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Molecular Cytotoxic Mechanisms of 1-(3,4,5-Trihydroxyphenyl)-dodecylbenzoate in Human Leukemia Cell Lines

Recent studies have shown that gallic acid and its alkylesters induce apoptosis in different cell lines. Since new compounds with biological activity and less cytotoxicity to normal cells are necessary for cancer therapy, the aim of this study was to evaluate the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. The cell viability was determined by MTT method. The apoptosis induction was assessed by bromide and acridine orange staining and by Annexin V-FITC Apoptosis Detection kit. The cell cycle analysis was carried out by flow cytometry using propidium iodide. Cytometric analysis was also performed to evaluate the expression of the following proteins: AIF, p53, Bcl-2 and Bax. The mitochondrial potential was also assessed by flow cytometry using MitoView633 kit. The results showed that the compound significantly reduced the cell viability of K562 and Jurkat cells in a concentration and time dependent manner (IC50 of 30 μM). The compound induced cell cycle arrest in G0/G1phase and significantly increased the proportion of cells in the sub-G0/G1phase. Apoptosis was confirmed by the sight of morphological characteristics of apoptosis and by phosphatidylserine externalization (73.47±5.71% of cells expressing annexin). The results also showed that the compound promotes a modification in Bax:Bcl-2 ratio and increases p53 expression. Thus, it is possible to conclude that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate induces apoptosis by inhibiting the antiapoptotic protein Bcl-2 and by increasing the release of AIF, Bax and p53. In addition, it blocks the cell cycle at G0/G1, stopping cell proliferation. So far, the results suggest that this compound may have a potential therapeutic effect against leukemia cells.

The homeostasis of normal cells is the result of a balance between proliferation, differentiation and programmed cell death (apoptosis), whereas malignant processes are characterized by the excessive expansion of tumor cells due to defects in one or more of these processes. Therefore, understanding the mechanisms of cell cycle regulation, the molecular apoptotic machinery and its defects in acute leukemia is a critical step for the identification of new targets in anticancer therapy [13,14] . Thus, the aim of this study was to assess the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on human leukemia cell lines (K562 and Jurkat cells) and investigate some of its mechanisms in order to discuss the interest of this compound as an antitumor agent.

MATERIALS AND METHODS
Cell culture medium, fetal bovine serum and antibiotics were purchased from GIBCO (São Paulo, Brazil) and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The compound 1-(3,4,5-trihydroxyphenyl)dodecylbenzoate was synthesized and characterized as described elsewhere [9] . To prepare the stock solution, the compound was dissolved in 100% dimethyl sulfoxide (DMSO), and then diluted in cell culture medium to a final concentration of 0.01% of DMSO.

Cell culture and viability assay (MTT assay):
Human acute myeloid leukemia (AML) K562 cells and human acute lymphoblastic leukemia (ALL) Jurkat cells (ATCC, Manassas, USA) were maintained in Roswell Park Memorial Institute Medium (RPMI; GIBCO, São Paulo, Brazil) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 10 mm HEPES, pH 7.4. The cells were maintained in plastic culture flasks at 37 o at a 5% CO 2 humidified atmosphere. Cells were seeded at a density of 10 6 cells per bottle and medium was changed every three days. Cell viability was assessed by MTT method (3-(4,5-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide, Sigma Chemical Co., St Louis, USA) [15] . Briefly, the compound was added to K562 and Jurkat cells at different concentrations (1-100 µM) and incubated for 24, 48 or 72 h. The same volume of DMSO was added to control wells. MTT solution was added to each well (10% v/v) and the plates were incubated for 3 h. After incubation, the supernatant was discarded, the formazan precipitated was dissolved with 100 ml of an isopropyl alcohol-HCl 0.04 N solution and the absorbance was determined at 540 nm. All assays were performed in triplicate and the IC 50 values (50% inhibitory concentration) were calculated using GraphPad Prism software package for Windows (v. 5.0 GraphPad Prism Software Inc, San Diego, CA).

Cell cycle analysis:
The compound's effect on cell cycle was investigated by PI/RNase solution kit (Immunostep, Salamanca, Spain). Cells (1×10 6 cells/well) were incubated with vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC 50 . After 24 h of incubation, cells were harvested and cell cycle analysis was assessed according to the kit protocol. Briefly, cells were harvested, washed with PBS and centrifuged for 5 min at 300 g. The supernatant was removed and cells were fixed with 70% ethanol for 30 min at 4°. Cells were washed once with PBS supplemented with 2% bovine albumin, centrifuged for 5 min at 300 g and then the supernatant was removed. To the cell pellet it was added 500 µl of PI/RNase solution followed by 15 min incubation at room temperature. Finally, the analysis was performed by flow cytometry as previously described.

Evaluation of apoptosis:
Apoptotic cell death was assessed by ethidium bromide (EB) and acridine orange (AO) assay and by Annexin V-FITC Apoptosis Detection kit. For the first method, 40 µl of a mixture of AO (10 µg/ml) and EB (5 µg/ml) was added to cells treated at their respective IC 50 for 12, 24 and 48 h. Cells with no treatment were used as a control group. The cells were observed in a fluorescence microscope (Olympus BX-FLA) and representative fields were photographed by digital camera (Olympus BX40, Japan). The www.ijpsonline.com Annexin V-FITC Apoptosis Detection kit was used according to the manufacturer's instructions. Briefly, 1×10 6 cells/well were incubated with the vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC 50 . After 16 h of incubation, cells were harvested, washed with PBS or annexin buffer (1:10) and double-stained with Annexin V-FITC solution and PI/RNase solution. After incubation, 300 µl of annexin buffer was added and fluorescence was analyzed by flow cytometry. Analysis was performed by FACSCantoII TM , Becton Dickinson Immunocytometry Systems. The data were analyzed by Infinicyt Software version 1.7.0 (Cytognos S.L., Salamanca, Spain).

Evaluation of the mitochondrial membrane potential:
The evaluation of the mitochondrial membrane potential was performed using MitoView633 TM kit (Biotium, Inc., Hayward, USA). K562 and Jurkat cells (1.0×10 6 cells/well) were incubated with the vehicle or 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate at their respectively IC 50 for 12 h. Cells were labeled as described by the manufacturer. Briefly, cells were collected by centrifugation and washed twice with PBS. Then, they were resuspended in 100 µl of MitoView 633 solution (1:10000). After incubation in the dark for 30 min, cells were washed and resuspended with 1 ml PBS. Analyses were performed by flow cytometry as previously described.

Statistical analysis:
Analyses were performed using GraphPad Prism software package for Windows (v. 5.0 GraphPad Prism Software Inc, San Diego, CA). A mixed model analysis of variance (ANOVA) was used in combination with post hoc testing (Bonferroni) to compare the cytotoxic effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on K562 and Jurkat cells. The level of significance was P<0.05 for all statistical analyses.

Effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on apoptosis:
To evaluate whether K562 and Jurkat cells treated with 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate and located at the sub-G 0 /G 1 phase were undergoing an apoptotic or necrotic pathway, two different methods were used. First, the cells were treated with the compound at their respective IC 50 for 12, 24 and 48 h and then stained with EB/AO to be observed in a fluorescence microscope. AO permeates all cells and stains them with green www.ijpsonline.com bright green nuclei with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; and necrotic cells have a structurally-normal orange nucleus [16] . As it may be seen in fig. 4, 1-(3

Effect of 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate on the expression of AIF, p53, Bcl-2, Bax and FasR proteins:
As shown in fig. 7, 1-(3,4,5-trihydroxyphenyl)dodecylbenzoate increased the expression of proapoptotic protein Bax in Jurkat cells after 4, 8 and 12 h treatment, which confirms the involvement of the intrinsic apoptotic pathway in this cell line. Again, no significant difference was observed on Bax or Bcl-2 expression on K562 cells. Interestingly, the compound increased the expression of AIF in both K562 and Jurkat cell lines after 12 h treatment. Fig. 7 also demonstrates that 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate decreased the expression of FasR and increased the expression of p53 on K562 cells.

DISCUSSION
Gallic acid and its alkyl esters have been studied for their cytotoxic activity on different tumor cell lines [7,12,[15][16][17] . According to the literature, these www.ijpsonline.com compounds also inhibit the proliferation of HL60 [18] , JY, MOLT-4, HT29 [12] and other cell lines [19] . Moreover, it is known that alkyl esters of gallic acid with lipophilic group such as octyl and dodecyl (lauryl), exhibit a potential antiproliferative effect between 50 and 250 times greater than the actual gallic acid [8,9,15,20] . Furthermore, the cytotoxic effect of alkyl esters of gallic acid is much lower in normal cells than in cancer cells [9,15] , which demonstrates selectivity towards neoplastic cells.
The results presented in this paper are consistent with the data described in the literature, since we demonstrated that 1-(3,4,5-trihydroxyphenyl)dodecylbenzoate, an alkylester derivated from gallic acid, was cytotoxic against AML K562 and ALL Jurkat cell lines in a concentration and time-dependent manner ( fig. 2). Furthermore, we demonstrated that the compound was slightly more cytotoxic against ALL cells.
The mechanisms of antileukemia compounds are based primarily on the inhibition of cell proliferation and on apoptosis induction. Apoptosis is a programmed cell death characterized by cell shrinkage, chromatin condensation and formation of apoptotic bodies [22] . Changes in the cell cycle and on regulatory proteins are often associated with the induction of apoptosis and are crucial to the development of new therapies. Many of the apoptotic effects of phenolic compounds, including 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate, have been attributed to disorders in cell cycle [23] . Ours results clearly demonstrate a decreased number of cells in G 2 M phase after 24h incubation with the compound and an increased number of cells in sub-G 0 /G 1 phase (fig. 3). This may indicate that the mechanism by which 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate inhibits the growth of leukemia cells is related to the cell cycle.
Several studies have shown that derivatives of gallic acid with lipophilic groups such as 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate present  an ability to induce apoptosis on human leukemia cells [8,15,16,22,24] . This information was consistent to our findings, since typical morphological changes of apoptosis could be observed on K562 and Jurkat cells treated with the compound for 12, According to the literature, the first sign of apoptosis is the externalization of phosphatidylserine residues, which bind to Annexin V after incubation, thus indicating cell death by apoptosis [12,20,24,25] .
The mechanisms that regulate apoptosis involve the activation of different signaling pathways and inhibitory components that culminate in cellular self-destruction [26] . A common feature of apoptosis is the activation of proteins called caspases, which act as effectors in cell death [22] . Other proteins such as p53, Bax and Bcl-2 are also involved in apoptosis regulation. The tumor suppressor protein p53 acts in the cell cycle, at the first checkpoint located between G 1 and S phases, through the activation of several pro-apoptotic effectors genes or inhibitors of growth [13,28] . Our results demonstrate that there was an increased expression of p53 after treatment with 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate. Bax and Bcl-2 are members of the Bcl-2 family [27] . The protein Bcl-2 has been described in the literature for its antiapoptotic effect on different cell lines [27,28] . Studies show that Bcl-2 retards the induction of apoptosis by 1-(3,4,5-trihydroxyphenyl)dodecylbenzoate in WEHI231 and MCF7 cells [20] . Furthermore, it is known that the overexpression of Bcl-2 acts preventing the mitochondrial release of AIF [29] . Our results show that K562 and Jurkat cells express antiapoptotic protein Bcl-2 but its expression was not decreased by 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate. This result suggests the involvement of other proteins in the induction of apoptosis. However, the compound increased the expression of proapoptotic Bax protein which may contribute to cell death. In addition, the compound strongly increased the expression of AIF in both K562 and Jurkat cells. AIF is a mitochondrial protein described in literature by its proapoptotic activities, which is important to the understanding of mitochondrial dysfunction and apoptosis regulation [30] .
According to the literature, members of the Bcl-2 family such as Bax and Bcl-2 regulate the mitochondrial membrane permeability and potential. After 12 h treatment, 1-(3,4,5-trihydroxyphenyl)dodecylbenzoate reduced the number of cells with intact mithocondrial membrane potential. This result, combined with the changes in expression of Bax and AIF, indicate that the mechanism of apoptosis induced by this compound involve the intrinsic pathway.
The evidences obtained so far suggest that one of the likely mechanisms of cytotoxicity induced by 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate occurs by the increased expression of Bax, p53 and AIF proteins, by the blockage of cell cycle and by the reduction of the mithocondrial membrane potential. The different results observed for K562 and Jurkat cell lines might be explained by the differences in the etiopathology of AML and ALL, respectively. Although further studies are needed in order to completely understand its mechanism of action, the inhibitory effect of1-(3,4,5-trihydroxyphenyl)dodecylbenzoate brings new options to the development of new drugs.
In conclusion, the compound 1-(3,4,5-t rihydroxyphenyl)-dodecylbenzoate significantly reduced the cell viability of K562 and Jurkat cells in a concentration-and-time dependent manner. In addition, leukemia cells treated with this compound revealed an increase in p53 expression and, regarding the cell cycle, a decreased population in G 2 M phase and an increased number of cells in sub-G 0 /G 1 phase. The increased expression of Bax by 1-(3,4,5-trihydroxyphenyl)-dodecylbenzoate results in a loss of mitochondrial membrane potential, which allows the release of AIF by mitochondria and promotes the induction of apoptosis. These results suggest that this compound is effective against AML and ALL and may be used as a new therapeutic strategy on acute leukemia.