Transcript Document

MODULATION OF INTRACELLULAR CERAMIDE USING POLYMERIC NANOPARTICLES
TO OVERCOME MULTIDRUG RESISTANCE IN CANCER CELLS
Lilian E. van
1
Vlerken ,
Dinesh
1
Shenoy ,
Zhenfeng
2
Duan ,
Michael
2
Seiden ,
Shashi
3
Mehta ,
and Mansoor
1
M. Amiji
1Department
of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston MA 02115
2Department of Hematology and Oncology, Massachusetts General Hospital, Harvard Medical School, Boston MA 02114
3Department of Pathology, Roger Williams Medical Center, Providence, RI 02908
Abstract
The development of multidrug resistance (MDR) in many tumor
types is a major barrier to successful anti-cancer therapy. One of
the mechanisms that leads to such chemoresistance is inhibition
of apoptotic signaling in MDR cancer cells through glycosylation
of the apoptotic mediator ceramide. The purpose of this study
was to investigate whether MDR could be reverted by coadministering exogenous Ceramide with a chemotherapeutic
(Paclitaxel), co-encapsulated in polymeric nanoparticles to
produce a multifunctional anticancer therapy. For starters,
accumulation and localization of the nanoparticles intracellularly
was observed by fluorescent microscopy. Next, to determine
efficacy of this novel therapeutic strategy, the percent cell death
of drug sensitive vs. multidrug resistant cancer cells in response
to the nanoparticle treatment was quantified. Lastly, apoptotic
activity in response to the paclitaxel/ceramide co-therapy was
measured. Results indicate that nanoparticle delivery of the cotherapy reduces chemoresistance of the MDR cells to paclitaxel
100-fold, to produce a chemosensitivity profile in the MDR cells
that is similar to their drug-sensitive counterpart cell-line. Cotherapy of ceramide with paclitaxel appeared to increase
apoptotic activity 2 fold in the MDR cells, suggesting that delivery
of exogenous ceramide reinstates the apoptotic signal to
resensitize MDR cells to chemotherapy. In conclusion, a
combination therapy of paclitaxel with ceramide, delivered in
polymeric nanoparticles, appears to greatly re-sensitize drug
resistant ovarian tumor cells to chemotherapy. The results
demonstrate the promising potential for clinical use of this
therapeutic strategy to overcome MDR.
Objective
Materials and Methods
The purpose of this study was develop a novel therapeutic
approach using polymeric nanoparticles for co-administration of
ceramide with the chemotherapeutic paclitaxel, to overcome
MDR in ovarian cancer.
› PEO-PCL nanoparticles were prepared by the controlled
› Intracellular
Introduction
a cross-resistance to a multitude of structurally and
functionally unrelated drugs.
The development of MDR poses a great problem in cancer
therapy, where tumors survive despite invasive
chemotherapy.
Among the many mechanisms responsible for
development of MDR in the cancer cell, alterations in
apoptotic signaling appears to greatly contribute to the
phenomenon
Many MDR cancer cells overexpress Glucosylceramide
Synthase (GCS), which converts the apoptotic signaling
mediator ceramide to an inactive form (glucosylceramide),
rendering the apoptotic signal incomplete.
Exogenously administering ceramide to MDR cells should
overcome this blockade and reinstate apoptotic signaling
initiated by chemotherapeutic stress.
Nanoparticles are useful drug delivery vehicles for cancer
therapy, since they 1) protect the drug from inactivation
and metabolism inside the body, 2) preferentially
accumulate at the tumor site by enhanced permeability
and retention, and 3) deposit the drug, or a combination of
drugs, intracellularly.
›
›
›
›
›
120
C.
› PEO-PCL nanoparticles are spherical in shape with a
mean diameter of 211.6 ±1.8 nm (figure 1a)
› PTX and CER nanoparticles are readily endocytosed by
›
›
SKOV3 and SKOV3TR cells to release the drug load
intracellularly by 6 hours (figure 1b, 1c)
CER co-treatment not only greatly re-sensitizes the MDR
cells to PTX chemotherapy, but also increases cell-kill
efficacy in DS cells. Delivery of the drugs encapsulated in
PEO-PCL nanoparticles enhances chemosensitization of
both DS and MDR cells (figure 3a,3b).
PTX/CER co-treatment increases apoptotic activity 2-fold in
both the MDR and DS cells (figure 4a,4b), suggesting that
exogenous CER reinstates apoptotic signaling, triggered by
PTX, in the MDR cells.
% cell viability
100
SKOV3TR
80
Figure 2 -. Dose
response
relationship
of
SKOV3
vs.
SKOV3TR cells
to PTX.
60
40
0
0.0001
0.001
0.01
0.1
1
10
100
› The wildtype (drug sensitive (DS)) human ovarian cancer
cell line SKOV3 was maintained in culture alongside an
SKOV3TR subculture selected for MDR in the presence of
PTX.
› For cytotoxicity studies, the DS and MDR cells were
subjected to dose-response studies against PTX, CER and
PTX combined with CER, delivered as free drugs in
solution or delivered in PEO-PCL nanoparticles. The Pglycoprotein inhibitor Verapimil (VPM) was used as a
positive control for MDR modulation. Resulting cell
death/viability after 6 days of treatment was measured by
the MTS (formazan) assay.
› Apoptotic activity in response to PTX/CER co-treatment
was measured by Yo-Pro-1 and propidium iodide staining
(Vybrant #7, Invitrogen), and quantitated by Laser
Scanning Cytometry and fluorescence microscopy.
Conclusion
A therapeutic strategy that co-administers PTX with CER,
delivered in polymeric nanoparticles significantly re-sensitizes
drug resistant tumor cells to chemotherapy, while also increasing
chemosensitivity of DS cancer cells. The results demonstrate the
promising potential for clinical use of this therapeutic strategy as
an anticancer treatment for MDR as well as non-MDR cancer
types.
[PTX] (mM)
SKOV3
A.
PTX
PTX + CER
120
100
80
60
40
20
0
p<0.001
120
100
80
60
40
20
0
p<0.001
control CER
60
A.
SKOV3TR
B.
% cell viability
B.
SKOV3
20
% cell viability
accumulation of the PEO-PCL
nanoparticles
loaded
with
rhodamine-PTX (red) and NBDCER (green) into SKOV3 (B) and
SKOV3TR (C) cells after 6 hours
incubation.
% apoptotic cells
Figure 1 – Imaging of PEO-PCL
nanoparticles by Scanning Electron
Microscopy (A), and intracellular
nanoparticle trafficking was performed by
loading the PEO-PCL nanoparticles with rhodamine-PTX
(0.1% w/w) or NBD-C6-CER (0.1% w/w), and incubating
the particles with the DS and MDR cells for 0.5, 1, 2, or 6
hours. Cells were fixed, and imaged by fluorescence
microscopy.
› Multidrug Resistance (MDR) refers to the development of
Results
A.
solvent displacement and loaded with 10% w/w paclitaxel
(PTX) or 20% w/w C6-ceramide (CER).
PTX 10 nM
PTX 100 nM
p<0.05
B.
120
100
80
60
40
20
0
PTX
PTX + CER
PTX + VPM
p<0.001
*
*
140
120
100
80
60
40
20
0
-20
p<0.001
p<0.001
*
control CER PTX 100 nM
p<0.05
40
*
Figure 3 - Therapeutic efficacy of
PTX/CER nanoparticle treatment.
Percent cell viability of SKOV3 (A)
and SKOV3TR (B) after 6 days of
PTX, CER, or PTX/CER treatment
delivered as free drug (top panel) or
within
PEO-PCL
nanoparticles
(bottom panel). PTX/VPM treatment
(SKOV3TR) is a positive control for
MDR modulation.
(* indicates
statistical significance of p<0.001
between PTX and PTX/VPM
treatment; n=8 samples/group)
PTX 1000 nM
Acknowledgements
20
0
control
CER
PTX PTX + CER
control
CER
PTX PTX + CER
Figure 4 - Percent apoptotic SKOV3 (A) and SKOV3TR (B) cells in response to the
CER, PTX or the PTX/CER treatment after 24 hours. SKOV3 cells (A) received 10 nM
PTX and/or 10 mM CER, while SKOV3TR cells (B) received 1 mM PTX and/or 10 mM
CER. (n=6 samples/group)
L.E. van Vlerken is a recipient of an NSF IGERT fellowship in
nanomedical science and technology.
This study was
supported by NIH grants CA-095522 and CA-119617.