An Endoplasmic Reticulum Specific Pro‐amplifier of Reactive Oxygen Species in Cancer Cells

Abstract

The folding and export of proteins and hydrolysis of unfolded proteins are disbalanced in the endoplasmic reticulum (ER) of cancer cells, leading to so-called ER stress. Agents further augmenting this effect are used as anticancer drugs including clinically approved proteasome inhibitors bortezomib and carfilzomib. However, these drugs can affect normal cells, which also rely strongly on ER functions, leading, for example, to accumulation of reactive oxygen species (ROS). To address this problem, we have developed ERtargeted prodrugs activated only in cancer cells in the presence of elevated ROS amounts. These compounds are conjugates of cholic acid with N-alkylaminoferrocene-based prodrugs. We confirmed their accumulation in the ER of cancer cells, their anticancer efficacy, and cancer cell specificity. These prodrugs induce ER stress, attenuate mitochondrial membrane potential, and generate mitochondrial ROS leading to cell death via necrosis. We also demonstrated that the new prodrugs are activated in vivo in Nemeth-Kellner lymphoma (NK/Ly) murine model.

Intracellular Amplifiers of Reactive Oxygen Species Affecting Mitochondria as Radiosensitizers

“…We confirmed that all these cells contain the high level of ROS. Therefore, they were expected to be sensitive to ROS amplifying drugs [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Due to the slow kinetics of action of 2c observed in A2780 cells ( Table S2, SI ), incubation times of 48–96 h were selected.…”

“…These data thus confirm the high mitochondria-specificity of 2c. Since 2c is an NAAF prodrug, another expected effect would be the amplification of ROS in cells [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. To evaluate whether this is the case, we used three different probes: the nonspecific probe 5-(6-)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-DCFH-DA) for the measurement of total ROS (tROS), the mitochondria-specific probe Red Mitochondrial Superoxide Indicator (MitoSOX) for the measurement of mitochondrial ROS (mROS) and 7-(diethylamino)coumarin-3-cerbohydrazide (DCCH) for monitoring ROS-induced formation of intracellular carbonyl-derivatives ( Figure 4 ).…”

“…The numbers in the brackets correspond to prodrug concentrations leading to aggregate formation (48 h incubation) determined by dynamic light scattering ( SI ). iii Solubility of prodrug 1 in Roswell Park Memorial Institute (RPMI) 1640 medium (Biochrom GmbH, Germany) supplemented with fetal bovine serum (FBS, 5%), l -glutamine (1%) Penicillin/Streptomycin (1%, Biochrom GmbH, Germany) and containing dimethylsulfoxide (DMSO, 1%, v / v ) was determined as described previously [ 29 ]. iv ROS release efficacy, expressed as relative acceleration of the rate of fluorescence increase (λ ex = 501 nm, λ em = 523 nm) in the mixture of 2′,7′-dichlorodihydrofluorescein (DCFH) and H 2 O 2 in aqueous solution buffered at pH 7.4.…”

“…For example, cancer cells have the higher level of ROS, whereas the level of ROS in normal cells at homeostatic conditions is negligible [ 16 , 17 , 18 ]. Known ROS activated prodrugs include N -alkylaminoferrocene-based (NAAF)-prodrugs [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ], pro-DNA alkylators [ 30 , 31 ], hydroxyferrocifens and their analogues [ 32 , 33 ], organochalcogen-based redox catalysts [ 34 ] and oligoferrocenes [ 35 ]. Unexpectedly, we have found that early NAAF prodrugs [ 18 , 19 , 21 ] exhibit no radiosensitizing effects (unpublished results) indicating that the intracellular ROS amplification alone is not sufficient to achieve the synergy with RT.…”

“…Recently, we have found conditions for a CuAAC reaction in non-aqueous buffers compatible with compounds containing B-C bonds. Using the single CuAAC, we have already prepared a series of conjugates of NAAF prodrugs with fluorescent dyes [ 22 , 25 , 28 , 29 ] and radio-tracers [ 27 , 43 ]. Here we applied both single (prodrugs 2a, 2b) and, for the first time, double sequential CuAAC reactions (prodrugs 2c, 2d) to generate a small library of derivatives of NAAF prodrug 1 ( Figure 1 ).…”