Patent Application
20180172712
This document relates to methods and materials for assessing hydrogen peroxide accumulation within cells exposed to one or more test agents. For example, this document provides methods and materials for determining whether or not cancer cells from a mammal accumulate hydrogen peroxide following contact with a test agent.
Multiple myeloma (MM) is a cancer that develops from the malignant proliferation of plasma cells in the bone marrow. MM can be treated with an immunomodulatory drug (IMID), which may be administered alone or in combination with other agents such as proteasome inhibitors.
This document provides methods and materials for assessing hydrogen peroxide accumulation within cells (e.g., cancer cells) exposed to one or more test agents. For example, this document provides methods and materials for determining whether or not cancer cells (e.g., MM cells) from a mammal (e.g., a human) accumulate hydrogen peroxide following contact with a test agent (e.g., an IMID) and exogenous H2O2. As described herein, cancer cells in solution can be exposed to a test agent and exogenous H2O2. Following this exposure, the solution containing the cells can be examined (e.g., visually examined) for the presence, absence, or level of O2 or bubble formation. Those test agents that result in the formation of detectable (e.g., visually detectable) O2 or bubbles can be identified as being a test agent with little or no treatment potential for the mammal from which the cells were obtained. Those test agents that result in the absence of or minimal formation of detectable (e.g., visually detectable) O2 or bubbles can be identified as being a test agent with treatment potential for the mammal from which the cells were obtained. In some cases, a particular IMID such as lenalidomide can be identified as having little or no treatment potential in one human based on the formation of detectable (e.g., visually detectable) O2 or bubbles using that human's cancer cells, while that same IMID (e.g., lenalidomide) can be identified as having treatment potential in a different human based on the lack of formation of detectable (e.g., visually detectable) O2 or bubbles using that different human's cancer cells. Once identified as having treatment potential for a particular mammal (e.g., a particular human), then that identified agent can be administered to that particular mammal to treat cancer within that mammal.
Having the ability to contact cells (e.g., cancer cells) with a test agent and exogenous H2O2 and then to inspect (e.g., inspect visually) the solution containing those cells for evidence of O2 or bubble formation to determine whether or not the test agent results in an accumulation of hydrogen peroxide within the cells can allow clinicians and patients to identify appropriate treatment options (e.g., an effective IMID for that particular patient) in a quick and accurate manner. In some cases, quantification of H2O2 mediated oxidized FAD and NAD(P) can be used in a manner that is more quantitative and accurate to determine cellular anti-oxidative capacity than O2 bubbles.
In general, one aspect of this document features a method for measuring H2O2 accumulation in a cell following exposure to an agent. The method comprises, or consists essentially of, (a) contacting cells in a solution with the agent and exogenous H2O2; and (b) determining whether or not O2 forms in the solution, wherein formation of the O2 in the solution indicates that the agent does not cause accumulation of H2O2 in the cells, and wherein a lack of formation of the O2 in the solution indicates that the agent causes accumulation of H2O2 in the cells. The agent can be an IMID. The IMID can be thalidomide, lenalidomide, pomalidomide, or apremilast. The exogenous H2O2 can be provided in an amount from about 20 μM to about 150 μM. The exogenous H2O2 can be provided in an amount of about 100 μM. The solution can be phosphate buffered saline (PBS). The cells can be cancer cells. The cancer cells can be myelodysplastic syndrome cells, erythema nodosum leprosum cells, multiple myeloma cells, Hodgkin's lymphoma cells, light chain-associated amyloidosis cells, primary myelofibrosis cells, acute myeloid leukaemia cells, prostate cancer cells, or metastatic renal cell carcinoma cells. The determining whether or not O2 forms in the solution can comprise (i) determining whether or not bubbles form in the solution, (ii) determining whether or not autofluorescence of FAD increases, or (iii) determining whether or not autofluorescence of NAD(P)H decreases. The determining step can comprise visually detecting formation of the bubbles.
In another aspect, this document features a method for identifying a cancer treatment agent for a mammal having cancer. The method comprises, or consists essentially of, (a) obtaining cancer cells from the mammal; (b) contacting the cells in a solution with a test agent and exogenous H2O2; and (c) detecting the absence of O2 formation in the solution; wherein the absence of the O2 formation in the solution indicates that the test agent is a potential cancer treatment agent for the mammal. The mammal can be a human. The agent can be an IMID. The IMID can be thalidomide, lenalidomide, pomalidomide, or apremilast. The exogenous H2O2 can be provided in an amount from about 20 μM to about 150 μM. The exogenous H2O2 can be provided in an amount of about 100 μM. The solution can be PBS. The cancer cells can be myelodysplastic syndrome cells, erythema nodosum leprosum cells, multiple myeloma cells, Hodgkin's lymphoma cells, light chain-associated amyloidosis cells, primary myelofibrosis cells, acute myeloid leukaemia cells, prostate cancer cells, metastatic renal cell carcinoma cells, or metastatic renal cell carcinoma cells. The detecting the absence of O2 formation in the solution can comprise (i) detecting the absence of bubble formation in the solution, (ii) detecting the absence of an increase in autofluorescence of FAD, or (iii) detecting the absence of a decrease in autofluorescence of NAD(P)H. The detecting step can comprise visually detecting the absence of formation of the bubbles.
In another aspect, this document features a method for treating cancer, wherein the method comprises, or consists essentially of, (a) obtaining cancer cells from a mammal having cancer; (b) contacting the cancer cells in a solution with an agent and exogenous H2O2; (c) detecting an absence of O2 formation in the solution; and (d) administering the agent to the mammal under conditions wherein the number of cancer cells within the mammal is reduced. The mammal can be a human. The agent can be an IMID. The IMID can be thalidomide, lenalidomide, pomalidomide, or apremilast. The exogenous H2O2 can be provided in an amount from about 20 μM to about 150 μM. The exogenous H2O2 can be provided in an amount of about 100 μM. The solution can be PBS. The cancer can be myelodysplastic syndrome, erythema nodosum leprosum, multiple myeloma, Hodgkin's lymphoma, light chain-associated amyloidosis, primary myelofibrosis, acute myeloid leukaemia, prostate cancer, or metastatic renal cell carcinoma cells. The detecting the absence of O2 formation in the solution can comprise (i) detecting the absence of bubble formation in the solution, (ii) detecting the absence of an increase in autofluorescence of FAD, or (iii) detecting the absence of a decrease in autofluorescence of NAD(P)H. The detecting step can comprise visually detecting the absence of formation of the bubbles.
In another aspect, this document features a method for treating cancer, wherein the method comprises, or consists essentially of, (a) obtaining cancer cells from a mammal having cancer; (b) contacting at least a portion of the cancer cells in a first solution with a first agent and exogenous H2O2; (c) detecting the presence of O2 formation in the first solution; (d) contacting at least a portion of the cancer cells in a second solution with a second agent and exogenous H2O2; (e) detecting the absence of O2 formation in the second solution; and (0 administering the second agent to the mammal under conditions wherein the number of cancer cells within the mammal is reduced. The mammal can be a human. The first agent can be an IMID. The second agent can be an IMID. The IMID can be thalidomide, lenalidomide, pomalidomide, or apremilast. The exogenous H2O2 can be provided in an amount from about 20 μM to about 150 μM. The exogenous H2O2 can be provided in an amount of about 100 μM. The method of claim 31, wherein the first solution can be phosphate buffered saline (PBS), and wherein the second solution is PBS. The cancer can be myelodysplastic syndrome, erythema nodosum leprosum, multiple myeloma, Hodgkin's lymphoma, light chain-associated amyloidosis, primary myelofibrosis, acute myeloid leukaemia, prostate cancer, or metastatic renal cell carcinoma. The detecting the presence of O2 formation in the first solution can comprise (i) detecting the presence of bubble formation in the first solution, (ii) detecting an increase in autofluorescence of FAD in the first solution, or (iii) detecting a decrease in autofluorescence of NAD(P)H in the first solution. The detecting the absence of O2 formation in the second solution can comprise (i) detecting the absence of bubble formation in the second solution, (ii) detecting the absence of an increase in autofluorescence of FAD in the second solution, or (iii) detecting the absence of a decrease in autofluorescence of NAD(P)H in the second solution. The detecting steps can comprise visually detecting the presence or absence of formation of the bubbles.
In another aspect, this document features a method for treating cancer, wherein the method comprises, or consists essentially of, (a) obtaining cancer cells from a mammal having cancer; (b) placing a portion of the cancers into a plurality of different containers in solution; (c) adding a different test agent to each of the plurality of different containers; (d) adding exogenous H2O2 to each of the plurality of different containers; (e) detecting the level of O2 formation in the solution of each of the plurality of different containers; (f) selecting the test agent present in one of the plurality of different containers that resulted in minimal O2 formation as compared to the level observed in at least one other of the plurality of different containers, thereby identifying the selected test agent as a treatment agent for the mammal; and (g) administering the treatment agent to the mammal under conditions wherein the number of cancer cells within the mammal is reduced. The mammal can be a human. At least one of the test agents can be an IMID. The IMID can be thalidomide, lenalidomide, pomalidomide, or apremilast. The exogenous H2O2 can be provided in an amount from about 20 μM to about 150 μM. The exogenous H2O2 can be provided in an amount of about 100 μM. The solution can be PBS. The detecting the level of O2 formation in the solution of each of the plurality of different containers can comprise (i) detecting the level of bubble formation in the solution of each of the plurality of different containers, (ii) detecting the level of autofluorescence of FAD in the solution of each of the plurality of different containers, or (iii) detecting the level of autofluorescence of NAD(P)H in the solution of each of the plurality of different containers. The detecting step can comprise visually detecting the level of formation of the bubbles.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for assessing hydrogen peroxide accumulation within cells (e.g., cancer cells) exposed to one or more test agents. For example, this document provides methods and materials for determining whether or not cancer cells (e.g., MM cells) from a mammal (e.g., a human) accumulate hydrogen peroxide following contact with a test agent (e.g., an IMID) and exogenous H2O2.
In some cases, cells (e.g., cancer cells) in solution can be exposed to a test agent and exogenous H2O2. If the cells (e.g., cancer cells) possess the ability to degrade H2O2 even in the presence of the test agent, then water and O2 will be formed. This O2 can be detected, thereby providing an indication that little to no H2O2 is accumulating within those particular cells (e.g., cancer cells) when contacted with the test agent. If the cells (e.g., cancer cells) lack the ability to degrade H2O2 in the presence of the test agent, then water and O2 will not be formed from the added H2O2. This lack of O2 can be detected, thereby providing an indication that H2O2 is accumulating within those particular cells (e.g., cancer cells) when contacted with the test agent.
Those test agents having the ability to prevent or reduce O2 formation from added H2O2 for particular cancer cells can be used as a cancer treatment agents. For example, a test agent found to have the ability to prevent the formation of visible bubbles from added H2O2 by cancer cells obtained from a particular human cancer patient can be administered to that particular patient to treat cancer.
Any appropriate method can be used to detect the formation of O2 from added H2O2. For example, measuring increases in autofluorescence of FAD (FITC fluorescence spectrum) and/or measuring decreases in autofluorescence of NAD(P)H (UV-blue fluorescence spectrum), which can correlate with O2 bubble formation, can be used to measure O2 formation within a solution. In some cases, the solution containing the cells can be examined (e.g., visually examined) for the presence, absence, or level of bubble formation (e.g., formation of O2-containing bubbles).
Any appropriate compound can be used as a test agent. For example, IMIDs can be used as a test agent. Examples of IMIDs include, without limitation, thalidomide, lenalidomide, pomalidomide, and apremilast.
The methods and materials provided herein can be used with any appropriate cell. For example, exogenous H2O2 and a test agent can be added to cancer cells in solution to determine if those cancer cells have the ability degrade or accumulate H2O2. Examples of cancer cells that can be used include, without limitation, myelodysplastic syndrome cells, erythema nodosum leprosum cells, multiple myeloma cells, Hodgkin's lymphoma cells, light chain-associated amyloidosis cells, primary myelofibrosis cells, acute myeloid leukaemia cells, prostate cancer cells, and metastatic renal cell carcinoma cells. Any appropriate number of cells can be used. For example, from about 1×104 to about 1×109 cells (e.g., from about 1×105 to about 1×109 cells, from about 1×106 to about 1×109 cells, from about 1×107 to about 1×109 cells, from about 1×108 to about 1×109 cells, from about 1×105 to about 1×108 cells, or from about 1×106 to about 1×108 cells) per mL of solution can be contacted with exogenous H2O2 and a test agent. Any appropriate amount of exogenous H2O2 and test agent can be used. For example, from about 0.001 mM to about 10 M (e.g., from about 0.001 mM to about 5 M, from about 0.001 mM to about 1 M, from about 0.001 mM to about 750 mM, from about 0.001 mM to about 500 mM, from about 0.001 mM to about 250 mM, from about 0.001 mM to about 100 mM, from about 0.001 mM to about 50 mM, from about 0.001 mM to about 25 mM, from about 0.01 mM to about 750 mM, from about 0.05 mM to about 750 mM, from about 0.1 mM to about 750 mM, from about 1 mM to about 750 mM, from about 1 mM to about 50 mM, or from about 1 mM to about 10 mM) of H2O2 can be used. In some cases, from about 0.001 mM to about 10 mM of test agent can be used.
The cells can be contacted with exogenous H2O2 and a test agent in any appropriate solution. For example, cells within PBS can be contacted with exogenous H2O2 and a test agent, and the solution assessed for O2 production and/or bubble formation.
Once the cells are contacted with exogenous H2O2 and a test agent, the solution can be assessed after about 1 minute to about 180 minutes (e.g., after about 5 minute to about 180 minutes, after about 10 minute to about 180 minutes, after about 15 minute to about 180 minutes, after about 25 minute to about 180 minutes, after about 60 minute to about 180 minutes, after about 5 minute to about 120 minutes, after about 5 minute to about 75 minutes, after about 5 minute to about 60 minutes, after about 10 minute to about 75 minutes, after about 10 minute to about 60 minutes, or after about 25 minute to about 60 minutes) for the presence, absence, or level of O2 formation and/or bubble formation (e.g., formation of O2-containing bubbles).
In some cases, the methods and materials provided herein can be used to assess IMID responsiveness in a mammal to be treated for cancer. Accumulation of intracellular H2O2 produced by an IMID can lead to apoptosis. Cells, however, may have an antioxidant defense system to decompose H2O2, thereby combating excessive production of H2O2. A cell's ability to decompose H2O2 is referred to as its anti-oxidative capacity and is indicative of IMID responsiveness. A cell with higher anti-oxidative capacity (efficiently decomposes H2O2) is indicative of IMID resistance, and a cell with a lower anti-oxidative capacity (inefficiently decomposes H2O2) is indicative of IMID sensitivity.
In some cases, the methods and materials described herein can be used to grade cancers for low anti-oxidative capacity, medium anti-oxidative capacity, or high anti-oxidative capacity. This can allow clinicians to select different treatment strategies for particular patients. In some cases, determining the total anti-oxidative capacity of cancer cells during therapy can be used as a prognostic marker. For example, the methods and materials provided herein can be used to determine whether or not a mammal (e.g., a human) having cancer is responding to a particular IMID based at least in part on the anti-oxidative capacity of cancer cells obtained from the mammal at different treatment time points.
As described herein, exogenously added H2O2 can result in the production of water and oxygen when contacted with cells. The qualitative estimate of oxygen bubbles generated from H2O2 can indicate IMID responsiveness.
Any appropriate mammal can be assessed and/or treated as described herein. For example, humans, non-human primates, monkeys, horses, bovine species, porcine species, dogs, cats, mice, and rats having cancer can be assessed to determine whether or not the mammal is likely to respond to an IMID and/or likely to be treated for cancer with a particular agent. In some cases, a mammal having any appropriate type of cancer can be assessed and/or treated as described herein. For example, mammals with myelodysplastic syndrome, erythema nodosum leprosum, multiple myeloma, Hodgkin's lymphoma, light chain-associated amyloidosis, primary myelofibrosis, acute myeloid leukaemia, prostate cancer, and metastatic renal cell carcinoma can be assessed to determine whether or not the mammal is likely to respond to an IMID and/or is a candidate for a particular cancer treatment.
In some cases, the methods and materials provided herein can be used to identify agents that have the ability to increase intracellular H2O2 accumulation in cells (e.g., cancer cells). For example, a test agent can be incubated with cells in solution in the presence of exogenous H2O2, and the solution can be assessed as described herein for the presence of O2 formation and/or bubble formation (e.g., increase oxidized FAD autofluorescence and/or decreased NAD(P)H autofluorescence). Those test agents that increase oxidized FAD autofluorescence and/or decreased NAD(P)H autofluorescence can be identified as being an agent that increases intracellular H2O2 accumulation.
In some cases, test agents can be assessed to determine if they inhibit intracellular H2O2 decomposition, thereby increasing intracellular H2O2. Test agents having this ability can be used for cancer therapy. Examples of agents having the ability to inhibit intracellular H2O2 decomposition include, without limitation, H2O2 analogues (D2O2 and HDO2), glutathione peroxidase inhibitors including glutathione analogs, NADPH peroxidase inhibitors and NADPH analogues, catalase inhibitors, thioredoxin peroxidase inhibitors, haem peroxidase inhibitors, peroxidase substrates that inhibit H2O2 decomposition, homocysteine, cysteine analogs that can inhibit H2O2 decomposition, and hydrogen peroxide stabilizers including inorganic phosphate. In some cases, a test agent can be assessed for the ability to increase intracellular H2O2 production in cancer cells by altering cancer cell metabolism and/or promoting the production of intracellular H2O2. Examples of agents having the ability to increase intracellular production of H2O2 include, without limitation, mitochondrial respiration activators (e.g., lipoamide, (R)-(+)-α-lipoic acid, and (S)-(−)-α-lipoic acid), citrate, ATP, NADH, agents that induce fatty acid and lipid biosynthesis such as second-generation antipsychotics (SGA) (e.g., clozapine, olanzapine, and dihydrotestosterone), dexamethasone, 3-isobutyl-1-methylxanthine (ibmx), oxidized L-glutathione, malonyl-CoA, acetyl coenzyme A, coenzyme A, retinal (also called retinaldehyde), vitamin A aldehydes, and trans-retinals. Fatty-acid oxidation mediated generation of H2O2 can be achieved by supplementing with fatty acids that undergo rapid intracellular oxidation and generation of H2O2. In some cases, redox cycling compounds (e.g., pyrroloquinoline quinone (PQQ), anisaldehyde, and veratraldehyde) can be used to generate intracellular H2O2. In some cases, a combination of at least one agent having the ability to inhibit intracellular H2O2 decomposition and at least one agent having the ability to increase intracellular production of H2O2 can be administered to a mammal (e.g., a human) having cancer to reduce the number of cancer cells within the mammal.
As described herein, lenalidomide and other IMIDs can inhibit thioredoxin reductase and induce the accumulation of intracellular H2O2 in cancer cells (e.g., myeloma cells). In some cases, thioredoxin reductase inhibitors can be used to increase intracellular H2O2 in cancer cells (e.g., myeloma cells) and can be used alone or in combination with other anti-cancer drugs to treat cancer. Examples of thioredoxin reductase inhibitors that can be used to treat cancer as described herein include, without limitation, auranofin, aurothiomalate, alantolactone, phosphine gold(I), GoPI ({1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl), gold(I)carbene complexes, gold(III)-dithiocarbamato complexes, AuBiPy, AuXil, AuPy, terpyridine-Pt(II), pyocyanin (5-methylphenazin-1(5H)-one), cisplatin (cis-diaminodichloroPt(II)), carboplatin, terpyridine-platinum(II), arsenic trioxide, methyl As(III), 2,4-Dihydroxybenzylamine, 13-cis retinoic acid, nitrosoureas, dinitrohalobenzenes, Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), fluoro-analogue of a menadione derivative, bromo-isophosphoramide, peroxynitrite, dinitrosoglutathione, S-nitrosoglutathione, EGCG (epigallocatechin-3-O-gallate), n-butyl 2-imidazolyl disulfide, 1-methylpropyl 2-imidazolyl disulfide, n-decyl 2-imidazolyl disulfide, xanthene (6-hydroxy-3-oxo-3H-xanthene-9-propionic acid), and safranin (3,7-diamino-2,8-dimethyl-5-phenyl-phenazinium chloride).
In some cases, thioredoxin inhibitors can be used to increase intracellular H2O2 in cancer cells (e.g., myeloma cells) and can be used alone or in combination with other anti-cancer drugs to treat cancer. Examples of thioredoxin inhibitors that can be used to treat cancer as described herein include, without limitation, PX-12 (1-methylpropyl 2-imidazolyl disulfide), PMX464, DTNB (5,5′-dithiobis-(2-nitrobenzoic acid) and its analogs, and 4-hydroxy-2-nonenal.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
The human multiple myeloma cell line (HMCLs) OPM2, MM.1S, MM.1Sres, KMS18, JJN3, KMS11, and OCIMY5 were studied. All exhibited different degrees of sensitivity to lenalidomide. HMCLs were maintained in RPMI-1640, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine. All HMCLs were grown at 37° C. in a 5% CO2 incubator.
Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cells were seeded in 96-well plates in 100 μL complete medium at a density of 20,000 cell/well and incubated with serial doses of lenalidomide (Chem-Pacific), and bortezomib (millennium pharmaceuticals) for different periods. The data were normalized to the DMSO-treated group. Each experimental condition was performed in triplicate and repeated at least once. Thalidomide was obtained from Sigma, and pomalidomide was obtained from Selleckchem.
Whole-cell lysates were prepared from cell pellets using cell lysis buffer (Cell Signaling Technology). Equal amounts of protein extracts were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad). Most gels were run under reducing conditions (by adding dithiothreitol), but analysis of immunoglobulin light chain (IgL) dimers required non-reducing conditions (without dithiothreitol). After blocking with 5% milk for 1 hour at room temperature, membranes were washed and probed with primary antibodies overnight at 4° C. Blots were washed with 0.1% Tris-buffered saline and Tween 20 and incubated with appropriate horseradish peroxidase-labeled secondary antibodies. Blots were developed using a chemiluminescent detection system (ECL, PerkinElmer). β-actin or GADPH were used as loading control.
Antibodies included those against p53 (DO-1, Santa Cruz), XBP-1 (Santa Cruz), cereblon (CRBN) (Sigma), β-actin (Sigma), λ light chain, κ light chain (Abcam), Bip (Cell Signaling Technology), Bim (Cell Signaling Technology), and poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology). Other antibodies were obtained from Cell Signaling Technology. The secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit immunoglobulin G (Cell Signaling Technology).
Total RNA from HMCLs treated with lenalidomide was isolated using the Qiagen RNeasy mini kit. RNA (1 μg) was used for cDNA synthesis using iScript Reverse Transcription Supermix (Bio-Rad). Real-time polymerase chain reaction (PCR) was performed using the SYBR green method. The following primers were used for amplification:
The fold change of the mRNA expression was calculated from the difference between treated and untreated cells, after normalizing to an endogenous control (β-actin). Primers encompassing the spliced sequences in XBP1 mRNA were used for PCR amplification, and products were separated by electrophoresis through a 2.5% agarose gel and visualized by ethidium bromide staining. All reactions were conducted in triplicate.
Treated cells were harvested and stained for flow cytometry with Annexin V-fluorescein isothiocyanate and propidium iodide (BD Pharmingen). Stained cells were analyzed with a BD LSRII flow cytometer, and the data were analyzed with BD fluorescence-activated cell sorting (FACs) DIVA software.
CRBN knockdown cells were used as described elsewhere (Zhu et al., Blood, 118(18):4771-9 (2011)). For Bim knockdown, lentiviral constructs expressing non-targeting (shCtrl) and Bim short hairpin RNAs (shRNAs) (Sigma-Aldrich) were used. TRC-vectors were cotransfected into 293T cells using a calcium phosphate precipitation method with the psPAX2 packaging plasmid and pMD2.G, a plasmid encoding the lentivirus envelope. Supernatants containing pseudotyped lentivirus were collected at 48 and 72 hours and were used to infect HMCLs. Four lentiviruses targeting Bim were screened to identify shRNA that optimally suppressed Bim. Forty-eight hours after transfection, cells were selected with puromycin, and OPM2 lysates were immunoblotted to confirm down-regulation of Bim. Clones #73 and #75, which optimally suppressed Bim, were used for subsequent experiments. IgL-λ knockdown was performed by using piLenti-siRNA-GFP to target the constant region of IgL-λ. IgL-κ knockdown was performed by using lentiviral-mediated siRNA to target the constant region of IgL-κ.
Human CRBN cDNA was obtained from Thermo Scientific and subcloned into a lentiviral expression vector, pCDH-CMV-MCS-EF1-copGFP (System Bioscience). Lentivirus harboring control vector and CRBN cDNA constructs were prepared and used to infect the multiple myeloma (MM) cell line OCIMY5. Infection efficiency was measured by FACScan analysis of GFP expression 3 days after infection. The cells were sorted for GFP expression 14 days after infection. CRBN overexpression was confirmed by immunoblotting.
HRP/Amplex Red in-vitro assay performed in 100 μL HBSS final reaction volume contain HRP (1 unit/mL) and Amplex Red (50 μM) and 10 μM concentration of drug (thalidomide, lenalidomide, and pomalidomide) or DMSO control with 5 μM concentration of H2O2 and kept for reaction at 37° C. for 30 minutes. After 30 minutes, plates were read for fluorescence at 530 nm excitation and 590 nm emission with plate reader (Biotek Cytation3). For determining intra-cellular peroxidase activity by Amplex Red, cells were washed and mixed with Amplex Red reagent (50 μM) in HBSS buffer and plated 100,000 cells per well into 96 well plate, four wells for each condition. After plating, the cells were immediately treated with H2O2 (100 μM) alone or together with thalidomide (20 μM), lenalidomide (20 μM), or pomalidomide (10 μM). The DMSO control included H2O2 (100 μM). The cells were incubated at 37° C. for 40 to 60 minutes for analyzing IMIDs ability to inhibit intracellular peroxidase activity and 30 minutes after H2O2 treatment for determining IMIDs ability to mediate inhibition of extracellular H2O2 decomposition. After specific time periods, the plates were read for fluorescence intensity using plate reader.
HMCLs cells (1 million cells/1 mL PBS) treated or not treated with 100 μM concentration of H2O2 were immediately analyzed for autofluoresnece of FAD (FITC-A channel) and NAD(P)H (UV Blue-A channel) with multicolor flow cytometry (BD LSRFORTESSA). Flowjo histogram normalization was used to overlay untreated versus treated samples.
Post-treatment reactive oxygen species (ROS) levels were determined using the cell-permeable fluorogenic probe DCFDA (Invitrogen Biosciences). Briefly, million cells per 2 mL (2 million cells in 4 mL medium) were cultured overnight. DCFDA (50 μM) was added to suspended cells and incubated for 30 minutes in the dark. Cells were collected and washed once with phosphate-buffered saline (PBS) and split into 2 FACS tubes. One tube contained vehicle (dimethyl sulfoxide), and the other contained lenalidomide (10 μM). Cells were analyzed using a FACSCalibur system (Becton and Dickinson), with excitation and emission spectra set at 488 and 530 nm, respectively. CellQuest software was used to calculate H2O2 production by measuring the increase in mean fluorescence.
A biochemical test was used to determine the total anti-oxidative capacity of MM cells by evaluating their ability to decompose exogenous H2O2 to water and oxygen. Exponentially growing MM cells (sub-cultured for 12 hours) were counted 3 times to ensure that an equal number of cells (1×106 cells) were suspended in equal volumes of PBS. An equal amount (500 μL) of 33% H2O2 was added directly to the cells, and the newly formed oxygen bubbles were qualitatively assessed after 15 minutes. This test was sufficiently sensitive to detect differences in cell lines with varying sensitivity to lenalidomide.
IMIDs Inhibit Peroxidase Mediated Decomposition of H2O2 in MM Cells
Lenalidomide was tested to determine if it could induce oxidative stress in HMCLs. Cell line MM.1S is highly sensitive to lenalidomide in vitro. Cells were pretreated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) for 30 minutes before exposure to lenalidomide or vehicle control. Lenalidomide-exposed MM.1S cells exhibited increasing intracellular H2O2, as evidenced by the fluorescent product 2′,7′-dichlorofluorescein (DCF) detected by fluorescence-activated cell sorting (FACS) analysis (
Thalidomide, lenalidomide, and pomalidomide were examined for the ability to inhibit peroxidase activity in vitro using the highly specific peroxidase substrate Amplex Red. In an in vitro assay, decomposition of H2O2 by horseradish peroxidase oxidizes Amplex Red to resorufin (oxidized fluorescent product). This was inhibited by immunomodulatory drugs (IMIDs) (thalidomide, lenalidomide, and pomalidomide;
To confirm IMIDs inhibit H2O2 decomposition, different HMCLs were treated with external H2O2. External H2O2 decomposition by cellular peroxidases was inhibited by IMIDs (
A downstream effect of elevated intracellular H2O2 is the induction of protein dimerization by disulfide bonds (Linke et al., Antioxid Redox Signal. 5(4):425-34 (2003); Piwkowska et al., J Cell Physiol. 227(3):1004-16 (2012); Reczek et al., Curr Opin Cell Biol. 33C:8-13 (2014); van der Wijk et al., J Biol Chem. 279(43):44355-61 (2004)). It is hypothesized that intracellular accumulation of H2O2 after lenalidomide treatment induces dimerization of proteins such as IKZF1. Indeed, lenalidomide-induced IKZF1 dimerization increased over 75 minutes and later decreased because of protein degradation (
H2O2 Effectively Degraded IKZF1 and IKZF3 in MM Cells Expressing CRBN
It is believed that lenalidomide-bound CRBN acquires the ability to target IKZF1 and IKZF3 for proteasomal degradation (Fischer et al., Nature. 512(7512):49-53 (2014); Kronke et al., Science. 343(6168):301-5 (2014); Lu et al., Science. 343(6168):305-9 (2014)). After demonstrating that lenalidomide inhibit decomposition of intracellular H2O2, it was hypothesized that lenalidomide-induced IKZF1 and IKZF3 degradation was mediated via oxidative stress. MM.1S cells were treated with different drugs that induce oxidative stress. H2O2 and lenalidomide degraded IKZF1 and IKZF3 most effectively (degradation was evident within 3 hours;
To confirm the central role of CRBN in IKZF1 and IKZF3 degradation by H2O2-induced oxidative stress, CRBN-knockdown OPM2 isogeneic cells and the CRBN-overexpressing OCIMY-5 cell line (transfected with wild-type CRBN) were examined. OPM2-NT (nontarget short hairpin RNA (shRNA) control) and OPM2-shCRBN (silencing CRBN) cells were treated with lenalidomide (10 μM) and two concentrations of H2O2 (25 or 50 μM) for 3 hours. H2O2 similarly mediated IKZF1 and IKZF3 degradation in a CRBN-dependent fashion (
OCIMY-5 cells overexpressing CRBN also exhibited enhanced IKZF1 and IKZF3 degradation within 3 hours of lenalidomide treatment or increasing concentrations of H2O2 (
MM Cells with Lower Antioxidative Capacity were More Vulnerable to Lenalidomide-Mediated Cytotoxicity
MM cells with similar levels of CRBN expression can exhibit differential sensitivity to lenalidomide and pomalidomide, suggesting other mechanisms of cytotoxicity. It was hypothesized that the differential capacity to combat H2O2 might affect sensitivity to IMIDs. The cellular anti-oxidative capacity as a predictor of lenalidomide sensitivity was analyzed.
The capacity of MM cells to decompose H2O2 was measured via a biochemical test that qualitatively measured the amount of oxygen bubbles formed in vitro after H2O2 exposure. MM.1S (hypersensitive to lenalidomide) and RPMI-8226 (resistant to lenalidomide) cell lines were tested (
For the development of more feasible and quantitative assay for determining cellular anti-oxidative capacity, a new strategy was developed to measure total cellular oxidation of FADH2 and NAD(P)H after H2O2 treatment. Cells with a high anti-oxidative capacity generate more oxidized FAD and NAD(P) after H2O2 treatment, but cells with a lower anti-oxidative capacity (already under high oxidative state) have less oxidation of FADH2 and NAD(P)H after H2O2 treatment. By taking advantage of auto-fluorescent properties of oxidized FAD and reduced NAD(P)H, H2O2 treatment increased oxidized FAD and NAD(P) with increased and decreased autofluoresensce, respectively. In addition, cells with more anti-oxidative capacity and resistance to lenalidomide exhibited a greater increase in FAD autofluoresence and decreased NAD(P)H autofluorescence after 100 μM H2O2 treatment (RPMI-8226 and JJN3) than cells with lower anti-oxidative capacity and sensitivity to lenalidomide (MM.1S and KMS11;
Lenalidomide-induced degradation of IKZF1 and IKZF3 was described elsewhere (Kronke et al., Science. 343(6168):301-5 (2014); Lu et al., Science. 343(6168):305-9 (2014)), but this is not necessarily predictive of cytotoxicity. It was hypothesized that lenalidomide-mediated cytotoxicity in MM is attributable to oxidative damage of intracellular immunoglobulin proteins. Intracellular immunoglobulin light chain (IgL) κ and λ exist in monomeric and dimeric forms (Kaplan et al., Scientific World Journal. 11:726-35 (2011)), and proper folding of IgL is a prerequisite for secretion (Leitzgen et al., J Biol Chem. 272(5):3117-23 (1997); Magrangeas et al., Blood. 103(10):3869-75 (2004)).
Using MM.1S cells, increased formation of IgL-λ dimers was observed after 3 hours of treatment with lenalidomide or H2O2 (
The assay was repeated with lenalidomide-resistant MM.1Sres cells, which were generated by culturing MM.1S in gradually increasing concentrations of lenalidomide (Bjorklund et al., J Biol Chem. 286(13):11009-20 (2011)). CRBN expression in MM.1Sres diminished as lenalidomide resistance increased as described elsewhere (Zhu et al., Blood. 118(18):4771-9 (2011)). MM.1S and MM.1Sres cells were treated with lenalidomide for 3 days, and IgL-λ dimers were observed only in MM.1S (
By using other sets of isogenic cells positive and negative for CRBN, it was confirmed that lenalidomide treatment caused accumulation of IgL-λ dimers only in CRBN-positive cells (
It was postulated that the intracellular accumulation of IgG-λ led to an endoplasmic reticulum (ER) stress response in CRBN-positive cells. After 3 days of treatment with increasing concentrations of lenalidomide, an ER stress response occurred in OPM2-NT cells, but not in CRBN-knockdown cells (
Other isogenic HMCLs MM.1S (lenalidomide sensitive) and MM.1Sres (lenalidomide resistant), as well as KMS18-NT and KMS18-shCRBN, were analyzed. Lenalidomide induced ER stress-mediated accumulation of Bip protein in CRBN-positive cells, but not in CRBN-negative cells (
Another IgL-λ knockdown clone was generated by using the stable shRNA method. This clone also had lenalidomide resistance compared with controls (
Bim activation induced apoptosis after lenalidomide treatment in CRBN-positive MM cells. CRBN-expressing and CRBN-knockdown OPM2 cells were treated with lenalidomide for 3 days, and cell lysates were immunoblotted and probed for various proapoptotic and antiapoptotic proteins. BH3-only protein Bim was activated after lenalidomide-induced ER stress (
Accumulation of Bim was observed, especially BimEL, after lenalidomide treatment in CRBN-positive, lenalidomide-sensitive cells. Mcl1 and Bcl2 antiapoptotic proteins did not change markedly after lenalidomide treatment (
To confirm Bim involvement in lenalidomide-induced apoptosis, stable shRNA expression was used to knock down Bim in OPM2 cells. Two different OPM2 clones (#73 and #75) with downregulated Bim were established and treated with lenalidomide. Because lenalidomide induced late apoptosis in OPM2 cells, a day-4 MTT assay for cell viability was performed. Both Bim knockdown clones were less sensitive to lenalidomide than control cells (
Pretreatment with Lenalidomide Enhanced Bortezomib Sensitivity in MM
From the above, it was postulated that lenalidomide-mediated ER stress would positively enhance bortezomib-mediated cytotoxicity in MM. To translate these findings to clinical applications, MM cells were pretreated with lenalidomide and then treated with bortezomib. OPM2 cells pretreated with lenalidomide for two days clearly exhibited increased sensitivity to bortezomib-induced apoptosis compared with cells that were not pretreated (
The results provided herein demonstrate that IMIDs inhibit peroxidase mediated H2O2 decomposition in MM cells, that H2O2 induces degradation of IKZF1 and IKZF3 in cereblon-positive cells, that cellular antioxidative capacity determines sensitivity to lenalidomide, and that elevated H2O2 mediates immunoglobulin dimerization and intracellular stress.
In another test, treatment with amplex red (a fluorescent substrate for peroxidases) and exogenous H2O2 treatment was used to detect the anti-oxidative capacity of cancer cells (
In another study, cancer patients (e.g., myeloma patients) were identified as being sensitive to IMIDs by assessing oxidation of NAD(P)H and FADH2. Briefly, an anti-oxidative capacity assay was used to identify two patients as being sensitive to IMIDs (lenalidomide) because after H2O2 treatment, no further increase in oxidation of NAD(P)H and FADH2 was observed (
RPMI-8226 myeloma cells are very resistant to IMIDs (lenalidomide) because they exhibit high anti-oxidative capacity. External H2O2 treatment induced high oxidation of FADH2 (
These results demonstrate that lipoic acid or lopoamide can be used to induce high oxidized states of FAD and NAD(P) and thereby sensitize cells to anti-cancer drugs (e.g., cancer treatments using IMIDs such as lenalidomide).
OCIMY5-Vector and OCIMY-CRBN cells were treated with lenalidomide, a thioredoxin reductase inhibitor (sodium aurothiomalate; ATM), or a thioredoxin inhibitor (PX12) for 72 hours, and cell viability was assessed using an MTT assay. ATM did not inhibit cell proliferation in both cell lines after 3 days of drug treatment (
Another drug, auranofin, which inhibits thioredoxin reductase, was effective at accumulating intracellular H2O2 in myeloma cells and reducing cancer cell viability (
Dehydroascorbic acid (DHA) is a substrate for thioredoxin reductase and high concentrations of DHA inhibited myeloma cell proliferation (
Myeloma cell line harboring CRBN or without CRBN were treated with NAD, NADH, NADP, or NADPH alone or NAD, NADH, NADP, or NADPH in combination with lenalidomide and analyzed for cell survival. Treatment with NAD, NADH, NADP, or NADPH cofactors alone or in combination with lenalidomide induced cell death in myeloma cells (
In addition, bortezomib (a proteasome inhibitors) worked in synergy with auranofin (
The following was performed to determine whether inhibition of thioredoxin reductase itself with aurothiomalate (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims priority to U.S. Application Ser. No. 62/180,327, filed on Jun. 16, 2015. The disclosure of the prior application is considered part of the disclosure of this application, and is incorporated in its entirety into this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US16/37899 | 6/16/2016 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62180327 | Jun 2015 | US |
