Noha et al. 2006 and Herr, Gassler et

Noha A. MohamedID: 1601262 Thesis Protocol 1.  Title:Effect of Dexamethasone on Doxorubicin-induced apoptosis of BreastCancer Cells????? ?????????????? ??? ??? ??????? ??????? ????  ????? ???????????? ?? ????? ????? ?????2.  Research Problem:Glucocorticoids(Gcs) are important for the treatment of lymphoid malignancy because they have spectacular effects on cell cycle progression andapoptosis. (Pirotte et al. 1997, Sionov et al.

2008).  In the solid tumours therapy regimens (GCs)  have remarkable effect as a co-medication eitherbecause of their efficiency  in treatingthe malignancy, or to alleviate oedema, pain, electrolyte imbalance, nausea andemesis or to reduce cytotoxic effect of the combined chemotherapeutic agents(Rutz 2002, Rutz & Herr 2004) Theglucocorticoid receptor’s (GR) transcriptional activityare various in the different tissuesleading to different effects. This results in diverse and evencontradictory effects on the response to the chemotherapeutic agent in manysolid tumors e.g breast, brain, bone, melanoma, cervix and neuroblastoma (Zhang, Beckermann et al. 2006,Moutsatsou P and Papavassiliou AG. 2008).

In spite of the  distinctive effect of the glucocorticoideither pro- or anti-apoptotic, the underlying mechanisms of glucocorticoidreceptor  signaling still poorly defined .The suggested mechanism of antiproliferative effect is inhibition of anti-apoptotic effect ofNF-{kappa}.In the other hand, the molecular mechanisms of the GC-mediated inhibition ofchemotherapy-induced apoptosis not fully identified. GR upregulated theanti-apoptotic genes SGK-1 and MKP-1.

This leads to acutelyinhibition of  mitogen-activated proteinkinase (MAPK) phosphorylation, thereby reduce the effictiviness ofchemotherapy-induced apoptosis. In addition down regulation of the genesrepresenting the pro-apoptosis function in the breast cancer cells  such as Bid and TRAIL  (Wu, Chaudhuri et al. 2004, Pang, Kocherginskyet al. 2006 and Herr, Gassler et al. 2007)  These controversial data and as dexamethasoneis used usually as conjunctive therapy in conventional breast cancer treatmentregimens that may alter  thechemosensitization effect to the chemotherapeuitic agent . Therefore, it isimportant to study the exact impact of dexamethasone on breast  cancer cells response to differentchemotherapeutic agents3.

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   ResearchObjectives:In this study our aim is to examine the possible effect ofDexamethasone treatment in enhancing the antitumor activity of Doxorubicin inbreast cancer cells, to investigate the sensitizing effect of dexamethasone forinducing apoptosis and disturbing the cell cycle phase distribution in breastcancer cells  and to overcome themultidrug resistance of Doxorubicin via dexamethasone induced apoptosis throughincreasing caspase-9 and the ratio of Bax/Bcl-2. 4.  Importanceof the Research:BreastCancer consider the most common and the fourth cause of mortality among cancertypes worldwide(Nickels et al., 2013) . The studies show that the incidence ofbreast cancer is increased significantly among Saudi patients and at an earlierage than western community(Al-Rikabi and Husain, 2012). The estimated percentfor  incidence and mortality by 2025, expected toincrease by about 350% and 160%, respectively. Doxorubicinconsider a primary chemotherapeutic agent for breast cancer with a responserate of 40-50% as a single agent and 60-80% in combination.

Its mechanism is mitochondrial-dependentapoptosis induction through upregulation of Bax/Bcl-2 ratio and caspase-9   by (Harashima et al., 1999,Sharifi, 2015). Obviously,the balance between pro- and anti-apoptosis effect of the glucocorticoid isimportant leading to cell death or survival and further studies are needed(Gruver-Yates and Cidlowski 2013).

Since Dexamethasone is in use in BCtreatment regimen several years ago, the probable interactions among thesedrugs need more studies.So it is important to estimate the direct role of GCsand the effect of Dex GC on the (Doxurobicin) in  breast cancer cell line MCF-7.5.   Literature Review:In1987, Benckhuijsan. et al.

proved that that glucorticoids enhanced theanticancer effect of alkylating agent Melphalan in the human melanoma celllines . They explained such  interactionbased on stimulation the transcription by GC which be easily attack byalkylating agent(Benckhuijsen, Osman et al. 1987). In 2011, Dobos,Kenessey et al.

approved that dexamethasone inhibit the growth of WM983A humanmelanoma cells. This due to apoptosis induction result in enhancement of thechemotherapeutic (Dobos, Kenessey et al. 2011). Moreover Buxant, Kindt et al.

2015. found that there is significant inhibition in cell proliferation(30–35%)followed administration of dexamethasone to MCF-7 cells (Buxant, F., et al.2015) . Pre-treatment of breast cancer xenograft with dexamethasone may greatlyreduce the effect of paclitaxel through apoptosis inhibition of tumor cells (Huang,Y et al 2000, Fan W. et al 2004, Sui M. et al 2006, Pang D et al 2006,Gruver-Yates and Cidlowski 2013 Hou W .

et al 2014, Crozier M, Porter LA.2015).Dexamethasone has anti apoptosis effect of human ovarian cancer  cells induced by Paclitaxil and they suggestthat Dexamethasone increase expression of anti-apoptotic Bcl-2 family (Bcl-2and Bcl-XL) and members of IAP family (survivin). (Hou W . et al 2014) . In2005, Lu YS. et al found that the effects of glucocorticoid in thechemosensetivity ofcancer cell to Cisplatin are heterogenous  (Lu YS.

Et al 2005) . In 2006,Zhang, C., et al discovered that dexamethasone induce cisplatin and5flurouracil resistance of primary tumor cells from bone, brain, cervix, breast,neuroblastoma and melanoma (Zhang, C., et al. 2006).  In 2011, Yang et alapproved that Dexamethasone reduce the cisplatin-induced apoptosis leading todecrease its effect in hepatocellular cancer (Yang N. et al 2011).

Dexamethasonereduce the effect of cisplatin in lung cancer xenograft (Li H.,et al.2012, CaoF. et al. 2014). In 2016, Li.

Et al. suggest mechanism of inducingresistance to docetaxel and cisplatin by increasing Krüppel-like factor 5 in triple-negativebreast cancer. (Li, Dong et al.

2016). In the other hand, Lu YS. et al studiedthe effect of Glucocorticoids in the human cervical carcinoma cell line SiHaand they found that GCs increase  cisplatin cytotoxicity through inhibition ofNF-{kappa}B activation (Lu YS.

et al . 2006). Wang et al has discovered thatcoadministration of dexamethasone increase the cytotoxicity effect ofCarboplatin and Gemcitabin in MCF7 in two studies (Wang, Li et al. 2004). Astudy done by Wu. et al in 2004 and they found that the dexamethasone causeinhibition of paclitaxel and doxorubicin induced apoptosis in  two breast cancer cell lines MCF-7 andMDA-MB-231 and this sound to be mediated byglucocorticoid receptor activation(Wu, 2004 #188.

Dexamethasoneenhances the effects of Adryamicin (Doxorubicin) on inducing cell death  and antiproliferative effect; and itsuppresses nuclear NF-kB a possibly via up-regulation of IkappaB. These resultsindecate that Dexamethasone can be used as to enhance the chemosensitivity and asa chemoprotectant (Wang H et al, 2007). 6.  ResearchHypothesisDexamethasonemay potentiate the effect of Doxorubicin as it has stimulatory transcriptioneffect and  antiproliferative activity 7.  ResearchMethodology and procedures:-Drugsand chemicals Doxorubicinhydrochloride, phosphate buffered saline (PBS), Trypsin, Dullbecco ModifiedEagle Medium (DMEM) High Glucose, Penicillin G / Steptomycin antibiotics, FetalBovin Serum (FBS), propodium iodide SulphoRhodamine- B (SRB), Trizma base  and Annexin V-FITC apoptosis detection kit.

Purchasing  of all these chemicals will be from Sigma AldrichCo . -Cellcultures Thehuman breast cancer cell line MCF-7, will be obtained  from National Cancer Institute, CairoUniversity, Egypt. The adherent cells will grow as”monolayer culture” andcultured in the incubator mimic the human environment ( 37°C in a 5% CO2atmosphere ) and the passage will be every 4 or 5 days.-MethodMethods and procedures will be done in Pharmacology department,Faculty of medicineand King Fahad Research Center, king Abdul-Aziz University, Kingdomof Saudi Arabia.  A.Assessment of Cytotoxic ActivityThe Sulforhodamine(SRB) colorimetric assay will be used to measurethe  cytotoxicity of alone and combination doses of Doxurobicin and Dexamethasone according to Skehan et alprotocol.

In day one, the MCF7 Cells will b e seeded in 96 well plates at aconcentration of 30 × cells/well in the DMEM high glucose medium and incubatedfor 24 hr in 37°C in a 5% CO2 atmosphere. Then various concentrations ofDoxorubicin  and dexamethsone treatmentwill be added for additional 48 hr. Then we will perform cells fixation with 50%TCA and incubate the plate for 1 h at 4°C. Then we will wash the wells 3-4times with distilled water  to remove theexcess fixative and serum proteins and we will air dry the plate. After thatstaining the wells with SRB dye solution and incubate for 30 min at roomtemperature in the dark then we will wash the wells four times with 1% aceticacid and air dry the plate.Then Solubilization of the dye by adding 10 mM Trisbase.

Finaly, We will Measure the absorbance at (490–530 nm) wavelength usingMicroplate Reader from BioTek Instruments,Inc (Winooski,VT, U.S.A.

) And we willcalculate the IC50 (the concentration of DOXrequired for 50% decrease in cell viability) from linear equation of thesurvival fraction curve.B.Analysis of Cell Cycle Status Cell cycleanalysis will be done following  Pozarowskiand Darzynkiewicz(Pozarowski and Darzynkiewicz 2004) method. First, MCF7 Cells will be plated in 12-well plates at cell density of 6–8×105cells/well in and incubate for 24 hours, then treatment with dexamethasone  and various concentrations of doxorubicin andincubate the cells for additional 48 hour. Then harvest and pellet cellsby washing the wells with Phosphate Buffer Saline (PBS) and centrifuging,resuspending the cells with PBS after discarding the supernatant. Then we willadd 70% cold ethanol(-20°C) and stored at -20°C.

After that we will stain thecells with Ki-67 antibody and fluroscent DNA dyes. Finally we will perform theflow cytometry using the flowcytometer  (BectonDicknoson (BD) FACSCalbur,USA) and adjust it on 488 nm .C.Analysis of reactive Oxygen species production Caspase-3, -8 and-9 productionThe caspases level will be measured using Enzyme-LinkedImmunoSorbent Assay (Platinum ELISA; eBioscience©) .D.

Assessment of doxorubicin cellular accumulation Doxorubicin cellular accumulation in MCF-7 cells will be performed bymeasuring spectrofluorometer following Kitagawa et al method (Kitagawa,2004) using excitation and emissionwavelengths of ? ex = 496 nm and ? em = 592 nm to measure the doxorubicinflurescence intensity.8.    Statistics Statisticalanalysis will be done using SPSS (statistical package version 16). One wayanalysisof variance (ANOVA),  post hocanalysis, will be measured. the value of P <0.05 will be counted as statistically significantResearchLimitation:The  dexamethasone antiproliferative effect onMCF-7, estrogen receptor positive and progesterone receptor positive , isreassuring, yet, it does not guarantee the safe use of Dexamethasone in hormone-dependentbreast cancer cells, so further investigations are neededThereare several breast cancer cell lines, in our study the available  one is MCF79.

    Study’s overall structure:The study will take about 2 yearsdevided as following Activity No. Tasks Months 1 Approval and fund 6 2 Field work 8 3 Data Analysis 4 4 Report writing 3 5 Submitting report 3  10.    ExpectedFindingDexamethasone treatmentinhibits epidermal growth factor (EGF)- induced SK-1 mRNA leading to decreasein  EGF-induced proliferation andmigration of BC cells expression .

So it may potentiate the therapeutic effectof  Doxorubicin against the breast cancercells growth.11.    Definition of Terms:FBS:fetal bovine serumGCs:GlucocorticoidsDMEM:Dullbecco’s modified Eagles Medium PI:Propidium Iodide PBS:phosphate buffered saline SRB:SulphoRhodamine- B