{"id":17557,"date":"2017-12-21T11:54:15","date_gmt":"2017-12-21T11:54:15","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=17557"},"modified":"2018-08-18T06:18:14","modified_gmt":"2018-08-18T06:18:14","slug":"diallyl-disulfide-dads-retards-the-growth-of-breast-cancer-cells-in-vitro-and-in-vivo-through-apoptosis-induction","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol10no4\/diallyl-disulfide-dads-retards-the-growth-of-breast-cancer-cells-in-vitro-and-in-vivo-through-apoptosis-induction\/","title":{"rendered":"Diallyl Disulfide (DADS) Retards the Growth of Breast Cancer Cells in Vitro and in Vivo Through Apoptosis Induction"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

Diallyl disulfide (DADS) is an organosulfur compound derived from garlic (Allium sativum) with proven health beneficial effects.1<\/sup> Various cell-based assays determining the anticancer activity of DADS have shown the ability of this compound to inhibit proliferation of cell lines representing carcinomas of breast, prostate, lung and liver.1,2, 3<\/sup> Mechanistic studies delineating the pathways involved in cancer cell growth inhibition have demonstrated (a) upregulation of apoptotic signaling, especially caspase-3 and cleaved PARP; (b) induction of cell cycle arrest at G0\/G1 and G2\/M phases; (c) inhibition of proliferation cascades (d) downregulation of cell migration and (e) activation of anti-oxidant response genes including Nrf2 and its target proteins NQO1 etc.1,4,5<\/sup>\u00a0 Likewise, recent studies have identified induction of p53 and p21 in esophageal squamous cell carcinomas.5<\/sup><\/p>\n

In animals, DADS was shown to retard the growth of subcutaneous xenografted tumors.6<\/sup>\u00a0 Few studies have also demonstrated the ability of DADS to retard EAC cell growth in the peritoneal cavity of mice.\u00a0 However, the molecular mechanisms responsible for DADS-induced EAC cell death are not known.\u00a0\u00a0 A separate study has demonstrated the ability of Garlic Oil (rich in DADS) to halt the development of EAC solid tumors.7<\/sup>\u00a0 This study has shown inhibition of HDAC and induction of antioxidant catalase (CAT), glutathione-S-transferase (GST) and glutathione (GSH) levels.\u00a0 But, not much is known about which master regulator is responsible for these molecular changes occurred due to Garlic Oil administration.7<\/sup>\u00a0 Further it is also not studied whether these molecular changes are due to DADS or any other sulfur containing compound present in garlic oil.8<\/sup><\/p>\n

Therefore, in this study we have determined the efficacy of DADS to inhibit triple negative breast cancer cell line MDA-MB-468 and compared the potential with normal lung epithelial cell line BEAS-2B.\u00a0 Next, the ability of DADS to inhibit intraperitoneally growing EAC cells was assessed in mice.\u00a0 Effect of DADS on the expression of proteins such as NQO1, SOD and Caspase, which are involved in antioxidant activity and apoptosis induction were also measured.\u00a0\u00a0 In summary, we could demonstrate that DADS inhibit breast cancer cell growth in vitro as well as in vivo by promoting apoptosis as well as by triggering antioxidant proteins, which subsequently stabilize the oxidative degradation of tumor suppressor proteins such as p53.\u00a0 Stabilized p53 promotes apoptosis in cells leading to tumor growth inhibition.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Materials<\/strong><\/p>\n

85% pure (by HPLC) diallyl disulfide (IUPAC Name: 4, 5-dithia-1, 7-octadiene; Mol. Wt: 146.28g\/mol) was procured in oil form from Alfa Aesar Haverhill, Massachusetts USA.\u00a0 Cancer cell line MDA-MB-468 (ER, PR and HER2 negative) was procured from National Center for Cell Science, Pune, Maharashtra, India.\u00a0 Cell culture consumables including DMEM with phenol red, with 4.5g\/L glucose, but lacking L-glutamine and sodium bicarbonate, fetal bovine serum,\u00a0 trypsin, glutamax, phosphate buffered saline, pen-strep antibiotic mixture were procured from Life Technologies, USA.\u00a0 All other reagents were of analytical grade. Swiss Albino mice were procured from central animal facility, JSS University, Mysuru.<\/p>\n

Methods<\/strong><\/p>\n

Purity of DADS<\/strong><\/p>\n

Purity of DADS was assessed by injecting 10\u00b5L of DADS from 0.01mg\/mL to 10.0 mg\/mL on Shim-Pack VP-ODS (C18 column, 250mm x 4.5mm), and by eluting the loaded sample using Acetonitrile : Water (75:25) as mobile phase at a flow rate of 1mL\/min.\u00a0 The eluted DADS was detected at 240nm.<\/p>\n

Determination of anti-cancer Activity of DADS<\/strong><\/p>\n

The anti-cancer activity of DADS was measured according to Madhunapantula et al., 2008 (9).\u00a0 In brief 0.5 X 104<\/sup> MDA-MB-468 cells (representing carcinomas of breast) in 100\u00b5L DMEM supplemented with 10% FBS were seeded in a 96-well plate and incubated at 37o<\/sup>C in a cell culture incubator maintained at 5% CO2<\/sub> and 95% relative humidity.\u00a0 After 48h, the cells were exposed to increasing concentration (62.5\u00b5M to 1000\u00b5M) of DADS (dissolved in DMSO and diluted in DMEM-10% FBS medium) for 24 and 48h and the viability of cells measured using sulforhodamine-B and MTT assays. Camptothecin (40\u00b5M) was used as positive control for drug treatment<\/p>\n

Measurement of Cell Viability using Sulforhodamine-B assay (SRB assay)<\/strong><\/p>\n

SRB assay was performed according to Skehan et al., 1990.10<\/sup>\u00a0 Experimentally, cells were fixed in 1\/4th volume of cold 50% (w\/v) TCA for 4\u02daC.\u00a0 After 1h, the media was removed and the wells washed with water (200\u00b5L X 4 times) to remove TCA and serum proteins.\u00a0 The plates were dried, incubated with 100\u00b5L 0.4% SRB (in 1% acetic acid) for 30.0 minutes to stain the cellular proteins.\u00a0 Unbound SRB was removed by washing quickly with 1% acetic acid (200\u00b5L X 4 times).\u00a0 The bound SRB was solubilized in 10.0mM Tris base solution (100\u00b5L\/well) and the absorbance measured in a multimode plate reader operating at 490nm.\u00a0\u00a0 Percentage cell viability was calculated using the following equation<\/p>\n

% Viability = 100- {(OD of Control \u2013 OD of Sample) \/ OD of Control) x 100}<\/p>\n

Measurement of Cell Viability using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide Assay<\/strong><\/p>\n

First, a stock of 12mM MTT was prepared by adding 1mL of sterile PBS to 5mg of MTT.\u00a0 For measuring the viability 20\u00b5L 12mM MTT was added to cells (untreated and treated) growing complete medium.\u00a0 The plate was incubated for 1h 10minutes in CO2<\/sub> incubator at 37o<\/sup>C added centrifuged at 3500rpm for 5miniutes. The supernatant was discarded and the pellet was dissolved in 100\u00b5L \u00a0of DMSO and read\u00a0 at 570nm in a multimode plate reader.<\/p>\n

Detection of Apoptosis by Acridine Orange and Ethidium Bromide Staining<\/strong><\/p>\n

Apoptosis detection using acridine orange and ethidium bromide staining method was carried out as described by Shailasree et al., 2015.11<\/sup>\u00a0 In brief, first, 0.3 x 106 <\/sup>MDA-MB-468 cells were plated in 6-well plates and allowed to grow for ~36h.\u00a0 The exponentially growing cells were exposed to increasing concentrations of DADS (500\u00b5M, 1000\u00b5M and 1500\u00b5M) for about 48h. The control and treated cells were trypsinized and mixed thoroughly to obtain a single cell suspension.\u00a0 Trypsin was neutralized by the addition of complete medium. An about 20.0\u03bcL cell suspension was incubated with 10.0\u03bcL ethidium bromide (100.0\u00b5g\/mL) and 10.0\u03bcL of acridine orange (100.0\u00b5g\/mL) mixture for 10.0 minutes.\u00a0\u00a0 The cells were imaged using the fluorescence microscope using TRITC and FITC filters. The images obtained using 2 different channels were merged to obtain a combined image, which emitted green and orange cells.11<\/sup>\u00a0 The live cells take up acridine orange (which stains the cells green), while the apoptotic cells, (whose membrane integrity is lost and nucleus is exposed) take up the ethidium bromide and appear orange when photomicrographed under fluorescence microscope.<\/p>\n

Evaluation of the Efficacy of Diallyl Disulfide for Retarding EAC Cells Growing in the Peritoneal Cavity of Mice<\/strong><\/p>\n

The study was carried out after receiving the approval from the Institutional Animal Ethics Committee, JSS College of Pharmacy (208\/2016), Mysuru.\u00a0 Experimentally, first, 6-8 weeks old female Swiss albino mice (n = 18) weighing around 25-28g were injected with 1 x 106 <\/sup>viable EAC cells in to the peritoneal cavity.\u00a0 The EAC cells were collected from ascites fluid of a mouse already harboring healthy EAC cells.\u00a0 The collected EAC cells were diluted with PBS, and number of viable cells quantitated using Tryphan blue exclusion method.12\u00a0<\/sup> After 24h of tumor cells injection, the mice were divided in to 3 groups (n=6\/group).\u00a0 Animals in Group-II (injected with EAC cells) were administered with vehicle 50% DMSO in PBS (50\u00b5L\/mouse, intra peritoneal), the animals in Group-III received DADS 50mg\/kg body weight (50\u00b5L\/mouse intra peritoneal).\u00a0 A set of 6 animals not injected with EAC cells (Group-I) were used as controls for measuring the normal body weight gain (No tumor).\u00a0 These animals were, however, injected with 50% DMSO in PBS (50\u00b5L\/mouse) to measure whether DMSO vehicle alone has any influence on body weight gain. \u00a0The treatment was continued for 18 days by injecting the drugs on alternative days. Body weight was recorded every other day. On 19th<\/sup> day mice were sacrificed by cervical dislocation and the ascites collected were used for estimation of SOD, GSH, and Caspase-3 activity. The vital organs were collected and the H and E staining was carried out.13<\/sup><\/p>\n

Colorimetric Estimation of Caspase-3 Activity using A Chromogen-Coupled DEVD Substrate<\/strong><\/p>\n

The caspase-3\/CPP32 colorimetric assay kit was used to measure the caspase-3 activity [http:\/\/www.biovision.com\/manuals\/K106.pdf] in EAC cells collected from tumor kinetics experiment.\u00a0 Protein lysates from EAC cells collected from control and DADS treated animals were prepared using 100\u03bcL of lysis buffer provided in the kit.\u00a0 Total protein content in the cell lysates was estimated using BCA method, and caspase-3 activity determined by incubating 100.0\u03bcg of total protein in a total volume of 50.0\u03bcL cell lysis buffer with 5.0\u03bcL 4.0mM DEVD-pNA substrate (200.0\u03bcm final concentration) and 50\u03bcL of 2X reaction buffer containing 10.0mM DTT at 37\u02daC for 3h.\u00a0 The developed color was read at 405nm using a multimode plate reader (PerkinElmer). The fold change compared to control untreated cells was calculated and plotted against compound concentration.<\/p>\n

Evaluation of Antioxidant Enzyme SOD and Reduced Glutathione (GSH) Levels<\/strong><\/p>\n

The levels of antioxidant enzyme SOD and the intracellular metabolite – reduced glutathione (GSH) was measured in EAC cells collected from mice treated with experimental DADS and control 50% DMSO vehicle, according to method described by Sharma et al 2009 and Sun Y et al 1988.14,15<\/sup> Experimentally, EAC cells were washed thrice with PBS to remove the traces of peritoneal fluid. To the pellet 100-150 \u00b5L of ice-cold RIPA buffer was added and incubated for 30 minutes on ice. The lysed cells were centrifuged at 14000 rpm for 30 minutes and the supernatant collected.\u00a0 Total protein in the collected protein lysates was estimated using the BCA method.<\/p>\n

Measurement of SOD: Superoxide dismutase enzyme catalyzes the conversion of super oxide anions into less destructive molecules thus making them an important antioxidant defense system.16<\/sup> The levels of SOD in EAC cells collected from mice treated with\u00a0 experimental DADS and control 50% DMSO vehicle was measured by the method detailed by Sun Y et al 1988.15<\/sup>\u00a0 First, cell lysates containing 2,4,6,8 and 10 \u00b5g of total protein in a final volume of 10.0\u00b5L were incubated with 250\u00b5L of 12mM methionine. Next, 30.0\u00b5L of 0.5mM riboflavin and 10\u00b5L of 3.4mM NBT, were mixed and exposed to illumination for 5 minutes in an illumination chamber lined with aluminium foil, and fitted with a 15W fluorescent lamp.\u00a0 Immediately after illumination, the optical density of reaction mixture was read at 560nm. SOD substrate alone, exposed to illumination, was used as standard while a substrate without NBT served as blank. A control without NBT for each sample was also maintained and the control values were subtracted from the test values. Decrease in OD was calculated using the formula<\/p>\n

Decrease in OD=(S-B)-(T-C)<\/p>\n

S= Standard, B= Blank, T=Test, C=Control<\/p>\n

SOD was expressed as units\/mg protein using the formula<\/p>\n

Formula<\/strong><\/p>\n

Measurement of reduced glutathione (GSH): Glutathione is a tripeptide antioxidant in the biological system.17<\/sup> The sulphydryl group of the GSH reacts with 2-nitro benzoic acid to form a stable yellow color, which was measured at 412nm.14<\/sup> 100\u00b5g of the cell lysate, collected from EAC cells harvested as described above, was incubated with 100\u00b5L of the disodium hydrogen phosphate (0.3M) and 0.04% Dithiobis nitro benzoic acid (DNTB) (prepared in 0.3M disodium hydrogen phosphate) and read immediately at 412nm using the multimode plate reader. The concentration of glutathione in the samples was calculated using the calibration graph constructed using GSH (0-160\u00b5M) and expressed in \u00b5M14<\/sup><\/p>\n

Statistical Analysis<\/strong><\/p>\n

All experiments were conducted at least with 3 replicates (intra experimental) and the results were expressed as mean of 3 independent experiments +<\/u> SEM calculated using GraphPad Prism version 6.0. The results were subjected to One-Way or Two-Way ANOVA to analyze differences between controls \/ positive controls and DADS treated samples. Tukey\u2019s post hoc test was used after ANOVA and the \u201cp\u201d value of < 0.05 was considered significant.<\/p>\n

Results<\/strong><\/p>\n

Diallyldisulfide Utilized in This Study is Pure<\/strong><\/p>\n

Diallyldisulfide (DADS, IUPAC Name: 4, 5-dithia-1, 7-octadiene; Mol. Wt: 146.28g\/mol) was procured in the form of an oil with a density of 1.008.\u00a0 Purity of DADS was assessed by injecting 10\u00b5L of DADS from 0.01mg\/mL to 10.0 mg\/mL on Shim-Pack VP-ODS (C18 column, 250mm x 4.5mm), and by eluting the loaded sample using Acetonitrile : Water (75:25) as mobile phase at a flow rate of 1mL\/min.\u00a0 The eluted DADS was detected at 240nm and the peak area was plotted against concentration to determine the linearity of standard graph (Figure 1A and 1B).\u00a0 A single symmetrical peak with a retention of 6.11minutes and a purity percentage of 85% was observed in the chromatogram.\u00a0 Two minor peaks 5.269 and 7.669 were also observed with an about 5.01% and 8.89% contribution of total area, respectively (Figure 1A).\u00a0 R2<\/sup> value of prepared standard graph was 0.998 indicating good linearity in the concentration ranges tested (Figure 1B)<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 1:<\/strong> Analysis of DADS using HPLC.<\/strong><\/p>\n

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(A) Representative chromatogram showing the elution profile of DADS. DADS was eluted as a predominant symmetrical peak at 8.2min.\u00a0 (B) The concentration curve of DADS.\u00a0 Increasing concentration of (1.0mM to 10mM) DADS was injected to C18 column and the eluted peak area plotted against concentration of DADS.\u00a0 A linear curve with an R2<\/sup> of 0.95 was obtained.<\/p>\n

DADS Inhibited the Growth of MDA-MB-468 Breast Cancer Cell Line More Effectively Compared to Normal Lung Epithelial Cell Line in Vitro<\/strong><\/p>\n

To determine the anti-cancer potential of DADS against a triple negative breast cancer cell line MDA-MB-468, 1.0X104<\/sup> cells were plated in 100\u00b5L DMEM supplemented with 10% FBS medium and allowed to grow for about 36h in a humidified carbon dioxide incubator.\u00a0 Exponentially growing cells were treated with increasing concentration of DADS (46.85\u00b5M to 1500.0\u00b5M) for 24h and 48h and the viability compared with vehicle DMSO (1% final concentration) exposed cells (Figure 2A).\u00a0 Vehicle DMSO had only minimal effect on cell viability (<10% cell death) compared to untreated cells (Figure 2A).\u00a0 A dose dependent decrease in cell viability was observed with increasing concentration of DADS.\u00a0 At 1500.0\u00b5M concentration an about 37% cell death was observed at 24h treatment (Figure 2A).\u00a0 Increasing the treatment time to 48h enhanced the efficacy of DADS as an about 51% decrease in the number of viable cells was observed (Figure 2B).<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 2:<\/strong> DADS inhibit breast cancer cell growth in vitro<\/strong><\/p>\n

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(A) Inhibition of cell growth at 24h: A dose dependent reduction in cell viability was observed when, MDA-MB-468 breast cancer cells were treated with increasing concentration of DADS (from 46.8 to 1500\u00b5M) for 24h. (B) When cells were further exposed to MDA-MB-468 for 48h the cellular viability further decreased with increase in dose as determined by SRB assay.<\/p>\n

Since DADS inhibited the triple negative breast cancer cell line MDA-MB-468, next, the efficacy on normal lung epithelial cell line BEAS-2B was tested to determine the selectivity. First, 1.0 X 104<\/sup> BEAS-2B cells were plated\/well in 100\u00b5L culturing medium and allowed to grow for 48h in a carbon dioxide incubator.\u00a0 Next, the cells were treated with increasing concentration of DADS (375.0\u00b5M, 750.0\u00b5M, 1500.0\u00b5M and 2000.0\u00b5M) for 24h and viability determined using MTT reagent as detailed in materials and methods.\u00a0 Analysis of the data showed a marginal cell death of 22.5% even at 1500.0\u00b5M compared to control untreated cells.\u00a0 DMSO (1%) alone also had an about 13.1% cell growth inhibition (Figure 3).\u00a0 Further increase in the concentration of DADS to 2000.0\u00b5M reduced the number of viable cells to 20% (ie., 80% inhibition) (Figure 3).<\/p>\n\n\n\n
\"Figure<\/td>\nFigure 3: DADS inhibit BEAS-2B cells relatively at a higher dose compared to MDA-MB-468.<\/strong><\/p>\n

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To assess whether DADS is selective to cancer cells or does it kill normal cells as well, human normal lung epithelial cells BEAS-2B were treated with increasing concentration of DADS (350.0\u00b5M, 750.0\u00b5M, 1500.0\u00b5M and 2000.0\u00b5M) for 24h and the viability measured.\u00a0 A significant decrease in number of viable cells was observed only at 2000.0\u00b5M DADS concentration.\u00a0 Not much cell death was noticed at other concentrations tested indicating that DADS has selectivity toward cancer cells<\/p>\n

Comparison of the cell growth inhibitory potential of DADS between normal BEAS-2B cell (Figure 3) line with MDA-MB-468 cancer cell line showed more selectivity of this sulfur-containing compound to cancer cells.\u00a0 For example, at 24h of treatment, 1500.0\u00b5M DADS inhibited the growth of MDA-MB-468 cells by 37% (DMSO alone had no effect on cell growth), compared to normal BEAS-2B cells, where the cell growth inhibition was only 22.5% (DMSO alone had ~13% effect, resulting in an overall effect of 9.5%).<\/p>\n

DADS-Induced MDA-MB-468 Cell Growth Inhibition Was Mediated by NQO1 Activation and Apoptosis Induction<\/strong><\/p>\n

To evaluate the mechanism of DADS induced cell growth inhibition, the MDA-MB-468 cells were exposed to acridine orange and ethidium bromide and imaged using fluorescence microscope. Live cells\u00a0 with intact membrane take up only acridine orange and emit green light while the dead cell where membrane integrity is lost are stained by ethidium bromide which appear red indicating apoptosis (Figure 4A) \u00a0Analysis of the data showed that DADS induced dose dependent apoptosis as evident by increased orange cells (73% at 1250\u03bcM) compared to 1% DMSO treated cells (4%). In addition the DADS also elevated NQO1 activity at 250, 750 and 1250\u03bcM compared to the DMSO treatment. The increase in activity was dose dependent with 1250\u03bcM showing highest activity of 76.1% compared to DMSO which showed 52% activity (Figure 4B) Elevated NQO1 stabilizes the P53 and thereby induce apoptosis.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 4: DADS-induced MDA-MB-468 cell growth inhibition was mediated by NQO1 activation and apoptosis induction.<\/strong><\/p>\n

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(A) DADS treatment increased the NQO1 activity in MDA-MB-468 cell line.\u00a0 A dose dependent increase in NQO1 activity upon treatment with increasing concentration of DADS was observed in MDA-MB-468 cells indicating that NQO1 might be having a role in cell death induction.\u00a0 (B) Treatment of MDA-MB-468 cells with DADS elevated the levels of Caspase-3.\u00a0 To check whether DADS treatment induced apoptosis in breast cancer cell line, the cells were exposed to increasing concentration of DADS for 48h and the treated cells stained with Acridine Orange (AO) and Ethidium Bromide (EtBr).\u00a0 Analysis of stained cells using Fluorescence Microscope showed both green (Live Cells) and Red (Dead Cells) cells.\u00a0 A dose dependent increase in dead cell population was observed.<\/p>\n

Intra Peritoneal Administration of DADS Retard the Growth of EAC Cells in Mice<\/strong><\/p>\n

Based on the in vitro efficacy data demonstrating the ability of DADS to inhibit breast cancer cells growth, an in vivo study using EAC tumor model was carried out to determine whether intra peritoneal administration of DADS could also retard the proliferation of cancer cells in animals.\u00a0 EAC is a good representative of breast cancer model in mice.18\u00a0<\/sup> EAC cells are undifferentiated; lack tumor-specific transplantable antigen (TSTA) and respond to drug treatment.18<\/sup> Therefore, EAC cells are considered to resemble human cancer cell lines.18\u00a0<\/sup> Hence, in this study we have determined the efficacy of DADS for inhibiting EAC cells proliferation in vivo using female Swiss albino mice.\u00a0 Experimentally, first, mice were pretreated with DADS 50mg\/kg body weight (i.p.) for 2 alternative days prior to induction of tumor. \u00a0Next, on 3rd<\/sup> day 1.0 x 106<\/sup> EAC cells were injected to all animals and treatment with DADS (for a group of 6 animals bearing EAC cells) was continued till 19th<\/sup> day by intraperitoneal injection on every alternative days. \u00a0Average weight gain compared to untreated control was recorded (Figure 5).\u00a0 Administration of DADS showed an about 33% decrease in body weight gain of animals, compared to vehicle treated mice (Figure 5).<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 5: Intraperitoneal administration of DADS retarded the growth of EAC cells in mice.<\/strong><\/p>\n

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To check whether intraperitoneal administration of DADS (50mg\/kg body weight) retards the growth of EAC cells growing in the peritoneal cavity of mice, DADS was administered every other day and animal body weight measured.\u00a0 Whereas the control mice that received the vehicle 50% DMSO showed a significant body weight gain (an indicator of tumor cell growth) the mice that received 50mg\/kg body weight DADS showed an about 30% reduction in body weight gain, indicating that DADS has anti-tumor activity.<\/p>\n

DADS-Mediated Tumor Growth Inhibition Was Due to the Induction of Apoptosis and NQO1 Activity in Mice<\/strong><\/p>\n

Since intraperitoneal administration of DADS retarded the growth of EAC cells in mice, the mechanism(s) responsible for this effect was studied next.\u00a0 Experimentally, EAC cells were collected at the end of EAC tumor kinetics experiment, and protein lysates harvested by centrifugation.\u00a0 Caspase-3, NQO1, activity and GSH levels were measured as detailed in the materials and methods and the data compared with vehicle DMSO treated mice.\u00a0 \u00a0A 4.49 fold increase in caspase-3 levels were observed in DADS treated mice compared to vehicle treated mice indicating that DADS induced caspase-3 mediated apoptosis (Figure 6A). In addition DADS significantly increased NQO1 activity in 50mg\/kg body weight DADS treated animals (from 17.55\u00b5moles\/min\/mg total protein to 26.35\u00b5moles\/min\/mg total protein) (Figure 6B).\u00a0 Even the cellular glutathione (GSH) level, although not significant statistically, was increased upon treatment of mice with 50mg\/kg DADS (Figure 6C).\u00a0 Corroborating with these results, a significant 3.36 fold increase in superoxide dismutase levels (from 41.19 Units in control animals to 138.29 Units) was observed in the EAC cell lysate collected from DADS treated animals (Figure 6D).<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\nFigure 6: Elevated NQO1, SOD and Caspase-3 enzymes, and GSH mediate DADS-induced tumor cell inhibition.<\/strong><\/p>\n

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To delineate the mechanisms leading to EAC cell growth inhibition, the ascites fluid from control DMSO treated and experimental DADS administered mice were collected and cell lysates prepared as detailed in materials and methods.\u00a0 Equal protein (from control and experimental groups) was analyzed for measuring (A) caspase-3; (B) NQO1; (C) GSH; and (D) SOD. \u00a0A significant increase in NQO1, caspase-3 and SOD level was observed with DADS treatment.\u00a0 However, GSH showed a non-significant increase upon treatment with DADS.\u00a0 This data indicate that the cell death induced by DADS is in part mediated by the induction of key oxidative stress enzymes and reduced metabolite GSH<\/p>\n

Discussion<\/strong><\/p>\n

DADS is an organo-sulfur compound derived from aged garlic.1<\/sup>\u00a0 Many studies have demonstrated the anti-cancer potential of DADS and pointed that the cell death induced by DADS is mediated through the activation of apoptosis, cell cycle arrest and inhibition of proliferation and angiogenesis.19<\/sup>\u00a0 However, these effects are dose dependent and the treatment outcomes and the mechanisms being operated in cancer cells vary with type of tumor being treated as well as the stage at which it is exposed to DADS.19<\/sup>\u00a0 For example, consumption of aged garlic (rich in DADS) prevents the transformation of normal cells to cancerous ones by protecting the cells from oxidative stress through the induction antioxidant enzymes NQO1, SOD and GST.1\u00a0<\/sup> Supporting this observation, many studies have shown the induction of transcription factor Nrf2 upon treatment of cells with DADS.4<\/sup> Hence, DADS is considered as a good cancer preventive agent.<\/p>\n

Recent studies have demonstrated the ability of DADS to treat advanced- and drug resistant cancer types.20\u00a0<\/sup> Anticancer ability of DADS is not only due to its ability to activate Nrf2 target genes such as NQO1 but also because of the potential of DADS to trigger apoptotic cell death and cell cycle arrest through the upregulation of proteins such as p53.21\u00a0<\/sup> Induction of NQO1 in tumor cells promotes the conversion of inactive anticancer agents in to active molecules thereby increase cell death.22<\/sup>\u00a0 For example, a study by Pink, J.J. et al., 2000 has demonstrated that NQO1 is the key determinant of b-lapachone activity in breast cancers.23<\/sup>\u00a0 Cells expressing NQO1 are more sensitive to\u00a0 b-lapachone treatment compared to NQO1 null or silenced cells.23\u00a0<\/sup> Therefore activation of NQO1 in cells lacking this protein sensitizes them to chemotherapeutic agents.\u00a0 However, it is not known how induction of NQO1 and other antioxidant proteins and metabolites is promoting cancer cell death as observed in this study.\u00a0 One possible explanation is that elevated expression of these proteins in DADS treated cells is a defensive mechanism of cells to avoid cell death induced by DADS through the induction of reactive oxygen species.24<\/sup> Treatment of cancer cells with DADS elevates ROS levels in cells.24\u00a0<\/sup> A significant dose dependent increase in ROS level was observed upon treatment of breast cancer cells MDA-MB-231 and MCF-7 with DADS (data not shown).\u00a0 Elevated cellular ROS promotes apoptosis in cells by damaging key constituents such as DNA, proteins and lipids.24<\/sup> A significant increase in the cellular apoptosis was observed when MDA-MB-468 cells were treated with DADS .\u00a0 Induction of apoptosis is not only due to elevated caspase-3 as observed in EAC-bearing mice treated with DADS but also could be due to enhanced p53 expression and stabilization.\u00a0 Elevated NQO1 is known to stabilize p53 protein in cells.25\u00a0<\/sup> Therefore, breast cancer cells treated with DADS are undergoing death due to induction of apoptosis, which is mediated by enhanced expression of caspase-3 proteins as well as stabilization of p53 through upregulated NQO1.<\/p>\n

Conclusions<\/strong><\/p>\n

DADS is a potent anti-breast cancer agent, which works by promoting apoptosis through caspase-3 induction and possibly through the p53 expression.\u00a0 However, further studies are warranted to determine the association between NQO1 expression with p53 stabilization.<\/p>\n

Acknowledgements<\/strong><\/p>\n

Authors would like to acknowledge DST-FIST for supporting the Department of Biochemistry, JSS Medical College, which helped us to procure key equipment utilized in this study.<\/p>\n

Conflict of Interest<\/strong><\/p>\n

Authors have no conflict of interest<\/p>\n

Funding Source<\/strong><\/p>\n

The Work was not supported by any Funding agency<\/p>\n

References<\/strong><\/p>\n

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