This study used an earlier characterized nigericin metabolite from a novel indigenous species of Streptomyces strain DASNCL-29, which was isolated from a plant-associated soil sample collected from Unkeshwar, Maharashtra, India. A seven-day log-phase culture of DASNCL-29 was used for fermentation to produce the bioactive compound nigericin. The fermentation was carried out in a 10-L lab-scale fermenter (Eppendorf BioFlow^® CelliGen^® 115, Hamburg, Germany). The harvested fermentation broth was used to extract the metabolite, and purification was carried out by gradient column chromatography and High-Performance Liquid Chromatography (HPLC, UltiMate 3000, ThermoFisher Scientific, Waltham, MA, USA). Further, structural elucidation was done with standard Nuclear Magnetic Resonance techniques (NMR) (Bruker Avance III HD 400 MHz spectrometer, Bruker BioSpin GmbH, Silberstreifen 4, 76287, Rheinstetten, Germany). The structural characteristics of purified nigericin were analyzed using spectral techniques, including Heteronuclear Multiple Bond Correlation (HMBC), Nuclear Overhauser Effect Spectroscopy (NOESY), and Correlation Spectroscopy (COSY) [35].

The anticancer effects of nigericin were evaluated against 17 cancer cell lines including U-937 (human myeloid leukemia), MDA-MB-468 (human breast cancer), HOP-62 (human lung cancer), SCC-40 (human oral squamous cell carcinoma), HL-60 (human leukemia), HT-29 (human colon cancer), COLO-205 (human colon cancer), HeLa (human cervical cancer), JURKAT (human T-cell leukemia), SiHa (human cervical cancer), DU-145 (human prostate cancer), Hep-G2 (human hepatocellular carcinoma), K-562 (human chronic myelogenous leukemia), MCF-7 (human breast cancer), A-549 (human lung carcinoma), PC-3 (human prostate cancer), and MDA-MB-231 (human breast cancer) with Vero cell line as a control. (Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai, India) using the sulforhodamine B (SRB) (0.4 % (w/v) in 1 % acetic acid) (Cat No. 230162, Sigma-Aldrich, St. Louis, MO, USA) assay. All the cell lines used in this experiment were authenticated using short tandem repeat (STR) profiling, and their mycoplasma contamination status was routinely monitored to ensure experimental validity. Cells were seeded in 96-well plates (5000 cells/well) in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine (SLM-240, Sigma-Aldrich), followed by 24 h incubation at 37°C and 5% CO₂. Nigericin and Doxorubicin, dissolved in DMSO (10⁻² M stock), were serially diluted to final concentrations of 100, 10, 1.0, 0.10 µM. After 48 h of treatment, cell viability was assessed using the SRB.

Percentage Growth was calculated using the equation PercentageGrowth(%)=[(Ti−Tz)C−Tz]×100 where C represents the control absorbance, Ti the test sample absorbance, and Tz the baseline (time zero) absorbance.

Synthetic compounds showing Growth inhibition of 50% (GI₅₀) (calculated from [(Ti-Tz)/(C-Tz)] × 100 = 50) ≤10 μg/mL were considered to be exhibiting significant anticancer activity.

The MOLM13 paired sensitive and venetoclax-resistant cell lines were procured from the European Institute of Oncology, Milan, Italy, and HL60 paired sensitive and Cytarabine-resistant cells from the University Hospital, Leipzig, Germany. The authentication of cell lines was confirmed through STR profiling in February 2024, with concurrent verification of mycoplasma contamination-free status. The procured cells were maintained in RPMI medium (Gibco, Grand Island, CA, USA) supplemented with 10% fetal bovine serum (Gibco) and penicillin-streptomycin (Himedia, Mumbai, India). The cultures were incubated under standard conditions, and subculturing was performed every three to five days once the cells reached 60%–80% confluency.

1 × 10⁴ MOLM13 sensitive and venetoclax-resistant and 1 × 10⁴ HL60 sensitive and cytarabine-resistant cells were seeded per well in a 96-well plate, with different concentrations (0, 12.5, 25, 50, 100, and 200 nM) of nigericin. The plates were incubated for 48 h under standard conditions. After the incubation period, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Himedia) at 1 mg/mL. The plates were further incubated for an additional 5 h to allow formazan crystal formation. The crystals were solubilized using a solubilizing buffer (Isopropanol, Triton X-100, and concentrated HCl), and the plate was left overnight at 37°C to ensure complete solubilization of the crystals. Absorbance was recorded at 570 nm using a microplate reader (BioTek, Cytation 5, Winooski, VT, USA). The absorbance readings in each treatment group were normalized to the untreated group to acquire percentage viabilities. The concentrations were transformed into a logarithmic scale, and a non-linear regression curve for log (inhibitor) vs. normalized response-variable slope was used to calculate the IC_50.

MOLM13 sensitive cells were labelled with the membrane-permeable fluorescent dye Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) (Cat No. C34554, Invitrogen, ThermoFisher Scientific) prior to nigericin treatment. CFSE (2 µM in DMSO) was freshly diluted 100-fold in 1× PBS (pH 7.4), and the cells were incubated with the dye for 10 min in the dark at room temperature. Complete RPMI medium was added to terminate the reaction, followed by incubation at 37°C for 10 min. Afterward, 1.0 × 10⁶ cells/mL in fresh RPMI medium were harvested and seeded. After a 24-h incubation, the cells were washed with PBS, resuspended, and analysed by flow cytometry using the Attune NxT Flow Cytometer (ThermoFisher Scientific).

1 × 10⁶ MOLM13 sensitive (with or without 5 µM. MitoTempo (MedChemExpress, Cat No. HY-112879, Monmouth Junction, NJ, USA) pretreatment) and MOLM13 and venetoclax-resistant cells were seeded in a 6-well plate with 5 mL of complete RPMI and treated with different concentrations of nigericin. After a 24-h incubation, the cells were harvested, washed with 1× PBS (pH 7.4) and resuspended in 1X annexin binding buffer and stained with Annexin V conjugated with Alexa Fluor 488 and Propidium Iodide (Cat No. V13241, Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions and acquired on an Attune NxT Flow Cytometer.

MOLM13 sensitive and venetoclax-resistant cells were treated with nigericin (20, 40, and 80 nM) for 24 h, then harvested and lysed using RIPA buffer (Cat No. R0278, Sigma-Aldrich) on ice for 45 min. Protein concentration was measured using the Bradford reagent (Himedia) according to the manufacturer’s instructions. A total of 40 µg of protein was loaded onto 10% or 12% SDS-PAGE gels, resolved, and transferred to nitrocellulose membranes (Cytiva, Marlborough, MA, USA). The membranes were blocked with 5% skimmed milk (Himedia) or BSA (Himedia), incubated overnight with primary antibodies including Succinate Dehydrogenase [Ubiquinone] Flavoprotein Subunit A (SDHA) (Cat No. PA5-27482, dilution 1:1000, ThermoFisher Scientific), Full length PARP (Cat No. 9532, dilution 1:1000, Cell Signaling Technology, Danvers, MA, USA), Cleaved PARP (Cat No. 32563, dilution 1:1000, Cell Signaling Technology), Cleaved Caspase-3 (Cat No. 9664, dilution 1:1000, Cell Signaling Technology), γH2AX (Cat No. 2577, dilution 1:1000, Cell Signaling Technology), MCL-1 (Cat No. 94296, dilution 1:1000, Cell Signaling Technology) at 4°C overnight, and then incubated with HRP-conjugated secondary antibody (Cat No. 7074, dilution 1:3000, Cell Signaling Technology) at room temperature for 1 h. Unbound primary and secondary antibodies were washed with TBS-T buffer (1M Tris pH 8, 2.5M NaCl, Tween 20, and double-distilled water) thrice for 15 min. After washing, protein bands were visualized using Cytiva ECL select™ western blotting detection reagent (Cat. No. RPN2235) and Bio-Rad Chemidoc system, California, USA (12003153).

Approximately 2 × 10⁶ MOLM13 sensitive cells, both untreated and 100 nM nigericin-treated, were used for protein isolation. The cells were lysed in a native buffer containing 20 mM Tris, 150 mM NaCl, 10% glycerol, and 0.1% NP-40, adjusted to pH 7.5. Following lysis, proteins were subjected to reduction with 10 mM Dithiothreitol (DTT) and alkylation using 20 mM iodoacetamide (IAA). Subsequent to reduction and alkylation, the proteins were digested overnight with trypsin at 37°C. The completeness of digestion was verified by SDS-PAGE. The digested samples were then cleaned using C18 ZipTips (Cat No. ZTC18S096, Merck Millipore, Burlington, MA, USA) prior to analysis by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) (nLc-ESI-Q-TOF, Triple TOF 5600 plus, SCIEX, Framingham, MA, USA).

Mitochondrial ROS levels were quantified using the MitoSOX™ Red probe (Cat No. M36008 ThermoFisher Scientific). MOLM13 sensitive and venetoclax-resistant cells (1 × 10⁶ cells/well) were treated with 20, 40, and 80 nM concentrations of nigericin or left untreated as a control, in a 6-well plate. After 24 h of incubation, the cells were harvested, washed with PBS and stained with 5 μM of MitoSOX Red for 15 min in the dark. The cells were then washed with PBS and analysed using flow cytometry.

1 × 10⁶ MOLM13 sensitive and venetoclax-resistant cells were seeded in a 6-well plate. After 24 h of incubation, the cells were harvested, washed with 1× PBS (pH 7.4), and stained with JC-1 probe (Cat No. HY-15534, MedChemExpress) according to the manufacturer’s instructions. The cells were then washed with 1× PBS (pH 7.4) and analysed using an Attune NxT flow cytometer.

1 × 10⁶ MOLM13 sensitive cells were seeded and treated with nigericin for 24 h. Post-treatment, the cells were collected and washed with 1× PBS (pH 7.4). Subsequently, the cells were incubated with 50 nM Mitotracker Green (Cat No. M6514, Invitrogen, ThermoFisher Scientific) at 37°C for 30 min. Following this staining, the cells were washed with PBS and then stained with Hoechst 33342 (Cat No. 62249, Invitrogen, ThermoFisher Scientific) for 10 min at 37°C. Finally, the cells were transferred to poly-L-lysine-coated glass-bottom dishes and visualized using a Nikon AX Eclipse Ti2 confocal microscope (New York, NY, USA). The mean fluorescence intensity of at least 30 cells per concentration was measured using the Image J version 1.54 g software, National Institutes of Health (Bethesda, MD, USA).

The data are represented as mean ± standard deviation (SD) unless otherwise specified. Statistical analyses were conducted using GraphPad Prism (version 8.4.2 (679), 8 April 2020, San Diego, CA, USA). An unpaired Student’s t-test was employed to evaluate differences in cell apoptosis, proliferation, mitochondrial ROS production, mitochondrial membrane potential, and mitochondrial mass. For cell viability assessments, non-linear regression analysis was applied, while simple linear regression was used for the Bradford assay. A significance level of p < 0.05 was considered statistically significant.

Submerged fermentation of Streptomyces strain DASNCL-29 yielded 25.0 g of a pure white crystalline compound from 75.0 g of crude extract, which translates to a 33% (w/w) yield. Further, the structural characteristics analysis via the mass spectrum of nigericin from strain DASNCL-29 supported its structural integrity, in comparison with the standard nigericin.

The in vitro cytotoxicity of nigericin was evaluated using the sulforhodamine B assay across seventeen human cancer cell lines representing various histological origins. Comprehensive anticancer screening data are presented in Fig. A1. The anticancer potential of nigericin, including its effects on AML cells, was investigated (Table A1). Notably, nigericin induced morphological changes, including cell shrinkage and detachment, as illustrated in Fig. A2, indicative of cell death. These findings emphasize nigericin’s ability to disrupt cellular viability in AML.

MOLM13 sensitive and venetoclax-resistant cells, and HL60 sensitive cells and cytarabine-resistant cells were treated with increasing concentrations of nigericin (12.5–200 nM) for 48 h, and an MTT assay was performed. The results indicated that nigericin reduced the viability of the treated AML cells in a dose-dependent manner, with IC₅₀ values of 57.02 and 35.29 nM in MOLM13 sensitive and venetoclax-resistant cells and 20.49 and 1.197 nM in HL60 sensitive cells and cytarabine-resistant cells (Fig. 1A). Further, MOLM13-sensitive and Venetoclax-resistant cells were incubated with different concentrations of nigericin for 24 h, and an Annexin V/PI assay was done. Nigericin was found to significantly increase the percentage of apoptotic cells (early and late) in a dose-dependent manner, whereas the percentage of necrotic cells was not significantly altered (Fig. 1B). Additionally, immunoblotting was performed to validate the induction of apoptosis. Upregulation of apoptotic markers, including cleaved PARP, cleaved caspase-3, and subsequent downregulation of full-length PARP and pro-survival protein MCL-1, was observed. Also, DNA double-strand breakage was confirmed by the upregulation of the protein γH2AX with increasing concentrations of nigericin (Fig. 1C). Next, the CFSE assay was done to check the effect of nigericin on AML cell proliferation. MOLM13 sensitive cells were stained with CFSE dye and treated with or without nigericin for 48 h. The results indicated a reduction in the percentage of cells divided in the 100nM nigericin-treated group, and a peak shift was observed in the control group, compared to nigericin-treated MOLM13 sensitive cells, reflecting the reduced proliferation of treated cells (Fig. 1D).

Proteomic profiling of MOLM13 sensitive cells treated with 100 nM nigericin identified 785 proteins, of which 264 were significantly dysregulated (p < 0.05) compared to controls. Among these, 115 proteins were upregulated, with 45 showing a fold change greater than 5.0, while 149 proteins were downregulated, with 39 exhibiting a fold change less than 0.2. Six proteins were further selected based on log-fold changes and statistical significance (Table 1). Among these, Succinate Dehydrogenase [Ubiquinone] Flavoprotein Subunit A (SDHA), an essential enzyme in the mitochondrial respiratory chain, was found to be upregulated and stood out as the protein of interest due to the previously reported role of nigericin to induce mitochondrial dysfunction in breast cancer cells. Further, immunoblotting revealed an upward trend in SDHA expression in nigericin-treated cells, though the increase did not reach statistical significance (Fig. 2).

Mitochondrial ROS levels were quantified in MOLM13 sensitive and venetoclax-resistant cells treated with nigericin for 24 h using the MitoSOX Red probe, which selectively detects mitochondrial superoxide production. The results demonstrated a significant increase in mitochondrial ROS, indicating elevated oxidative stress with increasing nigericin concentrations (Fig. 3A). Subsequently, mitochondrial membrane potential (Δψm) was assessed in both MOLM13-sensitive and Venetoclax-resistant cells using the JC-1 probe. Under normal conditions, JC-1 accumulates within mitochondria and forms red-fluorescent J-aggregates, indicative of an intact membrane potential. In contrast, a shift to green fluorescence occurs when Δψm is disrupted, as JC-1 remains in its monomeric form. Our findings revealed a concentration-dependent shift from red to green fluorescence, confirming that nigericin progressively decreases mitochondrial membrane potential (Fig. 3B). Finally, mitochondrial mass was evaluated in MOLM13 sensitive cells via confocal microscopy using the MitoTracker Green probe. A substantial reduction in mitochondrial mass was observed with increasing nigericin concentrations, further supporting the evidence of mitochondrial dysfunction at higher nigericin concentrations (Fig. 3C).

An apoptosis assay was performed using Annexin V/PI staining in MOLM13 sensitive cells. They were pretreated with or without 5 µM MitoTempo, a mitochondria-targeted ROS scavenger, followed by treatment with different concentrations of nigericin. Pretreatment with MitoTempo was found to attenuate nigericin-induced cell death, with a marked reduction in the percentage of dead cells compared to cells treated with nigericin alone. This functional linkage demonstrates that mitochondrial ROS elevation is one of the mechanistic drivers of apoptosis induction by nigericin in AML (Fig. 4).