Download Bisphosphonamidate Clodronate Prodrug Exhibits Selective

Published OnlineFirst December 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0315
Molecular
Cancer
Therapeutics
Small Molecule Therapeutics
Bisphosphonamidate Clodronate Prodrug Exhibits Selective
Cytotoxic Activity against Melanoma Cell Lines
Marie R. Webster1,4, Chandrashekhar Kamat2, Nick Connis2, Ming Zhao2,3, Ashani T. Weeraratna4,
Michelle A. Rudek2,3, Christine L. Hann2, and Caren L. Freel Meyers1
Abstract
Bisphosphonates are used clinically to treat disorders of calcium metabolism and malignant bone disease
and are known to inhibit cancer cell growth, adhesion, and invasion. However, clinical use of these agents for
the treatment of extraskeletal disease is limited because of low cell permeability. We recently described a
bisphosphonamidate prodrug strategy for efficient intracellular release of bisphosphonates, including clodronate (CLO), in non–small cell lung cancer cells. To evaluate anticancer activity of this prodrug class across
many cancer cell types, the bisphosphonamidate clodronate prodrug (CLO prodrug) was screened against the
NCI-60 cell line panel, and was found to exhibit selectivity toward melanoma cell lines. Here, we confirm
efficient cellular uptake and intracellular activation of this prodrug class in melanoma cells. We further
demonstrate inhibition of melanoma cell proliferation, induction of apoptosis, and an antitumor effect of CLO
prodrug in a xenograft model. These data suggest a novel therapeutic application for the CLO prodrug and
potential to selectively target melanoma cells. Mol Cancer Ther; 13(2); 297–306. 2013 AACR.
Introduction
Bisphosphonates are stable analogs of pyrophosphate
bearing a P-C-P linkage, and are used clinically to treat
disorders of calcium metabolism (1, 2). The two classes of
bisphosphonates, non-nitrogen containing bisphosphonates (NNBP) and nitrogen-containing bisphosphonates
(NBP), differ structurally in the substitution pattern at the
bridging carbon, and display distinct mechanisms of
action. The NBP class, bearing nitrogen-containing substituents at the bridging carbon, acts through inhibition of
isoprenoid biosynthesis enzymes, including farnesyl
pyrophosphate synthase (FPPS; refs. 3 and 4), to reduce
FPP levels and prevent downstream protein prenylation
events essential for normal protein function. The NNBP
class, which lacks a nitrogen-containing substituent at the
bridging carbon, is known to undergo conversion to
nonhydrolyzable ATP analogs (i.e., AppCCl2p). These
metabolites are thought to inhibit ATP-dependent processes including the ATP/ADP translocase at the mito-
Authors' Affiliations: Departments of 1Pharmacology and Molecular
Sciences and 2Oncology; 3Analytical Pharmacology Core of the Sidney
Kimmel Comprehensive Cancer Center, Johns Hopkins University School
of Medicine, Baltimore, Maryland; and 4The Wistar Institute, Philadelphia,
Pennsylvania
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Current address for M.R. Webster: The Wistar Institute, Philadelphia, PA.
Corresponding Authors: Caren L. Freel Meyers, WBSB 307B, 725 N.
Wolfe St., Baltimore, MD 21205. Phone: 410-502-4807; Fax: 410-9553023; E-mail: [email protected] or Christine Hann, [email protected]
doi: 10.1158/1535-7163.MCT-13-0315
2013 American Association for Cancer Research.
chondrial membrane, leading to apoptosis in osteoclasts
(5, 6).
Osteolytic lesions develop in a majority of patients with
multiple myeloma, and bone metastases are common in
patients with breast, prostate, and lung cancers (7).
Bisphosphonates with antiresorptive activity are used in
adjuvant therapies to effectively treat malignant bone
disease from advanced cancers. The ability of NBPs to
inhibit cancer cell invasion and adhesion, and exert
growth inhibitory activity in the bone microenvironment
is well documented (7–10). Consequently, there is increasing interest to evaluate clinically used bisphosphonates as
potential therapeutic agents for extraskeletal cancers (1).
The most potent NBP, zoledronate, displays variable
anticancer activity against several cancer cell lines (IC50s
ranging from 14 to >100 mmol/L; refs. 11–15). Inhibition of
FPPS by NBPs leads to a reduction in prenylation of small
G proteins, which are essential for migration, adhesion,
invasion, and proliferation. Furthermore, accumulation of
isopentenyl pyrophosphate (IPP) as a result of FPPS
inhibition leads to immune cell activation (10, 16) and
production of cytotoxic ATP analogs, which induce apoptosis (10, 17). In vitro studies in human melanoma cell
lines showed that the NBPs zoledronate and pamidronate
inhibit proliferation and induce apoptosis (15, 18); however, zoledronate, which was the more potent of the two,
displayed a modest IC50 100 mmol/L in the cell lines
tested. Contributing to these activities in melanoma is the
finding that NBPs inhibit several matrix metalloproteinases (MMP), which are important for tumor invasion and
metastasis (19, 20).
Relevant to this study, the NNBP clodronate is minimally active against breast, ovarian, and thyroid cancer
cell lines (high mmol/L to low mmol/L IC50s; refs. 12, 21,
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and 22), and is inactive against melanoma (15), although it
is reported to inhibit MMPs important for tumor invasion
in melanoma cells (23, 24). In contrast to the limited
preclinical activity observed, results from clinical trials
suggest potential benefits of adjuvant therapy with clodronate for breast cancer in older patients. A recent
clinical trial suggests clodronate increases bone metastasis–free and non-bone metastasis–free intervals, and
recurrence-free interval in postmenopausal women (25).
These results are consistent with two earlier studies showing increases in bone metastasis–free interval and an
overall survival benefit in postmenopausal women (26),
and a disease-free benefit in older patients (27). A study by
Saarto and colleagues (28) suggested no benefit of adjuvant clodronate; however, this study was carried out in a
predominantly premenopausal population.
A primary limitation of the use of bisphosphonates in
treating extraskeletal tumors is poor cell permeability, a
consequence of the highly polar, polyanionic structure of
bisphosphonates under physiological conditions. To
address poor cellular uptake of bisphosphonates, we have
developed a bisphosphonamidate prodrug strategy for
efficient intracellular delivery of CLO and methylene
bisphosphonate (MBP) in non–small cell lung cancer
(NSCLC) cells (29). In each case, the neutral prodrug incorporates two biodegradable nitroaryl groups and two halobutyl amine masking groups (Fig. 1). After cellular uptake,
the nitroaryl delivery groups undergo intracellular activation via an enzymatically driven nitroreduction, resulting
in release of a halobutyl phosphonamidate anion. This
phosphonamidate anion is chemically labile, undergoing
a series of spontaneous cyclization and P-N hydrolysis
reactions to release the fully unmasked bisphosphonate
(Fig. 1). The CLO and MBP prodrugs exhibited significantly
improved anticancer activity against NSCLC cells (low
mmol/L IC50s), compared with the parent bisphosphonates
(no activity up to low mmol/L concentrations), with the
CLO prodrug showing the most potent activity (29).
The CLO prodrug was submitted for analysis against
the NCI-60 panel to evaluate its activity across multiple
tumors types. Here we report the activity of the CLO
prodrug against 9 different cancer cell types, and further
characterization of our CLO prodrug in 2 melanoma cell
lines, SK-Mel-5 and UACC-62. Our results support efficient intracellular activation of bisphosphonamidate
prodrugs in melanoma cells, and demonstrate growth
inhibition (GI) and induction of apoptosis by the CLO
prodrug. We further demonstrate that intratumoral
injection of the CLO prodrug in subcutaneous SK-Mel5 xenografts causes tumor growth inhibition.
Materials and Methods
Cell culture and LC/MS-MS
UACC-62 cells were acquired from the National Cancer
Institute DTP catalog. SK-Mel-5 melanoma cells were
acquired from American Type Culture Collection. Both
cell lines were immediately expanded and multiple ali-
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Mol Cancer Ther; 13(2) February 2014
Figure 1. Bisphosphonamidate prodrugs of CLO and MBP.
quots were cryopreserved. The cell lines were used within
6 months of resuscitation. Peripheral blood mononuclear
cells were a generous gift from Dr. J. Conejo-Garcia (The
Wistar Institute, Philadelphia, PA) and were obtained by
leukapheresis/elutriation from healthy de-identified
donors, under institutional review board’s approval
(EX2100250-2). Keratinocytes were maintained in keratinocyte-SRM (1) media (Gibco; 17005-042). Keratinocytes
were isolated from neonatal foreskins after routine circumcision (EX21212264-1a; The Wistar Institute). UACC62 cells were maintained in RPMI 1640 with 10% FBS, 1%
pen/strep, 1% glutamate, and 1% sodium pyruvate. SKMel-5 cells were maintained in Eagle’s Minimum Essential Media (EMEM) with 10% FBS, 1% pen/strep, and 1%
glutamine. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) experiments were conducted
using a Quadrapole ABI5500 mass spectrometric detector
(Applied Biosystems). The instrument was equipped with
an electrospray interface in positive ion mode, and controlled using the Analyst version 1.2 software (Applied
Biosystems).
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Clodronate Prodrug Is Cytotoxic against Melanoma
NCI-60 cell line screen
The effect of clodronate prodrug on the NCI-60 cell
lines was determined using an assay to detect total
protein. After 48 hours, the assay was quenched with
the addition of 50 mL of cold TCA, and incubation at 4 C
for 60 minutes. The supernatant was removed, the plates
were washed 5 times with tap water, and then air dried.
Sulforhodamine B (SRB, 100 mL) solution [0.4% (w/v) in
1% acetic acids, 100 mL] was added to each well. The
plates were incubated at room temperature (RT) for 10
minutes, unbound stain was removed by washing 5
times with 1% acetic acid, and the plates were allowed
to air dry. Bound stain was solubilized with 10 mmol/L
trizma base, and the absorbance at 515 nm was read
using a plate reader (described on the DTP-NCI website:
dtp.nci.nih.gov).
that caused a 50% decrease in number of cells compared
with control.
Cell-cycle analysis
Cell-cycle distribution was determined using a BD
FACSCalibur flow cytometer. SKMel-5 and UACC-62
melanoma cells were plated at 6.7 104 cells per well in
flat bottom 6-well plates. Cells were dosed as described
earlier. At 24, 48, or 72 hours following drug treatment, the
cells were analyzed as previously described (29). DNA
content was analyzed using a BD FACSCalibur cytometer
with 10,000 propidium iodide (PI)-positive gated events
recorded per sample using CellQuest Pro software.
Cell-proliferation assay
Cell proliferation was determined using the Promega
CellTiter 96 Aqueous One Solution Cell Proliferation
Assay MTS assay. SK-Mel-5 and UACC-62 cells were
plated at 6.0 103 and 2.5 103 cells per well, respectively,
in flat bottom 96-well plates in 99 mL of media and allowed
to adhere overnight. The drugs were serially diluted in
100% dimethyl sulfoxide (DMSO). For each drug treatment group, 1 mL of a 100 stock solution was added to
each well for a final DMSO concentration of 1% (v/v).
Cells were treated for 24, 48, or 72 hours, and analyzed as
previously described (29).
Measurement of caspase-3 activity
SK-Mel-5 and UACC-62 melanoma cells were plated at
6.7 104 cells per well in flat bottom 6-well plates. Cells
were dosed as described earlier. At 24 or 48 hours following drug treatment, the media was collected and the
cells were washed with 1 mL PBS. Each well was trypsinized with 600 mL trypsin for 3 to 5 minutes. The trypsin
reaction was quenched with an equal volume of media.
All supernatants and washes were combined and centrifuged at 1,100 rpm for 5 minutes. The supernatant was
removed, the cells were washed with 1 mL FBS with 1%
PBS. The cells were centrifuged at 1,100 rpm for 5 minutes.
Cells were resuspended in 250 mL Cytofix/Cytoperm
(Pharmingen Kit #2075KK) and incubated at room temperature for 20 minutes. After 20 minutes, cells were
centrifuged at 1,100 rpm for 5 minutes. Cells were washed
2 with Perm/wash solution, centrifuging, and decanting
after each wash. Cells were resuspended in 50 mL Perm/
wash solution, 20 mL FITC Rabbit antiactive caspase-3
antibody (BD Pharmigen; 559341) was added, and samples were incubated at RT away from light. Cells were
spun at 1,100 rpm for 5 minutes. Cells were washed with 1
mL Perm/Wash, spun at 1,100 rpm for 5 minutes, and
resuspend in 300 mL Perm/Wash solution. The cells were
analyzed by flow cytometry and compared with control.
Cells not treated with antibody were used as a negative
control, cells treated with 500 nmol/L velcade (bortezomib) were used as a positive control. Quadrants were set
using the positive control, negative control and 0 mmol/L
clodronate prodrug samples. The percentage of cells in the
upper and lower left was determined by Cell Quest Pro
software.
Trypan blue cell viability assay
SK-Mel-5 and UACC-62 melanoma cells were plated at
7.1 104 and 3 104 cells per well, respectively, in flat
bottom 12-well plates. Cells were dosed as described
earlier. At 24, 48, or 72 hours following drug treatment,
the cells were collected and analyzed as previously
described (29). The absolute number of cells was determined at each drug concentration. The cell number for
each concentration was converted to percentage of control
for each time point, and plotted using GraphPad Prism
4.0. The EC50 was calculated as the concentration of drug
In Vivo Efficacy Study
Female, athymic nude mice (3–4 weeks old, purchased
from Taconic) were injected subcutaneously with 4.5 106
of SK-Mel-5 cells suspended in 100 mL of 1:1 PBS/matrigel.
After tumors reached a volume of 200 mm3, mice were
randomized into treatment groups as follows: treatment
group 1 (4 mice): vehicle twice per week; treatment group
2 (5 mice): drug twice per week. Prodrug was dissolved in
corn oil and administered intratumorally at a final dose of
200 mg/kg. Mice were weighed and tumor volume was
measured twice per week and monitored according to
Detection of intracellular MBP prodrug and
methylenebisphosphonate
SK-Mel-5 and UACC-62 melanoma cells were plated
at 1.3 105 cells per well in flat bottom 6-well plates.
Cells were dosed as described earlier. At 2, 4, or 8 hours
following drug treatment, the media was removed, and
the cells were washed with 1 mL PBS. To each well, 600
mL of trypsin was added and incubated for 3 to 5
minutes. The trypsin reaction was quenched with an
equal volume of media. The cells were transferred to a
15 mL conical and centrifuged at 1,100 rpm for 5 minutes. Supernatant was removed, and the cells were
resuspended in 200 mL of media. The cells were then
treated and analyzed using LC/MS-MS for intracellular
levels of MBP prodrug and derivatized MBP as previously described (29).
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ACUC protocols. Tumor length (mm) and tumor width
(mm) were measured and used to calculate tumor
volumes: [tumor length (mm) (tumor width tumor
width) (mm)2]/2. A 2-tailed paired t test was used to
determine significant difference (P < 0.05) between vehicle- and drug-treated groups.
Results
NCI-60 cell line panel screening reveals selective
toxicity of the CLO prodrug against melanoma cell
lines
The CLO prodrug was submitted to the National Cancer Institute Developmental Therapeutics Program for
screening against the NCI-60 cell line panel. The results
from the NCI-60 5-dose screen were analyzed for 3 different endpoints, including growth inhibition, total
growth inhibition (TGI), and lethal concentration (LC).
The CLO prodrug exhibits general growth inhibitory
activity against all the cell lines tested (Supplementary
Table S1). A TGI concentration, where growth was inhibited 100%, was reported for 18 of 60 cell lines (Table 1), and
the CLO prodrug was shown to be cytotoxic (LC50) against
4 cell lines. A concentration of the CLO prodrug where
50% of the cells died (LC50) could be measured for 4 cell
lines (Table 1); 3 of these were melanoma cell lines (LOX
IMVI, SK-MEL-5, and UACC-62), suggesting that the CLO
Table 1. Results from NCI-60 screen of the CLO
prodrug
Leukemia
NSCLC
Colon cancer
CNS cancer
Melanoma
Renal cancer
Breast cancer
Cell line
TGI
(mmol/L)a
LC50
(mmol/L)b
RMPI-8826
NCI-H522
COLO 205c
HT29
SF-295
SNB-75
LOX IMVIc
MDA-MB-4 35
SK-MEL-2
SK-MEL-5c
UACC257
UACC-62c
A498
RXF 393
MCF7
HS 578T
BT-549
MDA-MB-468
9.12
7.76
7.94
22.2
16.98
6.76
4.64
22.6
6.57
4.24
7.89
5.03
6.82
4.13
1.52
9.21
9.31
8.59
>100
>100
34.4
>100
>100
>100
54.4
>100
>100
8.88
>100
78.1
>100
>100
>100
>100
>100
>100
a
TGI is the minimum concentration of the CLO prodrug
required for total growth inhibition.
b
LC50 is the concentration of the CLO prodrug required to
cause 50% cell death compared with 0 hour.
c
Cell lines where an LC50 could be measured.
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Mol Cancer Ther; 13(2) February 2014
prodrug may exhibit selective cytotoxicity against melanoma. We selected 2 of these cell lines, SK-Mel-5 and
UACC-62, to further evaluate the cellular uptake, intracellular activation, and anticancer activity of the CLO
prodrug.
Bisphosphonamidate prodrugs show enhanced cell
permeability and undergo intracellular activation in
SK-Mel-5 and UACC-62 cells
The activity of the CLO prodrug in melanoma cells is
consistent with increased cell permeability of the bisphosphonamidate prodrug. In a previous study, we demonstrated the chemistry of prodrug activation as well as
intracellular prodrug activation through a combination
of 31P NMR and LC/MS-MS experiments carried out with
the structurally related methylenebisphosphonate (MBP)
prodrug as a model for this prodrug class (29). Likewise,
we have evaluated cellular uptake and intracellular activation of the same model MBP prodrug in SK-Mel-5 and
UACC-62 melanoma cells. Briefly, intracellular levels of
bisphosphonate prodrug were measured in SK-Mel-5 and
UACC-62 melanoma cells using the LC/MS-MS method
previously reported by our group (29). Methylenebisphosphonate released following intracellular activation of the
MBP prodrug (Fig. 2 and Supplementary Fig. S1A) is itself
difficult to detect by mass spectrometry; however, the
corresponding TBS ester (TBS-MBP), generated by derivatization of MBP with N-t-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), is easily detected by LC/
MS-MS. Thus, SK-Mel-5 and UACC-62 melanoma cell
lines were treated with the MBP prodrug at varying
concentrations (0, 10, or 30 mmol/L) for 2, 4, and 8 hours
(Fig. 2 and Supplementary Fig. S1). LC/MS-MS analysis
of cell lysates indicated that intracellular prodrug levels
are achieved in a concentration-dependent manner; at 8
hours, MBP prodrug levels of 175.6 and 408.3 ng/104 cells
were observed in SK-Mel-5 cells treated with 10 and 30
mmol/L MBP prodrug, respectively (Fig. 2B). A similar
observation was made in UACC-62 cells treated with the
MBP prodrug at 8 hours (Supplementary Fig. S1B). The
presence of intracellular bisphosphonate, in SK-Mel-5 and
UACC-62 cells treated with 10 or 30 mmol/L of the MBP
prodrug or 30 mmol/L bisphosphonate, was determined
by detection of the corresponding TBS ester (TBS-MBP)
formed following treatment of cell lysate with MTBSTFA
(Fig. 2C and Supplementary Fig. S1C). Intracellular
bisphosphonate is detected as early as 2 hours in SKMel-5 and UACC-62 cells treated with 10 and 30 mmol/L
MBP prodrug (Fig. 2A and Supplementary Fig. S1A).
CLO prodrug is cytotoxic to SK-Mel-5 and UACC-62
melanoma cell lines
Clodronate has shown little or no activity against multiple cancer cell lines, including melanoma (18); however,
we have previously demonstrated that the cell-permeable
bisphosphonamidate prodrug of CLO displays dramatically increased activity against the A549 (NSCLC) cell line
(29). Results from the NCI-60 screen suggests that the CLO
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Clodronate Prodrug Is Cytotoxic against Melanoma
Figure 2. The bisphosphonamidate
prodrug class is cell permeable and
undergoes intracellular activation
in SK-Mel-5 cells. A, MBP prodrug
permeability and intracellular
activation confirmed by LC/MSMS analysis. B, ion
chromatograms showing absence
of MBP prodrug in untreated cells
(a), and detection of MBP prodrug
in cells treated with 10 mmol/L
4
(175.6 ng/10 cells) (b) and 30
4
mmol/L (408.3 ng/10 cells) (c) MBP
prodrug. C, ion chromatograms
showing absence of tetra-silyl
bisphosphonate following
MTBSTFA reaction of lysate from
untreated cells (a), and detection of
TBS-MBP in cells treated with 30
mmol/L MBP (b), cells treated with
10 mmol/L MBP prodrug (c), and
cells treated with 30 mmol/L MBP
prodrug (d). Ion chromatograms
are offset in the y-dimension for
clarity.
prodrug exhibits a more pronounced affect against SKMel-5 and UACC-62 melanoma cell lines compared with
A549 cells. Growth inhibition was measured in the A549
cells with a GI50 of 4.25 mmol/L (50% less growth compared with control), but TGI and cell death were not
observed. In contrast, TGI and cell death were observed
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in SK-MEL-5 and UACC-62 cells treated with the CLO
prodrug (Table 1). To further characterize this activity, we
evaluated the effect of the CLO prodrug on proliferation
and viability of SK-Mel-5 and UACC-62 melanoma cells,
using the MTS and cell number assays. Dose–response
curves were generated using drug concentrations ranging
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Figure 3. CLO prodrug is
cytotoxic to SK-Mel-5 and
UACC-62 melanoma cell lines. A
and B, MTS assay of SK-Mel-5
(A) or UACC-62 (B) melanoma
cells treated with clodronate for
72 hours (*), or CLO prodrug for
24 hours (&), 48 hours (~), and
72 hours (^); C and D, cell
number, determined using
trypan blue, of SK-Mel-5 (C) or
UACC-62 (D) treated with CLO
prodrug at 24 hours (&), 48 hours
(~), and 72 hours (^).
from 0.3 to 300 mmol/L, and cell proliferation was measured at 24, 48, and 72 hours after drug treatment (Fig. 3A
and B and Supplementary Table S2), using the MTS assay.
As expected, the CLO prodrug displays a remarkable
enhancement in activity compared with clodronate in
both cell lines. The IC50s of CLO prodrug against melanoma cell lines, SK-Mel-5 (7 1 mmol/L) and UACC-62
(11.7 3 mmol/L) at 72 hours, are comparable with the
IC50 values previously determined in A549 cells (4.4 2
mmol/L), suggesting comparable uptake across these cell
lines. In both melanoma cell lines, there is a full antiproliferative effect by 48 hours, whereas in A549 cells a full
effect is not observed until 72 hours. To correlate the
growth inhibition of melanoma cells with viability, absolute cell number at varying drug concentration was determined using a trypan blue exclusion assay. The number of
viable cells at each dose was measured at 0, 24, 48, and 72
hours following drug treatment and plotted as a percentage of control (Fig. 3C and D). The EC50 of the CLO
prodrug in SK-Mel-5 cells is 6.3 3 mmol/L, which is
similar to its IC50 of 6.1 1 mmol/L (determined at 48
hours by the MTS assay; Supplementary Table S2). Similarly, the EC50 of 12.7 5 mmol/L for the CLO prodrug in
UACC-62 cells is comparable with its IC50 of 8 4 mmol/L
at 48 hours; however, in both cell lines there seems to be a
greater antiproliferative effect at 24 hours compared with
the effect on cell viability at the same drug treatment time.
This difference at 24 hours could reflect the time required
for prodrug activation and conversion of released bisphosphonate to the active metabolite. As a measure of general
toxicity, we assessed the effect of CLO prodrug on normal
cells and determined that CLO prodrug does not affect the
viability of PBMCs or primary keratinocytes (Supplementary Fig. S2).
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Mol Cancer Ther; 13(2) February 2014
CLO prodrug induces apoptosis in melanoma cell
lines
To further characterize the mechanism of cytoxicity
induced by the CLO prodrug in SK-Mel-5 and UACC62 cells, we evaluated the effect of drug exposure on cellcycle distribution and induction of apoptosis (Fig. 4A–C).
The cell-cycle profile of SK-Mel-5 and UACC-62 cells
treated with increasing concentrations of CLO prodrug
was analyzed at 24, 48, and 72 hours. A pronounced subG1 peak was observed in cells treated with 10 mmol/L CLO
prodrug after 48 hours, characteristic of caspase-activated
DNase (CAD) activity (Fig. 4C). CAD is downstream of
caspase-3, which catalyzes cleavage of ICAD to CAD (30);
thus, the increased percentage of cells in sub-G0–G1 phase
suggests the activation of caspase-3 (Fig. 4D and E).
Strikingly, the percentage of cells with active CAD
nearly doubles at each time point in SK-Mel-5 cells treated
with 10 mmol/L CLO prodrug, and in UACC-62 cells
treated with >30 mmol/L CLO prodrug. To confirm this,
SK-Mel-5 and UACC-62 cells were treated with increasing
concentrations of the CLO prodrug and assayed for activated caspase-3 at 24 and 48 hours by flow cytometry using
an antibody specific for cleaved caspase-3 (Fig. 4F and G).
Velcade (bortezomib) was used as a positive control for
activated caspase-3–mediated apoptosis. In both cell lines,
significant caspase-3 activity was measured at >3 mmol/L
CLO prodrug, providing support for a mechanism involving induction of apoptosis by CLO prodrug.
Intratumoral delivery of CLO prodrug causes tumor
growth inhibition
On the basis of the promising cytotoxicity of CLO
prodrug in vitro, we sought to determine its activity in a
mouse xenograft model. As a proof of concept that CLO
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Clodronate Prodrug Is Cytotoxic against Melanoma
Figure 4. CLO prodrug induces
apoptosis in melanoma cell lines.
A, cell-cycle analysis of SK-Mel-5
cells after 48 hours of treatment
with 0 mmol/L (A), 3 mmol/L (B), and
10 mmol/L (C) CLO prodrug. D and
E, quantification of percent SKMel-5 (D) or UACC-62 (E) cells in
sub-G0–G1 at 24, 48, and 72 hours
when treated with 0, 1, 3, 10, 30,
and 100 mmol/L CLO prodrug. F
and G, percentage of SK-Mel-5 (F)
or UACC-62 (G) cells positive for
activated caspase-3 at 24 and 48
hours posttreatment with CLO
prodrug. Velcade (500 nmol/L) was
used as a positive control at 24 and
48 hours. Cells (–) drug and (–)
antibody was used as a negative
control at 24 and 48 hours, to
determine background cellular
fluorescence.
prodrug exhibits antitumor effects in vivo, CLO prodrug
was delivered by intratumoral injection into SK-Mel-5
subcutaneous xenografts. Tumor growth was compared
with vehicle control as fold change in tumor size (Fig. 5A).
After 17-day treatment, as compared with a vehicle control
group, statistically significant (P < 0.05) decrease in tumor
growth was observed with twice weekly injection of the
CLO prodrug, compared with control. This difference
further increased after the 19th day (P < 0.001), suggesting
that if good bioavailability in tumor tissues is achieved, an
antitumor effect will be observed. Mouse weight was
measured twice weekly as a measure of general toxicity
(Fig. 5B). In contrast, IT administration of a similar dose of
clodronate did not have a significant effect on tumor
growth of SK-Mel-5 xenografts (Supplementary Fig. S3).
Discussion
Poor cellular uptake has limited the use of bisphosphonates in extraskeletal diseases. Previously, we described a
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prodrug strategy for efficient intracellular delivery of
bisphosphonates to overcome this limitation. Here, we
report the results from evaluation of a bisphosphonamidate clodronate prodrug (CLO prodrug) against the NCI60 cell line panel, a widely used resource, which has been
well-characterized molecularly and pharmacologically.
Results from the screen indicate that the CLO prodrug
seems selectively cytotoxic against melanoma cells lines,
displaying LC50 values in the micromolar range for only 4
cell lines, 3 of which are melanoma lines. Further investigation of CLO prodrug activity against 2 susceptible cell
lines, SK-Mel-5 and UACC-62, confirms remarkably
enhanced activity of the CLO prodrug compared with
clodronate, which presumably results from enhanced cell
permeability and consequently increased intracellular
clodronate concentration. This notion is further supported
by the LC/MS-MS detection of high intracellular levels of
the structurally related model MBP prodrug and released
MBP in both cell lines; however, cytotoxic effects of the
Mol Cancer Ther; 13(2) February 2014
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Webster et al.
Figure 5. Intratumoral delivery of CLO prodrug causes tumor growth inhibition of SK-Mel-5 xenografts. A, tumor size of CLO prodrug-treated mice compared
with control was followed for 4 weeks. There was a significant TGI of CLO prodrug (&) compared with control (*) in mice dosed twice per week. B, weights of
nude mice bearing xenografts treated with IT CLO prodrug (&) were compared with control (*). A statistically significant (P < 0.05) decrease in tumor growth
was observed with the CLO prodrug, compared with control. , this difference further increased after the 19th day (P < 0.001).
prodrug itself cannot be ruled out. Interestingly, the CLO
prodrug seems to exert its anticancer effects by a distinct
mechanism in melanoma cells. Our previous investigation of the CLO prodrug in A549 cells revealed a somewhat less potent effect on cell viability compared with
growth inhibition, and these effects generally correlated
with a mechanism involving G1-phase cell-cycle arrest. In
contrast, the low micromolar activity of the CLO prodrug
in growth inhibition and cell viability assays in melanoma
cells seems to correlate with induction of apoptosis in both
cell lines, as evidenced by the appearance of a sub-G0–G1
peak in cell-cycle analysis experiments and increased
activation of caspase-3 in both cell lines. Induction of
apoptosis is consistent with a mechanism involving CLO
prodrug activation to release CLO and subsequent conversion of CLO to the nonhydrolyzable ATP analog
(AppCl2p), a known inhibitor of mitochondrial ADP/
ATP translocase (5, 6).
Interestingly, although clodronate exhibits minimal
activity against cancer cell lines, studies by Yang and
colleagues highlight induction of apoptosis in human
thyroid cancer cell lines correlating with calcium release
from mitochondrial and endoplasmic reticulum that leads
to membrane depolarization of mitochondria (21, 22).
Nevertheless, CLO exhibits only weak growth inhibitory
activity in these cells lines, presumably as a result of poor
cellular uptake. It is tempting to speculate that a similar
effect on intracellular calcium signaling may underlie the
potent, selective cytotoxicity of the CLO prodrug against
melanoma cells, as calcium signaling is known to be
crucial in melanoma progression (31–33); however, further investigation is required to determine the molecular
mechanism of action of the CLO prodrug in melanoma
cells.
The surprising selective cytotoxicity of the CLO prodrug against SK-Mel-5 and UACC-62 cells suggests a new
application of bisphosphonates in melanoma therapy and
offers a solution to the problem of poor cellular uptake of
this class. Indeed, preliminary evaluation of the in vivo
antitumor effect of the prodrug is promising, as intratu-
304
Mol Cancer Ther; 13(2) February 2014
moral injection of SK-Mel-5 xenografts causes significant
tumor growth inhibition.
Advances in molecular targeting and immunotherapy
have added to the current treatment options for patients
with metastatic melanoma. BRAF, a key member of the
MAPK signaling cascade, is mutated at the V600 residue
in 45% of metastatic melanoma cases and is predictive for
responsiveness to the tyrosine kinase inhibitor, vemurafenib (34). Notably, the cell lines on the NCI-60 panel, most
sensitive to the CLO prodrug (Supplementary Table S1)
all harbor a BRAFV600E mutation, which could suggest that
the CLO prodrug may have an effect on RAF function;
however, we note that other cell lines harboring the
BRAFV600E mutation (Supplementary Table S1) are not
susceptible to the cell killing effects of CLO prodrug. A
second newly approved therapy, Ipilimumab, is a monoclonal antibody that targets the immune checkpoint,
CTLA-4, and was shown to significantly improve overall
survival; a favorable response was observed in 20% of
patients (35, 36). In 2011, both vemurafenib and ipilimumab received approval of the U.S. Food and Drug Administration for the treatment of patients with metastatic
melanoma. Although each represents an improvement
for treatment of patients with melanoma, their use is
restricted to specific subsets of patients with metastatic
melanoma, and neither agent is curative. Thus, for
patients who are not candidates for either therapy, or for
those who progress while on therapy, treatment with
DNA damaging agents, such as dacarbazine and temozolomide, remain the only therapeutic options (37, 38).
Response rates to these agents are low, and long-term
benefit is rare; thus, there continues to be great need for the
development of melanoma-specific treatments.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: M.R. Webster, C.L. Hann, C.L. Freel Meyers
Development of methodology: M.R. Webster, C. Kamat, M. Zhao, M.A.
Rudek, C.L. Hann
Molecular Cancer Therapeutics
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Published OnlineFirst December 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0315
Clodronate Prodrug Is Cytotoxic against Melanoma
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M.R. Webster, C. Kamat, N. Connis, A.T. Weeraratna, M.A. Rudek, C.L. Hann
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M.R Webster, C. Kamat, M. Zhao, C.L.
Hann, C.L. Freel Meyers
Writing, review, and/or revision of the manuscript: M.R. Webster, C.
Kamat, N. Connis, A.T. Weeraratna, M.A. Rudek, C.L. Hann, C.L. Freel
Meyers
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.R. Webster
Study supervision: C. Kamat, C.L. Hann, C.L. Freel Meyers
Acknowledgments
The authors thank Dr. M. Herlyn (The Wistar Institute) for supplying
keratinocytes and Dr. J. Conejo-Garcia (The Wistar Institute) for supplying
PBMCs for general toxicity studies and I. Dobromilskaya (Johns Hopkins
University) for technical help with in vivo studies.
Grant Support
C.L. Hann received an award from the Flight Attendants Medical
Research Institute. M.A. Rudek was supported by the Analytical Pharmacology Core of the Sidney Kimmel Comprehensive Cancer Center at
Johns Hopkins University (NIH grants P30 CA006973 and UL1 RR025005),
the Shared Instrument Grant (1S10RR026824-01) and an award from
the Johns Hopkins FAMRI Center of Excellence. The project described
was supported in part by grant number UL1 RR025005 from the National
Center for Research Resources (NCRR), a component of the National
Institutes of Health (NIH) and NIH Roadmap for Medical Research.
C.L. Freel Meyers was supported by NIH grant AI099704 and an award
from the Johns Hopkins FAMRI Center of Excellence. M.R. Webster was
supported by an award from the Johns Hopkins FAMRI Center of Excellence and training grant (2 T32 CA 9171-36) at The Wistar Institute.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received April 26, 2013; revised October 14, 2013; accepted November
17, 2013; published OnlineFirst December 5, 2013.
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Molecular Cancer Therapeutics
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Published OnlineFirst December 5, 2013; DOI: 10.1158/1535-7163.MCT-13-0315
Bisphosphonamidate Clodronate Prodrug Exhibits Selective
Cytotoxic Activity against Melanoma Cell Lines
Marie R. Webster, Chandrashekhar Kamat, Nick Connis, et al.
Mol Cancer Ther 2014;13:297-306. Published OnlineFirst December 5, 2013.
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