Multifunctional thiosemicarbazones and deconstructed analogues as a strategy to study the involvement of metal chelation, Sigma-2 (σ2) receptor and P-gp protein in the cytotoxic action: In vitro and in vivo activity in pancreatic tumors

Article abstract of DOI:10.1016/j.ejmech.2017.12.024

The aggressiveness of pancreatic cancer urgently requires more efficient treatment options. Because the sigma-2 (σ₂) receptor was recently proposed as a promising target for pancreatic cancer therapy, we explored our previously developed multifunctional thiosemicarbazones, designed to synergistically impair cell energy levels, by targeting σ₂ and P-gp proteins and chelating Iron. A deconstruction approach was herein applied by removing one function at a time from the potent multifunctional thiosemicarbazones 1 and 2, to investigate the contribution to cytotoxicity of each target involved. The results from in vitro (panel of pancreatic tumor cells) and in vivo experiments (C57BL/6 bearing KP02 tumor), suggest that while the multifunctional activity was not required for the antitumor activity of these thiosemicarbazones, σ₂-targeting appeared to allow alternative tumor cell death mechanisms, leading to potent and less toxic off-targets toxicities compared to other thiosemicarbazones devoid of σ₂-targeting.

Full text of DOI:10.1016/j.ejmech.2017.12.024

Accepted Manuscript
Multifunctional thiosemicarbazones and deconstructed analogues as a strategy to
study the involvement of metal chelation, Sigma-2 (σ ) receptor and P-gp protein in
2
the cytotoxic action: In vitro and in vivo activity in pancreatic tumors
Maria Laura Pati, Mauro Niso, Dirk Spitzer, Francesco Berardi, Marialessandra
Contino, Chiara Riganti, William G. Hawkins, Carmen Abate
PII:
S0223-5234(17)31030-9
10.1016/j.ejmech.2017.12.024
EJMECH 10002
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 August 2017
Revised Date: 17 November 2017
Accepted Date: 7 December 2017
Please cite this article as: M.L. Pati, M. Niso, D. Spitzer, F. Berardi, M. Contino, C. Riganti, W.G.
Hawkins, C. Abate, Multifunctional thiosemicarbazones and deconstructed analogues as a strategy to
study the involvement of metal chelation, Sigma-2 (σ ) receptor and P-gp protein in the cytotoxic action:
2
In vitro and in vivo activity in pancreatic tumors, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2017.12.024.
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ACCEPTED MANUSCRIPT
Multifunctional Thiosemicarbazones and Deconstructed Analogues as
a Strategy to Study the Involvement of Metal Chelation, Sigma-2 (σ2)
Receptor and P-gp Protein in the Cytotoxic Action: in vitro and in vivo
Activity in Pancreatic Tumors
Maria Laura Pati,a,b Mauro Niso,a Dirk Spitzer,b Francesco Berardi,a Marialessandra Contino,a
Chiara Riganti,c William G Hawkins,b,d and Carmen Abatea,d*
aDipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via
Orabona 4, I-70125 Bari, Italy.
bDepartment of Surgery, Division of Hepatobiliary, Pancreatic, and Gastrointestinal Surgery,
Washington University School of Medicine, St. Louis, MO, USA.
cDipartimento di Oncologia, Università degli Studi di Torino, via Santena 5/bis, I-10153, Torino,
Italy.
dCo-last authors
*To whom correspondence should be addressed. Tel.: +39-080-5442231. E-mail:
carmen.abate@uniba.it
Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea
nitrogen; Cr, Creatinine; i.p, intraperitoneal; ROS, reactive oxygen species;
Supplementary Information: Synthesis of intermediate compound 12 and final compounds 13 and
14. Western blot for P-gp detection in KP02 cell. ROS involvement, caspase-3 activity and
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mitochondrial superoxide detection in AsPC1 and Panc02 cell lines. Flow cytometry curves for σ1
binding of references compounds and 1, 2 and 7.
Abstract
The aggressiveness of pancreatic cancer urgently requires more efficient treatment options. Because
the sigma-2 (σ2) receptor was recently proposed as a promising target for pancreatic cancer therapy,
we explored our previously developed multifunctional thiosemicarbazones, designed to
synergistically impair cell energy levels, by targeting σ2 and P-gp proteins and chelating Iron. A
deconstruction approach was herein applied by removing one function at a time from the potent
multifunctional thiosemicarbazones 1 and 2, to investigate the contribution to cytotoxicity of each
target involved. The results from in vitro (panel of pancreatic tumor cells) and in vivo experiments
(C57BL/6 bearing KP02 tumor), suggest that while the multifunctional activity was not required for
the antitumor activity of these thiosemicarbazones, σ2-targeting appeared to allow alternative
tumor cell death mechanisms, leading to potent and less toxic off-targets toxicities compared to
other thiosemicarbazones devoid of σ2-targeting.
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1. Introduction
According to the latest Cancer Statistics’ report [1], cancer death rates have been decreasing by
23% since 1991. Despite that, cancer overtook cardiovascular diseases becoming the first cause of
death in 21 states within US, while death rates are increasing for cancers of the liver and pancreas.
Pancreatic cancer is one of the most aggressive diseases characterized by a rapid progression, high
probability of local recurrence and occurence of early liver metastases. [2] It represents the fourth
cause of death for cancer related mortalities with a very poor prognosis [3], and less than 8% five
years survival rate. Currently, for patients that are diagnosed early enough, surgery represents the
preferred treatment option, further supported by radiation- and gemcitabine-based chemotherapy,
but all of these treatment regimens generally result in heterogeneous response rates with a high
probability of developing recurrent disease [4-6]. Pancreatic cancer ultimately develops resistance
to gemcitabine and thus requires an urgent need of identifying more efficient treatment options as
well as better understanding the deranged molecular pathways characteristic for this debilitating
malignancy.
In recent years, many attempts to harness the σ2 receptor as a tumor-specific molecular target for
pancreatic cancer therapy have emerged. The σ2 receptor belongs to the σ receptors family, divided
in σ1 and σ2 proteins. After being proposed as a histone [7,8], the σ2 receptor was later idientified
as the progesterone receptor membrane component 1 (PGRMC1) protein complex [9]. However,
mounting evidence suggested that the σ2 receptor might actually not be related to PGRMC1 [10-12]
and has thus been recently proposed to be the endoplasmic reticulum ER-resident membrane protein
TMEM97 [13]. Despite such a controversial identification, the σ2 receptor has been increasingly
studied due to its overexpression and activity in a number of human tumors. σ2 Ligands are able to
selectively induce tumor cells death through mechanisms that may involve caspase-dependent and -
independent apoptosis, lysosomal membrane permeabilization, generation of reactive oxygen
species (ROS), and autophagy [14-20].
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Recently, the mitochondrial superoxide pathway has been recognized as σ2 receptor-activated
process in pancreatic cancer cells [21]. Importantly, certain σ2 receptor agonists have shown
efficacy in preclinical tumor models of pancreatic cancer [22-25] highlighting the notion that this
receptor is a promising target for cancer therapy. In an effort to develop new drugs that target
multiple biologic functions with a single molecule (i.e. multi-target approach), we recently
developed a novel class of σ2 receptor ligands, capable of chelating metal ions through their
thiosemicarbazone moiety [20]. These molecules were also able to interact with the P-glycoprotein
(P-gp) efflux pump, i.e. the most well-described ABC ‘polyspecific’ transporter, whose
overexpression is associated with poor prognosis and poor quality of life in cancer patients. Our
choice to focus on these particular cellular targets was made based on recent evidence that cancer
cells are exquisitely vulnerable to changes in energy levels due to their increased metabolism to
sustain rapid cell proliferation. Accordingly, a simultaneous action on the three targets would
synergistically compromise cells energy levels. Accordingly, engaging these three drug targets
simultaneously was projected to compromise the cellular energy levels in a synergistic manner,
because i) σ2 receptor ligand cytotoxicity is correlated with lysosomal membrane permeabilization
and an increase in ROS production [18,25]; ii) metal chelators that complex endogenous ironII/III
are known as redox cycling agents that also result in increased production of ROS; and iii) P-gp
modulation with activation of the futile ATP-cycle would lead to increases in oxidative
phosphorylation, subsequently increasing the level of ROS production [26]. Therefore, our
multifunctional thiosemicarbazones were anticipated to combine the cytotoxic properties of σ2
agonists with the ironII/III chelating and potentially P-gp modulating capacity within a single drug
conjugate, thereby creating a highly lethal and cancer-directed therapy option.
Among all the novel σ2 thiosemicarbazone ligands, two compounds, i.e. (Z)-2-(1-(4-(6,7-
Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-oxoindolin-3-ylidene)-N,N-
dimethylhydrazinecarbothioamide 1 and
(Z)-2-[1-[4-(4-Cyclohexylpiperazin-1-yl)butyl]-2-
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oxoindolin-3-ylidene]-N,N-dimethylhydrazinecarbothioamide 2 (Figure 1), stood out due to their
promising cytotoxic activity in two cell line pairs: MCF7 breast cancer cells, A549 lung cancer cells
and their corresponding doxorubicin-resistant cell lines, namely MCF7dx and A549dx, both
overexpressing P-gp (EC50s, Figure 1) [20]. With the aim of contributing to the proposal of
alternative strategies for treating pancreatic cancer, herein we studied the effects of 1 and 2 in
diverse pancreatic cancer cells. Because of the multifunctional nature of these molecules, we aimed
to study the impact of each target involved to the overall effect, as well as on the respective
mechanism leading to cell death. In order to do that, we applied a structural deconstruction
approach on thiosemicarbazones 1 and 2. Structural modifications were carried out so that the metal
chelating moiety or the interaction with σ2 receptor and/or with P-gp were alternatively removed.
Besides their affinity to the σ2 receptor, all ‘deconstructed’ analogues were evaluated for their
interaction capacity with P-gp, primarily because it has been recently shown that P-gp could play a
critical role in the acquired resistance of pancreatic cancer to gemcitabine [27]. For all of the
compounds, cytotoxic activity and pathways activated (e.g. caspase-3 activity, ROS and
mitochondrial superoxide generation) were studied in a panel of pancreatic cancer cells, with the
attempt to shed new light on the contribution of the targets involved in the mechanisms of cells
death which also depend on the different sensitivities of diverse cells. The most promising
compounds were tested in a pre-clinical tumor model of syngeneic KP02 pancreatic cancer cells,
which are known for closely mimicking the human disease with regard to a highly fibrotic tumor
stroma, providing valuable hints about the beneficial effects of a multifunctional, σ2 ligand-based
cancer therapeutics.
2. Results and Discussion
2.1. Chemistry
The synthetic scheme for the synthesis of isatin-β-thiosemicarbazones 7, 8 and 9 is depicted in
Scheme 1. 3,4-Dihydroisoquinolin-1(2H)-one 3 [28] was alkylated with 1-bromo-4-chlorobutane
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using NaH as a base to afford 2-(3-chlorobutyl)-3,4-dihydroisoquinolin-1(2H)-one 4 which was
used to alkylate isatin 5 providing intermediate 6. Isatins 5 or 6, were dissolved in hot ethanol and
treated with thiosemicarbazide or N,N-dimethyl-3-thiosemicarbazide to provide the final
thiosemicarbazones 7, 8 and 9 respectively. In Scheme 2 the synthesis of the final compounds 10,
11, 13 and 14 is illustrated. The already known compounds 13 and 14 [29] were obtained through a
slightly modified procedure. Treatment of indole with 1-bromo-4-chlorobutane in the presence of
KOH gave the intermediate 12. Final compounds 10-14 were obtained upon alkylation of 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline or 1-cyclohexylpiperazine with 1-bromo-butane or with
intermediate 12 in the presence of K2CO3.
2.2. σ2 Receptor Binding
For all the compounds, σ2 receptor affinity was evaluated by radioligand binding assays and the
results are reported as inhibition constants (Ki values) in Table 1. Removal of the N-butyl linked
basic moieties (7 and 8), as well as elimination of the basic properties of the N-atom (9),
dramatically reduced the σ2 receptor affinity (7, 8 and 9, Ki >10000). This outcome was anticipated
because of the lack of structural molecule domains that are essential for an interaction with σ2
receptor (i.e. basic moieties). On the other hand, butyl-6,7-dimethoxytetrahydroisoquinoline and
butyl-cyclohexylpiperazine showed appreciable σ2 affinity values (10 and 11, Ki = 15.0 nM and
65.2 nM respectively) despite the lack of an important hydrophobic portion required for the optimal
interaction with the σ2 receptors, according to the σ2 receptors pharmacophoric models [30-32]. The
presence of the indole hydrophobic portion in the 6,7-dimethoxytetrahydroisoquinoline 13 and
cyclohexylpiperazine 14 derivatives, that mimicks the isatin ring in 1 and 2, led a 3- to 30-fold
increase in the σ2 affinity (Ki = 3.66 nM and 1.90 nM respectively) compared to 10 and 11, in
agreement with the σ2 receptor’s pharmacophore.
2.3. Calcein-AM assay
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For all the novel deconstructed analogues of thiosemicarbazones 1 and 2, interaction with P-gp was
investigated employing the Calcein-AM assay. Results as EC50 values are reported in Table 1. Lead
compounds 1 and 2 moderately inhibited P-gp (EC50 = 3.04 µM and 2.83 µM, respectively) [20],
whereas the absence of the N-butyl-linked basic moiety at the isatin nucleus (7 and 8) completely
abolished the interaction with the P-gp, demonstrating how the sole isatin-thiosemicarbazone
portion is unable to interact with the efflux pump (Table 1). In accordance with previous SAfiR
analyses [33], removal of the basic character of the N-atom in the tetrahydroisoquinoline ring
through the replacement of 6,7-dimethoxytetrahydroisoquinoline with the corresponding amide 3,4-
dihydrisoquinolin-(2H)-1-one (9), kept a moderate interaction with the efflux pump (EC50 = 11.0
µM) [33]. On the other hand, 2-butyl-6,7-dimethoxytetrahydroisoquinoline (10) and 1-butyl-
cyclohexylpiperazine (11) respectively showed modest (EC50 = 76.4 µM) or a complete loss of
interaction with P-gp. Conversly, the corresponding indole derivatives 13 and 14 modulated the
efflux pump in the low micromolar or submicromolar range (EC50 = 6.09 µM and 0.46 µM,
respectively), indicative of a key importance of the the hydrophobic portion within these derivatives
for the interaction with P-gp.
2.4. Cytotoxic activity
The cytotoxic activities of 1 and 2 and their deconstructed analogues were in vitro measured in a
panel of human (MIAPaCa-2, BxPC3, AsPC1 and Panc-1) and mouse (Panc02, KP02 and KCKO)
pancreatic cancer cell lines, which were anticipated to display differential sensitivities to
structurally diverse σ2 receptor ligands, as already shown [21]. Cytotoxic activities of the ligands
are expressed as EC50 values in Table 2. Thiosemicarbazones 1 and 2 generated potent cytotoxic
effects in all the pancreatic cells studied, in line with the potent efficacy showed in the
MCF7/MCF7dx and A549/A549dx cell lines pairs (Figure 1) [20]. These compounds displayed a
potent activity even in the human pancreatic cancer cell line PANC-1 for which other σ2 ligands
previously developed, exhibited no or only minimal cytotoxic activity [21]. Therefore, combination
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of the σ2/P-gp targeting moieties with ironII/III chelator properties of the thiosemicarbazone group
in a single structure (1 and 2) resulted in cytotoxic agents with potent activity in pancreatic tumors
(EC50 values ranging from 1.17 µM to 14.66 µM). In order to evaluate the contribution of the metal
chelator properties alone on the overall activity profile of 1 and 2, the σ2/P-gp targeting moiety (i.e.
N-butyl-linked portions) was removed from the isatin-N-atom, and the thiosemicarbazone structure
was kept, in the N,N-dimethylated (7), and non-dimethylated forms (8), both inactive at σ2 receptor
and at P-gp. Compound 7 determined a strong cytotoxic activity in most of the cell lines studied
(EC50 values ranging from 1.21 µM to 3.14 µM) except for MIAPaCa-2 (EC50 > 18.3 µM) and
Panc-1, where curiously no cytotoxic activity was detected (EC50 > 100 µM). In contrast, the not-
dimethylated compound 8 did not show cytotoxic activity in the cell lines tested (EC50 > 100 µM).
This result is consistent with our previous data, in which N-terminal-dimethylation in
thiosemicarbazones resulted in enhanced antitumor activity in comparison to the un-substituted and
mono-substituted variants, similarly to what has been reported for other thiosemicarbazones [34-
36]. Therefore, independently from the presence of the σ2 targeting moiety, the absence of the
dimethyl substitution at the thiosemicarbazone moiety abolished the cytotoxic activity also in
pancreatic cancer cells. Removal of the sole σ2-mediated activity from the overall action of 1, was
obtained in compound 9, in which the metal chelator and P-gp targeting moieties were retained.
Thiosemicarbazone 9 displayed good efficacy in all the pancreatic cancer cell lines tested (EC50
values ranging from 2.18 µM to 10.30 µM). MIAPaCa-2 and Panc-1 responded to this compound
the least, which was not unexpected given the lack of sensitivity to compounds 1 and 2 and suggests
that these cell lines are less sensitive to thiosemicarbazones in general. The sole contribution of the
σ2 targeting moieties with respect to the overall activity profile of 1 and 2 was studied through the
N-butyl-6,7-dimethoxytetrahydroisoquinoline and 1-butyl-4-cyclohexylpiperazine (10 and 11), and
more reliably through their indole-bearing analogues (13 and 14), which are characterized by
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higher σ2 receptor affinities. While 13 and 14 displayed only a weak activity profile in KP02,
KCKO and MIAPaCa-2 cells (EC50 values ranging from 40 µM to 79 µM), and even further
reduced activity in the other pancreatic cancer cell lines studied (EC50 > 100 µM), 10 and 11 were
inactive in all the cell lines tested in our current study (EC50 > 100 µM). Since a number of other σ2
ligands devoid of metal chelator moieties show cytotoxic activity in pancreatic cancer cells [21,23],
the lack of cytotoxic effects in these σ2 ligands may suggest an antagonist activity at the σ2 receptor
(generally referred as lack of activity).
In summary, the results obtained in pancreatic cancer with our lead thiosemicarbazones 1 and 2 and
their deconstructed analogues show that while different pancreatic cell lines respond with
differential sensitivity to structurally diverse σ2 ligands, their σ2 receptor affinity and/or their
capacity to modulate P-gp does not seem to be required for their cytotoxic activity profiles. In these
molecules, the N,N-dimethyl-substituted-thiosemicarbazone appears to play a dominant role for
their augmented cytotoxic action, likely because of a good compromise in metal chelation and cell
permeation. In any case, it appears that a multitarget approach represents a beneficial constellation,
as the σ2 receptor is overexpressed in a number of diverse cancers and, in addition to the direct
cytotoxic effects of its ligands toward the cancer cells, the σ2 receptor could be harnessed for the
targeted delivery of drugs to tumor foci. In this regard, the combination of metal chelation and σ2-
targeting may be a promising strategy for the treatment of human malignancies.
2.5. Studies on cell mechanisms involved in the activity
We next investigated the contribution of the diverse targets hit by the thiosemicarbazones 1 and 2 to
the pathways activated that eventually lead to tumor cell death. Therefore, thiosemicarbazones 1
and 2 and their analogues displaying relevant cytotoxic activity (7 and 9) were evaluated using
mouse KP02 pancreatic cancer cells, characterized by forming a stroma-rich tumor
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microenvironment in vivo and thus more closely mimic the human disease.
2.5.1. P-gp expression in KP02 tumor cells
In this cell line, where σ2 ligands previously displayed promising in vitro and in vivo efficacy [37],
we assessed the expression level of P-gp as a mean to determine what role, if any, this efflux pump
plays in the context of the cytotoxicity of our drugs. Western blot analysis clearly showed no
expression of the efflux pump in KP02 cells, so that activity at the P-gp of these compounds did not
seem to be involved in their action in this cell line (Figure S1, Supporting Information).
Nevertheless, knowledge about the interaction of these compounds with P-gp may provide useful
information in case of their administration in P-gp overexpressing tumor cells.
2.5.2 ROS involvement in the antiproliferative activity
We next explored ROS involvement in KP02 tumor cells employing lipid antioxidant α-tocopherol
and the hydrophilic precursor of glutathione N-acetyl-L-cysteine (NAC). The effect of antioxidants
was evaluated 48 hours after treatment with 4 µM of 1, 2, 7 and 9 (Figure 2, panel A). Addition of
100 µM α-tocopherol 1 hour prior to drug treatment, rescued the pancreatic cancer cells, thus
effectively reducing cell death caused by 1, 2 and 9. By contrast, no cells were rescued by α-
tocopherol upon treatment with 7, with no differences between the α-tocopherol treated and
untreated cells. The same results were also obtained in AsPC1 and Panc02 tumor cells upon
treatment with the same compounds (Figure S2, Supporting Information). Interestingly, ROS
production was reduced following treatment with 1, 2 and 9 in the presence of α-tocopherol but not
in the presence of NAC. Cell death caused by the four compounds administered at a 4 µM
concentration was not rescued by ΝΑC in the KP02 cell line, and even higher levels of cell death
were recorded when cells were treated with NAC and 7 or 9 (Figure 2, panel A). These results are
in agreement with data obtained upon treatment of pancreatic cancer cells with σ2 ligands and NAC
[21]. We used two different antioxidants on the basis of their different mechanisms: while NAC is a
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hydrophilic antioxidant, precursor of glutathione that provides SH-groups to prevent oxidation, α-
tocopherol is a chain-breaking lipophilic antioxidant localized in cellular membranes responsible of
their integrity through inhibition of lipid peroxidation. Taken together, these results support the
hypothesis that generation of ROS is at least partially responsible for the mechanism of action of
these compounds in all the cell lines studied, except for 7. Therefore, the strong cytotoxic activity of
7 can only be explained with a mechanism of action that does not seem to involve ROS production.
These data also suggest that, despite of sharing the same thiosemicarbazone-β-isatin structure, ROS
generating pathways do not depend on it, as the presence of the N-butyl-linked moieties in
thiosemicarbazones 1, 2 and 9 triggers a pathway that is different from that of compound 7.
2.5.3. Caspase-3 activation
σ2 Ligands have been reported to activate mechanisms that are cell and ligand specific and can
cause in some cases caspase-dependent and in others caspase-independent apoptosis [21,25,38-40].
Therefore, activation of caspase-3 was evaluated for thiosemicarbazones 1, 2, 7 and 9 in mouse
KP02 tumor cells [41]. Pancreatic mouse adenocarcinoma cells were treated with the ligands (25
µM) for 5 hours and assayed for cleavage of the proluminescent caspase-3 substrate and subsequent
generation of a glow-type luminescent signal. Compound 2 induced a good activation of caspase-3,
increasing caspase-3 activity by 6-fold (p < 0.001) while compounds 1, 7, and 9 did not activate the
caspase-3 at all (Figure 2, panel B). Despite targeting the same biological targets,
thiosemicarbazones 1 and 2, which both increase ROS, did not share caspase-3 activation, in
accordance with previously reported σ2 ligands, whose activation of caspase-3 appeared to be both
cell dependent and molecule dependent. Similar results were obtained in AsPC1 and Panc02 tumor
cells upon the treatment with the same compounds (Figure S3, Supporting Information).
2.5.4. Superoxide radical detection in the mitochondria of KP02
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Since mitochondrial superoxide production was recently identified as a novel mechanism of σ2
receptor-mediated cell death [21], we wished to study superoxide radical production in
mitochondria of KP02 pancreatic cancer cell lines. MitoSOX™ Red reagent is a novel fluorogenic
dye specifically targeted to mitochondria in live cells, which is readily oxidized by superoxide but
not by other ROS- or reactive nitrogen species (RNS)- generating systems (e.g. such as peroxides,
hydroxyl radical, singlet oxygen, nitric oxide and peroxynitrite). KP02 cells were treated with 50
µM of 1, 2, 7 and 9 for 2 hours (Figure 2, panel C). While σ2 ligands 1 and 2 induced a strong
mitochondrial ROS production with the mean fluorescence intensities increasing by 6-fold and 20-
fold respectively, thiosemicarbazones 7 and 9 were not capable of superoxide radical production in
the mitochondria of KP02 tumor cells. With the aim of obtaining a more complete picture, we
assessed the mitochondrial superoxide production in the presence of the lipid antioxidant α-
tocopherol, and found that it was strongly reduced when cells were treated with σ2 ligands in the
presence of α-tocopherol, according to the rescue of cell viability displayed in the MTT assay
(Figure 2, panel A). Using the same compounds, similar results were obtained in Panc02 and
AsPC1 cells (Figure S4, Supporting Information). Taken together, these results demonstrate that the
cytotoxic activity of thiosemicarbazones 7 and 9, devoid of σ2 affinity, does not rely on superoxide
radical production in the mitochondria. On the other hand, we have confirmed that the
mitochondrial superoxide production is as a common pathway shared by the two σ2-targeting
thiosemicarbazones 1 and 2 in pancreatic cancer cells, in accordance with the results shown by
other σ2 ligands in the same cells [21].
2.6. Thiosemicarbazones reduces tumor volume in preclinical model of pancreatic cancer
Based on the strong in vitro antitumor activity and diversity of death pathways activation patterns
induced by the most promising thiosemicarbazones 1, 2 and 7, we next investigated these promising
drug candidates for efficacy in a syngeneic, genetically-engineered murine stroma-dense cancer
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model, closely mimicking the human disease. Using this configuration, the in vivo efficacy of the
two σ2/P-gp mixed ligands and iron chelators 1 and 2 (that share ROS and mithocondrial
superoxide production capacity, but not caspase-3 activation), could be compared with that of the
iron chelator 7, that does not act through ROS increase. For these three compounds, interaction with
the σ1 receptor, which is also endowed with antitumor properties, was ruled out through a flow
cytometry experiment previously set up (Figure S6, Supporting Information) [42].
C57BL/6 female mice were inoculated subcutaneously with 2.5 × 105 KP02 cells and a week later,
when tumors reached ~ 5-6 mm in diameter, mice were randomized into control and treatments
groups of (n = 7-10). Vehicle consisting of 25% Cremophor in water, multifunctional compounds 1
and 2 (750 nmol/100µL vehicle), or 7 (750 nmol/100µL vehicle) were given by i.p. injection daily
for two weeks. After conclusion of treatment, tumors were smaller for mice treated with iron
chelator 7 (mean = 328 mm3), compared to vehicle (mean = 596 mm3) (Figure 3 panel A; p <
0.0001). Mice treated with compounds 1 and 2 had both tumor volumes that were statistically
similar to vehicle, without experiencing any treatment-related deaths. Therefore, we repeated the
experiment by employing higher concentrations of compounds 1 and 2 (1500 nmol/100µL vehicle).
At this concentration, both multifunctional compounds were capable of reducing the mean tumor
volume (mean = 196 mm3 for compound 2 and mean = 205 mm3 for compound 1) compared to
vehicle treated group (mean = 592 mm3) (Figure 3, panel B; p > 0.001). Moreover, compounds 1
and 2 turned out to be more effective in reducing tumor burden than gemcitabine when compared to
mice bearing a KP02 orthotopic tumor and treated twice weekly by i.p. injections with 20 mg/kg
gemcitabine [37].
Importantly, we did not observe treatment-related deaths or gross abnormalities in mouse behavior.
In order to assess for more subtle toxicities, serum chemistries (AST, ALT, BUN, total protein,
glucose, and Cr) and complete blood counts were analyzed with no significant differences noted
when compared to the control group (Table 3). Necropsy revealed no difference in mouse weights
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(vehicle: 19.5± 0.5 grams; 7: 19.5 ± 0.5 grams; 1: 17.5 ± 0.5 grams; 2: 19 ± 1 grams; Figure 4; p >
0.05). There were no significant lesions in the brain, heart, alimentary tract, kidneys, liver, or
pancreas. Only mild peritonitis was identified at the site of repeated drug injections. However, the
compound 7 treated group revealed foci of pulmonary metastases of the implanted tumor and a mild
chronic progressive nephropathy that were not noticed in mice treated with compounds 1 and 2.
3. Conclusion
Based on the encouraging activity profiles of the multifunctional thiosemicarbazones 1 and 2 that
target the σ2 receptor and the P-gp efflux pump and have Iron chelating properties, a deconstruction
approach was applied to investigate the contribution of each target hit to the overall action of these
molecules. Because of the urgent need of novel treatments for pancreatic cancers, lead compounds
1 and 2 and the deconstructed analogues (7-11, 13 and 14) were investigated in a panel of
pancreatic tumor cells. Among the compounds, N,N-dimethyl-thiosemicarbazone-bearing
derivatives (1, 2, 7, 9) showed potent activity in most of the pancreatic cell lines studied, also in the
absence of the σ2-targeting moiety (7 and 9), so that the iron chelating structural portion (N,N-
dimethyl-thiosemicarbazone) appeared to be mainly responsible for the cytotoxic activity in these
cells (likely because it confers a good balance between activity and cell permeability). Nevertheless,
we showed that the death pathways engaged by these compounds (1, 2, 7 and 9) are not governed
by the common N,N-dimethyl-thiosemicarbazones portion since: i) ROS increase was shown by 1,
2, and 9 but not by 7; ii) caspase-3 activity was only increased by σ2-targeting thiosemicarbazone 2;
iii) mitochondrial superoxide production was only detected with the two σ2-targeted
thiosemicarbazones 1 and 2. Our deconstructive approach showed that while σ2 receptor targeting is
not necessary for the in vitro cytotoxicity of these multifunctional thiosemicarbazones (except for
Panc-1 cells), the presence of the N-butyl linked basic moieties triggers alternative death pathways
in comparison to those compounds that only contain metal chelating features, thus allowing for
differential sensitization levels of cell death, which we believe could be useful for developing
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tailored treatments. Importantly, besides a contribution to the cytotoxic activity (as in Panc-1 cells),
σ2 receptor binding may be beneficial for the targeted delivery to σ2 receptors overexepressing
tumors, according to a promising recent approach [39]. In vivo administration of the multifunctional
σ2 ligands 1, 2 or the metal chelator 7 markedly slowed the growth rate in a mouse stroma dense
model of pancreatic cancer (C57BL/6 bearing KP02 tumors), demonstrating that these compounds
reach their target and exert antitumor activity. While mice treated with the iron chlelator 7 revealed
foci of pulmonary metastases of the implanted tumor and a mild chronic nephropathy, mice treated
with multifunctional compounds 1 and 2 better tolerated the treatment without showing signs of off-
target toxicity.
Despite the suggested role that P-gp may play in the resistance of pancreatic cancer to gemcitabine,
the absence of the efflux pump in KP02 cells did not allow us to evaluate our compounds in the
context of P-gp expression.
Overall, the results from our deconstructive approach from in vitro and in vivo experiments suggest
that while the multifunctional drug composition is not necessary for the antitumor activity of these
thiosemicarbazones, σ2-targeting may allow for a more specific targeted delivery to tumor foci in
vivo (σ2 overexpressing tumors) with less toxic off-targets effects, together with the activation of
alternative tumor cell death mechanisms. Such multifunctional molecules endowed with potent in
vivo antitumor activity and reduced off-site toxicity are certainly worth studying further in the
oncology field.
Acknowledgements
This work was funded in part by a National Institute of Health R01 grant (US NIH
5R01CA16376402) (W.G. Hawkins). This work was conducted under the Agreement regulating
cultural and scientific cooperation between the University of Bari ALDO MORO (Italy) and
Washington University School of Medicine in St. Louis (USA).
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4. Experimental Section
4.1. Chemistry
Both column chromatography and flash column chromatography were performed with 60 Å pore
size silica gel as the stationary phase (1:30 w/w, 63−200 µm particle size, from ICN, and 1:15 w/w,
15−40 µm particle size, from Merck, respectively). Melting points were determined in open
capillaries on a Gallenkamp electrothermal apparatus. High-performance liquid chromatography
(HPLC) on an Agilent Infinity 1260 system equipped with diode array with a multiwavelenght
UV/vis detector set at λ = 230 nm, 254 nm and 280 nm, through a Phenomenex Gemini RP-18
column (250 × 4.6 mm, 5 µm particle size) was performed on target compounds confirming ≥ 95%
1
purity. H NMR spectra were recorded on a Mercury Varian 300 MHz or on a 500-vnmrs500
Agilent spectrometer (499.801 MHz). The following data were reported: chemical shift (δ) in parts
per million (ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), integration, and
13
coupling constant(s) in hertz. C NMR (125 MHz) were recorded on a 500-vnmrs500 Agilent
spectrometer (499.801 MHz) on novel final compounds: chemical shifts in ppm were reported.
Recording of mass spectra was done on an Agilent 1100 series LCMSD trap system VL mass
spectrometer; only significant m/z peaks, with their percentage of relative intensity in parentheses,
are reported. High resolution mass spectroscopy (HRMS) was performed on a Agilent 6530
Accurate-Mass Q-TOF LC/MS spectrometer. For final compounds 10 and 11, 13C NMR and HRMS
spectra were recorded on their hydrochloride salts. Chemicals were from Aldrich, TCI and Alpha
Aesar and were used without any further purification.
4.2. 2-(4-Chlorobutyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-1(2H)-one (4)
To a suspension of NaH (0.123 g, 5.12 mmol) in dry DMF (2 mL) a solution of 3 (0.425 g, 2.05
mmol) in dry DMF (3 mL) was added in a dropwise manner. The mixture was stirred at 0 °C for 15
min. Then a solution of 1-bromo-4-chlorobutane (0.3 ml, 2.66 mmol) in dry DMF (2 mL) was
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added and the resulting mixture was stirred for 4 h at room temperature. After cooling at 0 °C, the
reaction mixture was quenched with water and the solvent concentrated under reduced pressure.
The residue was dissolved in H2O and extracted with CH2Cl2 (3 ×10 mL). The collected organic
layers were dried over Na2SO4 and the solvent was evaporated under reduced pressure to afford the
crude as a yellow oil. Purification through column chromatography with CH2Cl2 as eluent afforded
the title compounds as colorless oil (0.426 g, 70% yield). GC-MS m/z: 297 (M+, 20), 220 (100).
4.3. 1-(4-(6,7-Dimethoxy-1-oxo-3,4-dihydroisoquinolin-2(1H)-yl)butyl)indoline-2,3-dione (6)
A solution of 4 (0.426 g, 1.43 mmol) in CH3CN (10 mL) was added with K2CO3 (2.87 mmol, 0.396
g) and isatin 5 (0.192 g, 1.3 mmol). The resulting mixture was refluxed overnight under stirring.
After the removal of the solvent under reduced pressure the residue was taken up with H2O and
extracted with AcOEt (3 × 10 mL). The collected organic layers were dried (Na2SO4) and
evaporated under reduced pressure to afford a crude oil, which was purified by column
chromatography (CH2Cl2/AcOEt 1:1) to give the title compound (0.28 g, 50% yield).
1H NMR (500 MHz, CDCl3) δ 1.72-1.81 (m, 4H, CH2CH2CH2N), 2.92 (t, 2H, J = 6.85 Hz,
NCH2CH2Ar), 3.53 (t, 2H, J = 6.85 Hz, NCH2CH2Ar), 3.64 (t, 2H, J = 6.85 Hz,
NCH2CH2CH2CH2N), 3.81 (t, 2H, J = 6.85 Hz, NCH2CH2CH2CH2N), 3.90 (s, 3H, OCH3), 3.92 (s,
3H, OCH3), 6.64 (s, 1H, aromatic), 7.00 (d, 1H, J = 7.83 Hz, aromatic), 7.10 (t, 1 H, J = 7.34 Hz,
aromatic), 7.54-7.61 (m, 3H, aromatic).
4.4. General procedure for the synthesis of final compounds 7-9
To a solution of isatins 5 or 6 (0.68 mmol) in Ethanol (10 mL) 4,4-dimethyl-3-thiosemicarbazide
(0.68 mmol) was added and the resulting mixture was refluxed overnight. Upon cooling,
precipitation of the final product was achieved.
4.4.1. (Z)-N,N-Dimethyl-2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide (7) Crystallization
from H2O/EtOH afforded the title compound as orange crystals (0.084g, 50% yield); mp = 250-250
1
°C; H NMR (500 MHz, DMSO-d6) δ 3.35 (s, 6H, CH3), 6.92-6.94 (m, 1H, aromatic), 7.06-7.09
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(m, 1H, aromatic), 7.32-7.35 (m, 1H, aromatic), 7.51 -7.53 (m, 1 H, aromatic), 11.28 (s, 1H, isatin
NH), 13.41 (s, 1H, NHCS); 13C NMR (500 MHz, DMSO-d6) 43.75; 43.83; 117.96; 119.45; 124.34;
129.54; 131.31; 134.22; 141.23; 169.51; 177.83. LC-MS (ESI+) m/z: 271 [M+Na]+; LC-MS-MS
271: 226; LC-MS (ESI-) m/z 247 [M-H]-; LC-MS-MS 247: 204, 174; QTOF (m/z) Calcd for
C11H12N4OS [M+Na]+: 271.0630, found: 271.0621.Compound was > 98% pure by HPLC analysis
performed with MeOH/H2O, 80 : 20 v/v, at a flow rate 0.8 mL min -1.
4.4.2. (Z)-2-(2-Oxoindolin-3-ylidene)hydrazinecarbothioamide (8) Crystallization from
H2O/EtOH afforded the title compound as orange crystals (0.07g, 50% yield); mp = 253 – 255 °C;
1H NMR (500 MHz, DMSO-d6) δ 6.90-6.92 (m, 1H, aromatic), 7.06-7.09 (m, 1H, aromatic), 7.32-
7.36 (m, 1H, aromatic), 7.63 -7.65 (m, 1 H, aromatic), 8.67 (s, 1H, NHH), 9.03 (s, 1H, NHH), 11.19
(s, 1H, isatin NH), 12.46 (s, 1H, NHCS); 13C NMR (500 MHz, DMSO-d6) 117.66; 119.51; 124.54;
129.44; 131.25; 134.65; 141.32; 168.72; 180.82. LC-MS (ESI+) m/z: 243 [M+Na]+; LC-MS-MS
243: 226, 185; LC-MS (ESI-) m/z 219 [M-H]-; LC-MS-MS 219: 160; QTOF (m/z) Calcd for
C9H8N4OS [M+Na]+: 243.0317, found: 243.0307. Compound was > 98% pure by HPLC analysis
performed with MeOH/H2O, 80 : 20 v/v, at a flow rate 0.8 mL min -1.
4.4.3. (Z)-2-(1-(4-(6,7-Dimethoxy-1-oxo-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-oxoindolin-
3-ylidene)-N,N-dimethylhydrazinecarbothioamide (9) Crystallization from H2O/EtOH afforded
the title compound as yellow crystals (0.173 g, 50% yield); mp = 201-202 °C; 1H NMR (500 MHz,
CDCl3) δ 1.67-1.83 (m, 4H, CH2CH2CH2N), 2.88 (t, 2H, J = 6.85 Hz, NCH2CH2Ar), 3.48-3.54 (m,
8H, CH2NCH2CH2Ar and N(CH3)2), 3.58-3.64 (m, 2H, CH2NCH2CH2Ar), 3.80-3.87 (m, 2H,
NCH2CH2CH2CH2N), 3.92 (s, 3 H, OCH3), 3.93 (s, 3H, OCH3), 6.63 (s, 1H, aromatic), 6.96 (d, 1H,
J = 7.34 Hz, aromatic), 7.12 (t, 1 H, J = 7.34 Hz, aromatic), 7.33 (d, 1 H, J = 7.34 Hz, aromatic),
7.60 (s, 1H, aromatic), 7.83 (t, 1H, J = 7.34 Hz, aromatic), 13.62 (s, 1H, NH); 13C NMR (500 MHz,
CDCl3) 24.74; 25.01; 27.72; 39.44; 46.17; 46.35; 56.04; 56.08; 109.23; 109.33; 110.44; 119.96;
121.66; 121.85; 123.27; 130.76; 131.54; 134.77; 142.04; 147.98; 151.88; 161.78; 164.62; 180.13.
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QTOF (m/z) Calcd for C26H31N5SO4 [M+Na]+: 532.1994, found: 532.1988. Compound was > 98%
pure by HPLC analysis performed with MeOH/H2O, 80 : 20 v/v, at a flow rate 0.8 mL min -1.
4.5. General procedure for the synthesis of 10-11
A solution of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline or 1-cyclohexylpiperazine (2.90 mmol)
in CH3CN was added with K2CO3 (0.49 g, 3.50 mmol) and 1-bromobutane (0.38 ml, 3.50 mmol).
The resulting mixture was refluxed overnight under stirring. After the removal of the solvent under
reduced pressure the residue was taken up with H2O and extracted with CH2Cl2 (3 × 10 mL). The
collected organic layers were dried (Na2SO4) and evaporated under reduced pressure to afford a
crude oil which were purified by column chromatography (CH2Cl2/MeOH 95:5) to give the title
compounds which were transformed into the corresponding hydrocloride salts, recrystallized from
MeOH/Et2O.
4.5.1. N-Butyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (10) was obtained as white crystals
1
(0.5 g, 90% yield); mp = 191-193 °C. H NMR (500 MHz, MeOH-d4) δ 1.03 (t, 3H, J = 7.3 Hz,
NCH2CH2CH2CH3), 1.44-1.49 (m, 2H, NCH2CH2CH2CH3), 1.78-1.84 (m, 2H, NCH2CH2CH2CH3),
3.10-3.12 (m, 2H, NCH2CH2CH2CH3), 3.23-3.26 (m, 2 H, ArCH2CH2NCH2), 3.29-3.31 (m, 2H,
ArCH2CH2NCH2), 3.80 (s, 6H, 2 OCH3), 4.86 (s, 2H, ArCH2NCH2CH2CH2CH3), 6.77 (s, 1H,
13
aromatic), 6.82 (s, 1 H, aromatic); C NMR (500 MHz, MeOH-d4) 13.83; 18.75; 23.73; 25.74;
51.43; 53.67; 56.10; 59.56; 111.41; 111.63; 116.76; 130.45; 146.71; 146.92; GC-MS m/z: 248 (M+,
0.5), 206 (100); QTOF (m/z) Calcd for C15H23NO2 [M+H]+: 250.1807, found: 250.1800. Compound
was > 98% pure by HPLC analysis performed with CH3CN/HCOONH4 (20 mM, pH = 5) 75 : 25
v/v, at a flow rate 1.0 mL min -1.
4.5.2. 1-Butyl-4-cyclohexyl-piperazine (11) was obtained as white crystals (0.5 g, 77% yield); mp
1
= 250-252 °C. H NMR (500 MHz, MeOH-d4) δ 1.01 (t, 3H, J = 7.34 Hz, NCH2CH2CH2CH3),
1.19-2.20 (m, 14H, cyclohexyl CH2 and NCH2CH2CH2CH3), 3.20-3.66 (m, 11H, piperazine CH and
13
CH2 and NCH2CH2CH2CH3); C NMR (500 MHz, MeOH-d4) 13.82; 18.90; 24.32; 25.74; 25.65;
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28.86; 50.10; 52.43; 53.82; 59.46; GC-MS m/z: 224 (M+, 30), 181 (100); QTOF (m/z) Calcd for
C14H29N2 [M+H]+: 225.2331, found: 225.2324. Compound was > 98% pure by HPLC analysis
performed with CH3CN/HCOONH4 (20 mM, pH = 5) 75 : 25 v/v, at a flow rate 1.0 mL min -1.
4.6. Biology
4.6.1. Materials
[3H]-DTG (50 Ci/mmol) and CulturePlate 96/wells plates were purchased from PerkinElmer Life
and Analytical Sciences (Boston, MA, USA). DTG was purchased from Tocris Cookson Ltd, UK.
(+)-Pentazocine and calcein-AM were obtained from Sigma-Aldrich-RBI s.r.l. (Milan, Italy). Male
Dunkin guinea-pigs (200-250 g) and Wistar Hannover rats (250-300 g) were from Harlan, Italy.
Cell culture reagents were purchased from EuroClone (Milan, Italy). Protease inhibitor cocktail,
was obtained from Sigma-Aldrich (Milan, Italy). Anti-P-Glycoprotein antibody produced in mouse
(C219) was purchased from Calbiochem (Merck-Millipore, Germany). Anti-β-actin, secondary
peroxidase antibodies and all reagents for western blotting were purchased from Life Technologies
Italia (Monza, Italy).
4.6.2. Compounds
σ2 Receptor ligands 1 [(Z)-2-(1-(4-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-
oxoindolin-3-ylidene)-N,N-dimethylhydrazinecarbothioamide hydrochloride], 2 [(Z)-2-[1-[4-(4-
Cyclohexylpiperazin-1-yl)butyl]-2-oxoindolin-3-ylidene]-N,N-dimethylhydrazinecarbothioamide
di-hydrochloride], intermediate 3 [6,7-dimethoxy-3,4-dihydroisoquinolin-1(2H)-one], and σ1
fluorescent
ligand
5-(dimethylamino)-2-(6-((5-(4-(4-methylpiperidin-1-yl)butyl)-5,6,7,8-
tetrahydronaphthalen-2-yl)oxy)hexyl)isoindoline-1,3-dione were synthesized in our laboratories
according to published methods [20,33,42]. Caspase-3 inhibitor Z-DEVD-FMK was purchased
from Tocris Bioscience, α-tocopherol and N-Acetyl-L-cysteine from Sigma-Aldrich (St. Louis,
MO). Compounds were dissolved in DMSO with final concentrations less than 0.3%.
4.6.3. Cell Culture
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Human pancreas cancer cell lines BxPC3, AsPC1, MiaPaCa-2, and Panc1 were obtained from
American Type Culture Collection (ATCC, Bethesda, MD). Murine pancreas adenocarcinoma
Panc02 was a gift from Bryan Clary (Duke University). The mouse KCKO cell line isolated from a
spontaneously developing pancreatic cancer overexpressing human MUC1 [41] was kindly
provided by Dr. Pinku Mukherjee (University of North Carolina, Charlotte, NC). The mouse KP02
line was derived from pancreatic cancer tumor tissue obtained from p48-CRE/LSL-
KrasG12D/p53flox/+ mice (backcrossed C57BL/6, n = 6). The MCF7σ1 was produced in our laboratory
[43]. AsPC-1, BxPC-3 and Panc02 cells were cultured in RPMI-1940 medium with 10% fetal
bovine serum (FBS). MIAPaCa-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) with 10% FBS and 2.5% horse serum. PANC-1 and MCF7σ1 cells were cultured in
DMEM with 10% FBS. KCKO cells were cultured in RPMI-1940 medium with 10% FBS, 1%
sodium pyruvate, 1% HEPES buffer, and 1% L-glutamine. KP02 cells were cultured in 1:1 mixture
of DMEM and Ham’s F-12 Nutrient Mixture with 10% FBS. Penicillin (100 mg/mL) and
streptomycin (100 mg/mL) were added to all media; cells were maintained in a humidified
incubator at 37 ºC with 5% CO2.
4.6.4. Competition σ2 Binding Assays
All the procedures for the binding assays were previously described. σ2 Receptor binding were
carried out according to Berardi et al [44]. The specific radioligand and tissue sources was: [3H]-
DTG in the presence of 1 µM (+)-pentazocine to mask σ1 receptors, rat liver membranes. The
following compounds were used to define the specific binding reported in parentheses: DTG (85-
96%). Concentrations required to inhibit 50% of radioligand specific binding (IC50) were
determined by using six to nine different concentrations of the drug studied in at least three
experiments with samples in duplicate. Scatchard parameters (Kd and Bmax) and apparent inhibition
constants (Ki) values were determined by nonlinear curve fitting, using the Prism, version 3.0,
GraphPad software [45].
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4.6.5. σ1 Binding by Flow Cytometry studies
The procedure for σ1 binding by flow cytometry studies were carried out according to Abate et al
2016 [42]. MCF7σ1 cells were incubated with increasing concentrations (0.1, 1, 10, and 100 nmol/L
and 1 and 10 µM) of (+)-pentazocine or PB212 [46-49] or thiosemicarbazones 1, 2 and 7, followed
by 100 nmol/L of either σ1 fluorescent compound (σ1FC, 5-(dimethylamino)-2-(6-((5-(4-(4-
methylpiperidin-1-yl)butyl)-5,6,7,8-tetrahydronaphthalen-2-yl)oxy)hexyl)isoindoline-1,3-dione) for
75 min at 37 °C. To mask σ2 receptors, 2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-
yl)propyl)-5-methoxy-3,4-dihydroisoquinolin-1(2H)-one F390 [28,42] (10 µM) was co-incubated.
At the end of the incubation periods, cells were washed twice with PBS, detached with 200 mL of
Cell Dissociation Solution (Sigma Chemical Co.) for 10 min at 37 °C, centrifuged at 13,000 g for 5
min and resuspended in 500 µL of PBS. The fluorescence was recorded using a Bio-Guava®
easyCyte™ 5 Flow Cytometry System (Millipore, Billerica, MA), with a 530 nm band pass filter.
For each analysis, 50,000 events were collected and analyzed with the InCyte software (Millipore).
4.6.6. Calcein-AM experiment
These experiments were carried out as already described [50]. MDCK-MDR1 cell line (50,000 cells
per well) was seeded into black CulturePlate 96/wells plate with 100 µL medium and allowed to
become confluent overnight. 100 µL of different concentrations of test compounds (0.1-100 µM)
were solubilized in culture medium and added to each well. The 96/wells plate was incubated at 37
°C for 30 min. 100 µL of Calcein-AM, solved in Phosphate Buffered Saline (PBS), was added to
each well to yield a final concentration of 2.5 µM, and the plate was incubated for 30 min. The plate
was washed 3 times with 100 mL ice cold PBS. Saline buffer (100 µL) was added to each well and
the plate was read by a PerkinElmer Victor3 spectrofluorimeter at excitation and emission
wavelengths of 485 nm and 535 nm, respectively. In these experimental conditions, Calcein cell
accumulation in the absence and in the presence of tested compounds was evaluated and
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fluorescence basal level was estimated by untreated cells. In treated wells the increase of
fluorescence with respect to basal level was measured. EC50 values were determined by fitting the
fluorescence increase percentage versus log[dose].
4.6.7. Cell Viability and ROS interference
Determination of cell growth was performed using the MTT assay at 48 h [51,21]. On day 1, 25,000
cells/well were seeded into 96-well plates in a volume of 100 µL. On day 2, the various drug
concentrations (1 µM-100 µM) were added. In all the experiments, the various drug-solvents
(EtOH, DMSO) were added in each control to evaluate a possible solvent cytotoxicity. After the
established incubation time with drugs (48 h), MTT (0.5 mg/mL) was added to each well, and after
3-4 h incubation at 37 °C, the supernatant was removed. The formazan crystals were solubilized
using 100 µl of DMSO/EtOH (1:1) and the absorbance values at 570 and 630 nm were determined
on the microplate reader Victor 3 from PerkinElmer Life Sciences. The interference of ROS in cell
viability was indirectly determined by MTT assay reported above at 24 h. On day 1, 25000 cells per
well were seeded into 96-well plates in the presence or absence of α-tocopherol (100 µM). On day
2, the drugs (1 µM-100 µM) were added alone and in combination with α-tocopherol (100 µM).
After incubation (24 h) with drugs, MTT assay was performed as above. The interference of ROS in
cell viability was indirectly determined by MTT assay reported above at 48 h. On day 1, cells were
plated at a density of 2 x 104 cells/well in opaque 96-well, clear-bottom plates 24 hours prior to
treatment. On day 2, cells were treated with the drugs in the presence or absence of α-tocopherol
(100 µM) or N-Acetyl-L-cysteine (100 µM). After incubation (48 h) with drugs, MTT assay was
performed as above.
4.6.8. Western blotting
The experiment was carried out according to Niso et al. with minor modification [52]. All cells
were washed twice with 10 ml phosphate-buffered saline (PBS), scraped in 1 ml PBS and
centrifuged for 1 min at 11,000 g. Proteins were extracted from cells by homogenization in cold
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RIPA buffer (Life Technologies) containing 1X protease inhibitor cocktail and centrifuged at
14,000 g for 15 min at 4°C. The supernatant was recovered and the protein concentration was
measured using the microLowry kit. 30 µg of protein extract was separated on 10% polyacrylamide
gel (Life Technologies) and then transferred onto a polyvinylidene difluoride membrane (PVDF) by
iBlot® Gel Transfer Device (Life Technologies). Membrane was blocked for 30 min at room
temperature with blocking buffer (1% BSA, 0.05% Tween 20 in Tris-buffered saline, TBS). The
membrane was then incubated overnight at 4°C with anti-P-Glycoprotein (1:500 mouse
monoclonal) or for 1h at room temperature anti-β-actin (1:1000 mouse monoclonal) antibodies,
diluted in blocking buffer. After incubation time, membrane was washed with washing buffer
(0.05% Tween 20 in Tris-buffered saline, TBS) for three times and incubated with a secondary
peroxidase antibody (1:2000 anti-mouse for P-glycoprotein and β-actin) for 1h at room temperature.
After washing, the membrane was treated with the enhanced chemiluminescence (ECL, Life
Technologies) according to the manufacturer's instructions and the blot was visualized by UVITEC
Cambridge (Life Technologies). The expression level was evaluated by densitometric analysis
using UVITEC Cambridge software (Life Technologies) and β-actin expression level was used to
normalize the sample values.
4.6.9. Detection of Caspase-3 activity in vitro
Caspase-3 activity was measured in KP02 cell lines with a Caspase-Glo® Assay Systems
(Promega) according to protocol in which the reagent contain luminogenic caspase substrates that
cleaved by activated caspase. Cells were seeded at a density of 1 × 104 in black 96-well, clear
bottom plates for 24 hours before treatment with 25 µM of compounds in presence or absence of α-
tocopherol (100 µM) or Z-DEVD-FMK (1µM) for 5 hours after treatment. The contents were then
mixed using plate shaker for 30 seconds, and incubated at room temperature for 90 minutes.
Luminescence signal was measured using multi-mode microplate reader (BioTek). Assay was
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performed in triplicates, and caspase activity was plotted compared cells treated with DMSO as a
control.
4.6.10. Detection of Mitochondrial Superoxide by flow cytometry and ROS interference
MitoSOX™ Red reagent is a novel fluorogenic dye specifically targeted to mitochondria in live
cells. Oxidation of MitoSOX™ Red reagent by superoxide produces red fluorescence.
Mitochondrial superoxide is generated as a byproduct of oxidative phosphorylation. In an otherwise
tightly coupled electron transport chain, approximately 1–3% of mitochondrial oxygen consumed is
incompletely reduced; those “leaky” electrons can quickly interact with molecular oxygen to form
superoxide anion, the predominant reactive oxygen species (ROS) in mitochondria [53-56].
MitoSOX™ Red reagent is readily oxidized by superoxide but not by other ROS- or reactive
nitrogen species (RNS)–generating systems. KP02 cells were seeded into 12-well plates 24 hours
before treatment with iron chelators (50 µM) for 2 hours at 37°C in presence or absence of α-
tocopherol (100 µM) followed by staining with MitoSOX™ Red (5 µM). Two hours after red dye
addition, the cells were washed twice with PBS and harvested with trypsin/EDTA buffer. The cells
were washed twice with PBS before analysis with FACSCalibur (BD Bioscience, San Jose, CA).
The oxidation product of MitoSOX™ Red by mitochondrial superoxide fluoresces with an emission
maximus of 580 nm and was detected in the FL3 channel. Experiment was performed in triplicates.
4.6.11. In vivo assessment of tumor growth
Animal studies were performed according to the animal studies protocol (20130073) approved by
the Washington University Institutional Animal Care Facility. In this pre-clinical model, we utilized
the KP02 cell line. In vivo studies with mice were performed to compare the effect of σ2 ligands
with iron chelator 7. Mice treated with vehicle alone (25% Cremophor in H2O) served as the control
cohort. Female C57BL/6 mice (8 weeks old, National Cancer Institute Laboratories) were injected
in the right flank with 200 µL of a single-cell suspension of KP02 cells in non-supplemented RPMI
medium (2.5 x 105 cells per mouse). Treatment began when the mean tumor diameter was ~ 5-6
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mm. Mice received daily intraperitoneal (i.p.) injections of the σ2 ligands 1 and 2 (750nmol), or 7
(750nmol) in 100 µL vehicle or vehicle alone (control) for 2 weeks. Tumors were measured three
times weekly in two dimensions with a digital caliper, and tumor volumes were calculated by the
standard formula of Tumor Volume = Length x Width2 x 0.5. All mice were euthanized when
tumors reached a diameter of 15 mm or had ulcerated. The experiment was repeated using a double
concentration of 1 and 2 following the same procedure described above. Several mice from each
treatment cohort were assessed for pathologic evaluation (Digestive Diseases Research Core Center
at Washington University School of Medicine, St. Louis, MO). Blood was collected for complete
blood count (CBC) and biochemical analysis (AST, ALT, BUN, total protein, glucose and Cr).
Organs were examined grossly and histologically.
4.6.12. Statistical analysis
Statistical analyses and data plotting were performed using GraphPad Prism software version 6.03
(San Diego, CA). Results were expressed as mean ± standard error of the mean of at least 3
biological replicates. EC50 values were calculated by curve fitting normalized viability versus drug
concentration. Differences in viability, caspase-3 activity, and tumor volume were analyzed using
two-way ANOVA to identify differences and confirmed with paired two tailed t-tests. Mann-
Whitney test was used to compare the difference in CBC and biochemistry analyses. Kaplan-Meier
survival analyses were used to assess differences between treatment groups and were compared
using a log-rank test. A p-value < 0.05 was considered significant for all analyses.
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