Balkan Journal of Medical Genetics

TWIST1 GENE EXPRESSION AS A BIOMARKER FOR PREDICTING PRIMARY DOXORUBICIN RESISTANCE IN BREAST CANCER
Demir S1,10,*, Müslümanoğlu MH2, Müslümanoğlu M3, Başaran S4, Çalay ZZ5, Aydıner A6, Vogt U7, Çakır T8, Kadıoğlu H9, Artan S1
*Corresponding Author: Selma Demir, Ph.D., Trakya University Faculty of Medicine, Department of Medical Genetics, 22030 Iskender, Edirne, Turkey. Tel: +90(284)2357642/2330. Fax: +90(284)2357652. E-mail: selmaulusal@trakya.edu.tr
page: 25

MATERIALS AND METHODS

Sample Collection. Twenty-six primary breast tumor samples taken from 26 different untreated breast cancer patients (26 women, mean age: 53.4) following permission of the institutional ethics board, were included in this study. Samples were taken from the breast tumors during initial breast surgery. Tumor cell content of collected samples was confirmed by imprint cytology. Samples were cut into two pieces of about 0.5 cm3. One piece of each tumor sample was put into an RNA stabilization reagent (RNAlater; Qiagen GmbH, Hilden, Germany) for protection of the RNA content until RNA isolation, and the other half was put directly into the 0.2% antimycotic/antibacterial added 7 mL transport medium [Dulbecco’s modified eagle medium (DMEM)], for transportation to the laboratory for adenosine triphosphate tumor chemo-sensitivity assay (ATP-TCA). Adenosine Triphosphate Tumor Chemo-Sensitivity Assay. A modified cell viability method of Andreotti et al. [13] was used to determine the responses of tumor cells to doxorubicin. Breast cancer tissue samples were mechanistically fragmented and treated for 12 hours with 10 mL sterile tumor dissociation enzyme reagent (TDR) in the incubator (37 °C, 5.0% CO2) before resuspension (1.5 × 105 cells/mL) in DMEM (Gibco Thermo Fisher Scientific, Waltham, MA, USA). Therapeutic drug preparations from commercial sources were stored and used before expiration dates according to the manufacturer’s instructions. Doxorubicin was prepared in six dilutions corresponding to 200.0, 100.0, 50.0, 25.0, 12.5 and 6.25%, respectively, of each standard test drug concentration (TDC). One hundred percent TDC value used for doxorubicin was 0.5 g/ mL [13-15]. Cultures of approximately 15,000-20,000 cells/ well were tested in 96-well microplates (Costar; Merck KGaA, Darmstadt, Germany) containing both 12 maximum inhibition control wells (i.e., negative controls containing no cells) and 12 no inhibition control wells. Cultures were incubated for 8 days at 37 °C in a >98.0% humidified, 95.0% air and 5.0% CO2 atmosphere; then cellular adenosine triphosphate (ATP) was extracted and stabilized by mixing cell lysing reagent (Merck KGaA) into each well. The ATP was measured in a BMG LUMIstar (BMG Labtech GmbH, Ortenberg, Germany) using the 1:1 mix containing cell lysate and Luciferin-Luciferase counting reagent (Merck KGaA). A 5 second-count integration with a 1 second delay was used. Each measurement was performed three times. After incubation for 6 days with the drug, percentages of breast tumor cell growth inhibition (BTGI) compared with control cultures was determined. The area under the curve (AUC) values were calculated using the trapezoidal rule [16]. RNA Isolation and Real-Time RT-PCR. Fresh tissue samples kept in RNAlater solution was lysed and homogenized using Tissue Lyser LT followed by manual RNA isolation performed with RNeasy Mini Kit (Qiagen GmbH) according to the manufacturer’s instructions. The RNA concentration was measured using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) using the Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany), 150 ng of total RNA was reverse transcribed with the combination of anchoredoligo( dT) and random hexamer primers included in the kit. TWIST gene expression of tumor samples was determined by real-time reverse-transcription polymerase chain reaction (RT-PCR) using β-actin gene expression as the reference ( Real-Time Ready Gene Expression Assay; Roche Diagnostics). All reactions were duplicated on a LightCycler® 480 (Roche Diagnostics). Gene expression differences of tumor samples were determined using the ΔΔCT (2–ΔΔCT) method [17]. The Statistical Package for the Social Sciences (SPSS®) version 11 (https://www. ibm.com) was used to determine the relationship between TWIST gene expression status and other tumor parameters and in vitro chemo-response of tumors to doxorubicin. The Mann-Whitney U test in the SPSS program has been used to determine if gene expression differences are related to chemo-response of tumors.

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