Graduate Category: Interdisciplinary Topics, Centers and Institutes ...
Graduate Category: Interdisciplinary Topics, Centers and Institutes Degree Level: PhD Bioengineering Abstract ID# 383
Nanoformulations of PARP Inhibitors for Cancer Therapy Paige Baldwin1, Shifalika Tangutoori1,2, Srinivas Sridhar1,2 1. Nanomedicine Science and Technology Center, Northeastern University, 2. Radiation Oncology, Dana Farber Cancer Institute
Abstract
Goal
100 μM Drug
50 μM Drug
10 μM Drug
1 μM Drug
500 nM Drug
100 nM Drug
50 nM Drug
No Drug
10 nM Drug
Dose Response
8000 cells/well
4000 cells/well
2000 cells/well
1000 cells/well
Doubling Cycle 500 cells/well
PARP inhibitors (Poly(ADP-ribose) polymerase inhibitors) such as Olaparib are among the most potent molecular inhibitors used in clinical trials for various types of cancers including prostate and ovarian cancers They act by inhibiting DNA damage repair and thus accumulating deleterious mutations leading to genetic instability as a function of number of cell replications . Thus, in this study, we thoroughly characterized and optimized the in vitro model of prostate cancer (PC3, LNCaP, FK01) and ovarian cancer (PA1), in a 96 well system. To compare and contrast the IC-50’s, of free drug and nanoformulated drug, we employed dose response curves generated for each cell line. Prior to this, it was crucial to develop growth rate curves for all cell lines and optimize the seeding density so as to ensure that cell death was not occurring because of over confluence and lack of nutrients, but rather due to the genetic instability caused by the treatment. These curves allow for each cell line to be exposed to the treatment of choice for 4 doubling cycles prior to analysis. Dose response was generated for 4 cell lines, with the 2 different drugs (Olaparib and nano-Olaparib). We determined the IC-50’S of these cells after 4 doubling cycles. These results allowed for comparison between the efficacy of the nanoformulation versus the free drug, thus determining the dose regimen for further invitro studies.
Results
Methods
A)
• Treatment with Olaparib and NanoOlaparib •Quantify the viable cells present after 4 doubling cycles
•MTS converted to Formazan by live cells •Quantify number of cells at varying time points •PC3, LNCaP,FKO1
Results
A)
3 2.5
24 hours
2
48 hours
1.5
72 hours
1
96 hours
0.5
3.5 3 2.5 2
24 hours
1.5
48 hours
1
72 hours
0.5
D)
0 500
1000
2000
4000
500
B)
8000
1000
2000
4000
Figure 2. IC-50’s comparing Olaparib and NanoOlaparib for (A) PC3, (B) FK01, and (C) PA-1. IC-50 for PC3 with Olaparib and NanoOlaparib is ~2.3 μM and ~1.3 μM, respectively. IC-50 for FK01 with Olaparib and NanoOlaparib is ~2.0 μM and ~2.5 μM, respectively. IC-50 for PA-1 with Olaparib and NanoOlaparib is ~2.3 μM and ~3.6 μM, respectively. Doubling time for FK01, PC3, PA-1, and LNCaP (D), based on a nonlinear fit of the normalized data from Figure 1.
8000
Conclusion and Future Work
LNCaP Absorbance
4
C)
Number of cells plated
Number of cells plated
Absorbance at 490 nm
• PARP-1 is a DNA repair protein which plays a role in • Base Excision Repair • Homologous Recombination • Non-Homologous End Joining • Transcriptional Regulation
3.5
0
FK01 Absorbance
4
Absorbance at 490 nm
•Cell lines: •PC3 (Prostate) ETS fusion negative •LNCaP (Prostate) ETS fusion postiveinteracts with PARP-1 •FK01 (Prostate) PTEN and p53 deficient • PA1 (Ovarian) genetic instabilityPARPi sensitive
Absorbance at 490 nm
Background
PC3 Absorbance
4
B)
3.5 3 2.5
72 hours 120 hours
2
168 hours
1.5
216 hours
1
264 hours
•Optimized experimental methods for IC-50’s of Olaparib and NanoOlaparib in 4 cell lines (PC3, FK01, LNCaP, PA-1) •Non-linear fit curves of different cell lines show doubling time of FK01 < PA-1 < PC3 < LNCaP •NanoOlaparib > Olaparib in some cell lines
0.5
C)
0 1000
2000
4000
8000
10000
Number of cells plated
Figure 1. Absorbance indicates the number of viable cells present at a given time. The absorbance of viable cells was quantified and used for further analysis to determine the doubling time for each cell line. The bar graphs above depict the viable cells for PC3 (A), FK01 (B), and LNCaP (C).
Acknowledgements Supported by IGERT grant NSF-DGE- 0965843 and Mazzone Foundation.
•Chemosensitization of Olaparib versus NanoOlaparib •Radiosensitization of Olaparib versus NanoOlaparib •In vivo prostate model testing Olaparib versus NanoOlaparib
References 1. Shifalika Tangutoori, Houari Korideck, Mike Makrigiorgos, Robert Cormack, Srinivas Sridhar. A Novel Nano-Formulation for Systemic Administration of PARPi-Olaparib (NANO-OLAPARIB) for Radiosensitization, Chemo-Sensitization and Combinatorial Therapy in Prostate Cancer. AACR Molecular Targets and Cancer Terapeutics, Boston, October 2013, 10/19/2013-10/23/2013 2. Chatterjee P, Choudhary GS, Sharma A, Singh K, Heston WD, et al. (2013) PARP Inhibition Sensitizes to Low Dose-Rate Radiation TMPRSS2-ERG Fusion Gene-Expressing and PTEN-Deficient Prostate Cancer Cells. PLoS ONE 8(4): e60408. doi:10.1371/journal.pone.0060408
Nanoformulations of PARP Inhibitors for Cancer Therapy Paige Baldwin1, Shifalika Tangutoori1,2, Srinivas Sridhar1,2 1. Nanomedicine Science and Technology Center, Northeastern University, 2. Radiation Oncology, Dana Farber Cancer Institute
Abstract
Goal
100 μM Drug
50 μM Drug
10 μM Drug
1 μM Drug
500 nM Drug
100 nM Drug
50 nM Drug
No Drug
10 nM Drug
Dose Response
8000 cells/well
4000 cells/well
2000 cells/well
1000 cells/well
Doubling Cycle 500 cells/well
PARP inhibitors (Poly(ADP-ribose) polymerase inhibitors) such as Olaparib are among the most potent molecular inhibitors used in clinical trials for various types of cancers including prostate and ovarian cancers They act by inhibiting DNA damage repair and thus accumulating deleterious mutations leading to genetic instability as a function of number of cell replications . Thus, in this study, we thoroughly characterized and optimized the in vitro model of prostate cancer (PC3, LNCaP, FK01) and ovarian cancer (PA1), in a 96 well system. To compare and contrast the IC-50’s, of free drug and nanoformulated drug, we employed dose response curves generated for each cell line. Prior to this, it was crucial to develop growth rate curves for all cell lines and optimize the seeding density so as to ensure that cell death was not occurring because of over confluence and lack of nutrients, but rather due to the genetic instability caused by the treatment. These curves allow for each cell line to be exposed to the treatment of choice for 4 doubling cycles prior to analysis. Dose response was generated for 4 cell lines, with the 2 different drugs (Olaparib and nano-Olaparib). We determined the IC-50’S of these cells after 4 doubling cycles. These results allowed for comparison between the efficacy of the nanoformulation versus the free drug, thus determining the dose regimen for further invitro studies.
Results
Methods
A)
• Treatment with Olaparib and NanoOlaparib •Quantify the viable cells present after 4 doubling cycles
•MTS converted to Formazan by live cells •Quantify number of cells at varying time points •PC3, LNCaP,FKO1
Results
A)
3 2.5
24 hours
2
48 hours
1.5
72 hours
1
96 hours
0.5
3.5 3 2.5 2
24 hours
1.5
48 hours
1
72 hours
0.5
D)
0 500
1000
2000
4000
500
B)
8000
1000
2000
4000
Figure 2. IC-50’s comparing Olaparib and NanoOlaparib for (A) PC3, (B) FK01, and (C) PA-1. IC-50 for PC3 with Olaparib and NanoOlaparib is ~2.3 μM and ~1.3 μM, respectively. IC-50 for FK01 with Olaparib and NanoOlaparib is ~2.0 μM and ~2.5 μM, respectively. IC-50 for PA-1 with Olaparib and NanoOlaparib is ~2.3 μM and ~3.6 μM, respectively. Doubling time for FK01, PC3, PA-1, and LNCaP (D), based on a nonlinear fit of the normalized data from Figure 1.
8000
Conclusion and Future Work
LNCaP Absorbance
4
C)
Number of cells plated
Number of cells plated
Absorbance at 490 nm
• PARP-1 is a DNA repair protein which plays a role in • Base Excision Repair • Homologous Recombination • Non-Homologous End Joining • Transcriptional Regulation
3.5
0
FK01 Absorbance
4
Absorbance at 490 nm
•Cell lines: •PC3 (Prostate) ETS fusion negative •LNCaP (Prostate) ETS fusion postiveinteracts with PARP-1 •FK01 (Prostate) PTEN and p53 deficient • PA1 (Ovarian) genetic instabilityPARPi sensitive
Absorbance at 490 nm
Background
PC3 Absorbance
4
B)
3.5 3 2.5
72 hours 120 hours
2
168 hours
1.5
216 hours
1
264 hours
•Optimized experimental methods for IC-50’s of Olaparib and NanoOlaparib in 4 cell lines (PC3, FK01, LNCaP, PA-1) •Non-linear fit curves of different cell lines show doubling time of FK01 < PA-1 < PC3 < LNCaP •NanoOlaparib > Olaparib in some cell lines
0.5
C)
0 1000
2000
4000
8000
10000
Number of cells plated
Figure 1. Absorbance indicates the number of viable cells present at a given time. The absorbance of viable cells was quantified and used for further analysis to determine the doubling time for each cell line. The bar graphs above depict the viable cells for PC3 (A), FK01 (B), and LNCaP (C).
Acknowledgements Supported by IGERT grant NSF-DGE- 0965843 and Mazzone Foundation.
•Chemosensitization of Olaparib versus NanoOlaparib •Radiosensitization of Olaparib versus NanoOlaparib •In vivo prostate model testing Olaparib versus NanoOlaparib
References 1. Shifalika Tangutoori, Houari Korideck, Mike Makrigiorgos, Robert Cormack, Srinivas Sridhar. A Novel Nano-Formulation for Systemic Administration of PARPi-Olaparib (NANO-OLAPARIB) for Radiosensitization, Chemo-Sensitization and Combinatorial Therapy in Prostate Cancer. AACR Molecular Targets and Cancer Terapeutics, Boston, October 2013, 10/19/2013-10/23/2013 2. Chatterjee P, Choudhary GS, Sharma A, Singh K, Heston WD, et al. (2013) PARP Inhibition Sensitizes to Low Dose-Rate Radiation TMPRSS2-ERG Fusion Gene-Expressing and PTEN-Deficient Prostate Cancer Cells. PLoS ONE 8(4): e60408. doi:10.1371/journal.pone.0060408
