In vitro Cytotoxic Activity of New Triphenyltin ( IV ) Alkyl-isopropyl-di-thiocarbamate Compounds on Human Acute T-Lymphoblastic Cell Line

Article history: Received on: 09/02/2015 Revised on: 17/02/2015 Accepted on: 11/03/2015 Available online: 15/05/2015 Cancer is one of the leading causes of mortality and morbidity worldwide. Various new metal-based compounds including organotin (IV) compounds have been actively synthesised due to their good cytotoxicity in cancerous cells. In this study, we tested the cytotoxicity of new triphenyltin (IV) dithiocarbamate compounds (triphenyltin (IV) benzylisopropyldithiocarbamate, compound 1; triphenyltin (IV) methylisopropyldithiocarbamate, compound 2; and triphenyltin (IV) ethylisopropyldithiocarbamate, compound 3) on the human acute T-lymphoblastic cell line, Jurkat E6.1 by MTT assay at three periods of time. The triphenyltin (IV) methylisopropyldithiocarbamate compound showed the lowest IC50 values (0.03 μM) after 24 h of treatment on Jurkat E6.1 cell lines. The IC50 values obtained for the three compounds for 24 h of treatment were 0.18 μM, 0.03 μM, and 0.42 μM, respectively. Next, for 48 h and 72 h of treatment, the IC50 values were 0.15 μM, 0.18 μM, and 0.40 μM and 0.18 μM, 0.19 μM, and 0.41 μM, respectively. These tested compounds were found to give cytotoxic activity against Jurkat E6.1 cell lines at micromolar doses. Observation on morphological changes of Jurkat E6.1 cell lines treated with IC50 values at 24 h of treatment showed morphological changes such as cell shrinkage and membrane blebbing, characterising the mode of cell death as apoptosis. Thus, further studies on the specific mechanisms of action of these compounds in human cells should be carried out to elucidate their potential as anticancer agents.


INTRODUCTION
Dithiocarbamates are versatile ligands capable of forming complexes with most existing elements.Dithiocarbamates are also able to stabilise transition metals in various oxidation states (Nabipour et al., 2010).The ability of these ligands to stabilise high oxidation states in metal complexes reflects the strong o-bonding characteristic.Although the sulphur atoms of dithiocarbamate ligands possess o-donor and n-back-donation characteristics of the same order of magnitude, these ligands also possess a special feature in that there is an additional n-electron flow from nitrogen to sulphur via planar de-localised π-orbital system, as shown below: This special feature results in strong electron donation, hence a high electron density on the metal, leading to its next higher oxidation state.However, while dithiocarbamate complexes have been known for over a century, with many thousands are being prepared, the vast majority of these complexes contain only simple alkyl substituents such as methyl and ethyl.A developing interest in the area of dithiocarbamate chemistry is the functionalisation of the backbone such that new applications and interactions can be developed.This area is still in its early stages, but interesting and potential applications have already been noted, including the functionalisation of gold nanoparticles, the stepwise build-up of multimetallic arrays, the synthesis of dithiocarbamate-containing N + N supramolecular systems that may be used for anion binding, and the development of technetium radiopharmaceuticals. Dithiocarbamates are a class of metal-chelating antioxidant compounds that have various applications in medicine, such as for the treatment of bacterial and fungal infections and possibly the treatment of AIDS (Buac et al., 2012).
Cancer is a prevalent cause of death worldwide.Every year, several natural and synthetic compounds are tested for various anticancer activities.Currently, research on the synthesis and application of metal-based antitumour drugs is considered one of the areas in biomedical and inorganic chemistry that are expanding at a quite fast rate (Gomez-Ruiz et al., 2008).More recently, various studies on the in vitro antitumour properties of organotin compounds against a wide panel of human tumour cell lines have been reported.
These compounds have been proven to be as effective as, or even better than, traditional heavy metal-based anticancer drugs (Kaluderovic et al., 2010).
One of the chemotherapeutic drugs currently used is the platinum-based compound, cisplatin, which is the first metalcontaining anticancer drug.However, its use in patients can potentially result in severe side effects.This disadvantage of cisplatin has spurred interest among researchers to identify safer metal-based compounds (Jamieson and Lippard, 1999).As platinum and tin atoms possess common chemical properties (Gielen, 2002), tin complexes have been proposed to be potential therapeutic alternatives to cisplatin and similar anticancer agents.
In our previous works, we have reported several new triphenyltin(IV) dithiocarbamate compounds and their cytotoxic effects on a few human cancerous cell lines (Awang et al., 2012;Awang et al., 2014).Interestingly, all of these compounds have a good potential to become anticancer agents against the cancerous cell lines, with their IC 50 less than 1 µM.Therefore, this study was conducted to evaluate the in vitro cytotoxicity of the newly synthesised triphenyltin(IV) dithiocarbamate compounds namely triphenyltin(IV) benzylisopropyldithiocarbamate (compound 1), triphenyltin(IV) methylisopropyldithiocarbamate (compound 2), and triphenyltin(IV) ethylisopropyldithiocarbamate (compound 3).The general chemical structure proposed for the three compounds is shown in Figure 1, where R = N-benzyl (compound 1); N-methyl (compound 2); and N-ethyl (compound 3).In this study, the cytotoxicity study of the compounds was done in human leukaemic cell lines, Jurkat E6.1.The observation of the morphological changes in the cell was also carried out.

Stock Preparation of the Three Compounds
The three compounds (0.12 µmol) were dissolved in 1.2 mL of dimethyl sulphoxide (DMSO) in order to obtain 100 mM of stock solution.This stock was stored at −20 °C, and serial dilution was freshly prepared using the culture media.

Jurkat E6.1 Cell Culture
Jurkat E6.1 cell lines were purchased from the American Type Culture Collection (ATCC).These cells were cultured in a T-75 culture flask containing Roswell Park Memorial Institute (RPMI 1640) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin to obtain complete growth media.The cell culture was incubated in an incubator at a temperature of 37 °C and humidified atmosphere containing 5% CO 2 .The subculture of cells was conducted every 2 to 3 days to maintain cell growth and healthiness.

MTT Cytotoxicity Assay
The viability of Jurkat E6.1 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazholium bromide (MTT) assay (Mossman 1983).The cells were treated with the compounds at three different times of exposure (24, 48, and 72 h) and different concentrations through the employment of the serial dilution method.Cells without treatment solution represented the negative control, whereas etoposide was used as the positive control.The cells were seeded in a sterile 96-well microplate at a density of 1 × 10 6 cells mL −1 with a fresh medium containing tested compounds at various concentrations ranging from 0 to 2 µM.The cells were incubated at 37 °C in 5% CO 2 for 24, 48, or 72 h.At the end of the treatment, 20 µL of 5 mg/mL MTT solution was added to each well prior to 4 h incubation.Approximately 180 µL media in each well were removed and replaced with 180 µL of DMSO to dissolve the crystal formazan.After 15 min of incubation, the plate was agitated using an orbital shaker for 5 min to ensure the crystal formazan was completely dissolved.The

Morphological Changes of Jurkat E6.1 Induced by Triphenyltin (IV) N-alkyl-N-isopropyldithiocarbamate Compounds
Figure 5 shows the morphology of the untreated Jurkat E6.1 cells.The cell morphology changes were assessed using the IC 50 concentration (0.18 µM, 0.03 µM, and 0.42 µM) of each compound (the three compounds) upon 24 h of treatment.The results of the observation showed that the cells shrank, and consequently apoptotic bodies formed (see Figure 6).

DISCUSSION
The organotin derivatives are greatly being synthesised, and their anticancer properties are extensively being studied.Organotin compounds have marked cytotoxicities in various types of cell lines of both human and animal origins.Several organotin compounds synthesised by our group also show significant cytotoxicity towards HepG2 hepatocarcinoma, Jurkat T lymphoblastic, chronic myelogenous leukaemia (K562), and thymoma murine (WEHI 7.2) cell lines (Awang et al., 2012;Awang et al., 2014).
Similarly, the triphenyltin (IV) N-alkyl-Nisopropyldithiocarbamate compounds may also be classified as very toxic due to their efficacy to inhibit the growth of the Jurkat E6.1 cell population at as low as 0.03 µM (for compound 2) at 24 h of treatment.As revealed by this present study, all the compounds showed cytotoxicity in Jurkat E6.1 cells, with the highest cytotoxicity was reported by compound 2 (0.03 µM) at 24 h of treatment.It is suggested that the molecular structure of the compound plays a significant role in determining its cytotoxicity.The shorter length of an alkyl substitution group in the compound increases its cytotoxicity (Biplob et al., 2008;Ray et al., 2000).This explanation probably reflects our findings that revealed compounds with methyl group showed higher cytotoxicity compared to compounds with benzyl and ethyl group as their dithiocarbamate ligand.
To further evaluate the cell changes upon treatment, the cell morphological observation was conducted in Jurkat E6.1 cells.The cells began to appear in visibly different form than the control at IC 50 concentration for the tested compounds.These results showed that these compounds gave a cytotoxic effect towards Jurkat E6.1 cells at very low concentrations.The cells started to lose their shape with more intercell spaces observed.

CONCLUSION
In conclusion, triphenyltin(IV) dithiocarbamate compounds tested in this study demonstrated cytotoxic properties.This finding warranted further investigation on the compounds as potential anticancer agents.
The synthesised compounds were next evaluated for their cytotoxicity against Jurkat E6.1 cell lines, and they were found to be highly potential.All of these compounds had high efficacy towards the tested cells.We conclude that the presence of aryl groups attached to the tin atom would be critical for their biological activities.Further studies are currently in progress in an attempt to define the important mechanisms of action of the mentioned compounds.
, ethyl, or methyl Fig. 1: General chemical structure of the three compounds.