Iranian Journal of Pharmaceutical Research (2019), 18 (4): 1959-1966 DOI: 10.22037/ijpr.2019.1100853 Received: September 2016 Accepted: December 2017 Original Article Effects of Baneh (Pistacia atlantica) Gum on Human Breast Cancer Cell Line (MCF-7) and Its Interaction with Anticancer Drug Doxorubicin Hamzeh Pasban-Aliabadia*, Vahid sobhania, Saeed Esmaeili-Mahanib, Hamid Najafipourc, Alireza Askaria and Hamidreza Jalaliand aExercise Physiology Research Center, life style institute, Baqiyatallah University of Medical Science, Tehran, Iran. bDepartment of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman. Kerman, Iran. cPhysiology Research Center and Department of Physiology and Pharmacology, Afzalipour Medical Faculty, Kerman University of Medical Sciences. Kerman, Iran. dDepartment of Hematology and Oncology, Baqiyatallah University of Medical Sciences, Tehran, Iran. Abstract Pistacia atlantica is one of the species of Anacardiaceae that grows in the wild in different regions of Iran. Traditionally, anacardiaceae family has antibacterial, fungicidal, and cytotoxic properties. Therefore, the present study was designed to investigate the possible cytotoxic and anti-proliferative properties of Baneh gum. Cytotoxicity of the plant gum was determined using MTT assay on MCF-7 human breast cancer cells. The cellular makers of apoptosis (caspase3 and P53) and cell proliferation (Cyclin-D1) were evaluated by western blotting. Doxorubicin was used as anticancer control drug in combination treatment. The result showed that Baneh gum (100 µg/mL) significantly induced cell damage, activated caspase3, and increased P53 protein level. In addition, Cyclin-D1 was significantly decreased in gum- incubated cells. Furthermore, combination treatment of cells with Baneh gum (25 µg/mL) and doxorubicin (200 nM) produced a significant cytotoxic effect as compared to each drug alone. In conclusion, Baneh gum (100 µg/mL) has a potential pro-apoptotic/anti-proliferative property against human breast cancer cells and its combination with doxorubicin in low doses may induce cell death effectively and be a potent modality to treat this type of cancer. Keywords: Baneh gum; Anti-apoptotic; Anti-proliferative; MCF-7 and doxorubicin. Introduction cancers. In addition, common cytotoxic therapies primarily target rapidly dividing cells Cancer is a fetal disease with no complete including malignant cells as well as certain therapy or cure, so it is critical to find beneficial normal cells, leading to significant morbidity solution to defeat this public health menace. and limited clinical benefits of troubled patients. Almost all chemotherapic drugs have unwanted Combination therapy is a useful method for and serious side effects. The development of the reduction of anticancer drug doses and resistance to chemotherapic drugs is a common subsequent side effects. clinical problem in the treatment of various Breast cancer is common well known cancer and is the main death reason of cancer * Corresponding author: in woman throughout the world. Each year E-mail: [email protected] over 1 million women are diagnosed with Pasban-Aliabadi H et al. / IJPR (2019), 18 (4): 1959-1966 breast cancer and nearly half of them die (1). cell line and its combinational therapy with At least one in nine women develops breast doxorubicin. cancer at some stages in their life. Currently, there is an urgent need for improvements in Experimental detection, diagnosis, and treatment of breast cancer. Unfortunately, the current classical Materials treatments (surgery, chemotherapy, and/or Cell culture reagents, penicillin-streptomycin radiotherapy) are impeded by side effects most solution, trypsin EDTA, and fetal bovine serum importantly development of tumor resistant, (FBS) were obtained from Biosera Co. (East loss of appetite, nausea and vomiting, weakness Sussex, UK). Culture flasks and dishes were and fatigue, mouth soreness, hair loss, weight acquired from SPL Lifesciences Inc. (Gyeonggi- gain, premature menopause, lowered resistance Do, South Korea). 3- [4, 5-Dimethyl-2- to infections, bleeding, and gastrointestinal thiazolyl]-2, 5-diphenyl-2-tetrazolium bromide illness (2).Therefore, finding novel and effective (MTT), doxorubicin and primary monoclonal therapies to prevent the development of breast anti- -actin antibody were purchased from cancer and lower mortality rates related to it, is Sigma (St. Louis, MI, USA). Primary monoclonal a current scientific challenge. anti-caspase3훽 antibody was purchased from Cell Over 7,000 species of plants grow in Iran Signaling Technology, Inc. (Beverly, MA, USA). among them 1000 species are estimated to have Primary polyclonal anti-P53, anti-Cyclin-D1 medicinal effects (3). The presence of various and secondary goat anti-rabbit, and goat anti- climatic conditions and ecological factors mouse antibodies were obtained from Santa provide an environment in which numerous Cruz Biotechnology, Inc. (Delaware Ave. Santa varieties of plants grow in differ ent regions Cruz, USA). of the country (4). The genus Pistacia belongs to the family of Anacardiaceae, and among Plant Extract 15 known species of pistachios. The Psitacia The gum of Baneh was collected from atlantica with the local name of Baneh has three Jebalbarez Mountain (Kerman province, sub-species: mutica, kurdica and cabulica (5). Southern part of Iran) in October. The voucher Baneh has been used in folk medicine for treating specimens (No. 2341) were deposited at the eczema, throat infection, renal stone, and asthma Herbarium of the Shahid Bahonar University of (6). It has been demonstrated that Baneh has Kerman (Kerman, Iran). The collected gum was Pro-apoptotic and Anti-proliferative effect on dried and powdered then dissolved in DMSO cancerous situation (7-9). The antioxidant (10), and used for treatment. The final percent of antimutagenic (11), antimicrobial and antiviral DMSO was less than 1%. (12), anti-inflammatory (13, 14), antidiabetic (15), antitumor (16), anti-hepatitis (15), anti- Cell Culture atherosclerosis (17), and anti-cholinesterase MCF-7 (human breast adenocarcinoma cell (18) effects of Baneh have been reported. line) cells were obtained from National Cell Previous studies used other parts (stem, leave, Bank of Iran (NCBI). The cells were grown pericarp and etc.) or different spesies (P. vera, with Dulbecco’s modified Eagle’s medium P. atlantica, P.terebinthus, P. khinjuk, and P. supplemented with 10% fetal bovine serum, lentiscus) or subspecies of baneh (cabulica penicillin (100 U/mL), and streptomycin (100 and mutica) instead of piatatia atlantica sub mg/mL). They were maintained at 37 °C in a 5% kurdica. CO2 atmosphere. Growth medium was changed Despite numerous reports regarding three times a week. The cells were plated at the cytotoxic effects of baneh, the effects of pistacia density of 5000 per well in a 96-microplate well atlantica sub kurdica gum alone and along with for the MTT assays. For protein extraction, the doxorubicin on breast cancer cell line have not cells were grown in a 6-plate well and permitted been yet reported. So, we decided to evaluate the to attach and grow for 24 h. Then the cells were cytotoxic effect of baneh gum on breast cancer incubated with different concentration of the 1960 Cytotoxic effect of Baneh gum on MCF-7 gum alone or in combination with anticancer and exposed to Lumi-Film chemiluminescent drugs doxorubicin. detection film (Roche, Germany). Lab Work analyzing software (UVP, UK) was used to Cell Viability Analysis analyze the intensity of the expression. β-actin Cellular viability was assessed by the immunoblotting (1: 1000) was used as loading reduction of 2-(4, 5-dimethylthiazol-2-yl)-2, control. The immunoblotting experiments for 5-diphenyltetrazolium bromide (MTT) to each protein were performed 3-4 independent formazan. MTT was dissolved in PBS and times. added to the culture at final concentration of 0.5mg/mL. After additional 2 h incubation at Statistical Analysis 37 °C, the media were carefully removed and The results are expressed as mean ± SEM. 100 µL DMSO was added to each well, and the The differences in mean cell viability and absorbance (OD) values were determined by blotting data between experimental groups were spectrophotometry at 490nm with microplate determined by one-way ANOVA, followed by reader (Eliza MAT 2000, DRG Instruments, Tukey test. < 0.05 was considered significant. GmbH). Each experiment was performed 5-6 independent times. The results were expressed 푃 Results as percentages of control. The effect of pistacia atlantica gum on cell Immunoblotting Analysis viability MCF-7 cells were homogenized in ice-cold At first, MCF-7 cell viability was analyzed by buffer containing 10mM Tris-HCl (pH 7.4), MTT assay. After 24 h attachment/grow period, 1mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, the cells were exposed to different concentration 1% NP-40 with protease inhibitors (1mM of pistacia atlantica gum (5, 25, 50, 100, and phenylmethylsulfonyl fluoride, 2.5 µg/mL of 200 µg/mL) for a 24 h period. Figure 1 shows leupeptin, 10 µg/mL of aprotinin), and 1mM that the gum could decrease MCF-7 cell viability sodium orthovanadate. The homogenate was in a dose dependent manner. The gum at doses centrifuged at 14000 g for 15min at 4 °C. The of 100 and 200 µg/mL potently elicited cell resulting supernatant was retained as the whole death after 24 h and had a moderate effect in 50 cell fraction. µg/mL, while it could not prevent cell damage in Protein concentrations were measured using dose of 5 and 25 µg/mL. the Bradford method and equal amounts of protein (40 µg) were resolved electrophoretically The effect of combinational treatment of gum on a 12% SDS-PAGE gel and then transferred and doxorubicin on cell viability to PVDF membranes (Hybond ECL, GE For this reason, the non-effective dose of Healthcare Bio-Sciences Corp. NJ, USA). After Baneh gum (25 µg/mL) was combined with overnight blocking at 4 °C with 5% nonfat doxorubicin. The data showed that doxorubicin dried milk in Tris-buffered saline with Tween at doses of 10 nM did not show a significant toxic 20 (blocking buffer, TBS-T, 150mM NaCl, effect on MCF-7 cells.