The in vitro cellular cytotoxicity of Sng alone and in combination with inhibitors against cSCC cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay, as described previously [26]. Firstly, Sng (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA) to make a stock solution and further diluted with the corresponding culture fluid to make a series of drug solutions. cSCC (10${}^4$) cells in the exponential phase were seeded into each well of a 96-well plate. Following incubation at 37 °C under a humidified atmosphere of 5% CO${}_2$ for 24 h, the cells were exposed to a series of concentrations of Sng (0.5, 1, 2, 4, 8, and 16 µM) and in combination with pharmacological inhibitors. Finally, 10 µL of CCK-8 reagent (Sigma-Aldrich, St. Louis, MO, USA) was added to each well according to the manufacturer’s protocol, and the spectrophotometric absorbance (optical density [OD]) was measured at 450 nm using a multifunction microplate reader (Tecan Bio Tek Instruments Inc., Morgan Hill, CA, USA). The percentage of cell viability was calculated as follows: OD of the experimental sample/OD of the control sample $\times$ 100.

The time- and dose-dependent cellular response profiles of Sng treatment were measured using the xCELLigence Real-Time Cell Analysis high-throughput detection platform. After setting up the program, 50 µL of cell culture medium was added to each well of an E-plate 16 (ACEA Biosciences, San Diego, CA, USA). After equilibration at 37 °C for 30 min, the E-plates were inserted into the xCELLigence RTCA station (ACEA Biosciences, San Diego, CA, USA) to check for appropriate electrical connections and gather the background impedance of the cell culture medium. A431 and A388 cells were resuspended in cell culture medium, seeded at the required cell density into each well of the E-plate, and incubated for 24 h at 37 °C with 5% CO${}_2$ in the RTCA cradle. Following incubation, the culture medium was removed, and the cells were treated with escalating concentrations (1 , 2 , 4 , 8 and 16 µM) of Sng. After adding Sng, the impedance signal (expressed as the cell index readout) was recorded every 15 min for approximately 250 h. The analysis and interpretation of the impedance pattern over time were performed using RTCA software (ACEA Biosciences, San Diego, CA, USA).

The cellular response to pharmacological inhibitors, with or without Sng, was assessed via the trypan blue dye exclusion assay [27]. Briefly, A388 cells were seeded in 60-mm culture plates and maintained for 24 h in complete DMEM medium. Post incubation, cells were pretreated with SB203580 (a p38 inhibitor; 20 µM) or U0126 (an ERK1/2 inhibitor; 20 µM) for 2 h followed by the treatment with Sng (4 µM) for 6 h. Following treatment, the cells were harvested, centrifuged, and resuspended in 1 mL PBS. Cells were then stained with 0.4% trypan blue solution (1:1; Sigma-Aldrich, St. Louis, MO, USA), and 10 µL of cell suspension was applied to the edge of the dual-chamber cell counting slide (Bio-Rad, Hercules, CA, USA). Finally, the slide was inserted into the TC20™ automated cell counter (Bio-Rad, Hercules, CA, USA), and the cell viability was estimated as the percentage of live cells in the sample via trypan blue exclusion.

The LIVE/DEAD® Viability Kit (Life Technologies Corporation, Carlsbad, CA, USA) was used according to the manufacturer’s instructions and as previously described [28] to visualize the distribution of viable and non-viable A431 and A388 cells 24 h post-treatment with Sng alone and in combination with inhibitors. Briefly, A388 and A431 cells were seeded at a density of 1 $\times$ 10${}^6$ cells per well in a 6-well plate and subjected to a 24 h treatment with varying concentrations of Sng alone (2, 4, and 8 µM), in combination with N-acetylcysteine (NAC; 10 mM; Sigma-Aldrich, St. Louis, MO, USA) as well as z-VAD-FMK (50 µM; Calbiochem, San Diego, CA, USA). Cells were then stained with the live/dead stain prepared by diluting 20 µL of ethidium homodimer-1 (EthD-1; final concentration: 4 µM) and 5 µL calcein AM (final concentration: 2 µM) in 10 mL of PBS. Green and red fluorescent signals from calcein-AM and EthD-1, respectively, were detected using the EVOS FLc cell imaging system (Invitrogen, Thermo Fisher Scientific, MA, USA).

The colony-forming assay was performed as described previously [29] to evaluate the proliferative and colony-forming abilities of A388 and A431 cells treated with Sng alone and in combination with inhibitors. Following optimization of the number of cells required to obtain the best size and distribution of colonies, A431 and A388 cells were seeded at a density of 10${}^4$ cells/well in a 6-well plate. After 24 h of incubation, cells were treated with varying concentrations of Sng alone and in combination with inhibitors (NAC, 10 mM; z-VAD-FMK, 50 µM) and incubated for 7 days in a humidified atmosphere at 37 °C with 5% CO${}_2$. After obtaining sufficiently sized colonies, cells were fixed with 10% (w/v) formaldehyde and stained with 0.1% (w/v) crystal violet solution for 30 min at room temperature. The staining solution was then carefully drained, and each well was washed with water until the excess dye was removed. Finally, colonies were visualized at 4$\times$ magnification using an EVOS FLc cell imaging system (Invitrogen, Thermo Fisher Scientific, MA, USA).

The quantification of apoptosis and necrosis after treatment with Sng alone and in combination with inhibitors (NAC, 10 mM; z-VAD-FMK, 50 µM; and SP600125, 30 µM) was performed using conventional Annexin V-FITC and Propidium iodide (PI) staining (Annexin V-FITC apoptosis detection kit, BD Biosciences, San Jose, CA, USA) followed by flow cytometric analysis. Approximately 1 $\times$ 10${}^6$ cells subjected to various treatments were centrifuged at 1500 rpm for 5 min at 4 °C before being washed in 1 mL PBS, centrifuged again, and diluted in 1$\times$ annexin binding buffer. Annexin V-FITC (10 µL) and PI (5 µL) were added to the suspension, and the tubes were incubated for 20 min in the dark at room temperature. Different cell populations were selected and viable and apoptotic cells were sorted by fluorescence-activated cell sorting using flow cytometry (BD LSR Fortessa cell analyzer; BD Biosciences, San Jose, CA, USA). The cells were identified and quantified as live (Annexin FITC${}^{\text{-ve}}$/PI${}^{\text{-ve}}$), early or primary apoptotic (Annexin FITC${}^{\text{+ve}}$/PI${}^{\text{-ve}}$), late apoptotic (Annexin FITC${}^{\text{+ve}}$/PI${}^{\text{+ve}}$), and necrotic (Annexin FITC${}^{\text{-ve}}$/PI${}^{\text{+ve}}$). Debris and cell aggregates were excluded from the analysis.

To evaluate the capacity of Sng to modulate the mitochondrial membrane potential ($\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$) in cSCC cells, the mitochondria-specific ratiometric fluorescent dye tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was used. Following 24 h treatment with Sng and in combination with inhibitors (NAC,10 mM; and z-VAD-FMK,50 µM), A388 and A431 cells were harvested, washed, resuspended in 1 mL PBS, and stained with JC-1 staining solution (BD Biosciences, San Jose, CA, USA) in the dark. Cells maintained on ice were then immediately analyzed for $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ using flow cytometry (BD LSR Fortessa analyzer, BD Biosciences, San Jose, CA, USA).

First, A388 and A431 cells were treated with Sng alone or in combination with z-VAD-FMK (50 µM) for 24 h. After treatment, cells were harvested, washed twice in PBS, and centrifuged. The cells were then fixed and permeabilized using a BD Cytofix/Cytoperm Plus Fixation kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Approximately 1 $\times$ 10${}^6$ cells were stained with 5 µL of BV605-tagged active caspase-3 and 5 µL of AF647-tagged cleaved PARP for 30 min and analyzed using flow cytometry (BD LSR Fortessa analyzer, BD Biosciences, USA).

To investigate Sng-induced oxidative stress, we pursued the generation of ROS in A388 and A431 cells treated with Sng alone or in combination with NAC (10 mM) or z-VAD-FMK (50 µM). 24 h post-treatment, the cells were harvested, washed using Hanks’ balanced salt solution (HBSS), and stained with CellROX™ Green Reagent (10 µM; to detect intracellular ROS) and MitoSOX Red reagent (5 µM; to detect mitochondrial superoxide) for 30 min at 37 °C. ROS levels were quantified using flow cytometry (BD LSR Fortessa analyzer, BD Biosciences, USA).

Sng-induced DNA double-strand breaks (DSBs) were analyzed by detecting phosphorylated histone H2AX Ser139 (p-H2AX${}^{\text{Ser139}}$) as previously described [30]. A431 and A388 cells were treated for 24 h with various concentrations of Sng alone or in combination with z-VAD-FMK (50 µM). After incubation, the cells were fixed and permeabilized using the BD Cytofix/Cytoperm plus fixation and permeabilization solution kit according to the manufacturer’s protocol. Approximately 1 $\times$ 10${}^6$ cells were stained with 5 µL f BV421 Mouse Anti-H2AX (pS139) antibody (BD Biosciences, San Jose, CA, USA) for 30 min at room temperature. Phosphorylated histone H2AX levels were then detected by flow cytometry using a BD LSR Fortessa analyzer (BD Biosciences, USA).

The effect of Sng on cell cycle distribution was analyzed as previously described [26]. A388 and A431 cells were seeded in 100-mm culture dishes and treated with gradient concentrations of Sng alone and in combination with NAC (10 mM) or z-VAD-FMK (50 µM) for 24 h. After treatment, cells were harvested, centrifuged, and fixed in ethanol (70%) for 2 h. For analysis, cells were resuspended in PBS, incubated with RNAse A solution (0.04 µg/mL) for 30 min at 37 °C, and then treated with PI DNA cell-cycle stain (40 µg/µL) for 30 min at 4 °C. By gating the forward and side scatter profiles of the samples, doublets, disintegrated nuclei, and other cell debris were excluded from the analysis. The gates were uniformly maintained across all the samples in each cycle. After that, the relative DNA content was determined, and the percentages of cells in the sub-G0/G1, G0/G1, S, and G2/M phases were quantified by flow cytometry (BD LSR Fortessa analyzer, BD Biosciences, USA) based on the amount of PI fluorescence.

Tumor-derived spheroids/tumorspheres from A388 cells were generated in non-adsorbent, ultra-low attachment, 6-well plates (Corning, USA). The two-dimensional cell culture was maintained for 24 h under standard culture conditions (37 °C with 5% CO${}_2$) in complete DMEM. After the two-dimensional cell culture was harvested, the cells were dissociated into a single-cell suspension through enzymatic digestion (trypsin). Five thousand cells were then added to each well of a 6-well plate and grown in complete cancer stem cell medium (3D Tumorsphere Medium XF, Promo Cell, Germany, C-28070). The plates were then placed in a humidified incubator at 37 °C with 5% CO${}_2$ until spheroids with 400–500 µm diameters were formed. The spheroids were treated with Sng alone or in combination with NAC (10 mM) or SP600125 (30 µM) for 7 days. Spheroid formation was monitored on days 0, 3, and 7 using the EVOS FLc cell imaging system (Invitrogen, Thermo Fisher Scientific) at 10$\times$ and 20$\times$ magnification.

Following cell lysis with a 2X Laemmli sample buffer (Bio-Rad, Hercules, CA, USA), the total protein from cells was extracted and quantified using a Nanodrop ND-100 spectrophotometer (Thermo Fisher Scientific). Approximately 30–100 µg of protein from each sample was loaded and resolved by SDS-PAGE (10–12%), transferred to a polyvinylidene difluoride (PVDF) membrane, and blotted with primary antibodies against p-p38 (Thr180/Tyr182), p38, p-p44/42 (Thr202/Tyr204), p44/42, p27, p21, CDK6, caspase-3 , caspase-9, cleaved caspase-8, PARP, BH3 interacting-domain death agonist (Bid), growth arrest-and DNA damage-induced 45 A (GADD45A), and p-H2AX${}^{\text{Ser139}}$ (Cell Signaling Technologies, Danvers, MA, USA) as well as p-JNK (Thr183/Tyr185), JNK, and Bcl-2-associated X protein (Bax) (Santa Cruz Biotechnology Inc., Dallas, TX, USA). After incubation with the corresponding secondary antibodies, the protein bands were detected using a Chemi-Doc System with an ECL substrate (Bio-Rad, Hercules, CA, USA). Internal controls such as GAPDH (Cell Signaling Technologies, Danvers, MA, USA), $\beta$-Actin, $\alpha$-tubulin, and HSP60 (Santa Cruz Biotechnology Inc., Dallas, TX, USA) were used.

The statistical significance between the treated and untreated samples was analyzed by one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism. All error bars represent the mean $\pm$ standard deviation (SD) of three independent experiments. p values 0.001 were considered statistically significant.

Several toxicological studies have demonstrated the anti-cancer potential of Sng in both in vitro and in vivo tumor models [31, 32, 33, 34, 35]. To obtain an accurate assessment of the Sng dose-response in cSCC cells, cell viability was assessed upon screening with escalating concentrations of Sng (0.5–16 µM) for 24 h. Sng began to show a response at 2 µM, evident from the significant decrease in the number of viable cells in both A388 and A431 cells (Fig. 1A,B). The RTCA system was also used to evaluate the cytotoxic actions of Sng. Being a high-throughput and quantitative technique, RTCA enables continuous, label-free and real-time monitoring of dose-dependent cellular responses such as cell proliferation and morphological alterations [36]. Dynamic real-time monitoring of cSCC cells treated with Sng confirmed the antiproliferative effect of the compound, and results were comparable to those obtained from the CCK-8 assay (Fig. 1C,D). Because the reduction in the number of viable cells obtained from the CCK-8 assay could have been caused either by cytotoxicity or inhibited cell proliferation, we performed a LIVE/DEAD® cell viability assay, which detects live and dead cells from the fluorescence emitted by calcein AM and EthD-1, respectively. The live/dead assay also confirmed the effective cytotoxicity exerted by Sng on A388 and A431 cells, as evidenced by the significant reduction in the number of viable cells (reflected by the green fluorescent signal) after Sng treatment compared to the control group. Moreover, it provided proof of apoptosis, as indicated by an increase in the relative number of dead cells (reflected by the red fluorescent signal) (Fig. 1E,F). To assess the effect of Sng on reproductive cell survival and growth in vitro, we used a conventional 2D clonogenic or colony formation assay. Fig. 1G,H show whole-well images of colony formation across all treatment groups and reflect the capacity of Sng to influence and antagonize the extent of clonogenic growth (reflected by the number and size of tumor cell colonies) of both primary and metastatic cSCC cells.

Fig. 1.

Sanguinarine (Sng) inhibits proliferation, impairs viability, and induces cell-cycle perturbations in cutaneous squamous cell carcinoma (cSCC) cells. (A–D) Metastatic (A388) and primary (A431) cSCC cells were treated with various concentrations of Sng (0.5–16 µM) for 24 h. The viability of the cells was determined by the CCK-8 assay (A,B) while the Real-Time Cell Analysis (RTCA) system was used to record the cell growth curves in response to different concentrations of Sng (1–16 µM) (C,D) Data represented as mean $\pm$ SD from three independent experiments. ns: not significant, ***p 0.001; one-way ANOVA followed by Tukey analysis. (E,F) The distribution of live (indicated by green fluorescence) and dead (indicated by red fluorescence) A388 and A431 cells following Sng treatment (2 µM, 4 µM, and 8 µM) for 24 h (magnification 4$\times$; scale bar: 1000 µm). (G,H) Colony-forming efficiency of A388 and A431 cells exposed to indicated concentrations of Sng (magnification 4$\times$; scale bar: 1000 µm). (I,J) Frequency histograms from flow cytometry showing the regulatory effect of Sng on cell-cycle distribution in A388 and A431 cells exposed to indicated concentrations of Sng for 24 h. Each histogram shows the quantified percentages of DNA content and cell-cycle distribution for sub-G0/G1, G0/G1, S, and G2/M phases. (K,L) Expression profile of cell-cycle regulatory proteins in A388 and A431 cells treated with increasing concentrations of Sng for 6 h. SD, standard deviation; CCK-8, Cell Counting Kit-8; ANOVA, analysis of variance.

In addition to the drug dose-response, it is crucial to quantitatively assess the growth-inhibitory effect of the drug as a function of exposure time. To track the cellular response to Sng across time, A388 and A431 cells were exposed to 2 µM Sng at different time points (0–48 h) and subjected to toxicity and viability measurements. Time-dependent analysis of cellular response from 0 to 48 h indicated a decline in cell viability (Supplementary Fig. 1a,b), as well as morphological and biochemical changes consistent with apoptosis, including activation of caspase-3, -8 and DNA fragmentation (Supplementary Fig. 1c–f). These results implied the dose- and time-dependent cytotoxic effects of Sng in cSCC cells.

Concerning the cytostatic assessment of Sng via fluorescent PI-based quantification of cell-cycle distribution, we determined that Sng alters the cell cycle machinery by markedly increasing the accumulation of the pro-apoptotic sub-G0/G1 fraction in both A431 and A388 cells in a concentration-dependent manner (Fig. 1I,J). Subsequently, western blot analysis was performed to delineate the molecular mechanisms underlying Sng-induced cell-cycle arrest. The analysis showed that Sng modulates cell cycle regulatory proteins, manifested by the upregulated expression of cyclin-dependent kinase inhibitors p21${}^{\text{WAF1/Cip1}}$ and p27${}^{\text{Kip1}}$ and the downregulated expression of CDK6. Notably, Sng induced a concentration-dependent increase in the expression of GADD45A in metastatic A388 cells (Fig. 1K,L). According to these results, Sng exerts its antiproliferative effects by modulating checkpoints at the sub-G1 phase of the cell cycle and inducing sub-G0/G1 phase arrest in cSCC cells.

Focusing on the mechanistic aspects of cytotoxicity induced by Sng, we sought to unveil and characterize the major regulated cell death (RCD) modality [37] initiated by perturbations in the intracellular or extracellular microenvironments of cSCC cells. Since apoptosis has been reported to be the most profound RCD program triggered by Sng [26, 38], we assessed concentration-dependent morphological response that is characteristic of apoptosis in cSCC cells following exposure to Sng for 24 h. As shown in Fig. 2A,B, the morphological hallmarks of apoptosis, including cell shrinkage, membrane blebbing, and membrane-bound apoptotic bodies, were observed. To quantify apoptotic rates and segregate the apoptotic population, cSCC cells were subjected to Annexin V/PI staining. As shown in Fig. 2C,D, the majority of the Sng-treated cell population was in a late apoptotic state, although there was a significant increase in the percentages of both early and late apoptotic A388 and A431 cells associated with increasing concentrations of Sng. Induction of apoptotic cell death typically occurs via two major pathways: an extrinsic death receptor pathway and an intrinsic/mitochondrial pathway. Each of these pathways requires specific initiating stimuli to activate specific initiator caspases [39] and, in turn, executioner caspase-3. To ascertain whether the extrinsic pathway was associated with Sng-induced apoptosis, we assessed the expression of caspase-8 and caspase-3 in A388 and A431 cells treated with increasing concentrations of Sng. Treatment with Sng increased the expression of both caspase-3 (Fig. 2E,F) and cleaved caspase-8 (Fig. 2G,H) in the A388 and A431 cells in a concentration-dependent manner. Given that PARP is the preferred cellular substrate for caspases, we analyzed the cleavage of PARP in response to caspase-3 activation induced by Sng. As shown in Fig. 2G,H, and Supplementary Fig. 2a,b, there was an obvious increase in the activity of PARP, represented by its cleaved form, in both A388 and A431 cells exposed to increasing concentrations of Sng. DNA fragmentation is a biochemical hallmark of apoptosis, which can be detected through the phosphorylation status and foci formation of H2AX in cells [40]. Exposure to Sng triggered a concentration-dependent increase in Ser139 phosphorylation of H2AX in A388 and A431 cells, indicating the significant genotoxic effect of Sng (Fig. 2G,H). These findings suggest the execution of a caspase-dependent extrinsic signaling pathway following the cell death stimulus generated by Sng.

Fig. 2.

Sanguinarine induces apoptosis in cSCC cells. (A,B) Microscopic images of cSCC cells showing morphological abnormalities induced by Sng (2 µM, 4 µM, and 8 µM) (magnification 20$\times$; scale bar: 200 µm). (C,D) Scatter plots presenting the kinetics of apoptosis in A388 and A431 cells treated with indicated concentrations of Sng. The percentages of apoptotic, necrotic and live cells were determined and analyzed by Annexin V-FITC/PI staining using flow cytometry after 24 h. The plot depicts segregation into four quadrants: the lower-left quadrant representing viable cells (Annexin V-FITC${}^{\text{-ve}}$/PI${}^{\text{-ve}}$); the lower-right quadrant representing cells that are in early apoptosis (Annexin V-FITC${}^{\text{+ve}}$/PI${}^{\text{-ve}}$); the upper-left quadrant representing cells that are in necrotic phase (Annexin V-FITC${}^{\text{-ve}}$/PI${}^{\text{+ve}}$); and the upper-right quadrant representing cells that are in late apoptosis (Annexin V-FITC${}^{\text{+ve}}$/PI${}^{\text{+ve}}$). (E,F) Scatter plots depicting the activity of caspase-3 in A388 and A431 cells treated with the indicated concentrations of Sng. A388 and A431 cells were treated with indicated concentrations of Sng for 24 h and then assessed for the activity of caspase-3 tagged with BV605 dye using flow cytometry. (G,H) Western blot analysis of cleaved caspase-8, Poly (ADP-ribose) Polymerase (PARP), and phosphorylated histone H2AX Ser139 (p-H2AX ${}^{\text{Ser139}}$) in A388 and A431 cells treated with indicated concentrations of Sng for 6 h. FITC, fluorescein isothiocyanate; PI, Propidium Iodide.

To substantiate the role of caspases in Sng-induced cellular apoptosis, A388 and A431 cells were preincubated with the pan-caspase inhibitor z-VAD-FMK (50 µM), followed by treatment with Sng (4 µM). z-VAD-FMK is a cell-permeable, irreversible fluoromethyl ketone (FMK)-derivatized peptide that acts as an efficient caspase inhibitor [41]. Applying z-VAD-FMK significantly restored the viability of the Sng-treated A388 and A431 cells (Fig. 3A,B). Evaluation of specific cell fractions by Annexin V/PI staining revealed that preventing the cleavage of caspases with z-VAD-FMK remarkably attenuated the accumulation of apoptotic cells relative to cells treated with Sng alone (Fig. 3C,D). The percentage of apoptotic (early and late) cells appeared as a readout of enhanced apoptosis in Sng-treated cells but was minimally detectable in z-VAD-FMK and Sng co-treated cells. Similarly, the Live/Dead assay also revealed an increase in the density of live cells in the z-VAD-FMK-treated group (Fig. 3E,F), corroborating the flow cytometry results. The clonogenic abilities of A388 and A431 cells were also restored upon pretreatment with z-VAD-FMK (Fig. 3G,H). Furthermore, co-treatment of cells with z-VAD-FMK and Sng significantly antagonized the expression of Sng-induced cleaved caspase-8 and PARP (Fig. 3I,J). These findings suggest that Sng induces caspase-dependent apoptosis in cSCC cells.

Fig. 3.

Sanguinarine induces caspase-mediated apoptosis in cSCC cells. (A,B) Pretreatment with the general caspase inhibitor z-VAD-FMK restores cell viability in A388 and A431 cells following exposure to Sng. A388 and A431 cells were pretreated with 50 µM z-VAD-FMK for 1 h before being challenged with 4 µM Sng. Cell viability was determined by CCK-8 analysis. Data presented as the mean $\pm$ SD from three independent experiments. ***p 0.001 compared with the Sng-treated group; one-way ANOVA followed by Tukey analysis. (C,D) Scatter plots presenting the kinetics of apoptosis in cells co-treated with z-VAD-FMK and Sng. A388 and A431 cells were pretreated with 50 µM z-VAD-FMK for 1 h before being challenged with 4 µM Sng for 24 h. (E,F) Representative fluorescence staining depicting the distribution of live (indicated by green fluorescence) and dead (indicated by red fluorescence) A388 and A431 cells following treatment with z-VAD-FMK (50 µM), either alone or in combination with Sng (4 µM) for 24 h (magnification 4$\times$; scale bar: 1000 µm). (G,H) Colony-forming efficiency of A388 and A431 cells co-treated with z-VAD-FMK (50 µM) and Sng (4 µM) for 24 h (magnification 4$\times$; scale bar: 1000 µm). (I,J) Western blot analysis of cleaved caspase-8 and PARP expression in A388 and A431 cells pretreated with z-VAD-FMK (50 µM) for 1 h followed by treatment with Sng (4 µM) for 6 h.

Delving deeper into the activity of z-VAD-FMK, we assessed whether z-VAD-FMK can reverse Sng-induced cell-cycle defects and found that z-VAD-FMK prevented the accumulation of sub-G0/G1 fraction in A388 cells (Supplementary Fig. 3c). Because Sng-treated cells expressed active caspase-3, we sought to determine its expression in response to pretreatment with z-VAD-FMK. z-VAD-FMK reduced the expression of active caspase-3 compared to that in the Sng-treated group (Supplementary Fig. 3e). Moreover, we quantified p-H2AX${}^{\text{Ser139}}$ levels and, thus, DNA damage in cells that were co-treated with z-VAD-FMK and Sng, considering that p-H2AX levels were distinctly elevated upon Sng treatment. Quantification of p-H2AX${}^{\text{Ser139}}$ levels by flow cytometry showed that the cell fraction from the co-treated group displayed p-H2AX levels comparable to those in the control group (Supplementary Fig. 3f).

ROS, a class of highly reactive molecules, are critical regulators of apoptosis. Considering the bidirectional nature of ROS [42], we determined the ability of Sng to manipulate levels of ROS in cSCC cells. Using CellROX and MITOSOX fluorogenic probes, we showed that Sng markedly increased intracellular (Fig. 4A,B) and mitochondrial (Supplementary Fig. 2c,d) ROS production in a concentration-dependent manner in both A388 and A431 cells. This ROS production was blocked by NAC (10 mM), a universal cytoprotective antioxidant that serves as a ROS scavenger and precursor of glutathione (GSH) synthesis in Sng-treated A388 (Fig. 4C; Supplementary Fig. 4a) and A431 cells (Fig. 4D). NAC also restored GSH levels (Supplementary Fig. 4d) that were elevated in A388 and A431 cells exposed to Sng (Supplementary Fig. 2e,f).

Fig. 4.

Sanguinarine induces reactive oxygen species (ROS)-dependent apoptosis in cSCC cells. (A,B) Frequency histograms showing the generation of intracellular ROS in A388 and A431 cells treated with increasing concentrations of Sng for 24 h. (C,D) Frequency histograms showing the effect on intracellular ROS in A388 and A431 cells following pretreatment with N-acetylcysteine (NAC) (10 mM) and then by Sng (4 µM) for 24 h. (E,H) Pretreatment with the ROS scavenger NAC restores cell viability in A388 and A431 cells following exposure to Sng. A388 and A431 cells were pretreated with 10 mM NAC for 1 h before being challenged with 4 µM Sng. Cell viability was determined by CCK-8 analysis. Data presented as the mean $\pm$ SD from three independent experiments. ***p 0.001 compared with the Sng-treated group; one-way ANOVA followed by Tukey analysis. (F,I) Representative fluorescence staining depicting the distribution of live (indicated by green fluorescence) and dead (indicated by red fluorescence) A388 and A431 cells following treatment with NAC (10 mM), either alone or in combination with Sng (4 µM) for 24 h (magnification 4$\times$; scale bar: 1000 µm). (G,J) Colony-forming efficiency of A388 and A431 cells co-treated with NAC (10 mM) and Sng (4 µM) (magnification 4$\times$; scale bar: 1000 µm). (K,M) Western blot analysis of cleaved caspase-8 and PARP in A388 and A431 cells co-treated with NAC (10 mM) and Sng (4 µM) for 6 h. (L) Inhibition of dissolution and apoptosis in A388 spheroids on pretreatment with NAC (magnification 20$\times$; scale bar: 200 µm).

Next, we investigated the role of ROS in the regulation of Sng-induced apoptosis. Pretreatment with NAC remarkably preserved cell viability and abrogated cytotoxicity in Sng-treated A388 (Fig. 4E,F) and A431 cells (Fig. 4H,I). The clonogenic assay in Fig. 4G,J further suggested that NAC restored cell survival and clonal expansion of A388 and A431 cells compared to cells treated with Sng alone. Furthermore, applying NAC decreased the sub-G0/G1 population (Supplementary Fig. 4b) of A388 cells. To determine whether ROS production precedes caspase-dependent apoptosis in cSCC cells, we determined cleaved caspase-8 and PARP expression levels in A388 and A431 cells cotreated with NAC and Sng. The expression of cleaved caspase-8 and PARP was markedly reduced upon pretreatment with NAC compared to cells treated with Sng alone (Fig. 4K,M). In 3D spheroid cultures, dissolution, the loss of well-circumscribed edges of normally compact cell aggregates, corresponds to the induction of apoptosis [43]. Interestingly, the dissolution of metastatic A388 cell-derived multicellular spheroids was also inhibited by NAC treatment alone or in combination with Sng (Fig. 4L).

To further investigate the role of ROS in caspase-mediated cell death, we analyzed changes in intracellular and mitochondrial ROS levels in A388 cells pretreated with z-VAD-FMK. Both intracellular and mitochondrial ROS production were significantly reduced upon pretreatment with z-VAD-FMK. In contrast, they remained more or less unchanged in cells treated with either z-VAD-FMK or Sng alone (Supplementary Fig. 3a,b). These findings emphasize the functional role of the caspase-dependent pathway in Sng-induced apoptosis, which is secondary to the modulation of ROS.

Sng has been previously shown to elevate ROS levels in neoplastic cells, thereby disrupting redox homeostasis and inducing oxidative stress [21, 26]. Because oxidative stress can occur at either a high $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ or low $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ [44], we assessed the capacity of Sng to alter $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ in A388 and A431 cells. As shown in Fig. 5A,B, Sng induces a progressive loss of $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ in cSCC cells, as manifested by the right-shift peak reflecting the increased number of cells with dissipated $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$. Additionally, co-treatment of A388 cells with NAC moderately restored $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ compared to the loss induced by Sng alone (Supplementary Fig. 4c). In addition, we ascertained whether z-VAD-FMK maintains $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$. Flow cytometric analysis showed that z-VAD-FMK pretreatment moderated the dissipation of $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ compared to the loss induced by Sng alone (Supplementary Fig. 3d).

Fig. 5.

Disruption of mitochondrial membrane potential in response to Sanguinarine in cSCC cells. (A,B) Distribution plots representing the percentage of cells exhibiting loss of $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ after exposure to indicated concentrations of Sng in A388 and A431 cells. (C,D) Western blot analysis of BH3 interacting-domain death agonist (Bid), Bcl-2-associated X protein (Bax), and caspase-9 expression in A388 and A431 cells subsequent to the loss of $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ induced by Sng.

The fact that alterations in mitochondrial energization, particularly the loss of $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$, trigger the translocation of the cytosolic pro-apoptotic Bcl-2 family member Bax to the mitochondria [45] prompted us to determine the levels of Bax in cSCC cells treated with Sng. We demonstrated that the exposure of A431 cells to increasing concentrations of Sng for 6 h increased the levels of Bax protein (Fig. 5D). However, in A388 cells, the expression of Bax gradually increased, with maximal expression at 4 µM Sng (Fig. 5C). Sng-treated cSCC cells were also examined for the expression of other intrinsic apoptotic death regulators involved in maintaining mitochondrial membrane integrity. Given that the pro-apoptotic effector Bid cooperates with Bax in mitochondrial dysfunction [46], we assessed the expression of Bid. In the case of A431 cells treated with indicated concentrations of Sng for 6 h, the expression of inactive full-length Bid gradually decreased with concentration, whereas maximal expression of the active large fragment of Bid (tBid) was observed in cells exposed to 4 µM of Sng (Fig. 5D). Similar to Bax expression in A388 cells, a gradual increase in the expression of inactive full-length Bid was observed, with maximal expression achieved post-treatment with 4 µM Sng (Fig. 5C) [47]. We also determined the expression of caspase-9 in response to Sng-induced mitochondrial membrane defects. As shown in Fig. 5C,D, an increase in caspase-9 activity in A388 (relatively higher at 4 µM Sng) and A431 (somewhat higher at 8 µM Sng) cells was observed. Taken together, Sng generates a potent intrinsic apoptotic stimulus and induces $\mathrm{\Delta }$$\mathrm{\Psi }$${}_{\text{m}}$ dysfunction, indicative of its mitochondrial-targeting potential.

Substantial evidence has highlighted the pivotal role of MAPKs in regulating stimulus-specific cellular responses, including cell growth, migration, proliferation, differentiation, and apoptosis [48]. To elucidate the possible link between apoptosis and the MAPK pathway in cSCC cells, we investigated the influence of Sng on the MAPK signaling pathways in A388 cells. Indeed, Sng promoted the phosphorylation and activation of JNK, p38, and ERK (p44/42 MAPK), with maximal phosphorylation (specifically that of JNK and ERK) observed at 8 µM Sng (Fig. 6A), thus corroborating the involvement of MAPKs in Sng-induced apoptosis in A388 cells. The expression of JNK, p38, and ERK remained unchanged. Following this observation, we investigated the impact of SB203580 (a p38 inhibitor), U0126 (an ERK1/2 inhibitor), and SP600125 (a JNK inhibitor) on MAPK signaling-mediated caspase-dependent apoptosis in A388 cells. According to the trypan blue assay, the Sng-induced decrease in viable A388 cells was not restored upon the pretreatment with SB203580 and U0126 (Supplementary Fig. 5a,b). SB203580/Sng co-treatment inhibited the expression of cleaved caspase-8 and PARP, whereas U0126/Sng co-treatment failed to inhibit the Sng-induced activity of these proteins in A388 cells. Moreover, inhibition of p38 and ERK did not attenuate the Sng-induced H2AX phosphorylation at Ser139, indicating no reversibility of DNA damage prompted by Sng in A388 cells (Supplementary Fig. 5c,d).

Fig. 6.

Activation of the JNK signaling pathway by Sanguinarine in A388 monolayer cells and spheroids. (A) Western blot analysis of JNK, p38, and ERK phosphorylation in A388 cells treated with indicated concentrations of Sng for 6 h. (B) Viability assessment of A388 cells treated with or without SP600125 (30 µM) by CCK-8 assay. Data presented as the mean $\pm$ SD from three independent experiments. ***p 0.001 compared with the Sng-treated group; one-way ANOVA followed by Tukey analysis. (C) Microscopic images of A388 cells treated with SP600125 (30 µM), either alone or in combination with Sng (4 µM) for 6 h (magnification 10$\times$; scale bar: 400 µm). (D) Representative fluorescence staining depicting the distribution of live (indicated by green fluorescence) and dead (indicated by red fluorescence) A388 cells following treatment with SP600125 (30 µM), either alone or in combination with Sng (4 µM) for 24 h (magnification 4$\times$; scale bar: 1000 µm). (E) Scatter plots presenting the kinetics of apoptosis in cells co-treated with SP600125 (30 µM) and Sng (4 µM). A388 cells were pretreated with SP600125 for 2 h before being challenged with Sng and then stained with Annexin V-FITC/PI after 24 h. The percentage of apoptotic (early and late) cells appears as a readout of enhanced apoptosis in Sng-treated cells but is only partially detectable in SP600125 and Sng co-treated cells. (F) Western blot analysis of apoptotic proteins, cell-cycle regulatory protein, and DNA double-strand break (DSB) indicator in A388 cells treated with SP600125 (30 µM) for 2 h prior to treatment with Sng (4 µM) for 6 h. (G) Western blot analysis of JNK phosphorylation in A388 cells in response to pretreatment with NAC (10 mM) followed by Sng (4 µM) for 6 h. (H) Inhibition of dissolution and apoptosis in A388 spontaneously-forming spheroids on pretreatment with SP600125 (magnification 10$\times$; scale bar: 400 µm).

Earlier studies have delineated the capacity of Sng to induce ROS production and effectively activate the JNK signaling pathway [49]. Therefore, we determined the functional role of JNK signaling in Sng-induced apoptosis in cSCC cells using the pharmacological JNK inhibitor SP600125. As shown in Fig. 6B, restoration of cell viability was observed when Sng-treated A388 cells were pre-exposed to SP600125 for 2 h. Viability assessment following inhibition of JNK activity in A431 cells also yielded similar results (data not shown). In A388 cells, pretreatment preserved the morphological characteristics (Fig. 6C) and markedly decreased the number of dead cells compared to the Sng-treated group (Fig. 6D). As derived from flow cytometry analysis (Fig. 6E), SP600125 pretreatment significantly attenuated the accumulation of apoptotic cells relative to cells treated with Sng alone. Furthermore, blocking JNK with SP600125 attenuated the Sng-induced expression of caspase-3, cleaved caspase-8, Bax, GADD45A, and p-H2AX${}^{\text{Ser139}}$ (Fig. 6F).

To elucidate whether ROS are involved in Sng-mediated JNK activation, A388 cells were treated with Sng with or without NAC. Co-treatment of A388 cells with Sng and NAC for 6 h blocked JNK activation relative to those treated with Sng alone (Fig. 6G). These results confirmed the participation of ROS-dependent JNK signaling in the pro-apoptotic effects induced by Sng cSCC cells.

Compared to rescuing parental monolayer cells from the apoptotic effect of Sng, SP600125 also prevented the Sng-induced dissolution of intact spheroids. Both SP600125 and SP600125-Sng co-treated spheroids maintained well-circumscribed edges comparable to those of the control (Fig. 6H), indicating spheroid viability.

In vitro, 3D tumor spheroid models have emerged as viable tools for drug screening. To analyze the cell response to Sng in 3D culture, we assessed the dissolution of metastatic A388 cell-derived spheroids. The so-called tumor-derived spheroids were generated in a 3D scaffold-free culture system using 2D A388 adherent cells that were grown as floating spheres in an ultra-low attachment 6-well plate and sensitized to increasing concentrations of Sng for 7 days. A388 cells formed dense globe-like aggregates with prominent core formation (Fig. 7A). Following a 7-day treatment period, the dissolution of the spheroids in each well was assessed. At a concentration of 2 µM, Sng showed no apoptotic response, as the spheroids retained their well-circumscribed edges identical to the control. This contrasts the higher concentrations of Sng (4 and 8 µM), which showed a mixed apoptotic response, characterized by the co-existence of spheroids with significantly distorted edges and single-cell populations (Fig. 7A).

Fig. 7.

Ablation of stemness potential by Sanguinarine in metastatic cSCC cells. (A) Representative optical images of A388 cell-derived spheroids that were systematically investigated for the effect of Sng on spheroidal dissolution and apoptosis at 0, 3, and 7 cultivation days (magnification 10$\times$; scale bar: 400 µm). (B) Expression profile of the stemness gene and CSC cell surface marker in A388 spheroids. (C) Expression profile of EMT-associated markers in A388 spheroids. CSC, cancer stem cell; EMT, Epithelial-Mesenchymal Transition.

To investigate the effect of Sng on cancer stem cells (CSCs) enriched in the formed spheroids, we assessed the expression of the key stemness gene Nanog and the cell surface marker aldehyde dehydrogenase A1 (ALDHA1). Sng-treated A388 spheroids displayed downregulation of both Nanog and ALDHA1 expression (Fig. 7B). Epithelial-mesenchymal transition (EMT) is a highly plastic, dynamic transitional process characterized by the loss of apical polarity and gain of mesenchymal properties, cell motility, and stemness in epithelial cells in a variety of cancerous tissues [50]. Considering that EMT confers stem cell-like traits to neoplastic cells, we evaluated the ability of Sng to abrogate the EMT phenotype in A388 spheroids. Western blot analysis demonstrated an increase in the expression levels of the epithelial marker E-cadherin, with concurrent downregulation in the levels of the mesenchymal markers N-cadherin and vimentin upon treatment with Sng (Fig. 7C).