Elucidating the Suppressive Capabilities of Novel Organotin(IV) Compounds Derived from N-Methyl-N-benzyl-dithiocarbamate Ligands: Assessing their Cytotoxicity Effects on Human Leukemic Cells (Jurkat E6.1)

Introduction

In 2020, leukemia contributed to roughly 2.5% of newly diagnosed cancer cases and was responsible for about 3.1% of global cancer mortality (Sung et al., 2021). Its incidence and death rates vary depending on age, gender, and geographic region (Lim et al., 2014). The peak incidence occurs in young children aged 0–5 years (9.24%) and older adults between 70–74 years (8.92%), while individuals over 60 account for more than half of leukemia-related deaths (57.56%) (Du et al., 2022). Although developed countries show higher diagnosis rates, the mortality burden tends to be greater in developing nations (Tebbi, 2021). Established risk factors include tobacco use, radiation exposure, contact with hazardous chemicals such as benzene, prior chemotherapy treatments, and inherited conditions like Down syndrome (Ansari et al., 2023).

Leukemia encompasses a group of rapidly progressing malignancies that affect the blood and bone marrow, compromising immune function (Dong et al., 2020). It may present as either acute or chronic, depending on disease progression and prognosis (Ahmed et al., 2019). Leukemia may present as acute or chronic, and is further classified by lymphoid or myeloid lineage, with ALL being the most prevalent pediatric type (Du et al., 2022; Kakaje et al., 2020). Acute lymphoblastic leukemia (ALL) was reported as the most prevalent pediatric cancer, often responds favorably to treatment. It arises from genetic alterations in B or T lymphoid progenitors, resulting in the excessive accumulation of immature blast cells in the bone marrow (Yasuj et al., 2020).

ALL treatment generally involves remission induction, consolidation, intensification, and maintenance phases. Chemotherapy is the cornerstone of initial treatment, often supplemented with targeted therapies throughout the process (Aureli et al., 2023). Chemotherapeutic agents disrupt cancer cell division through various mechanisms including DNA cross-linking inhibition, topoisomerase activity interference, and microtubule destabilization during mitosis (Wilkinson and Sumar, 2022). Daunorubicin, a frequently used anthracycline antibiotic, intercalates into DNA and hampers macromolecule synthesis (Al-Aamri et al., 2019). However, its side effects—including nausea, mucositis, hair loss, and diarrhea—can limit its efficacy (Roman Diaz et al., 2024). Moreover, resistance to chemotherapy presents a significant obstacle in treating T-cell acute lymphoblastic leukemia (T-ALL), with relapse rates of 20% in children and 40% in adults, and survival rates under 7% in relapsed cases (Fielding et al., 2007; Pui et al., 2008).

In light of these challenges, organotin(IV) compounds have garnered attention for their potent anticancer properties (Abd Aziz et al., 2023a). These compounds often demonstrate more favorable toxicity profiles compared to conventional platinum agents (Attanzio et al., 2020). Their catalytic and redox capabilities, structural versatility, and ability to interact with biologically active molecules contribute to their effectiveness (Rabiee et al., 2019). The main mechanism of exerting anticancer effects through organotin(IV) compounds is the induction of apoptosis (Jakšić, 2012; Stathopoulou et al., 2021; Syed Annuar et al., 2021, 2022). Due to their electrophilic properties, these compounds readily bind to biomolecules rich in electrons, such as phospholipids, ATP, and nucleic acids, disrupting essential cellular processes (Abd Aziz et al., 2023a). Additionally, organotin(IV) compounds can interact with DNA’s phosphate backbone, causing structural changes that lead to cell death (Yusof et al., 2019).

Dithiocarbamate ligands have emerged as promising agents in cancer treatment, owing to their capacity to influence critical proteins associated with apoptosis, oxidative stress, transcription, and protein degradation (Sofuoglu and Kosten, 2005). Besides, dithiocarbamates can inhibit catalase activity, leading to an increase in oxidative species and facilitating apoptosis (Fu et al., 2019). This research examines the cytotoxic properties of newly developed organotin(IV) compound derived from N-methyl-N-benzyldithiocarbamate ligands specifically on Jurkat E6.1 leukemia cells.

Figure 1: (a) The crystallographic structure of dibutyltin(IV) N-methyl-N-benzyldithiocarbamate (Compound 1) (Abd Aziz et al., 2023b), (b) The chemical structure of diphenyltin(IV) N-methyl-N-benzyldithiocarbamate (Compound 2),Click here to View Figure

Materials and Methods

Stock Solution Preparation 

To prepare the stock solutions, precise quantities of each compound were measured: 0.0125 g for Compound 1, 0.0109 g for Compound 3, and 0.0200 g for Compound 2. These were then dissolved in dimethyl sulfoxide (DMSO). Compounds 1 and 3 were each dissolved in 1 mL of DMSO, while Compound 2 required 6 mL of DMSO for complete dissolution. The resulting stock solutions had concentrations of 20 mM for Compounds 1 and 3, and 5 mM for Compound 2. All stock solutions were stored at 4 °C to preserve stability and purity until needed. Prior to experimentation, the stock solutions were diluted to the desired concentrations using the appropriate media.

Cell Line and Culture Conditions

Both Jurkat E6.1 and WIL-2NS cell lines were sourced from ATCC and cultured at the Biocompatibility and Biotechnology Laboratory, UKM, Kuala Lumpur. RPMI-1640 medium was used to culture both cells following ATCC guidelines, and incubation was carried out at 37 °C under humidified conditions with 5% CO₂.

MTT Cytotoxicity Assay 

MTT assay was employed to evaluate the cytotoxicity of the compounds on  leukemic cells (Jurkat E6.1) and non-leukemic cells (WIL-2NS), which were plated at 2 × 10⁶ cells/mL in 96-well flat-bottom plates. Compounds 1 and 2 were used at 5 μM, whereas Compound 3 was applied at 10 μM, with a 24-hour treatment duration. The untreated cells in this experiment served as the negative control, while menadione (20 μM) as the positive control. After treatment, the cells were incubated with the MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] for 4 hours, which viable cells reduce to purple insoluble formazan. Subsequently, the insoluble formazan crystals were solubilized using DMSO. The intensity of the color formed was measured  (570 nm) to evaluate cell viability, and IC₅₀ values were derived from the resulting data.

Cell Morphology Assessment

Jurkat E6.1 cells were treated based on their IC₅₀ concentrations determined from the MTT assay, with the objective of inducing approximately 50% cell death (CD₅₀) over a 24-hour period. The untreated cells in this experiment served as the negative control, while menadione as the positive control. Cell seeding followed the same protocol as the MTT assay but was performed in 6-well plates. After incubation, morphological changes associated with cell death were observed under an inverted light microscope at 40× magnification.

Selectivity Index Calculation

The selectivity index (SI) was determined by dividing the IC₅₀ value obtained for WIL2-NS cells by the corresponding IC₅₀ value for Jurkat E6.1 cells, as measured in the MTT assay. This ratio reflects the compound’s preference for targeting cancerous versus normal cells. A higher SI indicates improved selectivity towards cancer cells, whereas a lower SI suggests increased toxicity to normal cells. This parameter helps evaluate the therapeutic potential and safety margin of the tested agents.

Statistical Analysis

SPSS (v26.0) was used for data analysis, with one-way ANOVA employed to identify significant differences in cell viability percentages and compound concentrations among the various treatment groups. The findings were derived from three separate experiments and were deemed statistically significant when p was less than 0.05.

Results and Discussion 

The three compounds studied are novel derivatives of organotin(IV) N-methyl-N-benzyldithiocarbamate. The synthesis method used in this study follows the procedure previously described for Compound 1 in our earlier work (Abd Aziz et al., 2023b). As the focused of this study is to elucidate the cytotoxic effects of these compounds on K562 cells, we provide only a brief summary of their key spectroscopic features (¹H and ¹³C NMR) to support their novelty and confirmed their structures. The ¹H NMR spectra showed expected singlets for methylene protons (δ 5.060–5.148 ppm) and aromatic multiplets (δ 7.249–8.000 ppm) (Haezam et al., 2021). Signals for protons bound to Sn(IV) were observed in the ranges of δ 0.972–2.127 ppm (Compound 1), δ 7.270–7.522 ppm (Compound 2), and δ 7.247–7.491 ppm (Compound 3) (Mohamad et al., 2016). The ¹³C NMR spectra supported these findings, with the thioureide carbon resonating at δ 197–202 ppm, methylene carbon at δ 59.95–61.64 ppm, and methyl carbon at δ 41.77–42.90 ppm. Aromatic carbons appeared in the range of δ 127–151 ppm (Abd Aziz et al., 2023a). Additionally, the crystallographic data of Compound 1 confirmed a skewed trapezoidal bipyramidal geometry (Abd Aziz et al., 2023b), whereas Compound 3 exhibited a trigonal bipyramidal geometry. Full crystallographic data and detailed structural interpretation will be reported in a separate publication focused on the synthesis and molecular characterization of these compounds.

The cytotoxic impacts of these compounds on Jurkat E6.1 and WIL2-NS cells are illustrated in Figure 2. The median inhibitory concentrations (IC₅₀) for Compound 1 to 3 for both cell lines are detailed in Tables 1 and 2. Each of the compounds significantly reduced cell viability at concentrations of 5 μM and 10 μM after a 24-hour exposure. The MTT assay revealed IC₅₀ values of 0.23 μM, 0.58 μM, and 0.28 μM for Compounds 1, 2, and 3 in Jurkat E6.1 cells, respectively. For WIL2-NS cells, the IC₅₀ values were found to be 0.86 μM for Compound 1, 0.49 μM for Compound 2, and 0.39 μM for Compound 3. Bich-Loan et al. (2021) classified these compounds as highly toxic, supported by their low IC₅₀ values. These low IC₅₀ values classify the compounds as highly cytotoxic (Bich-Loan et al., 2021), suggesting they exert strong biological effects. 

Compounds 1 and 3 exhibited significant toxicity to Jurkat E6.1 cells across all tested concentrations: 0.08 µM to 5.00 µM for Compound 1 and 0.16 µM to 10.00 µM for Compound 3. Compound 2 showed significant toxicity from 0.16 µM to 5.00 µM. For WIL2-NS cells, Compound 1 was notably toxic at 0.63 µM and higher, while Compound 2 showed consistent toxicity across all concentrations tested. Compound 3’s toxicity was significant from 0.16 µM up to 10.00 µM. Among the three, Compound 1 exhibited the most potent cytotoxicity towards Jurkat E6.1 cells, reflected in its lowest IC₅₀ value of 0.23 μM. This heightened effect is attributed to its shorter alkyl chain on the tin(IV) atom, which enhances antioxidant activity and cytotoxic regulation (Koch, Basu Baul, and Chatterjee, 2009). Studies indicate that shorter alkyl chains improve both antioxidant properties and cytotoxicity (Verma et al., 2016). Similarly, Kamaludin et al. (2013) reported significant cytotoxic effects of dibutyltin(IV) butylphenyldithiocarbamate on Jurkat E6.1 and K562 cells, with IC₅₀ values falling below 5 µg/mL (<8.70 μM). In their research, the IC₅₀ values for Jurkat E6.1 cells treated with dibutyltin(IV) butylphenyldithiocarbamate ranged from 0.50 to 0.80 µM, while for K562 cells, they varied from 3.90 to 5.30 µM.

In contrast, Compound 3 showed greater cytotoxicity compared to Compound 2, likely due to its increased number of phenyl groups, which enhances lipophilicity. Increased lipophilicity facilitates stronger interactions with biological membranes, which are primarily composed of lipids, thereby promoting cellular uptake and amplifying cytotoxic effects (Hussain et al., 2015; Javed et al., 2016). This property also improves compound permeability across physiological barriers, such as the gastrointestinal epithelium, and enhances interaction with cellular lipids and proteins, potentially increasing bioavailability (Riley et al., 2001; Alavijeh et al., 2005). Such characteristics are crucial in drug development, where optimizing lipophilicity can improve therapeutic efficacy and minimize adverse effects.

While variations in the alkyl and aryl substituents influence lipophilicity and steric hindrance, the tin(IV) center itself plays a crucial role in the cytotoxic mechanism. Organotin(IV) compounds are recognized for their ability to interact with thiol-containing biomolecules, impairing cellular respiration, producing oxidative species, and activating apoptotic pathways. Notably, triorganotin(IV) compounds, such as Compound 3, may exert stronger biological activity due to their higher coordination flexibility and greater exposure of the Sn(IV) center, which enhances interactions with intracellular targets (Abd Aziz et al., 2023a; Iqbal et al., 2017). 

Figure 2: IC50 values for Jurkat E6.1 cells following treatment with a) menadione, b) Compound 1, c) Compound 2.Click here to View Figure

* Significant difference, a p-value of less than 0.05 

Table 1: IC₅₀ values of Compounds 1, 2, and 3 against Jurkat E6.1 cells

 Table 2: IC₅₀ values of Compounds 1, 2, and 3 against WIL2-NS cells

 The selectivity index (SI) of the compounds for cancer cells in comparison to normal cells was evaluated to identify potential cytotoxic agents, with an SI greater than 2 indicating a promising candidate (Badisa et al., 2009).  As shown in Table 3, Compound 1 demonstrated the highest selectivity toward Jurkat E6.1 cells with an SI of 3.74, followed by Compound 3 (1.39), while Compound 2 showed the lowest selectivity (0.84). These results indicate that Compound 1 may have tumor-specific properties, highlighting its potential as a promising candidate for further development. Overall, the data underscore the relevance of selective cytotoxicity in the search for effective anticancer agents.

Table 3: Values of selective index for Compounds 1, 2, and 3

A morphological evaluation was conducted on Jurkat E6.1 cells treated with the respective IC₅₀ concentrations of Compounds 1-3 (0.23 μM, 0.58 μM, and 0.28 μM, as determined from the MTT assay). Figure 3 illustrates the morphological alterations observed in Jurkat E6.1 cells after 24 hours of exposure to Compounds 1, 2, and 3, along with menadione, which served as the positive control.

Figure 3: Morphological alterations in Jurkat E6.1 cells were assessed after 24 hours using an inverted microscope (×40). Click here to View Figure

After 24 hours of treatment, all tested compounds—including menadione—induced morphological signs of apoptosis in Jurkat E6.1 cells, unlike untreated controls. This process is marked by chromatin condensation, DNA breakdown, cellular shrinkage, and membrane blebbing, eventually forming apoptotic bodies (Mills et al., 1998; Zhang et al., 2018). The observations in this study confirm that the compounds activated programmed cell death pathways, with consistent patterns observed across all treatments. These findings align with previous reports on organotin-induced apoptosis in leukemia models (Hamid et al., 2020).

Conclusion

This study confirms that organotin(IV) complexes of N-methyl-N-benzyldithiocarbamate possess cytotoxic potential against Jurkat E6.1 cells. Among the three, Compound 1 displayed the strongest effect and notable selectivity. Apoptotic features were prominent, and the compound’s selective action supports its further evaluation in cancer therapy.

Acknowledgment 

We express our heartfelt gratitude to the Ministry of Higher Education (MOHE), Malaysia, for sponsoring this research via the Fundamental Research Grant Scheme (FRGS) with grant number FGRS/1/2021/STG04/UKM/02/5. We also express our gratitude to the Faculty of Health Sciences, Universiti Kebangsaan Malaysia, for providingthe laboratory facilities that aided in the completion of this research.

Conflict of Interest

All researchers involved in this study have declared that they have no competing interests that could affect the results or interpretation of the findings.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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