Transcript Introduction - York College of Pennsylvania

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.