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. 2025 Dec 21;399(6):8101–8112. doi: 10.1007/s00210-025-04852-y

Mitocurcumin induces ROS-/JNK-mediated paraptosis to overcome chemoresistance in non-small cell lung cancer

Girish Ch Panigrahi 1,2, Amisha Joshi 1,2, Dinky Malhotra 1,3, Vikram Gota 1,2,✉
PMCID: PMC13086902  PMID: 41422457

Abstract

Lung cancer remains the leading cause of cancer-related mortality worldwide. A major barrier to effective treatment is the development of drug resistance, which contributes to poor patient survival. One key mechanism underlying this resistance is the ability of cancer cells to evade apoptotic cell death. Thus, there is an urgent need for novel therapeutic strategies to overcome chemoresistance in lung cancer. The ultrastructural features of mitochondria and the endoplasmic reticulum (ER) were assessed using transmission electron microscopy (TEM). Transcriptomic profiling of A549 cells was carried out through whole-exome sequencing. Protein expression levels were validated by western blot analysis, while mitochondrial calcium content was quantified using flow cytometry. Our study utilized mitocurcumin (mitoC) to study an alternative form of cell death in NSCLC. Mitochondria and ER vacuolation and swelling were observed upon mitocurcumin treatment. MitoC treatment upregulated ER and mitochondria stress protein levels in A549 and A549R cells. However, the inhibition of intracellular ROS and JNK pathway abrogated mitoC-induced mitochondria and ER stress proteins. Moreover, we observed that mitoC treatment enhanced mitochondrial calcium uptake in A549 and A549R cells, which gets abrogated upon ROS/JNK signaling inhibition. MitoC exerted organellar stress related to paraptosis in A549 and A549R cells through activation of the ROS-mediated JNK signaling pathway and induced mitochondrial calcium uptake.

Keywords: Mitocurcumin, Paraptosis, Organellar stress, JNK signaling, Calcium imbalance

Introduction

Lung cancer is the leading cause of cancer-related death globally, with an estimated 2.5 million new cases and 1.8 million fatalities (Bray et al. 2024). Non-small cell lung cancer (NSCLC) contributes to the major histological subtype, accounting for 85% of lung cancer (Khodabakhshi et al. 2021). Despite significant advances in treatment, including surgery, radiation, and chemotherapy with targeted and immune therapies, patient survival rates remain low, a challenge particularly evident in regions like India, where the five-year survival rate is just 3.7% (Noronha et al. 2024). A primary factor contributing to this poor prognosis is the development of resistance to existing therapies (Ashrafi et al. 2022). To ensure their survival, cancer cells develop diverse strategies to resist the cytotoxic impact of therapies, primarily by avoiding programmed cell death through apoptosis (Fulda 2009).

Current research efforts are increasingly focused on agents capable of inducing alternative, non-apoptotic modes of cell death (Chen et al. 2023; Jin et al. 2024). Paraptosis, a caspase-independent cell death pathway, is one such promising mechanism. It is characterized by extensive vacuolization of cytoplasmic organelles such as the endoplasmic reticulum (ER) and mitochondria, leading to cell death. This pathway is primarily triggered by ER stress, often caused by the accumulation of unfolded proteins and the overload of reactive oxygen species (ROS) or calcium (Ca2+) within the ER and mitochondria (Xu et al. 2024; Chang et al.2024).

Mitochondrial targeted curcumin or mitocurcumin (MitoC), a semi-synthetic derivative of curcumin, is known to selectively accumulate in cancer cell mitochondria (Reddy et al. 2014). MitoC exerts its anti-cancer activity in lung cancer by increasing mitochondrial reactive oxygen species (ROS) (Jayakumar et al. 2017). Given the central role of mitochondria in therapy resistance, mitochondrial targeting has emerged as a critical strategy in overcoming drug-refractory disease (Indran et al. 2011). Hence, our study aimed to investigate whether MitoC can induce an alternative form of cell death other than apoptosis in chemo-resistant NSCLC cells.

Materials and methods

Cell culture

A549 cell line was purchased from National Centre for Cell Science (NCCS), Pune, India. A549R (cisplatin-resistant cells) were developed as described previously (McDermott et al. 2014). Cells were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (Gibco, ThermoFisher Scientific) and 1% antibiotics (Gibco) and incubated at 37 °C with 5% CO2.

Cell proliferation assay

Cell viability and proliferation were determined using the MTT (Sigma-Aldrich) assay. Approximately 4000 cells were seeded per well in a 96-well plate (Tarsons). Cells were treated with 5 µM mitoC and 10 µM Z-VAD-FMK for 24 h. The media were then replaced with a 0.5 mg/mL MTT (Sigma-Aldrich) solution and incubated for 3 h. After removing the MTT solution, DMSO was added and incubated for 20 min at 37 °C. Absorbance was measured at 570 nm, using a microplate reader (Biotek).

CellTiter-Glo assay

Intracellular ATP levels were determined using the CellTiter-Glo luminescent assay. Briefly, cells were seeded in a white opaque 96-well plate (Corning) and treated with various concentrations of mitoC for 24 h. Following treatment, 100 μl of CellTiter-Glo reagent (Promega) was directly added to the cells cultured in 100 μl of growth medium and incubated for 30 min at room temperature with agitation. Luminescence intensity was measured using the Cytation 5 multimode reader (BioTek).

Transmission electron microscopy

For transmission electron microscopy, cells were treated with 5 µM mitoC or 10 µM curcumin for 24 h. Following this, cell pellets were initially fixed with 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Post-fixation was performed using 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 4 °C. The samples were then dehydrated in an ethanol series, embedded in Araldite resin, and polymerized for 24 h at 70 °C. Ultrathin Sects. (50–70 nm) were cut with a Leica UC7 ultramicrotome, mounted on 300-mesh copper grids, and stained with 10% uranyl acetate and lead citrate for enhanced contrast. Imaging was conducted on a JEOL 1400 plus transmission electron microscope at 120 kV with iTEM software and a Tengra camera. Analysis was done using the ImageJ software.

RNA sequencing

For transcriptomic analysis, RNA was extracted from untreated cells and cells treated with 5 µM mitoC for 24 h using the RNeasy mini kit (QIAGEN). To ensure quality, RNA concentration was measured using the Qubit RNA BR Assay (Invitrogen), and RNA integrity was assessed with RNA ScreenTapes (Agilent). Only samples with a RIN value > 7 were advanced to library preparation. and subsequently sequenced on the NovaSeqX Plus 25B platform, with a target of 60 million reads per sample. Following sequencing, data quality was checked with FastQC, and adapters were trimmed using fastq-mcf and cutadapt. High-quality reads were then aligned with the STAR aligner. After filtering out reads mapping to ribosomal and mitochondrial genomes, gene expression was quantified by estimating raw read counts with featureCounts and the data was normalized with DESeq2. Gene expression values were estimated in FPKM using cufflinks. Finally, DESeq2, a Bioconductor package in R, was used to perform the differential expression analysis.

Western blotting

Paraptosis protein markers were evaluated by treating NSCLC cells with various concentrations (1, 2.5, and 5 μM) of mitoC or curcumin (10 μM) for 24 h and with 5 μM mitoC at different time points (0, 6, 12, and 24 h). Further, cells were pretreated with 4 mM N-acetyl cysteine (NAC, Sigma-Aldrich) or 10 µM SP600125 (Sigma-Aldrich) for 2 h and then with 5 µM mitoC alone or in combination for 24 h. Following treatment, protein lysates were prepared from cells using RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitors (ThermoFisher Scientific). Protein concentration was determined via the Bradford assay (Sigma-Aldrich). A total of 20 µg of protein from each sample was separated using 10% polyacrylamide gels and subsequently transferred to PVDF membranes (Cytiva). The membranes were blocked for 1 h in 5% skim milk (HiMedia) before being incubated overnight at 4 °C with the appropriate primary antibodies listed in Table 1. Following washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Blots were developed using an ECL Select™ Western Blotting Detection Reagent (Cytiva) and imaged with a ChemiDocTMMP Imaging System (BioRad). Densitometric quantification of the bands was performed with Image Lab software (BioRad).

Table 1.

List primary western blotting antibodies and dilutions used

Antibody Dilution Cat no. (company)
Alix 1:1000 92,880 (Cell Signaling Technology)
P62/SQSTM1 1:1000 39,749 (Cell Signaling Technology)
CHOP 1:1000 2895 (Cell Signaling Technology)
BiP 1:1000 3177 (Cell Signaling Technology)
JNK 1:1000 9252 (Cell Signaling Technology)
P-JNK (Thr183/Tyr185) 1:1000 4668 (Cell Signaling Technology)
Beta-actin 1:2000 4970 (Cell Signaling Technology)
Phospho-ubiquitin (Ser65) 1:1000 62,802 (Cell Signaling Technology)
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Flow cytometry

For calcium level measurements, cells were seeded in 6-well plates (HiMedia) and treated with different concentrations (1, 2.5, and 5 μM) of mitoC at 12 h and 24 h time points. Further, cells were treated with 5 µM mitoC alone or in combination with 10 µM MCU-i4 (MedChemExpress) or 10 µM SP600125 (Sigma-Aldrich) or 4 mM NAC for 24 h. After incubation, cells were stained with Rhod-2 AM Dye (ThermoFisher Scientific) as per the manufacturer’s instructions. A minimum of 20,000 events per sample was analyzed by flow cytometry on an Attune NxT Flow Cytometer (Thermo Fisher), and data were analyzed using FlowJo software V.10.10.

Statistical analysis

All statistical analyses were conducted with GraphPad Prism Version 9. A Student’s two-tailed t-test was used for comparisons between two groups, while a one-way ANOVA was performed for comparing more than two groups. Statistical significance was established at a p-value < 0.05.

Results

MitoC disrupted the ultrastructure of mitochondria and ER in NSCLC cells

In terms of mechanism, mitoC is known to induce oxidative stress–driven apoptotic cell death in NSCLC (Jayakumar et al. 2017). Yet, its impact on intracellular organelle architecture following accumulation remains unclear. To address this, we examined ultrastructural changes in mitochondria and the ER of NSCLC cells using TEM after mitoC exposure. Our analysis revealed that mitoC induces pronounced swelling and dilation of mitochondria (Fig. 1a, ii and 1b, ii, black arrows) and ER (Fig. 1a, iv and 1b, iv, white arrows), leading to the formation of empty organellar vacuole-like structures termed vacuolation as compared to untreated A549 (Fig. 1a, i, iii) and A549R (Fig. 1b, i, iii) cells. Additionally, mitoC treatment led to disrupted mitochondrial cristae and the presence of dense intramitochondrial granules (Fig. 1a, ii and 1b, ii, yellow arrows). We have included the changes in mitochondrial and ER ultrastructure of A549 and A549R treated with curcumin as a positive control to confirm the paraptosis signature (Fig. 1a, v, vi and 1b, v, vi). Moreover, we investigated the cellular ATP levels of NSCLC cells following mitoC treatment. We observed that with increasing concentrations of mitoC, cellular ATP levels decrease in A549 and A549R cells, indicating functional disruption of mitochondria by mitoC (Fig. 1c–d). Collectively, these findings demonstrate that mitoC perturbs mitochondrial and ER ultrastructure and function in NSCLC cells.

Fig. 1.

Fig. 1

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Morphological changes in mitochondria and ER ultrastructure upon mitoC or curcumin treatment in NSCLC. a and b TEM images of a cross-section of A549 (a) and A549R (b) cells depicting swollen and dilated mitochondria ultrastructures (a, i, ii, v and b, i, ii, v, black arrows), accumulation of dark granules in mitochondria (a, ii, v and b, ii, v, yellow arrows) and swollen ER structures (a, iii, iv, vi and b, iii, iv, v, white arrows). Scale bar, 500 nm. c and d Quantification of cellular ATP levels by Rhod2-AM staining in A549 (c) and A549R (d) cells upon treatment with different concentrations of mitoC for 24 h

Transcriptomic profiling reveals increased expression of mitochondrial and ER stress–related genes in NSCLC cells following mitoC treatment

We next examined the impact of mitoC on cellular gene expression by performing whole-exome sequencing of A549 cells. Differential expression analysis identified the most significantly altered genes, with a heatmap of the top 50 displayed in (Fig. 2a). MitoC treatment notably influenced the expression of genes associated with mitochondrial and ER stress. Pathway enrichment analysis further revealed activation of ER stress pathways driven by the unfolded protein response (Fig. 2b), alongside suppression of key DNA replication–related pathways (Fig. 2c), implying a transition toward a sessile cellular phenotype. Together, these results indicate that mitoC transcriptionally reprograms mitochondrial and ER functions in NSCLC cells.

Fig. 2.

Fig. 2

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Transcriptomic profiling of mitoC-treated A549 cells revealed upregulation of mitochondrial and ER stress genes. a Heatmap illustrating the expression profiles of highly regulated genes related to ER and mitochondrial stress in control vs mitoC-treated cells. n = 3 independent biological replicates per group. b and c Pathway enrichment analysis highlighting significantly upregulated (b) and downregulated (c) biological processes in mitoC-treated A549 cells. (d) Log2 fold change expression of paraptosis-related genes analyzed from whole exome sequencing in mitoC-treated A549 cells. Abbreviations: SQSTM1, Sequestosome 1; PDCD6IP, programmed cell death 6 interacting protein; HSPA5, heat shock protein family A member 5; DDIT3, DNA damage-inducible transcript 3

MitoC-induced organellar stress associated with paraptosis markers in NSCLC

Structural changes in mitochondria and the ER, often linked to the activation of stress-related genes, are characteristic of paraptosis (Xu et al. 2024). To assess whether mitoC triggers organelle stress–mediated paraptotic death in NSCLC, we conducted transcriptomic profiling, which revealed increased expression of paraptosis-associated markers such as HSPA5 (BiP), DDIT3 (CHOP), and SQSTM1 (p62), alongside reduced expression of PDCD6IP (Alix) (Fig. 2d). These transcriptional alterations were further confirmed at the protein level by western blotting in both A549 and A549R cells. Consistently, mitoC enhanced BiP and CHOP expression while suppressing Alix (inhibitor of paraptosis) in a dose- and time-dependent manner (Fig. 3a–b). However, the induction of paraptosis markers was more prominent at higher concentrations and time points. Since disruption of proteostasis (protein homeostasis) is recognized as a key driver of paraptosis (Chang et al. 2024; Zhang et al. 2025), we next examined protein ubiquitination in NSCLC upon mitoC treatment. We found that mitoC increases poly-ubiquitination levels in A549 and A549R cells upon prolonged exposure (Fig. 3c). Moreover, we found curcumin (positive control) induced paraptosis markers in A549 and A549R cells (Fig. 3d), indicating the induction of true paraptosis phenotype by mitoC in NSCLC. Together, these findings demonstrated that mitoC induces paraptosis in NSCLC cells in both a dose and time-dependent manner.

Fig. 3.

Fig. 3

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MitoC-induced dose- and time-dependent expression of paraptosis markers in NSCLC. a Western blot analysis of dose-dependent induction of paraptosis markers in mitoC treated A549 and A549R cells. b Western blot analysis of time-dependent induction of paraptosis markers in A549 and A549R cells upon mitoC treatment (5 μM). c Immunoblotting analysis of protein ubiquitination levels in A549 and A549R cells treated with mitoC (5 μM) at different time points. d Immunoblotting analysis of induction of paraptosis marker upon curcumin treatment (10 μM) (positive control) in A549 and A549R cells. Abbreviations: mitoC, mitocurcumin; CHOP, C/EBP-homologous proteins; BiP, Binding immunoglobulin protein; Alix, Apoptosis linked gene 2-interacting protein X; JNK, Jun N-terminal Kinase

MitoC triggered organellar stress–associated paraptosis via ROS-dependent activation of the JNK signalling pathway in NSCLC

Previous studies have shown that mitoC promotes ROS-dependent activation of JNK in acute myeloid leukemia (Gaur et al. 2024), and enhanced JNK signalling has also been implicated in paraptosis (Chang et al. 2024). To explore whether mitoC induces paraptosis via JNK activation in NSCLC, we assessed JNK phosphorylation status. We observed that mitoC treatment increased phosphorylation of JNK at Thr183/Tyr185 in a dose- and time-dependent manner (Fig. 4a–b). In contrast, suppression of ROS with NAC or inhibition of JNK signaling using SP600125 attenuated mitoC-induced JNK phosphorylation in NSCLC cells, confirming ROS-mediated activation of the JNK pathway. Moreover, blockade of ROS or JNK signalling reversed the expression of paraptosis markers in A549 and A549R cells (Fig. 4c–d), underscoring the role of oxidative stress–driven JNK activation in paraptosis in NSCLC. MitoC is also known to trigger apoptosis in A549 cells (Jayakumar et al. 2017,); however, the contribution of paraptosis to its overall cytotoxicity remained unclear. To address this, we assessed mitoC-induced cytotoxicity in the presence of the pan-caspase inhibitor Z-VAD-FMK. Blocking apoptotic signaling did not rescue cell viability (Fig. 4e–f), suggesting the involvement of an alternative death pathway. These findings collectively suggested that mitoC drives paraptosis in NSCLC via ROS-induced JNK pathway activation.

Fig. 4.

Fig. 4

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MitoC-induced paraptosis via ROS-mediated JNK signalling in NSCLC. a Western blot analysis of the expression levels of p-JNK in A549 and A549R cells treated with different concentrations of mitoC. b Western blot analysis of p-JNK expression levels in A549 and A549R cells treated with mitoC (5 μM) at different time points. c Western blot analysis of key paraptosis markers in A549 and A549R cells treated with mitoC (5 μM) alone or in combination with NAC (ROS inhibitor) and SP600125 (JNK inhibitor) in A549 and A549R cells. d and e Percentage cell viability of A549 (d) and A549R (e) cells treated with mitoC alone or in combination with ZVAD-FMK (pan-caspase inhibitor). The data are presented as the mean ± SEM. Statistical significance was defined as a p < 0.0001 (#) or ns, non-significant. Abbreviations: mitoC, mitocurcumin; CHOP, C/EBP-homologous proteins; BiP, Binding immunoglobulin protein; Alix, Apoptosis-linked gene 2-interacting protein X; JNK, Jun N-terminal Kinase; ZVAD-FMK, pan caspase inhibitor

MitoC enhanced mitochondrial calcium overload through activation of the JNK signaling pathway in NSCLC

Mitochondrial swelling in paraptosis has been linked to intracellular calcium accumulation originating from the endoplasmic reticulum or cytosol, which drives excessive water influx into mitochondria (Chang et al. 2024). To determine whether mitoC elicits mitochondrial calcium in NSCLC, we assessed intracellular mitochondrial calcium levels following mitoC treatment at different time points. Flow cytometry analysis demonstrated that mitoC treatment markedly enhanced mitochondrial calcium uptake in A549 and A549R cells in a dose-dependent manner, profoundly at higher concentrations of mitoC (Fig. 5a–d). Pharmacological inhibition of the JNK signaling pathway with SP600125 alleviated this mitochondrial calcium overload. Likewise, blockade of the mitochondrial calcium uniporter (MCU) using MCUi4 effectively rescued mitoC-induced calcium accumulation, indicating involvement of MCU channels in mitoC-induced mitochondrial calcium uptake. Furthermore, scavenging intracellular ROS with NAC reduced mitochondrial calcium uptake under mitoC treatment in A549 and A549R cells (Fig. 5e–f), indicating involvement of ROS in mitochondrial calcium uptake. Collectively, these findings indicate that mitoC promotes mitochondrial calcium overload in NSCLC cells via MCU channels, primarily through activation of the JNK signaling cascade.

Fig. 5.

Fig. 5

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MitoC triggered mitochondrial calcium overload through activation of the JNK signalling pathway. a–d Quantification depicting changes in mitochondrial calcium at different concentrations and time points measured by Rhod-2 AM staining using flow cytometry in A549 (a and b) and A549R (c and d) cells. e and f Quantification depicting changes in mitochondrial calcium in A549 and A549R cells treated with mitoC alone (5 μM) or in combination with NAC, SP600125, and MCUi4 for 24 h. The data are presented as the mean ± SEM. Statistical significance was defined as a p < 0.05 (*) or p < 0.01 (**) or p < 0.001 (***) or p < 0.0001 (****) or ns, not significant. Abbreviations: mitoC, mitocurcumin; NAC, N-Acetyl Cysteine; MCUi4, Mitochondria calcium uniporter inhibitor; SP600125, JNK inhibitor

Discussion

Chemoresistance remains a major challenge in the treatment of non-small cell lung cancer (NSCLC), with resistance to apoptosis representing a central mechanism of therapeutic failure (Dhiman et al. 2025; Min and Lee 2021; Paul and Jones 2014). Therefore, the identification of alternative, non-apoptotic cell death pathways has gained increasing attention to thwart tumor survival strategies (Wang et al. 2022; Tait et al. 2014). Our study provides compelling evidence that mitocurcumin (mitoC) induces paraptosis in NSCLC cells, including cisplatin-resistant A549R cells.

Morphological and ultrastructural analyses revealed mitoC causes extensive endoplasmic reticulum (ER) and mitochondrial swelling, vacuolation, degeneration of mitochondrial cristae, and the presence of electron-dense deposits suggestive of calcium accumulation. The data are consistent with canonical descriptions of paraptosis (Xu et al. 2024), and further build upon earlier reports that emphasized apoptosis through mitochondrial ROS as the principal mechanism of mitoC cytotoxicity. Importantly, our findings revealed that mitoC activates a unique, caspase-independent mechanism, expanding its potential utility against tumors resistant to apoptosis.

With respect to mechanism, transcriptomic profiling of mitoC-treated NSCLC cells revealed robust activation of the unfolded protein response (UPR) and ER stress pathways, corroborating our morphological observations. Furthermore, mitochondrial calcium overload leading to paraptosis has been known previously and was recently reported for cannabinoids (Chang et al. 2024; de la Harpe et al. 2024). However, there is a dearth of studies supporting the mechanism underlying the calcium imbalance during paraptosis. Our study demonstrated activation of the ROS-mediated JNK signalling pathway leading to mitochondrial calcium overload via the mitochondrial calcium uniporter (MCU). While JNK signalling has been implicated in paraptosis in leukemia and hepatocellular carcinoma (Liu et al. 2021; Nakagawa and Maeda 2012), its role in NSCLC has remained poorly understood. Our findings therefore provide novel insights into the signalling circuitry underlying mitoC-induced paraptosis in NSCLC and highlight the involvement of ROS/JNK signalling in mitoC-induced mitochondrial calcium uptake (Fig. 6).

Fig. 6.

Fig. 6

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Proposed mechanism diagram of mitoC displaying induction of paraptosis in chemoresistance NSCLC. MitoC induce mitochondria and ER swelling and vacuolation in NSCLC. It upregulates mitochondrial and ER stress gene expression vis ROS mediated JNK activation. MitoC-induced JNK activation leads to mitochondrial calcium overload in NSCLC

The ability of mitoC to trigger paraptosis aligned with earlier studies on curcumin and its derivatives, such as dimethoxycurcumin and curcuminoid B63, which have been shown to induce paraptosis and proteasomal inhibition in breast and gastric cancer models (Yoon et al. 2010, 2012, 2014; Chen et al. 2019). These effects were mediated through ER stress, accumulation of polyubiquitinated proteins, mitochondrial calcium overload, and inhibition of thioredoxin reductase 1 (TrxR1). However, the clinical translation of curcumin-based therapies has been hampered by poor bioavailability (Dytrych et al. 2023; Yan et al. 2025). Recent research has shown that mitoC exhibits 25–50 times greater potency than curcumin, potentially overcoming the challenge of curcumin’s limited bioavailability (Jayakumar et al. 2017). The dual ability of mitoC to activate both apoptotic and non-apoptotic pathways represents a significant therapeutic advantage, offering a more comprehensive strategy to eliminate heterogeneous tumor cell populations.

Notably, our study demonstrates that mitoC-induced paraptosis was not restricted to treatment-naïve NSCLC cells but was also evident in cisplatin-resistant A549R cells, which is being reported for the first time. This suggests that mitoC can bypass conventional resistance mechanisms and may represent a promising therapeutic option for patients who have failed platinum-based regimens.

The study has few limitations. In-depth study is required to address the mechanism behind swelling and vacuolation of mitochondria and ER upon mitoC treatment in NSCLC. Moreover, pharmacological inhibition of MCU only suggests its involvement in mitochondrial calcium uptake. Knockdown and overexpression studies need to be conducted to establish the role of the MCU channel in the mitoC-induced paraptosis. Further, this study did not explore whether mitoC can induce other non-apoptotic death events other than paraptosis. Exploring this can highlight the more pleiotropic effect of mitoC. Moreover, to deepen the mechanistic insights of JNK regulation, the study warrants gene silencing experiments and to know which JNK phosphorylation is responsible for the induction of mitochondrial uptake. For future aspects, in vivo study needs to be done to further validate mitoC-induced paraptosis in NSCLC. In addition, a combinatorial therapeutic approach needs to be investigated to improve the translational impact of mitoC therapy.

Conclusions

In conclusion, our study establishes that mitoC induces organellar stress related to paraptosis in NSCLC cells through the ROS–mediated JNK signaling pathway. The ROS/JNK signaling is also responsible for inducing mitochondrial calcium uptake in NSCLC, which is one of the key events of paraptosis. Importantly, by activating a caspase-independent mechanism, mitoC potentially eludes one of the major therapeutic barriers in lung cancer, which is apoptosis resistance. Collectively, these findings position mitoC as a promising therapeutic candidate with the potential to overcome chemoresistance in NSCLC.

Acknowledgements

We thank BioRender for assistance in creating the illustration related to the mechanism of action of mitoC. We extend our gratitude to the intramural grant from the Department of Atomic Energy, India, for financial support, and finally, we would like to acknowledge ACTREC for the generous graduate fellowship awarded to Mr. Girish Ch. Panigrahi.

Author contributions

Vikram Gota and Girish Ch. Panigrahi conceptualized and designed the study. Girish Ch. Panigrahi, Amisha Joshi, and Dinky Malhotra carried out the experiments, analyzed the data, and prepared the figures. Girish Ch. Panigrahi and Amisha Joshi drafted the manuscript. Vikram Gota supervised the project and edited the manuscript. All authors approved the final manuscript. The authors declare that all data were generated in-house and that no paper mill was used.

Funding

Open access funding provided by Department of Atomic Energy. This study received an intramural grant from the Department of Atomic Energy, India (Grant number: 4598 C).

Data availability

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare that they have no competing interests.

Footnotes

This article is published as part of the special issue on “Innovative Therapy of Malignant Diseases.”

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

Articles from Naunyn-Schmiedeberg's Archives of Pharmacology are provided here courtesy of Springer

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