According to recent statistics, though the breast cancer death rate has decreased over the years, overall breast cancer incidence rates have increased among most races [1
]. The cause for breast cancer is still unknown though most of the evidence points toward genetic and epigenetic abnormalities. Epigenetic gene alterations are an important hallmark of breast cancer. A common cause of breast cancer is epigenetic modifications, especially increasing DNA methylation and histone deacetylation. Bioactive dietary compounds are of special interest because of their anticancer properties and numerous studies have suggested that these dietary compounds can alter abnormal epigenetic states in cancer [2
]. Regular consumption of bioactive nutritional compounds can alter the epigenome and play an active role in cancer prevention and therapy [4
]. Several dietary compounds administered singly or in conjunction with other dietary compounds or other chemotherapeutic agents can reverse abnormal gene activation or silencing in cancer cells [5
Combinatorial epigenetic marks are gaining importance as biomarkers for breast cancer diagnoses [6
]. Choosing an optimum concentration of dietary combination that can decrease DNA methylation and deacetylation levels may be effective to synergistically activate tumor suppressors [7
]. Thus, combinatorial regimens are gaining increasing popularity in cancer research because of their effectiveness in preventing and treating cancer. The combination of two or more compounds has been found to be more effective than compounds acting singly in many cases [8
] by the use of a quantitative measure named Combination Index. Combination Index measures the degree of interaction among drugs in the form of synergism or antagonism for a given end point of the effect measurement [10
]. The effectiveness of single compounds is too often performed at very high doses that can confer potential side effects. Lower doses of the single compounds in combination may have a better chemopreventive potential and translational impact.
Genistein (GEN), found in natural soybean, is an isoflavone that is a known DNA methyltransferase (DNMT) inhibitor while sulforaphane (SFN), which is enriched in cruciferous vegetables such as broccoli sprouts (BSp) and kale, has been shown to have histone deacetylase (HDAC) inhibiting properties [11
]. Figure 1
shows the structure of GEN and SFN. The inhibitory effects of GEN and SFN in tumor inhibition may be attributed to modulating epigenetic pathways by altering epigenetic marks in cancer cells. Our previous studies have shown that a combinatorial diet consisting of DNMT and HDAC inhibitors can alter the epigenetic machinery via direct and indirect inhibition of DNMTs and HDACs, thereby leading to modulating aberrations in DNA methylation and histone acetylation/deacetylation in tumor-related genes [13
]. SFN is an inhibitor of HDAC activity, which results in an increase in global and local histone acetylation of a number of genes [16
]. Unlike the single compounds, the combination of GEN and SFN has been understudied. GEN acts as an DNMT inhibitor reducing aberrant gene hypermethylation and SFN is an HDAC inhibitor that can remodel chromatin structure by affecting histone acetylation status in cancer cells and providing an open chromatin configuration.
We hypothesized that the optimal concentration of the two compounds should be effective in synergistically decreasing cancer cell viability. The correct combination of two or more dietary compounds that act synergistically can be very effective in preventing and treating breast cancer. Previous studies have shown that SFN exhibits chemopreventive activity by inducing cell cycle arrest and induction of cyclin-dependent kinase inhibitor 1A (p21 waf1/cip1) via KLF4 transcription factor mediation [17
The role of KLF4 in breast cancer is ambiguous [18
]. Downregulation of KLF4 in several cancers, such as colon, gastric, esophageal, prostate and lung may contribute to cellular hyperproliferation and malignant transformation, which is consistent with its role in cell cycle arrest and apoptosis [19
]. KLF4 is thought to act both as a tumor suppressor and as an oncogene in breast cancer. However, KLF4 is highly expressed in more than 70% of breast cancers and functions as an oncogene [21
]. It is also highly expressed in cancer stem cells [22
]. Previous studies have shown that KLF4 is essential for promoting migration and invasion, resulting in tumor formation in vivo
]. It is believed to promote tumor initiation through nuclear reprogramming [24
]. Moreover, KLF4 has been shown to act in concert with HDAC2 and HDAC3. Another study has shown that KLF4 recruits epigenetic modifiers HDAC2 and HDAC3 at the VEGF
]. KLF4 also activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in embryonic stem cells [26
]. Since we found an optimum dosage using combinatorial GEN and SFN for decreasing cellular viability, we investigated the effect of this combination on KLF4 at the transcriptional as well as protein level. Recent studies have shown that the anticancer properties of benzyl isothiocyanate, a chemopreventive constituent derived from cruciferous vegetables, were enhanced by KLF4 knockdown in breast cancer cells [27
]. We hypothesized that the combination of GEN and SFN causes breast cancer inhibition in vitro
and in vivo
via inhibition of KLF4 post-translationally as well as HDAC2 and HDAC3, which acts in conjunction with KLF4. Additionally, we sought to test whether the combination of GEN and SFN will also be effective in inhibiting hTERT, which is activated by recruitment of KLF4 to its promoter region. Moreover, we sought to evaluate whether a combinatorial GEN and SFN-enriched broccoli sprout diet could be effective in increasing tumor latency in spontaneous mouse models of mammary cancer and be more efficient in reducing tumor weight and volume as compared to mice groups administered a single compound or a control diet.
The side effects of toxic drugs and chemotherapy have led to a growing interest in compounds that provide non-toxic and safer therapy. The use of dietary compounds as chemotherapeutic and chemopreventive agents is becoming very popular as an additional form of therapy. A healthy diet still remains among the most effective chemoprevention methods [32
]. At the molecular level, a healthy diet consisting of bioactive nutritional compounds ensures proper epigenetic regulation of the system, which can facilitate prevention of cancer. Breast cancer is one of the leading causes of cancer death in women. The most prominent epigenetic features altered in breast cancer are DNA methylation patterns and chromatin states [33
]. Therefore, the study of bioactive dietary compounds has gained prominence because they may have the ability to modulate epigenetic aberrations in cancer and restore the normal epigenetic state of oncogenes or tumor suppressor genes [34
Breast cancer statistics have revealed that the incidence of breast cancer is much lower among Asians as compared to Caucasians and African Americans [35
]. Previous studies have attributed this variation in breast cancer incidence to differences in diet and lifestyle [36
]. The Asian diet in general is rich in soy and cruciferous vegetables and as a result may provide protection against breast cancer [37
]. Our current study focuses on assessing the impact of consumption of GEN and cruciferous vegetables in breast tumor prevention.
GEN, which is an isoflavone found in soybeans, is an anticancer agent due to its property of being a DNMT inhibitor. GEN has also shown HDAC inhibiting properties. Additionally, GEN has been known to resensitize breast cancer cells to estrogen-targeted chemotherapy by activation of estrogen receptor in ER-negative breast cancer [38
]. GEN has been known to block breast cancer cells in G2/M phase of the cell cycle by induction of p21 WAF1/CIP1 expression [39
] and increase acetylation of histones 3 and 4 and H3-lysine4 at the p21 WAF1/CIP1
and p16 INK4a
transcription start sites, mediated by induction of histone acetyltransferases (HATs) in prostate cancer cells [40
]. In renal cancer, GEN increases histone acetylation by enhancing HAT activity and causes acetylation of histones 3 and 4, dimethyl-H3K4 and trimethyl-H3K4 near the transcription start site at the BTG3
gene promoter [41
]. Our lab has recently shown that GEN alters the gut microbiome of breast cancer patients possibly via epigenetic modulation brought about by metabolites produced by bacteria residing in the gut [42
]. SFN, which is abundant in cruciferous vegetables such as broccoli or kale, is an HDAC inhibitor and is known to be an effective inhibitor of several class I and II HDAC inhibitors [43
]. It inhibits breast cancer stem cells in vitro
and in vivo
by suppressing the Wnt/ β-catenin pathway [44
]. Our earlier studies have shown that SFN can cause epigenetic repression of hTERT expression in breast cancer cells [46
]. It exhibits chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21WAF1/CIP1
). Higher levels of HDAC1 and HDAC2 expression are significantly associated with tumor differentiation [47
]. Previous studies have shown that SFN binds to the HDAC pocket and inhibits HDAC activity [48
]. In our current study, we used a combination of an HDAC inhibitor with the use of SFN along with a DNMT inhibitor with the use of GEN as this approach may have more tumor-inhibiting potential. Our rationale for using this combination is that the HDAC inhibitor will cause acetylation of histones and as a result a more open chromatin configuration and thus provide more accessibility to the DNA for the DNMT inhibitor to act upon.
Here, we report for the first time that at optimal concentrations, the combination of GEN and SFN can have synergistic effects in reducing the viability of breast cancer cells with no significant effect on the viability of non-cancerous control cells. We have used a much lower than usual concentration of GEN and SFN where they effectively decrease the viability of breast cancer cells. The study is particularly important as we are able to show that in MCF-7 ER (+) and MDA-MB-231 ER (−) breast cancer cells, the combination is much more effective than singly administered doses of these compounds both in vitro
and in vivo
studies. At a concentration of 5 µM SFN and 15 µM GEN, the cellular viability of breast cancer cells decreases synergistically. The combination is effective in promoting apoptosis much more effectively than separate doses. Cell cycle analyses reveal that the cells are arrested at G2/M phase for MDA-MB-231 cells and at G1 phase for MCF-7 cells. The molecular mechanisms reveal that at the transcriptional level, the combinatorial regimen can decrease the levels of HDACs and a similar observation can be seen at the post-translational level. Upon further assessing HDACs and HMTs, it can be observed that the activity of HDACs and HMTs is decreased with the combination therapy. However, the combination was not effective in synergistically downregulating DNMT activity. In addition to being a DNMT inhibitor, GEN is also known to possess histone modifying properties [40
]. Previous studies have shown that GEN increased acetylated histones 3, 4, and H3/K4 at the p21
transcription start sites and also increased the expression of histone acetyltransferases that function in transcriptional activation [50
]. Thus, the combination of GEN and SFN acting synergistically may be due to the histone acetyltransferase promoting activity of GEN that activates tumor suppressor genes such as p21
, whereas SFN may be acting as an HDAC inhibitor and suppressor of the activity of oncogenes. Together the combination of GEN and SFN is effective in altering epigenetic modifications in breast cancer cell lines. In our current study we observed the downregulation of kruppel like factor-KLF4 using this combination. Additionally, we have observed that in vivo
the combination was much more efficacious in reducing tumor volume and causing tumor latency in mice that that were fed the combinatorial diet as compared to the mice on a control diet or the diets consisting of the singly administered compounds.
In our study, we sought to assess the role of dietary phytochemicals in breast tumor inhibition. Therefore, our goal was to use compounds in their natural form. However, it was not possible to use soy to assess the efficacy of GEN since soy contains a mixture of compounds such as diadzein, glycetein and genistein. Therefore, it is difficult to elucidate the role of GEN in this complex mixture. Hence, we chose to use the purified compound. However, broccoli sprouts contain 50 times higher concentration of glucoraphanin, which is converted to isothiocyanate by the enzyme myrosinase [51
]. Thus, the use of broccoli sprout would be effective in assessing the role of SFN. Therefore, we used broccoli sprout instead of the purified compound. The dose of broccoli sprouts used is 13% BSp which is equivalent to 0.45% of SFN or 350 ppm. Studies in colorectal cancer have shown that dietary intake of 300 ppm SFN prevent adenoma formation in mice [53
]. Further studies have shown that in vivo
250 mg/kg GEN was effective in mammary cancer chemoprevention [54
Kruppel-like factors are transcriptional regulators that influence proliferation and several cellular functions in cancer. One family member, KLF4, can function both as a tumor suppressor and as an oncogene. KLF4 can activate and/or repress transcription depending on which transcription factors it interacts with and also on its target gene. Studies have indicated that p21 acts like a switch and modulates the activity of KLF4 [56
]. Some studies have reported that KLF4 activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in embryonic stem cells [57
]. Recent studies have indicated that poly (ADP-ribose) polymerase (PARP1) interacts with KLF4, where knockdown of PARP1 reduces hTERT expression [26
]. PARP1 recruits KLF4 to activate telomerase expression in cancer cells. GEN has been previously shown to decrease the expression of PARP1 [59
]. In our current study we observed a downregulation of KLF4
at the mRNA level for the MCF-7 cell line with the combination treatment. The combination was effective in downregulating KLF4 levels post-translationally for both MCF-7 and MDA-MB-231 cell lines. Previously, it has been reported that KLF4 expression is required to prevent epithelial-to-mesenchymal transition in MDA-MB-231 mammary cell line. The forced expression of KLF4 suppressed cell migration and invasion by restoring E-cadherin expression [60
]. Therefore, we assessed the level of E-cadherin in the treated cells and observed an upregulation of E-cadherin in the cells treated with the combinatorial regimen (Figure S1
). This may suggest that the upregulation could be independent of KLF4 downregulation. Previous studies have shown that GEN and SFN alone have increased the expression of E-cadherin through mechanisms not related to KLF4 regulation. GEN increases E-cadherin levels in vivo
by directly targeting it and regulating the Wnt signaling pathway and prevented accumulation of β-catenin in the cytoplasm [61
]. SFN has also been known to increase E-cadherin expression and inhibit prostate carcinogenesis and lung metastases in TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse models along with an increase in apoptotic bodies [62
]. Thus, the reports point towards an alternative pathway by the dietary phytochemicals to increase E-cadherin expression in cancer cells which may not necessarily involve KLF4 mediation. However, an in-depth study is warranted to study the association between KLF4 and E-cadherin expression in breast cancer cells.
The combination was also effective in downregulating HDAC2 and HDAC3 that act in concert with KLF4. Since KLF4 activates hTERT, with the downregulation of KLF4, hTERT should also be downregulated. Therefore, we assessed the levels of hTERT in breast cancer cells. As hypothesized, hTERT levels were significantly reduced using the combinatorial regimen in both of the breast cancer cell lines we tested.
The combinatorial dietary regimen of GEN and SFN was also administered to an ER (−) transgenic mammary cancer mouse model by orally feeding the mice with either GEN or SFN-enriched broccoli sprouts (BSps) diets alone or in combination. Our results showed combinatorial treatment with GEN and BSp diet led to the most effective response in both preventive and therapeutic effects on inhibition of mammary tumor in vivo
as compared to either non-combinatorial treatment or control diet. The dietary combinatorial regimen consists of relatively low concentrations of GEN and SFN that are physiologically accessible by daily consumption of soy and BSp [63
]. Thus, our novel combination dietary regimen shows an important bioavailability and translational potential toward chemoprevention and/or treatment of breast cancer. It would be interesting to design studies in the future to assess the influence of the combinatorial dietary regimen and compare their effects in ER positive, ER negative and BRCA1 mutated triple negative breast cancers.
4. Materials and Methods
: We used the ERα-positive MCF-7 and ERα-negative MDA-MB-231 breast cancer cells for this study. MCF10A human mammary epithelial cells were used as a non-cancerous control. All cell lines were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA). MCF-7 cells were derived from a 69-year-old Caucasian female from an excision of a chest wall nodule and from a pleural effusion [65
]. MDA-MB-231 cells are relatively aggressive in comparison to MCF-7 cells and have a mutated p53 status [66
]. MCF10A are non-tumorigenic cells derived from benign proliferative breast tissue and are spontaneously immortalized. They do not express estrogen receptors [67
Chemicals: Genistein (≥95% pure), C15H10O5 with a molecular weight of 270.241 g/mol was purchased from Sigma-Aldrich (St. Louis, MO, USA) and R,S-sulforaphane (≥98% pure), C6H11NOS2 with a molecular weight of 177.28 g/mol, was purchased from LKT Laboratories, Minneapolis, MN, USA. Each compound was diluted in DMSO and stored in stocks of 10 mmol/L at −20 °C.
Cell Culture and Treatment: The media to culture the MCF-7 and MDA-MB-231 cells was made using Dulbecco’s Modified Eagle’s Medium DMEM 1X (Corning cellgro-Thermo Fisher Scientific, Waltham, MA, USA), 10% by volume of fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA) and 1% by volume of 50X penicillin-streptomycin (Corning cellgro). The media for culturing MCF10A cells was made using DMEM F12 media (Corning cellgro-Thermo Fisher Scientific, Waltham, MA, USA), 5% Donor Horse Serum (Corning cellgro-Thermo Fisher Scientific, Waltham, MA, USA), 100 unit/mL penicillin streptomycin, 100 µL of 0.05 µg/mL hydrocortisone, 100 µL of 20 ng/mL EGF, 0.292 g of 2 mmol/L l-glutamine and 100 ng/mL cholera endotoxin. Incubation was done in an incubator that maintained a temperature of 37 °C and provided an environment having at 5% CO2 and 95% air. Cells were sub-cultured when they were about 90% confluent, counted using a hemocytometer and seeded into 6-well plates. After 24 h, they were treated with individual as well as combination doses of GEN and SFN for 3 days. DMSO was used as a control. Media and compounds were replaced every 24 h during the 3-day treatment.
Cell Density: For the cell density analyses, approximately 200,000 cells were plated in 6-well plates. They were incubated for 24 h and then treated with GEN and/or SFN each day at the same time for 72 h. Media was replaced each day. On day four, cells were viewed under a microscope and images were taken at 100× magnification.
MTT Assay: MTT assay was performed to assess the percentage of viability of breast cancer cells and the control cells after treatment with GEN and SFN. Tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide (MTT) was obtained from Sigma Aldrich (St. Louis, MO, USA) and diluted to 1mg/mL using PBS (Phosphate buffered saline). Cells were seeded in 96 well plates and treated with appropriate concentrations of GEN and SFN for 3 days. On the fourth day, 50 µL of MTT solution was added to each well and incubated for approximately 4 h at 37 °C. MTT solution was removed and DMSO was added and incubated for about 15 min. The color of the solution changed to shades of purple. The absorbance was measured using a spectrophotometer (BioRad; Hercules, CA, USA) at 595 nm.
RNA Isolation: RNA was isolated using the RNeasy kit from Qiagen (Valencia, CA, USA). MDA-MB-231 and MCF-7 cells were plated in 6-well plates and allowed to adhere to the plate for 24 h. The cells were then treated with individual and combination doses of GEN and SFN for 3 days. On the fifth day, the cells were collected using trypsin. After the cells detached from the plate, fresh media was added and then centrifuged at 300× g for 5 min. The pellets were used for further analysis using manufacturer’s protocol.
Quantitative Real-Time PCR
: cDNA synthesis kit (Bio-Rad) was used to reverse transcribe RNA (made using the protocol above) to cDNA. For the PCR setup, 1 µL of cDNA was used, along with 5 µL of SYBR green, 2 µL of nuclease-free water, 1 µL of reverse and forward primers specific for KLF4
. The total volume was made upto10 µL. PCR was performed using CFX Connect Real Time system (Bio-Rad) where the thermal cycling initiation (4 min at 94 °C) preceded 35 cycles of PCR (94 °C, 15 s; 60 °C, 30 s, 72 °C, 30 s). GAPDH (Glyceraldehyde 3-phospate dehydrogenase) was used as an endogenous control. Fold change was calculated using ΔΔCq method [68
]. All of the primers were purchased from Integrated DNA Technologies (Coralville, IA, USA). The primer sequences used were as follows:
GAPDH F 5′-ACC ACA GTC CAT GCC ATC AC-3′, R 5′-TCC ACC CTG TTG CTG TA-3′; KLF4 F 5′-CGA ACT CAC ACA GGC GAG AA-3′ R 5′-CGG AGC GGG CGA ATTT-3′; HDAC2 F 5′-CCG GAT CCA TGG CGT ACA GTC AAG GA-3′ R 5′-CCG CGG CCG CTC AAG GGT TGC TGA GT-3′; HDAC3 F 5′ TTG AGT TCT GCT CGC GTT ACA-3′ R 5′ CCC AGT TAA TGG CAA TAT CAC AGAT 3′
Cell Cycle Analysis: Approximate 5 × 104 cells were plated in 6-well plates. Furthermore, 2 mL of media was added to the wells and allowed to attach to the surface of the plate for 24 h. After 24 h, the cells were treated with GEN and SFN for 3 days at approximately the same time each day. On the fifth day, the cells were collected and fixed with 70% ethanol. Cells were prepared for analyses with a flow cytometer by treating them with propidium iodide (0.04 mg/mL), 0.1% TritonX-100 and RNase (100 mg/mL). Fluorescent signals were read at the UAB Comprehensive Flow Cytometry Core.
Annexin V Apoptosis Assay by Fluorescence-activated Cell Sorting (FACS): MCF-7 and MDA-MB-231 cells were seeded in 6-well plates. After 24 h, the cells were attached to the bottom of the well. The cells were then treated with the GEN and SFN for 3 days. On the fifth day, the cells were collected and prepared for apoptosis assay by using the Annexin V-conjugated Alexafluor 488 Apoptosis assay kit. Cells were treated with Alexa 488 and propidium iodide. The treated cells were then sent to UAB Comprehensive Flow Cytometry Core where they were analyzed using FACS-Caliber instrument (BD Biosciences, San Jose, CA, USA). The cells in Q1 phase were used in analysis of necrosis, whereas the ones in Q2 (early stage apoptosis) and Q4 (late stage apoptosis) phase were used for apoptotic analysis.
Western Blot Analysis: Breast cancer cells were treated with individual doses of GEN and SFN and their combination for 3 days, followed by extraction of protein by RIPA (Radioimmunoprecipitation buffer) lysis buffer. The concentration of protein was then determined by performing Bradford Assay using Bio-Rad protein assay kit (Bio-Rad; Hercules, CA, USA). 4–15% Tris-HCL gel was used for electrophoresis. The wells were loaded with equal concentration of protein mixed with loading buffer. After separation of the proteins by electrophoresis (200 V), the protein was transferred onto nitrocellulose membrane using the Trans Turbo Blot (BioRad). After successful transfer, the membrane was blocked with blocking buffer using a MilliPor SnapID. The milk buffer was made using dry milk powder (0.5%) and Tween (1%) dissolved in Tris Buffered Saline solution. Primary antibodies (Cell Signaling Technology, Danvers, MA, USA) specific to the genes of interest was incubated for 20–30 min and washed for four times using 30 mL of TBST (1X Tris-buffered saline solution with 1% Tween). Secondary antibodies were then added for 15–20 min followed by washing three-four times with TBST. Chemiluminescent substrate was added to the blot according to manufacturer’s suggestion and images were captured using an imager (BioRad Chemidoc XRX+ system).
HDAC Activity Assay: Breast cancer cells MCF-7 and MDA-MB-231 as well as control cells MCF10A were treated for 3 days with individual doses (Control, 5 µM SFN, 15 µM GEN) as well as their combination doses (5 µM SFN + 15 µM GEN). After treatment, the cells were collected and the nuclear extract was obtained using an EpiQuik Nuclear Extraction Kit from EpiGenTek (Farmingdale, NY, USA). An EpiQuik HDAC Activity/Inhibition Assay Kit obtained from EpiGentek (Catalog # P-4002) was used to colorimetrically measure HDAC activity using the protocol provided by the manufacturer.
HMT Activity Assay: Breast cancer cells MCF-7 and MDA-MB-231 as well as control cells MCF10A were treated for 3 days with single doses (Control, 5 µM SFN, 15 µM GEN) as well as their combination doses—5 µM SFN + 15 µM GEN. After treatment, the cells were collected and the nuclear extract was obtained using EpiQuik Nuclear Extraction Kit from EpiGenTek (Farmingdale, NY, USA). EpiQuik Histone Methyltransferase Activity/Inhibition Assay Kit (H3-K27) obtained from EpiGentek (Catalog # P-3005) was used to colorimetrically measure HMT activity using the protocol provided by the manufacturer.
Statistics and Data Analysis: Any conclusion in the study was derived from three independent experimental replicates. Data were represented as mean + SD. Statistics for the study were performed using Microsoft Excel. Significance was determined using a Student’s t-test in Microsoft Excel. p < 0.05 (indicated by *) is considered statistically significant. All comparisons were done with the DMSO control. For evaluating the nature of interaction between GEN and SFN, CompuSyn software was used. The dose of each compound was entered individually since they are non-constant ratio combos. The CompuSyn software gives combination index (CI) values. CI < 1 indicates synergism between the compounds, CI = 1 indicates additive interaction between the compounds and CI > 1 indicates antagonism between the compounds. The interaction between the dietary phytochemicals is represented in the form of a graph called isobologram. CI index analyses are similar to isobologram analysis and provide a quantitative assessment of the interaction between drugs. The isobologram equation is a special case of the combination index equation. The isobologram assesses the interaction between the compounds at median effect concentration.
Ethics: The in vivo experiments using mouse models were performed by strictly adhering to the rules and guidelines of UAB’s Institutional Animal Care and Use Committee (IACUC-131009970 21-Oct-2013).
Animal Experimental Design: The C3(1)-SV40 Tag transgenic mouse model (SV40) was used to study the efficacy of the combination treatment of GEN and SFN. The SV40 mouse model can spontaneously develop hormone-independent invasive mammary cancer (ERα-negative breast cancer) at an early age of around 20 weeks. The SV40 mice at 4 weeks of age were randomly divided into different experimental groups (9–12 mice/group) and diets were administered from 4 weeks of age and continued throughout the study.
The experimental groups were as follows: Group (1). Control group: Mice were fed with modified corn oil-based AIN-93G diet; Group (2). GEN group: Mice were fed with 250 mg/Kg GEN diet; Group (3). BSp group: mice were fed with 13% BSp diet (w/w); (4). GEN + BSp group: Mice were administered the combined GEN and BSp diet with concentrations as described above. All diets were prepared using a modified corn oil-based AIN-93G diet as a base by a commercially available company (TestDiet, St. Louis, MO, USA).
Tumor volumes and body weight were measured weekly. Tumor volumes were calculated using the formula: tumor volume (cm3
) = (length × width2
) × 0.523 [69
]. The experiment was terminated when the mean of tumor diameter in the control mice exceeded 1.0 cm. At the end of the experiment, the primary mammary tumors were excised, weighed and appropriately stored in liquid nitrogen for further analysis.