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Knockdown of MDR1 in ovarian cancer cells via delivery of siRNA using ceramide nanoliposomes Sydney Drury Department of Biological Sciences, York College of Pennsylvania https://www.atcc.org/Products/All/HTB-77.aspx http://www.keystonenano.com/platform/liposomes Introduction Overexpression of multi-drug resistance gene 1 (MDR1), an energy-dependent efflux drug transporter, is the cause of drug resistance in most recurring ovarian cancers (Yang et al., 2015). F lu o r e s c e n t L ip o s o m e Culture SKOV3-TRip2 Culture SKOV3 Fluorescence increased with longer incubation periods. Fluorescence significantly higher in the rhodamine liposome group Than the control. MTS Cell Proliferation Assay Make CNL (Kester formula) Figure 1. Fluorescence of Rhodamine-tagged liposomes and untagged liposomes within SKOV3 cells after liposomal uptake. A model of this drug resistance is the ovarian cancer cell line, SKOV3-TRip2, which overexpresses MDR1. Ceramide, a sphingolipid, is believed to cause apoptosis when in high concentrations, specifically in tumor cells (Zhai et al., 2015). Ceramide nanoliposomes (CNL), small ceramide-containing transport vesicles, can be used to deliver siRNA to cells (Kester et al., 2015). A clinically effective method for delivery of siRNA to knock down MDR1 and regain ovarian cancer sensitivity to chemotherapeutics has not yet been found. MDR1-targeting siRNA will be delivered to SKOV3-TRip2 cells to re-sensitize them to Taxol and prime them with increased amounts of apoptotic ceramide. Encapsulate siRNA: scramble, siRNA 1, siRNA 2 Y = - 0 .0 0 8 3 3 3 * X + 0 .9 1 6 7 50 0 30 No siRNA: Ghost 60 120 180 T im e ( m in ) -5 0 SKOV3-TRip2 shows no decrease in cell viability with increased Taxol concentrations. 150 Cellular Uptake Experiment (SKOV3) SKOV3 displays lower cell viability at all Taxol concentrations when compared to SKOV3-TRip2. 100 SKOV3 Y = -0.07877*X + 99.88 50 Figure 2. SKOV3 and SKOV3-TRip2 cell viability at increasing paclitaxel concentrations. A linear regression was performed for each cell line, showing the overall trend of the response to paclitaxel. 0 Incubate Cells, added: scramble, siRNA 1, siRNA 2, ghost, PBS 50 200 500 900 1000 T a x o l C o n c e n tr a tio n ( n M ) 3 Scramble siRNA knocked down proteins indiscriminately, therefore the exact competence of our siRNA 1 and 2 constructs is unknown. Detergent Compatible Protein Assay P-gp, the MDR1 protein, amounts appeared to be slightly higher in the MDR1-targeting siRNA-treated cells than in the cells not treated with siRNA. 2 1 0 C o n tro l s iR N A1 s iR N A2 G host C e lls O n ly T re a tm e n t Protein Quantification S K O V 3 - T R ip 2 Figure 3. CNL-delivered MDR1-targeting siRNA knockdown of MDR1 in SKOV3-TRip2 cells. A Kruskal-Wallis test was performed and found that the means of the liposome treatments are not significantly different (P= 0.3705, ns). • Knock down MDR1 expression in SKOV3-TRip2 with siRNA delivered via CNLs. Conclusions Literature Cited Ahn, S.J., Jeon, Y.H., Lee, Y.J., Lee, Y.L., Lee, S-W., Ahn, B-C., Ha, J-H., and Lee, J. 2010. Enhanced antitumor effects of combined MDR1 RNA interference and human sodium/iodide symporter (NIS) radioiodine gene therapy using an adenoviral system in a colon cancer model. Cancer Gene Therapy. 17: 492-500. Deng, J., Guo, Y., Jiang, Z., Yang, M., Li, H., and Wang, J. 2015. Enhancement of ovarian cancer chemotherapy by delivery of multidrug-resistance gene small interfering RNA using tumor targeting Salmonella. The Journal of Obstetrics and Gynaecology Research. 41(4): 615-622. Kester, M.,Bassler, J., Foxx, T.E., Carter, C.J., Davidson, J.A., and Parette, M.R. 2015. Preclinical development of a C6-ceramide NanoLiposome, a novel sphingolipid therapeutic. The Journal of Biological Chemistry. ooooo 396(6-7): 737-747. Sun, K., Jiao, J., Chen, S., Liu, B., Zhao, Y. 2015. MicroRNA-186 induces sensitivity of ovarian cancer cells to paclitaxel and cisplatin by targeting ABCB1. Journal of Ovarian Research. 8:80. Yang, X., Iyer, A.K., Singh, A., Choy, E., Hornicek, F.J., Amiji, M.M., and Duan, Z. 2015. MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer. Scientific Reports. [serial online] Zhai, L., Sun, N., Han, Z., Jin, H., and Zhang, B. 2015 Liposomal short-chain C6 ceramide induces potent antiosteosarcoma activity in vitro and in vivo. Biochemical and Biophysical Research Communications. 468(12): 274-280. N o n - flu o r e s c e n t L ip o s o m e Y = 0.006276*X + 126.8 Objectives • Demonstrate the difference in response to Taxol in SKOV3 and its MDR1-expressing mutant SKOV3-TRip2 100 S K O V 3 -T R ip 2 Western Blot • Show the competence of the CNLs as delivery vehicles 150 200 C e ll V ia b ility ( % ) Small interfering RNA (siRNA) are capable of selectively knocking down gene expression, such as MDR1, temporarily by degrading the specific gene’s mRNAs before they are translated (Ahn et al., 2010; Deng et al., 2015). Y = 0 .3 7 3 7 * X + 7 3 .0 5 200 P - g p C o n c e n tr a tio n ( % v o l) Despite the typical success of surgery and chemotherapy, ovarian cancer tends to recur in a form that is resistant to common chemotherapeutics (Sun et al., 2015; Yang et al., 2015). Results F lu o r e s c e n c e ( 5 6 0 : 5 8 3 n m ) Ovarian cancer is the fifth leading cause of cancer-related death among women in the United States. (Yang et al., 2015) Methods • The Kester lab CNLs are capable of getting inside the cells to deliver siRNA, whether they are endocytosed or merge with the cell membrane. • Taxol is less effective at killing the MDR1-expressing SKOV3TRip2 cells than SKOV3 cells. I would like to thank Dr. Mark Kester and Dr. Tye Deering of the Kester lab at the University of Virginia for inviting me into their lab, allowing me to pick up a project, assisting me in understanding the necessary procedures, and helping to improve my research ability every step along the way. • Our MDR1-targeting siRNA system must be further optimized to achieve complete knockdown of MDR1 in SKOV3-TRip2. I would also like to thank the Summer Research Internship Program of the University of Virginia for giving me the opportunity and funding to perform this research at their facilities. • Effectively re-sensitizing the MDR1-expressing SKOV3-TRip2 cells to chemotherapeutics could allow for the creation of an effective treatment for women afflicted by drug-resistant ovarian cancer. https://www.drvitaminsolutions.com/Nanosphere-Delivery/ Acknowledgements Furthermore, I would like to thank all of the members of the Kester lab, as I received help from each one of them at some point during my internship in their lab.