Results
Assessment of The Residence Time of AM4301 and AM9928 : In
vitro binding and unbinding of potent AM9928, as compared to another
MAGL inhibitor AM4301, carbamate-based inhibitors to human MAGL (hMAGL)
was monitored by 1H NMR spectroscopy using downfield1H NMR spectral pattern as a probe of conformational
changes (Zhu, Zhao et al., 2016; Tyukhtenko, Ma et al., 2020;
Tyukhtenko, Karageorgos et al., 2016) associated with binding. The
chemical shift perturbations (CSPs) induced by binding in the
characteristic downfield NMR pattern are shown in Figure 1. Addition of
equimolar amounts of inhibitor in both cases caused significant upfield
chemical shift (from 14.9 to ~13.9 ppm) for the
resonance peak corresponding to the catalytic His269 and smaller shifts
for other downfield resonances. Following the fast formation of hMAGL
covalent adducts with AM4301 (Figure 1a) and AM9928 (Figure 1b), slow
reactivation of the enzyme due to hydrolytic decomposition of adducts
was monitored by real-time NMR spectroscopy. The residence time
(lifetime) of ligand on a target is equal to the lifetime of covalent
adduct which is a period required for adduct to decrease to 1/e (0.367)
of its original concentration. The estimated residence time based on NMR
time course data for AM4301 is 1.4 hours whereas AM9928 residence time
is 46 hours. Significantly longer in vitro residence time of
AM9928 in comparison with AM4301 could be translated to its prolonged
pharmacodynamic effect. Based on these observations we selected AM9928
for our studies.
The toxicity effects and time kinetics of treatment with AM9928:The toxicity of AM9928 on TNBC cells were analyzed. The following doses
were examined: 10, 100, 250, or 500 nM, for durations of 24, 48 or 72
hours. No significant toxicity of AM9928 (Figure 2) was observed on the
tested cells.
MAGL mediates adhesion and transmigration of breast cancer cells
through human brain microvascular endothelial cells (HBMECs): To
determine the effects of MAGL on TNBCs co-cultured with HBMECs, in
vitro adhesion assay was performed using co-cultures of the
brain-seeking variant MDA-MB-BrM2 cells and HBMECs in the presence of
vehicle control or AM9928. MDA-MB-BrM2 cells adhered to the untreated
monolayer by as early as 10 minutes (Figure 3a). Pre-treatment of HBMECs
monolayers with AM9928 (100 ng/ml) blocked the adhesion of the tumor
cells at time points of 10 and 30 minutes (Figure 3a). Next, we examined
the role of MAGL in transmigration of TNBC cells across HBMECs using anin vitro model where HBMECs were co-cultured with breast tumor
MDA-MB-231 cells or with MDA-MB-BrM2 cells (Avraham, Jiang et al.,
2014). Tumor cell transmigration across HBMECs was significantly
inhibited (about 50%) by AM9928 (Figure 3b).
Targeting MAGL in human breast cancer cells: Cytokines are
produced by both immune cells and cancer cells (Parker, Limebeer et al.,
2016; Nagarsheth, Wicha et al., 2017). To characterize the potential
activity of MAGL inhibitor AM9928 on the inflammatory responses in
breast cancer, we have used TNBC cells, the highly invasive MDA-MB-231
and the brain- seeking variant MDA-MB-BrM2 cells, as compared to the
immortalized normal breast epithelial MCF-10A cells.
First, we examined the effects of AM9928 as compared to the vehicle
control on secretion of cytokines/chemokines in cells treated with
either the vehicle control or with AM9928 (250nM) for 72 hours. The
supernatants were collected and analyzed by specific ELISA assays for
IL-6, IL-4, INF-alpha2, TGF-alpha, TNF-alpha, and IL-8. We observed
significant inhibitory effects of AM9928 on IL-6 and IL-8 (Figure
4a-4b). Both inflammatory cytokines are important in tumor-associated
inflammation (Omabe, Ezeani et al., 2015; King, Mir et al., 2017;
Masjedi, Hashemi et al., 2018; Banerjee & Resat, 2016; Atretkhany,
Drutskaya et al., 2016).
Second, we analyzed the effects of AM9928 on the angiogenic factor
VEGF-A secretion and observed significant inhibitory effects of AM9928
on VEGF-A secretion (Figure 4c).
Third, to determine if chemokines/cytokines are directly involved in
activation of HBMECs, without the contact of tumor cells, we prepared
TNBC-derived exosomes untreated or treated with AM9928 and tested their
direct effects on HBMECs. MDA-MB-231 and MDA-MB-BrM2 -derived exosomes
caused activation of HBMECs leading to secretion of elevated levels of
the pro-inflammatory IL-8 and VEGF-A. AM9928 treated TNBC-derived
exosomes significantly inhibited activation of HBMECs, as observed by
the secretion levels of IL-8 and VEGF-A (Figure 4d-4e).
In vivo analysis of tumor growth in mammary fat pads and
colonization of TNBCs in brain: To examine the in vivo effects
of AM9928 on TNBC tumor growth and BBB integrity, GFP-4T1-BrM5 cells
were administered (5 x 104 cells) into the mammary fat
pads of BALB/c mice and were injected with either the vehicle control or
with AM9928 as shown in Figure 5a (n = 10 per group per
treatment). The GFP-4T1-BrM5 tumor cell growth in mammary fat pads and
tumor cell seeding in the brain was observed by in vivo imaging
and immunohistochemistry.
Tumor growth in mammary fat pads was significantly reduced in mice
treated with AM9928 as compared to mice treated with vehicle control
(Figure 5b). Quantitation of GFP–4T1‐BrM5 tumor cell colonization in
brain was determined by ex vivo imaging of the brain and by
immunohistochemistry using GFP antibody. The expression of GFP positive
tumor cells was significantly lower in the mice group treated with
AM9928, while the presence of GFP–4T1‐BrM5 tumor cells in brain was
significantly higher in the untreated GFP-4T1BrM5 cells and the vehicle
control treated mice (Figure 5c, 5d).
Increased in BBB permeability was found in mice administered with tumor
cells or with vehicle treated mice, while mice treated with AM9928 had
lower BBB permeability changes as compared to control mice at day 28
(Figure 6a). We then analyzed BMEC-tight junction proteins’ expression
of ZO-1 and Claudin-5 in vivo in normal brain, as compared to TJs
in tumor brains from syngeneic GFP-4T1-BrM5 mammary tumor cell model.
ZO-1 is the main scaffolding protein and claudin-5 is known as the TJ
sealer protein of the BBB. As shown in Figure 6b, both expression of
ZO-1 and claudin-5 were abundant in the normal brain vasculature.
Following GFP–4T1‐BrM5 cells administration to the mammary fat pads,
the GFP-tagged tumor cells migrated across the BBB after about 4 weeks.
The GFP tagged tumor cells were detected by immunostaining with either
Monoclonal Pan-Cytokeratin (Pan-CK) antibodies or GFP antibodies. No
staining of normal brain sections with Pan-ck antibodies was observed
(data not shown). These tumor cells were usually located around the
brain capillaries and in-close proximity to BMECs (BMECS were detected
with CD31 antibodies) (figure 6c). Both ZO-1 and claudin-5 structures
were damaged in the brain tumor vasculature (Figure 6d) as compared to
the control brain (Figure 6b). In some images, both ZO-1 and claudin-5
structures withing BMECs were seen to colocalized with tumor cells,
indicating the proximity of BMECs to the tumor cells in vivo.
Interestingly, following treatment with AM9928, the expression of both
ZO-1 and claudin-5 was less impaired as compared to the mice treated
with vehicle control (Figure 6d).
The number of CD31+ vessels expressing TJ proteins,
ZO-1, and claudin-5, was performed and images were analyzed using Adobe
Photoshop CS2. The expression of ZO-1, as determined by average sum of
intensity was 582,096 and following treatment with AM9928 the average
sum of intensity was 610,920 (Figure 6e). The average sum of intensity
of Claudin-5 expression in untreated group was 518,599 and following
treatment with AM9928 the average sum of intensity was 681,457 (Figure
6f). Although these differences in TJ numbers were not statistically
significant between the untreated mice and the treated AM9928 mice, both
ZO-1 and claudin-5 were observed as less damage’s structures in the
AM9928 treated mice, as compared to the vehicle treated mice.
In summary, the number of mice positive with mammary tumors and brain
tumors was significantly higher in the vehicle treated group as compared
to AM9928 treated mice (Table 1). Importantly, the number of alive mice
at day 28 was higher in the AM9928 treated mice as compared to the
vehicle control group (Table 1). Thus, AM9928 significantly inhibited
changes in the BBB permeability and reduced TNBC colonization in brain.