The suppression of rotenone-treated human breast cancer stem cell survival using survivin inhibitor YM155 related to oxidative stress modulation Septelia Inawati Wanandi 1 *, Resda Akhra Syahrani 2 , Elvira Yunita 3 , Go Aulia 4 1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia 1,2 Molecular Biology and Proteomics Core Facilities, IMERI Faculty of Medicine, Universitas Indonesia 3,4 Master Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia *Corresponding Author septelia@gmail.com, septelia@ui.ac.id Despite the recent progress in molecular-targeted therapies, breast cancer remains the first-leading cause of cancer-death among women over the world. Nowadays, breast cancer stem cells (BCSCs) is believed to be responsible for therapy resistance and recurrency of cancer. Very recently, we have demonstrated that human BCSCs (CD24-/CD44+) could survive better than their counterpart non- BCSCs (CD24-/CD44-) when treated with rotenone, which might be due to lower ROS levels production, high expression of antioxidant manganese superoxide dismutase (MnSOD) and anti-apoptosis survivin. The aim of this study was to verify the role of survivin on the survival of human BCSCs under oxidative stress modulation by suppressing its expression using a survivin inhibitor YM155. Human BCSCs (ALDH+ cells) were firstly treated with YM155 for 24 hours prior to rotenone treatment for another 6 hours. We determined the intracellular superoxide level using dihydroethidium assay, MnSOD expression using qRT-PCR, as well as cell viability using trypan blue exclusion and acridine orange/ ethidium bromide apoptosis assay. This study found that the suppression of survivin expression using YM155 could reduce the survival of rotenone-treated BCSCs, which may be associated with the oxidative stress modulation, as shown by the increased ROS levels and decreased MnSOD expression. In conclusion, we confirm that survivin is responsible for maintaining the BCSC survival under oxidative stress modulation. Furthermore, the present study reported for the first time that YM155 could modulate the oxidative stress in BCSCs by reducing the MnSOD expression and increasing the ROS levels. Thus, we propose that YM155 treatment may overcome the BCSC resistance to oxidative stress-based anti-cancer. Key Words: Breast cancer, Stem cell, YM155, Survivin Introduction The cancer stem cell (CSC) hypothesis proposes that tumors are initiated and maintained by a small fraction of cells, but the origin of these tumorigenic cells are actually not known. The stem cell theory of cancer proposes two major concepts. One theory claims that CSCs arise from mutated stem cells which then expand in such a manner that the mutation is shared by many of the descendants. 1 An alternative theory proposes that transformed and differentiated cells acquire stem cell-like characteristics. 2 It is also believed that CSCs may be responsible for resistance against therapeutic approaches and tumor recurrence. 3 Breast cancer is the most common cancer and the second leading cause of cancer-related mortalities among women after lung cancer. Breast cancer treatments include chemotherapy, hormone therapy, as well as surgery and radiotherapy. Despite the recent progress in molecular- targeted therapies, breast cancer remains the principal cause of cancer death in women due to high incidence of recurrence. 4 Breast CSCs (BCSCs) are heterogeneous and could be identified based on the presence of several surface antigen markers, such as CD44+, CD24−, ESA+, and CD133+. 5,6 As well as other CSCs, BCSCs are considered responsible for resistance to chemo-radiation therapy, disease recurrence, and metastasis. 7,8,9 Consequently, the development of effective breast cancer therapy should pay more attention on targeting the eradication of BCSCs. 8 One of the general strategies of breast cancer therapy is to treat cancer cells excessively with free radicals. 9 As a consequence, the balance between free radicals and endogenous antioxidant defense mechanisms in cancer cells will be disturbed, known as oxidative stress, leading to various types of cell death such as apoptosis and autophagy. 10,11 Chemo-radiation therapy modulates oxidative stress in cancer cells which induces cellular adaption responses such as cell survival and antioxidant defense mechanisms. 12,13,14 Our recent study has demonstrated that human BCSCs (CD24-/CD44+) could survive better than their counterpart non-BCSCs (CD24-/CD44-) when treated with rotenone, which might be due to lower ROS levels production, high expression of antioxidant MnSOD and anti- apoptosis survivin. 15 Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Retrospective studies revealed that up- regulation of survivin correlates with decreased survival rates, increased relapse, and higher frequency of metastases in breast cancer patients. 16,17 Moreover, survivin is highly expressed in other cancer cells in which its expression confers resistance to apoptosis induced by various chemotherapeutic agents. 18,19 Previous study has demonstrated that a novel small molecule, YM155, could suppress survivin expression with minimal effect on expression levels of other IAP family members or Bcl-2 related proteins. 20 In this study, we aimed to verify the role of survivin on the survival of human BCSCs under oxidative stress modulation by suppressing its expression using a survivin inhibitor YM155. Additionally, the in vitro antitumor efficacy of