Ph.D. Abstract ID # 1875
Graduate Category :Engineering and Technology Degree Seeking : Ph.D. Abstract ID # 1875
The Therapeutic Effect of Epigenetic Drug-encapsulating-lipid Nanoemulsions for Triple Negative Breast Cancer Cells Bumjun Kim1, Debra Auguste1, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115 Dept. of Chemical Engineering Opportunity
Approach
The plasticity of cancer epigenetics make them plausible candidates for therapeutic intervention. Decitabine (DAC) and Panobinostat (PAN) were shown to reverse abnormal methylation of DNA and altered chromatin structure, respectively, leading to the increased expression of tumor suppressor genes. Although DAC and PAN have therapeutic benefits, their use is limited by chemical instability and hydrophobicity to achieve effective therapeutic doses. We took advantage of elevated expression of lysophosphatidic acid receptors (LPARs) in advanced breast cancer tissues to target DAC and PAN to breast cancer cells. Herein, we present LPAR1- targeted-lipid nanoemulsions (LNEs) encapsulating both DAC and PAN. Our results showed that the uptake of LNEs was dependent on LPAR1 expression in triple negative breast cancer (TNBC) cell lines. DAC/PAN-LNEs were significantly more cytotoxic to TNBC cells than non-TNBCs. DAC/PAN-LNEs were effective in inhibiting the growth and migration of mesenchymal breast cancer cells that overexpress mFOXM1 by restoring mCDH1/E-cadherin and suppressing mFOXM1 expression while ineffective to epithelial breast cancer cells that inherently express low mFOXM1 and high mCDH1. Overall, we successfully designed LPAR1-targeted LNEs that can selectively suppress mCDH1(low)/mFOXM1(high) TNBC cell lines.
Lipid nanoemulsions (LNEs) were comprised of an oleic acid, cholesteryl oleate as the core, and lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC) and ly1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] as emulsifiers. Solvent-injection method was adopted to fabricate the stable LNE. Malvern Zeta sizer, Brookheaven Zeta Potential Analyzer, and TEM(Jeol2100) were used for the characterization of LNE. The encapsulation efficiency was measured using LC/MS. LNE was dispersed in DMEM culture media with 10% FBS and incubated at 37°C and 95% O2 / 5% CO2. The size and the zeta potential of LNE were tracked for 3 days. Three different ratios of LPC and LPA(1:0, 1:1, and 0:1) were used to fabricate LNEs and the binding affinity was evaluated. Briefly, cells were seeded into 6 well plates (3*105 cells/well) and preincubated overnight. Each cell line was treated with three different formulations of Rhodamine123 encapsulating LNEs (0.12 mg/ml) for two hours, and the uptake efficiency was quantified by flow cytometry. For cell viability, cells were seeded into 96 well plates (5000 cells/well) and applied with the different concentration of drugs and LNE after overnight pre-incubation. After 48 hrs incubation, Alamar Blue assay was performed and cell viability was examined with a microplate reader. For the cell proliferation, MDA-MB231 were plated on 6 well plates(half million cells/well) and different drugs and LNE were applied following day. After 24 hrs incubation with different drugs and LNE, cells in each group were passaged on 96 well plates and proliferation was observed for 5 days. All the medium and drugs were replaced every two days. All the gene analysis was performed using qPCR after 20 hours of LNEs treatment. To compare the surface E-cadherin levels of different breast cancer cell lines, flow cytometry was used to detect the fluorescence from Alexa Fluor® 647 tagged anti-human CD324 on breast cancers.
Data or Results 4. Screening of the Methylation Status in the Promoter Regions of MDA-MB-231 after DAC/PAN LNEs Treatment
1. LPAR1 and G2A Expression Levels in Different Cell Lines and Uptake of Rho-LNEs presenting LPA and LPC Figure 1(B)
Figure 1(A)
In summary, we developed LPAR1-targeted LNEs encapsulating DAC and PAN to selectively induce cytotoxicity to TNBCs. For the first time, we showed that LPAR1 overexpressing TNBCs can be a targeted using LPA-presented LNEs. Nanopackaging improved the stability of DAC, further suppressing the cell growth of MDA-MB-231. DAC/PAN delivery through LNEs restored mCDH1/E-cadherin expression and suppressed the mFOXM1 expression in MDA-MB-231, leading to a decrease in cell migration and cell viability. We also showed that DAC/PAN-LNEs selectively killed mCDH1(low)/mFOXM1(high) aggressive TNBCs. In vivo experiment may be required to further validate the therapeutic efficacy of LPAR1-targeted LNEs encapsulating DAC and PAN. However, our study showed that DAC/PAN treatment using LNEs may be a good alternative to Dox treatment for TNBCs.
Figure 4(B)
Figure 4(A)
Impact
2. Charaterization of LNEs Figure 2(A)
Acknowledgements
Figure 2(B) Size(nm)
PDI
Zeta potential(mV)
BLANK-E
188.0±0.7
0.118
-14.8±1.0
DAC-E
192.4±0.5
0.170
-12.2±0.6
PAN-E
199.3±2.2
0.136
-13.7±0.1
200.5±1.0 1(A)
0.103
-14.7±1.8
COCK-E Figure
DAC Encapsulation Efficiency(%)
PAN Encapsulation Efficiency(%)
I would like to acknowledge my mentor Dr. Debra Auguste for allowing me to have a chance to work on this project. I also would like to thank Auguste Lab members for helping me, CUNY Advanced Science Research Center for mass spectral data, RCMI and CCNY Electron Microscopy Center for allowing me to use FACs, confocal microscope, and TEM, and finally thank National Institutes of Health(NIH) for supporting this work(DP2-CA174495-01).
33.0±3.7
5. Restoration of CDH1 and Surface E-Cadherin upon DAC/PAN LNEs Treatment
68.2±0.5 48.8±3.2
65.1±0.7
Figure 2(C)
Figure 5(A)
**
Figure 5(C)
Figure 5(B)
Figure 5(D)
**
1. Weigelt, B., et al., J Pathol, 2008. 216(2): p. 141-50. 2. Haffty, B.G., et al., J Clin Oncol, 2006. 24(36): p. 5652-7. 2. Haffty, B.G., et al., J Clin Oncol, 2006. 24(36): p. 5652-7. 3. Caram, M.E., et al., Breast Cancer Res Treat, 2015. 152(1): p. 163-72. 4. Raychaudhuri et al., Cancer Res July 1 2011 (71) (13) 4329-4333
** **
6. Suppression of FOXM1 upon DAC/PAN LNEs Treatment 3. The Effect of LNEs on Cell Viability on Migration Figure 3(A)
Figure 3(B)
Figure 6(A) **
Figure 3(D)
Figure 3(C)
Figure (C)
** **
**
* **
Figure 1(A)
Figure 6(B)
Figure 1(B)
References
**
The Therapeutic Effect of Epigenetic Drug-encapsulating-lipid Nanoemulsions for Triple Negative Breast Cancer Cells Bumjun Kim1, Debra Auguste1, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115 Dept. of Chemical Engineering Opportunity
Approach
The plasticity of cancer epigenetics make them plausible candidates for therapeutic intervention. Decitabine (DAC) and Panobinostat (PAN) were shown to reverse abnormal methylation of DNA and altered chromatin structure, respectively, leading to the increased expression of tumor suppressor genes. Although DAC and PAN have therapeutic benefits, their use is limited by chemical instability and hydrophobicity to achieve effective therapeutic doses. We took advantage of elevated expression of lysophosphatidic acid receptors (LPARs) in advanced breast cancer tissues to target DAC and PAN to breast cancer cells. Herein, we present LPAR1- targeted-lipid nanoemulsions (LNEs) encapsulating both DAC and PAN. Our results showed that the uptake of LNEs was dependent on LPAR1 expression in triple negative breast cancer (TNBC) cell lines. DAC/PAN-LNEs were significantly more cytotoxic to TNBC cells than non-TNBCs. DAC/PAN-LNEs were effective in inhibiting the growth and migration of mesenchymal breast cancer cells that overexpress mFOXM1 by restoring mCDH1/E-cadherin and suppressing mFOXM1 expression while ineffective to epithelial breast cancer cells that inherently express low mFOXM1 and high mCDH1. Overall, we successfully designed LPAR1-targeted LNEs that can selectively suppress mCDH1(low)/mFOXM1(high) TNBC cell lines.
Lipid nanoemulsions (LNEs) were comprised of an oleic acid, cholesteryl oleate as the core, and lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC) and ly1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] as emulsifiers. Solvent-injection method was adopted to fabricate the stable LNE. Malvern Zeta sizer, Brookheaven Zeta Potential Analyzer, and TEM(Jeol2100) were used for the characterization of LNE. The encapsulation efficiency was measured using LC/MS. LNE was dispersed in DMEM culture media with 10% FBS and incubated at 37°C and 95% O2 / 5% CO2. The size and the zeta potential of LNE were tracked for 3 days. Three different ratios of LPC and LPA(1:0, 1:1, and 0:1) were used to fabricate LNEs and the binding affinity was evaluated. Briefly, cells were seeded into 6 well plates (3*105 cells/well) and preincubated overnight. Each cell line was treated with three different formulations of Rhodamine123 encapsulating LNEs (0.12 mg/ml) for two hours, and the uptake efficiency was quantified by flow cytometry. For cell viability, cells were seeded into 96 well plates (5000 cells/well) and applied with the different concentration of drugs and LNE after overnight pre-incubation. After 48 hrs incubation, Alamar Blue assay was performed and cell viability was examined with a microplate reader. For the cell proliferation, MDA-MB231 were plated on 6 well plates(half million cells/well) and different drugs and LNE were applied following day. After 24 hrs incubation with different drugs and LNE, cells in each group were passaged on 96 well plates and proliferation was observed for 5 days. All the medium and drugs were replaced every two days. All the gene analysis was performed using qPCR after 20 hours of LNEs treatment. To compare the surface E-cadherin levels of different breast cancer cell lines, flow cytometry was used to detect the fluorescence from Alexa Fluor® 647 tagged anti-human CD324 on breast cancers.
Data or Results 4. Screening of the Methylation Status in the Promoter Regions of MDA-MB-231 after DAC/PAN LNEs Treatment
1. LPAR1 and G2A Expression Levels in Different Cell Lines and Uptake of Rho-LNEs presenting LPA and LPC Figure 1(B)
Figure 1(A)
In summary, we developed LPAR1-targeted LNEs encapsulating DAC and PAN to selectively induce cytotoxicity to TNBCs. For the first time, we showed that LPAR1 overexpressing TNBCs can be a targeted using LPA-presented LNEs. Nanopackaging improved the stability of DAC, further suppressing the cell growth of MDA-MB-231. DAC/PAN delivery through LNEs restored mCDH1/E-cadherin expression and suppressed the mFOXM1 expression in MDA-MB-231, leading to a decrease in cell migration and cell viability. We also showed that DAC/PAN-LNEs selectively killed mCDH1(low)/mFOXM1(high) aggressive TNBCs. In vivo experiment may be required to further validate the therapeutic efficacy of LPAR1-targeted LNEs encapsulating DAC and PAN. However, our study showed that DAC/PAN treatment using LNEs may be a good alternative to Dox treatment for TNBCs.
Figure 4(B)
Figure 4(A)
Impact
2. Charaterization of LNEs Figure 2(A)
Acknowledgements
Figure 2(B) Size(nm)
PDI
Zeta potential(mV)
BLANK-E
188.0±0.7
0.118
-14.8±1.0
DAC-E
192.4±0.5
0.170
-12.2±0.6
PAN-E
199.3±2.2
0.136
-13.7±0.1
200.5±1.0 1(A)
0.103
-14.7±1.8
COCK-E Figure
DAC Encapsulation Efficiency(%)
PAN Encapsulation Efficiency(%)
I would like to acknowledge my mentor Dr. Debra Auguste for allowing me to have a chance to work on this project. I also would like to thank Auguste Lab members for helping me, CUNY Advanced Science Research Center for mass spectral data, RCMI and CCNY Electron Microscopy Center for allowing me to use FACs, confocal microscope, and TEM, and finally thank National Institutes of Health(NIH) for supporting this work(DP2-CA174495-01).
33.0±3.7
5. Restoration of CDH1 and Surface E-Cadherin upon DAC/PAN LNEs Treatment
68.2±0.5 48.8±3.2
65.1±0.7
Figure 2(C)
Figure 5(A)
**
Figure 5(C)
Figure 5(B)
Figure 5(D)
**
1. Weigelt, B., et al., J Pathol, 2008. 216(2): p. 141-50. 2. Haffty, B.G., et al., J Clin Oncol, 2006. 24(36): p. 5652-7. 2. Haffty, B.G., et al., J Clin Oncol, 2006. 24(36): p. 5652-7. 3. Caram, M.E., et al., Breast Cancer Res Treat, 2015. 152(1): p. 163-72. 4. Raychaudhuri et al., Cancer Res July 1 2011 (71) (13) 4329-4333
** **
6. Suppression of FOXM1 upon DAC/PAN LNEs Treatment 3. The Effect of LNEs on Cell Viability on Migration Figure 3(A)
Figure 3(B)
Figure 6(A) **
Figure 3(D)
Figure 3(C)
Figure (C)
** **
**
* **
Figure 1(A)
Figure 6(B)
Figure 1(B)
References
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