Monoamine oxidase a inhibitor-near-infrared dye conjugate reduces prostate tumor growth

Article abstract of DOI:10.1021/ja512613j

Development of anti-cancer agents with high tumor-targeting specificity and efficacy is critical for modern multidisciplinary cancer research. Monoamine oxidase A (MAOA), a mitochondria-bound enzyme, degrades monoamine neurotransmitters and dietary monoamines. Recent evidence suggests a correlation between increased MAOA expression and prostate cancer (PCa) progression with poor outcomes for patients. MAOA induces epithelial-mesenchymal transition (EMT) and augments hypoxic effects by producing excess reactive oxygen species. Thus, development of MAOA inhibitors which selectively target tumors becomes an important goal in cancer pharmacology. Here we describe the design, synthesis, and in vitro and in vivo evaluation of NMI, a conjugate that combines a near-infrared dye for tumor targeting with the moiety derived from the MAOA inhibitor clorgyline. NMI inhibits MAOA with low micromolar IC₅₀, suppresses PCa cell proliferation and colony formation, and reduces migration and invasion. In mouse PCa xenografts, NMI targets tumors with no detectable accumulation in normal tissues, providing effective reduction of the tumor burden. Analysis of tumor specimens shows reduction in Ki-67⁺ and CD31⁺ cells, suggesting a decrease of cell proliferation and angiogenesis and an increase in M30⁺ cells, indicating increased apoptosis. Gene expression profiles of tumors treated with NMI demonstrate reduced expression of oncogenes FOS, JUN, NFKB, and MYC and cell cycle regulators CCND1, CCNE1, and CDK4/6, along with increases in the levels of tumor suppressor gene TP53, cell cycle inhibitors CDKN1A and CDKN2A, and MAOA-downstream genes that promote EMT, tumor hypoxia, cancer cell migration, and invasion. These data suggest that NMI exerts its effect through tumor-targeted delivery of a MAOA-inactivating group, making NMI a valuable anti-tumor agent.

Full text of DOI:10.1021/ja512613j

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Article
Monoamine Oxidase A Inhibitor – Near-Infrared
Dye Conjugate Reduces Prostate Tumor Growth
Jason Boyang Wu, Tzu-Ping Lin, John David Gallagher, Swati Kushal,
Leland W. K. Chung, Haiyen Zhau, Bogdan Z Olenyuk, and Jean C. Shih
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja512613j • Publication Date (Web): 13 Jan 2015
Downloaded from http://pubs.acs.org on January 21, 2015
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Monoamine Oxidase A Inhibitor – NearꢀInfrared
Dye Conjugate Reduces Prostate Tumor Growth
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Jason Boyang Wu,† TzuꢀPing Lin,§,‡ John D. Gallagher, ‡ Swati Kushal,‡
Leland W.K. Chung,† Haiyen E. Zhau,*,† Bogdan Z. Olenyuk,*, ‡,ǁ and Jean C. Shih*, ‡,
⊥
Addresses:
†UroꢀOncology Research Program, Department of Medicine, Samuel Oschin Comprehensive
Cancer Institute, CedarsꢀSinai Medical Center, Los Angeles, CA 90048, U.S.A.
‡Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of
Southern California, Los Angeles, CA 90089, U.S.A.
⊥Department of Cell and Neurobiology, Keck School of Medicine, University of Southern
California, 1975 Zonal Ave., Los Angeles, CA 90033, U.S.A.
ǁUSC Norris Comprehensive Cancer Center, 1441 Eastlake Avenue, Los Angeles, CA 90033,
U.S.A.
§Current addresses: Department of Urology, Taipei Veterans General Hospital, Taipei, Taiwan
11217, R.O.C., and Department of Urology, School of Medicine, and ShuꢀTien Urological
Research Center, National YangꢀMing University, Taipei, Taiwan 11217, R.O.C.
*Corresponding authors:
Jean C. Shih, Department of Pharmacology and Pharmaceutical Sciences, 1985 Zonal Ave.,
PSC 518, Los Angeles, CA 90089, U.S.A. Tel.: +1ꢀ323ꢀ442ꢀ1441; Eꢀmail: jcshih@usc.edu
Bogdan Z. Olenyuk, Department of Pharmacology and Pharmaceutical Sciences, 1985 Zonal
Ave., PSC B15C, Los Angeles, CA 90089, U.S.A. Tel.: +1ꢀ323ꢀ442ꢀ1390; Eꢀmail:
bogdan@usc.edu.
Haiyen E. Zhau, UroꢀOncology Research Program, Department of Medicine, Samuel Oschin
Comprehensive Cancer Institute, CedarsꢀSinai Medical Center, Los Angeles, CA 90048, U.S.A.
Tel.: +1ꢀ310ꢀ423ꢀ5992; Eꢀmail: haiyen.zhau@cshs.org
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Abstract
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Development of anticancer agents with high tumorꢀtargeting specificity and efficacy are critical
goals of modern multidisciplinary cancer research. Monoamine oxidase A (MAOA), a
mitochondriaꢀbound enzyme, degrades monoamine neurotransmitters and dietary monoamines.
Recent evidence suggests correlation between increased MAOA expression and prostate cancer
(PCa) progression with poor outcomes for patients. MAOA induces epithelialꢀmesenchymal
transition (EMT) and augments hypoxic effects by producing excess reactive oxygen species.
Thus, development of MAOA inhibitors which selectively target tumors becomes an important
goal in cancer pharmacology. Here we describe the design, synthesis, in vitro and in vivo
evaluation of NMI, a conjugate that combines a nearꢀinfrared dye for tumor targeting with the
moiety derived from MAOA inhibitor clorgyline. NMI inhibits MAOA with low micromolar
IC50, suppresses PCa cell proliferation, colony formation and reduces migration and invasion. In
mouse PCa xenografts, NMI targets tumor with no detectable accumulation in normal tissues,
providing effective reduction of the tumor burden. Analysis of tumor specimens shows
reduction in Kiꢀ67+ and CD31+ cells, suggesting decrease of cell proliferation and angiogenesis,
and increase in M30+ cells, indicating increased apoptosis. Gene expression profiles of tumors
treated with NMI demonstrate reduced expression of oncogenes FOS, JUN, NFKB, MYC, and
cell cycle regulators CCND1, CCNE1, CDK4/6, and increase in the levels of tumor suppressor
gene TP53, cell cycle inhibitors CDKN1A and CDKN2A and MAOAꢀdownstream genes that
promote EMT, tumor hypoxia, cancer cell migration and invasion. These data suggest that NMI
exerts its effect through tumorꢀtargeted delivery of MAOAꢀinactivating group, making NMI a
valuable antiꢀtumor agent.
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Introduction
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The search for new targets and development of anticancer agents with high tumor targeting
specificity and efficacy are critical goals underpinning the urgent, unmet need for more effective,
mechanismꢀbased cancer therapies. Monoamine oxidase A (MAOA) is a mitochondriaꢀbound
enzyme which catalyzes the degradation of monoamine neurotransmitters and dietary amines by
oxidative deamination.1,2 This process is accompanied by production of hydrogen peroxide
(H2O2), a major source of reactive oxygen species (ROS), which can predispose cancer cells to
DNA damage and can be a main cause of tumor initiation and progression.3,4 Recent studies
performed by us5 and others6 have shown that increased MAOA levels are associated with
prostate cancer (PCa) progression and poor prognosis for patients,7 and pharmacological
inhibition of MAOA reduces the growth of PCa cells in vitro and tumor xenografts in vivo.5,8
PCa is the second most common cause of death from cancer in American men of all ages.9
Despite its widespread occurrence, current treatments that include hormonal therapy,10 radiation
therapy11 and surgery12 are beneficial only for patients in the early stages of the disease and
result in undesired side effects. These treatments have limited effectiveness for patients with
advanced stages of castrationꢀresistant and metastatic PCa. The urgent, unmet need for novel,
effective mechanismꢀbased therapies with reduced side effects facilitated search for novel targets
in PCa and their pharmacological inhibitors.
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We found that overexpression of MAOA in human PCa cell lines results in a loss of cell polarity,
cellꢀcell adhesion, and gain of migratory and invasive properties, indicating that MAOA induces
epithelialꢀmesenchymal transition (EMT).5 Moreover, MAOAꢀdependent activation of
oncogenic pathways was consistently detected in highꢀgrade PCa specimens, and knockdown of
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MAOA reduced or even eliminated prostate tumor growth and metastasis in a variety of PCa
xenograft mouse models. By conducting siteꢀdirected mutagenesis we found that MAOA
catalytic activity is the major reason for enhancing growth and metastasis of PCa.5 MAOA
produces hydrogen peroxide as a byꢀproduct of oxidative deamination reactions taking place in
the outer membrane of mitochondria. Hydrogen peroxide and products of its conversion as other
forms of ROS stabilize hypoxiaꢀinducible factor 1 α (HIF1α).13ꢀ15 HIF1α increases tumor
angiogenesis, survival responses as well as invasion and metastasis through the overexpression
of hypoxiaꢀinducible genes.16,17 These results are fully consistent with the clinical data, where
elevated MAOA expression in PCa tissues is correlated with poor prognosis for PCa patients.7
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In keeping with these observations, we investigated MAOA as a potential novel pharmacological
target for the treatment of human PCa. MAOA inhibitors, many of which are currently on the
market as antidepressants,1 target central nervous system and other peripheral tissues where
MAOA is present. This reduces effective systemic concentration of MAOA inhibitors limiting
their delivery into the tumor where they could be most effective. To circumvent these problems
we designed a novel tumorꢀtargeted MAOA inhibitor that would preferentially accumulate in the
cancerous lesions. This inhibitor contains tumorꢀtargeting nearꢀinfrared (NIR) dye and a moiety
of MAOA inhibitor. We reasoned that including an NIR imaging functionality could be useful
for measuring uptake and cellular localization of the conjugate and possibly, for future imageꢀ
guided diagnosis and drug delivery. We chose small molecule clorgyline as a MAOAꢀtargeting
functionality because of the high affinity and selectivity of this compound towards MAOA. The
presence of highꢀresolution crystal structure of clorgylineꢀMAOA complex18 facilitated our
design. For tumorꢀtargeting and NIRꢀimaging we chose a recently developed class of nonꢀtoxic,
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fluorescent heptamethine carbocyanine dyes with NIR emission maxima.19ꢀ21 The high
selectivity in targeting of these dyes to tumors, mediated by tumor hypoxia and organic anionꢀ
transporting polypeptides (OATPs),22 has been demonstrated for many types of cancers,
including human PCa.23 Here we report the first study on PCa targeting and inhibition of
xenograft growth in mice with designed dualꢀfunction NIR dye – MAOA inhibitor conjugate
(Figure 1A), abbreviated as NMI.
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Figure 1. Structure and in vitro characterization of NMI.
(A) Chemical structure of NMI. (B) Confocal images of a single LNCaP cell incubated with
NMI. DAPI and MitoTracker agent were used to stain the nucleus and mitochondria of cells,
respectively. Scale bar: 10 µm. (C) Inhibition of MAOA activity was determined in C4ꢀ2B
cells (see Experimental Section). Clorgyline (Clg) or NMI were preꢀincubated at 37°C for 20
min at indicated doses and IC50 were determined. The assay was performed in triplicate.
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Results
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Design and synthesis of NMI.
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NMI has been synthesized in a sequence of steps outlined in Scheme 1. The synthesis started
with commercially available 3ꢀbromopropylamine hydrobromide 1. This compound was
converted into tꢀbutyl (3ꢀbromopropyl)carbamate 2, which was used in the subsequent step to
alkylate the commercially available 2,4ꢀdichlorophenol, giving an intermediate 4. Deprotonation
of the amide in 4 was carried out with sodium hydride, followed by alkylation with propargyl
bromide 5, producing Bocꢀprotected alkyne 6. The protecting group was removed under acidic
conditions using TFA in dichloromethane. The product 7 was alkylated with 1ꢀbromoꢀ3ꢀ
thioacetylpropane 8, resulting in the formation of compound 9. Removal of the acetyl protective
group in 9 was carried out in methanolic HCl, affording an intermediate 10. This intermediate
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Scheme 1. Synthesis of NIR dye – MAOA inhibitor conjugate NMI.
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was then coupled to MHIꢀ148 dye 11 using EDCI and 4ꢀDMAP to give the product NMI in good
yield (Scheme 1). The compound was purified on preparative TLC and its identity and purity
were confirmed by NMR and mass spectrometry.
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NMI localizes in PCa cells and inhibits MAOA.
Recent reports suggested that the NIR hepatamethine carbocyanine dyes IRꢀ783 and MHIꢀ148
can be retained in cancer cells but not normal cells, in tumor xenografts and in spontaneous
tumors in transgenic mice.23 The two dyes have strong emission maxima at 820ꢀ860 nm upon
excitation at 750ꢀ780 nm, which can be easily detected by NIR imaging. MAOA inhibitorꢀ
monomethine carbocyanine conjugates have similar fluorescence properties as the NIR dye
MHIꢀ148 itself. Therefore, laserꢀscanning confocal microscope equipped with the appropriate
laser and filters for NIR imaging was used to examine the cellular uptake of NMI in human PCa
LNCaP cells which have high MAOA levels. Images of a single cell treated with NMI are shown
in Figure 1B (vide supra). This compound rapidly accumulated in LNCaP cells and localized in
the mitochondria, as determined by the coꢀstaining with the mitochondriaꢀspecific dye
MitoTracker Green. In order to test the inhibitory activity of NMI, an MAOA activity assay was
carried out in LNCaPꢀderived C4ꢀ2B cells using radiolabeled MAOAꢀspecific substrate
serotonin. The results indicate that NMI inhibits MAOA activity with the mean IC50 of (3.8
±0.3) × 10ꢀ6 M (Figure 1C).
NMI reduces colony formation, migration and invasion of PCa cells.
PCa LNCaP, C4ꢀ2B and MAOAꢀoverexpressing PCꢀ3 cells5 were used for cell viability (Figure
2A) and cell proliferation assays (Figure 2B). Treatment with clorgyline produced dose response
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curves with 50% inhibitory concentrations (IC50) of 80.7 ± 8.8 µM in LNCaP, 113.5 ± 8.0 µM in
C4ꢀ2B, and 129.3 ± 9.6 µM in MAOAꢀoverexpressing PCꢀ3 cells. By comparison, treatment
with NMI produced curves with IC50 of 5.1 ± 0.1 µM in LNCaP, 5.6 ± 0.1 µM in C4ꢀ2B, and 6.1
± 0.2 µM in MAOAꢀoverexpressing PCꢀ3 cells, indicating 12ꢀ20 times higher efficacy for NMI
in inhibiting PCa cells growth as compared to clorgyline (Figure 2A).
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Most PCa cell lines, including LNCaP, C4ꢀ2B and MAOAꢀoverexpressing PCꢀ3, possess low,
undetectable levels of monoamine oxidase B (MAOB), the second known MAO isoform.24
However, given the important role MAOB plays in normal physiology of the central nervous
system and peripheral tissues,2 we first decided to ascertain that MAOA is selectively inhibited
with minimal interference to MAOB. Given that clorgyline is selective for MAOA and produces
doseꢀsensitive inhibition,2 in our preliminary cellꢀbased assays we used a range of clorgyline
concentrations (10 nM – 10 µM) that are inhibitory for MAOA but below the IC50 for MAOB
inhibition.25 We determined 1 ꢁM concentration as the lowest required for clorgyline showing
efficacy in suppressing cell proliferation. This concentration was also consistent with the data
from other reported study.24 Thus, we chose 1 ꢁM as the concentration at which the efficacies of
both clorgyline and NMI could be assessed and compared in the subsequent in vitro studies.
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In cell number counting assays we observed that both clorgyline and NMI reduced the number
of proliferating cells after 12 days. NMI also showed higher efficacy as compared to clorgyline
(Figure 2B). Colony formation assays were performed in LNCaP cells treated with clorgyline or
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Figure 2. NMI inhibits PCa cell proliferation and colony formation.
(A) Effect of Clg and NMI on cell viability in LNCaP, C4ꢀ2B and MAOAꢀoverexpressing
PCꢀ3 cells as measured by an MTS assay. (B) Cell number counting assay with LNCaP, C4ꢀ
2B and MAOAꢀoverexpressing PCꢀ3 cells treated with Clg or NMI (1 ꢁM) for 12 days.
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Compoundꢀadded medium was replenished every 3 days, PBS was used as the vehicle.
p<0.05. (C) Colony formation assays in either LNCaP cells treated with Clg or NMI (1 ꢁM,
left panel) or LNCaP cells targeted by either a scrambled shRNA (shCon) or a MAOAꢀ
targeting shRNA (shMAOA). Representative colonies are shown (original magnification,
×100, scale bar: 50 ꢁm). * p<0.05, ** p<0.01.
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NMI (Figure 2C). In a parallel setup, LNCaP cells were targeted by either MAOAꢀtargeting
shRNA (shMAOA) or a scrambled shRNA (shCon). Treatment with clorgyline and NMI
resulted in a reduction of the colony number by as much as 45%, although in this assay the
difference between the activities of clorgyline and NMI was not statistically significant (left
panel). Treatment with MAOAꢀtargeting shRNA reduced colony number by only 25%, as
compared to treatment with scrambled shRNA (right panel). Because clorgyline has consistently
shown lower efficacy in these experiments, we focused on NMI in the subsequent studies.
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We tested the ability of NMI to inhibit migration of LNCaP and C4ꢀ2B cells. After treatment
with compounds at 1 µM concentration for 48 hours the LNCaP cells showed statistically
significant reduction in migration of 35% for NMI (Figure 3A, left panel). A similar result was
Figure 3. NMI reduces the migration and invasion of PCa cells.
(A) Migration assays of LNCaP and C4ꢀ2B cells treated with NMI (1 ꢁM, 48 hours).
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p<0.01. Representative images from LNCaP cells are shown. Original magnification,
×200, scale bar: 200 ꢁm. (B) Invasion assays of LNCaP and C4ꢀ2B cells treated with NMI (1
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ꢁM, 48 hours).
p<0.01. Representative images from LNCaP cells are shown. Original
magnification, ×200, scale bar: 200 ꢁm.
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observed for C4ꢀ2B cells (Figure 3A, right panel). In invasion assays, cells treated with NMI
also showed 50% reduction in LNCaP and 40ꢀ45% in C4ꢀ2B cells (Figure 3B).
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NMI reduces the rate of growth of PCa xenografts in nude mice.
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In order to assess the efficacy of NMI in vivo, subcutaneous tumor xenograft mouse models were
used. After being implanted subcutaneously into male nude mice, C4ꢀ2B cells formed tumors in
3ꢀ4 weeks. After tumors reached 200 mm3, mice were randomly assigned into two groups to
receive treatments every other day: 1) control and 2) NMI. Two routes of administration were
used to test the tumorꢀspecific targeting ability of NMI: intratumoral and intraperitoneal. Tumors
were measured with calipers and tumor volume was calculated every three days during the 21ꢀ
Figure 4. NMI inhibits the growth of C4ꢀ2B tumor xenografts in mice.
(A) C4ꢀ2B cells were subcutaneously injected into male nude mice (n=5 for each group) to
establish tumor xenografts. After tumor size reached 200 mm3, mice were given intratumoral
treatments (saline and NMI; 750 nmol/mouse) every other day for a 21ꢀday period. Tumor
size was determined by calipers and tumor volume calculated as described in Supporting
*
**
Information. p<0.05, p<0.01. (B) Serum PSA levels in mice were determined on day 11
*
since the treatment by ELISA assay. p<0.05. (C) Tumor weight, determined at the time of
*
**
euthanasia of mice. p<0.05, p<0.01. (D) In vivo and ex vivo NIR imaging of mice given
NMI treatment. Representative images are shown. NIR fluorescence color scales denote ×109
and ×108 for in vivo and ex vivo imaging, respectively, in the units of radiant efficiency
([p/sec/cm2/sr]/[ꢁW/cm2]). (E) Tumor MAOA catalytic activity was determined at the time of
euthanasia. * p<0.05. (F) Mice body weight, determined every week since tumor implantation.
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day treatment. Serum PSA levels in mice were determined on day 11, the middle of treatment
course, and tumor MAOA activity was determined at the end of treatment. Mice body weights
were monitored on a weekly basis since the time of inoculation. At the experiment endpoint mice
were euthanized, tumors were excised, and tumor weights were determined. NMIꢀtreated mice
showed significant delays in tumor growth (Figure 4A), reduction in PSA levels (Figure 4B) and
decreases in tumor weight as compared to control mice (Figure 4C). NIR imaging of the whole
body in vivo and individual tumor and normal organs ex vivo clearly showed NMI localization
within the tumor (Figure 4D). Measurements of MAOA activity in tumors showed its significant
reduction in NMIꢀtreated mice (Figure 4E). All mice in treated and control groups showed
similar changes in body weight that did not exceed 18% throughout the entire duration of
experiment (Figure 4F), suggesting that this treatment regimen was well tolerated by the animals.
We next performed hematoxylin and eosin staining (H&E) and immunohistochemical analysis of
protein expression patterns of Kiꢀ67 (a cell proliferation marker), M30 (a cell apoptosis marker)
and CD31 (an angiogenesis marker) in tumor specimens from control and treated groups (Figure
5A). H&E staining showed a decrease in the nucleus to cytoplasm ratios in cells from tumors
treated by NMI as compared to control group, suggesting reduced malignancy in treated
tumors.26 Kiꢀ67 staining of tumor specimens revealed a 30%–35% decrease of Kiꢀ67+ cells in
NMIꢀtreated tumors (Figure 5B, left panel). We observed 10ꢀ12 fold increase of M30+ area
(middle panel) and 30ꢀ40% decrease in CD31+ area (right panel) in the treated specimens as
compared to controls (Figure 5B), suggesting increased apoptosis and reduced angiogenesis
occurring in treated tumors.
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Figure 5. Analysis of tumor specimens obtained from treated and control mice.
(A) H&E and immunohistochemical analysis of Kiꢀ67, M30 and CD31 expression in
respective tumor specimens. Representative images are shown. Original magnification, ×400,
scale bars: 20 ꢁm. (B) Quantification of percent of Kiꢀ67+, M30+ and CD31+ tumor cells or
**
areas of five distinct images from each group. The data represent mean ± SD. p<0.01. (C)
Heat map depicting gene expression profiling of tumor samples from each group. Red and
blue colors indicate high and low relative expression levels, respectively, with the numbers
indicating log2ꢀtransformed ratios.
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Gene expression profiling further indicated downregulation in expression of such protoꢀ
oncogenes or oncogenes as FOS, JUN NFKB1 and MYC and upregulation of TP53 tumor
suppressor gene expression in response to treatment. Cellꢀcycle regulator genes that activate cell
cycle progression, such as CCND1, CCNE1, CDK4, and CDK6, were also downregulated,
whereas expression of select cellꢀcycle inhibitors, including CDKN1A and CDKN2A, has
increased in the NMIꢀtreated tumors as compared to controls. In contrast, decreased expression
of antiꢀapoptotic BCL2 gene was revealed in treated tumor samples. In addition, genes involved
in MAOAꢀdownstream signaling demonstrated to promote EMT (VIM, SNAI1, SNAI2 and
TWIST1), tumor hypoxia (VEGFA and GLUT1) and cancer cell migration, invasion and
metastasis (IL6, IL8, MMP2, MMP9 and MET)5 all showed reduced expression by treatment
(Figure 5C).
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To further evaluate tumorꢀtargeting properties of NMI, C4ꢀ2B cells were subcutaneously
implanted contralaterally into both flanks of male nude mice. After the formation of two tumor
xenografts in each mouse, NMI was injected intratumorally into one of the lesions. NIR imaging
conducted after three injections performed every other day showed localization of the conjugate
in both tumors (Figure 6A), which reveals tumorꢀtargeting properties of NMI. Remarkably, the
intensity of the NIR signals originating from the injected and contralateral tumor was
comparable, suggesting rapid redistribution of the injected compound between two tumors.
Figure 6B further shows that NMI selectively targets two tumors when administered in an
intraperitoneal injection. NIR imaging shows no observable NIR signals in other body parts,
further confirming the tumorꢀspecific targeting of NMI and its systemic circulation.
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Figure 6. Tumorꢀtargeting properties of NMI in mouse xenografts.
(A) C4ꢀ2B cells were subcutaneously implanted into both flanks of male nude mice to form
tumor xenografts, and the tumor only in one flank (right #525 or left #546) was given
intratumoral injection of NMI (12 nmol/mouse) every other day for three times followed by
NIR imaging. Representative images are shown. (B) Mice bearing C4ꢀ2B tumor xenografts as
described in (A) were intraperitoneally injected with NMI (50 nmol/mouse), and 24 hours
later, subjected to NIR imaging. One representative image is shown. Tumors are indicated by
the arrows. NIR fluorescence color scales denote ×109 and ×1010 for intratumoral and
intraperitoneal injections, respectively, in the unit of radiant efficiency
([p/sec/cm2/sr]/[ꢁW/cm2]).
Discussion
The search for effective therapies based on pharmacological targeting and inhibition of increased
MAOA expression in PCa and potentially other cancers is in its infancy. This study is our first
report on the design, synthesis, in vitro and in vivo evaluation of the therapeutic efficacy of NMI,
a novel conjugate that targets tumors and reduces MAOA activity in mouse PCa xenograft
models. The conjugate consists of the MAOA targeting functionality and a NIR dye. It was
synthesized in 8 steps in a scalable procedure from commercially available starting materials to
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give hundreds of milligrams of material. The conjugate was shown to target PCa cells, localize in
mitochondria and inhibit MAOA activity in the low micromolar IC50 range. It has shown
efficacy that exceeds clorgyline, a known MAOA inhibitor, in suppressing the growth of three
PCa cell lines with high levels of MAOA: LNCaP, C4ꢀ2B and MAOAꢀoverexpressing PCꢀ3. It
inhibited colony formation for LNCaP cells similarly to that of MAOAꢀtargeting shRNA. NMI
also inhibited invasion and migration of PCa cells, which is consistent with the demonstrated
mechanism that high MAOA activity is associated with aggressive cell behavior and induced
EMT. The diminished levels of an epithelial marker Eꢀcadherin and increased expression of a
mesenchymal marker vimentin indicates an EMT suppression. By conducting a comparative
study using NMI and clorgyline, we have shown that the tumorꢀtargeting property of NMI
makes it a superior pharmacological inhibitor, as compared to clorgyline, in nearly all cases.
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Our in vivo studies using PCa xenograft mouse models show that NMI localizes in the tumors,
reduces the rate of tumor growth, and decreases levels of PSA and MAOA catalytic activity in
mice engrafted with C4ꢀ2B cells. At a final dose of 6 nmol per mouse NMI shows significant
inhibitory efficacy on tumor xenograft growth. One of the unexpected findings is the discovery
that an intratumorally injected compound would redistribute itself into the neighboring tumors,
suggesting that its redistribution in vivo is facilitated by the rapid circulation in the bloodstream.
This is further supported by the observation of tumorꢀspecific targeting made by an
intraperitoneal administration.
We previously shown that tumor hypoxia is one of the major factors that mediates an uptake of
heptamethine carbocyanine NIR dyes by tumor cells.22 Hypoxia, through stabilization of HIF1α,
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also underlies one of the key MAOA functions in promoting tumor progression and metastasis in
PCa.5 The increased uptake and retention of NMI in tumors is likely due to tumor hypoxia
through an activated HIF1α/OATPs signaling axis,22 which is supported by our observation that
tumorꢀtargeting effect of NMI could be enhanced in PCa expressing high levels of MAOA
associated with increased tumor hypoxia. Indeed, the higher accumulation of NMI in MAOAꢀ
overexpressing PCꢀ3 tumors as compared to control tumor is paralleled by the increase in HIF1α
immunostaining in those tumor samples with elevated expression of MAOA (see Supporting
Information, Figure S1). These observations suggest that a synergistic strategy based on the use
of tumorꢀtargeting MAOA inhibitor such as NMI and inhibitors of hypoxiaꢀinducible
transcription could result in an improved efficacy and reduced side effects in treating hypoxic,
highꢀgrade PCa.
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Conclusion
We have demonstrated that tumorꢀtargeting NIR dye ꢀ MAOA inhibitor conjugate has the
highest efficacy among all the MAOA inhibitors tested in PCa and it may become a novel
pharmacological agent for treatment and diagnosis of this type of cancer. Such an agent, after the
proper preclinical development, could become an important platform for future generation of
anticancer therapeutics. Such conjugate also possesses a diagnostic potential, by differentiating
the prostate tumors with high MAOA activity, high hypoxia and elevated malignant potential,
from the indolent neoplasms. Its antiꢀtumor effect, largely derived through MAOA targeting, is
making it a valuable addition to the anticancer therapeutic armamentarium.
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Experimental Section
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General. All reagents and solvents were obtained from commercial sources and were used as
received unless otherwise stated. Molecular biology grade salts and buffers were obtained from
SigmaꢀAldrich. Mitotracker Green and DAPI dyes were obtained from Life Technologies. All
reactions involving moistureꢀsensitive reagents were conducted under argon atmosphere with
anhydrous solvents and flameꢀdried glassware. Hygroscopic liquids were transferred via a
syringe and were introduced into reaction vessels through rubber septa. Reaction product
solutions were concentrated using a rotary evaporator at 30ꢀ150 mm Hg. Column
chromatography was performed on silica gel (230ꢀ400 mesh) using reagent grade solvents.
Analytical thinꢀlayer chromatography (TLC) was performed on glassꢀbacked, preꢀcoated plates
(0.25 mm, silica gel 60, Fꢀ254, EM Science). Analytical HPLC were performed on Microsorbꢀ
MV C8 reverseꢀphase column (250 × 4.6 mm, Varian) using Shimadzu LCꢀ10A VP pump and
Shimadzu SPD 10A VP UVꢀvis variableꢀwavelength detector. Preparative HPLC purifications
were carried out with C8 reverse phase preparative column (Grace Davison). The flow rate for
preparative reverseꢀphase HPLC was 4 mL/min. In all cases, 5%ꢀ95% gradients of acetonitrile in
0.1% aqueous trifluoroacetic acid (TFA) were used as eluents. Water (18 Mꢀ) was obtained
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from a Barnstead water purification system, and all buffers were 0.2 ꢁm filtered. Nuclear
magnetic resonance (NMR) spectra were collected on instruments in the indicated solvents. The
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identity and purity of each intermediate and the final product were confirmed by H and C
NMR (Varian Mercury 400 MHz) and mass spectrometry (Agilent 6520 timeꢀofꢀflight system).
Synthesis of Compounds.
t-Butyl (3-bromopropyl)carbamate (2). To a 100ꢀmL roundꢀbottom flask equipped with a
magnetic stirrer was added 3ꢀbromopropylamine hydrobromide (1, 1.19 g, 5.44 mmol, 1.0 eq.)
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and dichloromethane (30 mL). To the resultant solution was added diꢀtertꢀbutyl dicarbonate
(2.16 g, 9.90 mmol, 1.8 eq.) in dichloromethane (20 mL), followed by triethylamine (0.866 mL,
6.21 mmol, 1.1 eq.). The solution was stirred at room temperature for 75 minutes. The reaction
was diluted with dichloromethane (50 mL), and washed twice with sodium bicarbonate (50 mL)
and once with brine (50 mL). The organic phase was dried over sodium sulfate, filtered, and the
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solvent was removed in vacuo to yield 2 (1.35 g, 80% yield). H NMR (400 MHz, CDCl3) 4.65
(s, 1H), 3.37 (dt, 2H, J1 = 6.4 Hz, J2 = 1.2 Hz), 3.21 (dd, 2H, J1 = 12.4 Hz, J2 = 6.4 Hz), 1.98 (m,
2H), 1.37 (s, 9H).
t-Butyl 3-(2,4-dichlorophenoxy)propylcarbamate (4). To a 50ꢀmL roundꢀbottom flask
equipped with a magnetic stirrer was added 2,4ꢀdichlorophenol (3, 452 mg, 2.77 mmol, 1.0 eq.)
followed by dimethylformamide (3 mL). To the resultant solution was added sodium hydride
portionꢀwise (111 mg, 2.77 mmol, 1.0 eq.) followed by 4 (661 mg, 2.77 mmol, 1.0 eq.) in DMF
(2 mL). The mixture was stirred at 60°C for 3 hours. The reaction was diluted with
dichloromethane (50 mL), and washed three times with 10% sodium hydroxide (15 mL), once
with HCl (25 mL), and once with brine (50 mL). The organic phase was dried over sodium
sulfate, filtered, and the solvent was removed in vacuo to yield 4 (431 mg, 48% yield). 1H NMR
(400 MHz, CDCl3) 7.36 (d, 1H, J = 2.8 Hz), 7.18 (dd, 1H, J1 = 8.8 Hz, J2 = 2.8 Hz), 6.83 (d, 1H,
J = 8.4 Hz), 5.16 (s, 1H), 4.07 (t, 2H, J = 5.6 Hz), 3.38 (m, 2H), 2.03 (m, 2H), 1.44 (s, 9H).
t-Butyl 3-(2,4-dichlorophenoxy)propyl(prop-2-ynyl)carbamate (6). To a 20ꢀmL scintillation
flask equipped with a magnetic stirrer was added 4 (273 mg, 0.858 mmol, 1.0 eq.) followed by
dimethylformamide (3 mL). To the resultant solution was added sodium hydride portionꢀwise
(35 mg, 0.858 mmol, 1.0 eq.) followed by propargyl bromide (5, 130 ꢁL, 0.858 mmol, 1.0 eq.).
The mixture was stirred at room temperature for 2 hours. The reaction was evaporated and the
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residue purified by column chromatography using a gradient of 2%ꢀ5%ꢀ50% EtOAC in hexanes
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to provide 6 (75.4 mg, 21% yield). H NMR (400 MHz, CDCl3) 7.36 (d, 1H, J = 2.8 Hz), 7.17
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(dd, 1H, J1 = 8.4 Hz, J2 = 2.4 Hz), 6.83 (d, 1H, J = 8.8 Hz), 4.07 (t, 2H, J = 6.0 Hz), 4.04 (t, 1H,
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J = 5.6 Hz), 3.56 (t, 2H, J = 6.8 Hz), 2.18 (t, 2H, J = 2.4 Hz), 2.03 (m, 2H), 1.57 (s, 2H), 1.44 (s,
9H).
N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium trifluoroacetate (7). To a 20ꢀmL
scintillation flask equipped with a magnetic stirrer was added 6 (72.6 mg, 0.858 mmol, 1.0 eq.)
followed by dichloromethane (4 mL). To the resultant solution was added trifluoroacetic acid (1
mL). The mixture was stirred at room temperature for 8 minutes. The reaction was evaporated
and dried in vacuo to provide 7 without further purification (75 mg, quantitative yield).
S-3-bromopropyl ethanethioate (8). A 250ꢀmL threeꢀneck roundꢀbottom flask equipped with a
thermocouple in a glass sleeve, a magnetic stirrer, a vigreux column with an Argon inlet (middle
stem) and a sleeved rubber septum stopper was assembled and dried with a heat gun under flow
of argon. Then, approximately 110ꢀ120 mL of anhydrous DMF was added via a cannula.
Potassium thioacetate (11.68 g, 102.3 mmol) was added by portions into the flask while cooled
with iceꢀcold MeOH bath. The reaction was stirred for 7 hours at about ꢀ10°C. Then iceꢀcold
MeOH bath was removed after quenching the reaction by adding 165 mL of water. The reaction
mixture was partitioned with 300 mL MTBE and 700 mL water. The water layer was washed by
200 mL MTBE. The MTBE layers were washed sequentially with water, saline and NaHCO3,
dried over MgSO4, filtered and evaporated to yield 8 (19.1 g, yield 98.7%). 1H NMR (400 MHz,
CDCl3) 3.45 (t, 2H, J = 6.4 Hz), 3.01 (t, 2H, J = 7.2 Hz), 2.35 (s, 3H), 5.16 (s, 1H), 2.13 (m, 2H).
S-3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propyl ethanethioate (9). To a 20ꢀ
mL scintillation vial equipped with a magnetic stirrer was added 7 (12 mg, 0.032 mmol, 1.0 eq.)
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followed by acetonitrile (2.5 mL). To the resultant solution was added potassium carbonate (186
mg, 1.343 mmol, 10.0 eq) followed by 8 (12.6 mg, 5.66 mmol, 17.7 eq). The mixture was stirred
at 80°C overnight. The reaction was then filtered, the solvent was removed under reduced
pressure and the residue was dried in vacuo. The crude material was purified by preparative TLC
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using 5% ethyl acetate in hexanes as an eluent to give 9. Yield 4.7mg (39%). H NMR (400
MHz, CDCl3) 7.35 (d, 1H, J = 2.4 Hz), 7.16 (dd, 1H, J = 8.4 Hz, J = 2.4 Hz), 7.35 (d, 1H, J = 8.8
Hz), 4.07 (t, 2H, J = 6.0 Hz), 4.04 (t, 1H, J = 5.6 Hz), 3.40 (s, 2H), 2.87 (t, 2H, J = 6.8 Hz), 2.70
(t, 2H, J = 6.4 Hz), 2.56 (t, 2H, J = 6.8 Hz), 2.30 (m, 3H), 2.17 (s, 1H), 1.95 (m, 2H), 1.71 (m,
2H).
3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propane-1-thiol (10). A solution of
Sꢀ3ꢀ((3ꢀ(2,4ꢀdichlorophenoxy)propyl)(propꢀ2ꢀynyl)amino)propyl ethanethioate 9 (1.17 mg, 3.10
ꢁmol) in 200 ꢁL ACN was added into a 20 mL vial equipped with a stir bar, evaporated and then
coꢀevaporated with MeOH 3 times to remove ACN. MeOH/HCl (200 ꢁL) was added into the
vial and then the vial was heated on an oil bath at 85°C for 6 hours. The reaction mixture was coꢀ
evaporated sequentially with MeOH 3 times and ACN 3 times to give 10 which was used in the
next step without further purification.
6-((Z)-2-((E)-2-(2-chloro-3-((E)-2-(1-(6-((3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-yn-1-
yl)amino)propyl)thio)-6-oxohexyl)-3,3-dimethyl-3H-indol-1-ium-2-yl)vinyl)cyclohex-2-en-1-
ylidene)ethylidene)-3,3-dimethylindolin-1-yl)hexanoate (NMI). MHIꢀ148 11 (170 mg, 242
ꢁmol, 1.10 eq.), dry dichloromethane (10 mL), EDCI hydrochloride (45 mg, 235 ꢁmol, 1.04 eq.),
and DMAP (8 mg, 6.5 ꢁmol, 0.03 eq.) were added to a 20ꢀmL roundꢀbottom flask equipped with
a stir bar and stirred for 15 minutes. Compound 10, obtained from the previous step (75 mg, 226
ꢁmol, 1.00 eq.) was added to the mixture as a solution in acetonitrile (2 mL), and the reaction
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was stirred at room temperature overnight. The solvent was removed under reduced pressure.
The compound was dissolved in 5 mL of dichloromethane and purified on preparative silica gel
plates using 7.5% MeOH in dichloromethane as an eluent. Yield 83.2 mg (37%). Alternatively,
the reaction mixture aliquot was dissolved in methanol and purified by HPLC using YMC HPLC
column, (C18 5ꢀꢁm column, 50 mm length × 20 mm I.D.) and a 5% ꢀ 95% gradient of ACN in
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0.1% aqueous trifluoroacetic acid (TFA) over 30 min to yield NMI (0.144 mg recovery). H
NMR (400 MHz, CDCl3): δ 1.49 (m, 2H, γꢀCH2(COOH)), 1.56 (m, 2H, γꢀCH2(COSCH2ꢀ)), 1.67
(s, 6H,CH3), 1.71 (s, 6H,CH3), 1.80 (m, 2H, βꢀCH2), 1.82 (m, 2H, βꢀCH2), 1.86 (m, 2H, δꢀCH2),
1.88 (m, 2H, δꢀCH2), 1.94 (s, 2H, OꢀCH2CH2CH2Nꢀ), 1.97 (s, 2H, SꢀCH2CH2CH2Nꢀ), 2.09 (s,
2H, CH2), 2.56ꢀ2.57 (m, 4H, αꢀCH2), 2.68ꢀ2.70 (m, 4H, CH2NCH2), 2.71 (s, 2H, CH2C=), 2.75
(s, 2H, CH2C=), 2.85 (m, 2H, ꢀSCH2), 4.04 (t, 1H, HCΞCꢀ), 4.05 (t, 4H, NꢀCH2), 4.05 (t, 2H, Oꢀ
CH2), 4.07 (t, 2H, CH2CΞ), 6.04 (d, 1H, CH=CH), 6.32 (d, 1H, CH=CH), 6.85 (d, 1H, ArꢀH),
7.05 (d, 1H, ArꢀH), 7.17 – 7.42 (m, 9H, ArꢀH), 8.28 (d, 1H, CH=CH), 8.40 (d, 1H, CH=CH).
HRMS calcd. for C57H69Cl3N3O4S m/z 996.4074; observed m/z 996.4068. λmax absorption and
fluorescence wavelengths in methanol were 780 and 802 nm, respectively.
Cell lines and cell culture conditions. Human PCa LNCaP cell line was obtained from
American Type Culture Collection. Human PCa C4ꢀ2B cell line was established from LNCaP as
described previously.27 Human PCa MAOAꢀoverexpressing PCꢀ3 cell line was established as
described previously.5 LNCaP and C4ꢀ2B cells were grown in RPMI 1640 medium (Life
Technologies) and MAOAꢀoverexpressing PCꢀ3 cells were grown in Tꢀmedium (Life
Technologies), with both media supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin.
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Laser-scanning confocal microscopy. Cells were plated in glass bottom microscopy dishes
(MatTek) at a density of 20,000 cells in 400 µl of medium supplemented with 10% FBS and
were allowed to form a monolayer over 20 hours. NMI at a concentration of 5 µM in 400 µL of
fresh medium containing 10% FBS, MitoTracker Green (1×) and DAPI (1×) were added to the
media and the cells were incubated at 37°C and 5% CO2 for additional 4 hours. Imaging was
performed on a Zeiss LSM 510 inverted laserꢀscanning confocal microscope, equipped with an
oilꢀimmersion ×63 objective lens. Excitation wavelengths were set at λmax = 488 nm (DAPI, blue
excitation), 514 nm (MitoTracker Green, greenꢀyellow excitation) and 790 nm (NMI, red
excitation). The data were acquired in a multiꢀtrack mode. Images were taken using the pinholes
of 130ꢀ200 µM in order to capture fluorescence signals on a thin focus plane.
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MAOA catalytic activity assay. MAOA catalytic activity was determined in PCa cells and
tumor samples treated with clorgyline or NMI at different concentrations. One hundred
micrograms of total protein (~1 × 106 cells) were incubated with 1 mM 14Cꢀ5ꢀhydroxytryptamine
(5ꢀHT) in the assay buffer (50 mM sodium phosphate buffer, pH 7.4) at 37oC for 20 minutes, and
the reaction was terminated by the addition of 100 ꢁL of 6N HCl. The reaction products were
extracted with benzene/ethyl acetate and centrifuged. The organic phase containing the reaction
products was extracted, and the radioactivity was determined by liquid scintillation spectroscopy.
Cell proliferation assays. Cells were seeded on 96ꢀwell plates in triplicate and treated with
vehicle, clorgyline or NMI at different concentrations for 96 hours. Cell proliferation was
determined by MTS assay (Promega) following the manufacturer’s instructions. For cell number
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counting assays, cells were seeded on 6ꢀwell plates (2 × 104 cells/well) and treated with vehicle,
clorgyline (1 ꢁM) or NMI (1 ꢁM) for 12 days with compoundꢀadded medium replenished every
3 days. Cell numbers from triplicate wells were counted by hemocytometer.
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Colony formation assay. Cells were suspended in the culture media containing 0.3% agarose
(FMC BioProducts) with vehicle, clorgyline (1 ꢁM) or NMI (1 ꢁM), and placed on top of
solidified 0.6% agarose in 6ꢀwell plates. The developed colonies were counted and recorded
under a microscope after 3ꢀweek incubation.
Migration and invasion assays. Assays were performed using 6.5 mm transwell (8 ꢁm pore
size) coated with either collagen I or Growth Factor Reduced Matrigel (BD Biosciences) for the
migration and invasion assays, respectively. Cells were serumꢀstarved overnight before seeding
to eliminate the interference of proliferative effect with cell migration or invasion. Cells were
seeded inside transwell inserts containing culture medium without serum and with treatment.
After 18ꢀ24 hours, the cells that translocated to the lower surface of the filters were fixed in 4%
formaldehyde. The fixed membranes were stained using crystal violet (1% solution). Assays
were quantified by counting of the number of stained nuclei in 5 independent fields in each
transwell.
Mouse subcutaneous tumor xenograft studies. Male 4ꢀ to 6ꢀweekꢀold athymic nude mice were
purchased from Taconic, Inc., housed in the animal research facility at University of Southern
California (USC), and fed a normal diet. For xenograft studies, 2 × 106 C4ꢀ2B cells were mixed
1:1 with Matrigel (BD Biosciences) and injected subcutaneously into nude mice with each
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mouse bearing one tumor on the right flank. After tumors reached a size of 100ꢀ200 mm3, mice
were randomly assigned to two groups (saline or NMI, n=5 per group). For the treatment group,
intratumoral injections of NMI (2 µg/tumor, 250 nmol/mouse) were given to mice every other
day for first 10 days, then the dose was increased to 6 µg/tumor (750 nmol/mouse). Intratumoral
injections of saline were given to control group. Tumor size was measured every 3 days by
calipers, and tumor volume was calculated by the formula of length × width2 × 0.52.28 Tumors
were dissected and weighted when mice were euthanized on day 21. Mice body weight was
measured on a weekly basis since tumor implantation. All animal studies received prior approval
by the USC IACUC and were conducted in compliance with its recommendations.
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Analysis of tumor uptake of NMI by NIR fluorescence optical imaging. Mice bearing C4ꢀ2B
subcutaneous tumors, when tumor size reached 2ꢀ6 mm in diameter assessed by palpation, were
injected intratumorally (3 consecutive injections every other day) with NMI at a dose of 12
nmol/mouse or intraperitoneally (1 injection) at 50 nmol/mouse, respectively. Wholeꢀbody NIR
fluorescence optical imaging was taken at 24 hour after the last injection using an IVIS Lumina
XR imaging system (PerkinElmer) equipped with fluorescence filter sets (excitation/emission,
783/840 nm) as described previously.29 During imaging, mice were maintained in an
anesthetized state.
Immunohistochemistry. Immunohistochemical analysis of tumor xenograft samples was
performed using antibodies against Kiꢀ67 (Abcam), M30 (DiaPharma), or CD31 (Santa Cruz)
following our published protocol30 with minor modifications. Briefly, formalinꢀfixed paraffinꢀ
embedded sections (4 ꢁm) were deparaffinized, rehydrated, and subjected to antigen retrieval.
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After incubation in Dual Endogenous Enzyme Block solution (Dako) for 10 minutes, the section
was treated with primary antibody diluted by different folds with Antibody Diluent solution
(Dako) at 4oC overnight. The section was then washed 3 times in PBST (PBS containing 0.2%
Tweenꢀ20) for 5 minutes per washing. To detect specific staining, the section was treated for 30
minutes with EnVision + Dual Link SystemꢀHRP (Dako), which contained HRPꢀconjugated goat
antibodies against mouse or rabbit IgG. The section was washed 3 times for 5 minutes each, and
specific stains were developed with 3’3ꢀdiaminobenzidine (Dako). Imaging acquisition was
performed using a Nikon camera and software. Magnification was ×400 (scale bars ~20 ꢁm).
Percent of Kiꢀ67+ cells and M30+ and CD31+ area was analyzed by ImageJ software (NIH).
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Statistical analysis. Data are presented as the mean ± SD as indicated in the figure legends. All
comparisons were analyzed by unpaired 2ꢀtailed Student’s tꢀtest. A pꢀvalue less than 0.05 was
considered to be statistically significant.
Acknowledgments. This work was supported by the US Department of Defense Prostate
Cancer Research Program grant W81XWHꢀ12ꢀ1ꢀ0282 (to J.C. Shih, B.Z. Olenyuk and H.E.
Zhau), the Daniel Tsai Family Fund (to J.C. Shih), Boyd and Elsie Welin Professorship (to J.C.
Shih), NIH/National Cancer Institute grants 5P01CA098912, R01CA122602, Board of
Governors Endowed Cancer Chair and Margaret E. Early Medical Research Trust Award (to
L.W.K. Chung), and by the University of Southern California (to B.Z. Olenyuk). We thank Bin
Qian (Department of Pharmacology and Pharmaceutical Sciences, University of Southern
California, Los Angeles, CA), YiꢀTing Chen (UroꢀOncology Research Program, Department of
Medicine, CedarsꢀSinai Medical Center, Los Angeles, CA) for technical assistance. We also
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thank Dr. Ramin Dubey, Dr. Vladimir Neschadimenko, and Ms. Li Zhou (Department of
Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles,
CA) for help in synthesizing NMI and precursors.
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Supporting Information Available. Figure S1, detailed description of reverse transcription and
quantitative realꢀtime PCR assays and Table S1. This material is available free of charge via the
Internet at http://pubs.acs.org/.
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