treatment with DAC and AZA, respectively. Other mechanisms involved in the development of resistance common for both drugs involved: i. increased DNA repair (caused for example by constitutive activation of the ATM/BRCA1 pathway and inhibition of p53-dependent apoptosis); ii. changes in the regulation of apoptosis/disrupted apoptotic pathways (specifically increased levels of the anti-apoptotic protein BCL2) and iii. increased resilience of leukemic stem cells to multiple drugs including HMAs. Despite intense research on the resistance of MDS and AML patients to HMAs, the mechanisms that may reduce the response of these cells to HMAs are not known in detail. We herein highlight the most important directions that future research should take.

Highly stereocontrolled total synthesis of racemic codonopsinol B through isoxazolidine-4,5-diol vinylation.

A new highly diastereoselective synthesis of the polyhydroxylated pyrrolidine alkaloid (±)-codonopsinol B and its N-nor-methyl analogue, starting from achiral materials, is presented. The strategy relies on the trans-stereoselective epoxidation of 2,3-dihydroisoxazole with in situ-generated DMDO, the syn-selective α-chelation-controlled addition of vinyl-MgBr/CeCl3 to the isoxazolidine-4,5-diol intermediate, and the substrate-directed epoxidation of the terminal double bond of the corresponding γ-amino-α,ß-diol with aqueous hydrogen peroxide catalyzed by phosphotungstic heteropoly acid. Each of the key reactions proceeded with an excellent diastereoselectivity (dr > 95:5). (±)-Codonopsinol B was prepared in 10 steps with overall 8.4% yield. The antiproliferative effect of (±)-codonopsinol B and its N-nor-methyl analogue was evaluated using several cell line models.

Development of Multidrug Resistance in Acute Myeloid Leukemia Is Associated with Alterations of the LPHN1/GAL-9/TIM-3 Signaling Pathway.

P-glycoprotein (known as ABCB1 transporter) expression in myeloid blasts of acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) leads to the commonly observed multidrug resistance. Overexpression of latrophilin-1 was detected in leukemic cells from AML patients. In a previous study, we showed that ABCB1 overexpression is associated with decreased latrophilin-1 expression in MOLM-13/VCR and SKM-1/VCR AML cell variants derived from MOLM-13 and SKM-1 cells by vincristine selection/adaptation. In the present study, we found that if ABCB1 overexpression occurs in myeloid blasts of newly diagnosed MDS patients, latrophilin-1 expression is attenuated. Latrophilin-1 may initiate TIM-3- and galectin-9-mediated immune escape. We demonstrated changes in the expression of both proteins by comparing ABCB1-positive cell variants (MOLM-13/VCR, SKM-1/VCR) with their ABCB1-negative counterparts. Galectin-9 was present in our cell lines in eight protein isoforms for which we identified the respective transcription variants resulting from alternative splicing, and we verified their structure by sequencing. The isoform profile of galectin-9 was different between ABCB1-positive and ABCB1-negative cell variants. The interaction partner of galectin-9 is CD44, and its expression was altered in the ABCB1-positive variants MOLM-13/VCR and SKM-1/VCR compared to their ABCB1-negative counterparts.

Changes in Apoptotic Pathways in MOLM-13 Cell Lines after Induction of Resistance to Hypomethylating Agents.

We established the following two variants of the MOLM-13 human acute myeloid leukemia (AML) cell line: (i) MOLM-13/DAC cells are resistant to 5-aza-2'-deoxycytidine (DAC), and (ii) MOLM-13/AZA are resistant to 5-azacytidine (AZA). Both cell variants were obtained through a six-month selection/adaptation procedure with a stepwise increase in the concentration of either DAC or AZA. MOLM-13/DAC cells are resistant to DAC, and MOLM-13/AZA cells are resistant to AZA (approximately 50-fold and 20-fold, respectively), but cross-resistance of MOLM-13/DAC to AZA and of MOLM-13/AZA to DAC was not detected. By measuring the cell retention of fluorescein-linked annexin V and propidium iodide, we showed an apoptotic mode of death for MOLM-13 cells after treatment with either DAC or AZA, for MOLM-13/DAC cells after treatment with AZA, and for MOLM-13/AZA cells after treatment with DAC. When cells progressed to apoptosis, via JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide) assay, we detected a reduction in the mitochondrial membrane potential. Furthermore, we characterized promoter methylation levels for some genes encoding proteins regulating apoptosis and the relation of this methylation to the expression of the respective genes. In addition, we focused on determining the expression levels and activity of intrinsic and extrinsic apoptosis pathway proteins.

The expression of P-glycoprotein in leukemia cells is associated with the upregulated expression of nestin, a class 6 filament protein.

Multidrug resistance (MDR) is a serious obstacle to the effective chemotherapeutic treatment of leukemia. Expression of plasma membrane P-glycoprotein (P-gp), a transporter involved in drug efflux, is the most frequently observed molecular causality of MDR. We observed the coexpression of P-gp and the filament protein nestin in the acute myeloid leukemia (AML) cell lines SKM-1 and MOLM-13 following the induction of P-gp expression using vincristine. Nestin is considered a marker of neural stem cells and neural progenitor cells. The aim of this study was to determine whether there is causal relationship between the expression of P-glycoprotein and the expression of nestin in both of these AML cell lines. The expression of P-gp was induced in SKM-1 cells by selective pressure using vincristine (VCR), mitoxantrone (MTX), azacytidine (AzaC) and lenalidomide (LEN). Whereas the selective pressure of VCR, MTX and AzaC also induced P-gp expression in MOLM-13 cells, LEN was found to be ineffective in this regard. In all cases in which P-gp expression was induced in SKM-1 and MOLM-13 cells, its expression was associated with the induction of nestin mRNA expression and the presence of a 200-220kDa nestin-immunoreactive protein band in western blots. Silencing P-gp expression using s10418 siRNA (known as the P-gp silencer) was associated with the downregulation of the nestin transcript level, demonstrated using RT-PCR. Nestin mRNA was also observed in two P-gp-positive variants of L1210 cells that were obtained either by selection with VCR or by transfection with a retrovirus encoding human P-gp. Detectable levels of nestin transcripts were not observed in P-gp-negative parental L1210 cells. Taken together, these results indicated that the induction of P-gp expression is causally associated with the expression of nestin in leukemia cells.

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.

Acute myeloid leukemia cells MOLM-13 and SKM-1 established for resistance by azacytidine are crossresistant to P-glycoprotein substrates.

Establishment of the acute myeloid leukemia cells SKM-1 and MOLM-13 for resistance by azacytidine (AzaC) resulted in SKM-1/AzaC and MOLM-13/AzaC cell variants with reduced sensitivity to AzaC. Despite the fact that AzaC is not substrate of P-glycoprotein (P-gp), the adaptation procedure resulted in an induction in P-gp expression/efflux activity that confers crossresistance to P-gp substrates in both resistant cell variants. While the resistance to P-gp substrates in SKM-1/AzaC and MOLM-13/AzaC cells could be reversed by the P-gp inhibitors, resistance to AzaC was insensitive to these inhibitors in both resistant cell variants. In addition, NF-κB and the antiapoptotic protein Bcl-2 were downregulated and the proapoptotic proteins Bax and p53 were upregulated in both resistant cell variants when compared with their sensitive counterparts. Moreover, at least five times the elevation in overall glutathione S-transferase activity was measured with 1-chloro-2, 5-dinitrobenzene as a substrate in the resistant variant of both cell lines. Taken together, the findings of the present study indicate that the treatment of AML cells with AzaC might lead to a drug resistance phenotype that may be associated with cross resistance to P-gp substrates and substrates of glutathione S-transferases.

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).

Reduced UDP-glucose Levels Are Associated with P-glycoprotein Over-expression in L1210 Cells and Limit Glucosylceramide Synthase Activity.

BACKGROUND/AIM: P-glycoprotein (Pgp) expression in neoplastic cells is known to reduce cell sensitivity to several cytotoxic Pgp substrates. A member of the ABC transporter family, Pgp, represents the most frequently described membrane efflux pump and its expression in neoplastic cells is responsible for multi-drug resistance. Several lines of evidence indicate that the expression and increased function of both Pgp and glucosylceramide synthase (GCS, an enzyme responsible for ceramide pathway de-activation in the regulation of apoptosis progression) enhance the resistance of Pgp-positive cells. Previously, we described a reduction in the uridine diphosphate (UDP)-glucose contents of mouse leukemia cells (R) expressing Pgp due to vincristine selection compared to parental L1210 cells (S). The reduced availability of UDP-glucose as a glucose donor in R cell glycosylation reactions could limit GCS-catalyzed ceramide glycosylation. Consequently, the over-expression of Pgp in Pgp-positive L1210 cells may be associated with reduced ceramide glycosylation. MATERIALS AND METHODS: To test this idea, we measured the expression and activities of Pgp and GCS, UDP-glucose levels, cellular uptake of C12-NBD-ceramide (a fluorescent analogue of ceramide) and ceramide-induced cell death in S and R cells. T-cells, another Pgp-positive variant of L1210 cells that express Pgp due to their transfection with a gene encoding human Pgp were also used in this study. RESULTS: We detected significantly reduced levels of C12-NBD-ceramide glycosylation and reduced UDP-glucose contents in Pgp-positive R and T-cells compared to S cells. C12-NBD-ceramide uptake assays revealed nearly identical dynamics of uptake time-dependency curves. The Pgp-positive L1210 variants (R and T) are more sensitive than Pgp-negative S cells to ceramide-induced cell damage, as measured by an fluorescein isothiocyanate-labeled annexin V and propidium iodide apoptosis necrosis kit. Short chain C2-ceramide was more effective at inducing cell damage than ceramide analogues with longer chains. CONCLUSION: These evidence indicates that the down-regulation of UDP-glucose contents in Pgp-positive L1210 cells is responsible for their collateral sensitivity to ceramide-induced apoptosis.

Vincristine-induced expression of P-glycoprotein in MOLM-13 and SKM-1 acute myeloid leukemia cell lines is associated with coexpression of nestin transcript.

Nestin is a class 6 filament protein typically expressed in neural stem/progenitor cells. However, nestin expression has been observed in other tissues during mammalian embryogenesis. In human neural stem/progenitor cells, coexpression of nestin and P-glycoprotein (P-gp, ABCB1 member of the ABC transporter family) was detected. P-gp-mediated drug efflux is the most common molecular cause of multidrug resistance in neoplastic cells. Nestin expression has also been detected in various human solid tumours as well as in the corresponding established cell lines. Interestingly, expression of nestin in different leukemia cells has been recently reported. Here, we show that expression of P-gp is associated with the simultaneous expression of nestin in acute myeloid leukemia cell lines (MOLM-13 and SKM-1) under the selective pressure of vincristine, a substance that may induce P-gp expression in neoplastic cells.

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.