Mitochondrial apoptosis and G₀/G₁-phase blockade: key mechanisms underlying triphenyltin(IV) dithiocarbamate-mediated cytotoxicity in human lymphoblastic leukemia cells

Abstract

Recent findings highlight organotin(IV) compounds as effective anticancer agents, especially for leukemia, offering a potential advancement in cancer therapy by inhibiting cell proliferation with reduced toxicity and resistance issues. Five novel di- and triphenyltin(IV) dithiocarbamate compounds (OC1-OC5) were tested against Jurkat E6.1 human lymphoblastic leukemia cells. All compounds produced potent cytotoxic effects by inducing 55% − 86% of apoptotic events at their respective IC₅₀ values, 7.1 µM (OC1), 0.1 µM (OC2), 2.8 µM (OC3), 0.2 µM (OC4) and 0.35 µM (OC5). OC2 [Ph₃Sn(N,N-diisopropyldithiocarbamate)] was the most potent, with an IC₅₀ of 0.1 µM and a selectivity index (SI) of 5.5, leading to further investigation. Mechanistic studies revealed that OC2 triggers apoptosis in Jurkat cells via the intrinsic pathway by disrupting mitochondrial membranes. This was initiated by DNA damage, leading to excessive reactive oxygen species (ROS) production and activation of caspase cascade (−9, −8, −3). Pretreatment with the antioxidant N-acetyl-L-cysteine (NAC) significantly reduced apoptosis, confirming oxidative stress involvement. OC2 also caused G₀/G₁ cell cycle arrest and upregulated cleaved-PARP and p21. These findings reveal the anti-proliferative ability of OC2 in a Jurkat T lymphoblastic leukemia cell model, which warrants further investigation for its antileukemic potential and therapeutic value.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-31015-z.

Introduction

Cancer is characterized by dysregulated cell proliferation. A thorough understanding of apoptosis, a regulated cell death process, is crucial for comprehending cancer progression^1,2. Mutations in genes can lead to changes in the cellular phenotype, disrupting the normal apoptotic response^1,2. This abnormal cellular activity creates a microenvironment that fosters genetic instability and DNA mutations, accelerating the progression of cancer. As a result, therapies that promote apoptosis, like chemotherapy, hold potential in battling this disease². Apoptosis is preferred over necrosis as a means of cell death because the latter can trigger inflammation, which can harm nearby healthy cells³.

Apoptosis is a precisely regulated and evolutionarily preserved process of cellular demise, wherein a cell undergoes self-destruction^4,5. It plays a critical role in multicellular organisms by eliminating unnecessary or excessive cells during development and neutralizing cells with DNA damage, thus hindering the progression towards cancer^5,6. Proper regulation of apoptosis is vital for preserving the normal equilibrium and integrity of cells. While apoptosis is crucial for maintaining a healthy body, malfunctions in its regulation can lead to a domino effect. Uncontrolled cell death can trigger chronic inflammation, contribute to the hardening of arteries (atherosclerosis) and even be a factor in the onset of cancers and respiratory diseases. Additionally, it’s implicated in neurodegenerative conditions like Alzheimer’s and Parkinson’s⁶.

Organotin(IV) compounds hold promise for various medical applications due to their ability to act as effective drugs when paired with specific molecules called ligands. Research suggests they have potential as anticancer agents^7,8, anti-inflammatory drugs⁹, antifungals¹⁰ and antibacterials¹¹. This versatility stems from the synergistic effects observed when ligands amplify the biological effects of these compounds. These findings suggest that organotin(IV) compounds hold promise as potent and adaptable therapeutic agents across various medical domains. Among various organotin(IV) compounds, organotin(IV) dithiocarbamates stand out due to their wide range of uses. They are valuable in agriculture, biology and even as catalysts. Additionally, they can be used to create nanoparticles made of tin sulfide. Additionally, these derivatives are valued for their capacity to maintain distinct stereochemistry within their complexes^12–14.

Dithiocarbamate is classified as a mono-anionic ligand that can create enduring complexes with a wide range of transition metals, as well as most main group elements, lanthanides and actinides. Additionally, its cationic characteristics make dithiocarbamate an effective solvent in both water and organic solvents¹⁵. The use of dithiocarbamate is widespread due to its potential as a chemotherapeutic agent, pesticide and fungicide¹⁶. According to Awang et al.⁷, dithiocarbamate ligands are prized in inorganic chemistry for their versatility in bonding with a wide range of metals. This chelating ability is attributed, in part, to the presence of two sulfur atoms within the ligand’s structure.

In a recent investigation conducted by Annuar et al.¹⁷, we documented the antitumor activities of three series of phenyltin(IV) dithiocarbamate compounds against the K562 human erythroleukemia cell line. Triphenyltin(IV) diisopropyl dithiocarbamate (OC2) exhibited the highest level of cytotoxicity among the compounds tested, while also showing moderate selectivity towards K562 cells. OC2 induced intrinsic mitochondria-mediated apoptosis by activating the caspase cascade and blocking cell cycle progression at the S phase checkpoint. This promising outcome motivates us to explore the potential of phenyltin(IV) dithiocarbamate compounds as antileukemic drugs. Consequently, this study aims to investigate the cytotoxic effects of these compounds in another human leukemia cell line.

The Jurkat cell line originates from a patient diagnosed with T-cell acute lymphoblastic leukemia (ALL). Acute lymphoblastic leukemia (ALL) is a set of conditions that affect blood cells with lymphoblastic lineage. T-cell ALL (T-ALL) and B-cell ALL (B-ALL) are two subtypes of ALL that can be distinguished based on the lymphoblastic lineage that is impacted. The survival of T-ALL patients has increased because of substantial advancements in T-ALL treatment during the past few years. However, a significant part of pediatric T-ALL patients and an even higher percentage of adult patients still encounter therapeutic failure^18,19. Immortalized T cell lines, including Jurkat cells, T-cell clones, and hybridomas, are popular in vitro models for studying apoptosis²⁰. Furthermore, in this study we present an investigation into the molecular mechanism of cytotoxicity, specifically examining the role of the mitochondrial pathway involves in cell death and cell growth regulation caused by organotin(IV) in the Jurkat human leukemia cell model. These findings enhance the potential value of organotin as a promising antileukemic agent.

Materials and methods

Compounds

Five novel compounds namely, di- and triphenyltin(IV) dithiocarbamate that represented as Ph_nSnL₂ (where Ph = C₄H₉, C₆H₅; n = 2,3; L = N,N-dithiocarbamate), Ph₂Sn (N,N-diisopropyldithiocarbamate) (OC1), Ph₃Sn (N,N-diisopropyldithiocarbamate) (OC2), Ph₂Sn (N,N-diallyldithiocarbamate) (OC3), Ph₃Sn (N,N-diallyldithiocarbamate) (OC4), and Ph₂Sn (N,N-diethyldithiocarbamate) (OC5), were synthesized and characterized as described previously¹⁷. Table 1 represents the chemical structures of OC1 – OC5, based on their corresponding ligands.

Table 1.

Chemical structures of OC1-OC5.

Di- and triphenyltin(IV) dithiocarbamate	Chemical structure
Compound 1 Diphenyltin(IV) diisopropyl dithiocarbamate MW: 625.26 g/mol
Compound 2 Triphenyltin(IV) diisopropyl dithiocarbamate21 MW: 526.11 g/mol
Compound 3 Diphenyltin(IV) diallyl dithiocarbamate22 MW: 617.26 g/mol
Compound 4 Triphenyltin(IV) diallyl dithiocarbamate22 MW: 522.11 g/mol
Compound 5 Triphenyltin(IV) diethyldithiocarbamate MW: 498.09 g/mol

Cell culture

Jurkat T lymphoblastic leukemia cells (clone E6-1) were obtained from the American Type Culture Collection (ATCC, TIB-152) and maintained in RPMI-1640 medium supplemented with L-glutamine, 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. The cells were cultured at 37 °C in a humidified incubator containing 5% CO₂.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay

The viability of Jurkat cells was determined using an MTT assay, following a modified protocol from Mosmann²³. This colorimetric assay provides an indirect measure of cell metabolic activity. OC1 and OC3 were tested at a maximum concentration of 10 µM, while OC2, OC4 and OC5 were examined at 5 µM. A serial dilution of the compounds was prepared in culture media by adding 200 µL of compound solution to row A of a 96-well plate, followed by serial dilution (1:2) across the subsequent wells (rows B-H) using 100 µL of culture medium. Jurkat cells were prepared at a density of 2 × 10⁶ cells/mL, and 100 µL of the cell suspension was added to each well containing 100 µL of compound dilution, resulting in a final cell density of 1 × 10⁶ cells/mL per well in a total volume of 200 µL. Plates were then incubated at 37 °C with 5% CO₂ for 24 h. After incubation, an MTT assay was performed to measure cell viability. Briefly, 20 µL of MTT solution (5 mg/mL) was added to each well containing 200 µL of cell suspension, yielding a final MTT concentration of 0.45 mg/mL. The plates were then incubated for 4 h at 37 °C to allow the formation of formazan crystals. The medium (180 µL) was then carefully aspirated and replaced with 180 µL of DMSO to solubilize the formazan crystals, followed by gentle agitation and a 15-minute incubation. As Jurkat is a suspension cell line, careful handling was required during aspiration to minimize cell loss, a known limitation of the MTT assay in non-adherent cultures. The optical density (OD) was measured at 570 nm using an ELISA microplate reader. The IC₅₀, the concentration inhibiting 50% of cell growth, was determined for each compound based on the methodology by Annuar et al.¹⁷.

Selectivity index (SI)

Effective anticancer drugs should preferentially target cancer cells²⁴. To assess this preferential targeting, we compared the concentration required to inhibit growth by half in normal-human B lymphocyte cells (WIL2-NS, ATCC, CRL-815) to the concentration required for the same effect in Jurkat cells.

Annexin V-FITC/propidium iodide labelling

Annexin V-FITC/PI staining was used to assess the specific cell death pathway triggered by the compound. This assay employs established protocols with minor modifications¹⁷. Each compound was applied to Jurkat cells (1 × 10⁶ cells/mL) at a concentration previously determined to inhibit their growth by 50% (IC₅₀). This incubation lasted for 24 h. Following incubation, the cells were harvested by centrifugation (220 x g, 5 min, 4 °C) and washed with chilled PBS to remove residual media. Following a subsequent centrifugation, the cell pellet was resuspended in 150 µL Annexin V Binding Buffer and stained with 2 µL Annexin V-FITC, a marker for phosphatidylserine exposure during early apoptosis, for 13 min at room temperature in the dark. Next, 10 µL Propidium Iodide (PI), a marker for necrotic and late apoptotic cells with compromised membranes, was then added and incubated for an additional 2 min. After incubation in the dark, the samples were diluted with 350 µL Annexin V Binding Buffer and analyzed using FACSCanto II flow cytometry (Becton Dickinson, USA).

Cell cycle checkpoint analysis

To evaluate the compound’s effect on cell cycle distribution, propidium iodide (PI) staining with RNase treatment was performed, adapting a published protocol²⁵. Jurkat cells (1 × 10⁶ cells/mL) were exposed to the IC₅₀ concentration for two and four hours. Following harvest, cells were fixed in 70% ethanol overnight at -20 °C to permeabilize membranes and preserve DNA. After washing, cells were stained with PI and RNase to differentiate between DNA content in G₀/G₁, S, and G₂/M phases. Flow cytometry (FACSCanto II, Becton Dickinson, USA) then quantified the distribution of at least 10,000 cells across the cell cycle.

Alkaline comet assay

DNA damage induced by the compound was assessed using a modified alkaline Comet assay based on the protocol by Singh et al.²⁶. Jurkat cells (1 × 10⁶ cells/mL) were treated with the IC₅₀ concentration for various time points (0.5–4 h). After incubation, cells were collected by centrifugation, washed and suspended in 0.6% low-melting-point agarose in PBS. This mixture was spread onto slides pre-coated with 0.6% normal-melting-point agarose and allowed to solidify at 4 °C.

Cells were then lysed overnight at 4 °C in lysis buffer consisting of 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (pH 10) and 1% Triton X-100. After lysis, slides were placed in freshly prepared alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) for 20 min to allow DNA unwinding, followed by electrophoresis at 25 V and 300 mA for 20 min at 4 °C. This run time was optimized in preliminary experiments to achieve clear comet tail formation without excessive DNA migration.

Following electrophoresis, slides were neutralized in 0.4 M Tris buffer (pH 7.5), stained with ethidium bromide (50 µL) and examined under a fluorescence microscope. A minimum of 100 randomly selected comets per treatment group were scored from each of three biological replicates using Cometscore III software (Tritek Inc.). Tail moment was used as the primary parameter to quantify DNA damage, with longer tail moments indicating greater DNA strand breakage.

Flow cytometric analysis of mitochondrial function and oxidative stress in response to OC2

Intracellular ROS levels and mitochondrial membrane potential (Δψm) were assessed using flow cytometry after treatment with the compound at its IC₅₀ for varying time points (1 to 6 h). We employed established methods for dihydroethidium (DHE) and tetramethylrhodamine ethyl ester (TMRE) staining, incorporating minor adjustments based on previous works by^17,27. Briefly, cells were harvested, washed and resuspended in 1000 µL fresh media. Then 1 µL of either 10 mM DHE or 50 µM TMRE was then added for staining, followed by 15-minute incubation at 37 °C in the dark. After washing, cells were analyzed using a FACSCanto II flow cytometer (Becton Dickinson, USA).

Cells pre-treated with N-acetyl-L-cysteine (NAC)

The involvement of intracellular oxidative stress was evaluated by pre-treating cells with the antioxidant NAC. Cells were exposed to either 20 µM or 50 µM NAC for 1 h, followed by treatment with the compound at its IC₅₀ concentration for 24 h. Subsequent steps for cell harvesting and analysis mirrored the Annexin V-FITC/PI assay protocol.

Determination of caspases − 3, -8 and − 9 activities

To identify the specific cell death pathway induced by the compound, Jurkat cells (1 × 10⁶ cells/mL) were treated with the compound’s IC₅₀ concentration for 2 and 4 h. A CaspGLOW fluorescein active caspase staining kit (BioVision) was then employed to analyze caspase activation, a key indicator of apoptosis. Following incubation, cells were collected, washed and resuspended in fresh media. Specific caspase activation (caspase-8, -9 or -3) was assessed by staining with 1 µL of the corresponding FITC-conjugated inhibitor (FITC-IETD-FMK, FITC-LEHD-FMK or FITC-DEVD-FMK) for 1 h at 37 °C. Cells were then washed, resuspended in buffer and analyzed using a BD FACSCanto II flow cytometer.

Western blot analysis

To investigate protein expression changes, Jurkat cells (1 × 10⁶ cells/mL) were treated with the compound at its IC₅₀ concentration for 0.5 to 6 h. Following treatment, cells were washed with ice-cold PBS and lysed using RIPA buffer. After centrifugation (13500 rpm, 30 min, 4 °C), protein lysates were collected and stored at -80 °C. Protein concentration was determined using a Bradford assay. For Western blot analysis, protein lysates were prepared at 1 mg/mL and mixed 1:1 with Laemmli buffer, yielding a final protein concentration of 0.5 mg/mL; 25 µg of protein per lane were loaded onto SDS-PAGE gels and separated by electrophoresis, followed by transfer to PVDF membranes. Membranes were blocked with 5% BSA in TBS-T for 1.5 h and incubated overnight at 4 °C with primary antibodies. HRP-conjugated secondary antibody (anti-rabbit) was then added for 1 h. β-actin served as a loading control. Protein bands were visualized using an ECL kit.

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons, using SPSS software (version 25.0). Differences between groups were considered statistically significant at p < 0.05.

Results

Differential cytotoxicity of di- and tri-phenyltin(IV) dithiocarbamates against Jurkat leukemia and WIL2-NS cells

Jurkat cells exhibited pronounced cytotoxicity after 24-hour treatment with the tested compounds (Figs. 1 and 2). Table 2 summarizes the IC₅₀ values, demonstrating potent cytotoxic activity against Jurkat cells. Etoposide (ETO), the positive control, had an IC₅₀ of 0.87 µM. Notably, Table 3 highlights the selectivity of these compounds. Among them, OC2 displayed the highest selectivity towards Jurkat cells based on its IC₅₀ value on the non-cancerous WIL2-NS cell line and the corresponding selectivity index (SI).

Fig. 1.

Jurkat cells viability after 24-hour treatment with OC1 and OC3. Data represent mean cell viability (%) ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

Fig. 2.

Jurkat cells viability after 24-hour treatment with OC2, OC4 and OC5. Data represent mean cell viability (%) ± SEM from three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

Table 2.

Half-maximal inhibitory concentration (IC₅₀) values of compounds for Jurkat cell proliferation.

	ETO	OC1	OC2		OC3		OC4	OC5
IC50 (µM) ± S.E.M	0.87 ± 0.06	7.1 ± 0.6	0.1 ± 0.01		2.8 ± 0.13		0.2 ± 0.01	0.35 ± 0.01

Table 3.

IC₅₀ values and selectivity index (SI) of compounds in WIL2-NS cells.

	OC1	OC2		OC3	OC4	OC5
IC50 (µM) ± S.E.M	2.6 ± 0.3	0.55 ± 0.02		1.2 ± 0.14	0.2 ± 0.016	0.2 ± 0.03
SI	0.37	5.5		0.42	1	0.57

Apoptosis induction by di- and tri-phenyltin(IV) dithiocarbamates

To determine if the observed cytotoxicity involved apoptosis, Jurkat cells were treated with the compounds for 24 h. They were then stained with FITC-annexin V and propidium iodide (PI) for flow cytometric analysis. The findings depicted in Fig. 3 demonstrate that all compounds induced apoptosis, leading to a range of apoptotic cell percentages from 55% to 86%. The treatment groups exhibited the following percentages of apoptotic events: 68.1 ± 4.2% at 7.1 µM OC1, 55.6 ± 5.0% at 0.1 µM OC2, 86.7 ± 2.9% at 2.8 µM OC3, 84.5 ± 5.3 at 0.2 µM OC4 and 73.4 ± 4.8% at 0.35 µM OC5. In comparison, the negative control displayed a significantly lower percentage of apoptotic events at only 15.0 ± 2.5% (p < 0.05).

Fig. 3.

Jurkat cell viability, apoptosis and necrosis after 24-hour treatment with compounds at IC₅₀. Data represent mean ± SEM from three independent replicates. Representative dot plots are provided in Supplementary Figure S1. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

OC2 treatment disrupts cell cycle progression

Triphenyltin(IV) diisopropyl dithiocarbamate (OC2) exhibited the most potent antiproliferative effects on Jurkat cells among the tested compounds, characterized by its potent cytotoxicity and ability to induce apoptosis. Notably, OC2 triggered 55% apoptotic events at its lowest IC₅₀ concentration, highlighting its high efficacy. Furthermore, its selectivity for Jurkat cells was superior, as reflected by a selectivity index (SI) of 5.5. Consequently, OC2 (IC₅₀ concentration) was chosen for further mechanistic investigation of phenyltin(IV) dithiocarbamates in this study. As shown in Fig. 4, OC2 effectively halted the Jurkat cell cycle in the G₀/G₁ phase within 2–4 h, with a significantly higher proportion of stained cells compared to the control.

Fig. 4.

Jurkat cell cycle distribution after OC2 treatment for 2 and 4 h. Cells were fixed, stained with propidium iodide (PI) and analyzed by flow cytometry. The proportions of cells in G₀/G₁, S and G₂/M phases were determined using ModFit LT software (Verity Software House, USA). Data represent mean ± SEM from 3 independent replicates. Representative flow cytometry histograms for the cell cycle analysis are provided in Supplementary Figure S2. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

OC2 treatment induces early-stage DNA damage

The alkaline Comet assay revealed DNA damage in Jurkat cells treated with OC2. As shown in Fig. 5A, damaged cells displayed comet-like tails due to DNA fragmentation following electrophoresis. OC2 induced this DNA damage in a time-dependent manner, with visible effects as early as 30 min post-treatment. Furthermore, the length of the comet tails increased with longer OC2 exposure (Fig. 5B), suggesting a progressive accumulation of DNA breaks.

Fig. 5.

Jurkat cell DNA damage after OC2 treatment (0.5 to 4 h). (A) Representative fluorescence microscopy images with EtBr staining: (i) control, (ii) etoposide (positive control) and (iii-vi) OC2 treatment for increasing durations. (B) Quantification of DNA damage by tail moment (mean ± SEM, 3 replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

Mechanism of OC2 mitochondrial membrane integrity

Figure 6 shows the changes in mitochondrial membrane potential (ΔΨm) induced by OC2 in Jurkat cells treated with IC₅₀ concentrations over time. The TMRE staining assay quantified the percentage of TMRE-negative cells, representing the population with reduced TMRE fluorescence and thus loss of ΔΨm. A slight increase in TMRE-negative cells was observed after 1 h of treatment, suggesting early mitochondrial depolarization, although the change was not statistically significant (p > 0.05) compared to the negative control. Prolonged exposure to OC2 for 2, 4 and 6 h resulted in a significant increase in TMRE-negative cells (p < 0.05), indicating progressive mitochondrial depolarization. Specifically, the proportion of TMRE-negative cells increased to 33.0 ± 4.5%, 37.4 ± 2.4% and 43.2 ± 5.5% at 2, 4 and 6 h, respectively, compared to untreated cells.

Fig. 6.

The loss of ΔΨm in OC2-treated Jurkat cells for 1–6 h of treatment. The data are presented as mean (± S.E.M) values obtained from three independent experimental replicates. Representative fluorescence histograms are shown in Supplementary Figure S3. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

Oxidative stress in OC2-mediated cytotoxicity

To explore the potential contribution of oxidative stress to OC2-induced apoptosis, we measured intracellular ROS levels. Jurkat cells treated with OC2 displayed a time-dependent increase in ROS production compared to the negative control (Fig. 7A). This suggests OC2 promotes ROS generation, potentially linking it to the observed mitochondrial dysfunction (ΔΨm loss). To further explore this connection, cells were pre-treated with the antioxidant NAC before OC2 exposure. Pre-treating cells with NAC significantly decreased the number of apoptotic cells compared to the control (Fig. 7B). These findings suggest that ROS contribute to the apoptotic pathway triggered by OC2.

Fig. 7.

(A) The ROS production in Jurkat cells after being exposed with OC2 for 1–6 h. The data are presented as mean (± S.E.M) values obtained from three independent experimental replicates. Representative DHE fluorescence histograms can be found in Supplementary Figure S4. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control. (B) Jurkat cell viability, apoptosis, and necrosis following pre-treatment with NAC (20 µM and 50 µM). Data represent mean ± SEM from 3 independent replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Different letters indicate significant differences between groups (p < 0.05): a = vs. negative control; b = vs. 20 µM NAC + OC2; c = vs. 50 µM NAC + OC2.

Caspase cascade activation during OC2-mediated apoptosis

The study assessed caspase activation, a hallmark of apoptosis, using fluorescence-tagged caspase-specific substrates. Jurkat cells treated with OC2 exhibited time-dependent activation of caspase-9, a key executioner caspase, as shown in Fig. 8. The percentage of cells with active caspase-9 steadily increased from 2 to 4 h post-treatment. Notably, caspase-8 activation, associated with the extrinsic apoptotic pathway, remained unchanged compared to the control up to 4 h. However, a significant increase in caspase-3 positive cells, another executioner caspase, was observed after 4 h of OC2 treatment, suggesting its involvement in the later stages of apoptosis.

Fig. 8.

Jurkat cell caspase activity after OC2 treatment (2 and 4 h). Caspase-8, -9, and − 3 activation are shown. Data represent mean ± SEM from 3 independent replicates. Representative fluorescence histograms for caspase-9, -8 and − 3 activation are shown in Supplementary Figure S5. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

Analysis of protein expression p21 and cleaved-PARP

Western blot analysis revealed increased p21 and cleaved-PARP protein expression in OC2-treated Jurkat cells, as shown in Fig. 9. While a slight increase in p21 was observed after 2 h, it became statistically significant (p < 0.05) at later time points (4 and 6 h) compared to the control. Cleaved-PARP expression also exhibited a similar pattern, showing a marked rise (p < 0.05) at 6 h compared to the control. These findings suggest OC2-induced apoptosis through the mitochondrial pathway, involving caspase activation and p21 upregulation, potentially contributing to cell cycle disturbance in G₁/G₀ phase.

Fig. 9.

The expression of p21 protein and cleaved-PARP in Jurkat cells treated with OC2 at IC₅₀ dose. (A) illustrates the protein bands of p21 and cleaved-PARP expression. Meanwhile, (B) presents the bar graph of p21 protein and cleaved-PARP expression (arb. unit). The data are presented as mean (± S.E.M) values obtained from three independent experimental replicates. Uncropped western blot images corresponding to Fig. 9A can be found in Supplementary Figure S6. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05 vs. negative control.

Discussion

The success of cisplatin, a platinum-based chemotherapeutic drug, has highlighted the potential of metal compounds in revolutionizing cancer treatment. Its potent antitumor activity has proven effective against a wide range of cancers, significantly improving patient outcomes. However, cisplatin’s effectiveness is hampered by significant limitations. These limitations include the drug’s ability to cause DNA damage in healthy cells along with the concerning development of drug resistance in cancer cells over time^14,27,28. This has driven the exploration of alternative metal-based drugs, particularly non-platinum compounds, with the goal of achieving improved efficacy and reduced side effects.

Beyond platinum, other metals like copper, gold²⁹, gallium, germanium, tin, ruthenium, iridium, and lanthanum³⁰ demonstrate promising activity in cancer treatment. However, to date, none of them have entered the clinical trial stage¹⁴. Gold and tin derivatives have gained significant interest among researchers due to their demonstrated high potential as anticancer drugs³¹. In a prior study, our group³², demonstrated the cytotoxic activity of a novel phenyltin(IV) diallyldithiocarbamate compound in human colon adenocarcinoma cells (HT-29). This compound displayed selectivity towards HT-29 cells, with a high selectivity index (SI) of 9.03 and induced apoptosis-mediated cell death. Awang et al.³³ also revealed that all three triphenyltin(IV) dithiocarbamate compounds studied were able to inhibit 50% of CCL-119 human leukemia cells’ growth at low IC₅₀ concentrations (< 1 µM). Moreover, all of these compounds induced apoptosis within 24 h of treatment at the IC₅₀ concentration. While phenyltin(IV) compounds have demonstrated significant cytotoxic effects on various types of human tumor cells, their specific mechanisms of action have not been thoroughly studied yet.

Di- and triphenyltin(IV) dithiocarbamate compounds demonstrated a potent capacity to suppress the proliferation of Jurkat cells, effectively halting their growth at low micromolar concentrations ranging from 0.1 µM to 7.1 µM, as determined by MTT assays. This potent dose-dependent inhibition of Jurkat cell growth classifies these compounds as highly potent (IC₅₀ < 7.34 µM) according to established guidelines^8,34. Notably, the observed order of potency was OC1 < OC3 < OC5 < OC4 < OC2, suggesting that structural variations may influence their efficacy. This sequence clearly demonstrates that triphenyltin(IV) compounds have stronger cytotoxic effects compared to diphenyltin(IV), consistent with studies reported by Abd Aziz et al.³⁵, Hadi et al.³⁶ and Sirajuddin et al.³⁷, where groups treated with triorganotin(IV)-substituents demonstrated the most potent toxicity. Adeyemi et al.³⁸ also observed that organotin(IV) compounds with longer alkyl or aryl chains exhibit greater biological activity.

The presence of phenyl groups, known for their hydrophobic nature, likely enhances the lipophilicity of these compounds. This property can influence their cellular uptake and distribution within the organism³⁹. This can be explained by the Tweedy chelation theory, which suggests that the metal ion’s polarity decreases when it forms a complex, leading to increased lipophilicity of the complex. Moreover, this increased lipophilicity allows the complex to penetrate the cellular membrane more easily⁴⁰. Therefore, as the number of aryl substituents increases from two in OC1 and OC3 to three in OC2, OC4 and OC5, the polarity decreases, resulting in increased lipophilicity of the complexes. Referring to previous studies, the degree of lipophilicity is associated with cytotoxic activity, where higher lipophilicity of a compound corresponds to stronger cytotoxic effects^41–43.

Several other researchers have also stated that the relationship between the structure of a compound and its activity depends on the ligand and the substituent groups -R attached to the tin atom^44,45. The importance of ligands in modulating the cytotoxic properties of a complex is crucial. The lipophilicity and stability of metal complexes are highly dependent on the properties of the ligand system⁴⁶. Therefore, the design of metal-based drugs and the selection of suitable ligand systems have significant implications in specificity, toxicity control and bioavailability⁴⁷. To achieve this goal, numerous ligand systems have been developed, among which dithiocarbamate has emerged as a favored option for diverse medical applications. These include its use as carbonic anhydrase (CA) inhibitors and its significance as a compound in cellular metabolism⁴⁸. Johnson and colleagues⁴⁹ also stated that dithiocarbamate compounds are non-organic compounds that can bind to metal ions. Moreover, they function as ligands in in vitro models used as antioxidants, inhibitors or inducers of apoptosis, as well as enzyme inhibitors. Furthermore, the property of dithiocarbamate compounds, which exhibit greater solubility in organic solvents (lipophilic) compared to water, facilitates the transportation of organotin(IV) compounds across cell membranes. The coordination of dithiocarbamate has also been reported to have potential in chemoprotection⁵⁰, treatment of bacterial and fungal infections, as well as addressing HIV-AIDS and cancer⁵¹.

Our data from the Annexin V-FITC/PI assay demonstrate that OC1-OC5 induce apoptosis. A previous study conducted by Ray et al.⁵² also found that tri-n-butyltin(IV) benzoate compound induced apoptosis-mediated cell death in K562 cells. Our results are also corroborated by a previous study by Kamaludin et al.⁵³, which showed that organotin(IV) compounds induced a substantial amount of apoptotic cells in several human leukemia cell lines, including Jurkat E6.1, K562 and HL-60. Costa et al.⁵⁴ further strengthened the evidence for organotin(IV) compounds as apoptosis inducers in malignant melanoma. Their study showed that both diorganotin(IV) and triorganotin(IV) compounds triggered apoptosis in A375 human melanoma cells. Additionally, Awang et al.⁵⁵ also reported that triphenyltin(IV) dithiocarbamate compound triggers apoptosis in human colon adenocarcinoma cells, HT-29. Organotin(IV) derivatives have demonstrated efficacy as potential anticancer agents, effectively triggering apoptosis in diverse cancer cell lines^25,56.

OC2 has shown the most potent cytotoxic effects on both K562¹⁷ and Jurkat leukemia cell lines, inhibiting 50% of cell growth at very low doses of 0.55 µM (K562 cells)¹⁷ and 0.1 µM (Jurkat cells). Furthermore, at IC₅₀ concentration, OC2 was able to induce apoptotic cells within K562¹⁷ and Jurkat cells, accounting for 57.1 ± 4.5% and 55.6 ± 4.95%, respectively. Therefore, OC2 was selected for a more detailed investigation of its mechanism of action in influencing cytotoxicity on Jurkat cells. This selection was not only based on the MTT and Annexin V-FITC/PI assays data, but also on the OC2’s ability to exhibit good selectivity towards both leukemia cell lines, with a selectivity index (SI) value of 1 for K562 cells¹⁷ and 5.5 for Jurkat cells. OC2 stands out as a promising candidate for development as an antileukemic drug, demonstrating potent antiproliferative activity against leukemia cells.

The cell cycle is regulated by a dual mechanism involving protein phosphorylation and degradation. Enzymes called cyclin-dependent kinases (CDKs) phosphorylate specific proteins, triggering events like DNA replication or cell division. Phosphatases then remove these phosphates, allowing the proteins to return to their inactive state. Additionally, the cell employs a cleanup crew called the ubiquitin-proteasome system, which targets and breaks down proteins no longer needed, ensuring a smooth cell cycle progression as described by Bai et al.⁵⁷. These regulatory mechanisms prevent cells with DNA damage in the G1 phase from progressing into the S phase, allowing time for DNA repair before entering the M phase. Consequently, chromosomes can be properly replicated before cell division occurs⁵⁷. Apoptosis induction is a common feature of cytotoxic drugs and DNA-damaging agents. These agents achieve this by halting the cell cycle at different stages, such as G₀/G₁, S, or G₂/M phases²⁴.

Cell cycle analysis revealed that OC2 treatment for 4 h induced cell cycle arrest in Jurkat cells at the G1/G0 phase. This finding is consistent with a recent study by Attanzio et al.⁵⁸, which reported that the studied organotin(IV) compound disrupted the cell cycle at the G₁/G₀ phase. The G₁/G₀ phase arrest suggests that cancer cells undergo DNA damage by inhibiting DNA replication and transcription, ultimately leading to apoptosis⁵⁸. Additionally, several other organotin(IV) derivatives have been shown to interfere with the development of various human cancer cell cycles at the G₀/G₁ and G₂/M phases^59–62. The disruptions in different phases of the cell cycle emphasize the importance of both the organotin(IV) compound and its ligand in determining its biological activity.

In this study, OC2 was able to induce DNA damage as early as the 30th minute after treatment in Jurkat cells. DNA damage plays a crucial role in determining cell fate. Mild damage, as reported by Jiang et al.⁶³ and Surova and Zhivotovsky⁶⁴, can disrupt cell cycle progression, potentially allowing for repair mechanisms. However, extensive DNA damage can overwhelm these mechanisms, triggering apoptosis signaling pathways to eliminate the compromised cell. In some cases, severe damage can even lead to necrosis, a form of uncontrolled cell death. In line with previous findings⁶³, OC2 treatment induced time-dependent DNA damage in Jurkat cells, as evidenced by comet tail formation in treated cells. DNA damage can occur due to the action of ROS, random errors during replication or recombination and exposure to environmental or therapeutic genotoxic substances⁶⁵. Single-celled (unicellular) organisms react to DNA damage by triggering cell cycle checkpoints and repair processes, whereas multicellular organisms have additional potential to eliminate damaged cells by inducing cell death⁶⁵. The DNA damage inflicted by genotoxic anticancer drugs, including methylating agents, ionizing radiation and cisplatin, activates apoptosis⁶⁶. These lesions disrupt essential cellular functions like transcription and DNA replication. Studies involving cells with deficiencies in DNA repair mechanisms due to mutations or the blockade of essential repair proteins, as well as studies involving cytotoxic modified nucleotide precursors incorporated into DNA, have demonstrated the ability of DNA lesions to trigger apoptosis⁶⁶.

OC2 treatment rapidly impaired mitochondrial function in Jurkat cells, with Δψm significantly dropping (p < 0.05) within 2 h. This early event was followed by a rise in intracellular ROS production after 4 h, suggesting a potential link between mitochondrial dysfunction and ROS generation in the OC2-induced apoptotic pathway. Based on TMRE and DHE staining data, OC2 showed slight differences compared to most previous studies in signaling the apoptotic pathway, where OC2 was found to affect mitochondria-mediated apoptosis followed by ROS generation both in K562¹⁷ and Jurkat human leukemia cell lines. Studies conducted by Attanzio et al.⁵⁸, Wang et al.⁶², Ge et al.⁶⁷, Girasolo et al.⁶⁸, Yusof et al.⁶⁹ and Zhang et al.⁷⁰ have reported that organotin(IV) derivative compounds used induced apoptosis through ROS-mediated dysfunction of mitochondria, similar to the mechanism of action of cisplatin⁷¹. This study, alongside Annuar et al.¹⁷, provides the first glimpses into the action mechanism of organotin(IV) compounds in human leukemia models. We demonstrate that these compounds trigger apoptosis by regulating ΔΨm, likely initiated by DNA damage.

Interestingly, the OC2-induced apoptotic pathway observed in both K562¹⁷ and Jurkat cells correlates with the findings of Roslie et al.⁷². The stilbene compound used by Roslie et al.⁷² to treat K562 leukemia cells targeted mitochondria directly, resulting in a decrease in ΔΨm followed by ROS expression, leading to cytochrome-c release and ultimately triggering the apoptotic pathway. This indicates that apoptotic regulators can control different signaling pathways of apoptosis. According to Circu and Aw⁷³, it has been proven the significant involvement of mitochondria within living cells in the generation of free radicals. In the energy conversion process within the Krebs cycle, a fraction of electrons is spontaneously released to oxygen, leading to the formation of superoxide free radicals⁷⁴. It is known that ROS are a natural byproduct of regular mitochondrial respiration, as highlighted by Chance et al.⁷⁵. Approximately 1–3% of electrons within the mitochondrial electron transport chain are inadvertently leaked during the reduction of oxygen to water, resulting in the production of superoxide radicals⁷⁶.

Pretreatment with NAC, an antioxidant that scavenges ROS, notably attenuated in Jurkat E6.1 cells (p < 0.05). This suggests ROS plays a role in OC2-induced apoptosis. Similar findings were reported by Li et al.⁷⁷ where NAC reduced ROS and apoptosis in gastric cancer cells treated with di-n-butyl-di-(4-chlorobenzohydroxamato)tin(IV). Consistent with our findings, Annuar et al.¹⁷ reported a notable decrease in glutathione (GSH) levels (p < 0.05) in K562 cells treated with the IC₅₀ dose of OC2 for 6 h. This depletion of GSH was accompanied by a substantial elevation in ROS levels (p < 0.05) during the same treatment period. These observations suggest a potential link between GSH depletion, ROS production, and OC2-induced apoptosis. GSH is a recognized direct antioxidant and serves as a substrate for various antioxidant enzymes. The delayed rise in ROS production compared to the early decrease in Δψm suggests that ROS generation might be a downstream consequence of mitochondrial dysfunction in OC2-mediated apoptosis. NAC exerts its antioxidant activity through at least three mechanisms: (i) directly scavenging specific reactive oxygen species (ROS), (ii) indirectly boosting glutathione (GSH) synthesis by providing cysteine, a crucial precursor and (iii) breaking disulfide bonds in proteins, thereby regenerating the pool of essential antioxidant thiol groups⁷⁸.

OC2 treatment for 4 h stimulated the enzymatic activity of caspase-9 and − 3 (p < 0.05) in Jurkat cells, indicative of the initiation of intrinsic mitochondrial apoptosis. Conversely, caspase-8 activation remained unchanged. Consistent with DNA damage, TMRE loss, ROS production and this caspase profile, OC2 likely triggers apoptosis through an intrinsic, DNA damage-initiated mitochondrial pathway.

Organotin(IV) compounds, especially those containing tin(IV), have shown promise as potential anticancer agents due to their ability to trigger apoptosis in cancer cells through the activation of specific enzymes called caspases. For example, tributyltin (TBT) induces apoptosis by activating caspases-3 and − 7⁷⁹. Additionally, studies have demonstrated that organotin(IV) compounds with specific structures, such as 4-nitro-N-phthaloylglutamine, can activate caspases-3 and − 9 in a dose-dependent manner, suggesting the involvement of the mitochondrial pathway in their ability to induce apoptosis in liver cancer cells (HepG2 cells)⁸⁰. Furthermore, these compounds have been shown to interact with DNA, potentially contributing to their apoptotic effects. Similarly, trimethyltin(IV) compound (TMT) reported by Jenkins and Barone⁸¹ is capable of inducing apoptosis through caspase-3 and caspase-9, along with p38 protein activity in PC12 cells. A study by Anasamy et al.⁸² explored a tribenzyltin(IV) carboxylate derivative and its effects on various breast cancer cell lines (MCF-7, MDA-MB231, and 4T1). The compound significantly induced caspase-3 and − 7 activation, hallmarks of the apoptotic (cell death) pathway. Interestingly, it also caused a rise in caspase-9 activity, indicative of mitochondrial pathway involvement, and a slight increase in caspase-8 activity at certain points, suggesting potential crosstalk between apoptotic pathways. This suggests the compound may trigger apoptosis in these cancer cells through two different pathways: the intrinsic pathway (internal cellular signals) and the extrinsic pathway (external signals).

PARP-1 acts as a target for caspases during apoptosis. Caspase-3 and caspase-7 specifically cleave PARP-1, leaving behind a signature fragment indicative of apoptosis. While most caspases can modify PARP-1 in vitro, caspases 3 and 7 appear to be the primary executioners in cellular contexts⁸³. We evaluated the influence of OC2 on cleaved-PARP protein levels over time. While no significant rise was observed from 0.5 to 4 h, a noteworthy increase in cleaved-PARP was evident at the 6-hour mark. This time-dependent effect aligns with the findings of Stridh et al.⁷⁹, where tributyltin(IV) exposure also induced a gradual increase in cleaved-PARP protein. Moreover, the induction of cleaved-PARP expression by OC2 closely correlates with the level of caspase-3 activity, which showed an increase throughout the treatment period. The data obtained from this study demonstrate that the apoptotic checkpoint occurs downstream through important nuclear effector molecules.

Playing a critical role alongside p53, the tumor suppressor protein p21 acts as a conductor of cell division. It accomplishes this by putting the brakes on cyclin-dependent kinases (CDKs), essential proteins that drive cell cycle progression⁸⁴. Therefore, the expression levels of p21 protein were determined in this study. Treatment with OC2 for just 4 h significantly boosted p21 protein levels in Jurkat cells. This rise in p21 coincided with cell cycle arrest at the G₁/G₀ phase, observed during the same timeframe. These findings suggest that OC2 triggers p21 activation, leading to cell cycle inhibition. This aligns with previous studies by Li et al.⁷⁷ and Fanil et al.⁸⁵, where similar p21 upregulation was seen in human breast and gastric cancer cells upon treatment with N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzohydrazidatoaquatin(IV) chloride and di-n-butyl-di-(4-chlorobenzohydroxamato)tin, respectively.

In silico prediction using ADMETlab¹⁷ suggests favorable pharmacokinetic properties for OC2. Both the triphenyltin(IV) and diisopropylthiocarbamate moieties contribute to its ability to permeate cell membranes, potentially due to the hydrophobic nature of organotin(IV) compounds⁸⁶. This characteristic allows them to interact with various intracellular targets. The predicted CaCo2 permeability test results indicate that OC2¹⁷ might possess good intestinal absorption. CaCo2 cells, a model for intestinal drug absorption, express transporter and efflux proteins alongside Phase II conjugation enzymes, reflecting potential absorption and metabolic pathways⁸⁷.

Moreover, OC2 is unlikely to be a substrate or inhibitor of P-gp, potentially allowing for blood-brain barrier penetration^17,88. Additionally, OC2 is predicted not to inhibit any major CYP450 enzymes, potentially minimizing drug interactions¹⁷. Furthermore, in silico models suggest that OC2 may lack mutagenic and carcinogenic potential and might not cause skin sensitization or liver injury¹⁷. However, in vitro and in vivo studies are necessary to confirm these predictions.

Our investigation revealed potent cytotoxic activity for all five synthesized di- and triphenyltin(IV) dithiocarbamates, with OC2 exhibiting particular selectivity towards Jurkat leukemia cells. OC2 triggered apoptosis via the mitochondrial pathway and halting cell division at the G₁/G₀ checkpoints in Jurkat cells. Interestingly, OC2’s mechanism appears to differ from some reported pathways, where DNA damage initiates the process. In our study, OC2 caused a decrease in Δψm followed by ROS production, potentially linking mitochondrial dysfunction to ROS generation, as observed by Sinha et al.⁸⁹. This imbalance in the Bax/Bcl-2 ratio disrupts mitochondrial permeability, leading to cytochrome c release and apoptosome formation. Activated caspase cascade (caspase-9, caspase-3, and PARP cleavage) ultimately leads to apoptosis. The proposed mechanism of action for OC2 at its IC₅₀ concentration is illustrated in Fig. 10. Notably, OC2 also triggered p21 upregulation, potentially contributing to cell cycle arrest.

Fig. 10.

Model of OC2-induced apoptosis in Jurkat cells, involving mitochondrial dysfunction and cell cycle regulation.

Conclusion

The five newly synthesized derivatives of phenyltin(IV) dithiocarbamate tested in this study effectively exhibited cytotoxicity against Jurkat E6.1 leukemia cell line at low micromolar concentrations, primarily through apoptosis. Notably, OC2 displayed selectivity towards Jurkat cells compared to normal lymphocytes. OC2’s mechanism appears to involve initial DNA damage, leading to a cascade of events. It disrupts mitochondrial function (ΔΨm), triggers ROS production and activates the Bax/Bcl-2 pathway. This ultimately results in cytochrome c release, apoptosome formation and caspase activation, culminating in cell death. Interestingly, OC2 also modulates p21 protein, causing cell cycle arrest within hours. These findings suggest organotin(IV) compounds hold promise as novel anticancer agents. Further exploration involving structural modifications and in-depth molecular characterization is warranted to develop effective and selective cancer therapies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

SNSA carried out the experiment. NFK is the principal investigator of the project. SNSA wrote the manuscript with support and supervision from NFK, NA and KMC. All authors provided critical feedback and helped shape the context of the paper.

Funding

This study was supported by a research grant from the Malaysian Ministry of Higher Education (MOHE), FRGS/1/2019/STG04/UKM/03/3.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.