α-Tocopheryl Succinate Induces ErbB2-expressing Breast Cancer Cell Apoptosis Via The NfκB Pathway

Objectives: Vitamin E (VE) analogs, a novel class of VE compounds withstrong pro-apoptotic activity and epitomized by α-tocopheryl succinate(α-TOS), have been shown to trigger apoptosis in a variety of cancer cells, andhighly selective for malignant cells, but not toxic to normal cells, making it apromising cancer prevention and treatment. Breast cancer is one of the mostcommon malignancies of women, the incidence rate of31%of women intumors,10%-34%of erbB2expression in breast cancer. Conventionaltreatment methods include surgery, chemotherapy and endocrine therapy forbreast cancer. The erbB2positive breast cancer, a higher degree of malignancy,and is not sensitive to many chemotherapeutic drugs. The major complicationassociated with erbB2expression is linked to auto-activation of downstreamsignaling pathway(s) such as phosphatidylinositol3-kinase (PI3K), whichleads to the activation of Akt, a serine/threonine kinase that promotes cellularsurvival. Akt causes activation of the transcription factor nuclear factor-κB(NFκB), so that the tumor cells resistant to apoptosis. Therefore, agents thatinhibit NFκB activation in erbB2-expressing cancer cells are of clinicalinterest. Recently, we found that α-TOS induced apoptosis in breast cancercells expressing erbB2. In this study, we use human breast cancer cells such asMCF-7and MDA-MB-453cells as modle to study the molecular mechanismsby which α-TOS induces apoptosis in erbB2-positive breast cancer cells. Wedetermined whether the NFκB pathway plays critical role in α-TOS inducingerbB2-positive breast cancer cell apoptosis.Methods:1Cell culture and treatment Human breast cancer cell linesMCF7and MDA-MB-453were routinely cultured in DMEM with10%FCS,100U/ml penicillin and100μg/ml streptomycin at37℃in a humidifiedatmosphere of5%CO2. At approximately50-70%confluence, cells were treated with up to100μmol/L α-TOS. After exposure to the drugs for therequired period of time, cells were harvested and assessed using differentmethods.2MTT assay MDA-MB-453cells and MCF-7Cells were plated in96-well culture plates, allowed to attach overnight, and treated with differentconcentrations of α-TOS for24h and48h. Each dose established six holes.Following exposure of cells to the agents,5mg/ml MTT was added, and afterincubation for4hours at37℃, the medium was removed and combined with200μl DMSO. Absorbance was read at490nm using an ELISA plate reader.SPSS software was used for data analysis to calculate the half maximalinhibitory concentration (IC50) value.3Hoechst staining Cells were placed on glass slides in6-well plates,after treatment, cells were fixed with fixer for10min and stained with Hoechst33258for5min and then observed under fluorescence microscopy.4Western blotting Whole cell lysates were obtained using a kit.Nuclear and cytoplasmic fractions were obtained using the Nuclear andCytoplasmic Protein Extraction Kit. Protein samples were denatured, resolvedusing10%SDS-polyacrylamide gels and transferred onto nitrocellulosemembranes. The membrane was blocked and incubated with primaryantibodies then incubated further with horseradish peroxidase-conjugatedsecondary IgG. Anti-β-actin or Anti-histone1was used as a control for proteinloading.5RT-PCR Total RNA was extracted from cells using Trizol Reagent.Reverse transcription was carried out using AMV reverse transcriptase andoligo (dT)18primer. One to three microliters of the cDNA was used foramplification and PCR was run for30cycles. The specific oligonucleotideprimer pairs for certain genes and the expected size of PCR products are asfollows: c-IAP1FW,5′-GAA TAC TCC CTG TGA TTA ATG GTG CCGTGG-3′and RV,5′-TCT CTT GCT TGT AAA GAC GTC TGT GTC TTC-3′,230bp; FLIP FW,5′-CAT ACT GAG ATG CAA GAA TT-3′and RV,5′-GCTGAA GTC ATC CAT GAG GT-3′,875bp; β-actin FW,5′-TGA CGG GGT CAC CCA CAC TGT GCC-3′and RV,5′-CTA GAA GCA TTT GCG GTGGAC GAT GGA GGG-3′,662bp. The conditions used for PCR amplificationwere as follows:94oC for30sec,65oC for30sec, and72oC for1min,30cycles in a25μl reaction.6Caspase3and caspase8activity assay The activity of caspase3andcaspase8was measured by cleavage of the chromogenic caspase substrates,Ac-DEVD-pNA (acetyl-Asp-Glu-Val-Asp p-nitroanilide) and Ac-IETD-pNA(acetyl-Ile-Glu-Thr-Asp p-nitroanilid), respectively Approximately30μg oftotal protein was added to reaction buffer containing Ac-DEVD-pNA (2mM)or Ac-IETD-pNA (2mM) and incubated for2h at37℃.The absorbance ofyellow pNA cleaved from its corresponding precursor was measured using aspectrometer at405nm. The specific caspase activity was normalized for totalprotein of the cell lysates and then expressed as the fold increase of thebaseline caspase activity of control cells cultured in DMEM with10%FBS.7Immunocytochemistry Cells were placed on sterile coverslips in12-well plates overnight and fixed with100%methanol for3min,1%H2O2-methanol for5min,95%methanol for3min and50%methanol for3min. The cells were then exposed to anti-p65IgG, followed by thebiotinylated secondary antibody. After washing with PBS, the cells wereincubated with streptavidin-HRP. Finally, protein expression was detectedusing DAB solution and observed by microscopy.8Statistics Data were obtained from at least three different experimentsand the results are presented as X±SD. Significance between data points wasassessed using the Student’s t-test and data were considered significantlydifferent when p<0.05.Results:1α-TOS inhibited MCF7cells and MDA-MB-453cells growthThe concentration of α-TOS is as the abscissa, inhibition rates are as thevertical coordinate. Inhibition rates of cell groeth show that α-TOS of variousconcentrations can inhibit MCF-7and MDA-MB-453cells growth and canresult in dose-and time-dependent induction of proliferation inhibition. TheIC50values were calculated using SPSS software. IC50values of α-TOS on MCF-7cells were:111.72μg/ml (24h),44.29μg/ml (48h); the IC50values ofα-TOS on the MDA-MB-453cells were:113.23μg/ml (24h),47.20μg/ml(48h).2α-TOS induced apoptosis of MCF-7cells and MDA-MB-453cellsMCF-7and MDA-MB-453cells were treated with α-TOS for48h and thenstained using Hoechst. The chromatin condensation was examined in cellstreated with α-TOS. In contrast, no apoptotic cells were observed in control.3α-TOS inhibited NFκB nuclear translocation in breast cancer cellsexpressing erbB23.1Western blotting We assessed the level of erbB2andphosphorylated Akt (p-Akt) in MDA-MB-453and MCF7cell lines anddetermined that both cell lines express erbB2and pAkt and similar levels ofAkt. The western blotting results showed that p65protein decreased in thenuclear fraction and increased in the cytoplasmic fraction after treatment withα-TOS in both cell lines.3.2Immunocytochemical staining of the p65protein (a subunit of NFκB)showed much weaker nuclear staining in MDA-MB-453and MCF7(data notshown) cells after treatment with100μmol/L α-TOS than in cells withoutα-TOS treatment.4α-TOS inhibited Akt activation we examined the amounts of Akt andp-Akt in MDA-MB-453and MCF7cell lines after α-TOS treatment withdifferent time.The western blotting results showed that α-TOS treatmentdecreased the level of p-Akt in MCF7and MDA-MB-453cells. The level oftotal Akt was not altered in either cell lines after treatment with α-TOS.5α-TOS suppressed c-IAP1and FLIP expression in erbB2-positivebreast cancer cells5.1The effects of α-TOS on c-IAP1and FLIP protein expression inerbB2-positive breast cancer cells Result shows that α-TOS reduced c-IAP1protein expression in both cell lines. Result shows that α-TOS reduced FLIPprotein expression in both cell lines.5.2The effects of α-TOS on c-IAP1and FLIP RNA expression in erbB2-positive breast cancer cells shows that α-TOS reduced the amount ofc-IAP1RNA in MDA-MB-453and MCF7cells in a dose-and time-dependentmanner. A decrease in the amount of FLIP RNA was also found inMDA-MB-453and MCF7cells after treatment with α-TOS.6α-TOS augmented the activity of caspases We found that MCF7cellsdo not express caspase3, shows that α-TOS induced an increase in caspase8activity in MCF7cells.Results show that α-TOS increased the activity ofcaspase3and caspase8in MDA-MB-453cells.Conclusions:1α-TOS inhibited MCF7cells and MDA-MB-453cellsgrowth.2α-TOS induces erbB2-expressing breast cancer cell apoptosis3α-TOS induces erbB2-expressing breast cancer cell apoptosis via theNFκB pathway...