Content uploaded by Ivana Damnjanovic
Author content
All content in this area was uploaded by Ivana Damnjanovic on Apr 04, 2019
Content may be subject to copyright.
FARMACIA, 2019, Vol. 67, 2
226
https://doi.org/10.31925/farmacia.2019.2.5
ORIGINAL ARTICLE
POSSIBLE MOLECULAR MECHANISMS AND PATHWAYS INVOLVED
IN BH3 MIMETIC ACTIVITY OF ALPHA-LIPOIC ACID ON HUMAN
COLON CANCER CELL LINE
IVANA DAMNJANOVIC 1*, GORDANA KOCIC 2, STEVO NAJMAN 3, SANJA STOJANOVIC 3,
KATARINA TOMOVIC 1, BUDIMIR ILIC 4, ANDREJ VELJKOVIC 2, SRDJAN PESIC 5, ANDRIJA
SMELCEROVIC 1,4
1Department of Pharmacy, Faculty of Medicine, University of Nis, Blvd. Dr Zorana Djindjica 81, 1800 Nis, Serbia
2Department of Biochemistry, Faculty of Medicine, University of Nis, Blvd. Dr Zorana Djindjica 81, 18000 Nis, Serbia
3Department for Cell and Tissue Engineering, Institute of Biology and Human Genetics, Faculty of Medicine, University of
Nis, Blvd. Dr Zorana Djindjica 81, 18000 Nis, Serbia
4Department of Chemistry, Faculty of Medicine, University of Nis, Blvd. Dr Zorana Djindjica 81, 18000 Nis, Serbia
5Department of Pharmacology, Faculty of Medicine, University of Nis, Blvd. Dr Zorana Djindjica 81, 18000 Nis, Serbia
*corresponding author: ivana.damnjanovic@medfak.ni.ac.rs
Manuscript received: March 2018
Abstract
Alpha-lipoic acid (ALA), a naturally-occurring antioxidant, inhibits proliferation and induces apoptosis in various cancer cell
lines without effects on normal non-transformed cells. The aim of this study was to examine the effects of alpha-lipoic acid
(ALA), alone and combined with 5-fluorouracil (5-FU), on Bcl-2/Bax expression in human colon cancer Caco-2 cell line as
well as to investigate possible molecular mechanisms and pathways involved in ALA-mediated effects. In the present study
ALA and 5-FU showed a tendency to decrease Bcl-2 and increase Bax expression. ALA exerted higher inhibitory effects on
Bcl-2 expression, while the significant increase of Bax expression was shown after the treatment with the combination of
ALA and 5-FU. The binding modes of ALA and 5-FU with both targets were shown to be closely similar, and some
interactions the same like those of known BH3 mimetics. Thus, ALA may be considered as potential BH3 mimetic.
Additionally, with in silico calculated physico-chemical properties taken into account, it was confirmed that ALA may easily
be delivered to its intracellular and membrane targets.
Rezumat
Acidul alfa-lipoic (ALA), un antioxidant natural, inhibă proliferarea și induce apoptoza la nivelul diferitelor linii celulare
canceroase, fără efect asupra celulelor normale netransformate. Scopul acestui studiu a fost de a evalua efectele acidului alfa-
lipoic (ALA), singur și combinat cu 5-fluorouracil (5-FU), asupra exprimării Bcl-2 / Bax în linia celulară Caco-2, de origine
din colonul uman, ca să investigheze posibilele mecanisme moleculare și căile implicate în efectele mediate de ALA.În
studiul de față ALA și 5-FU au arătat o tendință de scădere a Bcl-2 și creșterea expresiei Bax. ALA a exercitat un efect
inhibitor mai mare asupra exprimării Bcl-2, în timp ce creșterea semnificativă a expresiei Bax a fost demonstrată după
tratamentul combinat ALA și 5-FU. Modelele de legare a ALA și 5-FU de ambele ținte moleculare s-au dovedit a fi foarte
apropiate, iar unele interacțiuni sunt identice cu cele cunoscute pentru mimeticele BH3. Astfel, ALA poate fi considerat ca
potențial mimetic BH3. În plus, proprietățile fizico-chimice evaluate in silico au confirmat că ALA are un tropism deosebit
pentru țintele intracelulare și membranare ale acțiunii sale.
Keywords: alpha-lipoic acid, BH3 mimetic, colon cancer, molecular mechanisms
Introduction
Alpha-lipoic acid (ALA), a small dithiol molecule
derived from octanoic acid, may act as a powerful
micronutrient with diverse pharmacological and anti-
oxidant properties [1]. This naturally-occurring anti-
oxidant, known as a vitamin, co-factor of some
mitochondrial enzymes (pyruvate dehydrogenase, α-
ketoglutarate dehydrogenase, glycine decarboxylase),
potent free radical scavenger and drug candidate, may
be de novo synthesized in small amounts by animals
and humans [2, 3]. It can also be absorbed from
dietary sources such as red meat, potatoes and may
be present in wheat and, to a lesser degree, in fruits
and vegetables [4, 5]. Namely, high ALA contents
(0.55 to 2.36 ppm) are present in food of animal
origin (mainly in liver and muscles), whereas plants
contain very little (0.09 ppm) or no detectable ALA
[6]. It has been used like a dietary supplement in the
prevention or treatment of stroke, diabetes, neuro-
degenerative and hepatic disorders [3]. Moreover, it
has been reported that ALA inhibits proliferation
and induces apoptosis in various cancer cell lines
including human lung epithelial cancer NCI-H460
cells [7], human cervical carcinoma HeLa S3 cells [8],
FARMACIA, 2019, Vol. 67, 2
227
ovarian carcinoma cell lines IGROV1 and IGROV1-
R10 [9], SMMC-7721 human hepatoma cell line [10],
MDA-MB-231 breast cancer cells [11], T24 human
bladder cancer cell line [12] and human promyelocytic
HL-60 cells [3], without effects on normal non-
transformed cells [7]. The cytotoxicity of ALA at
millimolar concentrations was shown on SW620 human
colon carcinoma cells as well as the enhancement of
this effect in a synergistic manner in the combination
with ascorbate [13]. It has been determined that ALA
is able to effectively induce apoptosis in human colon
cancer HT-29 cells via increased ROS (reactive
oxygen species) production in mitochondria [14].
ALA has been reported to induce cell death in HT-29
and Caco-2 cell lines mediated by the activation of
caspase-9, -3 and -7, and potentiate the cytotoxicity
of 5-fluorouracil (5-FU) in these cells [15]. ALA
may be considered as potential novel drug candidate
for cancer therapy, but mechanisms of its chemo-
preventive effects need to be better clarified.
Continuing research on chemopreventive potential of
ALA, after testing its effects alone or in combination
with 5-FU or cisplatin on the proliferation of colon
cancer cell lines and proved anti-proliferative activity
[8], the aim of this study was to assess the effects of
ALA, alone and combined with 5-FU, on Bcl-2/Bax
expression in human colon cancer Caco-2 cell line as
well as to investigate possible molecular mechanisms
and pathways involved in ALA-mediated effects.
Molecular docking studies were performed to gain an
insight into the binding modes of possible interactions
of ALA and 5-FU with Bcl-2 and Bax proteins as
checkpoints of intrinsic apoptotic pathway.
Additionally, an in silico study using Molinspiration
tool [16] was performed in order to gain a better
insight into the physico-chemical properties of ALA.
Materials and Methods
Chemicals
Alpha-lipoic acid (ALA) and 5-fluorouracil (5-FU) were
purchased for experiments as follows: ALA (Berlition
ED 300, Berlin-Chemie, Germany, 300 mg/12 mL)
and 5-FU (Fluorouracil Teva, Pharmachemie BV -
Netherlands, 50 mg/mL). DMEM (Dulbecco's Modified
Eagle Medium), FBS (Foetal Bovine Serum), antibiotic/
antimycotic solution, L-glutamine and Trypsin-EDTA
solution were purchased from PAA Laboratories (PAA
Laboratories, Austria). Trypan blue for cell staining
was purchased from Invitrogen. Primary anti-Bcl-2
and anti-Bax antibodies and secondary antibodies
were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Examined cytostatic drug 5-FU is
used in the treatment of colon carcinoma according
to the protocol [17].
Cell line
In this study we used Caco-2 cell line (human colon
cancer cells) which was obtained from ATCC. Cells
were cultured in DMEM supplemented with 10%
FBS, 2 mM L-glutamine and antibiotic/antimycotic
solution at 37ºC in an atmosphere with 5% CO2 and
saturated humidity. Replacement of the culture medium
was performed every 2 to 3 days.
Treatment of cells
Confluent culture of Caco-2 cells was harvested using
Trypsin-EDTA solution, washed in buffer solution
and the total number of cells was determined by
Trypan blue dye exclusion test. Cells were seeded
in 96-well plates (Greiner Bio-One, Germany) at a
density of 3 x 104 cells per well and cultured for 24 h
under standard cell culture conditions. After that, the
examined compounds, alone or in combination, were
added to the cells. ALA and 5-FU were diluted in
DMEM and three concentrations of each of these
compounds were tested (group 1 – the lowest
concentration, group 2 – middle concentration and
group 3 – the highest concentration). Final concentrations
of the assayed compounds were 10, 100 and 1000 µM.
Combining of ALA and 5-FU was performed using
the same concentrations as the following: group 1
with group 1, group 2 with group 2, and group 3
with group 3 in the ratio 1:1, so that the effective
concentrations of compounds in combinations were
twice less than the concentrations of compounds that
were applied alone. As control we used cells that
were incubated only with completed cell culture
medium, DMEM, without the assayed compounds.
Cells were incubated with ALA and 5-FU for the next
48 h. After that, the level of Bcl-2 and Bax proteins
was measured.
Measurement of Bcl-2 and Bax protein levels
For determining the levels of Bcl-2 and Bax proteins,
the cells were treated as it was described in the section
“Treatment of cells”. After 48 h of incubation with
the assayed compounds cells were further processed
according to the protocol by Kocic et al. [18]. Briefly,
the cells were washed with phosphate-buffered saline
(PBS), fixed by using 70% methanol and permeabilized
with 0.1% Triton in PBS. The cells were incubated
with the primary anti-Bax and anti-Bcl-2 antibodies,
washed three times and incubated with the FITC-
conjugated secondary antibodies. The mean fluorescence
intensity (MFI; logarithmic scale) was determined
and analysed on a Victor™ multiplate reader (Perkin
Elmer-Wallace, Wellesley, MA). The presented results
were obtained following the subtraction of blank
values obtained by the treatment with the secondary
antibodies only.
Ligand and receptor preparation and validation of
docking protocol
The molecular docking study was performed using
MOE 2014.0901 to understand the ligand protein
interactions in detail. The X-ray crystallographic
structures of Bcl-2 protein complexed with inhibitor
(PDB code: 4IEH) and Bax protein complexed with
FARMACIA, 2019, Vol. 67, 2
228
activator (PDB code: 2K7W) were obtained from
the Protein Data Bank [19, 20].
Statistical analysis
The data were analysed by the commercially available
statistics software package (SPSS for Windows®, v.
17.0, Chicago, USA) using the Students' t-test and the
ANOVA test. The results are presented as percentage
of control. The statistical significance was set to p <
0.05.
Results and Discussion
Figure 1 shows the levels of Bcl-2 and Bax quantitative
expression in Caco-2 cell culture treated with different
concentrations of ALA and 5-FU under the given
experimental conditions. Both tested compounds
showed a tendency to decrease Bcl-2 and increase
Bax expression levels, compared with control samples.
It was shown that ALA exerts a significant inhibitory
effect on Bcl-2 expression at the concentration of
1000 µM, while the effect of 5-FU at the same
concentration was lower. The significant increase of
Bax expression was shown after the treatment with 5-
FU alone and its combination with ALA, especially at
the highest tested concentrations of both compounds.
Literature data showed that 5-FU-induced apoptosis
was accompanied by an increased expression of Bax
and Bak without consistent modulation of other Bcl-
2 family proteins [21].
Figure 1.
The effects of ALA, 5-FU and their combination on Bcl-2 and Bax expression in Caco-2 cell culture
The apoptosis has been shown to be a major barrier
to cancer cells [22]. Various factors such as Bcl-2
family members play a major role in the intrinsic
mitochondrial apoptotic cascade [23]. The members
of Bcl-2 family are divided in two main groups, pro-
apoptotic (Bax, Bad, Bak, Bim, Bid, Bik, Noxa, Puma)
and antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, Mcl-1), which
are characterized by sharing at least one region of
Bcl-2 homology sequence termed BH1-BH4 [24, 25].
The over expression of survival proteins not only
contributes to the progression of cancer, but also
confers resistance to the therapeutic treatments [25].
Nowadays, the importance of apoptosis, its signalling
pathways, checkpoints, mediators and modulators in
the pathogenesis and prognosis of colorectal carcinoma
is being increasingly recognized. Molecules involved
in these pathways represent potential diagnostic
markers and therapeutic targets and consequently are
the focus of numerous research efforts [26]. Bax/Bcl-2
ratio can act as a rheostat which determines cell
susceptibility to apoptosis as well as affects tumour
progression and aggressiveness [27]. It has been
reported that antiapoptotic Bcl-2 protein plays a role
in colorectal precancerous and cancerous lesions
[28]. On the other hand, the observed loss of Bcl-2
expression in high-grade tumours may lead to tumour
progression to a deregulated state where Bcl-2 would
not be required for the cell survival [29]. Indicating
the involvement of Bax expression in tumour
differentiation and metastatic progression, it has
been demonstrated that Bax expression is decreased
not only from primary to metastatic but also well/
moderately to poorly differentiated tumours, what
leads to more infiltrative growth pattern, more distant
metastases, and a weak trend toward poor prognosis
[27]. The absence of Bax expression in colorectal
cancer cells might induce resistance to apoptosis
triggered by different chemotherapeutic agents [30].
The expression of Bcl-2 and Bax has been shown to
predict the response to 5-FU based adjuvant therapy
in colorectal carcinomas. Namely, the patients with
low Bax/Bcl-2 ratio would benefit from this therapy
[31]. Over 50 years have passed since 5-FU was
developed, but it still plays a key role in chemo-
therapeutic regimens in the treatment of breast, colon
and pancreatic cancer [32]. Although, 5-FU is a
commonly used effective chemotherapeutic agent for
treating a wide variety of malignant tumours, the
effectiveness of the chemotherapy may be limited
because of acquired or intrinsic drug resistance [33].
FARMACIA, 2019, Vol. 67, 2
229
Small BH3-only members, with proapoptotic activity,
may be helpful in elucidating alterations in Bax and
Bcl-2 expression level in human colorectal carcinoma
[27]. Proapoptotic and prosurvival Bcl-2 family
proteins contain shared Bcl-2 homology domains
BH1-3, that are involved in the formation of a
hydrophobic BH3 docking groove harbouring binding
sites for only BH3 containing proteins [34]. It has
been demonstrated that peptides containing only BH3
domain of proapoptotic Bcl-2 family members are
able to bind and inhibit antiapoptotic proteins [25].
Some BH3-only proteins such as Bim may directly
bind to and activate Bax, what leads to the
oligomerisation of this proapoptotic protein,
permeabilization of the mitochondrial outer membrane,
cytochrome c release and activation of caspase-9, -3,
-6 and -7 [35, 36]. It has been suggested that Bim
downregulation is important for tumour-genesis,
especially for metastatic ability [36]. Recently, it has
been shown that ALA upregulates Bim in ovarian
carcinoma cells [9]. Bim phosphorylation by extra-
cellular signal regulated kinase 1/2 (ERK1/2) promotes
its degradation through the proteasome-ubiquitination
system [36]. Some preclinical studies have pointed
out that Bim induction by inhibition of the ERK
pathway plays a key role in apoptosis of oncogene-
addicted solid cancer cells including BRAF - mutant
colorectal ones [37]. Moreover, it has been recently
reported that BH3 mimetic ABT-737 may markedly
suppress the ERK1/2 phosphorylation levels in a
concentration-dependent manner and induce pro-
apoptotic Bim signalling pathway in human oral
squamous carcinoma HN22 cells [38]. Contrary to
Bim, the phosphorylation of Bcl-2 mediated by ERK2
might be of substantial importance for Bcl-2 stability
and cell survival. Bcl-2 dephosphorylation induces its
degradation through the proteasome-ubiquitination
system and subsequent cell death [39]. It has been
documented that ALA induces downregulation of
Bcl-2 through its enhanced proteasomal degradation
in human lung epithelial cancer cells [7]. The activation
of ERK, c-Jun N-terminal kinase (JNK) and p38
kinase in colon tissues was shown to be significantly
inhibited by ALA treatment [40]. Thus, by the
inhibition of mitogen-activated protein kinase (MAPK)/
ERK, ALA may be considered to upregulate Bim
and downregulate Bcl-2 levels in cancer cells.
Phosphorylation of Bcl-2 family members has appeared
to be a generalized phenomenon that occurs in pro-
apoptotic and antiapoptotic family members [41].
Bax conformational change and its translocation to the
mitochondrial membrane is inhibited by phosphorylation
in an Akt-dependent manner. After the phosphorylation,
Bax effects on the mitochondria are inhibited by
remaining its inactive heterodimerized form with
antiapoptotic Bcl-2 family members in cytoplasm.
Akt (protein kinase B, PKB), also, phosphorylates and
inhibits the protease activity of caspase-9 [41-44].
It has been shown on hepatoma and human breast
cancer cells that ALA may inhibit Akt activity [10,
11]. In that way, by the inhibition of Akt, ALA may
potentiate Bax proapoptotic actions. Recent study on
bladder cancer cells represents one more confirmation
of the ERK1/2 and Akt downregulation caused by
ALA [12]. Generally, the kinase activities of Akt and
ERK1/2 were shown to be significantly upregulated in
CD133+ primary colon cancer cells. The clonogenic
growth, proliferation and differentiation of these cells
was greatly reduced by the inhibition of Akt and
ERK1/2 activity. The reported involvement of Akt and
MAPK pathways in the tumorigenesis of CD133+
colon cancer cells, suggest that molecules in these
two pathways might be potential targets in the future
therapy [45]. Moreover, the fate of Caco-2 cells has
been shown to be regulated by MAPK and Akt
pathways [46, 47]. Thus, the potential mechanism of
proapoptotic effect of ALA concretely in Caco-2 cells
might be explained on the basis of these mentioned
pathways.
The activation of intrinsic mitochondrial pathway of
apoptosis in cancer cells, mediated by Bcl-2 family
members as critical checkpoints, represents the most
frequent mechanism of action of anticancer drugs
[48]. Antiapoptotic family members are now a major
target in the development of novel anticancer drug
candidates [49]. Many structurally different Bcl-2
inhibitors and some Bax activators have been discovered
in recent years [50, 51]. A large number of pre-
clinical data suggest that BH3 mimetics may be highly
useful for applying in synergistic therapies with
conventional anticancer drugs and radiotherapy [35].
Despite this, the use of BH3 peptides as therapeutic
agents may be limited by their unsatisfactory bio-
availability, including poor cellular permeability,
solubility and metabolic instability in vivo. It might
be feasible to develop agents by "BH3 profiling" with
individual prosurvival Bcl-2 family members like
targets [52].
The search of protein targets of ALA as well as docking
of the binding site is necessary to understand and
support its physiological roles and pharmacological
effects [2]. Considering the promising biological
results in order to evaluate the mode of possible
interaction of ALA and 5-FU with Bcl-2 and Bax
targets, molecular docking of these compounds and
some known Bcl-2 inhibitors and Bax activators was
carried out and results were compared. The structures
of known inhibitors/activators of these targets are
various and were used as starting points to allow the
evaluation of the binding modes of ALA and 5-FU.
The binding modes of the two assayed compounds
resembled those found by the docking study on
complexes of Bcl-2 and Bax with their inhibitors
and activators, respectively.
Our docking study indicated that the binding mode
of two BH3 mimetics with reported high affinity,
FARMACIA, 2019, Vol. 67, 2
230
synthesized, orally available ABT-263 (navitoclax)
and its synthetic derivative ABT-199, includes the
interaction with Arg66 residue of Bcl-2 target. Namely,
the interactions of the sulfonyl oxygen of ABT-263
and the carbonyl and sulfonyl oxygen and oxygen
of the nitro group of highly specific and potent Bcl-
2 inhibitor ABT-199 with Arg66 residue were
established. Importantly, the interaction of carbonyl
oxygen of ALA with Arg66 residue was found in
the present study. The common interaction of the
binding modes of ALA and 5-FU with Bcl-2 protein
was shown to be the arene-hydrogen interaction with
Tyr161, which was shown to be involved also in the
binding of BH3 mimetic BI97D6, the new compound
of gossypol family with modest binding affinity and
reported in vivo antitumor activity. Most of the amino
acid residues of the Bcl-2 binding site for ALA and
5-FU overlapped with those involved in the binding
pockets for the known and assayed Bcl-2inhibitors
(Figure 2).
Figure 2.
Docking conformation and 2D representation of ALA (A) and 5-FU (B) interactions in the binding site of Bcl-2
target (PDB code: 4IEH)
Carbonyl oxygen of ALA was found to be responsible
also for the binding of Bax target. Namely, the hydrogen
bond interaction between carbonyl oxygen of ALA
and 5-FU with the backbone structure of Asp53 was
found to be involved in their binding for this pro-
apoptotic target. The amino acid residues of the Bax
binding site for ALA and 5-FU mainly overlapped
with those of the binding cavity for Bax activators
BAM-7 and BTC-8, but the arene-hydrogen interactions
involved in the binding mode of BAM-7 and BTC-8,
were found absent in the binding of ALA and 5-FU
(Figure 3).
Molecular docking was performed to evaluate the
probable mechanism of proapoptotic action of ALA in
cancer cells. It was found that some interactions with
the certain amino acid residues of Bcl-2 and Bax
binding sites might play a role in the mechanism of
Bcl-2 downregulation and Bax upregulation caused
by ALA. Moreover, the binding modes of ALA and
5-FU with both targets were shown to be closely
similar, and some interactions the same like those of
standard known BH3 mimetics. Therefore, this may
be a new possible mechanism of the proapoptotic
effect of these compounds.
FARMACIA, 2019, Vol. 67, 2
231
Figure 3.
Docking conformation and 2D representation of ALA (A) and 5-FU (B) interactions in the binding site of Bax
target (PDB code: 2K7W)
In order to open new possibilities of this targeted
medical application of ALA and to determine the
potential advantages with respect to existing agents
that influence the apoptotic markers and pathways in
cancer cells, the calculated physico-chemical properties
of this compound were taken into account. Summarizing
the physico-chemical properties, calculated by using
Molinspiration tool [16], it was concluded that this
compound obeys the Lipinski "Rule of five" and
meets all criteria for good solubility, permeability and
conformational flexibility. More than one violation
of this rule is the critical limit for acceptable drug-
likeness [53]. It might be concluded that ALA also
obey the Veber rules [54] and might be considered to
possess the ability to penetrate biological membranes,
that is a common requirement for bioavailability.
The lipophilicity of compounds is a common property
used to estimate the membrane permeability of a
molecule. Thus, ALA may easily cross biological
membranes, and reach all the compartments of the
cell. Therefore, this is one more confirmation that
ALA would be easily delivered to the targets inside
the cell or on the cell membranes.
Transcription nuclear factor kappa B (NF-κB) is
present in the cytoplasm and translocates into the
nucleus in response to various inflammatory stimuli. It
is known that NF-κB regulates the expression of genes
which play a role in the development and progression
of cancer such as cell proliferation, migration and
apoptosis [55]. Expression of inducible transcription
factor NF-κB and mitochondrial markers may be
helpful in predicting clinical outcome and good
predictor of cellular response to a given chemo-
therapeutic agent. As indicated in our previous studies,
the inhibition of growth of cancer HeLa and Caco-2
cell lines associated with the inhibition of NF-κB,
this powerful transcription factor in the regulation
of cell fate might also be one of the potential ways
through which an antiproliferative effect of ALA is
performed [8].
To determine the chemo-preventive potential and
prognostic relevance of biomarkers involved in the
apoptotic pathways in colorectal cancer, multiple
markers that reflect the apoptotic status should be
studied together. According to our results, ALA may
be considered as a promising therapeutic agent in
colon cancer due to its efficiency and significant
chemopreventive potential. An ideal therapeutic agent
would specifically target the cancer cells without
causing serious cytotoxicity and systemic damage.
FARMACIA, 2019, Vol. 67, 2
232
However, in the case of conventional chemotherapeutic
agents, the reason of serious adverse effects is the
presence of their targets in normal healthy cells.
High selective or specific actions with high efficacy
are needed for a good drug candidate that would be
nontoxic to the patient [38]. The advantage of ALA
from this point of view is the evidence that it may
inhibit proliferation and induce apoptosis in various
cancer cell lines without effect on normal non-
transformed cells [7].
Conclusions
Our findings suggest that the possible mechanism of
proapoptotic effect of ALA in human colon cancer
cells may be the inhibition of Bcl-2 or the activation
of Bax apoptotic checkpoints and regulators. Thus,
ALA may be considered as potential BH3 mimetic.
Moreover, the possible mechanism may involve the
inhibition of ERK1/2 and Akt pathways. The supposed
mechanisms may act synergistic in exerting pro-
apoptotic effects of ALA in cancer cells. Considering
the physico-chemical properties taken into account, it
may be concluded that ALA can be easily delivered
to the potential intracellular targets, such as Bcl-2,
Bax, ERK1/2, Akt or NF-κB. These findings may be a
part of the explanation of possible mechanisms that
contribute to the beneficial effects of this readily
available dietary supplement in cancer therapy. The
observed mechanisms represent a step forward that
will be helpful to further investigation. The obtained
results are only the beginning of the examination of the
possible effects of ALA on cancer chemoprevention
and its effects on standard cytostatics. Moreover,
further research from different aspects may be useful
in the way of clarification of these suggested or
some new potential mechanisms of ALA-mediated
antiproliferative and proapoptotic effects in the
various types of cancer cells.
Acknowledgement
The study was supported by the Ministry of Education,
Science and Technological Development of the Republic
of Serbia (Grants TR 31060, III 41017 and OI 172044)
and Internal project of the Faculty of Medicine,
University of Nis (No 31). The authors would like to
thank Chemical Computing Group for providing us
the MOE academic license free of cost for this study.
References
1. Smith AR, Shenvi SV, Widlansky M, Suh JH, Hagen
TM, Lipoic acid as a potential therapy for chronic
diseases associated with oxidative stress. Curr Med
Chem., 2004; 11: 1135-1146.
2. Maldonado-Rojas W, Olivero-Verbel J, Ortega-Zuniga
C, Searching of protein targets for alpha lipoic acid.
J Braz Chem Soc., 2011; 22: 2250-2259.
3. Selvakumar E, Hsieh T, Regulation of cell cycle
transition and induction of apoptosis in HL-60 leukemia
cells by lipoic acid: role in cancer prevention and
therapy. J Hematol Oncol., 2008; 1: 1-8.
4. Petersen Shay K, Moreau RF, Smith EJ, Smith AR,
Hagen TM, Alpha-lipoic acid as a dietary supplement:
Molecular mechanisms and therapeutic potential.
Biochim Biophys Acta, 2009; 1790: 1149-1160.
5. Bernini R, Crisante F, Merendino N, Molinari R,
Soldatelli MC, Velotti F, Synthesis of a novel ester
of hydroxytyrosol and α-lipoic acid exhibiting an
antiproliferative effect on human colon cancer HT-
29 cells. Eur J Med Chem., 2011; 46: 439-446.
6. Kim DC, Jun DW, Jang EC, Kim SH, Kim EK, Lee
SP, Lee KN, Lee HL, Lee OY, Yoon BC, Lipoic acid
prevents the changes of intracellular lipid partitioning
by free fatty acid. Gut Liver, 2013; 7: 221-227.
7. Moungjaroen J, Nimmannit U, Callery PS, Wang L,
Azad N, Lipipun V, Chanvorachote P, Rojanasakul Y,
Reactive oxygen species mediate caspase activation
and apoptosis induced by lipoic acid in human lung
epithelial cancer cells through Bcl-2 down-regulation.
J Pharmacol Exp Ther., 2006; 319: 1062-1069.
8. Damnjanovic I, Kocic G, Najman S, Stojanovic S,
Stojanovic D, Veljkovic A, Conic I, Langerholc T,
Pesic S, Chemopreventive potential of alpha-lipoic
acid in the treatment of colon and cervix cancer cell
lines. Bratisl Lek Listy., 2014; 115: 611-616.
9. Kafara P, Icard P, Guillamin M, Schwartz L, Lincet H,
Lipoic acid decreases Mcl-1, Bcl-xL and up regulates
Bim on ovarian carcinoma cells leading to cell death.
J Ovarian Res., 2015; 8: 1-13.
10. Shi D, Liu H, Stern JS, Yu P, Liu S, Alpha-lipoic acid
induces apoptosis in hepatoma cells via the PTEN/
Akt pathway. FEBS Lett., 2008; 582: 1667-1671.
11. Na MH, Seo EY, Kim WK, Effects of α-lipoic acid on
cell proliferation and apoptosis in MDA-MB-231
human breast cells. Nutr Res Pract., 2009; 3: 265-271.
12. Yamasaki M, Iwase M, Kawano K, Sakakibara Y,
Suiko M, Ikeda M, Nishiyama K, α-Lipoic acid
suppresses migration and invasion via downregulation
of cell surface ß1-integrin expression in bladder cancer
cells. J Clin Biochem Nutr., 2014; 54: 18-25.
13. Casciari JJ, Riordan NH, Schmidt TL, Meng XL,
Jackson JA, Riordan HD, Cytotoxicity of ascorbate,
lipoic acid, and other antioxidants in hollow fibre in
vitro tumours. Brit J Cancer., 2001; 84: 1544-1550.
14. Wenzel U, Nickel A, Daniel H, α-lipoic acid induces
apoptosis in human colon cancer cells by increasing
mitochondrial respiration with a concomitant O2-
generation. Apoptosis, 2005; 10: 359-368.
15. Dӧrsam B, Gӧder A, Seiwert N, Kaina B, Fahrer J,
Lipoic acid induces p53-independent cell death in
colorectal cancer cells and potentiates the cytotoxicity
of 5-fluorouracil. Arch Toxicol., 2015; 89: 1829-1846.
16. Molinspiration Cheminformatics v2014.11; Molin-
spiration property engine: www.molinspiration.com.
17. Labianca R, Nordlinger B, Beretta GD, Brouquet A,
Cervantes A, Primary colon cancer: ESMO Clinical
Practice Guidelines for diagnosis, adjuvant treatment
and follow-up. Ann Oncol., 2010; 21: 70-77.
18. Kocic G, Pavlovic R, Najman S, Nikolic G, Sokolovic
D, Jevtovic-Stoimenov T, Musovic D, Veljkovic A,
Kocic R, Djindjic N, Circulating ribonucleic acids and
FARMACIA, 2019, Vol. 67, 2
233
metabolic stress parameters may reflect progression of
autoimmune or inflammatory conditions in juvenile
type 1 diabetes. Sci World J., 2011; 11: 1496-1508.
19. Touré BB, Miller-Moslin K, Yusuff N, Perez L, Doré
M, Joud C, Michael W, Dipietro L, Van Der Plas S,
McEwan M, Lenoir F, Hoe M, Karki R, Springer C,
Sullivan J, Levine K, Fiorilla C, Xie X, Kulathila R,
Herlihy K, Porter D, Visser M, The role of the acidity
of N-heteroaryl sulfonamides as inhibitors of Bcl-2
family protein-protein interactions. ACS Med Chem
Lett., 2013; 4: 186-190.
20. Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird
GH, Katz SG, Tu HC, Kim H, Cheng N, Tjandra LD,
BAX activation is initiated at a novel interaction site.
Nature, 2008; 455: 1076-1081.
21. Mirjolet JF, Barberi-Heyob M, Didelot C, Peyrat JP,
Abecassis J, Millon R, Merlin JL, Bcl-2/Bax protein
ratio predicts 5-fluorouracil sensitivity independently
of p53 status. Br J Cancer, 2000; 83: 1380-1386.
22. Hanahan D, Weinberg RA, The hallmarks of cancer.
Cell, 2000; 100: 57-70.
23. Ocker M, Höpfner M, Apoptosis-modulating drugs
for improved cancer therapy. Eur Surg Res., 2012;
48: 111-120.
24. Thomadaki H, Scorilas A, Bcl-2 family of apoptosis-
related genes: functions and clinical implications in
cancer. Crit Rev Clin Lab Sci., 2006; 43: 1-67.
25. Delgado-Soler L, Pinto M, Tanaka-Gil K, Rubio-
Martinez J, Molecular determinants of Bim (BH3)
peptide binding to pro-survival proteins. J Chem Inf
Model., 2012; 52: 2107-2118.
26. Alcaide J, Funez R, Rueda A, Perez-Ruiz E, Pereda T,
Rodrigo I, Coveñas R, Muñoz M, Redondo M, The
role and prognostic value of apoptosis in colorectal
carcinoma. BMC Clin Pathol., 2013; 13: 1-7.
27. Khodapasand E, Jafarzadeh N, Farrokhi F,
Kamalidehghan B, Houshmand M, Is Bax/Bcl-2 ratio
considered as a prognostic marker with age and tumor
location in colorectal cancer?. Iran Biomed J., 2015;
19: 69-75.
28. Saleh HA, Jackson H, Khatib G, Banerjee M,
Correlation of Bcl-2 oncoprotein immunohistochemical
expression with proliferation index and histopathologic
parameters in colorectal neoplasia. Pathol Oncol Res.,
1999; 5: 273-279.
29. Tsamandas AC, Kardamakis D, Petsas T, Zolota V,
Vassiliou V, Matatsoris T, Kalofonos CE, Vagianos
CD, Scopa CD, Bcl-2, bax and p53 expression in rectal
adenocarcinoma. Correlation with classic pathologic
prognostic factors and patients' outcome. In Vivo,
2007; 21: 113-118.
30. Rashmi R, Kumar S, Karunagaran D, Human colon
cancer cells lacking Bax resist curcumin-induced
apoptosis and Bax requirement is dispensable with
ectopic expression of Smac or downregulation of
Bcl-xL. Carcinogenesis, 2005; 26: 713-723.
31. Katkoori VR, Suarez-Cuervo C, Shanmugam C, Jhala
NC, Callens T, Messiaen L, Posey J3rd, Bumpers HL,
Meleth S, Grizzle WE, Manne U, Bax expression is a
candidate prognostic and predictive marker of colo-
rectal cancer. J Gastrointest Oncol., 2010; 1: 76-89.
32. Xu ZY, Tang JN, Xie HX, Du YA, Huang L, Yu PF,
Cheng XD, 5-Fluorouracil chemotherapy of gastric
cancer generates residual cells with properties of
cancer stem cells. Int J Biol Sci., 2015; 11: 284-294.
33. Uchibori K, Kasamatsu A, Sunaga M, Yokota S,
Sakurada T, Kobayashi E, Yoshikawa M, Uzawa
K, Ueda S, Tanzawa H, Sato N, Establishment and
characterization of two 5-fluorouracil-resistant hepato-
cellular carcinoma cell lines. Int J Oncol., 2012; 40:
1005-1010.
34. Bodur C, Basaga H, Bcl-2 inhibitors: Emerging drugs
in cancer therapy. Curr Med Chem., 2012; 19: 1804-
1820.
35. Kogel D, Exploiting BH3 mimetics for cancer therapy.
In: Neuzil J, Pervaiz S, Fulda S, (editors). Mitochondria:
The anti-cancer target for the third millennium. Chapter
2. Dordrecht: Springer Science + Business Media;
2014. 39-58.
36. Akiyama T, Dass CR, Choong PFM, Bim-targeted
cancer therapy: A link between drug action and
underlying molecular changes. Mol Cancer Ther.,
2009; 8: 3173-3180.
37. Cragg MS, Jansen ES, Cook M, Harris C, Strasser A,
Scott CL, Treatment of B-RAF mutant human tumor
cells with a MEK inhibitor requires Bim and is
enhanced by a BH3 mimetic. J Clin Invest., 2008;
118: 3651-3659.
38. Shin JA, Kim LH, Lee SJ, Jeong JH, Jung JY, Lee HN,
Hong IS, Cho SD, Targeting ERK1/2-Bim signaling
cascades by BH3-mimetic ABT-737 as an alternative
therapeutic strategy for oral cancer. Oncotarget, 2015;
6: 35667-35683.
39. Breitschopf K, Haendeler J, Malchow P, Zeiher
AM, Dimmeler S, Posttranslational modification of
Bcl-2 facilitates its proteasome-dependent degradation:
Molecular characterization of the involved signaling
pathway. Mol Cell Biol., 2000; 20: 1886-1896.
40. Sun J, Zhang H, Guan L, Zhou H, Sun M, Alpha-
lipoic acid attenuates trinitrobenzene sulfonic acid-
induced ulcerative colitis in mice. Int J Clin Exp Med.,
2015; 8: 358-367.
41. Gardai SJ, Hildeman DA, Frankel SK, Whitlock BB,
Courtney FS, Borregaard N, Marrack P, Bratton LD,
Henson PM, Phosphorylation of Bax Ser184 by Akt
regulates its activity and apoptosis in neutrophils. J
Biol Chem., 2004; 279: 21085-21095.
42. Yamaguchi H, Wang HG, The protein kinase PKB/
Akt regulates cell survival and apoptosis by inhibiting
Bax conformational change. Oncogene, 2001; 20:
7779-7786.
43. Sadidi M, Lentz SI, Feldman EL, Hydrogen peroxide-
induced Akt phosphorylation regulates Bax activation.
Biochimie, 2009; 91: 577-585.
44. Tsuruta F, Masuyama N, Gotoh Y, The phosphatidyl-
inositol 3-kinase (PI3K)-Akt pathway suppresses Bax
translocation to mitochondria. J Biol Chem., 2002;
277: 14040-14047.
45. Wang YK, Zhu YL, Qiu FM, Zhang T, Chen ZG,
Zheng S, Huang J, Activation of Akt and MAPK
pathways enhances the tumorigenicity of CD133
primary colon cancer cells. Carcinogenesis, 2010; 31:
1376-1380.
46. Bocca C, Bozzo F, Gabriel L, Miglietta A, Conjugated
linoleic acid inhibits Caco-2 cell growth via ERK-
MAPK signaling pathway. J Nutr Biochem., 2007;
18: 332-340.
FARMACIA, 2019, Vol. 67, 2
234
47. Calvo N, Russo de BA, Gentili C, PTH inactivates
the AKT survival pathway in the colonic cell line
Caco-2. Biochim Biophys Acta., 2010; 1803: 343-351.
48. Danial NN, Korsmeyer SJ, Cell death: critical control
points. Cell, 2004; 116: 205-219.
49. Weyhenmeyer B, Murphy AC, Prehn JHM, Murphy
BM, Targeting the anti-apoptotic Bcl-2 family members
for the treatment of cancer. Exp Oncol., 2012; 34:
192-199.
50. Billard C, BH3 Mimetics: Status of the field and new
developments. Mol Cancer Ther., 2013; 12: 1691-1700.
51. Stornaiuolo M, La Regina G, Passacantilli S, Grassia G,
Coluccia A, La Pietra V, Giustiniano M, Cassese G,
Di Maro S, Brancaccio D, Taliani S, Ialenti A, Silvestri
R, Martini C, Novellino E, Marinelli L, Structure-based
lead optimization and biological evaluation of BAX
direct activators as novel potential anticancer agents.
J Med Chem., 2015; 58: 2135-2148.
52. Zhang L, Ming L, Yu J, BH3 mimetics to improve
cancer therapy; mechanisms and examples. Drug Resist
Updat., 2007; 10: 207-217.
53. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ,
Experimental and computational approaches to estimate
solubility and permeability in drug discovery and
development settings. Adv Drug Deliver Rev., 2012;
64: 4-17.
54. Veber DF, Johnson SR, Cheng HY, Smith BR, Ward
KW, Kopple KD, Molecular properties that influence
the oral bioavailability of drug candidates. J Med
Chem., 2002; 45: 2615-2623.
55. Dolcet X, Llobet D, Pallares J, Matias-Guiu X, NF-kB
in development and progression of human cancer.
Virchows Arch., 2005; 446: 475-482.
