Abstract
In breast cancer (BC) care, radiotherapy is considered an efficient
treatment, prescribed both for controlling localized tumors or as a
therapeutic option in case of inoperable, incompletely resected or
recurrent tumors. However, approximately 90% of BC-related deaths are
due to the metastatic tumor progression. Then, it is strongly desirable
to improve tumor radiosensitivity using molecules with synergistic
action. The main aim of this study is to develop curcumin-loaded solid
nanoparticles (Cur-SLN) in order to increase curcumin bioavailability
and to evaluate their radiosensitizing ability in comparison to free
curcumin (free-Cur), by using an in vitro approach on BC cell lines. In
addition, transcriptomic and metabolomic profiles, induced by Cur-SLN
treatments, highlighted networks involved in this radiosensitization
ability. The non tumorigenic MCF10A and the tumorigenic MCF7 and
MDA-MB-231 BC cell lines were used. Curcumin-loaded solid nanoparticles
were prepared using ethanolic precipitation and the loading capacity
was evaluated by UV spectrophotometer analysis. Cell survival after
treatments was evaluated by clonogenic assay. Dose–response curves were
generated testing three concentrations of free-Cur and Cur-SLN in
combination with increasing doses of IR (2–9 Gy). IC[50] value and Dose
Modifying Factor (DMF) was measured to quantify the sensitivity to
curcumin and to combined treatments. A multi-“omic” approach was used
to explain the Cur-SLN radiosensitizer effect by microarray and
metobolomic analysis. We have shown the efficacy of the Cur-SLN
formulation as radiosensitizer on three BC cell lines. The DMFs values,
calculated at the isoeffect of SF = 50%, showed that the Luminal A MCF7
resulted sensitive to the combined treatments using increasing
concentration of vehicled curcumin Cur-SLN (DMF: 1,78 with 10 µM
Cur-SLN.) Instead, triple negative MDA-MB-231 cells were more sensitive
to free-Cur, although these cells also receive a radiosensitization
effect by combination with Cur-SLN (DMF: 1.38 with 10 µM Cur-SLN). The
Cur-SLN radiosensitizing function, evaluated by transcriptomic and
metabolomic approach, revealed anti-oxidant and anti-tumor effects.
Curcumin loaded- SLN can be suggested in future preclinical and
clinical studies to test its concomitant use during radiotherapy
treatments with the double implications of being a radiosensitizing
molecule against cancer cells, with a protective role against IR side
effects.
Subject terms: Radiotherapy, Systems biology
Introduction
Radiotherapy (RT) is a vital component of multimodal therapy for many
types of cancer. For example, in Breast Cancer (BC) care, RT is
considered an efficient treatment, prescribed as adjuvant therapy
strategy combined with surgery for controlling localized tumors, as the
last option for patients with inoperable cancers and also as the first
choice in cases of incompletely resected or recurrent tumors after
surgery^[56]1.
Overall BC women candidates for conservative surgery were also treated
with RT to consolidate surgical treatment and success rate. According
to the international BC treatment guidelines, the recommended total
dose to be administered to BC patients is 50–60, with a fractionation
of 2 Gy/die for 5 days/week^[57]2. Moreover, in some cases of high
recurrence risk (i.e. ductal invasive and microinvasive carcinoma), a
radiation boost of 10–20 Gy could be administered in order to improve
local control. Nonetheless in a phase III study involving 2657 BC
patients, the radiation boost after whole-breast irradiation had no
effect on long-term overall survival, with the largest absolute benefit
in young patients^[58]3–[59]5. In addition, approximately 90% of
BC-related deaths are due to the metastatic progression of the
tumor^[60]6. Then, it is strongly desirable to improve the tumor
radiosensitivity in order to further reduce cancer recurrence. In this
sense, one successful strategy could be to combine RT schedules with
targeted therapies by using molecules with synergistic and
radiosensitizing action.
Among these molecules, characterized by specific anti-cancer actions,
the nutraceutical compounds often available in medicinal plants, have
recieved a recent strong interest by several authors. Among these, a
prominent role is reserved to the Curcuma Longa plant for its
potentially anti-cancer multitarget effect and its active compounds,
which are represented by curcuminoids. The curcuminoids belong to the
polyphenol family, and are known to play key roles in the control of
inflammation and oxidative stress in many conditions^[61]7. In
particular, among these compounds, curcumin is known to play multiple
pleiotropic effects (i.e. anti-bacterial, anti-fungal, antiviral,
anti-oxidant, anti-inflammatory), thanks to its ability to interact and
regulate multiple molecular targets such as transcription factors
(TFs), growth factors, kinases, pro-inflammatory cytokines, adhesion
molecules, etc. In turn, literature data highlight its potential
therapeutic effects useful for the prevention and treatment of various
diseases, including BC^[62]8.
On the other hand, thanks to its low toxicity and great
anti-inflammatory effects, curcumin has recently been studied as a
radiosensitizing molecule against tumor cells and radioprotective agent
for healthy tissues^[63]9. In particular, the radiosensitizing role is
supposed to be related to curcumin-induced inhibition of NF-KB, AP-1
and STAT3 transcription factors (TFs), highly constitutively expressed
in numerous cancers and also transiently induced after ionizing
radiation (IR) exposure. Moreover, curcumin is described to induce
radiosensitization through the inhibition of such genes involved in
several processes such as survival (Bcl-2, Bcl-XL), proliferation
(COX-2, cyclin D1, and c-Myc), angiogenesis (VEGF and IL-8), invasion
(MM9) and metastasis (ICAM-1, VCAM-1, ELAM-1). Overall, all these
factors could contribute to the development of radioresistance in
neoplastic cells^[64]10,[65]11.
On the other hand, literature data report that RT treatments combined
with curcumin inhibit proliferation and induce apoptosis of neoplastic
cells, significantly improving the effects of RT, thanks to the
suppression of cyclin D1, implicated in the G1/S cell cycle
transition^[66]12 or by enhancing the effect of RT to arrest cell cycle
in G2/M^[67]13. In this phase, cells are about three times more
radiosensitive because, once they pass the G2 checkpoint, they are
unable to repair DNA damage. Interestingly, the effect of
radiosensitization is also mediated by increasing the intrinsic and
extrinsic apoptosis induced by ionizing radiation (IR), through an
increase of p53 expression by the MDM2 down-regulation mediated by
Akt.^[68]14. Regarding the intrinsic apoptosis process, curcumin has
been found to increase the mitochondrial Ca^2+ level and Reactive
oxygen species (ROS) production, resulting in an increased permeability
of the mitochondrial membrane, allowing cytochrome C to translocate
into the cytosol and bind Apaf-1 to form the apoptosome complex which
triggers the caspase cascade (by activation of caspase-9) and
subsequent cell death^[69]15. On the other hand, curcumin induces
extrinsic apoptosis of neoplastic cells by increasing the expression
levels of the “death activators” Fas ligand, TRAIL and TNF-α and,
consequently, causing the activation of caspases 8 and 3^[70]16.
Although tumor cell radiosensitization is considered a promising idea
to fight cancer diseases, it is equally important to reduce IR toxic
effects in healthy tissues surrounding tumors. So, since one of the
most prominent cancer pathogenic factors are of an inflammatory nature,
it follows that the curcumin anti-inflammatory powerful effects could
down regulate inflammation and ROS production, through to the
inhibition of NF-kB. This mechanism is also involved in the reduction
of the RT induced fibrosis, driven by TGFβ which is also under the
NF-kB transcription control, so that curcumin could even contribute in
the reduction of normal tissue toxicities following RT^[71]17. Then,
curcumin could play a key interesting role in
radiosensitivity/radioresistance cell balance. However, considering its
chemical characteristics, it is necessary to study new strategies to
increase its absorption in cells. In particular, curcumin is a
lipophilic and hydrophobic compound, and therefore insoluble in water
and soluble in ethanol, dimethyl sulfoxide (DMSO) and acetone. It is
poorly absorbed by the body, so much so that its serum concentrations
after oral administration are particularly low. Curcumin stability is
pH-dependent and in a basic environment it is degraded very
quickly^[72]8. After oral administration, curcumin is metabolized by
reduction and conjugation, producing metabolites vehicled by glucuronic
acid and sulfate in plasma, whose biological activities are strongly
reduced compared to that of curcumin. In this study, to facilitate its
absorption, we have trapped curcumin into a Solid Lipid Nanoparticle
carrier (SLN), in order to realize a molecule having a better in vivo
biodistribution and an improved bioavailability and stability.
Summarizing, the principal aim of this study was to evaluate the
radiosensitizing ability of curcumin loaded SLN (Cur-SLN), in
comparison to free curcumin (free-Cur) by using an in vitro approach on
BC models. Here we used the MCF10A immortalized non tumorigenic mammary
epithelial cell line and MCF7 (ER+/PR+/HER2−), MDA-MB-231
(ER−/PR−/HER2−) tumorigenic immortalized cell lines, characterized by
different tumorigenic aggressive phenotypes^[73]18. In addition, we
also reported in a descriptive way, transcriptomic and metabolomic
profiles and a quantification of oxidative stress induced by Cur-SLN
treatments, highlighting intracellular networks modulated sustaining
the radiosensitizing Cur-SLN role in irradiated breast cell lines.
We trust that all the data here reported may encourage the use of
curcumin-loaded SLN in preclinical studies to test the combination with
the radiotherapy treatment plans and its application in clinical
trials.
Methods
Curcumin nanoparticles preparation
Empty and curcumin-loaded nanoparticles were prepared using an
ethanolic precipitation technique followed by ultraturrax
homogenization. Briefly, 118 mg of Precirol ATO was heated at 5–10 °C
above its melting point (56 °C). In order to obtain drug-loaded
nanoparticles, curcumin (20 mg) was added, under mechanical stirring,
to the melted lipid phase. An ethanolic solution (2 ml) containing
Pluronic F68 (200 mg) and then the cationic surfactant
dimethyldioctadecyl-ammonium bromide (30 mg) was added to the melted
lipid phase. Finally, the resulting warm organic solution was dispersed
into the bidistilled water (100 ml) at 80 ± 1 °C and homogenized by
Ultraturrax T25 (IKA Labortechnik, Germany) at 17,500 rpm for 10 min.
The obtained emulsion was quickly cooled using an ice bath for 30 min
and then purified by exhaustive dialysis (12,000–14,000 Dalton cutoff
membranes, Spectra/Por®, USA). After purification, to prevent any
nanoparticle aggregation, a cryoprotector (trehalose) to nanoparticle
suspensions was added, using nanoparticles:cryoprotector ratio of 1:2
(w/w). Finally, curcumin nanoparticles were freeze-dried by using a
lyophilizer (FreeZone® 4.5 Freeze Dry System, Labconco Corporation, MO,
USA) and stored at room temperature for successive characterization.
Particle size analysis and Zeta-potential measurements
The hydrodynamic diameter (z-average) of empty and curcumin loaded
nanoparticles was determined by Photon Correlation Spectroscopy (PCS)
by using a Zetasizer Nano ZS (Malvern Instrument Ltd, UK), as reported
in literature^[74]19. The samples were diluted in bidistilled water
until the appropriate concentration and then the measurements were
carried out at 25 ± 1 °C, at a fixed angle of 173° (NIBS = non-invasive
backscattering detection) in respect to the incident beam. Each sample
was kept in a cuvette and analyzed in triplicate. Zeta potential
(ζ-potential) values were measured at 25 ± 1 °C using samples
appropriately diluted in bidistilled water. The instrument setting
conditions were equal to those described above for size measurements.
Each sample was analyzed in triplicate.
Drug loading (DL%) and Entrapment Efficiency (EE%) determination
Curcumin loading capacity was evaluated by UV spectrophotometer
analysis. The nanoparticles were solubilized using organic mixtures
until the appropriate concentrations
[dichloromethane/acetonitrile/ethanol 4/4/2 (v/v/v)], filtered through
0.45 μm cellulose acetate membrane filters (VWR, Milan, Italy) and
analyzed by UV spectrophotometer (UV-1800 Shimadzu, Kyoto, Japan), at
423 nm. The peak areas were compared with the one obtained by analyzing
standard solutions of curcumin at known concentrations. The
straight-line equation was: y 132.8x + 0.0084 and the linear regression
value was: r2 = 0.9992. The linearity of the method was studied for
curcumin concentrations in the range of 1–20 μg/ml. DL% results were
expressed as the weight percent ratio between the curcumin loaded and
the total dried sample weight. Entrapment efficiency (EE%) was
expressed as the weight percent ratio between the amount of curcumin
entrapped into nanoparticles and the total amount of curcumin used to
prepare the particles. Below, the equations used for DL% and EE%
determination are reported.
[MATH: DL(%)=Wdrug/WNp×100 :MATH]
Wdrug represents the amount of curcumin entrapped into the
nanoparticles and WNp represents the weight of the drug-loaded
nanoparticles.
[MATH: EE(%)=Wf/Wi×100 :MATH]
Wf represents the amount of curcumin entrapped and Wi represents the
amount of curcumin used to prepare the curcumin-loaded nanoparticles.
Cell cultures and intracellular and extracellular concentrations of free-Cur
and Cur-SLN
The human non-tumorigenic breast epithelial MCF10A cell line and the
human breast adenocarcinoma MCF7 and MDA-MB-231 cell lines, were
purchased from the American Type Culture Collection (ATCC, Manassas,
VA) and cultured according to the ATCC’s specifications. Cells were
maintained in an exponentially growing culture condition in an
incubator at 37 °C in a humidified atmosphere (95% air and 5% CO[2])
and were routinely subcultured in 25-cm^2 (T25) and 75-cm^2 T75)
standard tissue culture flasks. Cell lines were cultured in the
presence and in the absence of 2.5 µM of free- Cur or Cur-SNL for 24
hrs. At the end of the stimulation, cells and supernatants were
collected to assess the intracellular and extracellular content of
curcumin by UV analysis. In particular, the supernatants were sucked
from the wells and then collected by centrifugation at 1,300 rpm for
10 min. Cells were detached from the wells by tripsin, washed with PBS
and stored as dry pellets at −80 °C.
Clonogenic survival assay and IC50 determination
Cell survival after treatments was evaluated by clonogenic assay
performed as previously described^[75]20,[76]21. Briefly, 48 hours
before treatments cells were detached, counted by haemocytometer and
re-plated in triplicate at opportune densities in 6-well plates to
assay the surviving fraction (SF). As control (basal), untreated cells
were seeded in the same conditions in order to evaluate the plating
efficiency (PE). All cell survival data post-treatment were normalized
to the untreated sample. Colonies were allowed to grow under normal
cell culture conditions for 10–15 days after treatments and then were
fixed and stained with 50% methanol and 0.5% crystal violet (both from
Sigma-Aldrich, St. Louis, MO, USA). Colonies with more than 50 cells
were counted manually under a Zeiss Axiovert phase-contrast microscope
(Carl Zeiss, Göttingen, Germany) and also automatically with a
computer-assisted methodology^[77]22.
Empty SLN has been tested for cellular toxicity on the three cell lines
under study using the concentrations of 2.5, 5, 10, 25, 50 and 100 μM
and showing not to be toxic, as no significant reduction of cell
survival has been observed even at the higher concentration of 100 μM
(data not shown). In order to obtain the IC[50] value of free-Cur and
Cur-SLN for each breast cell line, which represents the concentration
required to obtain the 50% of in vitro inhibition, cells were treated
in triplicates using a wide range of free-Cur and Cur-SLN
concentrations (2.5, 5, 10, 25, 50 and 100 μM) in 6-well plates. 24 hrs
after treatments, the medium was replaced and the cells were maintained
in standard growth conditions until processing according to the
clonogenic assay protocol. The IC[50] was calculated from the two drug
dose–response curves obtained by the increase of concentrations of
free-Cur and Cur-SLN, using a 2nd order polynomial fitting analysis.
Moreover, dose-response curves were generated by means of the 2nd order
polynomial fitting of FS% vs increasing IR dose for each concentration
of free-Cur and Cur-SLN used.These curves were used to obtain the Dose
Modifying Factor (DMF), arbitrarily calculated at the SF of 50% (i.e.
the relative dose of irradiation required to obtain the isoeffect of
SF = 50% with radiation treatment alone respect to combined treatments
with a defined concentration of free-Cur or Cur-SLN^[78]23.
Radiation treatments
A medical linear accelerator, Siemens Primus (Siemens Medical Systems,
Concord, CA, USA) emitting photon beams (X-rays) of 6 MV nominal
energy, was used to perform cell irradiations. The Linac was calibrated
under reference conditions defined by the International Atomic Energy
Agency Technical Reports Series No. 398 “Adsorbed Dose Determination in
External Beam Radiotherapy”^[79]24. The irradiation setup and the dose
distribution were studied using the Pinnacle Treatment Planning System
(Philips Medical Systems). Cell irradiations were carried out using
doses of 2, 4, 6 and 9 Gy to evaluate the radiation efficacy through a
dose-response curve.
Oxidative stress quantification
A quantification of cellular oxidative stress has been performed
measuring the intracellular reactive oxygen species (ROS) in the
control (untreated), 2 Gy, 2 Gy + Cur-SLN, Cur-SLN samples of the three
cell lines studied. The production of ROS was evaluated using
2′,7′-dichlorodiidrofluorescineacetate (DCFH-DA) (Molecular Probes,
Eugene, OR). The DCFH-DA is de-esterified intracellularly and turns
highly fluorescent 2′,7′-dichlorofluorescein upon oxidation, allowing a
sensitive and rapid quantitation of oxygen reactive species in response
to oxidative metabolism. 24 hours after treatments, cells were
trypsinized, washed with PBS and incubated with 5 μM DCFH-DA reagent
working solution according to manufacturing protocol. For consistency,
10^5 cells were analyzed by fluorimeter for each determination
performed in triplicate. The oxidation reaction was evaluated using the
GloMax® Discover System (Promega) at the excitation wavelength of
475 nm and emission wavelength of 555 nm. The data shown are obtained
from three independent experiments and are expressed as the
mean ± standard error of the mean (SEM). The significance level respect
to the control sample was set to *P < 0.05.
Whole genome cDNA microarray expression analysis
We decided to evaluate only gene expression profiles (GEP) generated by
the administration of Cur-SLN, as this formulation could have a clinic
interest. Furthermore, we decided to evaluate the lowest
dose/concentration combination (2 Gy/2.5 µM), a synergistic
radiosensitizing treatment aims to obtain the maximum efficacy by
administering the minimum doses of a drug and RI. Furthermore, it
should be remembered that 2 Gy is the daily dose delivered in
fractionated radiotherapy treatments.
To analyze GEP induced by 2.5 µM Cur-SLN in MCF10A, MCF7 and MDA-MB-231
cell lines treated with 2 Gy of photon beams, we used whole human
genome 4 × 44 K cDNA microarray expression analyses. In particular, we
analyzed GEP of the following configurations: MCF10A Cur-SLN 2 Gy
normalized with MCF10A 2 Gy; MCF7 Cur-SLN 2 Gy normalized with MCF7
2 Gy; MDA-MB-231 Cur-SLN 2 Gy normalized with MDA-MB-231 2 Gy.
Total RNA extraction and its qualitative (expressed in term of RNA
integrity number evaluation, RIN) and quantitative analyses were
conducted as previously described^[80]20,[81]21. Microarray experiments
were performed according to the Agilent Two-Color Microarray-Based Gene
Expression Analysis protocol (Agilent Technologies, Santa Clara, CA,
USA). In turn, 6 replicates for each configuration assayed were
performed. Statistical data analysis, background correction,
normalization and summary of expression measures were conducted with
GeneSpring GX 10.0.2 software (Agilent Technologies). Finally, genes
were identified as being differentially expressed if they showed a fold
change (FC) of at least 1.5 with a p-value < 0.05 compared to
irradiated cells of the same cell line analyzed, that were used as
reference samples. The data discussed in this publication, in
compliance with Minimum Information About a Microarray Experiment
(MIAME) standards, have been deposited in the National Center for
Biotechnology Information Gene Expression Omnibus (GEO)^[82]25 and are
accessible through the following GEO Series accession number:
[83]GSE118061.
In addition, the GEPs obtained in this work were also analyzed by
pathway analysis using the Database for Annotation, Visualization and
Integrated Discovery (DAVID) network building tool that provides a
comprehensive set of functional annotation tools for investigators to
understand the biological meaning behind a long list of genes
([84]https://david.ncifcrf.gov/tools.jsp). The most representative
significantly changed networks and pathways were selected and analyzed.
Metabolite profiling of cell culture samples
Metabolites from the same treatments described in the above
transcriptomic section (2 Gy and 2.5 µM Cur-SLN) were extracted and
analyzed by gas chromatography-mass spectrometry (GC-MS), as previously
described^[85]26. Derivatization was performed using automated sample
prep WorkBench instrument (Agilent Technologies). Dried polar
metabolites were dissolved in 60 μl of 2% methoxyamine hydrochloride in
pyridine (Pierce) and held at 40 °C for 6 hrs. After dissolution and
reaction, 90 μl of MSTFA (N-Methyl-N-(trimethylsilyl)
trifluoroacetamid) were added and samples were incubated at 60 °C for
1 h. GC/MS analysis was performed using 7200 accurate-mass Q-TOF GC/MS
(Agilent Technologies) equipped with a 40-m DB-5MS capillary column
operating under electron impact (EI) ionization at 70 eV. Samples
(2 μl) were injected in a splitless mode at 250 °C, using helium as the
carrier gas at a flow rate of 1 ml/min. The GC oven temperature was
held at 100 °C for 2 min and increased to 325 °C at 10 °C/min.
GC/MS data processing was performed using Agilent Muss Hunter software
and statistical analyses were performed using Mass Profile Professional
(MPP) software (Musharraf et al., 2016). Relative metabolites abundance
was carried out after normalization to internal standard d27-myristic
acid and cell number.
Ethics approval and consent to participate
This study does not require ethical approval and informed consent, as
the research project uses commercial immortalized cell lines samples
from the American Type Culture Collection (ATCC, Manassas, VA, USA).
Results
Curcumin loaded nanoparticles preparation and characterization
In literature is widely reported the potential application of curcumin
but it was heavily limited in biomedicine because of its poor
solubility in water^[86]27–[87]29. The oral bioavailability of
curcuminoids in healthy humans is markedly enhanced by micellar
solubilisation but not further improved by simultaneous ingestion of
sesamin, ferulic acid, naringenin and xanthohumol^[88]30. To date, to
increase the bioavailability of curcumin several methods were
developed. In the present work, empty and curcumin-loaded solid lipid
nanoparticles (SLNs) were successfully prepared by using the ethanolic
precipitation technique and then, stored at room temperature for
successive characterization in terms of mean size, polydispersity index
(PDI) and ζ-potential in bidistilled water (Table [89]1).
Table 1.
Size, PDI, and ζ-potential of empty and curcumin loaded SLNs in
bidistilled water.
Systems Z-average (nm) PDI ζ-potential (mV) ± SD
empty SLNs 159.0 0.275 +44.9 ± 7.5
Curcumin SLNs 302.5 0.544 +41.4 ± 4.6
[90]Open in a new tab
Table [91]1 shows that the curcumin presence inside the nanoparticle
system caused an increase of the mean size (from 159 nm to 302 nm) and
polidispersity index (PDI). The zeta potential values, instead, were
similar in both the systems considered and they ensured an adequate
repulsion of particles.
Drug loading (DL%) and Entrapment Efficiency (EE%) determination
In order to investigate the amount of curcumin entrapped into the SLNs,
UV spectrophotometrics analysis was performed, as reported in the
Materials and Methods section. The drug loading value of SLNs prepared,
expressed as weight percent ratio between entrapped curcumin and the
total dried sample weight (DL% w/w) and the Entrapment Efficiency
(EE%), expressed as the weight percent ratio between the amount of
curcumin entrapped into SLNs and the total amount of curcumin used to
prepare the particles, were 1.64% and 68.62% respectively.
IC50 determination
In order to evaluate the cytotoxicity ability of free curcumin and
curcumin-loaded SLN (Cur-SLN) in term of concentration that determined
the 50% of growth inhibition (IC[50]), MCF10A, MCF7 and MDA-MB-213 cell
lines were treated with increasing doses of free-Cur and Cur-SLN,
ranging from 2.5 to 100 μM for 24 hours and subjected to long-term
clonogenic assay.
Free-curcumin and Cur-SLN significantly inhibited the viability of MCF7
cells, which showed greater sensitivity to Cur-SLN than free-Cur,
displaying IC[50] values of 2.45 μM and 3.80 μM, respectively. The
cytotoxic effect of Cur-SLN was comparable to that of free curcumin in
MDA-MB-231 cells, where IC[50] values were of 7.60 μM and 6.63 μM,
respectively. Finally, MCF10A cells were more sensitive to Cur-SLN than
to free curcumin, displaying IC[50] values of 6.81 μM and 10.58 μM,
respectively.
Cell radiosensitization following combined treatments with free-Cur and
Cur-SLN
To evaluate the radiosensitizing ability of Cur-SLN in comparison to
free-Cur, we investigated the combined effects of each of these two
molecules on BC cells exposed to different ionizing radiation (IR)
doses.
MCF10A, MCF7 and MDA-MB-231 cell lines were treated with only RT with
increasing photon doses of 2, 4, 6, 9 Gy or were irradiated after a
pretreatment with 2.5, 5, 10 µM of free-Cur and Cur-SLN for 24 hrs. The
surviving fraction (SF) values, obtained by clonogenic assay after
treatments, were used in order to generate dose-response curves in
absence or presence of a certain concentration of free-Cur or Cur-SLN
for the three BC cell lines (See Additional file 1). This, in turn, led
to define the dose modifying factor (DMF), arbitrarily calculated at
the SF of 50% by means of a 2nd order polynomial fitting from the dose
response curves (i.e. the relative dose of irradiation required to
obtain the isoeffect of SF = 50% delivering the radiation treatment
alone respect to combined treatments with different concentration of
free-Cur or Cur-SLN) (Table [92]2)^[93]23. These values show an overall
increasing radiosensitization effect, rising with the concentration of
both the two drugs. However, the three cell lines show a different
level of sensitivity to free-Cur and Cur-SLN. Indeed, the normal
epithelial MCF10A showed to be the most sensitive to all the
radiosensitizer combined treatments, in comparison with the other two
tumorigenic cell lines, being more sensitive to the vehicle curcumin
Cur-SLN for each concentrations tested. On the other hand, although a
lesser radiosensitizer effect can be observed for the two tumorigenic
cell lines, MCF7 resulted more sensitive to the treatment with
increasing concentration of vehicled curcumin Cur-SLN, whereas
MDA-MB-231 have proved to be more sensitive to free-Cur, reaching the
maximum of radiosensitizer effect using 10 µM of free-Cur.
Table 2.
Dose Modifying factors (DMF) for increasing concentrations of free-Cur
and Cur-SLN.
MCF10A MCF7 MDA-MB-231
DMF[50_]Photons/Photons + free-Cur 2,5 µM 1,29 0,99 0,93
5 µM 1,44 0,92 1,08
10 µM 2,28 1,68 1,72
DMF[50_]Photons/Photons + Cur-SLN 2,5 µM 1,65 1,06 0,81
5 µM 1,65 1,58 1,03
10 µM 2,76 1,78 1,31
[94]Open in a new tab
Table [95]2 displays the DMFs, arbitrarily calculated at the SF of 50%,
by means of a polynomial fitting from the dose response curves.
As we decided to study GEP and metabolomic changes caused by the
administration of the minimum concentrations of Cur-SLN,
Figs [96]1–[97]3 display the relative effect of combined treatments
with 2,5 µM of free-Cur and Cur -SLN respect to that deriving from the
exclusive treatment with IR. A better response with the minimum dose of
2,5 µM of loaded Curcumin (Cur-SLN) is observable for MCF10A and MCF7,
whereas MDA-MD-231 receives a protective effect by low concentrations
of both free-Cur and Cur-SLN.
Figure 2.
Figure 2
[98]Open in a new tab
Dose-Response curves of MCF7. Dose-Response curves of MCF7 BC cells
subjected to increasing doses of IR with or without 2.5 µM of free-Cur
and Cur-SLN.
Figure 1.
Figure 1
[99]Open in a new tab
Dose-Response curves of MCF10A. Dose-Response curves of MCF10A cells
subjected to increasing doses of IR with or without 2.5 µM of free-Cur
and Cur-SLN.
Figure 3.
Figure 3
[100]Open in a new tab
Dose-Response curves of MDA-MB-231. Dose-Response curves of MDA-MB-231
BC cells subjected to increasing doses of IR with or without 2.5 µM of
free-Cur and Cur-SLN.
Oxidative stress quantification
As shown in Fig. [101]4 the result of this assay shows and confirms the
anti-oxidant properties of curcumin. Indeed, for all the three cell
lines analyzed, a significant increase in the oxidative stress caused
only by irradiation can be observed, whereas treatment with curcumin
alone or in combination with IR is able to lower the level of cellular
oxidative stress below the value of untreated cells (P < 0.05).
Moreover, in the combined treatments, the level of oxidative stress is
always significantly lower than that observed in samples subjected to
irradiation only, demonstrating the curcumin efficacy in lowering the
ROS levels produced following the administration of IR.
Figure 4.
Figure 4
[102]Open in a new tab
Determination of intracellular ROS production. Oxidative stress
quantification performed measuring intracellular ROS levels with the
DCFH-DA molecular probe in the MCF10A, MDA-MB-231 and MCF7 cell lines
24 hrs after treatments with 2 Gy, 2 Gy + Cur-SLN, Cur-SLN. The
significance level respect to the control sample was set to *P < 0.05.
Gene expression profiles and gene signatures after combined Cur-SLN and RI
treatment
As above described we evaluated only GEP generated by Cur-SLN, as this
formulation could have a clinic interest. Furthermore, we decided to
evaluate the lowest dose/concentration combination (2 Gy/2.5 µM), a
synergistic radiosensitizing treatment aims to obtain the maximum
efficacy by administering the minimum doses of a drug and RI.
Furthermore, it should be well-known that 2 Gy is the daily dose
delivered in fractionated radiotherapy treatments.
Here, we reported GEP data obtained applying a Two-Color
Microarray-Based Gene Expression Analysis (Agilent technologies) on
MCF10A, MCF7 and MDA-MB-231 breast cell lines treated with 2.5 µM
Cur-SLN and irradiated with photon beams using the IR dose of 2 Gy. In
particular, comparative differential gene-expression analysis revealed
that multiple deregulated genes (DEG) were significantly altered, by
1.5-fold or greater according to the specific experimental
configuration. MCF10A cells treated with Cur-SLN changed the expression
levels of 846 genes (189 down regulated and 657 up regulated). On the
other hand, 1265 DEGs were selected in MCF7 BC cell line Cur-SLN
treated and, among these, 752 were up regulated while 513 were down
regulated. Finally, in MDA-MB-231 BC cell lines treated with Cur-SLN we
selected 2301 DEGs (112 down regulated and 1179 up regulated).
Moreover, up and down regulated transcripts were selected and grouped
according to their involvement in specific biological networks using
Integrated pathway enrichment analysis with the DAVID tool^[103]25 and
the top molecular pathways of DEG datasets were selected and reported
in Tables [104]3–[105]5.
Table 3.
DAVID pathway analysis of the top pathways and related genes,
deregulated after Cur-SLN treatment in MCF10 cell line.
Pathway analysis of GEP induced by SLNB-Cur in MCF10 non tumorigenic
cell line
Term Genes count % P value Genes
1 Lysine degradation 7 0.0071 0.0075125 KMT2C, WHSC1L1, ASH1L, WHSC1,
SETD2, NSD1, SUV39H2
2 Transcriptional misregulation in cancer 12 0.0122 0.0255476 NFKBIZ,
CCR7, RXRA, TP53, CDK9, FOXO1, MDM2, MLLT1, WHSC1, ZBTB16, JMJD1C,
RUNX2
[106]Open in a new tab
Table 5.
DAVID pathway analysis of the top-5 pathways and related genes,
deregulated after Cur-SLN treatment in MDA-MB-231 BC cell line.
Pathway analysis of GEP induced by SLNB-Cur in MDA-MB-231 BC cell line
Term Genes count % P value Genes
1 FoxO signaling pathway 27 0.0094 0.0006 ATG12, FOXO1, TGFB2, IGF1R,
PRMT1, FBXO25, CAT, EGF, AGAP2, PIK3R1, EGFR, GABARAPL2, IL6, PRKAB2,
TGFBR2, SKP2, HOMER2, ATM, CDK2, BCL2L11, TNFSF10, PLK4, MAPK13,
MAPK14, FBXO32, GADD45B, GADD45A
2 Cell cycle 24 0.0084 0.0021 ANAPC1, E2F2, E2F5, RBL1, SKP2, TTK,
PRKDC, CDC20, WEE1, CDK2, ATM, TGFB2, MAD2L1, BUB1, TFDP2, YWHAQ,
BUB1B, ANAPC7, ABL1, GADD45B, GADD45A, CCNA2, BUB3, TFDP1
3 TGF-beta signaling pathway 18 0.0063 0.0030 SMAD9, LTBP1, ROCK1,
E2F5, FST, TGFBR2, RBL1, RPS6KB2, TGFB2, ID2, ID1, ZFYVE16, SMURF2,
SMURF1, BAMBI, BMPR1A, BMP6, TFDP1
4 TNF signaling pathway 19 0.0066 0.0152 TRAF1, ICAM1, IL6, CSF1,
CREB1, CXCL2, EDN1, NFKBIA, CREB5, CX3CL1, MMP14, CXCL10, RPS6KA5,
BAG4, ATF4, MAPK13, MAPK14, MAP2K6, PIK3R1
5 Phosphatidylinositol signaling system 17 0.0059 0.0299 PRKCA, INPP1,
PIK3C2A, PIK3C2B, SYNJ1, ITPKB, PI4K2B, PIP5K1A, DGKA, DGKE, PLCD4,
INPP5E, MTMR8, INPP5D, PLCB1, INPP5B, PIK3R1
[107]Open in a new tab
The result of this mapping revealed the involvement of a set of factors
controlling cellular processes described as follows.
Summarizing, as displayed in Table [108]3, only 2 statistically
relevant pathways were selected in MCF10A cell line treated with
Cur-SLN. In particular, lysine degradation pathway is known to be
mainly involved in the Acetyl-CoA production, modulated by curcumin
through mitochondrial fatty acid oxidation^[109]31, whereas
transcriptional misregulation in cancer signaling contains several
factors known to be deregulated in cancer cells because of their key
roles in controlling cell survival/death balance.
On the other hand, in MCF7 BC cell line Cur-SLN causes the deregulation
of molecules belonging to apoptosis induction, inflammation signaling
(sustained by cytokine and platelet activation pathways), tyrosine
metabolism known to be involved in cellular stress response and in
glucagon signaling pathway, recently described as modulated by curcumin
to improve glucose tolerance (Table [110]4)^[111]32–[112]37.
Table 4.
DAVID pathway analysis of the top-5 pathways and related genes,
deregulated after Cur-SLN treatment in MCF7 BC cell line.
Pathway analysis of GEP induced by SLNB-Cur in MCF7 BC cell line
Term Genes count % P value Genes
1 Cytokine-cytokine receptor interaction 21 0.0145 0.0257 TNFRSF6B,
TNF, OSMR, TNFRSF25, CSF1, LIFR, CD70, CX3CL1, CCL28, IL11RA, TGFB2,
LIF, AMH, IL17A, IL20RB, CCR3, IL2RG, LTB, LTA, IL3RA, BMPR1A
2 Apoptosis 9 0.0062 0.0186 TNF, BAX, PIK3CD, CASP8, TP53, IL3RA,
PIK3R1, AKT2, PIK3R2
3 Platelet activation 14 0.0097 0.0246 F2RL3, ADCY2, PIK3CD, ITGA2,
APBB1IP, COL5A2, JMJD7-PLA2G4B, P2RX1, PRKACA, COL1A1, COL11A2, PIK3R1,
PIK3R2, AKT2
4 Tyrosine metabolism 6 0.0041 0.0397 TYR, PNMT, MAOA, AOX1, HPD, AOC3
5 Glucagon signaling pathway 11 0.0076 0.0428 LDHB, CPT1B, ADCY2, PYGM,
GCK, PPP3R1, PRKACA, ACACB, PPARGC1A, GCGR, AKT2
[113]Open in a new tab
Finally, as displayed in Table [114]5, in MDA-MB-231 BC cell line the
Cur-SLN treatment activate specific set of genes controlling cell cycle
process, TGF-beta pathway, TNF signaling, phosphatidylinositol
signaling and FoxO signaling pathway.
Analysis of selected gene signatures Cur-SLN treatment induced
Finally, in order to evaluate the unique and common DEG lists and
cellular process modulated by these factors after Cur-SLN treatments in
MCF10A, MCF7 and MDA-MB-231 cell lines, we performed Venn diagrams as
shown in Fig. [115]5.
Figure 5.
[116]Figure 5
[117]Open in a new tab
Venn diagrams of unique and shared differentially expressed genes by
breast cell lines after combined treatments with IR/2,5 µM Cur-SLN. (A)
A 51-gene signature of shared deregulate genes after Cur-SLN treatment
was selected among MCF10A, MCF7 and MDA-MB-231 breast cell lines. The
top-GO Biological processes were also displayed. (B) A 263-gene
signature of common deregulated genes was selected for MCF7 and
MDA-MB-231 BC cell lines and the top-GO biological processes regulated
by these genes, were also reported in the figure.
As displayed in Fig. [118]5A, common and unique DEGs were selected
among all the configurations analyzed in this work. So, here we report
an overall cell-dependent gene expression deregulation after Cur-SLN
treatments. Moreover, as shown in Fig. [119]5A, 51 differentially
expressed genes (51-gene signature) were shared among the three cell
lines treated with Cur-SLN. Regarding these genes, GO Biological
processes analysis conducted by the DAVID tool, revealed the activation
of chromatin modification, cell migration and calcium signaling
pathways. Finally, as displayed in Fig. [120]5B, we compared the list
of DEGs of the two tumorigenic MCF7 and MDA-MB-231 BC cell lines
treated with Cur-SLN, producing Venn diagrams (Fig. [121]5B). In
particular, a 263-gene signature was selected and composed by DEGs
involved in cell migration, angiogenesis, G2/M transition of mitotic
cell cycle and calcium signaling pathways.
Moreover, in the Supplementary Files [122]1 and [123]2 we highlighted
the gene expression value details of 51- and 263 gene signatures,
respectively.
These data highlight the wide range of curcumin bioactivities, that
could help to obtain successful treatment plans in combination with RT.
Metabolomics change induced by Curcumin-Loaded SLN and irradiation treatments
To further understand better the effects induced by combined treatment
of 2.5 µM Curc-SLN and irradiation with 2 Gy of photon beams in MCF10A,
MCF7 and MDA-MB-231 human breast cell lines, we performed metabolomic
analysis using gas chromatography/mass spectrometry (GC/MS) and liquid
chromatography/mass spectrometry (LC/MS) systems.
The hierarchical clustering plot of metabolomic profiling revealed a
similar metabolic phenotype between the two human breast cancer cell
lines MCF7 and MDA-MB-231 under 2 Gy and 2 Gy/Cur-SLN treatments, as
compared to MCF10A normal breast cell line (compare the right vs left
side of the hierarchical clustering plot) (Fig. [124]6A). In
particular, untargeted metabolic profiling, corresponding to the upper
part of the hierarchical clustering plot, showed enrichment of
increased of metabolites levels involved in anti-oxidant response such
as: GSH metabolism, arginine and proline metabolism, pentose phosphate
pathway (PPP) and taurine and hypotaurine metabolism, as well as in
metabolites involved in fatty acids and nucleotide metabolism in MCF7
and MDA-MB-231 cell lines under 2 Gy and 2 Gy/Cur-SLN treatments,
respect to MCF10A cells under the same conditions (Fig. [125]6B). On
the contrary, the same cell lines (MCF7 and MDA-MB-231) under 2 Gy and
2 Gy/Cur-SLN treatments, showed significant decreased levels of
metabolites involved in amino acids metabolism, TCA cycle and
glycolysis as compared to MCF10A under the same conditions (see the
lower part of the hierarchical clustering plot) (Fig. [126]6C).
Moreover, the identification of the single branch clustering of MCF7
under 2 Gy/Cur-SLN treatments (yellow color) as compared to others
(Fig. [127]6A) prompted us to perform a deeper statistical analysis in
order to evaluate better the radiosensitising effect of loaded-curcumin
in respect to irradiation only for each cell line studied. Statistical
analysis of MCF10A exposed to 2 Gy vs 2 Gy/Cur-SLN treatments showed
significant metabolites involved mostly in amino acids metabolism,
highlighting increased significant levels of glucose and citrate in
MCF10A exposed to the combined treatment of irradiation plus loaded
curcumin (Fig. [128]7A). Similarly to MCF10A cells, comparison of
MDA-MB-231 cancer cell line exposed to 2GY vs 2 Gy/Cur-SLN treatments
showed significant metabolites involved both in amino acids metabolism
and in anti-oxidant processes such as taurine and hypotaurine
metabolism, fatty acids and TCA cycle (Fig. [129]7C). Finally,
consistently with previous results observed in MDA-MB-231 cells, also
MCF7 cells exposed to 2 Gy vs 2 Gy/Cur-SLN treatments showed
significant increased metabolites involved in amino acids metabolism,
in anti-oxidant action, fatty acids and TCA cycle probably also due to
the activation of autophagy mechanism (Fig. [130]7B).
Figure 6.
[131]Figure 6
[132]Open in a new tab
Comparative analysis of MCF10A, MCF7 and MDA-MB-231 by Hierarchical
Clustering and untargeted enrichment plots. (A) Comparative analysis of
MCF10A, MCF7 and MDA-MB-231 by Hierarchical Clustering. (B) Untargeted
enrichment plot displaying increased metabolites levels in MCF7 and
MDA-MB-231 under 2 Gy and 2 Gy/Cur-SLN treatments, respect to MCF10A
under the same condition. (C) Untargeted enrichment plot displaying
decreased metabolites levels in MCF7 and MDA-MB-231 under 2 Gy and
2 Gy/Cur-SLN treatments, respect to MCF10A under the same condition.
Figure 7.
[133]Figure 7
[134]Open in a new tab
Hierarchical Clustering analysis and untargeted enrichment plots of
MCF10A, MCF7 and MDA-MB-231 exposed to 2 Gy vs 2 Gy/Cur-SLN treatments.
(A) Hierarchical Clustering analysis and untargeted enrichment plots of
MCF10A, exposed to 2 Gy vs 2 Gy/Cur-SLN treatments. (B) Hierarchical
Clustering analysis and untargeted enrichment plots of MCF7, exposed to
2 Gy vs 2 Gy/Cur-SLN treatments. (C) Hierarchical Clustering analysis
and untargeted enrichment plots of MDA-MB-231, exposed to 2 Gy vs
2 Gy/Cur-SLN treatments.
Therefore, taken together, these results showed that the effect of
Cur-SNLB administration on breast cell lines exposed to irradiation is
to exert a protective role against oxidative stress-induced by
irradiation damage and to induce an antitumor effect through autophagy
mechanism activation^[135]38.
Discussion
The main aim of this research study was to evaluate the
radiosensitizing effects mediated by Cur-SLN in comparison to that
generated by free-Cur, using an in vitro approach on three immortalized
breast cell lines: the epithelial non-tumorigenic MCF10A cell line and
the two tumorigenic MCF7 (Luminal A: ER+/PR+/HER2−), and MDA-MB-231
(triple-negative: ER−/PR−/HER2−) cell lines. Specifically, these cell
lines were subjected to single or combined treatment using four doses
of IR (2, 4, 6 and 9 Gy) and 6 concentrations of free-Cur or Cur-SLN
(2.5, 5, 10, 25, 50 and 100 μM). Empty SLN has been also tested for
cellular toxicity on the three cell lines under study using the same
above reported concentrations, showing it not to be toxic. The IC[50]
evaluation showed that all the cell lines tested are sensitive to
treatment with free and curcumin loaded-SLN. However, the MCF7
tumorigenic cell line showed a strong sensitivity to treatment with
Cur-SLN and free-Cur, compared to the other two cell lines analysed,
with IC[50] values of 2.47 μM and 3.81 μM, respectively. On the other
hand, the two cell lines MCF10A and MDA-MB-231 displayed lower levels
of sensitivity to both the molecules. In particular, while in
MDA-MB-231 cells the cytotoxic effect of Cur-SLN was very similar to
that of free curcumin, with IC[50] values of 7.62 μM and 6.62 μM,
respectively, the MCF10A were proved to be more sensitive to Cur-SLN
than free curcumin by finding IC[50] values of 6.74 and 10.56 μM
respectively.
Based on the IC[50] values, we tested the radiosensitizing effect of
free-Cur and Cur-SLN, used at concentrations of 2.5, 5, 10 μM, in
combination with conventional radiotherapy treatment with increasing
doses of 2, 4, 6, 9 Gy, in order to generate dose/response curves for
all the treatment configurations tested.
The radiosensitizing effect was determined by calculating the dose
modifying factor (DMF), arbitrarily obtained at the SF of 50% (i.e. the
relative dose of irradiation required to obtain the isoeffect of
SF = 50% using the radiation treatment alone respect to combined
treatments with different concentration of free-Cur or Cur-SLN), in
order to highlight the capacity of enhancing tumor cells killing by the
combined treatments in respect to irradiation only^[136]23.
In other terms, a DMF value of 2, means that the isoeffect could be
obtained by the combined treatment with the half of IR dose, respect to
the dose necessary using IR only.
Overall, the DMFs value reported in Table [137]2 highlight a general
increasing radiosensitization effect, rising with the concentration of
both the two drugs, nonetheless differences can be identified in the
sensitivity of the three cell lines to free-Cur and Cur-SLN.
Indeed, the normal epithelial MCF10A received a strong radiosensitizer
effect by the combined treatments, although they were more sensitive to
the vehicle curcumin Cur-SLN for each concentrations tested. This means
that further studies are needed to understand if the use of curcumin as
radiosensitizer could increase normal tissues toxicity
post-irradiation. However, it should be noted that normal tissue
complications (NTCP) are of inflammatory nature and the powerful
anti-inflammatory effects of curcumin could reduce these toxicities in
vivo, reducing the inflammation and the reactive oxygen species
production, thanks to the down-regulation of NF-kB. This mechanism is
also involved in the reduction of fibrosis induced by radiotherapy, in
which a key role is played by TGFβ^[138]17. On these aspects, a
preclinical study evaluated the adverse effects of cutaneous toxicity
that occur following exposure to IR^[139]17, evaluating a possible
protective role exerted by curcumin. In this case, curcumin was
administered intragastric and intraperitoneally in C3H/HeN mice, 5 days
before RT or 5 days after RT. Skin damage was assessed at 15–21 days
(acute cutaneous toxicity) and at 90 days (chronic cutaneous toxicity)
following a single dose of 50 Gy of radiation in the posterior leg of
each mouse. The results showed that curcumin, administered before or
after radiotherapy, markedly reduces acute and chronic cutaneous
toxicity, significantly decreasing the expression of inflammatory IL-1,
IL-6, IL-18, IL-1Ra and fibrogenic (TGF-β) cytokines in irradiated skin
and muscles.
On the other hand, although a lesser radiosensitizer effect can be
observed for the two tumorigenic cell lines, MCF7 resulted more
sensitive than MDA-MB-231 to the treatment with increasing
concentration of vehicled curcumin Cur-SLN, reaching a DMF value of
1,78 using 10 µM of Cur-SLN. Instead, MDA-MB-231 were more sensitive to
free-Cur, reaching the maximum of radiosensitizer effect with 10 µM of
free-Cur (DMF = 1,72). However, a clinical administration of curcumin
is likely in the vehicle form, then the aggressive triple negative
MDA-MB-231 BC cell line could receive a radiosensitization effect by
Cur-SLN, as the DMF value is 1.38 with 10 µM of Cur-SLN. Thus, the
possibility to use drug/IR combined treatments permits to increase the
tumor control probability (TCP) even for radioresistant tumors such as
the triple negative BC.
Furthermore, in Figs [140]1–[141]3 the efficacy of combined treatments
is compared using the minimum concentration of 2,5 µM of free-Cur and
Cur -SLN in respect to that deriving from the exclusive treatment with
IR. A better response with the minimum dose of 2,5 µM of loaded
Curcumin (Cur-SLN) is observable for MCF10A and MCF7 cells, whereas
MDA-MB-231 receives a protective effect by low concentrations of both
free-Cur and Cur-SLN.
Moreover, in order to highlight the action mechanism of Cur-SLN and to
confirm the anti-oxidant properties of Cur-SLN, a quantification assay
of oxidative stress induced by the treatments has been performed
(Fig. [142]4). On the three cell lines analyzed, a significant increase
in oxidative stress caused by only irradiation can be observed, whereas
treatment with Cur-SLN alone or in combination with IR administration
is always able to lower the level of cellular oxidative stress
significantly below the values of untreated cells, demonstrating the
curcumin efficacy in lowering the ROS levels produced following the
administration of IR.
In addition, we carried out a “omic” study (transcriptomic and
metabolomic), in order to explore better this new formulation (Cur-SLN)
action mechanism in producing a radiosensitivity effect on the three
cell lines analyzed. This is thanks to its formulation which
facilitates its transport in an aqueous solution, it could be
administered in preclinical or clinical application. Moreover, we only
compared the effects of lower IR doses/drug concentrations, since the
combination with a radiosensitizer should, hopefully, allow the use of
lower IR doses maintaining the isoeffect of therapeutic efficacy and
reducing the side effects to the minimum. In addition, 2 Gy is the
daily dose administered in fractionated treatments with external beams.
Therefore, for the “omic” study the comparisons considered were: 2 Gy
vs 2 Gy + 2.5 μM Cur-SLN, for each cell line tested, analyzed with
“whole genome microarray” approach and “two-color” staining method. The
comparative gene expression analysis of MCF10A, MCF7 and MDA-MB-231
cells revealed that a large number of genes were deregulated (DEG) from
treatment with Cur-SLN with a fold change (FC) ≥ 1.5. Below are the
details of the genes deregulated in the three cell lines: 1) MCF10A:
846 DEG (189 down-regulated and 657 up-regulated); 2) MCF7: 1265 DEG
(513 down-regulated and 752 up-regulated); 3) MDA-MB-231: 2301 DEG (112
down-regulated and 1179 up-regulated).
Figure [143]5 shows the detail of deregulated genes specific or common
to the three cell lines analyzed, using a Venn diagram. As shown, the
molecular response to Cur-SLN treatment is predominantly cell-line
dependent, since most of the deregulated genes are specific of each
cell line (MCF10A: 555 specific DEG, MCF7: 883 specific DEG,
MDA-MB-231: 1917 specific DEG). On the other hand, the common response
of the three cell lines to Cur-SLN treatment is determined by a
signature of 51 genes, while the tumorigenic ones (MCF7 and MDA-MB-231)
share a specific signature of 263 genes.
Thanks to the use of the DAVID tool, these deregulated genes have been
grouped, based on their involvement in some biological networks and
cellular processes. This approach has allowed to better characterize
the unique and common molecular response among the analyzed cell lines.
Specifically, in the non-tumorigenic MCF10A cell line, the specific
molecular response to Cur-SLN treatment is characterized by the
statistically significant deregulation of “Transcriptional
misregulation in cancer”, which involves several key factors altered in
neoplastic cells and “lysine degradation process” (Table [144]3), the
latter involved in acetyl-CoA production, modulated by curcumin through
mitochondrial oxidation of fatty acids^[145]31.
On the other hand, in MCF7 cell line the Cur-SLN treatment resulted in
the deregulation of molecules involved in inflammatory processes,
induction of apoptosis, tyrosine metabolism which is implicated in the
response to cellular stress and glucagon signaling pathway, which is in
turn regulated by curcumin in response to low glucose levels
(Table [146]4)^[147]31–[148]37. Finally, in MDA-MB-231 cells,
deregulated genes involved in the cell cycle, in the TGF-β and in the
TNF pathway, in the pathways of phosphatidylinositol and FoxO were
identified (Table [149]5). Regarding the TGF- pathway, several authors
reported that curcumin inhibits cell proliferation and invasion by
preventing TGF-β1-induced phosphorylation of Smad2, ERK1/2, p38MAPK,
resulting in down-regulation of MMP metalloproteinases-9, thus limiting
the effect of cellular invasion^[150]34,[151]39. Furthermore, it is
also reported in the literature that curcumin is also able to trigger
the extrinsic apoptotic pathway, allowing the binding of TNF-α and Fas
Ligand “death activators” to their corresponding cell surface receptors
that, by the activation of caspase-8, induce the caspase
cascade^[152]40. In particular, these anti-tumor actions are largely
modulated by curcumin by the negative regulation of various growth
factors, inflammatory cytokines, transcription factors (TF), protein
kinases and other oncogene molecules. In this context, the
phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway,
usually activated in onset and neoplastic development, is inhibited by
curcumin. This process could constitute a key molecular target for
anti-cancer therapies as it represents a negative regulator of tumor
progression processes and metastasis^[153]32,[154]41,[155]42.
Several studies have found that curcumin is also involved in the
regulation of the FoxO pathway implicated in multiple cellular
processes, such as cell cycle arrest, apoptosis, DNA repair, glucose
metabolism and oxidative stress response. In particular, Fox TFs are
negatively regulated by the AKT survival signal, as normally
phospho-Akt prevents the nuclear localization of FOX and plays a key
role in regulating its activity. Furthermore, tetrahydrocurcumin (THC),
a metabolite of curcumin, regulates the oxidative stress response
through FOXO, promoting its nuclear translocation and regulating,
therefore, the expression of several genes involved in apoptosis, in
cell cycle progression, in DNA repair, in oxidative stress, in the
control of muscle growth, as well as in cell differentiation and
glucose metabolism. Based on these findings, FOXO TF could be used as
potential tumor suppressors^[156]43–[157]48.
Similar molecular findings have been described in an in vivo study
which evaluated the radiosensitizing effect of curcumin on nude mice
inoculated with colon cancer HCT 116 cells, performed by the group of
Kunnumakkara^[158]49. This study evaluated the effect of the combined
administration of IR (4 Gy, twice a week) and curcumin administered
orally 1 hour before IR exposure (1 g/kg). The effect was compared with
a control group and two groups corresponding to the two treatments
performed in single. The results showed that the tumor volume of mice
that received combined treatment was significantly reduced compared to
that of the control group of mice and those treated with curcumin alone
or with radiation alone. Furthermore, it has been found that curcumin
enhances the effect of RI, inhibiting the proliferation of neoplastic
cells and generating a significant reduction of the Ki-67 cell
proliferation marker and of CD31 (tumor density microvase marker) in
tumor tissues, compared to the group that received only RIs. The same
study showed a decrease in the expression levels of COX-2, VEGF, MMP-9,
Cyclin D1, c-Myc, associated with angiogenesis, invasion, metastasis
and proliferation and whose expression is generally increased following
exposure to IR; this down-regulation was observed in the group treated
with only curcumin and in the group subjected to the combined
treatment. In general, since these factors are regulated by NF-kB, it
can be concluded that curcumin exerts its powerful radiosensitizing
effect through the down-regulation of NF-kB and its transcriptional
targets, also allowing to reduce the toxic effects of inflammatory
nature induced by radiation in healthy tissues.
Regarding the 51-gene signature composed by DEG common to three cell
lines analyzed, the Gene Ontology analysis conducted using the DAVID
tool, showed that the genes were involved in cell migration, in the
calcium signaling pathways and in the activation of the chromatin
modification process.
Regarding the radiotherapy context, chromatin remodelling through
histones acetylation and deacetylation represents attractive target for
radiosensitization as this process plays a significant role in the
repair mechanisms of the double strand breaks (DSB) generated by IR.
In addition, preclinical studies have found that inhibitory molecules
of HAT (histone acetyltransferase) and HDAC (histone deacetylase), such
as curcumin, are able to radiosensitize neoplastic cells. In fact, it
has been found that curcumin is able to significantly reduce the
acetylation of H3/H4 histones and to increase the demethylation of
histone H3K9 at the level of the cytokines promoter regions, thus
inhibiting the expression of genes for proinflammatory
chemokines^[159]33,[160]50,[161]51. Several authors have reported that
curcumin limits the migration/proliferation process of neoplastic
breast cancer cells by inducing crosstalk between the anchoring
junctions and the Wnt pathway by means of the EPR-1 factor (Early
Growth Response 1), and by inhibiting the expression of several genes
involved in the epithelial-mesenchymal transition (Epithelial
mesenchymal transition-EMT) (such as β-catenin, Slug etc.), a process
involved in cancer progression, thus suggesting its powerful
anti-metastatic function^[162]52,[163]53. The antitumor effect of
curcumin is also related to the increase in intracellular calcium
levels (Ca2+) inducing intrinsic apoptosis, as described above.
Finally, the 263 gene-signature constituted by deregulated genes common
to the two tumorigenic MCF7 and MDA-MB-231 cell lines was found to
consist of DEGs implicated in the cell cycle G2/M transition, in cell
migration, in angiogenesis and in calcium signaling.
Indeed, various studies have found that the curcumin anti-tumor effect
is due to its ability to stop the cell cycle in G2/M phase and to
trigger apoptosis of neoplastic cells by increasing the expression
levels of FOX proteins, caspase 3 cleaved, Fas ligand (FasL) and the
reduction of expression levels of cyclin-dependent kinases^[164]54.
The metabolomic analysis allowed us to highlight the effects induced on
metabolism by Cur-SLN in the combined treatment on the three cell
lines.
In particular, the two tumorigenic cell lines MCF7 and MDA-MB-231
showed greater similarities between them in the activated metabolic
pathways, compared to the non-tumorigenic MCF10A cell line,
highlighting a tumor specific metabolic response. Indeed, the
hierarchical clustering reveals two defined area (the upper and the
downer one) where it can be observed a tendency for which the
up-regulated genes of tumorigenic cell lines are down-regulated in the
MF10A, and on the contrary, the down-regulated genes of tumorigenic
cell lines are up-regulated in the MCF10A (Fig. [165]6A). Specifically,
as evidenced by the two metabolic enrichment plots, in the two
tumorigenic cell lines MCF7 and MDA-MB-231, undergoing 2 Gy or
2 Gy/Cur-SNL treatments, an increase in the levels of metabolites
involved in the anti-oxidant response is observed (glutathione
metabolism (GSH), metabolism of arginine and proline, the pentose
phosphate (PPP) pathway and the metabolism of taurine and hypotaurine),
as well as of the metabolites involved in the metabolism of fatty acids
and nucleotides, compared to the non-tumorigenic MCF10A cell line under
the same conditions (Fig. [166]6B). In contrast, the same tumorigenic
cell lines (MCF7 and MDA-MB-231), subjected to 2 Gy and 2 Gy/Cur-SNL
treatments, showed significantly reduced levels of metabolites involved
in amino acid metabolism, Krebs cycle (TCA) and glycolysis compared to
the non-tumorigenic MCF10A cell line under the same treatment
conditions (Fig. [167]6C).
Furthermore, in order the specifically highlight the radiosensitizing
effect of Cur-SLN on each cell line tested, the comparison of metabolic
profilings deriving from 2 Gy vs 2 Gy + 2.5 µM Cur-SLN has been
analysed. In MCF10A cells, a group of metabolites significantly
involved in amino acid metabolism were found to be significantly
deregulated, showing a significant increase in glucose and citrate
(Fig. [168]7A). In contrast, in the MDA-MB-231 cell line the
significantly deregulated metabolites are involved in the metabolism of
amino acids and in antioxidant processes, such as metabolism taurine
and ipotaurine, fatty acids and the Krebs cycle (TCA) (Fig. [169]7C).
Finally, consistently with the results observed in MDA-MB-231, also for
MCF7 BC cells the comparison 2GY vs 2 Gy/Cur-SLN showed a significant
increase in metabolites involved in the metabolism of amino acids, in
antioxidant processes, in the metabolism of fatty acids and Krebs cycle
(TCA), probably due to the activation of the autophagy mechanism
(Fig. [170]7B). Then, the metabolomics analysis, in addition to the
quantification of oxidative stress, has revealed that the
administration of Curc-SNL on BC tumor cell lines exposed to
irradiation with 2 Gy of photon beam, is able to play a protective role
against oxidative stress induced by ionizing radiation and, at the same
time, in the opposite direction, to induce an antitumor effect through
activation of the autophagic mechanism^[171]38.
Based on the findings, the results obtained from this work have
corroborated the literature knowledge on the wide variety of curcumin
bioactivity that justify its suggested administration in combination
with other strategies such as radiotherapy. As curcumin is a natural
molecule with potent anti-inflammatory properties, its
co-administration during the course of a fractioned protocol of
irradiation with conventional radiotherapy, should enhance the cell
killing effect by protecting the closed area by the onset of normal
tissue complications which have inflammatory basis^[172]55.
Then, the formulation of a delivery system to increase the molecule
stability and bio-distribution was the main aim of this study and a
crucial step to move on preclinical studies and clinical trials. In
conclusion, here we have shown the safety and efficacy of a curcumin
delivery system (Cur-SLN) as radiosensitizer on three breast cell
lines, deeply describing its molecular and metabolic effects in
combined treatments.
Conclusion
In conclusion, the data here described indicate that SLN-curcumin
exerts a radiosensitizing effect, rising with its concentration
increasing, particularly, Dose Modifying Factors (DMFs), calculated at
the isoeffect of SF = 50%, showed that the Luminal A MCF7 resulted more
sensitive to the combined treatments using increasing concentration of
vehicled curcumin Cur-SLN, reaching a DMF value of 1,78 using 10 µM of
Cur-SLN. Instead, the more aggressive triple negative MDA-MB-231 cells
were more sensitive to free-Cur, reaching the maximum of
radiosensitizer effect using 10 µM of free-Cur (DMF = 1,72). However, a
clinical administration of curcumin is likely in the vehicle form, then
the aggressive triple negative MDA-MB-231 BC cell line could receive a
radiosensitization effect by Cur-SLN, as the DMF value is 1.38 with
10 µM of Cur-SLN. In addition, the oxidative stress quantification and
the “omic” study explained the radiosensitizing function of Cur-SLN,
confirming at the transcriptomic level many action mechanisms already
known for curcumin, which underline anti-oxidant and anti-tumor
effects, thanks to its ability to regulate various cellular processes.
Moreover, also the metabolomic study highlights, in synthesis, this
double action of curcumin, which on the one hand activates an
anti-oxidant metabolism with protective role against IR and, on the
other hand, exerts an anti-tumor role, stimulating autophagy.
Therefore, the use of curcumin loaded-SLN can be suggested in future
preclinical studies and clinical trials to further test the clinical
implications for the loaded curcumin concomitant use in the course of
fractionated radiotherapy treatments, with the double implications of
being a radiosensitizing molecule against cancer cells, with protective
effects against the onset of normal tissue complications.
Supplementary information
[173]Supplementary info^ (468.9KB, pdf)
[174]51 gene signature.^ (29KB, xls)
[175]263 gene signature^ (62KB, xls)
Acknowledgements