Attenuative Effect of Diallyl Trisulfide on Caspase Activity in TNF-α-induced Triple Negative Breast Cancer Cells

Abstract

Background/Aim:

Diallyl trisulfide (DATS) has been shown to prevent and inhibit carcinogenesis in cancer cells. We have previously shown DATS’s ability to decrease the percentage of viable cells, inhibit cell migration and modulate genes involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling.

Materials and Methods:

This study aimed to compare the efficacy of DATS in tumor necrosis factor alpha (TNF-α) induced MDA-MB-231 and MDA-MB-468 cells and investigate its role in cell-death signaling via cell cycle, flow cytometry, and caspase assay.

Results:

DATS exhibit a time-dependent accumulation of G₂/M phase cells in both cell lines, with higher effects in the MDA-MB-468 for all time points. DATS’s ability to decrease the percentage of viable cells in both MDA-MB-231 and MDA-MB-468 cells was shown by a significant but slight increase of early and late apoptosis in the presence of DATS compared to control. Moreover, MDA-MB-468 cells showed more sensitivity to the DATS effect, evidenced by the higher percentage of apoptosis than MDA-MB-231 cells. The caspase studies showed a significant increase in caspase 3 and 8 activity in the presence of DATS, compared to control, in both cell lines. DATS showed no significant increase in caspase 9 activity in both cell lines compared to the control.

Conclusion:

DATS-induced apoptosis in human breast cancer cells is mediated, at least in part, by cell cycle arrest and caspase activity. These findings provide information for future studies into the role of DATS in TNBC therapy and prevention.

In the United States, the incidence of Breast cancer (BC), the most commonly diagnosed cancer among women and the second most frequent cause of cancer-related deaths (1), has slightly increased in recent years, despite the previously documented decline in incidence and death rate since 2002. This highly heterogeneous disease is classified based on the histological and molecular subtypes. Triple-negative breast cancer (TNBC), the most invasive and aggressive of all molecular cancer subtypes, accounts for 15–20% of all cancer cases (1, 2). The poorer prognosis and health outcomes observed in African Americans, despite their lower incidence of BC, are more likely to result in the development of TNBC. Currently, there is a paucity of targeted therapy specific to TNBC treatment (3, 4) due to the heterogeneity in mutations within this molecular subtype and lack of biological targets. The available treatment options include traditional chemotherapy, such as paclitaxel or doxorubicin, which causes significant adverse effects or expensive immunotherapies (i.e., atezolizumab) that are selective for specific biomarkers not detected in all TNBCs. Due to the lack of targeted therapy for all types of breast cancer, it is imperative to identify biological markers for TNBC as well as phytochemical plants that can be utilized to prevent breast cancer and its progression. Although recent studies have produced promising results, there is still much work required to establish a potent anticancer drug effective against TNBC with minimal side effects; no compound currently on the market has accomplished this goal.

In a previous study, we examined the effect of diallyl trisulfide (DATS), an organosulfide of garlic (Allium sativum), and showed that it attenuates TNF-α induced CCL2 release in two ethnically and genetically different TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells). In that study, we demonstrated that DATS attenuated TNF-α induced CCL-2 production in MDA-MB-231 cells through NF-kappa B signaling, while DATS did not affect DATS induced CCL-2 production but did downregulate MAPK8 protein expression in MDA-MB-468 cells. The cytotoxicity results in the study also revealed that MDA-MB-468 cells appear to be more sensitive to DATS than MDA-MB-231 cells. These results have prompted us to determine if the cell death observed after exposure over time is due to DATS-induced apoptosis in these cell lines. Furthermore, we would like to understand the mechanism of apoptosis.

Signaling pathways that regulate cellular homeostasis are highly regulated. Various cancers have been associated with the aberrant activation or repression of these signaling pathways (5). Such abnormalities contribute to the proliferation, self-renewal, differentiation, and survival properties of cancer cells (5). Cytokines are a class of small proteins involved in cell signaling (6, 7). They have the ability to address multiple targets and physiological effects and thus are known as pleiotropic (8). Cytokines include tumor necrosis factor, chemokines, interleukins, lymphokines, and interferons, but generally do not include growth factors or hormones (9–16). Cytokines help maintain and re-establish homeostasis if these conditions are disrupted. Monocytes are cells that differentiate into macrophages once they enter the tissue and release a cytotoxic cytokine known as tumor necrosis factor-alpha (TNF-α) in the tumor microenvironment in an attempt to destroy the tumor (9–16). However, TNF-α mimics the characteristics of malignant cells when it is unable to destroy the tumor, resulting in the formation of tumor-associated macrophages (TAMs), allowing the progression of the tumor (9–16).

Upon extracellular signaling, TNF-α binds to its transmembrane receptors, forming a complex, leading to a conformational change followed by activation of adaptor proteins. The downstream signaling is determined by adaptor proteins that are activated (9–16). The adaptor proteins with a death domain lead to apoptosis, while those with activated mitogen-activated protein kinase pathway (MAPK) or nuclear-factor kappa B (NFκB) pathway lead to proliferation, survival and growth (9–16).

Researchers have expressed high interest in plants for proliferation, cell survival, and cancer promotion mechanisms because of their phytochemical products. Garlic (Allium sativum) is an herb grown throughout the world. It has been used worldwide as a culinary ingredient, spice, and food, as well as, in both traditional and alternative medicine. Garlic active ingredients, a group of oil-soluble compounds known as organosulfur compounds (OSC), are responsible for garlic’s therapeutic effectiveness. Diallyl trisulfide (DATS), the most active OSC found in garlic, has been shown to have the ability to reduce DNA strand breaks, arrest the cell cycle, impede angiogenesis, have antioxidant properties, and induce apoptosis (17–21).

Cells have evolved ways to repair cellular injuries to maintain homeostasis. When physiological damage occurs in the tissue, the cell is required to recognize, localize, and resolve the wound (22). Cell cycle or cell division cycle is defined as the process of duplicating DNA followed by segregation of the copies into genetically identical daughter cells, the process consists of interphase and mitotic periods (23). The cell cycle contains several checkpoints that are essential in preventing genomic instability, which can promote tumorigenesis (22). These checkpoints are cyclins and cyclin-dependent kinases (CDKs) that regulate the cell cycle by controlling the transition between the phases of the cell cycle. DATS has been shown to induce G₂/M cell-cycle arrest and mitochondrial apoptosis by triggering DNA damage in ATCC 8505C cells (24). Other studies have also reported similar findings of G₂/M cell-cycle arrest induced by DATS (25–28).

The various cell cycle phases are tightly regulated, and there are checkpoints to detect potential DNA damage and allow it to be repaired, in an attempt to prevent the transformation of normal cells into a neoplastic phenotype. If the damage cannot be repaired, a cell becomes targeted for apoptosis. DATS has been shown to induce cell cycle arrest and apoptosis in cultured cell lines, such as MCF-10A, MCF-7, MDA-MB-231, AGS human gastric carcinoma, and ATC 8505C cells (29–31). Apoptosis is defined as programmed cell death and plays a critical role in regulating cellular homeostasis (32). The deregulation of apoptosis may lead to the development of various diseases, such as neurodegenerative disorders or cancer (32, 33). The extrinsic and intrinsic pathways are the main apoptotic initiating pathways (18, 34). DATS was able to induce apoptosis in human prostate cancer cells and in 8505C cells (24, 35). Ji et al. found that DATS induced apoptosis in human nasopharyngeal carcinoma (CNE2) possibly through p38MAPK and Caspase-8 (36). Another study showed DATS’s ability to activate caspases 8 and 9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways, in T24 human bladder cancer cells (37). Yu et al. found that DATS was able to increase the levels of cytochrome c, Apaf-1, AIF, and caspase 3 and 9 in primary colorectal cancer cells (38–41).

Biochemical changes, such as the production of reactive oxygen species (ROS), increase in caspase activity, and loss of mitochondrial membrane potential (MMP) can lead to apoptosis (42). The mechanism by which DATS induces apoptosis is still not fully understood. This study attempted to compare the ability of DATS to induce apoptosis in both MDA-MB-231 and MDA-MB-468 breast cancer cells. In a previous study, we examined the effect of DATS in attenuating TNF-α induced CCL2 release in two ethnically and genetically different TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells). In the study, we demonstrated that DATS attenuated TNF-α induced CCL-2 production in MDA-MB-231 cells through NF-κB and MAPK8 signaling, while DATS did not affect TNF-α induced CCL-2 production but did downregulate NFκB and MAPK8 protein expression in MDA-MB-468 cells. The cytotoxicity results in the study also revealed that MDA-MB-468 cells appear to be more sensitive to DATS than MDA-MB-231 cells.

Materials and Methods

Cell lines, chemicals and reagents.

MDA-MB-468 (African American TNBC cell line) and MDA-MB-231 (Caucasian American TNBC cell line) cell lines were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagles Medium (DMEM) high glucose, Diallyl Trisulfide (DATS), and Dimethyl sulfoxide (DMSO) were acquired from Sigma (St. Louis, MO, USA). Penicillin/Streptomycin, trypsin-EDTA, and Hanks Balanced Salt Solution (HBSS) were acquired from Invitrogen (Carlsbad, CA, USA). Ethanol was obtained from Cruzan International (Deerfield, IL, USA). Tumor necrosis factor alpha (TNF-α) and Annexin V-FITC apoptosis Kit (Cat# 68FT-AnnV-S100) were purchased from RayBiotech (Norcross, Ga, USA). Caspase 3, 8 and 9 multiplex activity Assay kit (Fluorometric) (Lot# GR3387283–1) were purchased from Abcam (Cambridge, MA, USA).

Cell maintenance and treatments.

MDA-MB-468 and MDA-MB-231 TNBC cell lines were cultured in DMEM with fetal bovine serum (10%) and 1% penicillin (100 U/ml)/streptomycin (0.1 mg/ml) and incubated in an atmosphere of 5% CO₂ at 37°C. Every 3–4 days the complete medium was changed, and the cells were subcultured every 4–7 days. Cells were subcultured in T-175 flasks to 90% confluency prior to plating to begin the experiment. Each experiment was performed in triplicate. Cells were treated with 40 ng/ml TNF-α, 75 mM diallyl trisulfide (DATS), or co-treatment (CoTx; 40 ng/ml TNF-α and 75 mM DATS). 0.1% DMSO was used as a vehicle control.

Cell cycle assay.

To determine the effect of DATS on cell cycle distribution, we performed cell cycle analysis using flow cytometry. Cells were treated at 60–70% confluency in a T-75 flask. Each experiment was done in triplicate. Briefly, following sample treatment and incubation, cells were harvested, washed, and fixed with absolute ethanol and stored until ready for use. Samples were vortexed and then centrifuged at 3000 rpm for 5 minutes. Ethanol was removed, and cells were resuspended in a staining buffer (PBS with 25 μg/ml RNase A, 50 μg/ml Propidium Iodide). Stained cells were incubated at room temperature (30–60 minutes). FACSCalibur flow cytometer (B.D. Biosciences, San Jose, CA, USA) was used to determine the proportion of cells in each cell cycle stage within 2 h of staining. Before the analysis, the instrument was aligned with Calibrite beads (B.D. Biosciences). Samples were further analyzed using the ModFit LT 6.0 software (Verity Software House, Bedford, MA, USA).

Apoptosis assay.

The Annexin V-FITC Apoptosis assay Kit from RayBiotech was used to determine DATS’s ability to induce apoptosis in MDA-MB-231 and MDA-MB-468 cells. Each experiment was done in triplicate and performed as described by the manufacturer. Briefly, both cell lines were seeded at 5×10⁵ cells/well in a 6-well plate and incubated overnight. Cell-only samples were used as a negative control, while cells treated with 1 mM staurosporine were used as positive controls. DATS, TNF-α, and CoTx, as well as control samples, were brought to a final volume of 2 ml/well of experimental media to induce apoptosis. After a 72 h incubation period, cells were harvested, centrifuged, pelleted, and washed with PBS. Cell pellets were resuspended in 500 μl of 1X Annexin-V binding buffer, then 5 μl of Annexin V-FITC and propidium iodide (PI) was each added to the appropriate tube according to the manufacturer’s protocol. Finally, the apoptotic effect was quantified within 10 minutes by Sony SH800 Cell Sorter (San Jose, CA, USA). The Sony SH800 Cell Sorter was aligned with Sony SH800 and MA900 automatic setup beads, and the samples were analyzed within 1 h for data acquisition on Cell Sorter Software version 2.1.6. For each sample, 10×10³ cells were analyzed separately.

Caspase 3, 8, and 9 multiplex activity assay kit.

The Caspase activity assay was used to determine the effect of DATS on proteolytic enzymes and the role of caspases in controlling cell death and inflammation. Each experiment was done in triplicate. All materials and reagents were prepared at room temperature prior to use. Cells were plated at 2×10⁴ cells/90 μl per well in a 96-well plate and incubated overnight at an atmosphere of 5% CO₂ at 37°C. Cells were treated with various treatments [DATS, TNF-α, CoTx, control (cell and media alone), positive control (1 μm staurosporine)] at the various time points (4, 12 and 24 h) in a final volume of 100 μl/well of experimental media to induce apoptosis. At the end of treatment (4, 12, and 24 h), 100 μl/well of Caspase loading solution was added to each well. The plates were then incubated at room temperature for 30–60 minutes in the dark. Following the incubation period, the plates were read under a microplate reader at the following specific wavelengths: Ex/Em=535/620 nm (red) for Caspase 3, Ex/Em=490/525 nm (green) for Caspase 8, and Ex/Em=370/450 nm (blue) for Caspase 9.

Statistical analysis.

Data represent at least three biological replicates, tested in triplicate, and averaged, as mean±standard error of the mean (SEM) and analyzed using GraphPad Prism 9.0.2 (San Diego, CA, USA). Gene expression was analyzed using the CFX 3.1 Manager software (Bio-Rad, Hercules, CA, USA). Significant differences were determined by one-way ANOVA followed by Dunnett’s posthoc test or using a student’s t-test. Values of p<0.05 were considered statistically significant.

Results

Cell cycle analysis.

Cell cycle phases were assessed in cells treated with 40 ng TNF-α, 75 μM DATS or DATS/TNF-a CoTx at different time points (24, 48, and 72 h) and analyzed via flow cytometry. Our data showed that exposure to DATS and CoTx resulted in time-dependent accumulation of the G₂/M phase, which was accompanied by a reduction in cells in the G₁ and S phase in both cell lines (Figure 1) when compared to the control. The G₂/M phase cell cycle arrest was evident as early as 24 h after treatment and persisted for the 72 h duration in both cell lines. After 24 h of treatment, the percentage of cell accumulation at the G₂/M phase in MDA-MB-231 cells was significant (p<0.001) at 75 μM of DATS (26.80%±0.01%) and in the CoTx (23.83%±0.02%) compared to the control (9.14%±0.01%) (Figure 1A). Similarly, in MDA-MB-468 cells treated for 24 h, there was a significant increase (p<0.001) in G₂/M accumulation at 75 μM of DATS (53.61%±0.02%) and in the CoTx (42.17%±0.01%) compared to the control (12.70%±0.01%) (Figure 1D). The 48 h treatment of MDA-MB-231 cells resulted in a significant increase (p<0.001) in G₂/M accumulation at 75 μM DATS (55.63%±0.02%) and in the CoTx (42.15%±0.01%) compared to the control (12.32%±0.04%) (Figure 1B). There was a significant increase (p<0.01) in G₂/M accumulation in MDA-MB-468 cells treated with 75 μM DATS (71.38%±0.01%) and CoTx (69.18%±0.02%) for 48 h when compared to the control (59.80%±0.02%) (Figure 1E). The MDA-MB-231 cells that underwent 72 h treatment showed a significant increase (p<0.001) in cell G₂/M accumulation at 75 μM DATS (47.61%±3.63%) and in the CoTx (40.22%±1.37%), compared to control (1.75%±0.2%) (Figure 1E). In a similar pattern to the 48 h treatment, after 72 h of treatment, the MDA-MB-468 cells resulted in a significant increase in G₂/M accumulation at 75 μM of DATS alone (70.91%±0.01%; p<0.01) and CoTx (61.03%±0.02%; p<0.05) compared to control (39.12%±0.1%) (Figure 1F).

Figure 1.

(A-F) The effect of diallyl trisulfide (DATS) on tumor necrosis factor alpha (TNF-α) induced MDA-MB-231 and MDA-MB-468 breast cancer cells. Cell cycle analysis was used to examine the DATS effect on cell cycle phase in TNF-α induced MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of DATS on the cell cycle was determined via flow cytometry against various treatments [TNF-α (40 ng), 75 μM DATS or TNF-α/DATS (CoTx)] at different time points (24, 48 and 72 h). Percentages of cells in the G₁, S, and G₂/M phases are represented in the graph. Each experiment was performed at least three times. The data are presented as the mean±SEM. Statistically significant differences between control vs. treatments were evaluated by a one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test with *p<0.05, **p<0.01 and ***p<0.001.

Apoptosis assay.

Apoptosis is programmed cell death and is tightly regulated. In this study, we examined DATS’s ability to induce apoptosis via flow cytometry using Annexin V-FITC/PI staining in cells after 72 h of DATS exposure. We found that DATS was effective in inducing apoptosis in both MDA-MB-231 and MDA-MB-468 cell lines in comparison to the control. Our data showed the ability of DATS to induce a small but significant decrease in the percentage of viable cells when compared to the control in MDA-MB-231 (p<0.01) and MDA-MB-468 (p<0.001) cells (Figure 2A–D). In the TNF-α treated MDA-MB-231 and MDA-MB-468 cells, there was no significant difference in viable cells in comparison to the control (Figure 2A–D). While CoTx significantly decreased the number of viable cells in the MDA-MB-231 (p<0.01) and MDA-MB-468 (p<0.001) cells, TNF-α attenuated this increase in the CoTx when compared to DATS alone (Figure 2A–D). In addition, apoptosis observed in the CoTx was significantly greater in MDA-MB-468 in comparison to MDA-MB-231 cells. Thus, when comparing MDA-MB-231 (Table I) and MDA-MB-468 cells (Table II), MDA-MB-468 showed more sensitivity to DATS, as evidenced by the higher percentage of early and late apoptosis viable cells.

Figure 2.

The effect of diallyl trisulfide (DATS) on apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. Apoptosis assay was used to examine the DATS effect on apoptosis in tumor necrosis factor alpha (TNF-α) induced MDA-MB-231 breast cancer cells. The effect of DATS on apoptosis was determined via flow cytometry using Annexin V-FITC kit and flow cytometry to compare the apoptotic cell percentage to the control. Graphs A and B are indicative of the scatter plots for each cell line, illustrating cellular movement from the resting to the apoptotic state. Graphs C and D show the percentage of apoptosis compared to the control group. All experiments were performed at least 3 times. The data are presented as mean±SEM. Statistically significant differences between control vs. treatments were evaluated by a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test and student’s t-test was used to compare the results between the two cell lines with *p<0.05, **p<0.01 and ***p<0.001.

Table I.

MDA-MB-231 apoptosis after 72 hours.

Quadrants	Control	TNF-α	75 μM DATS	CoTx	+ Control
Lower left (L.L.)	99.64±0.07%	99.02±0.13%	91.05±2.67%	92.37±0.7%	28.74±0.26%
Lower right (L.R.) (early apoptosis)	0.35±0.02%	0.29±0.03%	5.63±1.73%	2.86±0.10%	55.17±1.06%
Upper right (U.R.) (late apoptosis)	0±0%	0.02±0.01%	1.69±0.44%	1.29±0.2%	13.81±0.86%
Upper left (U.L.) (necrosis)	0.32±0.01%	0.68±0.11%	1.64±0.5%	3.48±1%	2.28±0.06%

Table II.

MDA-MB-468 apoptosis after 72 hours.

Quadrants	Control	TNF-α	75 μM DATS	CoTx	+ Control
Lower left (L.L.)	99.3±0.3%	98.91±0.6%	87.39±0.69%	90.77±0.68%	7.49±1.52%
Lower right (L.R.) (early apoptosis)	0.41±0.13%	0.2±0.2%	10.31±0.13%	8.1±0.44%	54.14±6.57%
Upper right (U.R.) (late apoptosis)	0.15±0.2%	0.07±.0.7%	1.75±0.76%	0.52±0.06%	33.83±7.6%
Upper left (U.L.) (necrosis)	0.14±0.02%	0.83±0.4%	0.55±0.05%	0.64±0.18%	4.53±0.44%

Caspase 3, 8, and 9 multiplex activity assay kit.

Caspases are a family of protease enzymes that play a key role in apoptosis or programmed cell death and inflammation. In our study, we assessed the effect of DATS and/or TNF-α on caspase activity in these racially and genetically different breast cancer cell lines. DATS treatment induced apoptosis in both MDA-MB-231 and MDA-MB-468 cell lines in comparison to control. Following 4 h of exposure to DATS in MDA-MB-231(125.5%±2.46%) and MDA-MB-468 (138%±5.03%) cells, we found a small but significant increase in caspase 8 activity (p<0.05) (Figure 3B and E) in comparison to control; however, it showed no significant difference in caspase 3 and 9 activities (Figure 3A and C). Similarly, TNF-α and CoTx showed no significant difference in comparison to control after 4 h (Figure 3A–C and D–F). However, TNF-α and CoTx showed no significant difference in comparison to the control for any of the caspases following 4 hours of treatment in either cell line. Following 12 h of treatment in both MDA-MB-231 and MDA-MB-468 cells, DATS and CoTx treatment showed significant increases in caspase 3 (p<0.001 and p<0.01 respectively) (Figure 3G and J) and caspase 8 (p<0.01 and p<0.05, respectively; Figure 3H and K) activity in comparison to control but showed no significant difference in caspase 9 activity (Figure 3I). DATS and CoTx exposure in MDA-MB-231 and MDA-MB-468 cells showed a significant increase in caspase 3 (p<0.001 and p<0.01, respectively) and caspase 8 (p<0.01 and p<0.05, respectively) activity in comparison to the control (Figure 4G and H) but showed no significant difference in caspase 9 activity (Figure 3I and L).

Figure 3.

The effect of diallyl trisulfide (DATS) on Caspase 3, 8 and 9 activities in MDA-MB-231 and MDA-MB-468 breast cancer cells after 4 h. Caspase activity in relative fluorescence units (RFU) was used to examine DATS effect on apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of DATS on apoptosis was determined via caspase 3, 8 and 9 fluorescent activities compared to control cells. All experiments were performed at least 3 times. The data are presented as the mean±SEM. Statistically significant differences between control vs. treatments were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test and the student’s t-test was used to compare the results between the two cell lines with *p<0.05, **p<0.01 and ***p<0.001.

Figure 4.

The effect of diallyl trisulfide (DATS) on Caspase 3, 8 and 9 activities in MDA-MB-231 and MDA-MB-468 breast cancer cells after 12h. Caspase activity in relative fluorescent units (RFU) was used to examine DATS effect on apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of DATS on apoptosis was determined via caspase 3, 8 and 9 fluorescent activities compared to control cells. All experiments were performed at least 3 times. The data are presented as mean±SEM. Statistically significant differences between control vs. treatments were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test and student’s t-test was used to compare the results between the two cell lines with *p<0.05, **p<0.01 and ***p<0.001.

When both cell lines were compared after 12 h exposure to DATS, caspase 3 (p<0.01) and caspase 8 (p<0.05) activity significantly increased in MDA-MB-468 (81%±1%; 167%±2.5%) compared to MDA-MB-231 (62.75%±3.6%; 156±8.19%) cells. DATS significantly increased caspase 3 (71.50%±1.87%) (p<0.001) and caspase 8 (164%±2%) activities (p<0.001) (Figure 5M and N) when compared to control following 24 h exposure in MDA-MB-231 cells. Additionally, there was a significant increase in caspase 3 (63.71%±2.72%) (p<0.01) and caspase 8 (147.7%±3.53%) (p<0.01) (Figure 3M and N) activity following exposure to CoTx. There was no significant difference in caspase 9 activity following 24 h of exposure to DATS and CoTx (Figure 3O). In contrast, both DATS (127.5%±2.5%; 184.5%±1.55%) and CoTx (116.7%±.66%; 175.5%±3.5%) exposure showed a significant increase (p<0.001) in caspase 3 and 8 activity but showed no significant difference in caspase 9 activity in MDA-MB-468 cells. TNF-α showed no significant difference in caspase activity in comparison to the control in both MDA-MB-231 and MDA-MB-468 cells following 24 h exposure. Higher caspase 3 (p<0.001) and caspase 8 (p<0.01) activity was observed in MDA-MB-468 cells (127.5%±2.5%; 184.5%±1.55%) in comparison to MDA-MB-231 cells (71.50%±1.87%; 164%±2%) after 24 hours. These results suggest that the pro-apoptotic activity of DATS is probably regulated by a caspase-dependent cascade through the activation of the extrinsic signaling pathways.

Figure 5.

The effect of diallyl trisulfide (DATS) on Caspase 3, 8 and 9 activities in MDA-MB-231 and MDA-MB-468 breast cancer cells after 24h. Caspase activity in relative fluorescent units (RFU) was used to examine DATS effect on apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. The effect of DATS on apoptosis was determined via caspase 3, 8 and 9 fluorescent activities compared to control cells. All experiments were performed at least 3 times. The data are presented as mean±SEM. Statistically significant differences between control vs. treatments were evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test and student’s t-test was used to compare the result between the two cell lines with *p<0.05, **p<0.01 and ***p<0.001.

Discussion

Garlic is not only an important food seasoning but also serves as a traditional medicine. The therapeutic value of garlic resides in its organosulfides, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). DATS is the most effective organosulfide found in garlic due to its higher number of sulfur atoms. DATS has been shown to induce cell cycle arrest and apoptosis in cultured cell lines, such as MCF-7, MDA-MB-231, AGS human gastric carcinoma, and ATC 8505C cells (29, 31, 43). While DATS’s ability to act as an anticancer agent against breast cancer has been reported (29, 31, 43), the molecular mechanisms for its therapeutic action as an antitumor agent remain unclear. TNBC (estrogen, progesterone, and HER2 receptor negative), an aggressive subtype of cancer with a poor prognosis and with a mutation at the BRCA1 gene, is the most aggressive and invasive of all cancer subtypes. Tumor development initially begins with uncontrolled cell division. The cell cycle is the complete process of one cell division to the end of the next, which consists of interphase and mitotic periods. Interphase consists of the G₁, S, and G₂ phases. During interphase, the cell grows (G₁), accumulates the energy necessary for duplication, replicates cellular DNA (S), and prepares to divide (G₂). Each phase of the cell cycle is tightly regulated, and there are checkpoints to detect potential DNA damage and allow it to be repaired in an attempt to prevent the transformation of a normal cell into a cancer cell. If the damage cannot be repaired, a cell becomes targeted for apoptosis. G₁/S and G₂/M transformation are two very important phases in the cell cycle. Cells in these two stages experience a complex and active period of change and are particularly susceptible to environmental conditions. Currently, there is no clinical therapy specifically for TNBC patients (3, 4). In addition, due to the cell and genetic heterogeneity of cancers of the same subtype such as TNBC, tumor responsiveness to therapy varies.

Therefore, it is imperative to identify common biological markers for TNBC and plants that can be useful as both medicine and food in preventing breast cancer and its progression. Previously, we showed that DATS cytotoxicity was both dose- and time-dependent (44). DATS and TNF-α concentration was chosen based on the literature review as well as previous work done in our lab (9, 44). In the present study, we demonstrated DATS ability to induce cell cycle arrest at the G₂/M phase in genetically and ethnically different TNBC cells, specifically MDA-MB-231 and MDA-MB-468 cells, via flow cytometry. This study revealed cell cycle arrest after DATS and Co-Tx (40 ng TNF-α and 75 μM of DATS), in both cell lines in comparison to control. Additionally, 75 μM of DATS induced higher cell cycle arrest than CoTx. Treatment with DATS alone resulted in time-dependent accumulation of cells in the G₂/M phase in both cell lines (Figure 1). G₂/M phase cell cycle arrest was evident as early as 24 h after DATS treatment and persisted for 72 h in both cell lines. Higher sensitivity to DATS was observed in MDA-MB-468 cells, as denoted by the higher number of cells arrested in the G₂/M phase. Similarly, Zheng et al. demonstrated that DATS induced G₂/M cell-cycle arrest and mitochondrial apoptosis by triggering DNA damage in ATC 8505C cells (24). Other studies have also reported similar findings of G₂/M cell-cycle arrest induced by DATS (25–28).

Previous studies in this lab (44) have shown that DATS attenuated TNF-α induced cell migration and metastasis through the NF-κB and MAP kinase pathways. These studies found that DATS significantly induced cell death and inhibited cell migration via the downregulation of the above-mentioned pathways. The attenuation of these survival pathways most often occurs through apoptosis. Apoptosis, also known as programmed cell death, plays a key role in regulating cellular homeostasis (32). Studies have shown that the dysregulation of apoptosis may in part lead to the development of various diseases, such as neurodegenerative disorders or cancer (32, 33).

In the present study, an annexin v-propidium iodide assay was used to confirm that the cell death observed in the previous studies performed in our lab was by apoptosis. The results from this study showed TNF-α had no effect on apoptosis in either cell line. In contrast, DATS significantly induced and increased in late and early-stage apoptosis, when compared to control in MDA-MB-231 and MDA-MB-468 cells (Figure 2A–D). Additionally, it was observed that TNF-α alone had no effect on the number of viable cells while it attenuated DATS induced apoptosis in the CoTx in both cell lines; however, DATS apoptotic affect in CoTx was more evident in MDA-MB-468 in comparison to MDA-MB-231. Interestingly, MDA-MB-468 cells, a cell line of African American origin of basal a phenotype with a BRCA1 mutation and PTEN homodeletion, showed more sensitivity to DATS, evident by the higher percentage of early and late apoptosis cells when compared to the MDA-MB-231 cells, a Caucasian cell line of basal b phenotype with a p53, KRAS, BRAF, CDKN2A, PDGFRA, and NF2 mutations. These results indicate that MDA-MB-468 cells are more sensitive to DATS than MDA-MB-231 cells.

In our previous study, DATS attenuated TNF-α induced CCL2 release by attenuating the IkBKE, a classified oncogene of NF-κB pathway and MAPK8 expression in MDA-MB-231 cells. In contrast, DATS significantly attenuated TNF-α induced expression of IKBKE and MAPK8, while CCL2 release was not affected in MDA-MB-468 cells. There were also other proteins affected, including PDGF-BB and NT in MDA-MB-468 cells. Further studies must be done to determine the effect of DATS on these pathways in MDA-MB-468 cells. DATS was able to induce apoptosis in human prostate cancer cells and in 8505C cells (24, 35). These results indicate that the efficacy of DATS may be dependent on the mutations observed in TNBC due to the signaling pathways that impact receptors such as tumor necrosis receptor 1 and plasma-membrane death receptor (its extracellular ligand, Fas-L) (18, 34). The Fas/Fas-L complex recruits the Fas-associated death domain-containing protein (FADD) and pro-caspase-8, collectively becoming the death-inducing signaling complex (DISC) L (18, 34). Pro-caspase-8 is then activated and stimulates pro-caspase-3, leading to the apoptotic process (34). In the intrinsic pathway, cytochrome c is released from the mitochondria outer membrane through the actions of the pro-apoptotic proteins such as Bak or Bax. Once released, cytochrome c associates with Apoptotic protease activating factor – 1 (Apaf-1) and ATP, which then bind to pro-caspase-9 to form an apoptosome. The apoptosome activates the downstream stimulation of the caspase 9/3 signaling cascade (34, 45).

Caspases are a family of protease enzymes involved in programmed cell death. Caspases help maintain cellular homeostasis by regulating the degradation of cellular content within a cell (32, 46). Apoptosis can be separated into caspase-dependent and independent pathways (32, 46). Caspases are viewed as the central executors of the apoptotic pathway (32, 46). Caspase-3, an executioner caspase, has been reported to be the most frequently activated caspase protease in apoptotic cells, indicating its critical role in the apoptotic process (47). In this study, DATS effectively activated the expression of Caspase 3 and 8 in both cell lines; however, there was no significant difference in Caspase-9. Ji et al. found that DATS induced apoptosis in human nasopharyngeal carcinoma (CNE2), possibly through p38MAPK and Caspase-8 (36). Another study showed DATS’s ability to activate caspases 8 and 9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways, in T24 human bladder cancer cells in vitro (48). Yu et al. found that DATS was able to increase the levels of cytochrome c, Apaf-1, AIF and caspase-3 and caspase-9 in primary colorectal cancer cells (32).

In summary, our current investigation demonstrated DATS potential in cancer suppression of the two different TNBC cell lines: MDA-MB-231 and MDA-MB-468. We have previously shown DATS has higher cytotoxicity, and anti-migratory activity in MDA-MB-468 compared to MDA-MB-231 cells. In the current study, we verified that the higher cytotoxicity observed in our previous study following exposure to DATS may be due to apoptosis through the extrinsic pathway in both MDA-MB-231 and MDA-MB-468 cell lines.

Conclusion

In conclusion, this study indicates that DATS could induce G₂/M arrest, leading to apoptosis via the extrinsic pathway. DATS induced higher cellular death in MDA-MB-468 than MDA-MB-231 cells, suggesting a possible diverse response to DATS treatment in genetically different cell lines. Together, these findings suggest DATS potential anticancer properties in MDA-MB-231 and MDA-MB-468 cells may partly occur via cell cycle arrest and programmed cell death, which may occur through the extrinsic apoptosis pathway with involvement of the NF-κB and MAPK signaling pathways. DATS may be a potential candidate for TNBC therapy. In addition, these findings provide more evidence for DATS’s role in cancer prevention. Future research is needed to elucidate which proteins are essential in DATS’s induced cell cycle arrest and apoptosis.