In total, for survival selleck chemical Veliparib analyses we studied 16 distinct types of cancer. Details of each dataset, the number of samples with clinical details, the expression platform, and associated Pubmed IDs for the GEO datasets are in Additional file 13 Table S3. Gene expression microarray analysis Generation of Gene Expression Microarrays was previously described and data were deposited in Inhibitors,Modulators,Libraries ArrayExpress database. Gene Set Enrichment Analysis measures the enrich ment of a gene set within a GEM experiment. The enrich ment score is a metric Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries of the skew of a gene set within the rank of genes sorted by their GEM expression difference. The significance of enrichment is the proportion of true ES 1000 ES generated from random gene sets. Leading edge genes are the subset that contributes most to the ES.

Statistical analysis For Inhibitors,Modulators,Libraries survival analysis we used the R survival package. To survey for potential association between gene expression and survival we categorized samples as below or above median expression for each gene and then calculated the log rank P value comparison between the groups. For KIRC, SKCM and PRAD GSE21034 datasets with significant NFE2L2 log rank tests we also calculated the hazard ratio using the Cox proportional hazard model. Elsewhere data were analyzed using Students t test, Spearmans rho or log rank test as appropriate for the analysis. Values are given as mean SD. All statistical tests were two sided, and results were considered statistically sig nificant when P 0. 05. Background Prostate cancer is the most common non cutaneous malignancy in men in the Western world.

The etiology of prostate cancer is not well defined. however, the inhibition of various tumor suppressor genes and con comitant activation of oncogenes is a frequent occur rence in most cancers, including prostate cancer. DNA hypermethylation of promoters at CpG sequences often sterically hinders the binding of transcription factors, thereby represses gene Inhibitors,Modulators,Libraries transcription. The transfer of a methyl group to DNA at the fifth carbon position of cytosine residues by the DNA methyltransferase family of enzymes is one of the most common events for the establishment of epigenetic program. Three active DNMTs have been identified so far in mammalian cells. DNMT1, DNMT3A, and DNMT3B. DNMT1 is the most abundant and methylates hemimethylated CpG di nucleotides in the mammalian genome during DNA replication in a number of different cancer types.

DNMT3A and DNMT3B are methyltransferases involved in de novo methylation of DNA following replication. Numerous reports things have demonstrated the overexpres sion of DNMT1 in lung, hepatocellular, acute and chronic myelogenous leukemia, colorectal, gastric, breast and prostate cancers. Gravina and associates have shown that the hormone resistant prostate cancer phenotype is associated with an increase in DNMT expression and activity.

The total Ag content was determined using AAS according to the above mentioned procedure. The results were normalized according to the cell number and expressed as % of the controls. Results are presented as mean standard deviation of 2 replicates. Cell viability Lactate dehydrogenase www.selleckchem.com/products/AG-014699.html assay The LDH assay is used to evaluate the degree of cellular membrane damage associated with leakage of the cyto solic LDH enzyme. The Cytotox Non Radioactive Cytotoxicity Assay Kit was used in a 96 well plate format. The cells were exposed to the AgNP dis persions at particle doses ranging from 5 to 100 ugmL in 100 uL for 4 and 24 h. After exposure, 50 uL of the supernatant was transferred to a new 96 well plate. The rest of the supernatant was discarded and the cells were lysed with 100 uL Triton 1% for 30 min at 37 C.

50 uL of the lysate was transferred to a new 96 well plate and 50 uL of reconstituted substrate was added to both the supernatant and the cell Inhibitors,Modulators,Libraries lysate plates. After 20 min incu bation at dark conditions, reactions in both plates were terminated using 50 uL stop solution. Absorbance was measured at 495 nm using a plate reader. The absorbance of the supernatant corresponds to the LDH release, whereas the sum of the absorbance of the supernatant and cell lysate corresponds to the maximum LDH release. The cell viability was calculated by dividing the LDH release to the maximum LDH re lease for each well. The control was Inhibitors,Modulators,Libraries set to 100% viability and the results were expressed as percentage cell viabil ity. The experiments were performed at least three times in triplicate wells for each time point and AgNP dose.

The interference of AgNPs with the LDH assay was tested in an acellular system, as well as by incubating cell lysates with AgNPs before performing the assay. The acellular interference was performed by Inhibitors,Modulators,Libraries incubating different concentrations of particles with reconstituted LDH substrate. The interference was found to be non significant. The interference of the AgNPs with the LDH assay in terms of possible enzyme inhibition was investi gated by incubating cell lysates with AgNPs for 0, 4 and 24 h before performing the LDH Inhibitors,Modulators,Libraries assay. Alamar Blue assay The AB assay is used to assess cell viability based on the reduction potential of metabolically active cells. BEAS 2B cells were seeded in transparent 96 well plates and ex posed to the AgNP dispersions at concentrations ran ging from 5 to 100 ugmL for 4 and 24 h.

After exposure, 10 uL of AlamarBlue reagent was added in each well Inhibitors,Modulators,Libraries and incubated for 2 h at 37 C. The fluorescence was measured at 560 nm excitation and 590 nm emission wavelengths using a plate reader. Results were expressed as percent http://www.selleckchem.com/products/BAY-73-4506.html age cell viability versus the control. The experiments were performed at least three times in triplicate wells for each time point and AgNP dose.

The re sult of this experiment showed that the attachment of chondrocytes to fibronectin coated plates was obviously increased between 2 and 7 days after plating. Next, to determine the significance of 5B1 integrin in cell attachment, 7 day cultured chondrocytes, once harvested, were incubated with a function blocking anti selleckchem Bosutinib 5B1 integrin antibody or control IgG for 90 mi nutes at room temperature, and were then plated onto fibronectin coated plates. This experiment confirmed that the attachment of chondrocytes to fibronectin coated plates was primarily mediated by 5B1 integrin. Since the level of expression of 5BB1 integ rin changed little within that culture period, this result was considered to indicate an increase in the activity of 5B1 integrin.

Given this result, we next examined whether RRAS is indeed involved in the observed increase in integrin ac tivity. In the experiment, chondrocytes cultured in monolayers for Inhibitors,Modulators,Libraries 7 days were infected with the adenovi ruses carrying CA or DN mutants of five small GTPases, and the attachment of the cells to fibronectin coated plates Inhibitors,Modulators,Libraries was evaluated 3 days later. Inhibitors,Modulators,Libraries These five small GTPases are known to be involved in the regulation of integrin activity in certain types of cells. In this experiment, cell attachment was significantly increased by the overexpression of a CA mutant of RRAS, and tended to be reduced by that of a DN mu tant. Such a change in cell at tachment was not observed with any other small GTPases.

Induction of type I and type III procollagen expression and AKT phosphorylation was indeed regulated by RRAS in monolayer cultured chondrocytes The following experiments were performed to confirm the involvement of RRAS in the induction Inhibitors,Modulators,Libraries of type I and type III procollagen expression and AKT phosphory lation. If our above presumption is correct, phosphory lation of AKT should be modulated by RRAS through the change in the activity of 5B1 integrin. To examine this hypothesis, CA RRAS or DN Inhibitors,Modulators,Libraries RRAS was over expressed in monolayer cultured chondrocytes by means of adenoviral transduction, and phosphorylation of AKT was evaluated. As anticipated, the phosphorylation was enhanced by the overexpression of CA RRAS, and tended to be reduced by that of DN RRAS. Consistently, in those chondrocytes, the expression of type I and type III procollagen was significantly ele vated by the overexpression of CA RRAS. For further confirmation, we suppressed the expression of RRAS by RNAi and observed whether any changes occurred in AKT phosphorylation and noncartilaginous procollagen expression. In this experiment, AKT phos phorylation and procollagen expression were reduced, selleck bio as predicted, by the suppression of RRAS expression.

Sulforhodamine B assay One of the melanoma cell lines in the current study was found not to metabolise the MTS reagent. Therefore, we performed an SRB assay to calculate an IC50 to E6201 for this line. The SRB assay was per formed as previously described. Briefly, after drug treatment cells were then fixed with 25 uL of cold trichloroacetic acid for 60 selleck chem Veliparib minutes. Cells were subsequently washed five times with H2O and air dried. Next, cells were stained with 50 uL of 0. 04% SRB in 1% acetic acid and incubated at room temperature for 30 minutes. Unbound SRB was removed by washing five times with 1% acetic acid and air dried. Finally, bound SRB stain was solubilized in 100 uL of 10 mM Tris buf fer before taking an optical density measurement at 570 nm using the BioTek microplate reader.

PI3K and MAPK pathway activation Cell lines in the panel were plated at a density of 500,000 cells per well on day 0 in a 6 well plate. On day 1, cells were washed twice with PBS, serum starved in DMEM containing 0. 2% FBS and protein lysates were collected 16 hours after serum starvation. 50 ug of total protein were analysed Inhibitors,Modulators,Libraries on a 3 8% SDS PAGE. Phosphory lated Akt and phosphorylated ERK1/2 proteins were probed for with phospho specific antibodies from Cell Signaling Technology. Immunoblots were then stripped and re probed for total Akt and ERK1/2. The ratio of phosphorylated Akt or ERK1/2 to total Akt or ERK1/2 respectively was calculated by densitometry using Image J software and scored as follows negative 0 15%. 15 50%, 50 100%.100% of phosphorylated protein relative to total protein levels.

On additional Western blots, PTEN and GAPDH proteins were probed for with antibodies from Cell Signaling Technology and Abcam respectively. Cell cycle Inhibitors,Modulators,Libraries analysis Cells were plated in triplicate in 100 mm2 plates. The next day, cells were treated with 200 nM E6201 or 0. 01% DMSO. After 48 hours of treatment, cells were fixed in 80% ethanol for 2 hours, washed with ice cold PBS, and then resuspended in 500 uL cell cycle staining buffer. DNA content was evaluated by flow cytometry as an indicator of Inhibitors,Modulators,Libraries cell cycle progression. Cell cycle ana lysis was performed using ModFit software. The percentage of G1 arrest was calculated as the percent increase in cells in G1 relative to the percent of cells in G1 in DMSO con trol samples as follows x 100.

Cell death analysis by Annexin V staining Annexin V FITC staining was Inhibitors,Modulators,Libraries used to measure phospha tidylserine exposure on cells undergoing apoptosis according to the manufacturers instructions. Inhibitors,Modulators,Libraries http://www.selleckchem.com/products/U0126.html 2. 5 105 cells were plated per well in a 6 well plate. Cells were treated with 200 nM E6201 or 0. 01% DMSO 24 hours after plating. After 72 hours, floating and attached cells were collected and resuspended in Annexin binding buffer. Following the addition of 500 ng/mL Annexin V FITC and 1 ug/mL propidium iodide, cells were analysed for Annexin positive cells using a CyAn ADP flow cytometer and Summit software, version 4. 3.

Slides were Ruxolitinib mechanism washed in PBS and water, allowed to dry, and coverslipped with Aqua Poly/Mount. Slides were then stored at 4 C until fluorescent images were acquired using an Olympus BX50 Light Microscope with attached mercury epi fluorescence illumination. Affymetrix gene profiling Microarrays were performed on MM cell samples from 3 independent experiments as described previously. Each of the samples was analyzed on a separate array, i. e, N 3 arrays per MM line. A Human U133A 2. 0 array was scanned twice, the images overlaid, Inhibitors,Modulators,Libraries and the aver age intensities of each probe cell compiled. Microarray data were analyzed using GeneSifter software. This program used a t test for pairwise comparison and a Benjamini Hochberg test for false discovery rate to adjust for multiple comparisons.

A 2 fold cut off limit was used to assess statistical significance. Quantitative real time PCR To validate microarray profiles and PCR Array profiles of genes, qRT PCR was performed as described previously. Triplicate assays were per formed with RNA samples isolated from Inhibitors,Modulators,Libraries at least 3 inde pendent experiments. Fold changes in gene expression were calculated using the delta delta Ct method using hypoxanthine phosphoribosyl transferase as the normalization control. The Assay on Demand pri mers and Inhibitors,Modulators,Libraries probes used were purchased from Applied Biosystems. Assessment of Dox effects on human tumors appearing after injection of stably transfected shERK1, shERK2 and shControl MM lines in a mouse xenograft model HMESO cells stably transfected with either shERK1, shERK2 or shControl were injected into 4 subcutaneous sites on the dorsa of 6 wk old Fox Chase Severe Combined Immunodeficient mice.

All mice were weighed weekly Inhibitors,Modulators,Libraries and exam ined every other day for morbidity and tumor growth. Immediately after tumor appearance each group was divided in two subgroups, each containing Inhibitors,Modulators,Libraries 3 mice. Three mice in each group were given Dox 3X weekly. The remaining 3 mice from each group received saline 3X weekly. The Dox dose and frequency were previously determined to cause no toxicity to mice. After 6 wk of treatment, all mice were weighed and euthanized by intraperitoneal injection of sodium pentobarbital, necropsied to determine possible gross metastases, and major organs removed and stored in 4% paraformalde hyde before processing for histopathology.

Tumors were characterized using previously described histochemical criteria and karyotyped to prove that they were human in origin. Tumor volumes were calculated using formula /6. All experiments using mice were approved by the Institu tional Animal Care and Use Committee at the Univer sity of Vermont College of Medicine. customer review Statistical analyses In all in vitro assays, at least 3 independent samples were examined at each time point per group in dupli cate or triplicate experiments.

Similarly, there was no difference selleck Imatinib in the cell cycle distribution of cells transfected with scrambled or Sin3A siRNA and treated with estrogen. As expected, estrogen treatment resulted in an increase in the percentage of cells in the S/G2/M phases and a subsequent decrease in G0/G1 phases. Similar results were found with the 96 hour samples, shown in the right panel of Figure 3A, indicating that Sin3A is not involved in cell cycle progression of MCF7 cells. Knockdown of Sin3A under these conditions was veri fied and is shown in Figure 4B. The samples described above were analyzed simulta neously for Annexin V staining to assess the level of cellular apoptosis. At the 72 hour time point, there was no difference in apoptosis Inhibitors,Modulators,Libraries of scrambled and Sin3A siRNA transfected cells treated with vehicle ethanol.

Estrogen treatment in control transfected cells decreased the level of apoptosis compared to ethanol. How ever, this level of apoptosis was significantly increased Inhibitors,Modulators,Libraries with loss of Sin3A. By 96 hours, there was a significant increase in apoptosis in Sin3A siRNA knockdown cells both in the presence and absence of estrogen. This identifies Sin3A as a prosurvival factor in MCF7 cells that pro tects against apoptosis. Sin3A is required for maximum growth of ERa positive Inhibitors,Modulators,Libraries breast cancer cells Cell growth assays were performed to determine if the observed increase in apoptosis by Sin3A knockdown was sufficient to attenuate growth of breast cancer cells. Since the increase in apoptosis at 96 hours occurred both in the presence and absence of estrogen, the growth of ERa negative as well as ERa positive cell lines was analyzed.

Cells were transfected Inhibitors,Modulators,Libraries with either the negative control scrambled or Sin3A siRNA, treated with vehicle ethanol or estrogen, and the number of live cells was counted every 24 hours by trypan blue exclusion. MCF7 cells transfected with the scrambled negative control grew steadily in the presence of estrogen, but no cell growth was observed in the scrambled ethanol Inhibitors,Modulators,Libraries group, consistent with the fact that this is an estrogen dependent cell line. Notably, a significant decrease in estrogen induced growth of MCF7 cells was exhibited by those transfected with Sin3A siRNA. MCF7 cells transfected with the Sin3A siRNA but treated with ethanol also tended to have lower cell numbers than the corresponding scrambled control, but data were not statis tically significant.

T47D cells, another ERa positive cell line, also exhibited significant attenuation of estrogen induced growth in the presence of Sin3A siRNA. The growth defect was not as dramatic as that observed in MCF7 cells. It is of note that knockdown of Sin3A protein, DAPT secretase FDA shown by Western blot analysis in Figure 4D, was also less efficient in the T47D cells. In contrast to the ERa positive cell lines, ERa negative MDA MB 231 and Hs578T cells grew at the same rate in the presence and absence of Sin3A siRNA or estrogen.

During the past decade,various Rapamycin buy strategies have been used to deliver selleckbio therapeutic drugs selectively to brain tumors and injured brain,including,biodegradable polymers implanted into the tumor cavity,convection enhanced delivery,and BBB BTB disruption. Our labora tory has focused on pharmacologic modulations to increase BTB permeability and increase delivery of thera peutic drugs selectively to brain tumors with little or no drug delivery to normal brain tissue. This strategy exploits the function of certain vasomodulators that play a key role in modulation of BBB BTB permeability. It has been Inhibitors,Modulators,Libraries demonstrated that bradykinin,leukotriene,nitric oxide,c GMP,and potassium channel agonists can selectively increase capillary permeability in primary brain tumors,while leaving normal brain unaffected.

These findings Inhibitors,Modulators,Libraries have already been translated into clinical studies to increase drug delivery selectively to tumor tissue in brain tumor patients. Modulation of critical mole cules involved in Inhibitors,Modulators,Libraries selectively increasing BTB permeability could lead to the development Inhibitors,Modulators,Libraries of effective strategy to increase chemotherapy delivery to brain tumors. Large conductance calcium activated potassium channels are a unique class of ion channel coupling intra cellular chemical and electrical signaling. These channels give rise to outwardly rectifying potassium currents and respond not only to changes in membrane voltage,but also to changes in intracellular calcium. Recent studies suggest that KCa channel expression levels correlate posi tively with the malignancy grade of glioma.

KCa chan Inhibitors,Modulators,Libraries nels are also present in cerebral blood vessels,where they regulate cerebral blood vessel tone and,probably,BBB BTB permeability. Evidence from several studies further indicate that KCa channels play an impor tant role in vasodilation when it is mediated by bradyki nin,NO donors,and cyclic GMP. In response to the binding of bradykinin to its type 2 recep tors,intracellular Inhibitors,Modulators,Libraries Ca2 is increased either by mobi lization of Ca2 from internal sites and influx or by NO production from NO synthase activation. The increase in intracellular Ca2 level activates KCa channels and alters the Inhibitors,Modulators,Libraries membrane potential of cells.

Further more,previous Inhibitors,Modulators,Libraries studies have also shown that bradykinin induced KCa channel activation Inhibitors,Modulators,Libraries in endothelial cells is potentiated more by NS1619,a selective KCa channel agonist,and attenuated by a highly selective inhibitor,iberi otoxin.

We previously demonstrated that KCa channels are overexpressed in primary brain tumors and tumor microvessels,and Inhibitors,Modulators,Libraries such channels respond to NS1619,which selectively increases BTB permeability. The accelerated formation of pinocytotic vesicles appears to be the cellular mechanism by which KCa channels medi ate increases in BTB permeability. Moreover,in a rat brain selleck compound tumor model,we showed that the B2R expression level on brain tumors directly correlates with bradykinin induced BTB permeability increases.

Both inhibitors were found to be less effective at both inhibiting the Inhibitors,Modulators,Libraries growth of pancreatic cancer cell selleck chem Y-27632 lines compared to IGF IR inhibitor NVP AEW541, now with IC50s ranging from 2. 3 uM to 13. 7 uM for MAPKK in hibitor and 5. 5 uM to Inhibitors,Modulators,Libraries 11. 3 uM for the PI3K inhibitor. Interestingly, the most resistant cell lines to PI3K inhibition were also found to be resistant to anti MAPKK treatment. Cell cycle distribution Inhibitors,Modulators,Libraries analyses We used flow cytometry in order to determine the effect of NVP AEW541 on the cell cycle distri bution of the pancreatic cancer cell lines. We have reported recently that treatment with gemcitabine increased the percentage of cells in the sub G1 and S phase while afatinib increased the proportion of cells in the sub G1 and this was accompanied by a decrease in the population of cells in G0/G1.

Similarly, an increase in the sub G1 fraction, indicative of apoptosis, was observed in the majority of cell lines following NVP AEW541 treat ment and this was statistically significant in FA6, AsPC 1, PT45 and Capan 1 cells. An increase in the percentage Inhibitors,Modulators,Libraries of cells Inhibitors,Modulators,Libraries in G0/G1 phase was demonstrated only in five out of the seven cell lines and this increase Inhibitors,Modulators,Libraries was statistically significant in BxPc3 and PANC1. Effect of HER and IGF IR ligands in the presence or absence of inhibitors on downstream cell signaling molecules First we determined the effect of EGF and IGF I on the phosphorylation of AKT and MAPK in all pancreatic cancer cell lines included in this study and in all cell lines, with the exception of FA6 cells, EGF primarily induced to the activation of MAPK while it had low or Inhibitors,Modulators,Libraries no effect on AKT phosphorylation.

In contrast, Inhibitors,Modulators,Libraries IGF I was more potent in inducing the activation of AKT, while having no or minimal effect on MAPK Inhibitors,Modulators,Libraries phosphor ylation. Next, Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries we examined the effect of EGF, IGF I, IGF II, in sulin and NRG1 on the activation of downstream signaling pathways in BxPc3 cell line in the presence Inhibitors,Modulators,Libraries or absence of afatinib, Inhibitors,Modulators,Libraries NVP AEW541 or mAb ICR62. BxPc3 cell line was selected as the most appropriate model for investigating cell signaling events Inhibitors,Modulators,Libraries since the combination of afatinib with NVP AEW541 exhibited the highest synergistic effect in these cells.

In addition, BxPc3 cell line was positive for all HER family members and IGF IR with the exception of HER 4.

Of the HER ligands, EGF induced phophorylation of EGFR Inhibitors,Modulators,Libraries and MAPK while NRG1 induced first selleck chemical Trichostatin A phosphorylation of HER 3 and both of MAPK and AKT in BxPC 3 cells and these effects were blocked completely by afatinib. In addition, treatment with IGF IR ligands increased the level of p IGF IR and pAKT but not pMAPK. At 400 nM NVP AEW541 inhibited the IGF IR ligands induced phosphorylation of both Sorafenib Tosylate mw IGF IR and AKT but not completely.

Cell counting assays MDA MB 468, MDA MB 468shAhR, MCF7, MDA MB 231, Cal51, and Cal51shAhR selleck were seeded at 20,000 cells/ well and 15,000 cells/well, each in triplicate 12 well tissue culture plates in DMEM 10% FBS at 37 C and 5% CO2. AhR knockdown cells were pretreated with vehicle or Inhibitors,Modulators,Libraries 750 ng/mL Dox for seven days prior to seeding in 12 well tissue culture Inhibitors,Modulators,Libraries plates to achieve knockdown of AhR. During AF treatment, vehicle/Dox treatments were continued. All cell lines tested were treated with AF for seven days prior to analysis. Approxi mate GI50 value, which is the concentration of compound that inhibits cell growth by 50% compared to control, was calculated using GraphPad Prism Software and a three parameter log versus inhibition nonlinear regression. GI50 values are expressed as the 95% confidence interval.

Gene expression analysis MDA MB 468, MDA MB 468shAhR, Cal51, and Cal51 shAhR cells were cultured in phenol red free DMEM 10% charcoal Inhibitors,Modulators,Libraries stripped FBS at 37 C and 5% CO2 for three days prior to experiment to remove residual estrogens. Triplicate 80% confluent six cm tissue culture dishes of MDA MB 468 and Cal51 were treated with 0. 1% DMSO, 1 uM AF, or 1 uM BNF for six hours. MDA MB 468shAhR and Cal51shAhR were pretreated with vehicle or 750 ng/mL Dox for seven days prior to seeding onto triplicate six cm tissue culture dishes, and then treated with 0. 1% DMSO, 1 uM AF, or 1 uM BNF for six hours in the presence or absence of 750 ng/mL Dox. Total RNA was extracted using HP Total RNA Kit according to the manufacturers proto col.

Two micrograms of RNA were reverse transcribed using Superscript II RT according to the manufacturers protocol. Fast Start Universal SYBR Green Master Mix was used to perform qPCR for CYP1A1 on a BioRad CFX 96 instrument, using RPL13A as a housekeeping Inhibitors,Modulators,Libraries gene. The primer sequences Propidum iodide staining AFs ability to alter the cell cycle in MDA MB 468shAhR and Cal51shAhR cells was analyzed using a propidium iodide staining assay according to manufacturers pro tocols. Briefly, MDA MB 468shAhR cells were seeded into six well tissue culture plates and treated with 0. 1% DMSO or Inhibitors,Modulators,Libraries 25nM AF for 4, 24, 48, 72, or 120 hours. Cal51shAhR cells were seeded into six well tissue culture plates and treated with 0. 1% DMSO or 250nM AF for 24, 48, 72, 120, or 168 hours. Triplicate samples were col lected for all controls, and duplicate samples were col lected for all treatment groups.

Cells were harvested and fixed with selleck inhibitor EtOH up to a concentration of 70%, and kept at 4 C until PI staining. Samples were then analyzed by a FACScalibur instrument for cell cycle alterations. Data was analyzed using ModFitLT 3. 2. 1. Analysis of apoptosis and DNA damage AFs ability to induce apoptosis and DNA damage in MDA MB 468 and Cal51 cells was analyzed using an Apoptosis, DNA Damage, and Cell Proliferation flow cytometry kit, according to the manufac turers protocol.