Correlation between Sulforaphane and Glutathione Peroxides-1 Accelerates Breast Cancer Cell Apoptosis

Broccoli and broccoli sprouts have long been shown to contain large amounts of glucosinolates [1]. These compounds have been shown by numerous reports to be chemo-preventive against many cancer types. Such anti-cancer activity is due to the enzymatic hydrolysis of glucosinolates from isothiocyanates [2]. Sulforaphane (SFN) is produced from glucoraphanin which is a major glucosinolate found in broccoli/broccoli sprouts [3]. SFN blocks tumor genesis primarily by inhibiting enzymes that convert procarcinogens to carcinogens and inducing enzymes that promote excretion of carcinogens [2]. Furthermore, SFN inhibits tumor cell growth by modulating cellular activities such as apoptosis or cell cycle by regulating key molecules involved in each phenomenon [2,4]. Other anti-tumorigenic activities of SFN include downregulation of molecules such as VEGF, HIF-1α, MMP-2 and MMP-9 that promote angiogenesis and metastasis [4]. SFN is also effective against cancer stem cells that rely upon given growth pathways including Wnt/β-catenin, Hedgehog and Notch to self renews [57]. And promote cancer chemoand radio-resistance [8-10]. SFN has been shown to inhibit breast cancer stem cells (BCSCs) in size and number by down regulating their Wnt/β-catenin self-renewal pathway [11]. Glutathione peroxidase-1 (GPX-1) is a seleniumdependent antioxidant enzyme and GPX-1 gene polymorphisms and sequence alterations have been implicated in cancer development [12]. These changes minimize GPX-1 responsiveness to selenium and so reduce GPX-1 cytosolic activity ultimately leading to development of various cancers including breast cancer (BC) [12]. We have previously shown that GPX-1 can synergize with the natural turmeric compound curcumin beside anti-Her2 antibody herceptin in inducing BC cell apoptosis [13]. In this study, we examined function of GPX-1 in potentiating SFN in inducing MCF7 cell death and modifying expression levels of related molecules.


Introduction
Broccoli and broccoli sprouts have long been shown to contain large amounts of glucosinolates [1]. These compounds have been shown by numerous reports to be chemo-preventive against many cancer types. Such anti-cancer activity is due to the enzymatic hydrolysis of glucosinolates from isothiocyanates [2]. Sulforaphane (SFN) is produced from glucoraphanin which is a major glucosinolate found in broccoli/broccoli sprouts [3]. SFN blocks tumor genesis primarily by inhibiting enzymes that convert procarcinogens to carcinogens and inducing enzymes that promote excretion of carcinogens [2]. Furthermore, SFN inhibits tumor cell growth by modulating cellular activities such as apoptosis or cell cycle by regulating key molecules involved in each phenomenon [2,4]. Other anti-tumorigenic activities of SFN include downregulation of molecules such as VEGF, HIF-1α, MMP-2 and MMP-9 that promote angiogenesis and metastasis [4]. SFN is also effective against cancer stem cells that rely upon given growth pathways including Wnt/β-catenin, Hedgehog and Notch to self renews [5][6][7]. And promote cancer chemo-and radio-resistance [8][9][10]. SFN has been shown to inhibit breast cancer stem cells (BCSCs) in size and number by down regulating their Wnt/β-catenin self-renewal pathway [11]. Glutathione peroxidase-1 (GPX-1) is a seleniumdependent antioxidant enzyme and GPX-1 gene polymorphisms and sequence alterations have been implicated in cancer development [12]. These changes minimize GPX-1 responsiveness to selenium and so reduce GPX-1 cytosolic activity ultimately leading to development of various cancers including breast cancer (BC) [12]. We have previously shown that GPX-1 can synergize with the natural turmeric compound curcumin beside anti-Her2 antibody herceptin in inducing BC cell apoptosis [13]. In this study, we examined function of GPX-1 in potentiating SFN in inducing MCF-7 cell death and modifying expression levels of related molecules.

Cell Culture and Treatment
Human embryonic kidney cell line HEK293T and human BC cell line MCF-7 (Michigan Cancer Foundation-7) were all maintained in DMEM plus 10% FBS and expanded and split exactly as we have reported [13]. SFN (Sigma Aldrich, U.S.A.) was dissolved in DMSO for a stock solution of 10 mM and frozen in aliquots at -20°C. Further dilutions were made in complete medium to required concentrations between 0.1-40 μM for the treatment of MCF-7 cells.

Recombinant Lentivirus Production and MCF-7 Cell Infection
We used our lent virus vector pLV-GPX1 [14]. To generate recombinant lentivirus particles harboring the human GPX-1 expression cassette beside the pLV-EGFP empty vector control [15]. The virus stocks were titrated using fresh batches of wild-type (WT) HEK293T cells and according to standard protocols [16]. For transduction purposes. MCF-7 cells were seeded in 96-well plates with a number of 6000 cells/well and 24 hours later infected with pLV-GPX1 (and pLV-EGFP as a control) at a multiplicity of infection of 10 (MOI=10). We further applied the infection step to MCF-7 cells seeded in 6-well plates with a number of 3 X 105 cells/well for RNA extraction.

Reverse Transcription (RT) PCR
RT-PCR was carried out exactly as reported [15]. using primer pairs listed in (Table 1). PCR reactions were carried out for 30 cycles consisting of denaturation at 94 °C for 45", annealing at various temperatures (depending on primer set) for 1' and extension at 72 °C for 45". Gel electrophoresis and band intensity measurement were duplicated as reported [15].

Measurement of Intracellular GPX-1 Content
We used an ELISA Kit (AdipoGen, San Diego, CA) to measure GPX-1 levels within each cell group. In order to calculate GPX-1 concentrations, we duplicated the procedure that we have already reported [14].

Measurement of Anti-oxidant Enzymatic Activity
The rate of NADPH oxidation is considered an activity indicator of a set of anti-oxidant peroxidases including GPX-1. NADPH oxidation was determined based on a method, reported by St. Clair and Chow [17]. that we have previously adapted and reported [15].

Cell Death Analyses
The MTT viability assay was used exactly as we have reported [18]. To measure cell viability as a percentage of untreated WT control cells.

Cell Co-staining and Live Cell Count
Since acridine orange (AO) co-applied with ethidium bromide (EB) stains nuclear DNA green in live cells and orange in dead cells, we followed our reported co-staining method to measure the percent of viable cells [14]. Cell groups were suspended at a normal counting concentration from which 50 µL was mixed with 50 µL of the stain in 96-well plates and subjected to counting under fluorescent light. Images were captured using a fluorescence microscope coupled with a Nikon digital camera. Six random microscopic fields per well were selected to count an average of 120 cells counted for each cell group in triplicates (three wells per group). Green cells and orange cells were considered, respectively, as live and dead cells. The data collected from three independent experiments were then expressed as percent of live cells by dividing the number of live cells by the number of total cells counted (live + dead). For flow cytometry, we followed our previous report [13]. To make PBS suspension of ethanol-fixed cell samples and stained them with 10 µg/mL propidium iodide (Sigma) before measuring the fluorescence using a FACScan (Becton Dickinson FAC Star Plus flow cytometer).

Statistical Analyses
Data in the figures are represented as the mean ± standard error of the mean (SEM) of three or more separate experiments. Student's T-test was used to analyze differences between two groups. Differences among three or more groups were analyzed by one-way analysis of variance (ANOVA), followed by a post hoc Duncan Multiple-comparisons Test (P<0.05, statistically significant; P<0.01 or P<0.001, highly significant). For correlation analyses (Pearson, 2-tailed), we used data of triplicates or more repeats for each set of experiment, and comparisons were made only between relevant pairs of data.

Successful Lentivirus Production and MCF-7 Cell Transduction
Lentivirus construct pLV-GPX1 co-harboring GPX-1 and EGFP was used for virus production as reported previously [14,15]. Reporter gene expression at both post-transfection and postinfection levels indicated successful production of recombinant viruses and transduction of MCF-7 cells ( Figure 1A). We expanded this transduced cell line to be able to carry out our subsequent analyses.

Overexpression and Overactivity of GPX-1
Two weeks after infection, the RNA of MCF-7 cells was extracted and subjected to RT-PCR. A 387-bp GPX-1 gene fragment was PCR amplified and resolved using agarose gel electrophoresis ( Figure  1B). We found that the pLV-GPX1-infected MCF-7 cells expressed nearly threefold higher GPX-1 mRNA levels compared to the WT cells and empty vector pLV-EGFP controls ( Figure 1B) P<0.01; Duncan multiple-comparisons test). Amplification of a 206-bp mRNA fragment from the housekeeping β-actin gene ensured equality and integrity of all RNA samples. We further measured GPX-1 protein using a GPX1-specific ELISA kit and found parallel increase in the protein levels of the enzyme in parallel with increase in its mRNA (Table 2), pLV-GPX1 compared to pLV-EGFP and WT samples, P<0.001). Changes in intracellular peroxidase activity were monitored by detecting NADPH oxidation. As a result of this analysis, the enzymatic activity was found induced in correlation with the levels of GPX-1 mRNA and protein ( Table 3). The NADPH levels indicated the proxidase activity was over 2.5 folds up in the pLV-GPX1 cell group compared to WT and the empty vector controls (Table 3), P<0.01, Duncan multiple-comparisons test).

Synergistic Effect of SFN and GPX-1 on MCF-7 Cell Apoptosis
We first determined the LD50 for SFN by treating the wild-type MCF-7 cells with serial concentrations of the compound that stood at 24 M ( Figure 3A). We repeated the experiment for both pLV-EGFP empty vector and pLV-GPX1 cell groups at MOI=10. While the empty vector controls responded to treatment similar to the WT cell groups, the pLV-GPX1 groups showed stronger response to SFN doses so that the LD50 was declined from 24 M in controls to 14 M in these cells and their survival rate was reduced significantly ( Figure 3A), P<0.05). Figure 3B

The Apoptotic Nature of Cell Death Co-Induced by SFN and GPX-1
We looked at the nature of cell death among our treated samples first by AO/EB co-staining and then flow cytometry both determining percent of apoptosis cells. We treated cell groups with 10 µM SFN and then co-stained with AO/EB before collecting the fluorescent images. Figure 3A shows the microscopic images of the co-stained cell groups and indicates that while untreated WT and pLV-EGFP cell groups were unaffected, SFN induced apoptosis in pLV-EGFP cells and accelerated cell death in pLV-GPX-1 cell groups. We quantified apoptosis among these cells by treating them with SFN. Figure 3B shows that increasing concentrations of the compound induced apoptotic cell death and this induction occurred more significantly in pLV-GPX1 cell groups compared to their WT and pLV-EGFP counterparts (P<0.05 or 0.01).

Diminished Expression of Pro-Survival Genes and Increased Expression of Pro-Apoptotic Genes
RT-PCR analysis indicated that while the expression of antiapoptotic BCL-2 and pro-survival/pro-growth genes AKT and SRC has been diminished in treated cell groups, the pro-apoptotic Bax and p53 genes have been up regulated as a result of these treatments (Figure 3). GPX-1 overexpression per se did not greatly affect apoptosis genes but treatment of WT or empty vector controls with 24 M SFN significantly reduced levels of BCL-2, AKT and SRC while significantly inducing expression of BAX and p53 towards apoptosis ( Figure 3B), treated cell groups versus untreated counterparts, P<0.05). This trend was accelerated when pLV-GPX1overexpressing cell groups were subjected to SFN treatment. As a result, further reduction in anti-apoptotic/pro-survival genes and conversely further increase in expression of pro-apoptotic genes were detected that were in all cases significant ( Figure 3B), P<0.05).

Correlation between Cell Treatment and Apoptosis Rate
Comparison between RT-PCR data, on one hand, and data of ELISA as well as enzymatic activity, on the other hand, indicated that GPX-1 mRNA levels correlated with GPX-1 protein levels and peroxidase activity, respectively, by 98% and 96% (P<0.01; 2-tailed) whereas correlation between GPX-1 protein levels and its enzymatic activity stood at 95.5% (P<0.05; 2-tailed). Meanwhile, we found significant correlations between GPX-1 virus concentrations and apoptosis when the cells were treated with SFN, so that when we used GPX-1 MOI=5 and 14 µM SFN, the correlation with apoptosis rate was over 96% (P<0.01) whereas application of MOI=5 or 10 with 24 µM SFN caused 99% correlation with apoptosis rate (P<0.01) (Figure 4).

Figure 4:
Changes in mRNA expression levels of pro-apoptotic and pro-survival molecules. Samples were analyzed by RT-PCR and resolved by agarose gel electrophoresis. The intensity of gel images was then analyzed using Image software (http://rsbweb.nih.gov/ij/) as we have reported (Safi 13 et al 2011). The RT-PCR analysis was repeated three times as three independent experiments. Differences of band intensity between each cell group and wild-type control are shown by * or ** for increase and by ≠ for reduction.

Discussion
We have previously shown that glutathione peroxidase-1 can synergize with the natural turmeric compound curcumin beside anti-Her2 antibody herceptin in inducing BC cell apoptosis [13]. In this study, we examined the co-effect of SFN and GPX-1 in inducing MCF-7 cell death and modifying expression levels of related molecules. SFN per se induced apoptosis in the cells with its concentration of 24 µM determined as LD50 of the drug. However in the active presence of GPX-1, this figure was dropped to 14 µM, a significant reduction that indicated sensitization of SFN on MCF-7 cells by GPX-1 anti-oxidant activity. Correlation analysis indicated that GPX-1 mRNA and protein significantly match with GPX-1 intracellular activity and this activity significantly sensitizes the BC cells to the pro-apoptotic effects of SFN. SFN among other nutraceuticals has drawn attention in its potential to block tumor genesis by a variety of molecular mechanisms that contribute in many key steps of tumor development [19]. Human GPX-1, on the other hand, has been shown to play important role in prevention of BC whereas its mutations or polymorphisms promote tumor genesis [12]. We have previously shown in SKBR-3, a HER2-amplified BC cell line, that GPX-1 overexpression can induce apoptosis by up regulating BAX and p53 and reducing expression of BCL-2 [13]. Our current study shows that GPX-1 per se is unable to promote these changes in MCF-7 that has no HER-2 amplification and expresses moderate levels of the receptor [20]. On the other hand, MCF-7 treatment with SFN per se induces apoptosis by up regulating pro-apoptotic molecules while down regulating pro-survival genes. Furthermore, when the cells were transduced with GPX-1 lentivirus stocks and then treated with SFN, they showed significantly increased sensitivity to SFN indicating that increased activity of GPX-1 has potentiated the proapoptotic and anti-survival effects of the compound. These findings were confirmed by our correlation data.
Free radicals in the form of reactive oxygen species (ROS) play key role in initiation and progression of cancer by destabilizing cellular DNA [21]. In return, cancer cells are forced to counteract the toxic effects of ROS by adapting mechanisms for adjustment to survive that include anti-apoptotic and drug resistant measures. Furthermore, anti-oxidant enzymes including GPX-1 function against ROS by relying upon intracellular anti-oxidant defense system largely based on glutathione. In fact, GPX-1 is heavily dependent on intracellular glutathione content for its anti-oxidant function against reactive oxygen species [22]. These enzymes can affect cancer initiation and progression steps by substantially reducing intracellular ROS levels. Sulforaphane, on the other hand, accelerates cancer cell death by disrupting growth signaling pathways and inducing cell death pathways. Our findings in this study suggest that while GPX-1 reduces excessive levels of ROS that MCF-7 is exposed to upon it cancerous metabolism, in essence it facilitates the pro-apoptotic function of SFN. In order to test this hypothesis, the mechanism of GPX-1 action on MCF-7 cells needs to be studied by measuring ROS levels upon depletion of intracellular glutathione and exploring diverse molecules that play role in cancer cell metabolism including proliferation, autophagy and cancer stem cell reactions to treatment and co-treatment.

Conclusion
We showed that while GPX-1 alone is unable to produce a major shift in the cancerous state of MCF-7 cells, it potentiates the anti-survival activities of SFN. We demonstrated this change by examining cell phenotype and intracellular pathways of survival and death. The precise mechanism of SFN-GPX-1 synergy in inducing apoptosis in MCF-7 requires further investigations on cancer cell metabolism.