scholarly journals Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

2019 ◽  
Vol 9 (23) ◽  
pp. 5170 ◽  
Author(s):  
G.M. Azevedo ◽  
J.F.A. Valente ◽  
A. Sousa ◽  
A.Q. Pedro ◽  
P. Pereira ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Plasmid Dna ◽  
P53 Protein ◽  
Metabolic Stress ◽  
Suppressor Gene ◽  
Experimental Conditions ◽  
P53 Expression ◽  
Supercoiled Plasmid ◽  
Protein P53

The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.

Effect of suppression of bcl-2 with antisense oligonucleotides on p53 expression.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e16085-e16085
Author(s):  
Patrick Guinan ◽  
Marvin Rubenstein ◽  
Courtney M.P. Hollowell
Keyword(s):  
Suppressor Gene ◽  
Background Intensity ◽  
Tumor Resistance ◽  
Lncap Cells ◽  
Rt Pcr ◽  
Compensatory Mechanisms ◽  
P53 Expression ◽  
Regulatory Pathways ◽  
Antisense Oligos

e16085 Background: In theory gene therapy is specific but difficulties are encounted in practice. Tumors express altered patterns of expression and regulatory pathways provide many targets. However the actual activity of most genes are similar to normal. Resistance develops because pathways are complex, regulated by stimulatory and inhibitory factors, each affected by therapy. Tumors alter dependence on targeted gene products for growth by relying upon others, through compensation. Antisense oligos have targeted regulatory proteins in both in vivo and in vitro prostate cancer models. Cells treated with antisense directed against bcl-2 compensated by suppressing caspase-3 (an apoptosis promoter) and enhancing androgen receptor (AR), (co-activating) p300 and IL-6 expression. This suggests that in LNCaP a progression to increased androgen sensitivity accompanies bcl-2 suppression with a pattern of co-activation associated with more advanced prostate tumors. Methods: We evaluated mono- and bispecific oligos which targeted and equally suppressed bcl-2 expression in LNCaP cells. To further evaluate compensatory mechanisms related to tumor resistance we evaluated the level of the suppressor gene p53 employing RT-PCR and agarose gel quantification. Bands representing pcr product were photographed, converted to black and white and assessed by MIPAV software. Results: Comparable amounts of RNA from LNCaP cells treated with either mono- or bispecific oligos directed against bcl-2 (and EGFR in the bispecifics) were evaluated by RT-PCR using primers directed against p53. When background intensity was subtracted, the relative intensity of all bands corresponding to p53 and representing cells treated with MR4, MR24 and MR42 (compared to controls) were respectively increased 47.5% ± 42.3 (p = 0.02), 86.5% ± 35.3 (p = 0.00013) and 58.0% ± 42.0 (p = 0.0077). These results were pooled from both duplicate PCR runs and gels, and indicate each oligo targeting bcl-2 significantly enhanced p53 expression. Conclusions: Inhibition of bcl-2 activity mediated via antisense oligos enhances suppressor gene p53 expression. In contrast to the effects noted above, p53 enhancement would appear beneficial.

scholarly journals Perkembangan Terapi Kanker Terkait Senyawa Terpineol, P53 dan Caspase 3

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Maya E. W. Moningka
Keyword(s):  
Aspartic Acid ◽  
Cancer Cell ◽  
Anticancer Drug ◽  
Caspase 3 ◽  
Suppressor Gene ◽  
Acid Protease ◽  
Caspase 8 ◽  
Anticancer Activities ◽  
P53 Expression

Abstract: Recent anticancer drug development aims to molecular aspect with more specific target without harming healthy cells. Natural resources have been providing promising new anticancer drugs. Terpineol, an essential oil, is one of the anti-breast cancer candidates. Terpineol can be made from turpentine, which is non-wood product of pine tree latex. Alpha-terpineol isolated from terpineol has an anticancer potency and has been proven to inhibit the growth and induce cancer cell death in vitro by inhibiting NF-κB. P53 is a tumor suppressor gene which triggers apoptosis when irreparable DNA damage occurs. Activity of p53 can be altered and/or inhibited by mutation and inactivation of other oncogenes. The main mechanism underlying apoptosis is caspase (cysteine aspartic acid protease) activity. One of the caspases responsible for apoptosis is caspase 3. This caspase 3 can be activated by either intrinsic (mitochondrial signaling) or extrinsic (death ligand) mechanism; the latter involves caspase 8 and 9. Activated caspase 3 will execute the apoptosis inside the cells. Cytotoxic activity of α-terpineol and its involvement in apoptosis, p53 expression, and caspase 3 activities in cancer cell cultures are still being investigated to determine their anticancer activities and the possibility of anticancer drug development.Keywords: cancer therapy, terpineol, p53, caspase-3 Abstrak: Pengembangan obat antikanker saat ini lebih ditujukan pada aspek molekuler dengan adanya target terapi yang lebih spesifik sehingga lebih aman untuk sel-sel tubuh yang normal. Dewasa ini, eksplorasi terhadap bahan alam untuk kandidat obat antikanker semakin dilirik. Minyak esensial terpineol merupakan salah satu bahan pada komposisi obat antikanker payudara. Terpineol dapat dibuat dari terpentin yang merupakan hasil hutan non kayu dari pohon pinus, dengan cara mengambil getahnya. Dari terpineol diisolasi senyawa α-terpineol yang berpotensi sebagai antikanker serta telah terbukti dapat menghambat pertumbuhan dan menginduksi kematian sel tumor melalui mekanisme yang melibatkan inhibisi aktivitas NFкB. Gen p53 merupakan gen tumor supresor yang memicu terjadinya suatu kematian sel atau apoptosis bila terdapat kerusakan DNA dalam upayanya untuk mengatur proliferasi sel. Selain karena adanya mutasi gen p53, inaktivasi dapat terjadi oleh overekspresi onkogen yang nantinya berikatan dengan p53 dan menghambat kerja gen tersebut. Mekanisme utama yang juga mendasari terjadinya apoptosis ialah aktivitas cysteine aspartic acid protease (caspase). Salah satu caspase yang berperan dalam menginduksi apoptosis ialah caspase 3. Caspase ini dapat diaktifkan melalui mekanisme intrinsik (jalur mitokondrial) maupun ekstrinsik (death ligand), dengan bantuan caspase 8 dan caspase 9. Bila caspase 3 teraktifkan maka sebagai caspase eksekutor, akan melakukan tugasnya untuk mengapoptosis sel. Kajian aktivitas sitotoksik senyawa α-terpineol terhadap suatu cell line, pengaruh senyawa tersebut terhadap proses apoptosis, ekspresi p53, dan aktivitas caspase 3 pada berbagai macam kanker masih terus diteliti dalam perkembangannya sebagai obat anti kanker.Kata kunci: terapi kanker, terpineol, p53, caspase-3

scholarly journals Anti-apoptosis mechanism of triptolide based on network pharmacology in focal segmental glomerulosclerosis rats

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Yayu Li ◽  
Xue Jiang ◽  
Litao Song ◽  
Mengdie Yang ◽  
Jing Pan
Keyword(s):  
Focal Segmental Glomerulosclerosis ◽  
Cell Apoptosis ◽  
P53 Protein ◽  
Network Pharmacology ◽  
Tripterygium Wilfordii ◽  
P53 Expression ◽  
Chemical Structures ◽  
Segmental Glomerulosclerosis

Abstract Triptolide (TPL), the active component of Tripterygium wilfordii, exhibits anti-cancer and antioxidant functions. We aimed to explore the anti-apoptosis mechanism of TPL based on network pharmacology and in vivo and in vitro research validation using a rat model of focal segmental glomerulosclerosis (FSGS). The chemical structures and pharmacological activities of the compounds reported in T. wilfordii were determined and used to perform the network pharmacology analysis. The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) was then used to identify the network targets for 16 compounds from Tripterygium wilfordii. Our results showed that 47 overlapping genes obtained from the GeneCards and OMIM databases were involved in the occurrence and development of FSGS and used to construct the protein–protein interaction (PPI) network using the STRING database. Hub genes were identified via the MCODE plug-in of the Cytoscape software. IL4 was the target gene of TPL in FSGS and was mainly enriched in the cell apoptosis term and p53 signaling pathway, according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. TPL inhibited FSGS-induced cell apoptosis in rats and regulated IL4, nephrin, podocin, and p53 protein levels via using CCK8, TUNEL, and Western blot assays. The effects of IL4 overexpression, including inhibition of cell viability and promotion of apoptosis, were reversed by TPL. TPL treatment increased the expression of nephrin and podocin and decreased p53 expression in rat podocytes. In conclusion, TPL inhibited podocyte apoptosis by targeting IL4 to alleviate kidney injury in FSGS rats.

scholarly journals SAT-124 Optimization of Experimental Conditions for Mimicking Hypoxia in Cultured Breast Cancer Cells by Using Cobalt(II) Chloride (CoCl2)

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Karin Chen ◽  
Leo Satlof ◽  
Udithi Kothapalli ◽  
Noah Ziluck ◽  
Maribel Lema ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Apoptosis ◽  
Breast Cancer Cells ◽  
Nuclear Translocation ◽  
Breast Cancer Cell Lines ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Experimental Conditions

Abstract Hypoxia is a common phenomenon in solid tumor development caused by a decrease in either oxygen concentration or oxygen pressure as a result of rapid tumor cell growth. Hypoxia is characterized by stabilization of the alpha subunit of the hypoxia-inducible factor (HIF-1α) and its nuclear translocation and heterodimerization with HIF-1β. Activation of this signaling pathway involves multiple downstream effectors including carbonic anhydrase 9 (CA9, s. CAIX). A reliable method to mimic hypoxia utilizes cobalt(II) chloride (CoCl2), which directly induces the expression of HIF-1α. The aim of this study was to optimize the experimental conditions for CoCl2 treatment of breast cancer cells in vitro using three human breast cancer cell lines (MDA-MB-231, T-47D, and MCF-7 cells). We performed time- and concentration-response experiments, using various concentrations of CoCl2 (50, 100, 200, and 300 μM) for 24 and 48 hours, and measured the expression of HIF-1α and CA9 by qRT-PCR and Western blot analyses. Results demonstrated that CoCl2 downregulated HIF-1α mRNA levels but upregulated CA9 mRNA levels in a concentration- and time-dependent manner. Concomitantly, CoCl2 treatment resulted in a significant induction of HIF-1α protein levels. We further investigated the effect of the CoCl2 concentrations listed above on cell apoptosis using an in situ apoptosis detection kit. The results demonstrated that concentrations of CoCl2 up to 100 μM had no significant effect on cell apoptosis.

An oral second-generation proteasome inhibitor oprozomib significantly inhibits lung cancer in a p53 independent manner in vitro

2019 ◽  
Vol 51 (10) ◽  
pp. 1034-1040 ◽  
Author(s):  
Hongge Zhu ◽  
Tianhai Wang ◽  
Zhou Xin ◽  
Yiyi Zhan ◽  
Guoming Gu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Anticancer Agents ◽  
Proteasome Inhibitor ◽  
Second Generation ◽  
26S Proteasome ◽  
Lung Cancer Cells ◽  
Independent Manner ◽  
P53 Expression

Abstract The destruction of proteins via the ubiquitin–proteasome system is a multi-step, complex process involving polyubiquitination of substrate proteins, followed by proteolytic degradation by the macromolecular 26S proteasome complex. Inhibitors of the proteasome promote the accumulation of proteins that are deleterious to cell survival and are promising anticancer agents. Oprozomib (OPZ), an oral second-generation proteasome inhibitor, has been shown to inhibit the growth of several cancers in preclinical and clinical trials, including multiple myeloma and head and neck cancers, but its effects on lung cancer has not yet been determined. In this study, we evaluated the inhibitory effects of OPZ on lung cancer cell lines in vitro. The results showed that OPZ significantly suppressed cell proliferation and strongly induced apoptosis in both tested lung cancer cells independent of p53 expression. OPZ was able to cause obvious caspase 3 and PARP cleavages and stabilize p53 and its transcriptional targets p21, PUMA, and Noxa. Moreover, OPZ was capable of sensitizing lung cancer cells to the conventional chemotherapeutic drug cisplatin. Our study provides preclinical data and sheds light on the potential applications of proteasome inhibitor OPZ in lung cancer treatment.

scholarly journals KAI-1 and p53 expression in odontogenic cysts: An immunohistochemical (IHC) marker study

2017 ◽  
Vol 04 ◽  
pp. 1
Author(s):  
Namrata N. Patil ◽  
Vijay Wadhwan ◽  
Minal Chowdhary ◽  
Abhishek Singh Nayyar ◽  
◽  
...  
Keyword(s):  
P53 Protein ◽  
Suppressor Gene ◽  
Tumour Suppressor Gene ◽  
Odontogenic Cysts ◽  
P53 Expression ◽  
Odontogenic Keratocysts ◽  
P53 Antibodies ◽  
Dentigerous Cysts ◽  
Protein Functions ◽  
Marker Study

Background: KAI-1/CD82 is a tumour suppressor gene; decreased gene expression is associated with the increased invasive ability of oral squamous cell carcinoma (OSCC), as hypothesised for various odontogenic cysts and tumours. p53 protein functions in the G1-S phase of the cell cycle to allow repair of the damaged DNA. In the present study, p53 and KAI-1 expression was investigated by using monoclonal antibodies in the various odontogenic cysts. Aims: To detect KAI-1 and p53 expression in radicular cysts, dentigerous cysts and odontogenic keratocysts (OKCs) and to assess the relation between p53 and KAI-1 expression in the aforementioned cysts. Materials and Methods: The present study included histopathologically diagnosed cases of radicular cysts, dentigerous cysts and OKCs for the expression of KAI-1 and p53 antibodies. Results: Amongst odontogenic cysts, radicular cysts expressed a maximum positivity of KAI-1 (20.92%) while p53 positive cells were maximum in odontogenic keratocysts (4.04%). The correlation between KAI-1 and p53 expression in the various odontogenic cysts was not found to be significant. Conclusion: The increased KAI-1 expression in the radicular cysts and its downregulation in OKCs may be indicative of aggressive clinical behaviour and the fact that OKCs are hypothesised as neoplastic rather than being developmental in origin.

scholarly journals Dynamic variability in apoptotic threshold as a strategy for combating fractional killing

2018 ◽  
Author(s):  
Baohua Qiu ◽  
Jiajun Zhang ◽  
Tianshou Zhou
Keyword(s):  
Cancer Cells ◽  
First Passage Time ◽  
P53 Protein ◽  
P53 Gene ◽  
Threshold Level ◽  
Passage Time ◽  
Genetic Mutation ◽  
P53 Expression ◽  
Apoptosis Inducing Agents ◽  
Cell Variation

AbstractFractional killing, which is a significant impediment to successful chemotherapy, is observed even in a population of genetically identical cancer cells exposed to apoptosis-inducing agents. This phenomenon arises not from genetic mutation but from cell-to-cell variation in the activation timing and level of the proteins that regulate apoptosis. To understand the mechanism behind the phenomenon, we formulate complex fractional killing processes as a first-passage time (FPT) problem with a stochastically fluctuating boundary. Analytical calculations are performed for the FPT distribution in a toy model of stochastic p53 gene expression, where the cancer cell is killed only when the p53 expression level crosses an activity apoptotic threshold. Counterintuitively, we find that threshold fluctuations can effectively enhance cellular killing by significantly decreasing the mean time that the p53 protein reaches the threshold level for the first time. Moreover, faster fluctuations lead to the killing of more cells. These qualitative results imply that dynamic variability in threshold is an unneglectable stochastic source, and can be taken as a strategy for combating fractional killing of cancer cells.

Immunologically distinct p53 molecules generated by alternative splicing

1986 ◽  
Vol 6 (9) ◽  
pp. 3232-3239
Author(s):  
N Arai ◽  
D Nomura ◽  
K Yokota ◽  
D Wolf ◽  
E Brill ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Cdna Clone ◽  
P53 Protein ◽  
Lymphoid Cells ◽  
Antigenic Determinants ◽  
Cdna Clones ◽  
P53 Expression ◽  
Mrna Species

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

Rosemary Extract and Its Biogenic Silver Nanoparticles Induce Apoptosis and Arrest Cell Cycle in HT-29 Colon Cancer Cells

2021 ◽  
Vol 13 (1) ◽  
pp. 36-49
Author(s):  
Essam H. Ibrahim ◽  
Ali Alshehri ◽  
Hamed A. Ghramh ◽  
Mona Kilany ◽  
Kareem Morsy ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Silver Nanoparticles ◽  
Cancer Cells ◽  
P53 Protein ◽  
Acetone Extract ◽  
Rosemary Extract ◽  
Minute Amount ◽  
P53 Expression ◽  
Dividing Cells ◽  
Ht 29

Colorectal malignancy is a significant reason of morbidity and mortality. Rosmarinus officinalis is a medicinal plant and used as diet. Metal nanoparticles are utilized in various fields. This study aimed to investigate if R. officinalis leaves acetone extract (ROLAExt) can kill the abnormally fast dividing cells (cancer) and not normal fast diving cells (proliferation-activated) aided by nanoparticles. Silver nanoparticles (AgNPs) were prepared using ROLAExt and characterized with UV/Vis spectrophotometry, XRD and SEM. Functional groups found in the ROLAExt and ROLAExt containing AgNPs (ROLAExt + AgNPs) were explored using FTIR. Sugars, ROS and proteins in ROLAExt were investigated. Biological properties of ROLAExt and ROLAExt + AgNPs including cell cycle arrest, antibacterial, induction of p53 protein, and apoptotic capacity properties were tested. Results demonstrated that AgNPs are of 75 nm in diameter. There is active biomolecules, minute amount of sugars, no protein bands, and 175.2 pg/mL ROS in ROLAExt. AgNPs increased the antibacterial activity, p53 expression, apoptosis, arrestment of HT-29 cells at G2/M phase and did not affect the fast dividing cells. ROLAExt and ROLAExt + AgNPs were safe to the vital organs. In conclusion, R. officinalis acetone extract showed excellent effects on HT-29 cancer cells, but in the presence of AgNPs killed cancer cells and stimulated splenic cells.

Impact of combined MRP1 inhibition and mutant p53-targeting compound APR-246.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14712-e14712
Author(s):  
Anna Sophia Ceder ◽  
Sofi E. Eriksson ◽  
Emarndeena Haji Cheteh ◽  
Vladimir J.N. Bykov ◽  
Lars Abrahmsen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Cultured Cells ◽  
P53 Protein ◽  
Tumor Development ◽  
Glutamate Transporter ◽  
Suppressor Gene ◽  
Phase Iii ◽  
Mutant P53 ◽  
Phase Ii Studies

e14712 Background: The tumor suppressor gene TP53 is the most frequently mutated gene in cancer. Mutant p53 protein is often expressed at high levels and accompanied with gain-of-function activities that promote tumor development and resistance towards conventional treatment. APR-246 is a mutant p53-reactivating small molecule undergoing a Phase III clinical study in myelodysplastic syndrome (MDS), and several phase II studies. APR-246 is non-enzymatically converted to its active product methylene quinuclidinone (MQ) which binds to cysteine residues in p53. This stimulates proper folding of p53's DNA-binding core domain, leading to cell death. APR-246 also exhibits pro-oxidant activity as the electrophile MQ binds and inactivates important antioxidants such as glutathione and thioredoxin reductase, which both are essential for cellular defense against oxidative and electrophilic stress. Methods: All results are in vitro experiments by LC-MS as well as experiments in cultured cells, including 14C-APR-246/MQ detection, cell viability measurements, LC-MS analysis, enzyme recycling measurements and Western blotting. Results: We have utilized 14C-labelled APR-246 to investigate the effect of APR-246 and its active moiety MQ in tumor cell cultures upon inhibition of efflux transporter multidrug resistance protein 1 (MRP1) or cystine-glutamate transporter (xCT). Transient downregulation or small molecule inhibitors (MK-571, reversan, sulfasalazin) targeting MRP1 or xCT increased drug content and shifted intra- and extracellular thiol status. Missense mutant TP53-carrying cells exhibited higher sensitivity to APR-246 and combination therapies. MRP1 inhibition showed strong synergy with APR-246 and increased intracellular levels of MQ conjugated to glutathione (GS-MQ). We found that GS-MQ conjugate formation is reversible, and suggest that the intrinsic reversibility of MQ adduct formation is an important aspect of the mechanism of action of APR-246. This reversibility may also in part account for the benign safety profile reported from clinical studies with APR-246. Conclusions: Sensitivity to APR-246 is affected by MRP1 efflux activity and the redox status, reflected in ratios of cysteine/cystine and reduced/oxidized glutathione (GSH/GSSG).

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