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Lithium chloride increases sensitivity to photon irradiation treatment in primary mesenchymal colon cancer cells

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  • National Institutes of Health, Bethesda, United States

Abstract and Figures

Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide. It is also the second most common cause of cancer‑associated mortality; it accounted for about 9.2% of all cancer deaths in 2018, most of which were due to resistance to therapy. The main treatment for CRC is surgery, generally associated with chemotherapy, radiation therapy and combination therapy. However, while chemo‑radiotherapy kills differentiated cancer cells, mesenchymal stem‑like cells are resistant to this treatment, and this can give rise to therapy‑resistant tumors. Our previous study isolated T88 primary colon cancer cells from a patient with sporadic colon cancer. These cells exhibited mesenchymal and epithelial features, high levels of epithelial‑to‑mesenchymal transition transcription factors, and stemness markers. In addition, it was revealed that lithium chloride (LiCl), a specific glycogen synthase kinase (GSK)‑3β inhibitor, induced both the mesenchymal‑to‑epithelial transition and differentiation, and also reduced cell migration, stemness features and cell plasticity in these primary colon cancer cells. The aim of the present study was to investigate the effect of LiCl treatment on the viability of primary colon cancer cells exposed to 7 Gy delivered by high‑energy photon beams, which corresponds to 6 megavolts of energy. To achieve this aim, the viability of irradiated T88 cells was compared with that of irradiated T88 cells pre‑treated with LiCl. As expected, it was observed that LiCl sensitized primary colon cancer cells to high‑energy photon irradiation treatment. Notably, the decrease in cell viability was greater with combined therapy than with irradiation alone. To explore the molecular basis of this response, the effect of LiCl on the expression of Bax, p53 and Survivin, which are proteins involved in the apoptotic mechanism and in death escape, was analyzed. The present study revealed that LiCl upregulated the expression of pro‑apoptotic proteins and downregulated the expression of proteins involved in survival. These effects were enhanced by high‑energy photon irradiation, suggesting that LiCl could be used to sensitize colon cancer cells to radiation therapy.
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MOLECULAR MEDICINE REPORTS
Abstract. Colorectal cancer (CRC) is the third most prevalent
type of cancer worldwide. It is also the second most common
cause of cancer‑associated mortality; it accounted for about
9.2% of all cancer deaths in 2018, most of which were due to
resistance to therapy. The main treatment for CRC is surgery,
generally associated with chemotherapy, radiation therapy and
combination therapy. However, while chemo‑radiotherapy
kills differentiated cancer cells, mesenchymal stem‑like
cells are resistant to this treatment, and this can give rise to
therapy‑resistant tumors. Our previous study isolated T88
primary colon cancer cells from a patient with sporadic colon
cancer. These cells exhibited mesenchymal and epithelial
features, high levels of epithelial‑to‑mesenchymal transition
transcription factors, and stemness markers. In addition, it
was revealed that lithium chloride (LiCl), a specic glycogen
synthase kinase (GSK)‑3β inhibitor, induced both the mesen‑
chymal‑to‑epithelial transition and differentiation, and also
reduced cell migration, stemness features and cell plasticity in
these primary colon cancer cells. The aim of the present study
was to investigate the effect of LiCl treatment on the viability
of primary colon cancer cells exposed to 7 Gy delivered by
high‑energy photon beams, which corresponds to 6 megavolts
of energy. To achieve this aim, the viability of irradiated T88
cells was compared with that of irradiated T88 cells pre‑treated
with LiCl. As expected, it was observed that LiCl sensitized
primary colon cancer cells to high‑energy photon irradiation
treatment. Notably, the decrease in cell viability was greater
with combined therapy than with irradiation alone. To explore
the molecular basis of this response, the effect of LiCl on
the expression of Bax, p53 and Survivin, which are proteins
involved in the apoptotic mechanism and in death escape, was
analyzed. The present study revealed that LiCl upregulated
the expression of pro‑apoptotic proteins and downregulated
the expression of proteins involved in survival. These effects
were enhanced by high‑energy photon irradiation, suggesting
that LiCl could be used to sensitize colon cancer cells to
radiation ther a py.
Introduction
Colorectal cancer (CRC) is one of the main causes of cancer
deaths and the fourth most frequent cancer worldwide, with
about 1,096,601 newly diagnosed cases and 551,269 deaths in
2018 (1). Despite the increase in the incidence of CRC over
the last 20 years, CRC mortality has decreased in many coun‑
tries, probably due to prevention strategies, early detection
and more effective treatment (1). Surgery is the gold standard
treatment for early colorectal tumors: Indeed it is associated
with 5‑year cancer‑specic survival rates of 91.4 and 70.2%
in stage II and III forms, respectively (2). Treatment of CRC is
dened based on tumor stage. Very early tumors can be treated
using local excision, while neoadjuvant chemora‑diotherapy is
indicated in locally‑advanced rectal cancer (3,4). Metastatic
disease (stage IV) is usually treated with chemotherapy with a
combination of 5‑uorouracil and leucovorin (e.g. oxaliplatin‑
FOLFOX, irinotecan‑FOLFIRI). In addition, two monoclonal
antibodies against the epidermal growth factor receptor are
now used in combination with well‑established CRC‑treatment
schedules (3,5‑7). According to the American Cancer Society
Guidelines, radiation therapy can be considered for colon
cancer therapy to promote cancer reduction before surgery. It
can also be used after surgery in cases in which the tumor
Lithium chloride increases sensitivity to photon irradiation
treatment in primary mesenchymal colon cancer cells
FRANCESCA CAMMAROTA1*, ANDREA CONTE1,5*, ANTONIETTA AVERSANO1, PAOLO MUTO2,
GIANLUCA AMETRANO2, PATRIZIA RICCIO1, MIMMO TURANO3, VALERIA VALENTE1, PAOLO DELRIO4,
PAOLA IZZO1, GIOVANNA MARIA PIERANTONI1** and MARINA DE ROSA1**
1Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II;
2Radiation Oncology, Istituto Nazionale Tumori‑IRCCS Fondazione G. Pascale, I‑80131 Naples;
3Department of Biology, University of Naples Federico II, I‑80126 Naples; 4Department of Abdominal Oncology,
Colorectal Surgical Oncology Unit, Istituto Nazionale Tumori‑IRCCS Fondazione G. Pascale, I‑80131 Naples, Italy
Received July 25, 2019; Accepted November 26, 2019
DOI: 10. 3892/mmr.2020.10956
Correspondence to: Professor Marina De Rosa or Professor
Giovanna Maria Pierantoni, Department of Molecular Medicine
and Medical Biotechnology, University of Naples Federico II, Via S.
Pansini 5, I‑80131 Naples, Italy
E‑mail: marina.derosa@unina.it
E‑mail: gmpieran@unina.it
Present address: 5Lymphocyte Nuclear Biology Lab, National
Institutes of Health, Bethesda, MD 20892, USA
*,** Contributed equally
Key wo rds: lithium chloride, high energy photon irradiation,
mesenchymal colon cancer cells, cancer drug resistance, apoptosis
CAMMAROTA et al: LiCl AND RA DIOTHER APY IN CRC
2
adheres to other organs and cannot be totally removed surgi
cally. Moreover, the so‑called intraoperative radiation therapy
can be used during surgery to kill cancer cells in their loca
tion. Lastly, radiotherapy combined with chemotherapy can
be used for unresectable cancer or to attenuate symptoms in
advanced cancers and in case of metastases (8,9). Although
anticancer therapies have yielded a good success rate, in terms
of overall survival, they fail to kill cancer cells in over 90%
of patients with advanced CRC due to the development of
therapy resistance. Metastatic cancer cells are characterized
by mesenchymal and stemness features conferring them aber‑
rant survival capacity and evasion of apoptosis that represent
the major mechanisms responsible for cancer resistance to
th era py (10).
LiCl is the most well studied GSK‑3β inhibitor. It exerts
its effect through a direct and indirect mechanism. In the rst
case, it competes with the GSK‑3β cofactor Mg2+, thereby
inhibiting the enzyme's activity, whereas in the second case,
it increases the inhibition of phosphorylation of GSK‑3β
Ser9 (11). In addition to its role in the regulation of GSK‑3β,
LiCl emerged as a promising drug for various diseases, such
as neurological diseases, cancer, and inammation (12‑14).
We previously demonstrated that LiCl induces the differen
tiation and the mesenchymal‑to‑epithelial transition (MET)
of primary colon cancer cells, thereby reducing migration
and stemness features (15,16). Since mesenchymal and
stemness features are the main causes of aberrant survival
capacity and evasion of apoptosis during cancer progression,
we suggested that LiCl could sensitize colon cancer cells to
chemo‑radiotherapy. To address this issue, we investigated
the effects of LiCl treatment on the viability of primary
mesenchymal colon cancer cells in combination with radia
tion therapy. We observed that LiCl and high‑energy photon
irradiation had an additive effect both on the viability of
mesenchymal colon cancer cells, and on the induction of
apoptosis. Finally, at molecular level, we found that LiCl
induced strong Survivin down‑regulation and p53 and Bax
up‑regulation. We believe that these molecular changes could
contribute to LiCl‑mediated sensitization to high‑energy
photon irradiation in CRC.
Materials and methods
Sample collection. T88 primary CRC cells were previously
isolated and characterized (15,16). The patient study was
approved by the ethics committe of the University of Naples
Federico II, ‘Comitato etico per le attività Biomediche‑Carlo
Romano’, with protocol no. 35/17. The patient provided written
informed consent to the study. All methods were performed in
accordance with the relevant guidelines and regulations.
Cell culture and treatments. Primary T88 cells were cultured
as reported elsewhere (15,16). Subsequently, cell suspensions
(500 µl containing 240x103 cells) were plated on 100 mm
tissue culture treated plates and cells were alternatively incu‑
bated with LiCl (30 mmol/l) for 48 h, irradiated with 2 or 7 Gy
of high‑energy photon beams or irradiated and pretreated
with LiCl. Untreated cells were compared with treated cells
for subsequent cell analysis. RKO cells were from ATCC and
grown in Eagle's Minimum Essential Medium (M22791L,
Sigma) supplemented with 10% FBS, 100 U/ml penicillin and
100 µg /ml streptomycin.
MTT assay. Untreated cells, LiCl‑treated cells, irradiated cells
and cells irradiated after LiCl pretreatment were analyzed
immediately after (T0) and 48 hours after (T48 h) treat
ments. Cells were washed and incubated for three hours in
100 µl DMEM (ECB7501L; Euroclone) supplemented with
0.45 mg/ml MTT reagent; the medium was then replaced by
100 µl of 0.1 M HCl in isopropanol and cells were incubated
30 min for lysis. Insoluble formazan was resuspended, and
optical densities were measured at a wavelength of 570 nm
with a Microplate Reader (Biotek Synergy Microplate
Reader), according to the MTT manufacturer's protocol. Data
are expressed as mean ± SEM of three experiments.
Western blot analysis. Total protein extracts were isolated
from untreated T88 cells, LiCl treated cells, irradiated cells
and cells irradiated after LiCl pretreatment; all cells were lysed
at time T48h. Cell lysates were prepared as reported previ
ously (17), proteins were separated by SDS‑polyacrylamide
gel electrophoresis, and blots were prepared as reported
previously (18). Primary antibodies against Survivin (rabbit
polyclonal anti‑human; cat. no. 2803; dilution 1:1,000),
β‑catenin (rabbit polyclonal anti‑human; cat. no. 9562; dilution
1:1,000) p53 (rabbit monoclonal anti‑human; cat. no. 2527)
and Bax (rabbit monoclonal antihuman; cat. no. BK2772T)
were from Cell Signaling Technology; the anti‑GAPDH
(monoclonal anti‑mouse; sc‑393358) antibody was from Santa
Cruz Biotechnology. Membranes were probed with peroxi
dase‑conjugated secondary antibodies against rabbit or goat
IgG, and immunoreactive bands were detected as described
before (19). The experiment was repeated three times with
similar results. Densitometric analyses were performed using
Image J software.
Flow cytometry analysis. Untreated cells, LiCl‑treated cells,
irradiated cells and irradiated cells pretreated with LiCl, were
harvested at time T48h, xed and stored in ice‑cold ethanol
at 20˚C. Propidium iodide (PI) (Applichem) was used for
cell cycle analysis, as described previously (20). Briey, cells
were washed twice in ice‑cold PBS and then resuspended at a
concentration of about 1 million/ml of cells in 0.1% Na‑citrate,
50 mg/ml RNase, 50 mg/ml propidium iodide and incubated
for 30 min in the dark at room temperature. PI uorescence
intensity was measured using the BD C6 Accuri cytometer and
data were analyzed using the BD C6 Accuri Software (Becton
Dick inson).
Statistical analysis. All data were obtained from at least three
independent experiments and are reported as the mean ± SEM.
Statistical differences between groups was determined by the
t test and/or Kruskal‑Wallis test followed by a Dunn's post hoc
test at a signicance level of P<0.05.
Results
LiCl decreases the viability of irradiated cancer cells. Using
the MTT assay, we rst evaluated the effect of high‑energy
photon irradiation on the viability of T88 primary colon cancer
MOLECULAR MEDICINE REPORTS 3
cells and of commercially available RKO colon cancer cells.
We initially irradiated cells with 2 Gy, which is the dose gener
ally used in clinical practice. However, neither the T88 primary
colon cancer cells or the commercially available colon cancer
RKO cells responded to this treatment (data not shown). Thus,
we irradiated cells with 7 Gy of high‑energy photon beams,
and obtained a response, in terms of cell viability, only in
RKO cells. As shown in Fig. 1, the photon irradiation affected
the viability of RKO cells, but not that of T88 cells, which
appear completely unresponsive. To determine whether LiCl
would sensitize T88 cells to high‑energy photon irradiation,
we evaluated the viability of T88 cells after i) incubation with
LiCl, ii) high energy photon irradiation and iii) combined
treatment immediately after completion of treatment (T0) and
48 h after completion of treatment (T48). As shown in Fig. 2
and Table I, LiCl decreased the viability of T88 cells versus
untreated cells, at both T0 (Fig. 2A) and T48 h (Fig. 2B). As
expected, high‑energy photon irradiation affected cell viability
only at T48. Notably, the maximum effect on cell viability was
observed when cells were irradiated after LiCl pretreatment,
which suggests that LiCl and high‑energy photon irradiation
may have an additive effect.
LiCl sensitizes colon cancer cells to high energy photon
irradiation altering the balance between pro‑apoptotic and
survival signaling. Since both LiCl and high‑energy photonare
known to activate pro‑apoptotic signals in colon cancer cells,
we investigated the effects of each of these treatments alone
and in combination on the expression of proteins involved in
apoptosis and death escape, such as p53, Bax, and Survivin.
To this aim, we performed Western blot assay on total protein
extracts from untreated cells, cells treated with LiCl or high
energy photons, and cells irradiated with high‑energy photons
after pretreatment with LiCl. Samples were collected 48 h
after completion of treatments. As shown in Fig. 3A‑C, p53
and Bax protein expression was upregulated in both LiCl and
LiCl plus high‑energy photon treated cells, and the highest
increase was observed after combined treatment. On the
contrary, Survivin expression was greatly reduced after each
treatment (Fig. 3A and D). As LiCl induces GSK‑3β inhibi‑
tion, we investigated βcatenin expression level in treated and
untreated cells. As shown in Fig. 3A and in Fig. 3E, βcatenin
expression was stabilized in both LiCl‑treated cells and in cells
treated with LiCl plus high‑energy irradiation. Interestingly,
in LiCl‑treated cells, western blot immunostaining showed
upregulation of an isoform of β‑catenin a little higher in molec‑
ular weight than that observed in cells irradiated without LiCl
pre‑treatment. Additional experiments are necessary to shed
light on this result. To verify whether these molecular changes
result in differences in apoptosis levels, we also performed a
FACS analysis to evaluate the percentage of subdiploid cells.
As expected, the percentage of subdiploid cells increased
signicantly after the combined LiCl and high‑energy photon
treatment versus control cells (Fig. 4). Taken together, these
data indicate that LiCl sensitizes T88 primary CRC cells to
the effects of high‑energy photon treatment by altering the
expression pattern of pro‑apoptotic and pro‑survival proteins.
Discussion
The epithelial‑to‑mesenchymal transition, a biological process
by which epithelial cancer cells lose their epithelial phenotype
and acquire a mesenchymal phenotype, is a physiological
mechanism developed by cancer cells during cancer progres‑
sion and metastases. Indeed, cells that have undergone the
EMT‑transcription factors (EMT‑TFs) acquire all the features
needed to complete the metastatic process, such as, motility,
stem cell features, cell plasticity, resistance to apoptosis and
to therapy. This process is orchestrated by the EMT‑TFs
TWIST1 and Snail. It has been suggested that high levels
of these EMT‑TFs can also cause resistance to apoptosis by
altering the TGF‑β‑ and p53‑mediated programmed cell
death pathways (21‑24). Apoptosis is an energy‑dependent
mechanism of programmed cell death by which organisms
maintain tissue homeostasis (25). Several pathways regulate
apoptosis and several mechanisms are used by tumor cells to
survive, suppressing apoptotic program. The most frequent
apoptotic pathways are the extrinsic and intrinsic pathways.
Both are usually characterized by early activation of the
caspases proteolytic cascade that causes cleavage of cellular
proteins and of other components essential for cell survival,
thereby triggering programmed cell death. In the extrinsic
pathway, death domain‑containing proteins, such as the
tumor necrosis factor family of receptors, which are direct
Figure 1. Effect of high energy photon irradiation on RKO and T88 cells. An MTT assay was performed in untreated and high energy photon‑ irradiated
(A) RKO and (B) T88 cells. Bars represent the mean absorbance ± SEM of experiments performed in triplicate. *P<0.05.
CAMMAROTA et al: LiCl AND RA DIOTHER APY IN CRC
4
targets of caspase cleavage, are activated (26). In the intrinsic
pathway, mitochondria play an essential role in triggering
apoptotic signals by releasing cytochrome‑c into the cytosol,
which induces ‘apoptosome’ assembly. Subsequently, the
‘apoptosome’ complex is able to activate the caspases proteo‑
lytic cascade. This pathway is known as the ‘BCL‑2‑regulated
apoptotic pathway’, because the cell death program is trig
gered by the upregulation of the BCL‑2 protein family, that,
in turn, activates the cell death effectors Bax and Bak (27).
Cytochrome c can also induce the release of other proteins,
i.e., endonuclease G and apoptosis‑inducing factor, that may
promote caspase‑independent cell death (28,29).
The intrinsic pathway is altered in most cancer cells and
is closely regulated by cellular metabolism. Indeed metabolic
changes occurring in cancer cells, consequent to oncogenic
activation or stress‑induced therapy, promote resistance
to apoptosis and therapy via alterations of BCL‑2 family
expression (25). P53, a protein often altered in tumor progres
sion, has been implicated in the activation of the intrinsic
and extrinsic pathways, and its ability to induce apoptosis
depends on NF‑κB activation (30). Furthermore, alteration in
the extrinsic and intrinsic pathways could cause resistance to
anoikis, which is programmed cell death induced by detach
ment of cells from the extracellular matrix, that is often
altered in cancer cells with metastatic potential (31‑34). Upon
activation of proapoptotic cellular pathways, the survivin
protein, a member of a family of apoptosis inhibitors, is
released from mitochondria and inhibits caspase‑9 thereby
enhancing the effects of inhibitor of apoptosis proteins (35).
In this scenario, we previously isolated and characterized
epithelial colon cancer cells endowed with mesenchymal
features, and together with high EMT‑TFs expression, in the
attempt of identifying a therapeutic target able to sensitize colon
cancer cells to specic therapies. These cells were able to grow
for long periods in suspension as tumorspheres, which suggests
they are anoikis‑resistant (16). We have also demonstrated
that LiCl induces differentiation, MET, and downregulation
of the EMT‑TFs TWIST‑1 and Snail, in primary colon cancer
cells (15). In the present study, we demonstrate that T88
primary colon cancer cells are completely unresponsive to
high‑energy photon irradiation in terms of cell viability, and
that LiCl sensitizes them to such treatment.
Development of radiation resistance has been correlated
with two main mechanisms, one consisting in disequilibrium
between pro‑apoptotic signaling transduction pathways (medi
ated by p53 and Bax) and pathways mediating cell survival,
in which the protein Survivin plays an essential role (36‑38).
On the other hand, DNA double strand break repair pathways,
which maintain genomic integrity and prevent mis‑repair
and chromosomal rearrangements, could be responsible
for radio‑resistance and failure of radiation treatment.
Rouhani et al (39) reported that LiCl increases radio‑sensitivity
in breast cancer cells in vitro by abrogating DNA repair.
Indeed, in contrast to the expected effect of LiCl, i.e., GSK‑3β
inactivation and β‑catenin stabilization, the authors observed
GSK‑3β upregulation and β‑catenin down‑regulation together
with mRNA downregulation of its transcriptional target
MR11, which is a crucial protein of DSB repair system (39).
Figure 2. Effect of LiCl on high energy photon irradiation sensitivity of T88 cells. MTT assay performed in T88 cells (A) immediately after (T0) or (B) 48 h
(T48 h) after treatment with LiCl and/or high energy photons. Bars represent the mean absorbance ± SEM of a representative experiment performed in
triplicate. *P<0.05 vs. untreated cells, using Kruskal‑Wallis test. LiCl, lithium chloride.
Table I. Statistical analysis of MTT assay.
Statistic T0 T48h
Un. (mean ± SEM) 100.00 100.00
LiCl (mean ± SEM) 66.00±3.46 61.00±19.08
High energy photons (mean ± SEM) 91.33±8.41 83.00±10.60
LiCl + high energy photons (mean ± SEM) 63.00±3.00 31.00±14.18
P‑value: Un. vs. LiCl 0.1161 0.3312
P‑value: Un. vs. high energy photons >0.9999 >0.9999
P‑value: Un. vs. LiCl+high energy photons 0.0476a 0.0262a
Results are representative of three independent experiments. aP<0.05 (Kruskal‑Wallis test). Un, untreated; LiCl, lithium chloride.
MOLECULAR MEDICINE REPORTS 5
As expected, we observed that LiCl induces apoptosis by
activating cell death signaling and down‑regulating survival
signaling in T88 cells. Indeed, LiCl induced upregulation of
the p53 and Bax proteins and strong downregulation of the
survivin protein. In addition, combined cell treatment with LiCl
and high energy photons was more effective than high energy
photons or LiCl used alone in reducing the viability of colon
cancer cells. In fact, under combined treatment, cells show
the highest percentage of apoptotic subdiploid cells between
all treatments analyzed, associated with the highest expres
sion of p53 and Bax protein, and absence of survivin protein
expression. A diagrammatic summary representing a model
for the action of LiCl by which it sensitizes resistant colon
cancer cells to radiotherapy is shown in Fig. 5. In contrast to
breast cancer (39), but in accordance with the LiCl mechanism
of action, we observed stabilization of high molecular weight
β‑catenin isoforms in cells treated with LiCl alone and in cells
treated with LiCl plus high energy photons. We speculated
that these isoforms could be those phosphorylated by pyruvate
dehydrogenase kinase 1 in Thr112 and Thr120, which are selec
tively directed to the plasma membrane, where they interact
with the E‑cadherin protein (40). We previously observed that
the effect exerted by LiCl on the level of β‑catenin expression
was time‑dependent and LiCl promoted β‑catenin membrane
localization (15,16). Furthermore, β‑catenin is heterogeneously
distributed in CRC cells; indeed, well‑differentiated paren
chymal cells, located in the tumor center, retain β‑catenin
membranous expression comparable to that of normal colon
epithelium, while nuclear β‑catenin expression predominates
in tumor cells localized at the invasion front (41). It would be
interesting to investigate further the role of β‑catenin in LiCl
‑induced sensitization to photon irradiation therapy and, more
Figure 3. Effect of LiCl and high energy photon irradiation on p53, Bax, Survivin and βcatenin protein expression in T88 cells. (A) Representative western
blot images revealing the protein expression levels of p53, Bax and Survivin in treated and untreated T88 cells. GAPDH was used as the loading control.
The graph shows the densitometric analysis of (B) p53, (C) Bax, (D) Survivin and (E) β‑catenin compared with GAPDH. Bar graphs represent mean ± SEM
(3 independent experiments). *P<0.05 and ***P<0.0001 vs. untreated cells, using Kruskal‑Wallis test. LiCl, lithium chloride.
CAMMAROTA et al: LiCl AND RA DIOTHER APY IN CRC
6
Figure 5. Model of LiCl action in sensitizing resistant colon cancer cells to radiotherapy. Photon irradiation induces apoptosis, in the majority of differentiated
cancer cells a nd healthy cells, via p53 upregulation. Only few cells survive; the cells which under went EMT. Mesenchymal colon cancer cells show nuclear
localization of β‑cateni n and p53 alterations a nd, consequently, are resistant to apoptosis after irradiation. LiCl induces mesenchymal to epithelial transition
and differentiation of colon cancer cells. The EMT‑TFs are downregulated and β‑catenin is selectively directed to the plasma membrane. Consequently, photon
irradiation causes p53 upregulation and cancer cell death by apoptosis. LiCl, lithium chloride; EMT, epithelial‑mesenchyma l transition.
Figure 4. Effects of LiCl and high‑energy photon irradiation on the subdiploid‑apoptotic fraction of T88 cells. Propidium iodide was used to stain cellular DNA
and ow cytometry was performed to analyze cell cycle distribution. LiCl, lithium chloride.
MOLECULAR MEDICINE REPORTS 7
generally, in the antineoplastic effect of LiCl in colon cancer.
Could the LiCl‑sensitizing effect to photon irradiation on colon
cancer cells be also mediated by a decrease in the activity of
the DSB repair system, as observed in breast cancer cells?
Additional experiments are required to shed light on these
intriguing and controversial points.
As we discussed in a previous paper (15), cell culture
models obviously do not mimic the complex interactions that
occur in the intestinal mucosa, and a functional change in cell
cultures may not equate with the effect observed in vitro. On
the other hand, complex functions can be reproduced in cell
cultures and thus become amenable to investigation. However,
the data reported herein could have important clinical signi
cance and clinical applications and need, in a next future, to
be improved and reinforced with animal models experiments.
In conclusion, we demonstrate that T88 mesenchymal
colon cancer cells are resistant to radiotherapy, and that LiCl
sensitizes these cells to apoptosis in response to high‑energy
photons, probably by acting on the balance between pro‑apop‑
totic and survival signaling transduction pathways. In light of
our nding, we suggest that LiCl could be used to increase
sensitivity of resistant colon cancer cells to radiotherapy.
Acknowledgements
The authors would like to thank Dr Jaen Ann Gilder for the
text editing.
Funding
The present study was supported by a grant from Fondo
Straordinario di Ateneo‑2018, Università di Napoli Federico II;
POR Campania FESR 2014‑2020 ‘SATIN’.
Availability of data and materials
All data generated or analyzed during this study are included
in this published article.
Authors' contributions
GMP, MDR and PR designed the study. FC, AC, AA, VV and
MT performed the cellular and molecular experiments. PM,
GA and PR performed the high energy photon irradiation.
MDR and PR performed the statistical analysis of the data.
PD provided the surgical sample for primary cell isolation.
GMP and MDR coordinated the work. GMP, MDR, PI and PD
contributed to data interpretation. PI provided funding. MDR,
PI and PD wrote the manuscript. GMP and MT critically
revised the manuscript. All authors edited and approved the
nal version of the manuscript.
Ethics approval and consent to participate
The patient study was approved by the Ethics Committe of
the University of Naples Federico II, ‘Comitato etico per le
attività Biomediche‑Carlo Romano’ (no. 35/17). The patient
provided written informed consent for the study. All methods
were performed in accordance with the relevant guidelines
and regulations.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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... In light of this previous evidence, in this study, we have analyzed the effects of LPE using primary human colon T88 and T93 cancer cells, which have been previously characterized [30][31][32]. These primary colon cancer cells were isolated and established from the tumor tissues of two patients affected by sporadic colon adenocarcinoma. ...
... The T93 cell line exhibited a chromosomal instability (CIN) phenotype and the T88 cell line exhibited a microsatellite instability (MSI) phenotype. It has been previously demonstrated that these cells both underwent EMT from epithelial adenocarcinoma cells; indeed, they simultaneously expressed epithelial and mesenchymal markers, such as cytokeratin and Ecadherin, and Vimentin and N-cadherin, respectively, together with high levels expression of EMT-associated transcription factors, stemness markers, and the Cyclooxygenase-2 (Cox-2) enzyme [30][31][32]. ...
... Furthermore, the data reported here show that LPE is able to inhibit the rIL-6-dependent cell migration and invasiveness in human primary T88 and T93 colon cancer cells [30][31][32] via the up-regulation of MMP-2 expression, and that the observed effects correlate with the STAT3 phosphorylation levels. The protective effects showed by plant polyphenols against different types of cancer [36] include their anti-inflammatory and antioxidant properties [26][27][28][29]. ...
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... To set up the optimal conditions and discriminate apoptotic cells from live cells, we used RKO and HEK 293T untreated cells as internal controls in these experiments. When these cells were induced to undergo apoptosis by a 24 h treatment with 60 mM LiCl, a GSK3β inhibitor known to induce apoptosis in several human cell lines [35][36][37], they exhibited a high percentage of subdiploid cells ( Figure 3A). In contrast, when Dox-treated RKO and HEK 293T cells were analyzed for their DNA content, they presented only a negligible percentage of the subdiploid peak ( Figure 3A), indicating that they are not subjected to apoptosis. ...
... To set up the optimal conditions and discriminate apoptotic cells from live cells, we used RKO and HEK 293T untreated cells as internal controls in these experiments. When these cells were induced to undergo apoptosis by a 24 h treatment with 60mM LiCl, a GSK3β inhibitor known to induce apoptosis in several human cell lines [35][36][37], they exhibited a high percentage of subdiploid cells ( Figure 3A). In contrast, when Dox-treated RKO and HEK 293T cells were analyzed for their DNA content, they presented only a negligible percentage of the subdiploid peak ( Figure 3A), indicating that they are not subjected to apoptosis. ...
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Dyskerin is an evolutionarily conserved nucleolar protein implicated in a wide range of fundamental biological roles, including telomere maintenance and ribosome biogenesis. Germline mutations of DKC1, the human gene encoding dyskerin, cause the hereditary disorders known as X-linked dyskeratosis congenita (X-DC). Moreover, dyskerin is upregulated in several cancers. Due to the pleiotropic functions of dyskerin, the X-DC clinical features overlap with those of both telomeropathies and ribosomopathies. In this paper, we evaluate the telomerase-independent effects of dyskerin depletion on cellular physiology by using inducible DCK1 knockdown. This system allows the downregulation of DKC1 expression within a short timeframe. We report that, in these cellular systems, dyskerin depletion induces the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum, which in turn induces the activation of the PERK branch of the unfolded protein response. We also demonstrate that the PERK-eIF2a-ATF4-CHOP signaling pathway, activated by dyskerin downregulation, triggers a functional autophagic flux through the inhibition of the PI3K/AKT/mTOR pathway. By revealing a novel unpredicted connection between the loss of dyskerin, autophagy and UPR, our results establish a firm link between the lowering of dyskerin levels and the activation of the ER stress response, that plays a key role in the pathogenesis of several diseases.
... Furthermore, the rats treated with lithium chorlide and CCl 4 showed a significant increase in the apoptotic cell count compared to those treated with CCl 4 only. These results are in accordance with Zhang et al. [63], Yao et al. [64] and Cammarota et al. [65] who reported that lithium chloride up-regulated the expression of proapoptotic proteins such as apoptosis regulator BAX, p53, and Survivin and down-regulated the expression of proteins involved in survival. In addition, these rats showed significant up-regulation of α-SMA expression compared to the rats treated with CCl 4 only. ...
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We asked what preoperative radiotherapy/chemoradiotherapy (PRT/PCRT) has brought to patients in terms of perioperative and long-term outcomes over the past decades. A systematic review and meta-analysis was conducted using PubMed, Embase and Web of Science databases. All original comparative studies published in English that were related to PRT/PCRT and surgical resection and which analyzed survival, postoperative and quality of life outcomes were included. Data synthesis and statistical analysis were carried out using Stata software. Data from 106 comparative studies based on 80 different trials enrolling 41,121 patients were included in our study. Based on our overall analyses, PRT/PCRT significantly improved patients' local recurrence-free survival (LRFS), but neither overall survival (OS) nor metastasis-free survival (MFS) showed improvement. In addition, PRT significantly increased the postoperative morbidity and mortality but PCRT did not have a significant effect. Furthermore, PRT/PCRT significantly increased the risk of postoperative wound complications but not anastomotic leakage and bowel obstruction. Our comprehensive subgroup analyses further supported the aforementioned results. Meanwhile, long-term anorectal symptoms (impaired squeeze pressures, use of pads, incontinence and urgency) and erectile dysfunction were also significantly increased in patients after PRT/PCRT. The benefits of PRT/PCRT as applied over the last several decades have not been sufficient to improve OS. Metastases of primary tumor and postoperative adverse effects were the two primary obstacles for an improved OS. In fact, the greatest advantage of PRT/PCRT is still local tumor control and a significantly improved LRFS. This article is protected by copyright. All rights reserved.