br introduction of normal mitochondria into cancer cells
introduction of normal mitochondria into cancer Pentostatin could reverse their malignancy, growth, invasion and drug resistance (Kaipparettu et al., 2013). Therefore, increasing the mitochondrial oxidation of substrates may suppress cancer growth by denying the substrates re-quired for the Warburg eﬀect (Seyfried, 2015).
One approach to reverse the Warburg eﬀect by mitochondrial oxi-dation is to increase the capacity of uncoupling proteins (UCPs) located in the inner membrane of mitochondria because UCPs promote the oxidation of substrates independent of ATP synthesis. As it is broadly expressed in cancer cells (albeit at a lower level), UCP2 has been con-sidered an attractive target for the treatment of cancer (Imai et al., 2017; Donadelli et al., 2015). It has been reported that overexpression of UCP2 inhibits cancer cell proliferation and tumorigenesis in vivo whereas UCP2-null mice display increased incidence of tumors, such as colon tumors (Esteves et al., 2014; Derdak et al., 2006). An increased expression in UCP2 can eﬃciently switch the Warburg eﬀect to the
∗ Corresponding author. School of Pharmaceutical Sciences, Sun Yat-sen University Guangzhou, China. E-mail address: [email protected] (Y. Rao).
oxidation path and induces a metabolic reprogramming in cancer cells without toxicity (Derdak et al., 2006). However, till now, the under-lying mechanism involved in for the upregulation of UCP2 regulation has is not well understood. Bouchardatine (Bou) is an alkaloid rich in Rutaecae (B. Neurococca) which is commonly used in the treatment of diarrhea and inflammation in traditional Chinese medicine. Our previous studies have demon-strated that Bou can alleviate adipose tissue hyperplasia, dyslipidemia and insulin resistance through the promotion of mitochondrial oxida-tion capacity (Rao et al., 2017). As these metabolic eﬀects appear to compete against the Warburg eﬀect, we hypothesized that Bou may have therapeutic eﬀects for certain types of cancer. Therefore, the present study aimed to examine the therapeutic eﬀects of Bou on rectal cancer and to investigate the underlying mechanism for its molecular mode of action. We chose to rectal cancer for the present study because this type of cancer ranks among the most lethal cancers (the overall with 5-year survival is less than 5%). Despite various treatment ap-proaches, there is still an urgent need to improve the eﬃcacy of che-motherapy by developing new therapeutic agents.
2. Materials and methods
Bouchardatine (Bou), with a purity of 99% as determined by high-performance liquid chromatography (HPLC), was prepared and syn-thesized by our group as previously reported . 2-(N-(7-Nitrobenz-2-
oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) (sigma, Shanghai, Chian), reactive oxygen species probe (Beyotime, Jiangsu, China), mitochondria isolation kit, coimmunoprecipitation (CoIP) assay kit and chromatin immunoprecipitation (ChIP) assay Kit were from Thermo (Shanghai, China). Latate dehydrogenase (LDH) determination kit and trypan blue dye were from Beyotime (Jiangsu, China). The extracellular acidification rate (ECAR) and oxygen consumption ratio (OCR) assay kits and related metabolism inhibitor were from Agilent (USA). Dithiothreitol (DTT), 5-Aminoimidazole-4-carb-oxyamide ribo-nucleoside (AICAR) and nicotinamide (NAM) were from Selleck (Jiangsu, China). Mitochondrial assay kit was from Abcam and Jiancheng Bio (Nanjing, China). adenosine-triphosphate (ATP), nicoti-namide adenine dinucleotide (NADH) level and SIRT 1 activity assay kit were from Abnova (Wuhan, China). Cell cycle and apoptosis assay kit were from MultiScience (Guangzhou, China). Plasmids of UCP2 and PGC-1α and UCP2-Luciferase, PGC-1α-Luciferase, SIRT1 and SIRT1H355A were from Addgene (USA), AMPKα and SIRT1 siRNA were from Santa Cruz Biotechnology (USA). Antibodies information is listed in Table 1.
2.2. Cell lines and cell culture
Antibodies information used for Western blot assay.
Antibody name Company Dilution ratio Location
GAPDH Cell signaling Technology 1:1500 China CPT-1β Abcam 1:1000 China PGC-1α Abcam 1:1000 China ACSL Bioworld Technology 1:1000 China UCP2 Bioworld Technology 1:1000 China SIRT1 Cell signaling Technology 1:1000 China Ac-Lys Abcam 1:1000 China AMPKα Cell signaling Technology 1:1000 China p-AMPKα(Thr-172) Cell signaling Technology 1:1000 China p-ACC(Ser 89) Cell signaling Technology 1:1000 China
from American Type Culture Collection (Shanghai, China, ATCC). Cells were cultured in Dulbecco's modified Eagle's medium and 1640 culture medium supplemented with 10% fetal bovine serum (Invitrogen, Shanghai, China) and 10 U/mL penicillin/streptomycin at 37 °C in a humidified 5% CO2 incubator.