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ORIGINAL RESEARCH
published: 15 September 2020
doi: 10.3389/fonc.2020.01528
Frontiers in Oncology | www.frontiersin.org 1September 2020 | Volume 10 | Article 1528
Edited by:
Anna Sebestyén,
Semmelweis University, Hungary
Reviewed by:
Anna Maria Tokes,
Semmelweis University, Hungary
Chris Albanese,
Georgetown University, United States
*Correspondence:
Federica Sotgia
fsotgia@gmail.com
Michael P. Lisanti
michaelp.lisanti@gmail.com
Specialty section:
This article was submitted to
Cancer Metabolism,
a section of the journal
Frontiers in Oncology
Received: 23 May 2020
Accepted: 16 July 2020
Published: 15 September 2020
Citation:
Ózsvári B, Magalhães LG, Latimer J,
Kangasmetsa J, Sotgia F and
Lisanti MP (2020) A Myristoyl Amide
Derivative of Doxycycline Potently
Targets Cancer Stem Cells (CSCs)
and Prevents Spontaneous
Metastasis, Without Retaining
Antibiotic Activity.
Front. Oncol. 10:1528.
doi: 10.3389/fonc.2020.01528
A Myristoyl Amide Derivative of
Doxycycline Potently Targets Cancer
Stem Cells (CSCs) and Prevents
Spontaneous Metastasis, Without
Retaining Antibiotic Activity
Béla Ózsvári1, Luma G. Magalhães1, Joe Latimer2, Jussi Kangasmetsa3,
Federica Sotgia1,4*and Michael P. Lisanti1,4*
1Translational Medicine, School of Science, Engineering and Environment (SEE), University of Salford, Manchester,
United Kingdom, 2Salford Antibiotic Research Network, School of Science, Engineering and Environment (SEE), University of
Salford, Manchester, United Kingdom, 3Eurofins Integrated Discovery UK Ltd., Essex, United Kingdom, 4Lunella Biotech,
Inc., Ottawa, ON, Canada
Here, we describe the chemical synthesis and biological activity of a new Doxycycline
derivative, designed specifically to more effectively target cancer stem cells (CSCs).
In this analog, a myristic acid (14 carbon) moiety is covalently attached to the free
amino group of 9-amino-Doxycycline. First, we determined the IC50 of Doxy-Myr using
the 3D-mammosphere assay, to assess its ability to inhibit the anchorage-independent
growth of breast CSCs, using MCF7 cells as a model system. Our results indicate that
Doxy-Myr is >5-fold more potent than Doxycycline, as it appears to be better retained
in cells, within a peri-nuclear membranous compartment. Moreover, Doxy-Myr did not
affect the viability of the total MCF7 cancer cell population or normal fibroblasts grown
as 2D-monolayers, showing remarkable selectivity for CSCs. Using both gram-negative
and gram-positive bacterial strains, we also demonstrated that Doxy-Myr did not show
antibiotic activity, against Escherichia coli and Staphylococcus aureus. Interestingly,
other complementary Doxycycline amide derivatives, with longer (16 carbon; palmitic
acid) or shorter (12 carbon; lauric acid) fatty acid chain lengths, were both less potent
than Doxy-Myr for the targeting of CSCs. Finally, using MDA-MB-231 cells, we also
demonstrate that Doxy-Myr has no appreciable effect on tumor growth, but potently
inhibits tumor cell metastasis in vivo, with little or no toxicity. In summary, by using
9-amino-Doxycycline as a scaffold, here we have designed new chemical entities
for their further development as anti-cancer agents. These compounds selectively
target CSCs, e.g., Doxy-Myr, while effectively minimizing the risk of driving antibiotic
resistance. Taken together, our current studies provide proof-of-principle, that existing
FDA-approved drugs can be further modified and optimized, to successfully target the
anchorage-independent growth of CSCs and to prevent the process of spontaneous
tumor cell metastasis.
Keywords: cancer stem-like cells (CSCs), Doxycycline, myristic acid, fatty acylation, cancer cell metastasis,
prophylaxis of metastasis, 9-amino-Doxycycline, antimitoscins
Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
INTRODUCTION
Cancer stem cells (CSCs) are thought to be the root cause of
recurrence, metastasis, and drug-resistance, in a host of cancer
types (1–5). As a consequence, there is an unmet need to
develop new therapeutics to target and selectively kill CSCs,
while avoiding side effects, especially severe chemo-toxicity.
Metastasis is believed to be driven by this unique sub-population
of cancer-initiating cells (1–3). CSCs have the ability to generate
de novo tumors in immuno-deficient host organisms. Moreover,
they have the capacity to engage in anchorage-independent
growth, which facilitates their invasive spread throughout the
various tissues and organ systems, resulting in local, and distant
disseminated lesions (4,5). Remarkably, these metastatic lesions
are resistant to both chemo-therapy and radiation treatments.
Unfortunately, the Achilles’ heel of these pro-metastatic CSCs
remains largely unknown. As a consequence, currently there are
no anti-cancer drugs that are FDA-approved for the prevention
of metastasis.
Over the last 5 years, we identified that mitochondria in CSCs
may be a novel tractable therapeutic target, for inhibiting their
anchorage-independent growth (5). More specifically, increased
mitochondrial biogenesis may facilitate efficient high energy
production, resulting in the rapid propagation of CSCs (6–10).
In addition, within metastatic lymph nodes isolated from breast
cancer patients, disseminated cancer cells show elevated levels
of mitochondrial activity, especially Complex IV activation, as
revealed by functional activity assays (11).
Interestingly, mitochondrial biogenesis is critically linked
to the activity of mitochondrial ribosomal proteins, that
functionally translate key mitochondrial proteins, which are
genetically encoded by mitochondrial DNA (mt-DNA); this
includes 13 proteins that are necessary to functionally maintain
OXPHOS and mitochondrial ATP synthesis (6–13).
The reason that mitochondria have their own DNA and
specific machinery for protein translation is that they originally
evolved from engulfed aerobic bacteria, over the last 1.4
billion years, after invading eukaryotic cells. This evolutionary
symbiotic relationship has certain functional consequences
that could be safely exploited to achieve anti-mitochondrial
therapy, specifically targeting CSCs. For example, because of
the similarities between bacteria and mitochondria, a sub-
set of bacteriostatic antibiotics block mitochondrial protein
translation, as a manageable side effect (6,10,13). In this
context, Doxycycline inhibits the activity of the small mito-
ribosome, while Azithromycin blocks the large mito-ribosome,
both as off-target side effects (6,10,13). Similarly, Doxycycline
and Azithromycin both inhibit the propagation of CSCs, in an
anchorage-independent fashion, in numerous breast cancer cell
lines, as well as in cell lines derived from many other solid tumor
types (6,10,13). As such, we have suggested that these side-effects
could be re-purposed to clinically target and therapeutically
eradicate CSCs.
In direct support of this notion, a Phase II clinical trial
has documented that brief treatment with Doxycycline, in
early breast cancer patients, is indeed sufficient to significantly
decrease the content of CSCs in the tumor mass, by employing
CD44-staining as an established marker of CSCs in ER(+)
patients (14). Overall, the response rate approached 90%,
resulting in reductions of up to nearly 67% in CSC tumor burden
(14). Quantitatively similar results were obtained in HER2(+)
patients, using ALDH1 as a CSC marker. As such, blocking
mitochondrial protein synthesis may be a viable approach for
targeting and removing CSCs in vivo, prior to surgical excision,
possibly preventing the development of metastases.
Consistent with the above observations, other groups have
shown that Doxycycline treatment effectively reduces the
expression of a panel of CSC markers, in breast cancer cell
lines, such as CD44, ALDH, Oct4, Sox2, and Nanog (9,12).
Moreover, Doxycycline treatment functionally inhibits multiple
CSC signaling pathways, including Wnt, Notch, Hedgehog, and
STAT1/3-signaling (10).
Here, we describe a medicinal chemistry approach to design
novel therapeutics to more selectively target CSCs. More
specifically, we show that several new potent Doxycycline analogs
can be generated by attaching a fatty acid onto 9-amino-
Doxycycline, which is a relatively straightforward chemical
modification. Importantly, these analogs, such as Doxy-Myr, lack
antibiotic activity, but more potently target CSCs and effectively
prevent metastasis, in an in vivo pre-clinical model. Therefore,
Doxy-Myr could be used to eradicate CSCs, without exerting the
selective pressures required for the development of antimicrobial
resistance and without significant toxicity.
To better describe this general class of novel compounds
which are lipid-modified FDA-approved antibiotics, we propose
the term Antimitoscins, to specifically reflect their intrinsic anti-
mitochondrial activity.
MATERIALS AND METHODS
Materials
MCF7 and MDA-MB-231 cells were obtained from the American
Type Culture Collection (ATCC). hTERT-BJ1 fibroblasts were
as we previously described (13). Cells were cultured in DMEM,
supplemented with 10% fetal calf serum (FCS), Glutamine
and Pen/Strep.
Chemical Synthesis
Custom-chemical syntheses were performed by Eurofins
Integrated Discovery UK Ltd., (Essex, UK). Conventional
peptide synthesis methods were used to covalently attach each
free fatty acid to 9-amino-Doxycycline. The desired reaction
products were identified, chromatographically purified and the
chemical structures were validated, by using a combination
of NMR and mass spectrometry. The IUPAC names for the
chemical compounds are as follows.
Doxycycline
(4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-
pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-
4H-tetracene-2-carboxamide.
Doxycycline-Myr
(4S,5S,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-
pentahydroxy-6-methyl-1,11-dioxo-9-(tetradecanoylamino)-
4a,5,5a,6-tetrahydro-4H-tetracene-2-carboxamide.
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Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
Doxycycline-Laur
(4S,5S,6R,12aS)-4-(dimethylamino)-9-(dodecanoylamino)-
3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-
tetrahydro-4H-tetracene-2-carboxamide.
Doxycycline-Pal
(4S,5S,6R,12aS)-4-(dimethylamino)-9-(hexadecanoylamino)-
3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-
tetrahydro-4H-tetracene-2-carboxamide.
Doxycycline-TPP
[6-[[(5R,6S,7S,10aS)-9-carbamoyl-7-(dimethylamino)-
1,6,8,10a,11-pentahydroxy-5-methyl-10,12-dioxo-5a,6,6a,7-
tetrahydro-5H-tetracen-2-yl]amino]-6-oxo-hexyl]-triphenyl-
phosphonium oxalate.
9-Amino-Doxycycline
(4S,5S,6R,12aS)-9-amino-4-(dimethylamino)-3,5,10,12,12a-
pentahydroxy-6-methyl-1,11-dioxo-4a,5,5a,6-tetrahydro-4H-
tetracene-2-carboxamide.
9-Amino-Doxycycline was synthesized, essentially as
previously described (15).
See Supplemental Materials and Methods, and
Supplemental Figure 1, for further details on the chemical
synthesis of these Doxycycline derivatives.
3D-Mammosphere Assay
The mammosphere assay is considered as the gold-standard
for functionally measuring “stemness” and CSC propagation in
breast cancer cells. Single cells are first plated at low density
on low-attachment plates and >90% of “bulk” cancer cells die
under these conditions, in a process of programmed cell death,
termed anoikis. Only stem-like cells survive and propagate in
suspension. Each 3D mammosphere is formed from a single
CSC. Test compounds are added at the moment of single
cell plating and then the number of 3D mammospheres are
counted 5 days after plating. More specifically, a single cell
suspension of MCF7 cells was prepared using enzymatic (1x
Trypsin-EDTA, Sigma Aldrich) and manual disaggregation (25-
gauge needle) (16–19). Cells were then plated at a density of
500 cells/cm2in mammosphere medium (DMEM-F12/B27/ EGF
(20-ng/ml)/PenStrep) in non-adherent conditions, in culture
dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA,
Sigma). Cells were then grown for 5 days and maintained in a
humidified incubator at 37◦C at an atmospheric pressure in 5%
(v/v) carbon dioxide/air. After 5 days in culture, spheres >50 µm
were counted using an eye-piece graticule, and the percentage of
cells plated which formed spheres was calculated and is referred
to as percent mammosphere formation, normalized to vehicle-
alone treated controls. Mammosphere assays were performed in
triplicate and repeated three times independently.
Fluorescence Imaging
Fluorescent images were taken after 72 h of incubation of MCF7
cells treated with either Doxycycline or Doxy-Myr (both at
10 µM), or vehicle control. Cell cultures were imaged with the
EVOS Cell Imaging System (Thermo Fisher Scientific, Inc.),
using the GFP channel. No fluorescent dye was used before
imaging, therefore, any changes in signal were exclusively due to
the auto-fluorescent nature of the Doxycycline compounds.
Cell Viability Assay
The Sulphorhodamine (SRB) assay is based on the measurement
of cellular protein content (16–19). After treatment for 72 h
in 96-well plates (8,000 cells/well), cells were fixed with 10%
trichloroacetic acid (TCA) for 1 h in the cold room, and were
dried overnight at room temperature. Then, cells were incubated
with SRB for 15 min, washed twice with 1% acetic acid, and air
dried for at least 1 h. Finally, the protein-bound dye was dissolved
in a 10 mM Tris, pH 8.8, solution and read using the plate reader
at 540-nm.
Cell Proliferation
Briefly, MCF7 cells were seeded in each well (10,000 cells/well)
and employed to assess the efficacy of Doxycycline and Doxy-
Myr, using RTCA (real-time cell analysis), via the measurement
of cell-induced electrical impedance plate (Acea Biosciences Inc.)
(18). This approach allows the quantification of the onset and
kinetics of the cellular response. Experiments were repeated
several times independently, using quadruplicate samples for
each condition.
Cell Cycle Analysis
We performed cell-cycle analysis on MCF7 cells treated
with Doxycycline, Doxy-Myr, or vehicle-alone. Briefly, after
trypsinization, the re-suspended cells were incubated with
10 ng/ml of Hoechst solution (Thermo Fisher Scientific)
for 40 min at 37◦C under dark conditions. Following a
40 min period, the cells were washed and re-suspended
in PBS Ca/Mg for acquisition on the Attune NxT flow
cytometer (Thermo Scientific). We analyzed 10,000 events
per condition. Gated cells were manually-categorized into
cell-cycle stages (19).
Bacterial Growth Assays
Briefly, antibiotic activity was assessed using standard assay
systems (20–22). The antibiotic activity of Doxycycline analogs
was determined experimentally, using Resazurin (R7017; Sigma-
Aldrich, Inc.) as an indicator of bacterial metabolism/vitality, in
a 96-well plate format, using Escherichia coli and Staphylococcus
aureus. The minimum inhibitory concentration (MIC) for
the studied compounds was determined using the broth
microdilution method, the reference susceptibility test for rapidly
growing aerobic or facultative microorganism(s). The assays
were performed according to the Clinical and Laboratory
Standards Institute (CLSI) guidelines. The test compounds
and positive control (doxycycline, Sigma Aldrich #D1822)
stock solutions were prepared at 25 mM in DMSO and
serially diluted (2-fold dilution from 200 to 1.56 µM) in
cation adjusted Mueller Hinton Broth (MHB, Sigma Aldrich
#90922) in 96-well transparent plates (VWR #734-2781) into
a final volume of 50 µL/well. Staphylococcus aureus (ATCC
29213) and E. coli (ATCC 25922) cultures were maintained
on Mueller Hinton Agar (MHA, Sigma Aldrich #70191). A
single colony of each strain was then grown overnight at 37
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Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
◦C in MHB until OD600 ∼0.6–0.8 and diluted into MHB to
a concentration of 106 colony forming units (CFU)/mL, which
was equivalent to an OD600 ∼0.01. Diluted inocula (50 µL)
were transferred to the wells of the previously prepared 96-
well plates containing the test compounds, negative control
(1% DMSO in MHB) and positive control (Doxycycline).
Final wells volume was 100 µL, final concentrations for
the testing compounds were between 100 and 0.78 µM and
final microorganism concentration was 5 ×105CFU/mL.
Subsequently, 10 µL of one negative control well was plated
in a petri dish containing MHA to check CFU and the
purity of the cultures. The plates were incubated at 37◦C for
24 h after which 20 µL of resazurin solution (0.2 mg/mL)
was added to the wells followed by 1 h 30 min incubation at
37◦C. The OD570 and OD600 were measured in a microplate
reader (BMG FLUOstar Omega). The ratio between OD570
and OD600 was determined and the MIC represents the lowest
concentration of compound that inhibited bacterial growth
(OD570/OD600 ratio inferior to the average ratio determined for
negative control wells). MIC values were determined by three
independent experiments.
Assays for Tumor Growth, Metastasis, and
Embryo Toxicity
These xenograft assays were carried out, essentially as previously
described, without any major modifications (23–27).
Preparation of Chicken Embryos
Fertilized White Leghorn eggs were incubated at 37.5◦C with
50% relative humidity for 9 days. At that moment (E9), the
chorioallantoic membrane (CAM) was dropped down by drilling
a small hole through the eggshell into the air sac, and a 1 cm2
window was cut in the eggshell above the CAM.
Amplification and Grafting of Tumor Cells
The MDA-MB-231 tumor cell line was cultivated in
DMEM medium supplemented with 10% FBS and 1%
penicillin/streptomycin. On day E9, cells were detached
with trypsin, washed with complete medium and suspended in
graft medium. An inoculum of 1 ×106cells was added onto
the CAM of each egg (E9) and then eggs were randomized
into groups.
Tumor Growth Assays
At day 18 (E18), the upper portion of the CAM was removed
from each egg, washed in PBS and then directly transferred to
paraformaldehyde (fixation for 48 h) and weighed. For tumor
growth assays, at least 10 tumor samples were collected and
analyzed per group (n≥10).
Metastasis Assays
On day E18, a 1 cm2portion of the lower CAM was collected to
evaluate the number of metastatic cells in 8 samples per group (n
=8). Genomic DNA was extracted from the CAM (commercial
kit) and analyzed by qPCR with specific primers for Human
Alu sequences. Calculation of Cq for each sample, mean Cq,
and relative amounts of metastases for each group are directly
managed by the Bio-Rad R
CFX Maestro software. A one-way
ANOVA analysis with post-tests was performed on all the data.
Embryo Tolerability Assay
Before each administration, the treatment tolerability was
evaluated by scoring the number of dead embryos.
Statistical Analysis
Statistical significance was determined using the Student’s t-test,
values of <0.05 were considered significant. Data are shown as
the mean ±SEM, unless stated otherwise. Also, ANOVA was
conducted, where appropriate.
RESULTS
Generating New Analogs of Doxycycline
for Targeting CSCs: Doxy-Myr and
Doxy-TPP
Doxycycline is known to function as an inhibitor of the
propagation of CSCs, through its ability to inhibit the small
mitochondrial ribosome, which is an off-target side-effect
(6,10,13). Normally, Doxycycline is used as a broad-
spectrum bacteriostatic antibiotic to treat a wide range
of infections caused by gram-negative and gram-positive
bacteria. Therefore, we sought to optimize the ability of
Doxycycline for the targeting of CSCs, while minimizing its
antibiotic activity, to derive a new chemical entity to selectively
target CSCs.
As a first step, we synthesized 9-amino-Doxycycline, to which
we covalently attached either a 14 carbon fatty acid moiety
(myristic acid) or a six carbon spacer arm containing tri-phenyl-
phosphonium (TPP). The chemical structures of these two
Doxycycline analogs, as well as the parent compound, are all
shown in Figure 1. We speculated that the TPP-moiety would
better target Doxy-TPP to mitochondria in CSCs. In contrast,
the addition of myristic acid could act as a membrane targeting
signal, possibly leading to the increased retention of Doxy-
Myr within membranous compartments, such as the plasma
membrane, the endoplasmic reticulum (ER), the Golgi apparatus,
and/or mitochondria.
To determine the functional activity of these Doxycycline
analogs, we used the 3D-mammosphere assay, to assess their
ability to inhibit the 3D anchorage-independent propagation of
MCF7 CSCs. Interestingly, Doxy-TPP was not more potent that
Doxycycline itself, so further assays with Doxy-TPP were not
carried out (data not shown).
Figure 2 shows a direct comparison of Doxycycline with
Doxy-Myr. Remarkably, Doxy-Myr was >5-fold more potent
than Doxycycline, with an IC50 of 3.46 µM. In contrast,
Doxycycline had an IC50 of 18.1 µM. Therefore, Doxy-Myr
is more potent for targeting the 3D anchorage-independent
propagation of CSCs.
To experimentally test the hypothesis that Doxy-Myr was
better retained within cells, we took advantage of the observation
that Doxycycline is fluorescent (Ex. 390–425 nm/Em. 520–
560 nm). Doxy-Myr was more easily detected and retained in
monolayer MCF7 cells, when compared to Doxycycline or cells
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Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
FIGURE 1 | Chemical structures of two new Doxycycline derivatives: Doxy-Myr and Doxy-TPP. Note that Doxy-Myr contains a 14-carbon fatty acid (myristate)
covalently attached to 9-amino-Doxycycline and Doxy-TPP contains a TPP-moiety attached via a 6-carbon spacer to 9-amino-Doxycycline.
FIGURE 2 | Doxy-Myr more potently inhibits the anchorage-independent propagation of CSCs. (A) MCF7 cells were plated under anchorage-independent growth
conditions and the number of 3D-mammospheres were counted after 5 days. Note that Doxycycline and Doxy-Myr both inhibited 3D-mammosphere formation, all
relative to vehicle-alone controls. However, Doxy-Myr was >5-fold more potent than Doxycycline (IC-50 of 3.46 vs. 18.1 µM). (B) Representative phase images of
3D-mammospheres are shown. Bar =200 µm.
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Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
FIGURE 3 | Doxy-Myr is better retained within cells and reveals a peri-nuclear staining pattern. MCF7 cells were cultured for 72 h as 2D-monolayers, in the presence
of Doxycycline or Doxy-Myr, at a concentration of 10 µM. Vehicle-alone controls were processed in parallel. Then, MCF7 cells were washed twice with PBS and
subjected to live cell imaging to capture the auto-fluorescent signal retained within cells. Note that Doxy-Myr showed the strongest intracellular retention, and was
concentrated within peri-nuclear intracellular compartments. No nuclear staining was observed. Quantitation of mean pixel intensity, using Image J software, revealed
that relative to Doxycycline, Doxy-Myr showed a near 3-fold increase in intracellular fluorescence.
treated with vehicle alone (Figure 3). Doxy-Myr fluorescence
showed a peri-nuclear staining pattern, consistent with its
partitioning and retention within intracellular membranous
compartments. This observation could mechanistically explain
its increased potency. No nuclear staining for Doxy-Myr was
observed, indicating that it was predominantly excluded from
the nucleus.
Doxy-Myr Is Non-toxic in 2D-Monolayers of
MCF7 Cells and Normal Human Fibroblasts
To further assess the effects of Doxy-Myr on 2D-cell growth, we
next used MCF7 cells and normal human fibroblasts (hTERT-
BJ1) treated over a period of 3 days. Figure 4 shows that
both Doxycycline and Doxy-Myr had no appreciable effects
on cell viability, as determined over the concentration range
of 5–20 µM.
Potential 2D-effects on cell proliferation and the cell cycle
were also determined using MCF7 cell monolayers. Clearly,
Doxycycline and Doxy-Myr did not inhibit the proliferation
of MCF7 cells, as assessed using the xCELLigence (Figure 5).
Similarly, relative to the parent compound Doxycycline,
Doxy-Myr did not have any significant effects on reducing cell
cycle progression in 2D-monolayers of MCF7 cells (Figure 6).
Therefore, overall Doxy-Myr did not significantly reduce
the viability, proliferation or cell cycle progression of 2D-
monolayers of MCF7 cells, indicating that its effects were
specific for cell propagation under 3D anchorage-independent
growth conditions.
Doxy-Laur and Doxy-Pal Are Less Potent
Than Doxy-Myr in Targeting CSCs
We also synthesized two other new Doxycycline analogs to
study the influence of the fatty acid chain length on their
functional activity. These two analogs included Doxy-Laur
(harboring a 12 carbon fatty acid) and Doxy-Pal (harboring
a 16 carbon fatty acid) (Figure 7). Therefore, we directly
compared the functional inhibitory activity of Doxy-Myr, Doxy-
Laur, and Doxy-Pal in the 3D-mammosphere assay, using
MCF7 cells. Interestingly, Figure 8 demonstrates that the rank
order potency is: Doxy-Myr >Doxy-Laur >Doxy-Pal >
Doxycycline, with no direct correlation observed between chain
length and activity. As such, the addition of myristic acid
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Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
FIGURE 4 | Doxy-Myr does not affect the viability of MCF7 cells or normal fibroblasts, when grown as 2D-monolayers. To determine the effects of Doxy-Myr on cell
viability, we next treated MCF7 cells and normal human fibroblasts (hTERT-BJ1) over a period of 3 days. Note that Doxycycline and Doxy-Myr had no appreciable
effects on cell viability, in the concentration range of 5–20 µM. (A) MCF7 cells, (B) hTERT-BJ1 cells.
FIGURE 5 | Doxy-Myr does not inhibit the proliferation of MCF7 cell 2D-monolayers. Potential effects of Doxycycline and Doxy-Myr on cell proliferation were
determined using MCF7 cell monolayers. Note that Doxycycline and Doxy-Myr did not inhibit the proliferation of MCF7 cells, as assessed using the xCELLigence, in
the concentration range of 5–20 µM. *p<0.05.
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Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
FIGURE 6 | Doxy-Myr does not induce cell cycle arrest, in MCF7 2D-monolayers. MCF7 cells were cultured for 72 h as 2D-monolayers, in the presence of
Doxycycline or Doxy-Myr, at a concentration of 10 µM. Vehicle-alone controls were processed in parallel. Note that relative to the parent compound Doxycycline,
Doxy-Myr did not have any significant effects on reducing cell cycle progression in 2D-monolayers of MCF7 cells. Representative FACS cell cycle profiles are shown.
FIGURE 7 | Chemical structures of two other Doxycycline derivatives: Doxy-Laur and Doxy-Pal. We created two other new Doxycycline analogs by varying the chain
length of the fatty acid attached to 9-amino-Doxycycline. These two analogs included Doxy-Laur (harboring a 12 carbon fatty acid) and Doxy-Pal (harboring a 16
carbon fatty acid). Their chemical structures are as shown.
(a 14 carbon fatty acid) appears to be the optimal chain
length modification.
Doxy-Myr, Doxy-Laur, and Doxy-Pal Lack
Antibiotic Activity Against Common
Gram-Negative and Gram-Positive
Bacteria
Doxycycline is a well-established broad-spectrum antibiotic,
that is routinely used for therapeutically targeting both
gram-negative and gram-positive bacterial infections. As
a consequence, we also assessed the antibiotic activity
of the Doxycycline analogs, as compared to the parent
compound Doxycycline.
Figure 9 reveals that, as expected, Doxycycline potently and
effectively inhibits the growth of both gram-negative (E. coli) and
gram-positive (S. aureus) micro-organisms. Minimum inhibitory
concentrations were 3.125 µM (1.3 mg/L) and 12.5 µM (5.5
mg/L), respectively. However, in striking contrast, Doxy-Myr,
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FIGURE 8 | Rank order potency of the new Doxycycline derivatives. Note that Doxy-Myr is the most potent Doxycycline derivative for targeting CSC propagation, as
assayed using the 3D mammosphere assay to quantitatively measure anchorage-independent growth. The rank order potency is Doxy-Myr >Doxy-Laur >Doxy-Pal
>Doxycycline.
FIGURE 9 | Doxy-Myr, Doxy-Laur and Doxy-Pal do not show any residual antibiotic activity. The novel Doxycycline analogs (Doxy-Myr, Doxy-Laur, and Doxy-Pal) were
screened against a gram-positive (S. aureus; ATCC 29213) and a gram-negative (E. coli; ATCC 25922) strain of bacteria to verify the maintenance of the antibiotic
activity, when compared to the parent compound. While Doxycycline presented MIC values of 3.125 µM against E. coli and 12.5 µM against S. aureus, none of the
Doxycycline analogs inhibited bacterial growth up to the concentration of 100 µM.
Doxy-Laur, and Doxy-Pal did not show any obvious antibiotic
activity, in the same concentration range. Minimum inhibitory
concentrations of the Doxycycline analogs were >100 µM (>65
mg/L). The clinical breakpoints for tetracyclines against E. coli
and S. aureus are between 0.5–2.0 mg/L and 2.0–6.0 mg/L,
respectively (28).
Frontiers in Oncology | www.frontiersin.org 9September 2020 | Volume 10 | Article 1528
Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
Therefore, these simple chemical modifications of
Doxycycline have successfully removed its ability to act as
a functional antibiotic, while simultaneously increasing its
specificity for targeting CSCs.
FIGURE 10 | Doxycycline and Doxy-Myr have no effect on tumor growth.
MDA-MB-231 cells and the well-established chorio-allantoic membrane (CAM)
assay in chicken eggs were used to quantitatively measure tumor growth. An
inoculum of 1 ×106MDA-MB-231 cells was added onto the Upper CAM of
each egg (on Day E9) and then eggs were then randomized into groups. On
day E10, tumors were detectable and they were then treated daily for 8 days
with vehicle alone (1% DMSO in PBS), Doxycycline or Doxy-Myr. After 8 days
of drug administration, on day E18 all tumors were weighed. Note that
Doxycycline and Doxy-Myr did not have any significant effects on tumor
growth. Averages are shown ±SEM. NS, not significant.
Doxy-Myr Potently Inhibits Cancer Cell
Metastasis in vivo, Without Significant
Toxicity
To experimentally evaluate the functional effects of Doxycycline
and Doxy-Myr in vivo, we next used MDA-MB-231 cells and
the well-established chorio-allantoic membrane (CAM) assay
in chicken eggs, to quantitatively measure tumor growth and
metastasis (16–19). MDA-MB-231 breast cancer cells were used
for our in vivo studies, as they are estrogen-independent,
intrinsically more aggressive, form larger tumors and are
significantly more migratory, invasive and metastatic. As such,
they are a better in vivo model, for simultaneously evaluating
both tumor growth and spontaneous metastasis. Moreover, we
have previously demonstrated that Doxycycline also effectively
inhibits the 3D anchorage-independent growth of MDA-MB-231
cells (13).
Briefly, an inoculum of 1 ×106MDA-MB-231 cells was added
onto the Upper CAM of each egg (day E9) and then eggs were
randomized into groups. On day E10, tumors were detectable
and they were then treated daily for 8 days with vehicle alone
(1% DMSO in PBS), Doxycycline or Doxy-Myr. After 8 days
of drug administration, on day E18 all tumors were weighed,
and the Lower CAM was collected to evaluate the number of
metastatic cells, as analyzed by qPCR with specific primers for
Human Alu sequences.
Morphologically, the CAM is constructed of two opposing
sheets of epithelial cells, which are separated by a middle stromal
layer, containing blood vessels and lymphatics. One epithelial
layer is of ectodermal origin, while the other epithelial layer
is of mesodermal/endodermal origin. Importantly, movement
FIGURE 11 | Doxycycline and Doxy-Myr selectively target and prevent cancer metastasis. MDA-MB-231 cells and the well-established chorio-allantoic membrane
(CAM) assay in chicken eggs were used to quantitatively measure spontaneous tumor metastasis. An inoculum of 1 ×106MDA-MB-231 cells was added onto the
Upper CAM of each egg (on day E9) and then eggs were then randomized into groups. On day E10, tumors were detectable and they were then treated daily for 8
days with vehicle alone (1% DMSO in PBS), Doxycycline or Doxy-Myr. After 8 days of drug administration, the Lower CAM was collected to evaluate the number of
metastatic cells, as analyzed by qPCR with specific primers for Human Alu sequences. Note that Doxycycline and Doxy-Myr both showed significant effects on
MDA-MB-231 metastasis. However, Doxy-Myr was clearly more effective than Doxycycline in inhibiting metastasis. Averages are shown ±SEM. ****p<0.0001.
Frontiers in Oncology | www.frontiersin.org 10 September 2020 | Volume 10 | Article 1528
Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
TABLE 1 | Chick embryo toxicity of Doxycycline and Doxy-Myr.
Group. # Group description Total Alive Dead % Alive % Dead
1 Neg. Ctrl. 18 15 3 83.33 16.67
2 Doxy, 0.125 mM 13 11 2 84.62 15.38
3 Doxy, 0.250 mM 13 10 3 76.92 23.08
4 Doxy-Myr, 0.125 mM 14 11 3 78.57 21.43
5 Doxy-Myr, 0.250 mM 13 12 1 92.31 7.69
of metastatic MDA-MB-231 cells from the Upper CAM to the
Lower CAM involves their migration away from the primary
tumor, cellular invasion, intravasation, extravasation, and the
formation of a new distant lesion, all of the normal steps
that are key features of spontaneous tumor cell metastasis (see
Supplemental Figure 2).
Figure 10 shows the effects of Doxycycline and Doxy-Myr on
MDA-MB-231 tumor growth. Note that that they both did not
show any significant effects on tumor growth, as a result of the
8-day period of drug administration.
However, both Doxycycline and Doxy-Myr showed significant
effects on MDA-MB-231 cancer cell metastasis. Figure 11
illustrates that Doxycycline inhibited metastasis (by 44–57.5%).
In contrast, Doxy-Myr inhibited metastasis (by 85–87%), at the
same concentrations tested. Interestingly, the effects of Doxy-
Myr on metastasis were significantly more pronounced.
Surprisingly, little or no embryo toxicity was observed for
Doxycycline and Doxy-Myr (Table 1). Therefore, we conclude
that Doxy-Myr can be further developed as an anti-metastatic
agent, selectively inhibiting tumor metastasis, without showing
significant toxicity or antibiotic activity.
DISCUSSION
Here, we report the chemical synthesis and biological activity
of several new Doxycycline analogs, modified to increase their
effectiveness in the targeting of CSCs. The most promising
compound was Doxy-Myr, a Doxycycline analog in which a
myristic acid (14 carbon) moiety is covalently attached to
the free amino group of 9-amino-Doxycycline. We analyzed
the potency of Doxy-Myr, using the 3D-mammosphere assay,
to assess its potential inhibitory effects on the anchorage-
independent propagation of breast CSCs. Overall, we observed
that Doxy-Myr is >5-fold more potent than Doxycycline,
the parent compound. Moreover, Doxy-Myr showed better
intracellular retention, and was specifically localized within a
peri-nuclear membranous compartment. In striking contrast,
when MCF7 breast cancer cells or normal fibroblasts were
grown as 2D-monolayers, Doxy-Myr did not reveal any
effects on cell viability or proliferation, highlighting its unique
selectivity for targeting the 3D-propagation of CSCs. In addition,
we evaluated other Doxycycline analogs, with longer (16
carbon; palmitic acid) or shorter (12 carbon; lauric acid)
chain lengths (Figure 12). However, these two analogs were
less effective than Doxy-Myr for targeting of CSCs. Finally,
FIGURE 12 | Schematic diagram highlighting our systematic approach to
generating Doxycycline derivatives, to target CSCs and prevent metastasis.
Lipid moieties were covalently conjugated to 9-amino-Doxycycline.
Importantly, these three Doxycycline derivatives lack anti-microbial activity.
using MDA-MB-231 cells, we demonstrated that Doxy-Myr
has no appreciable effects on tumor growth, but potently
inhibits tumor cell metastasis in vivo, with little or no chick
embryo toxicity.
Our results also showed that the lipophilic amide substituents
in Doxycycline on the C9 of the Tetracycline (TC) skeleton
led to the loss of its antibacterial activity. Previously published
structure-activity relationship (SAR) studies have shown that
chemical modification of the TC skeleton on C9 can be tolerated,
leading to diverse antibacterial activity, as is exemplified by
the antibiotic Tigecycline. The lipophilicity of the TCs seems
to play a key role in the biological potency of this family
of drugs. There is a trend of decreased antibiotic activity
with the increase of lipophilicity, specifically against gram-
negative species, with eventual loss of activity when high
lipophilicity is achieved, which could partially explain the loss
of activity, we observed after the addition of fatty acids to the
Doxycycline scaffold.
Importantly, our improvement in the biological properties of
these Doxycycline analogs for targeting CSCs and the associated
loss of the anti-microbial activity, make these new analogs
extremely promising, because tetracycline resistance among
gram-negative and gram-positive pathogens requires exposure to
inhibitory concentrations as a selective pressure (28–30). MICs
exceeding 65 mg/L also suggest that these analogs might be non-
inhibitory to members of the human microbiome, the complex
community of microorganisms which exerts wide-ranging effects
on human immunity and disease (29,30).
Interestingly, a previous study successfully used the parent
compound, Doxycycline, to prevent bone metastasis in a mouse
model, by employing MDA-MD-231 cells (31). However, these
Frontiers in Oncology | www.frontiersin.org 11 September 2020 | Volume 10 | Article 1528
Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
FIGURE 13 | In vitro inhibition of 3D-growth predicts in vivo inhibition of metastasis. Here, we used two complementary breast cancer cell lines for our in vitro
screening (MCF7) and in vivo (MDA-MB-231) validation assays. More specifically, Doxy-Myr had no effect on 2D-growth in vitro and no effect on tumor growth in vivo.
Conversely, we showed that Doxy-Myr potently inhibited 3D-growth in vitro, which directly correlated with inhibition of metastasis in vivo. In support of this
observation, 3D anchorage-independent growth is thought to be a required step for metastasis in vivo. N.E., no effect.
FIGURE 14 | Summary: Properties of Doxy-Myr. Briefly, Doxy-Myr is a lipid modified Doxycycline derivative. Our results show that Doxy-Myr potently targets CSCs
and selectively prevents metastasis, without affecting tumor growth. Moreover, Doxy-Myr was non-toxic in the chick embryo assay and did not affect the viability of
normal cells, or MCF7 cells, grown as a 2D-monolayer. Importantly, Doxy-Myr lacked antibiotic activity, and did not affect the growth of gram-positive (E. coli) or
gram-negative (S. aureus) organisms.
Frontiers in Oncology | www.frontiersin.org 12 September 2020 | Volume 10 | Article 1528
Ózsvári et al. Doxycycline Derivatives for Metastasis Prevention
authors did not examine the effects of Doxycycline on tumor
growth, but only focused on bone metastasis. They attributed the
efficacy of Doxycycline to its tropism for bone and to its ability
to act as a protease inhibitor for lysosomal cysteine proteinases,
the cathepsins, and MMPs, because Doxycycline behaves as a
zinc chelator.
In contrast, herein, we have demonstrated that Doxycycline
and Doxy-Myr both act as inhibitors of metastasis, by targeting
the 3D anchorage-independent growth of CSCs, which is a
completely different molecular mechanism (Figures 13,14).
However, as predicted, Doxy-Myr was significantly more effective
than Doxycycline, at the same concentrations examined. As such,
based on these functional observations, we propose that this
overall lipid modification strategy may be generally applicable,
to facilitate the development and discovery of other drugs,
for effectively preventing tumor progression, recurrence, and
distant metastasis.
Moreover, our current results are consistent with recent
studies showing that prophylaxis with other classes of
mitochondrial inhibitors is indeed sufficient to prevent
metastasis, using the same pre-clinical xenograft model, with
little or no effect on tumor growth and minimal toxicity (32).
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the
article/Supplementary Material.
AUTHOR CONTRIBUTIONS
ML and FS conceived and initiated this project, they selected the
clinically-approved drug Doxycycline for chemical modification
and optimization by medicinal chemistry. JK performed the
custom-chemical syntheses. The phenotypic drug screening and
the majority of other wet-lab experiments described in this paper
were performed by BÓ. LM determined the antibiotic activity of
Doxycycline and its derivatives. BÓ, JK, and LM generated the
final figures for the paper. ML and FS wrote the first draft of the
manuscript, which was then further edited and approved by BÓ,
LM, JL, JK, FS, and ML. ML generated the schematic summary
diagrams. All authors contributed to the article and approved the
submitted version.
FUNDING
This work was supported by research grant funding, provided by
Lunella Biotech, Inc. The funder, Lunella Biotech, Inc., provided
the necessary monetary resources to carry out the current study.
ACKNOWLEDGMENTS
We are grateful to Rumana Rafiq, for her kind and dedicated
assistance, in keeping the Translational Medicine Laboratory
at Salford running smoothly. We would like to thank the
Foxpoint Foundation (Canada) and the Healthy Life Foundation
(UK) for their philanthropic donations toward new equipment
and infrastructure, in the Translational Medicine Laboratory at
the University of Salford. We are thankful to Inovotion, Inc.
(Grenoble, France), for independently performing the tumor
growth and metastasis studies, using the CAM assay, as well as
evaluating chicken embryo toxicity, through a research contract
with Lunella Biotech, Inc. (Ottawa, Canada).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fonc.
2020.01528/full#supplementary-material
Supplemental Figure 1 | Chemical synthesis of Doxycycline derivatives.
Supplemental Figure 2 | CAM model for measuring tumor growth, metastasis,
and embryo toxicity. On day E9, an inoculum of 1 million MDA-MB-231 breast
tumor cells was layered on top of the Upper CAM and was allowed to form a
primary tumor. Potential therapeutics were applied for a period of 8-days. Then,
on day E18, the primary tumor was harvested from the upper CAM and the
magnitude of distant metastases was quantitated in the Lower CAM, by
performing qPCR with specific primers for recognizing Human Alu sequences. In
order for the cells to metastasize, from the Upper CAM to the Lower CAM, it has
been established that they need to undergo migration, invasion, intravasation,
extravasation, and secondary lesion formation. Toxicity was measured by scoring
embryo viability on day E18. See Materials and Methods for further details.
Reproduced and modified, under a creative commons license, from the following
source (33).
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Conflict of Interest: JK is employed by the company Eurofins Integrated
Discovery UK Ltd. FS holds a part-time affiliation with Lunella Biotech, Inc. ML
holds a part-time affiliation with Lunella Biotech, Inc.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2020 Ózsvári, Magalhães, Latimer, Kangasmetsa, Sotgia and Lisanti.
This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms.
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