Nutrient deprivation enhances the cytotoxic effects of a sulfonamide-chalcone hybrid in PC-3 prostate cancer cells

Reagents and chemicals

SSC185 was synthesized by Claisen-Schmidt condensation at the Chemical Institute, Federal University of Goiás (UFG), Goiânia, Brazil. Its structure was elucidated through high-resolution mass spectroscopy and nuclear magnetic resonance (¹H and ¹³C NMR), as previously reported by [31, 32]. Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) were obtained from Exodo científica (Sumaré, SP, Brazil). The Panoptic staining kit was obtained from Laborclin^® (Pinhais, PR, Brazil). Neutral red was obtained from Inlab (São Paulo, SP, Brazil). Primary antibodies against phosphorylated PARP1 (#9542, RRID: AB_2160739), phospho-AKT (#4060, RRID: AB_2315049), AKT (#4691, RRID: AB_915783), phospho-RPS6 (#4858, RRID: AB_916156), RPS6 (#2217, RRID: AB_331355), phospho-4E-BP1 (#9455, RRID: AB_560835), 4E-BP1 (#9452, RRID: AB_331692), phospho-ERK1/2 (#9101, RRID: AB_331646), ERK1/2 (#9102, RRID: AB_330744), SQSTM1/p62 (#88588, RRID: AB_2800197), LC3B I/II (#2775, RRID: AB_915950), α-tubulin (#2144, RRID: AB_2210548), and GAPDH (#2118, RRID: AB_561053) were obtained from Cell Signaling Technology (Danvers, MA, USA). Secondary anti-rabbit (#7074, RRID: AB_2099233) and anti-mouse (#7076, RRID: AB_330924) antibodies conjugated with horseradish peroxidase were also obtained from Cell Signaling Technology (Danvers, MA, USA). Chloroquine (CQ), pan-caspase inhibitor Quinoline-Val-Asp-Difluorophenoxymethylketone (Q-VD-OPh), cycloheximide (CHX), N-acetylcysteine (NAC), curcumin, L-cysteine, and DTNB were purchased from Sigma (St. Louis, MO, USA). Propidium iodide and Rhodamine 123 were obtained from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA).

Cell lines and culture

The PC-3 cell line (RRID: CVCL_0035), obtained from the American Type Culture Collection (ATCC, CRL-1435), was maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Capricorn Scientific, Ebsdorfergrund, HE, Germany) and 1% penicillin (100 U/mL)-streptomycin (100 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA), in a humidified atmosphere containing 5% CO₂ at 37 °C. The deprivation of FBS induced nutrient deprivation of the PC-3 cell line for 24 h. Cells were seeded overnight, and the supplemented RPMI medium (FBS 10%) was removed. The cells were washed with Phosphate-buffered saline (PBS), and RPMI medium without supplementation was added 24 h before the assays.

MTT assay

Cell viability was assessed indirectly using the MTT reduction assay [33]. PC-3 cells were seeded in 96-well plates at a density of 5 × 10³ cells/well and allowed to adhere for 24 h. The cells were then treated with increasing concentrations ranging from 0.02 to 100 µM of SSC185 as an initial screening to determine IC₅₀ values at 72 h following [34]. After determining the IC₅₀ value at 72 h, the 24 h time point was evaluated, with concentrations ranging from 1.6 to 100 µM, to assess the effects of compound exposure over time under standard culture conditions (with FBS) or serum deprivation, in which cells were deprived of FBS for 24 h before treatment. For the inhibition studies, cells were pretreated for 4 h with CQ, Q-VD-OPh, CHX, or NAC. After pretreatment, the inhibitors were removed, and the cells were exposed to the compound for 24 h, both under standard conditions and under serum deprivation conditions. Control groups treated only with inhibitors were included. At the end of exposure to the compound, MTT (0.5 mg/mL) was added, and the cells were incubated for 3 h at 37 °C. Formazan crystals were solubilized with DMSO, and absorbance reading was performed in a microplate reader (Spectramax 190, Molecular devices, CA, USA) at 595 nm.

Intermittent exposure assay

For intermittent SSC185 exposure, the MTT assay was used. The assay was conducted as described previously [35]. PC-3 cells (5 × 10³ cells/well) were seeded in a 96-well plate. Cells were exposed to SSC185 (1.6 to 100 µM) for 3, 6, 12, or 24 h. After each predetermined time point, the medium containing SSC185 was removed, and the wells were washed with PBS, followed by incubation in compound-free media for 72 h. The MTT solution (0.5 mg/mL) was added to each well and incubated for 3 h. The MTT-formazan product dissolved in DMSO was quantified by measuring absorbance at 595 nm using a microplate reader Spectramax 190 (Molecular devices, CA, USA).

Colony formation assay

The clonogenic assay was conducted as described previously [34]. 500 cells per well of the PC-3 cell line were seeded in sterile 6-well plates. Concentrations of 4.8, 9.5, 19, 38, and 76 µM of SSC185 were added to the corresponding wells for 6 and 24 h. After the designated time, the cells were washed with PBS, and the medium was promptly replaced with compound-free RPMI for 8 days. The cells were then washed with PBS and stained with a solution of methanol and 1% crystal violet for 5 min. Images were acquired using the G: BOX Chemi XRQ (Syngene, Cambridge, UK) and analyzed using the “ColonyArea” plugin in ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cell morphology analysis

Cell morphology was assessed by optical microscopy. PC-3 cells were seeded at a density of 0.5 × 10⁵ cells per well in 24-well plates and allowed to adhere for 24 h. The cells were then treated with SSC185 at concentrations of 19 and 38 µM, using CQ (100 µM) as a control. After 6 h of exposure, the cells were either examined directly by optical microscopy or stained with the Panoptic rapid kit [9] or Neutral Red (40 µg/mL) [36], as previously described, to visualize morphological changes and intracellular vacuolization. Images were acquired using a Zeiss Primovert inverted microscope (Oberkochen, BW, DE) equipped with a 200× total magnification objective.

Cell cycle distribution and plasma membrane integrity analysis

Cell cycle and plasma membrane integrity analysis was performed by flow cytometry with propidium iodide (PI) DNA labeling following [37]. PC-3 cells were seeded in 12-well plates at a density of 1 × 10⁵ cells per well. Cells were exposed to SSC185 (19 µM and 38 µM) under standard conditions for 6 and 24 h or (4 µM) under starvation conditions for 24 h. The pellets were resuspended in 1 mL of PBS. A 200 µL aliquot of the cell suspension, both exposed and unexposed to the compound, was incubated with 200 µL of lysis solution (0.1% sodium citrate, 0.1% Triton X-100, and 2 µg/mL PI in PBS) for 30 min in the dark to analyze the distribution of DNA content. For the analysis of plasma membrane integrity, PC-3 cells were collected, washed with PBS, and incubated with PI at a final concentration of 25 µg/mL for 10 min at room temperature, protected from light. After incubation, the samples were analyzed immediately. Cells were considered PI-negative when they exhibited preserved plasma membrane integrity. Analyses were performed using a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA) and the data were analyzed using FlowJo v.X.0.7 software (Treestar, Inc., San Carlos, CA, USA).

Mitochondrial depolarization analysis

Mitochondrial depolarization analysis was performed by flow cytometry using Rhodamine 123 (Rhd 123), as described previously [38]. Briefly, PC-3 cells were seeded in 12-well plates at a density of 1 × 10⁵ cells per well. Cells were exposed to SSC185 (19 µM and 38 µM) under standard conditions for 6 and 24 h or (4 µM) under starvation conditions for 24 h. The pellets were resuspended in 1 mL of PBS. A 500 µL aliquot of cell suspensions exposed and unexposed to the compound was centrifuged in PBS for staining. Staining was performed with Rhd 123 added to the cell suspension at a final concentration of 1 µM. Samples were incubated for 15 min in the cold, protected from light. After incubation, the cells were washed with cold 1× PBS and centrifuged (2,000 rpm/5 minutes) to remove excess dye and resuspended in 500 µL of PBS for acquisition. The acquisition was performed using a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA), and the data were analyzed using the FlowJo software v.X.0.7 (Treestar, Inc., San Carlos, CA, USA).

Acidic vesicular organelles (AVOs) stained by acridine orange

The assessment of acidic vesicular organelles was performed as previously described [39]. PC-3 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells per well. Cells were exposed to SSC185 (19 µM and 38 µM) under standard conditions for 24 h or (4 µM) under nutrient deprivation conditions for 24 h. Cells were washed with PBS and incubated with a solution containing 0.1 µg/mL acridine orange (AO) (Sigma-Aldrich) in PBS for 15 min. After staining, cells were analyzed using GFP and RFP channels on a Lionheart FX automated fluorescence microscope (Agilent BioTek Instruments Inc., Santa Clara, CA, USA) at 200× magnification.

Western blotting

Protein expression was analyzed by Western blotting, as described previously [40]. Total protein extraction from PC-3 prostate cancer cells was performed using a buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 2 mM PMSF, 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, and 4 mM EDTA, from cells with or without exposure to SSC185 (9, 5, 19, and 38 µM) for 24 h. The extracted proteins were quantified using the Bradford method. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (30 µg), the proteins were transferred to nitrocellulose membranes (GE Healthcare, Chicago, IL, USA) and detected by incubating the membranes with specific primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected using the SuperSignalWest Dura Extended Duration substrate system (Thermo Fisher Scientific, Inc.) and a G: BOX Chemi XX6 gel documentation system (Syngene). Protein bands were normalized relative to tubulin or GAPDH.

Thiol reactivity assay

The thiol reactivity of SSC185 was evaluated using L-cysteine and the DTNB/Ellman reagent, as previously described [41], with minor modifications. SSC185 or curcumin (25 or 50 µM) was incubated with L-cysteine (250 µM) in 50 mM phosphate buffer, pH 7.4, at room temperature, in 96-well plates, for 0, 5, 10, 15, 30, 60, and 120 min. After each incubation period, DTNB was added to a final concentration of 500 µM, and the mixture was incubated for 5 to 10 min, protected from light. Absorbance was measured at 405 nm using a microplate reader (DR-200BN-BI, Kasuaki, Brazil distributor).

Wells containing only L-cysteine were used as free thiol controls with their respective incubation times, while wells containing phosphate buffer and DTNB were used for background signal correction. Additional wells containing SSC185 or curcumin, in the absence of L-cysteine, were included to correct for the intrinsic absorbance of the compound. Curcumin was used as an electrophilic reference compound. The remaining free L-cysteine was expressed as a percentage relative to the corresponding L-cysteine control, according to the following equation:

Statistical analysis

The statistical studies were performed using GraphPad Prism (Institutive Software for Science, San Diego, California, USA). MTT signal was expressed as a percentage of untreated controls, and IC₅₀ values were obtained by non-linear regression. The data were compared using analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Data are representative of three independent experiments. Bars, Standard Error Mean (S.E.M.); *p < 0.05, **p < 0.01, ***p < 0.001.

SSC185 reduces the MTT signal and clonogenic capacity of PC-3 cells in a time- and concentration-dependent manner

To investigate the biological effects of SSC185 in androgen-independent PC-3 prostate cancer cells, we first evaluated its effects using the MTT reduction assay. SSC185 reduced the MTT signal with an IC₅₀ of 19 µM after 72 h of continuous exposure (Fig. 1b). When PC-3 cells were treated for 24 h, SSC185 reduced the MTT signal in a concentration-dependent manner, with an IC₅₀ of 24.69 µM (Fig. 6a), indicating that the compound exerts a relevant effect in the first 24 h of exposure.

To further characterize the temporal dependence of SSC185 activity, an intermittent exposure assay was performed in which PC-3 cells were exposed to SSC185 for 3, 6, 12, or 24 h. The medium containing the compound was then replaced with compound-free medium until a total incubation time of 72 h was reached. The MTT signal was then quantified. Short exposure intervals of 3 and 6 h required markedly higher concentrations of SSC185 (IC₅₀ > 50 µM) to reduce the MTT signal (Fig. 1c, d). In contrast, exposures of 12 h and 24 h resulted in IC₅₀ values of 35.68 µM and 22.31 µM, respectively (Fig. 1c, d). These results indicate that the effect of SSC185 on MTT reduction depends on both exposure time and concentration, with longer exposure resulting in lower IC₅₀ values.

The long-term proliferative potential of PC-3 cells after transient exposure to SSC185 was evaluated using a clonogenic assay. Cells were treated with SSC185 (4.8, 9.5, 19, 38, and 76 µM) for 6–24 h, after which the medium was replaced with compound-free medium and colonies were allowed to form. The percentage of colony area was then quantified. After 6 h of treatment, a significant reduction in colony formation was observed only at 38 and 76 µM (p < 0.001), whereas no significant reduction was detected at 4.8, 9.5, or 19 µM (Fig. 1e).

In contrast, after 24 h of exposure, a significant reduction in colony formation was observed across a broader concentration range. Even the lowest concentration tested (4.8 µM) significantly reduced colony formation (p < 0.001), while complete suppression of colony formation was observed at concentrations ≥ 19 µM (p < 0.001; Fig. 1f).

SSC185 promotes morphological changes and reduces plasma membrane integrity

Next, the initial effects of SSC185 on the morphology and plasma membrane integrity of PC-3 cells were evaluated during the first 6 h of exposure. Plasma membrane integrity was assessed based on the proportion of PI− cells, which correspond to cells retaining the ability to exclude propidium iodide. Neutral red and rapid panoptic staining revealed concentration-dependent morphological changes. Treatment with SSC185 at 19 µM (IC₅₀) induced cytoplasmic vacuolization (Fig. 2a) and reduced the proportion of PI-negative (PI−) cells to 52.80% (p < 0.001; Fig. 2b), compared with 89.95% in the control group. At 38 µM (2× IC₅₀), cells exhibited retraction and prominent cytoplasmic vacuoles with intense basophilia (Fig. 2a), accompanied by a further reduction in the PI− population to 31.13% (p < 0.001; Fig. 2b). These findings indicate a concentration-dependent reduction in the proportion of cells retaining plasma membrane integrity. Although cytoplasmic vacuolization was observed at both 19 and 38 µM, the neutral red staining pattern differed from that observed following chloroquine treatment (CQ, 100 µM), indicating distinct patterns of vacuolar staining under these experimental conditions (Fig. 2a).

After 24 h of treatment, the proportion of PI− cells was reduced to 57.68% following exposure to 19 µM SSC185 (p < 0.001) and to 16.27% following exposure to 38 µM SSC185 (p < 0.001), compared with 94.47% in the control group (Fig. 2c). The marked reduction in the PI− population at 38 µM indicates a pronounced loss of plasma membrane integrity after prolonged exposure.

SSC185 induces time‑ and concentration‑dependent cell cycle alterations

Given that SSC185 impaired viability, morphology, and membrane integrity, its impact on cell cycle progression was evaluated in PC‑3 cells. Flow cytometry analysis demonstrated time- and concentration-dependent alterations in cell cycle distribution. After 6 h of treatment, SSC185 reduced the G₀/G₁ fraction and increased the proportion of cells with 4 C DNA content, consistent with accumulation in the G₂/M phase. The proportion of cells in G₂/M increased to 26.13% ± 1.00% at 19 µM and 23.93% ± 0.94% at 38 µM (p < 0.001), compared with 19.85% ± 0.39% in control cells (Fig. 3a). Correspondingly, the G₀/G₁ population decreased from 40.27% ± 1.31% in control cells to 31.04% ± 1.78% and 32.70% ± 1.72% at 19 and 38 µM, respectively (p < 0.01; Fig. 3a).

After 24 h, the effects diverged according to concentration. At 19 µM, the proportion of cells in G₂/M remained elevated and further increased to 40.68% ± 2.01% (p ≤ 0.01; Fig. 3b). In contrast, at 38 µM, cells shifted predominantly to G₀/G₁ (46.47% ± 3.01%, p ≤ 0.01), accompanied by a reduction in the S‑phase fraction (17.79% ± 1.64%, p ≤ 0.01; Fig. 3b). Notably, the sub‑G₀/G₁ population already increased within the first 6 h at 38 µM, from 7.76% ± 0.50% in control cells to 11.21% ± 1.08% (p < 0.05), suggesting early onset of cell death-associated DNA fragmentation at the higher concentration.

Together, these data indicate that prolonged exposure to 19 µM SSC185 is associated with a sustained increase in the proportion of cells in G₂/M, whereas treatment with 38 µM produces a distinct cell-cycle distribution characterized by increased G₀/G₁ and reduced S-phase fractions.

Exposure to SSC185 does not induce accumulation of acridine orange-stained acidic vesicles

Based on the cytoplasmic vacuolization observed after SSC185 treatment, acidic vesicular organelles were evaluated by acridine orange fluorescence microscopy. PC-3 cells were exposed to SSC185 at 19 and 38 µM for 24 h, while chloroquine (100 µM) was included as a reference condition (Fig. 4a). Quantitative analysis of the mean fluorescence intensity in the RFP channel showed no significant increase in SSC185-treated cells compared with the untreated control (p > 0.05; Fig. 4b). In contrast, chloroquine treatment produced a significant increase in RFP fluorescence (p < 0.01), indicating a greater accumulation of acidic vesicular compartments under this condition.

The expression of the autophagy-related proteins LC3B and SQSTM1/p62 was subsequently evaluated by Western blotting. SSC185 treatment significantly increased LC3B-I levels (p < 0.01; Fig. 4c), whereas no detectable alteration in LC3B-II levels was observed. Concurrently, SQSTM1/p62 expression was significantly increased following SSC185 exposure (p < 0.01; Fig. 4d). Collectively, these results demonstrate that SSC185 induces changes in autophagy-related markers characterized by increased LC3B-I and SQSTM1/p62 levels, without detectable LC3B-II accumulation or an increase in acridine orange-stained acidic vesicles under the experimental conditions evaluated.

SSC185 modulates pathways involved in cellular survival and metabolism

To gain an initial understanding of the mechanisms underlying SSC185-induced cytotoxicity, an exploratory screening of proteins associated with key cell death and survival pathways was performed. PC-3 cells were treated with SSC185 (9.5, 19, and 38 µM) for 24 h, and total protein extracts were analyzed by Western blot. Membranes were probed for PARP1, phosphorylated AKT (p-AKT), total AKT (pan-AKT), phosphorylated RPS6 (p-RPS6), total RPS6, phosphorylated 4E-BP1 (p-4E-BP1), and total 4E-BP1, using GAPDH and α-tubulin as loading controls. Representative blot images are shown in Fig. 5a, and densitometric quantifications are presented in Fig. 5b. In this exploratory analysis, no PARP1 cleavage was detected after 24 h of treatment, while decreases in p-AKT, pan-AKT, p-RPS6, and 4E-BP1 levels were observed. As these initial observations were obtained from a single biological experiment, they were considered exploratory and were not subjected to statistical analysis.

Based on these initial observations, RPS6 signaling was subsequently evaluated in three independent experiments, together with ERK1/2 (Fig. 5c) signaling, considering the central role of the PI3K/AKT/mTOR and MAPK/ERK pathways in maintaining cell survival and proliferative capacity. Western blot analysis (Fig. 5d) showed that exposure to SSC185 significantly reduced the levels of phosphorylated ribosomal protein S6 (p-RPS6) (p < 0.01), a downstream effector of the mTOR pathway, compared to the control group. Total S6 protein expression levels were not affected (Fig. 5e). In contrast, under the same treatment conditions, a significant increase in phosphorylated ERK1 (p-ERK1) was observed (Fig. 5f; p < 0.01), while the levels of phosphorylated ERK2 (p-ERK2) and total ERK1/2 remained unchanged (Fig. 5g, h, i).

Nutrient deprivation enhances the cellular response to SSC185 and increases its sensitivity to NAC pretreatment

Nutrient deprivation was used to assess whether the cellular response to SSC185 was influenced by nutritional conditions. Additionally, pharmacological modulators targeting processes related to autophagy (CQ), caspase activity (Q‑VD‑OPh), protein synthesis (CHX), and redox-sensitive (NAC) responses were employed to further characterize the response profile to SSC185 under standard and nutrient-deprived conditions.

Under standard culture conditions supplemented with FBS, pretreatment with these pharmacological inhibitors did not produce significant changes in IC₅₀ values compared with SSC185 alone (Fig. 6b, d). In contrast, when the assay was performed under starvation conditions, a marked reduction in IC₅₀ was observed, from 24.69 µM (Fig. 6a) to 4.20 µM (Fig. 6e), indicating that nutrient deprivation substantially enhances the effect of SSC185 on MTT reduction. Notably, under starvation, pretreatment with NAC partially attenuated this effect, increasing the IC₅₀ to 11.51 µM (p < 0.01) (Fig. 6f, h).

SSC185 reduces mitochondrial membrane potential under standard and nutrient-deprived conditions

To evaluate mitochondrial responses to SSC185, mitochondrial membrane potential (ΔΨm) was assessed using rhodamine 123. After 6 h of exposure, no statistically significant differences in the proportion of Rho123 − cells were observed between SSC185-treated groups and the control group (Fig. 7a). After 24 h of exposure, treatment with 38 µM SSC185 significantly increased the proportion of Rho123 − cells to 27.84% ± 3.52 (p < 0.01), indicating a reduction in mitochondrial membrane potential (Fig. 7b).

Under nutrient deprivation, treatment with 4 µM SSC185 also significantly increased the Rho123 − population to 23.39% ± 3.44 (p < 0.01), compared with cells maintained in complete medium (8.24% ± 0.74) and nutrient-deprived controls (10.25% ± 1.44). Co-treatment with chloroquine (CQ, 25 µM) did not prevent the increase in the Rho123 − population associated with SSC185 treatment (Fig. 7c), whereas CQ alone did not significantly alter the proportion of Rho123 − cells compared with the corresponding controls.

Nutrient deprivation dissociates the effects of SSC185 on the cell cycle of PC-3

Cell-cycle distribution and plasma membrane integrity were evaluated in PC-3 cells under nutrient-deprived conditions following treatment with SSC185. Plasma membrane integrity was assessed based on the proportion of PI− cells, which correspond to cells retaining the ability to exclude propidium iodide. The PI− population was 93.40% ± 0.39 in FBS-supplemented controls and 85.12% ± 1.99 in nutrient-deprived controls. Treatment with 4 µM SSC185 under nutrient deprivation significantly reduced the proportion of PI− cells to 63.70% ± 4.39 (p < 0.001), indicating a reduction in the population of cells with preserved plasma membrane integrity (Fig. 8a, c). CQ (25 µM) similarly reduced the PI− population to 69.80% ± 3.26 (p ≤ 0.01), while co-treatment with SSC185 and CQ further reduced this population to 54.25% ± 2.13 (p < 0.01) compared with CQ alone.

Analysis of cell-cycle distribution showed that treatment with 4 µM SSC185 did not significantly alter the proportion of cells in G₀/G₁, S, or G₂/M phases compared with the corresponding controls under either FBS-supplemented or nutrient-deprived conditions. In contrast, CQ treatment under nutrient deprivation increased the proportion of cells in G₀/G₁ (46.93% ± 2.38; p ≤ 0.05) and reduced the S-phase fraction (16.48% ± 2.33; p ≤ 0.05), indicating an altered cell-cycle distribution under this experimental condition.

SSC185 does not increase acidic vesicle accumulation under nutrient deprivation

To evaluate changes in acidic vesicular compartments under nutrient-deprived conditions, PC-3 cells were analyzed by acridine orange staining following treatment with SSC185. The fluorescence pattern was similar to that observed under standard culture conditions, as SSC185 did not significantly increase the mean fluorescence intensity in the RFP channel (Fig. 8e, f). In contrast, CQ treatment resulted in a significant increase in RFP fluorescence (p < 0.05), consistent with greater accumulation of acridine orange-stained acidic vesicular compartments. Co-treatment with SSC185 and CQ produced a non-significant increase in RFP fluorescence compared with the corresponding control condition. These findings indicate that SSC185 does not promote detectable accumulation of acidic vesicular compartments under nutrient deprivation.

SSC185 exhibits time-dependent thiol reactivity

To further assess the possibility of nonspecific electrophilic reactivity, a DTNB-based thiol-reactivity assay was performed with L-cysteine under physiologically relevant conditions. SSC185 induced a mild-to-moderate and time-dependent reduction in free L-cysteine levels, with 78.50% ± 2.78 of thiol content remaining after 120 min at 50 µM. A weaker effect was observed at 25 µM (87.55% ± 4.25), suggesting partial concentration dependence. Importantly, the overall profile was comparable to that observed for curcumin, the reference electrophilic compound (84.46% ± 4.53 and 87.28% ± 0.66 at concentrations of 50 µM and 25 µM, respectively). These findings indicate that SSC185 exhibits measurable but not rapid or exhaustive thiol reactivity under the tested conditions (Fig. 9).

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.