Therefore, inhibition of BRCA2 may be a useful strategy to decrease metastatic burden in patients treated with cisplatin. Kif15-IN-2 Funding This work is supported by the Ontario Center of Excellence C2C award #20159. Conflict of interest The authors have no conflict of interest to declare. Supporting information The following are the supplementary data related to this article: Supplementary Physique?1 BRCA2 knockdown modulates cisplatin\ or melphalan\induced A549 cell proliferation. ASO using a Student’s t\test (p?0.05). MOL2-8-1429-s002.pdf (203K) GUID:?C6FE04DF-E537-49C0-8074-0DF0CE2AC44A Abstract Tumor cells have unstable genomes relative to non\tumor cells. Decreased DNA integrity resulting from tumor cell instability is usually important in generating favorable therapeutic indices, and intact DNA repair mediates resistance to therapy. Targeting DNA repair to promote the action of anti\cancer agents is therefore an attractive therapeutic strategy. BRCA2 is usually involved in homologous recombination repair. BRCA2 defects increase malignancy risk but, paradoxically, cancer patients with BRCA2 mutations have better survival rates. We queried TCGA data and found that BRCA2 alterations led to increased survival in patients with ovarian and endometrial cancer. We developed a BRCA2\targeting second\generation antisense oligonucleotide (ASO), which sensitized human lung, ovarian, and breast malignancy cells to cisplatin by as much as 60%. BRCA2 ASO treatment overcame acquired cisplatin resistance in head and neck malignancy cells, but induced minimal cisplatin sensitivity in non\tumor cells. BRCA2 ASO plus cisplatin reduced respiration as an early event preceding cell death, concurrent with increased glucose uptake without a difference in glycolysis. BRCA2 ASO and cisplatin decreased metastatic frequency in?vivo by 77%. These results implicate BRCA2 as a regulator of metastatic frequency and cellular metabolic response following cisplatin treatment. BRCA2 ASO, in combination with cisplatin, is usually a potential therapeutic anti\cancer agent. colony formation was used as a more stringent measure of the long term effects of treatment on seeding potential (Hao et?al., 2012). Treatment with BRCA2 ASO and cisplatin decreased colony\formation ability in both HN\5a and HN\5a/carbo\15a cells (Physique?4d and e), suggesting that BRCA2 inhibition can enhance the ability of cisplatin to limit cancer cell proliferation and colony forming potential. 3.5. BRCA2 modulates tumor cell metabolism following cisplatin treatment Given the dependence of DNA maintenance Kif15-IN-2 and repair on functional metabolic processes (Jeong et?al., 2013), it was possible that part of the BRCA2 ASO\mediated increase in cisplatin cytotoxicity was due to alterations in cellular metabolism. In addition, cisplatin has been shown to preferentially target mitochondrial DNA in tumor cells (Yang et?al., 2006). To investigate, we measured changes in cell impedance, acidification and oxygen consumption associated with BRCA2 ASO and cisplatin treatment to determine monolayer integrity, cellular glycolytic activity, and respiration (Alborzinia et?al., 2011). After 24\h exposure to cisplatin, A549 tumor cells pre\treated with BRCA2 ASO had 39% less respiratory activity Rabbit polyclonal to BMPR2 than cells pre\treated with control ASO. Furthermore, in BRCA2 ASO\treated cells, the respiration decrease was evident 10?h after addition of cisplatin and 15?h earlier than in cells treated with control ASO (Figure?5a). Respiration began to decrease in response to cisplatin in BRCA2\treated cells 10?h prior to observable reduction in adhesion, suggesting that respiration reduction occurred independent of changes in cell number or viability (Physique?5b). However, no difference in acidification (a?measure of glycolysis) was observed between the BRCA2 ASO and control ASO Kif15-IN-2 groups treated with cisplatin (Physique?5c). Open in a separate window Physique 5 BRCA2 modulates tumor cell metabolic response following cisplatin treatment. A549 cells were exposed to cisplatin (6?M, 24?h) following 6?h of incubation in medium to determine baseline metabolic levels. At 24?h after addition of cisplatin, medium was exchanged for medium without cisplatin. Measurements of oxygen consumption (a), impedance (b), and changes in medium pH (c) were conducted. Magenta = Control ASO, Blue = BRCA2 ASO, Green = Control ASO + Cisplatin, Red = BRCA2 ASO + cisplatin. A549 cells were transfected with control or BRCA2 ASO and then treated with cisplatin (6?M). Mitotracker staining (d) and glucose uptake (e) were determined using flow cytometry. Unstained control = Red, Control ASO = blue, BRCA2 ASO = orange, Control ASO + Cisplatin = light green, BRCA2 ASO + cisplatin = dark green. *Different from cells treated with control ASO using a Student’s t\test (p?0.05). Changes in cellular respiration induced by BRCA2 ASO in conjunction with cisplatin suggested BRCA2 ASO\mediated inhibition of mitochondrial function. We used mitochondria\specific dye.