3,3′-Diselenodipropionic acid (DSePA) induces reductive stress in A549 cells triggering p53-independent apoptosis: A novel mechanism for diselenides
Graphical abstract
Introduction
Despite significant advancement in the treatment modalities, cancer is a global health problem requiring attention of researchers to develop newer anticancer drugs [1]. Among the cancer of different tissue origins, non-small cell lung cancer is considered to be highly aggressive, chemo-resistant and radio-resistant. It is also one among the top ten cancer types contributing to the overall cancer related mortality in the world [2]. Several reports have indicated that organoselenium compounds exhibit chemo-preventive as well as chemo-therapeutic activities in both cellular and in vivo models [[3], [4], [5], [6]]. Organodiselenide a class of organoselenium compounds are structurally defined as R-Se-Se-R, wherein R is alkyl or aryl group [7]. These molecules have shown to exhibit antioxidant activity by undergoing reduction either through NADPH in a thioredoxin reductase (TrxR) catalysed reaction or through glutathione (GSH) to form selenol (R-Se-H) or selone (R–SeC) [7]. The reduced species in turn can catalyze the reduction of hydrogen peroxide into water molecules by a glutathione peroxidase (GPx)-like catalytic mechanism. On the contrary, organodiselenides are also known to exhibit pro-oxidant activity by oxidizing cellular GSH in futile cycle and leading to generation of reactive oxygen species (ROS) [8]. These paradoxical behaviors of diselenides make them excellent candidates for dual activities of chemoprevention and chemotherapeutic applications respectively. In contradiction to these reports, our group has recently shown that aryl diselenides, specifically pyridine diselenide (Py2Se2) induces cytotoxicity in lung cancer cells (A549) by causing reductive stress rather than oxidative stress [7,9]. This was a new interesting observation with regard to the mechanism of anticancer activity of organodiselenides. Reductive stress is just opposite to oxidative stress and is characterized by a shift in the intracellular redox state towards reduction rather than oxidation [10]. The biomarkers of reductive stress are the elevated ratios of redox couples like GSH/GSSG, NADPH/NADP and Trxred/Trxoxi [10]. As per recent reports, reductive stress exhibits all the features of oxidative stress such as ROS generation, DNA damage, mitochondrial dysfunctions and cell death via apoptosis or endoplasmic reticulum (ER) stress but in a delayed manner which suggests that reductive stress is followed by oxidative stress [[11], [12], [13]]. Accordingly, reductive stress has been implicated in suppressing the proliferation and/or inducing cell death (apoptosis) in tumor cells [14]. Although, Py2Se2 exhibited potent cytotoxicity in A549 cells, it was also equally toxic to normal fibroblast cells and therefore cannot be considered as an ideal candidate for chemotherapy. Apart from aryl diselenides, our group has also been working on the pharmacological evaluation of aliphatic diselenides. In this context, we have identified a compound called 3,3′-diselenodipopionic acid (DSePA) showing multiple pharmacological activities like GPx-like catalytic activity, ROS scavenging and lung radioprotection against thoracic and whole-body irradiation [[15], [16], [17], [18], [19]]. The median lethal dose (LD50), pharmacokinetics and bio-distribution of this compound are also well established [20,21]. Further, several other research groups have also worked on this molecule and have reported its role in nitric oxide (NO) generation for wound healing, as an antioxidant to prevent neurotoxicity and to reduce radiation and chemotherapy side effects and also as a chemo-sensitizer to enhance the efficacy of chemotherapy [[22], [23], [24]]. Moreover, DSePA is shown to be orally bio-available with maximum uptake in lung compared to other organs [20,21]. With this background, it was hypothesized that DSePA might be effective in killing lung cancer cells without producing any significant side effects in the surrounding normal cells. Additionally, we further wanted to study whether DSePA being an aliphatic diselenide followed the same reductive mechanism as shown by aryl diselenide or acts by a pro-oxidant mechanism. In order to address the above said hypothesis, DSePA has been investigated in detail for redox modulatory effects in human cancer cell lines of lung tissue origin and its mechanism of action was also established. The chemical structure of DSePA is shown in Scheme 1.
Section snippets
Chemicals
The synthesis, purification and characterization of DSePA have been reported previously [19] Dimethyl sulfoxide (DMSO), propidium iodide (PI), 3-(4,5-Dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide (MTT), glutathione (GSH), β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetra sodium salt hydrate (NADPH), glutathione reductase (GR), cumene hydroperoxide, diethyl pyrocarbonate (DEPC), 2′,7′-dichlorofluorescin diacetate (DCFDA), dihydroethidium (DHE), dihydrorhodamine (DHR) 123,
Evaluation of the cytotoxicity of DSePA in lung cancer and normal cell lines
In vitro anticancer activity of DSePA against A549 lung cancer cells was evaluated by treating these cells with the increasing concentrations of DSePA (1–100 μM) for varying times such as 24 h, 48 h and 72 h and monitoring their viability by MTT assay. The percentage cytotoxicity (100 - % viability) obtained from these analyses was plotted against the concentrations of DSePA to obtain the IC50 value (Fig. 1A). The results indicated that DSePA treatment up to a concentration of 50 μM showed
Discussion
Previous studies have reported that aliphatic diselenides in specific selenocystine exhibits anticancer activity through pro-oxidant mechanism mediated by the oxidation of cellular thiols, inhibition of thioredoxin system, production of ROS, DNA damage and cell death [[38], [39], [40]] On the contrary, present study indicated that DSePA, which is a structural analogue of selenocystine lowered ROS level and increased the levels of reducing equivalents such as GSH and NADH at 6 h post treatment.
Author contribution statement
V.V. Gandhi: Conceptualization, Methodology, Investigation, Formal analysis, Validation, Writing; K.A. Gandhi: Methodology, Investigation, Formal analysis, Validation; L.B. Kumbhare: Resources; J.S. Goda: Investigation, Formal analysis, Resources; V. Gota: Investigation, Formal analysis, Resources; K.I. Priyadarsini: Resources; A. Kunwar: Conceptualization, Supervision, Writing - Review & Editing.
Patent disclosure
A part of the study titled “Use of 3,3′-diselenodipropionic acid (DSePA) as an anticancer agent” is under evaluation for USA patent (US App. No. 17/003420 dated 26-Aug-2020). The inventors of this patent application are A. Kunwar, V.V. Gandhi, K.A. Gandhi, V. Gota, J.S. Goda, J. Kode, L.B. Kumbhare, V.K. Jain, & K.I. Priyadarsini.
Declaration of competing interest
None.
Acknowledgement
The research work presented in the manuscript is a part of the doctoral thesis of Miss V.V. Gandhi. The authors acknowledge Dr. Pradip Choudhary, Dr. Jyoti Kode, and Dr. Venkatesh Pai from ACTREC for helping in animal studies. They also acknowledge Dr. Awadhesh Kumar, Head, RPC Division BARC, and Dr. A. K. Tyagi, Associate Director, Chemistry Group, BARC for their support and encouragement. Miss V.V. Gandhi acknowledges HBNI for awarding senior research fellowship to carry out this work.
References (59)
- et al.
Organic selenium compounds as potential chemotherapeutic agents for improved cancer treatment
Free Radical Biol. Med.
(2018) - et al.
Plasma, normal and tumor tissue selenium concentrations as a function of 5-methyl-selenocysteine dose in mice bearing human tumor xenografts
Biochem. Pharmacol.
(2007) - et al.
Increased reactive oxygen species production during reductive stress: the roles of mitochondrial glutathione and thioredoxin reductases
Biochim. Biophys. Acta
(2015) - et al.
Reductive Stress increases reactive oxygen species production in cardiac mitochondria: a key role of thioredoxin reductase
Biophys. J.
(2014) - et al.
In vivo radioprotection studies of 3,3′-diselenodipropionic acid, a selenocystine derivative
Free Radic. Biol. Med.
(2010) - et al.
Oral administration of 3,3′-diselenodipropionic acid prevents thoracic radiation induced pneumonitis in mice by suppressing NF-kB/IL-17/G-CSF/neutrophil axis
Free Radical Biol. Med.
(2019) - et al.
3,3′-Diselenodipropionic acid (DSePA): a redox active multifunctional molecule of biological relevance
Biochim. Biophys. Acta Gen. Subj.
(2021) - et al.
Toxicological safety evaluation of 3,3′-diselenodipropionic acid (DSePA), a pharmacologically important derivative of selenocystine
Regul. Toxicol. Pharmacol.
(2018) - et al.
Nitric oxide producing coating mimicking endothelium function for multifunctional vascular stents
Biomaterials
(2015) - et al.
Rapid spectrophotometric method for quantitation of cytochrome C release from isolated mitochondria or permeabilized cells revisited
Biochim. Biophys. Acta Bioenerg.
(2000)
A simple technique for quantitation of low levels of DNA damage in individual cells
Exp. Cell Res.
An introduction to death receptors in apoptosis
Int. J. Surg.
DNA-damaging agents in cancer chemotherapy: serendipity and chemical biology
Chem. Biol.
Activation of apoptosis signaling pathways by reactive oxygen species
Biochim. Biophys. Acta Mol. Cell Res.
Oxidative homeostasis regulates the response to reductive endoplasmic reticulum stress through translation control
Cell Rep.
Selenocystine induces caspase-independent apoptosis in MCF-7 human breast carcinoma cells with involvement of p53 phosphorylation and reactive oxygen species generation
Int. J. Biochem. Cell Biol.
Studies on analogues of L-cysteine and L-cystine. III. The effect of selenium cystine on leukemia
Blood
Diselenoamino acid derivatives as GPx mimics and as substrates of TrxR: in vitro and in silico studies
Org. Biomol. Chem.
A novel antioxidant mechanism of ebselen involving ebselendiselenide, a substrate of mammalian thioredoxin and thioredoxin reductase
J. Biol. Chem.
Methylseleninic acid promotes antitumour effects via nuclear FOXO3a translocation through Akt inhibition
Pharmacol. Res.
Regulation of glutathione synthesis
Mol. Aspect. Med.
Innovative approaches for cancer treatment: current perspectives and new challenges
ecancer
Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries
Ca Canc. J. Clin.
Selenides and diselenides: a review of their anticancer and chemo preventive activity
Molecules
Toxicology and pharmacology of selenium: emphasis on synthetic organoselenium compounds
Arch. Toxicol.
Paradoxical behavior of organodiselenides: pro-Oxidant to antioxidant
Chem. Process
Antioxidant-prooxidant properties of a new organoselenium compound library
Molecules
2,2′-Dipyridyl diselenide (Py2Se2) induces G1 arrest and apoptosis in human lung carcinoma (A549) cells through ROS scavenging and reductive stress
Metall
Proteostasis and REDOX state in the heart
Am. J. Physiol. Heart Circ. Physiol.
Cited by (15)
Preparation of protein nanoparticles using diselenide functionalized gelatin for redox responsive release of doxorubicin
2024, Journal of Drug Delivery Science and TechnologyInhibition of HAdV-14 induced apoptosis by selenocystine through ROS-mediated PARP and p53 signaling pathways
2023, Journal of Trace Elements in Medicine and BiologyGelatin-lecithin-F127 gel mediated self-assembly of curcumin vesicles for enhanced wound healing
2022, International Journal of Biological MacromoleculesCitation Excerpt :The total RNA was extracted from the tissue homogenate prepared in Trizol reagent as per manufacturer's instruction. Following this, 2 μg of total RNA was reverse transcribed using cDNA synthesis kit as per manufacturer's instruction and used as the template for RT-PCR using gene specific primers (Table - 1) and a previously optimized method [33]. The expressions of targets genes were normalized against the house-keeping gene β-actin according to previous method and represented as relative gene expression [34].