Elsevier

Free Radical Biology and Medicine

Volume 175, 1 November 2021, Pages 1-17
Free Radical Biology and Medicine

3,3′-Diselenodipropionic acid (DSePA) induces reductive stress in A549 cells triggering p53-independent apoptosis: A novel mechanism for diselenides

https://doi.org/10.1016/j.freeradbiomed.2021.08.017Get rights and content

Highlights

  • DSePA shows differential cytotoxicity between lung cancer cells and normal lung cells.

  • DSePA induces reductive stress followed by oxidative stress.

  • DSePA elicits intrinsic, extrinsic and ER-stress pathways of apoptosis.

  • DSePA shows significant antitumor activity in A549 xenograft tumor model.

Abstract

The aim of present study was to investigate the anticancer mechanisms of 3,3′-diselenodipropionic acid (DSePA), a redox-active organodiselenide in human lung cancer cells. DSePA elicited a significant concentration and time-dependent cytotoxicity in human lung cancer cell line A549 than in normal WI38 cells. The cytotoxic effect of DSePA was preceded by an acute decrease in the level of basal reactive oxygen species (ROS) and a concurrent increase in levels of reducing equivalents (like GSH/GSSG and NADH/NAD) within cells. Further, a series of experiments were performed to measure the markers of intrinsic (Bax, cytochrome c and caspase-9), extrinsic (TNFR, FADR and caspase-8) and endoplasmic reticulum (ER) stress (protein ubiquitylation, calcium flux, Bip, CHOP and caspase-12) pathways in DSePA treated cells. DSePA treatment significantly increased the levels of all the above markers. Moreover, DSePA did not alter the expression and phosphorylation (Ser15) of p53 but caused a significant damage to mitochondria. Pharmacological modulation of GSH level by BSO and NAC in DSePA treated cells led to partial abrogation and augmentation of cell kill respectively. This established the role of reductive stress as a trigger for the apoptosis induced by DSePA treatment. Finally, in vitro anticancer activity of DSePA was also corroborated by its in vivo efficacy of suppressing the growth of A549 derived xenograft tumor in SCID mice. In conclusion, above results suggest that DSePA induces apoptosis in a p53 independent manner by involving extrinsic and intrinsic pathways together with ER stress which can an interesting strategy for lung cancer therapy.

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–Sedouble bondC) [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.

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