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Structural tuning of organoruthenium compounds allows oxidative switch to control ER stress pathways and bypass multidrug resistance.
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Structural tuning of organoruthenium compounds
allows oxidative switch to control ER stress
pathways and bypass multidrug resistance†
Mun Juinn Chow,ab Cynthia Licona,cd Giorgia Pastorin,be Georg Mellitzer,cd
Wee Han Ang*ab and Christian Gaiddon*cd
Multidrug resistance (MDR) is a major impediment to the success of chemotherapy in many cancer types.
One particular MDR mechanism is the inherent or acquired adaptation of the cellular survival pathways that
render malignant cells resistant to apoptotic cell death. Since most drugs act through apoptosis,
compounds capable of inducing alternative forms of programmed cell death (PCD) can potentially be
harnessed to bypass MDR. We investigated two organoruthenium complexes, RAS-1H and RAS-1T, and
demonstrated that although they both induced non-apoptotic PCD through ER stress pathways, their
modes-of-action were drastically different despite modest structural variations. RAS-1T acted through
Received 20th January 2016
Accepted 25th February 2016
ROS-mediated ER stress while RAS-1H was ROS-independent. We further showed that they were more
efficacious against apoptosis-resistant cells compared to clinical drugs including oxaliplatin. This work
DOI: 10.1039/c6sc00268d
provides the basis for underpinning ER stress modulation using metal complexes to bypass apoptosis
www.rsc.org/chemicalscience
resistance.
Introduction
Chemotherapy remains one of the major treatment options for
many cancer types.1 However, the effectiveness of chemotherapeutic treatments is frequently diminished2 due to the multidrug-resistance (MDR) phenotype found in many cancers that
are associated with a poor clinical outcome such as gastric
cancer, the third and h leading cause of cancer mortality in
men and women worldwide, respectively.3–6 One dominant
MDR mechanism in these cancers was determined to be the
defective or selective adaptation of apoptotic pathways, which
results in resistance to apoptosis.7–11 In addition, the overexpression of the membrane-bound ‘efflux’ transporter P-gp,
one of the main causes of MDR, is also known to inhibit
apoptosis by preventing caspase activation.12–14 Given that most
clinically used drugs act by inducing apoptosis as their primary
mode-of-action,15,16 it is not surprising that the success rates of
chemotherapy in these cancers have been poor. One of the few
a
Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543
Singapore. E-mail: chmawh@nus.edu.sg; Tel: +65 6516 5131
b
NUS Graduate School for Integrative Sciences and Engineering, Singapore
c
U1113 INSERM, 3 Avenue Molière, Strasbourg 67200, France. E-mail: gaiddon@
unistra.fr; Tel: +33 68 52 53 56
d
Section Oncology, FMTS, Strasbourg University, Strasbourg, France
e
Department of Pharmacy, National University of Singapore, 18 Science Drive 4,
117543 Singapore
† Electronic supplementary information (ESI) available: Preparation,
characterization protocol and data for RAS-1H and RAS-1T, and the protocol
and data for in vitro studies. See DOI: 10.1039/c6sc00268d
This journal is © The Royal Society of Chemistry 2016
strategies to overcome this mechanism of MDR involves the
restoration of the expression or function of the pro-apoptotic
gene in cancer cells through chemical or genetic modulation.17,18 A more attractive strategy would involve directly
bypassing this mechanism of MDR entirely to induce cancer cell
death via non-apoptotic programmed cell death (PCD).19
With the discovery of the antitumoural activity of cisplatin
(CDDP), a widely used PtII-based clinical drug for cancer
chemotherapy, metallodrugs have received revived interest and
attention in recent years.20 Yet, as with most clinical drugs
including CDDP, the majority of the metallodrugs investigated
act via apoptotic pathways and are therefore subject to classical
MDR limitations. There have only been a handful of metallocomplexes shown to exert cytotoxicity via other alternative
forms of PCD. Several Au-, Ru- and Fe-based complexes have
exhibited the ability to induce type II autophagic cell death.21–23
A class of CuII thioxotriazole complexes and a CuI triazole–
phosphine complex have demonstrated the ability to induce
paraptosis in several cancer cell lines.24,25 More recently, two ReV
oxo complexes were reported to induce cell death via a form of
programmed necrosis called necroptosis.26 However, their
ability to overcome MDR mechanisms has never been validated.
Therefore, new metallocomplexes with well-delineated modes
of action, particularly via alternative PCD, could constitute
a new strategy to overcome MDR.
We earlier reported the combinatorial synthesis and
evaluation of a new class of water-soluble/stable half-sandwich
RuII arene Schiff-base (RAS) complexes via a coordinationdirected 3-component assembly.27 RAS-1T, which bore
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Scheme 1 Synthesis route for the preparation of Ruthenium(II) Schiffbase (RAS) complexes.
Differential ER stress pathway activation by RAS complexes
leads to an alternative (non-apoptotic) PCD that bypasses drug resistance mechanisms.
Fig. 1
triisopropylbenzene (TIPB) and iminoquinoline ligands, was
identied as a lead candidate as it was highly efficacious against
several cancer cell lines yet exhibited attributes distinct from
classical alkylating agents, such as CDDP and reported anticancer RuII complexes. For instance, RAS-1T was stable against
hydrolysis and did not interact directly with dGMP nucleotides.
In addition, it did not induce p53 expression in treated cells,
commonly associated with DNA damage. Using gastric cancer
as a model in this present study and in comparison with a newly
synthesized hexamethylbenzene (HMB) analogue, RAS-1H, we
demonstrate for the rst time that varying the facially-bound
arene ligands can have drastic effects on the compounds' modeof-action leading to cell death, activating either ROS-dependent
or ROS-independent ER stress pathways (Fig. 1). We show that
the independent activation of both pathways leads to nonapoptotic PCD in treated cells and that this strategy could be
harnessed to overcome apoptosis-resistance.
Results and discussion
the desired [Ru(arene)Cl2]2 precursor (0.5 equiv., arene ¼ HMB
or TIPB) in MeOH. Purication via ash column chromatography gave both compounds in good yields. The complexes were
characterized using 1H NMR and ESI-MS and their purity was
conrmed using RP-HPLC and elemental analysis.
We compared the physicochemical properties of both
complexes. Stability studies using UV-Vis spectroscopy showed
that both complexes were stable to aquation, and were not
prone to react with sulfur- or nitrogen-containing biomolecules
(Fig. S4†). The lipophilicity of RAS-1H and RAS-1T was determined from their extent of partitioning between n-octanol and
water. The log POW for RAS-1H and RAS-1T was determined to
be 1.40 and 0.85, respectively, showing that both
compounds were relatively hydrophilic despite having highly
hydrophobic ligands, presumably due to the cationic nature of
the compounds.
Distinct cytotoxicity proles of RAS-1H and RAS-1T
We investigated the potentially contrasting mode-of-action of
RAS-1H and RAS-1T by rst testing their efficacy against a panel
of gastric and colorectal cancer cell lines (Table S1†). As expected, RAS-1T displayed a low micromolar IC50 value in all 4
Synthesis and characterization
We previously reported the combinatorial synthesis of 450
distinct RAS complexes via the co-ordination of in situ assembled Schiff base ligands to organoruthenium scaffolds.27
Screening of this RAS library revealed RAS-1T to exhibit p53independent activity and to be a lead candidate for a metallocomplex with antiproliferative activity that is distinct from
classical alkylating agents, e.g. cisplatin, for further mode-ofaction elucidation. We postulated that minor structural variations could inuence cellular pathway activation, regardless of
the similarities in their physicochemical properties. In order to
discern how the structural variation of the arene ligand could
inuence the mode-of-action, we included the HMB-analogue
RAS-1H in the present study. Both RAS-1H and RAS-1T were
synthesized in good yields and purity (Scheme 1). The chelate
ligand was prepared directly from 4-methoxylaniline and 2formylquinoline via imine condensation and was treated with
4118 | Chem. Sci., 2016, 7, 4117–4124
Fig. 2 Varying the arene ligand on RAS complexes changes their
antiproliferative profiles. Cell viability curves of compounds RAS-1H
and RAS-1T. The curve represents the mean s.e.m. of three independent experiments. RAS-1H displayed a biphasic cytotoxicity curve
unlike RAS-1T, hinting at a different mode-of-action. Two equipotent
concentrations at low-dose (LD) and high-dose (HD) were chosen for
cell treatment in proteins and mRNA expression studies.
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cell lines tested. The IC50 of RAS-1T was also 34 times and 7
times lower than that of cisplatin in the gastric cancer cell lines
AGS and KATOIII, respectively. In comparison, RAS-1H
demonstrated slightly more modest activity with IC50 values 11
times lower than cisplatin in AGS and 1.5 times higher in
KATOIII. Closer scrutiny of the cytotoxicity curve of RAS-1H in
AGS revealed a bi-phasic prole that was different from that of
RAS-1T (Fig. 2). This suggested different modes-of-action for
RAS-1H and RAS-1T. We determined two equipotent concentrations at low dose (LD) and high dose (HD) for each complex
for cell treatment to further determine the difference in their
mode-of-action.
RAS-1H and RAS-1T induced early time-point ROS and
activated the antioxidant defense mechanism
To unravel the mechanism of action of RAS-1H and RAS-1T,
given the complex interplay of various possible pathways, we
examined oxidative stress levels in treated AGS gastric cancer
cells and the subsequent cellular response. Reactive oxygen
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species (ROS) generation has been implicated in the mode-ofaction of many RuII complexes given the low energy barrier of
the RuII/RuIII redox states, although it is not always clear that
ROS generation was the cause of the observed cytotoxicity.28–30
Cellular ROS levels were determined using a commercial cellpermeable ROS probe, carboxy-H2DCFDA. The ROS levels were
markedly increased in AGS cells treated with both RAS
complexes in a time- and concentration-dependent manner. In
cells treated with both complexes, ROS induction peaked at an
early time-point of 3 h at similar levels before decreasing as
indicated from the quantitative uorescence measurements
(Fig. 3a) and uorescence microscopy images (Fig. 3b and S5†).
To gain an insight into the impact of ROS on the cells at
relevant time-points (6 h and 24 h post-treatment), we examined
the cellular response by looking into the antioxidant defense
mechanism (Scheme S1†). Central to the antioxidant defense is
the transcription factor Nrf-2,31 which is responsible for the
regulation of several downstream target genes such as gclc,32
mrp2,33 and nqo1,34 each having various roles in the mediation
Fig. 3 Complexes RAS-1H and RAS-1T induce early time-point ROS and activate the cellular antioxidant defence mechanism. (a) Detection
of ROS with carboxy-H2DCFDA (20 mM) after treatment with RAS-1H and RAS-1T for 3 h, 6 h and 9 h using a microplate assay. Mean s.e.m.
(*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Student's t test). (b) Detection of ROS with a fluorescence microscope after treatment for 6 h.
(c) Western blot analysis of Nrf-2, a central protein in cellular antioxidant defence and (d) expression levels of the Nrf-2 target gene in AGS cells
after treatment with RAS-1H, RAS-1T and cisplatin at LD and HD for 6 h and 24 h. Homogeneous protein loading determined with reference to
actin and gene expression normalized against tbp levels.
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of oxidative stress. When ROS levels are elevated, Nrf-2 is activated and its target genes expression increases. Increased
expression of Nrf-2 was observed aer 6 h of treatment at HD for
both RAS-1H and RAS-1T (Fig. 3c). Similarly, mRNA levels for all
three Nrf-2 target genes increased aer 6 h of treatment at HD
(Fig. 3d). This was consistent with the induction of ROS
observed at early-time points. In contrast, cells treated with
CDDP remained at basal levels. However, there were differences
in the Nrf-2 expression and activity patterns for both
compounds. Firstly, the protein levels for Nrf-2 were higher aer
RAS-1H treatment compared to RAS-1T. Secondly, Nrf-2 induction remained elevated aer 24 h for both concentrations of
RAS-1H while Nrf-2 returned to basal levels aer 24 h for
RAS-1T. Lastly, the RNA level of gclc was more signicantly
increased by RAS-1H than RAS-1T. Taken together, the results
indicate that the antioxidant defense for RAS-1H was switched
on for an extended duration, underscoring the mechanistic
differences in oxidative stress induction between the RAS
complexes despite similar early time-point ROS production.
Differential induction of the ER stress pathway by RAS-1H and
RAS-1T
The relationship between ROS and endoplasmic reticulum (ER)
stress is well-established and ROS could occur either upstream
(cause) or downstream (product) of ER stress.35,36 ER stress can be
characterized by the unfolded protein response (UPR), which can
lead to recovery, cellular dysfunction or cell death.37 Three
distinct UPR signaling pathways have been identied, namely the
PERK/eif2a, IRE1a/XPB-1s and ATF6 pathways (Scheme S2†).38
Since elevated ROS levels point towards ER stress and UPR, we
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investigated if RAS-1H and RAS-1T induced ER stress biomarkers
and the implications to their antiproliferative activity. RAS-1H
and RAS-1T induced ER stress via the IRE1a/XPB-1s pathway as
seen from the increased accumulation of XBP-1s aer 6 h of
treatment at HD (Fig. S6†). The induction of the downstream
target CHOP aer both 6 h and 24 h of HD treatment also
conrmed the ER stress induction.39 While RAS-1H induced high
levels of CHOP expression, RAS-1T favored XBP-1s splicing,
alluding to different signaling pathways.
We therefore examined whether ROS production and the
subsequent ER stress were critical to the antiproliferative
activity of RAS-1H and RAS-1T on AGS cells. We employed
N-acetylcysteine (NAC) as a ROS quencher and determined the
cell viability and protein level expression of Nrf-2, XBP-1s and
CHOP under various conditions, in the absence or presence of
NAC. In the case of RAS-1T, cytotoxicity up to 2.5 mM (HD) was
completely removed by the co-treatment with NAC. At higher
levels of RAS-1T exposure, i.e. 10 mM which typically led to the
100% loss of viable cells, 50% cell viability was restored with
NAC co-treatment (Fig. 4a). Furthermore, Nrf-2 levels were low
in cells co-treated with NAC (Fig. 4b) indicating that the antioxidant defense was not switched on. In keeping with these
observations, ER stress markers XBP-1s and CHOP were also
suppressed in cells co-treated with NAC, suggesting a low UPR
and reduced ER stress. Taken together, the results point toward
ROS production being critical for the antiproliferative activity
of RAS-1T (causal) and it exerting its mode-of-action via
ROS-mediated ER stress. In contrast, ROS quenching did not
suppress RAS-1H-induced cell death at treatments of less than
30 mM (HD) (Fig. 4a). The NAC treatment did not block RAS-1Hinduced Nrf-2 protein levels aer 6 h of treatment and had only
Fig. 4 Differential activation of ROS-independent and ROS-mediated ER stress pathways by RAS-1H and RAS-1T. (a) Cell viability (%) of AGS cells
treated with RAS-1H and RAS-1T for 48 h with and without NAC (2 mM). Mean s.e.m. (**p < 0.01, ***p < 0.001, ****p < 0.0001; two-tailed
Student's t-test). (b) Western blot analysis of ER stress protein markers in AGS cells after treatment with RAS-1H and RAS-1T at HD, with and
without NAC (2 mM). Homogeneous protein loading determined with reference to actin.
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a partial effect at the 24 h time-point. Moreover, ROS quenching
also did not suppress XBP-1s or CHOP induction (Fig. 4b). In
fact, the co-treatment of RAS-1H with NAC increased ER stress
as indicated by the marked increase in XBP-1s and CHOP
accumulation. This suggested that the cell death induced by
RAS-1H was not dependent on the elevated ROS levels (Fig. 3a)
and that ROS production was not critical for its mode-of-action
(by-product).
RAS-1H and RAS-1T induce non-apoptotic programmed cell
death
The contrasting yet well-dened antiproliferative pathways
brought about by RAS-1H and RAS-1T, both of which invoked
ROS production, could be harnessed to bypass the conventional
apoptotic pathways induced by classical drugs such as CDDP.
We therefore compared the apoptosis biomarkers commonly
induced by CDDP treatment with the RAS complexes.40,41
Western blot analyses of AGS cells treated with CDDP revealed
signicant upregulation in p53 expression and cleavage of
caspase 3 and PARP-1 (Fig. 5a). A concomitant increase of bax
and decrease of bcl-2 gene expression was also observed aer
24 h (Fig. 5b), indicative of apoptosis induction. In contrast,
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cells treated with RAS-1H and RAS-1T did not exhibit such
expression proles, regardless of the concentration or duration
of treatment. Furthermore, the two distinct hallmarks of
apoptosis, namely (i) the activation of apoptosis-executor caspases, and (ii) morphological changes consistent with apoptosis
were also absent in cells treated with RAS-1H and RAS-1T.42 The
co-treatment of cells with a broad-spectrum caspase-inhibitor,
Z-VAD-FMK, did not reduce the efficacy of either RAS-1H or
RAS-1T, as seen from their unchanged IC50 values (Fig. 5c) but it
provided cytoprotection from CDDP. In addition, the cell death
morphology induced by RAS-1H and RAS-1T aer 24 h was
markedly different from that induced by CDDP (Fig. 5d and
S7†). For cells treated with cisplatin, the ‘budding’ and formation of smaller apoptotic bodies were observed. In contrast, cells
treated with RAS-1H and RAS-1T did not display the same
morphological changes commonly seen in apoptotic cell death.
To rule out the possibility of necrotic cell death, we also tested
the activity of RAS-1H and RAS-1T in the absence and presence
of IM-54, an inhibitor of oxidative stress-induced necrosis. The
cell viability of cells treated with various concentrations of
RAS-1H and RAS-1T did not change signicantly in the presence
of IM-54, indicating that necrosis was unlikely to be the mode of
Complexes RAS-1H and RAS-1T induce non-apoptotic cell death. (a) Western blot analysis of proteins related to the apoptosis pathway
and (b) expression levels of pro-apoptotic and anti-apoptotic genes in AGS cells after treatment with RAS-1H, RAS-1T and cisplatin at LD and HD
for 6 h and 24 h. Homogeneous protein loading determined with reference to actin and gene expression normalized against tbp levels. (c) IC50
values of RAS-1H, RAS-1T and cisplatin after 48 h of treatment with and without an apoptosis inhibitor, Z-VAD-FMK (5 mM). Mean s.e.m.
(*p < 0.05; two-tailed Student's t-test). (d) Microscope images of AGS cells treated with RAS-1H, RAS-1T and cisplatin for 24 h (scale bar ¼
100 mm). Examples of smaller apoptotic bodies due to ‘budding’ are pointed out with white arrows.
Fig. 5
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cell death (Fig. S8†). The lack of caspase involvement and the
marked difference in cell death morphology compared to
CDDP-treated cells suggested that RAS-1H and RAS-1T induced
a form of non-apoptotic PCD.
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RAS-1H and RAS-1T bypass the apoptosis resistance
mechanism in colorectal cancer cell lines
In order to validate the ability of the RAS complexes to bypass
apoptosis resistance, we employed the apoptosis-resistant TC7
cell line as a functional cell model. TC7 is a cell line cloned from
parental colorectal adenocarcinoma Caco-2 cells using limited
dilution and is found to be highly resistant to conventional drug
treatment.43 It has a p53-null status and a higher basal expression of anti-apoptotic Bcl-2 and Bcl-xL, as well as a lower
expression of pro-apoptotic Bax. A previous study done on the
activity of 5-uorouracil on a panel of colorectal cancer cell lines
including TC7 showed a positive correlation between their
resistance to 5-uorouracil treatment and their basal (Bcl-2 +
Bcl-xL)/Bax expression ratio,44 suggesting that the drug-resistance observed in TC7 was due in part to apoptosis-resistance.
The colorectal cancer cell lines HCT116 and HT-29 were used as
non-resistant models. The activities of RAS-1H and RAS-1T were
Fig. 6 RAS-1H and RAS-1T are least affected by the resistance
mechanism of drug-resistant TC7 cells, compared to other clinically
approved drugs. (a) Resistance factors are calculated by taking the ratio
of IC50 from 3 independent experiments in apoptosis-resistant TC7
over the mean IC50 in non-resistant HT29 and HCT116. Mean s.e.m.
(*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA test with Tukey
post-hoc test). #IC50 in TC7 > 1000 mM in all 3 experiments and the
resistance factor shown is the lowest possible. (b) Comparison of the
cell viability curves for RAS-1T and 5-FU in TC7 and HT-29.
4122 | Chem. Sci., 2016, 7, 4117–4124
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compared to the clinical drugs oxaliplatin (OXP), etoposide
(ETOP), 5-uorouracil (5-FU) and doxorubicin (DOX) using their
resistance factor (RF), expressed as a factor of IC50 [TC7] against
either IC50 [HT-29] or IC50 [HCT116]. OXP was used instead of
CDDP as it is the leading drug for the treatment of colorectal
cancer for which CDDP is poorly efficacious.
Both RAS-1H and RAS-1T were the least affected by TC7
compared to the frequently used clinical drugs (Fig. 6 and S9,
and Table S2†). In particular, RAS-1T exhibited the lowest
resistance factors (RFs) amongst all tested compounds of 4.5
(TC7/HT-29) and 2.8 (TC7/HCT116) while the RAS-1H values
were 9.2 and 9.0, respectively. In contrast, the RF values for OXP
were 26.9 and 10.2 while 5-FU exhibited RFs exceeding 111 and
123. Furthermore, RAS-1T also exhibited low micromolar IC50
values in TC7, highlighting its high efficacy in the apoptosisresistant cell lineage. These results suggested that the
apoptosis-resistance of TC7 did not signicantly impact the
effectiveness of both RAS complexes, unlike the tested clinical
drugs, thereby validating in a functional cell model the
hypothesis that RAS-1H and RAS-1T could bypass apoptosis
resistance via the induction of alternative PCDs (Scheme S3†).
Conclusion
The variation of the arene ligand in half-sandwich Ru
complexes is commonly used as a means to modulate physical
properties such as hydrophobicity and solubility. Limited
studies have been done on how arene ligands could inuence
the cellular mode-of-action. Two separate studies on a class of
Ru(II)–arene complexes bearing ethylenediamine ligands have
demonstrated that arene ligands, with varying hydrophobicity
and p-acidity, could be used to modulate the rate of hydrolysis
and extent of p-stacking of DNA bases.45,46 A separate study
showed that the functionalization of the arene with a maleimide
moiety allowed for the selective delivery of the complexes via
selective binding with thiol-containing biomolecules.47
However, these studies focused on how arene modication
could be used to modulate the physicochemical properties and
target-binding ability without demonstrating any differential
activation of cellular pathways.
In contrast, our current study represents one of the few such
studies, demonstrating that a subtle change in the arene
ligands on the RAS complexes had a drastic effect on its modeof-action, switching the ability of the drug to induce cell death
from a ROS-mediated ER stress pathway to a ROS-independent
pathway. Since the variation of the arene ligand did not
signicantly change the physiochemical properties such as
stability, reactivity to biomolecules or hydrophobicity, the
observed difference is most likely inuenced directly by the sitespecic structural changes of the arene ligand. Although the
induction of ER stress by other metal complexes has been
previously reported,30,48,49 mechanistic insights into ER stress
activation and the subsequent implications of structural factors
were not discussed. Our current study provides the rst
molecular basis for ER stress activation, highlighting the
complex relationship between the structure of the compound
and its impact on the mode-of-action via ER stress. It is
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noteworthy that the structural tuning also impacted its ability to
bypass cancer cell MDR mechanisms, as seen in the two-fold
difference in the resistance factor between RAS-1H and RAS-1T.
These factors should be taken into consideration when
designing such compounds as anticancer agents to improve the
treatment outcome for MDR cancers. Based on the current
study, we hypothesized that the structural change to the RAS
complexes could have affected the selectivity of binding to their
target, especially if the cellular target contains several homologous isoforms. This could have caused the observed differences in the mode-of-action between the two complexes.
Nevertheless, more studies are required to identify the cellular
target(s) of RAS-1H and RAS-1T before this can be validated.
Conflict of interest
The authors declare no competing nancial interest.
Acknowledgements
The authors acknowledge ARC, Ligue contre le cancer, CNRS,
European COST action CM1105 and the Singapore Ministry of
Education (R143-000-638-112) for funding as well as CMMACNUS for performing elemental analysis and ICP-OES analysis.
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