Effects of DNA damaging agents on cultured fibroblasts derived from patients with Cockayne syndrome.

The cytotoxic action of physical and chemical agents on 10 skin fibroblast strains in culture derived from individuals with Cockayne's syndrome was measured in terms of colony-forming ability. As compared to fibroblasts from normal donors, all Cockayne cell strains tested exhibited a significantly increased sensitivity to UV light and a normal sensitivity to X-rays. Cells from two sets of parents of unrelated Cockayne children showed an intermediate level of UV sensitivity. There was no effect of 0.5 mM caffeine on UV survival in normal and two Cockayne strains tested, indicating that postreplicational repair in Cockayne cells as measured by caffeine sensitivity was probably normal. Sensitivity of normal and Cockayne cells to the chemical carcinogens and mutagens 4NQO, N-AcO-AAF, ICR-170 and EMS was also compared. An increased sensitivity of Cockayne cells to 4NQO or N-AcO-AAF, but not the ICR-170 or EMS, was observed. However, unlike the intermediate UV sensitivity, the cell strains from two parents of Cockayne patients showed the same sensitivity to N-AcO-AAF or 4NQO as fibroblasts from normal individuals. Quantiation of damage to the DNA after 20 J . m-2 UV irradiation indicates normal levels of [3H] thymidine incorporation in the Cockayne cells, in contrast to UV-irradiated xeroderma pigmentosum cells (XP 12BE) in which there was a very low level of repari synthesis. Moreover, we have shown previously that excision of UV-induced pyrimidine dimers in 2 of the 10 Cockayne cell strains was normal.     

Base pair substitution and frameshift mutagenesis induced by apurinic sites and two fluorene derivatives in a recA441 lexA (Def) strain

Summary One of the consequences of the induction of the Escherichia coli SOS system is the increased ability of the cells to perform mutagenesis. Induction of the SOS system is the result of derepression of a set of genes through a regulatory mechanism controlled by LexA and RecA. In response to an inducing signal, RecA is activated in a form that facilitates the proteolytic cleavage of LexA repressor. Previous works have shown that activated RecA plays a second role, i.e. it is required for the establishment of base pair substitution mutations promoted by UV irradiation. Using a forward mutatonal assay and recA441 lexA(Def) host bacteria, we show that the result can be extended not only to other mutagens promoting base pair substitution mutations (Apurinic sites, Ap sites and N-hydroxy-N-2-aminofluorene, N-OH-AF) but also mutagens promoting frameshift mutations (N-Acetoxy-N-2-acetylaminofluorene, N-AcO-AAF). In the recA441 lexA(Def) strain all the genes which are part of the lexA regulon, including recA itself, are expressed constitutively. The recA441 mutation allows RecA to acquire its activated form when the bacteria are grown at 42° C. We show that in such strains Ap sites or N-OH-AF induce a high level of mutations only when the bacteria are grown at 42° C. On the other hand, we show that N-AcO-AAF can promote mutations even at 30° C; the number of mutations being increased when the bacteria were grown at 42° C. Analysis of the mutants obtained at 30° C indicate that they belong to both type of mutations, UmuC-dependent or UmuC-independent. The much higher ability of N-AcO-AAF to induce RecA as compared to N-OH-AF strongly suggests that the former mutagen is able to induce at least partially the activated form of RexA441 even at 30°C in a strain which overproduces RecA, [lexA(Def)]. Furthermore, we show that the UmuC-independent type of mutagenesis induced by N-AcO-AAF depends on gene(s) that are part of the lexA regulon.     

Comparison of the frequency of diphtheria toxin and thioguanine resistance induced by a series of carcinogens to analyze their mutational specificities in diploid human fibroblasts

The mutagenic specificities of ethylnitrosourea (ENU), X-rays (+/-)7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene (BPDE), ICR-191, and N-acetoxy-2-acetylaminofluorene (N-AcO-AAF) were analyzed and compared in diploid human fibroblasts and Salmonella typhimurium. In the human fibroblasts, we compared the frequency of diphtheria toxin (DT)-resistant mutants, presumably induced in the gene coding for elongation factor-2, with the frequency of 6-thioguanine (TG) resistance induced by mutations in the gene coding for hypoxanthine(guanine)phosphoribosyltransferase (HPRT). Recovery of DT-resistant (DTr) cells requires that the mutant EF-2 retain the ability to carry on protein synthesis since the normal EF-2 will be inactivated by DT selection. Therefore, the DTr mutation cannot involve major changes in the gene. In contrast, cells can acquire TG resistance by any mechanism which eliminates HPRT activity, e.g., base substitution, frameshift, deletion, loss of chromosomes. Each agent was assessed by calculating the ratio of the slopes of the dose-response plots (induced variant frequency as a function of dose of the agent used) for the two markers (DTr/TGr variants.). In S. typhimurium we examined the reversion frequency in four histidine-requiring strains bearing forward mutations of the frameshift (TA1538, TA98) or missense (TA1535, TA100) type. ENU, which was predominantly a base substitution mutagen in the bacteria, gave a ratio of DTr to TGr variants of 1.5. As expected of an agent inducing gross chromosomal changes, X-rays induced no revertants in bacteria and in human cells gave a ratio of 0.1. ICR-191 which was predominantly a frameshift mutagen in bacteria gave a ratio of 0.15. In the set of bacterial strains containing the plasmid pKM101, BPDE reverted both frameshift and base substitution mutations. It did not cause reversions in the other set of strains. In human cells BPDE gave a response similar to ENU, i.e., a ratio of DTr/TGr variants of 1.5. As reported by others, N-AcO-AAF was predominantly a frameshift mutagen in bacteria. However, in the human cells it gave a ratio of DTr/TGr variants of 1.5, similar to ENU and BPDE. These results suggest that in human cells, BPDE and N-AcO-AAF, like ENU, yield predominantly base substitutions, while ICR-191 and X-rays largely produce mutations by mechanisms which result in more extensive alterations in the gene.     

Ability of structurally related polycyclic aromatic carcinogens to induce homologous recombination between duplicated chromosomal sequences in mouse L cells

To investigate the role of DNA damage in the induction of homologous recombination in mammalian cells, a series of structurally related, polycyclic aromatic carcinogens, i.e., 1-nitrosopyrene (1-NOP), N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), and 4-nitroquinoline 1-oxide (4-NQO), were compared for their ability to cause intrachromosomal homologous recombination between two herpes simplex virus thymidine kinase (Htk) genes stably integrated in the genome of a tk- mouse L cell strain 333 M. Each Htk gene contains an 8-bp XhoI linker inserted at a unique site so that expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. Each carcinogen caused a dose-dependent increase in the frequency of recombination. The results were compared to what had been found previously for a structurally related carcinogen, (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). As a function of concentration, BPDE was the most active agent, followed by 4-NQO, and 1-NOP, and then N-AcO-AAF. When compared on the basis of equal cell killing, the most efficient carcinogen was 1-NOP, followed by N-AcO-AAF and BPDE, and then 4-NQO. Use of tritium-labeled compounds to determine the frequency of recombination as a function of the number of adducts initially bound to DNA showed that the most effective agent was BPDE, followed by 1-NOP and 4-NQO, and then N-AcO-AAF (ratio, 6.6:2.5:1.8:1.0). To determine if these differences in recombinagenic effectiveness reflected different rates of removal of the adducts from DNA, we measured the percentage of DNA adducts removed during the 24-h period post treatment and found that 1-NOP, 4-NQO and N-AcO-AAF residues were removed at approximately the same rate, i.e., 25%-30% off. Cellular analysis of a series of independent recombinants indicated that approximately 82% of the recombinational events induced by each agent were consistent with gene conversion. DNA-DNA hybridization analysis confirmed this, and showed that each recombinant tested contained an XhoI-resistant (wild-type) Htk gene; with the majority retaining the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids.     

Plasmodium chabaudi: Genetics of resistance to chloroquine

A high level of chloroquine resistance was developed in the rodent malaria parasite, Plasmodium chabaudi. This resistance was stable and its inheritance was shown to be multigenic; intermediate levels of resistance were obtained from a cross between highly resistant and sensitive parasites. Chloroquine resistance was shown to segregate independently of pyrimethamine resistance and enzyme markers.     

The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism

In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser⁶⁹, confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr⁷⁰⁵) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response.     

C-alkylation of N-acetoxy-N-2-acetylaminofluorene: effects on its reaction with DNA in vitro

In vitro reactions of DNA with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF), N-acetoxy-7-ethyl-N-2-acetylaminofluorene (N-AcO-EtAAF), N-acetoxy-7-n-butyl-N-2-acetylaminofluorene (N-AcO-But-AAF) are compared. C-alkylation of N-AcO-AAF affects the reactivity of the metabolite towards DNA. The electronic effect and the size of alkyl group seem to determine the reactivity of the metabolite. Although the adducts are about the same for the three metabolites, the proportion of guanine-C-8 adducts diminishes with an increase in the size of the alkyl group.     

Ca2+-dependent modulation of antibiotic resistance in Streptomyces lividans 66 and Streptomyces coelicolor A3(2)

The level of resistance to antibiotics of various chemical structure in actinobacteria of the genus Streptomyces is shown to be regulated by Ca²⁺ ions. The inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent serine/threonine protein kinases (STPK) are found to reduce antibiotic resistance of actinobacteria. The effect of Ca²⁺-dependent phosphorylation on the activity of the enzymatic aminoglycoside phosphotransferase system protecting actinobacteria from aminoglycoside antibiotics was studied. It is shown that inhibitors of Ca²⁺/calmodulin and Ca²⁺/phospholipid-dependent STPK reduced the Ca²⁺-induced kanamycin resistance in Streptomyces lividans cells transformed by a hybrid plasmid which contained the aminoglycoside phosphotransferase VIII (APHVIII) gene. In S. coelicolor A3(2) cells, the protein kinase PK25 responsible for APHVIII phosphorylation in vitro was identified. It is suggested that STPK play a major role in the regulation of antibiotic resistance in actinobacteria.     

Phosphorylation of chloramphenicol by a recombinant protein Yhr2 from Streptomyces avermitilis MA4680

Although phosphorylation of chloramphenicol has been shown to occur in the chloramphenicol producer, Streptomyces venezuelae, there are no reports on the existence of chloramphenicol phosphorylase in other Streptomyces species. In the present study, we report the modification of chloramphenicol by a recombinant protein, designated as Yhr2 (encoded by SAV_877), from Streptomyces avermitilis MA4680. Recombinant Yhr2 was expressed in Escherichia coli BL21 (DE3) and the cells expressing this recombinant protein were shown to phosphorylate chloramphenicol to a 3′-O-phosphoryl ester derivative, resulting in an inactivated form of the antibiotic. Expression of yhr2 conferred chloramphenicol resistance to E. coli cells up to 25 μg/mL and in an in vitro reaction, adenosine triphosphate (ATP), guanosine triphosphate (GTP), adenosine diphosphate (ADP) and guanosine diphosphate (GDP) were shown to be the phosphate donors for phosphorylation of chloramphenicol. This study highlights that antibiotic resistance conferring genes could be easily expressed and functionalized in other organisms that do not produce the respective antibiotic.     

Discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) that modulates ABCB1-mediated multidrug resistance (MDR)

Multidrug resistance (MDR) has been shown to reduce the effectiveness of chemotherapy. Strategies to overcoming MDR have been widely explored in the last decades, leading to a generation of numerous small molecules targeting ABC and MRP transporters. Among the ABC family, ABCB1 plays key roles in the development of drug resistance and is the most well studied. In this work, we report the discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) from our structurally diverse in-house compound collection that selectively modulates ABCB1-mediated multidrug resistance. WS-10 enhanced the intracellular accumulation of paclitaxel in SW620/Ad300 cells, but did not affect the expression of ABCB1 Protein and ABCB1 localization. The cellular thermal shift assay (CETSA) showed that WS-10 was able to bind to ABCB1, which could be responsible for the reversal effect of WS-10 toward paclitaxel and doxorubicin in SW620/Ad300 cells. Docking simulations were performed to show the possible binding modes of WS-10 within ABCB1 transporter. To conclude, WS-10 could be used as a template for designing new ABCB1 modulators to overcome ABCB1-mediated multidrug resistance.     

CUT Domains Stimulate Pol β Enzymatic Activities to Accelerate Completion of Base Excision Repair

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase β to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol β in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5′-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.     

Reversal effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone on multi-drug resistance in resistant human hepatocellular carcinoma cell line BEL-7402/5-FU

Multi drug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a chalcone, isolated from the buds of Cleistocalyx operculatus, has been shown to have antitumor effects on human carcinoma SMMC-7721 cells in vitro and in vivo. In this paper, we studied the reversal effect and the mechanism of DMC on human hepatocellular carcinoma drug-resistant cells BEL-7402/5-FU in vitro. Administration of DMC reversed the multi-drug resistance of human hepatocellular carcinoma BEL-7402/5-FU cells significantly. DMC enhanced the sensitivity of BEL-7402/5-FU cells to 5-fluorouracil (5-FU) and doxorubicin (DOX). Staining with Hoechst 33258 and flow cytometric analysis showed that DMC has apoptosis-inducing effect on BEL-7402/5-FU cells. It could also increase the concentration of 5-FU in the resistant multi-drug-resistant cells. We also observed that over-expression of the multi-drug resistance-associated protein (MRP1) and of the glutathione S-transferase π (GST-π) contributed to MDR in BEL-7402/5-FU cells. The mRNA expressions of MRP1 and GST-π and the protein expression of MRP1 were decreased by DMC. These data demonstrated that DMC could effectively reverse MDR in BEL-7402/5-FU cells.     

Temozolomide resistance in glioblastoma cells occurs partly through epidermal growth factor receptor-mediated induction of connexin 43

Glioblastoma Multiforme (GBM) is an aggressive adult primary brain tumor with poor prognosis. GBM patients develop resistance to the frontline chemotherapy, temozolomide (TMZ). As the connexins (Cx) have been shown to have a complex role in GBM, we investigated the role of Cx43 in TMZ resistance. Cx43 was increased in the TMZ-resistant low passage and cell lines. This correlated with the data in The Cancer Genome Atlas. Cx43 knockdown, reporter gene assays, chromatin immunoprecipitation assay, real-time PCR and western blots verified a role for Cx43 in TMZ resistance. This occurred by TMZ-resistant GBM cells being able to activate epidermal growth factor receptor (EGFR). In turn, EGFR activated the JNK-ERK1/2-AP-1 axis to induce Cx43. The increased Cx43 was functional as indicated by gap junctional intercellular communication among the resistant GBM cells. Cell therapy could be a potential method to deliver drugs, such as anti-EGF to tumor cells. Similar strategies could be used to reverse the expression of Cx43 to sensitize GBM cells to TMZ. The studies showed the potential for targeting EGF in immune therapy. These agents can be used in conjunction with stem cell therapy to treat GBM.     

Application of Measurements of Transepithelial Electrical Resistance of Intestinal Epithelial Cell Monolayers To Evaluate Probiotic Activity

Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.     

Further evidence that ouabain-resistant variants induced by chemical carcinogens in transformable C3H/10T1/2 Cl 8 mouse fibroblasts are mutants

Ouabain-resistant (Oua^r) variants were induced in C3H/10T1/2 Cl 8 cells by the chemical carcinogens, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF), and benzo[a]pyrene (BaP). The use of the Poisson calculation to determine Oua^r variant frequencies gave more linear dose-response curves than when variant frequencies were calculated from the observed number of Oua^r colonies. Increasing the Oua concentration from 3 to 6 mM decreased the frequency of Oua^r variants. When cloned Oua^r variants were mixed with wild-type cells, there was no metabolic cooperation and no loss of mutants when mock expression-time curves were determined. Oua^r variants remained Oua^r after prolonged cultivation in the absence of Oua. ⁸⁶Rubidium (⁸⁶Rb) uptake was at least 10-fold more resistant to inhibition by Oua in Oua^r variants than in wild-type cells. In one Oua^r clone, one-third of the ⁸⁶Rb uptake was not inhibited by Oua concentrations as high as 10 mM, indicating that C3H/10T1/2 Cl 8 cells might be triploid at the Oua^r locus. The relationship between the inhibition of ⁸⁶Rb uptake and the cytotoxicity caused by the same concentration of Oua was the same for 2 Oua^r clones and wild-type C3H/10T1/2 Cl 8 cells. Therefore, the Oua^r variants detected by this assay are most likely true mutants possessing an altered Na⁺K⁺ transport system, the Na⁺K⁺Mg²⁺-activated adenosine triphosphate (ATPase), that is more resistant to Oua inhibition than the ATPase in wild-type cells.     

The effect of serum starvation on tight junctional proteins and barrier formation in Caco-2 cells

Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm² but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4–6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm², which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutics.     

Expression of foreign genes in regenerated plants and in their progeny

Chimeric genes comprised of the nopaline synthase promoter and bacterial coding sequences specifying resistance to kanamycin, chloramphenicol or methotrexate, were inserted into the non-oncogenic Ti plasmid vector pGV3850 by recombination (through homologous pBR322 sequences present in the chimeric gene constructs and pGV3850). These co-integrates in Agrobacterium were used to infect single plant protoplasts of Nicotiana by co-cultivation. The resistance traits allowed the selection of transformed calli in tissue culture in the presence of the appropriate antibiotic. Furthermore, as a non-oncogenic Ti plasmid was used for the protoplast transformation, phenotypically normal and fertile plants could be regenerated from the resistant calli. We have shown that these fully differentiated plant tissues exhibit functional expression of resistance traits (Km^R and Cm^R). All plants carrying the chimeric genes developed normally, flowered, and set seeds. The inheritance of several of these resistance traits was analyzed and shown to be Mendelian. These results are model experiments to demonstrate that genes of interest can be systematically transferred to the genome of plants using non-oncogenic Ti plasmid derivatives; and that transformed plants are capable of normal growth and differentiation, thus providing a natural environment for the study of gene expression and development of plant cells.     

Leishmania mexicana: Inhibition of camptothecin-induced apoptosis of monocyte-derived dendritic cells

Macrophages (M?) and dendritic cells (DC) are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a method employed by multiple pathogens to ensure their survival in the infected cell. Leishmania has been shown to protect M? and neutrophils from both natural and induced apoptosis. As shown in this study, apoptosis in monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by coincubation with L. mexicana, as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low molecular weight DNA fragments, and annexin V binding to phosphatidylserine. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in moDC. The capacity of L. mexicana to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.     

The Nuclear Factor (Erythroid-derived 2)-like 2 and Proteasome Maturation Protein Axis Mediate Bortezomib Resistance in Multiple Myeloma

Resistance to the proteasome inhibitor bortezomib is an emerging clinical problem whose mechanisms have not been fully elucidated. We considered the possibility that this could be associated with enhanced proteasome activity in part through the action of the proteasome maturation protein (POMP). Bortezomib-resistant myeloma models were used to examine the correlation between POMP expression and bortezomib sensitivity. POMP expression was then modulated using genetic and pharmacologic approaches to determine the effects on proteasome inhibitor sensitivity in cell lines and in vivo models. Resistant cell lines were found to overexpress POMP, and while its suppression in cell lines enhanced bortezomib sensitivity, POMP overexpression in drug-naive cells conferred resistance. Overexpression of POMP was associated with increased levels of nuclear factor (erythroid-derived 2)-like (NRF2), and NRF2 was found to bind to and activate the POMP promoter. Knockdown of NRF2 in bortezomib-resistant cells reduced POMP levels and proteasome activity, whereas its overexpression in drug-naive cells increased POMP and proteasome activity. The NRF2 inhibitor all-trans-retinoic acid reduced cellular NRF2 levels and increased the anti-proliferative and pro-apoptotic activities of bortezomib in resistant cells, while decreasing proteasome capacity. Finally, the combination of all-trans-retinoic acid with bortezomib showed enhanced activity against primary patient samples and in a murine model of bortezomib-resistant myeloma. Taken together, these studies validate a role for the NRF2/POMP axis in bortezomib resistance and identify NRF2 and POMP as potentially attractive targets for chemosensitization to this proteasome inhibitor.     

Discovery of a non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) that modulates ABCB1-mediated multidrug resistance (MDR)

Multidrug resistance (MDR) has been shown to reduce the effectiveness of chemotherapy. Strategies to overcoming MDR have been widely explored in the last decades, leading to a generation of numerous small molecules targeting ABC and MRP transporters. Among the ABC family, ABCB1 plays key roles in the development of drug resistance and is the most well studied. In this work, we report the discovery of non-toxic [1,2,4]triazolo[1,5-a]pyrimidin-7-one (WS-10) from our structurally diverse in-house compound collection that selectively modulates ABCB1-mediated multidrug resistance. WS-10 enhanced the intracellular accumulation of paclitaxel in SW620/Ad300 cells, but did not affect the expression of ABCB1 Protein and ABCB1 localization. The cellular thermal shift assay (CETSA) showed that WS-10 was able to bind to ABCB1, which could be responsible for the reversal effect of WS-10 toward paclitaxel and doxorubicin in SW620/Ad300 cells. Docking simulations were performed to show the possible binding modes of WS-10 within ABCB1 transporter. To conclude, WS-10 could be used as a template for designing new ABCB1 modulators to overcome ABCB1-mediated multidrug resistance.