Cytotoxic effect, antitumour activity and toxicity of organotin derivatives with ortho- or para-hydroxy-benzoic acids

Article abstract of DOI:10.1007/s00044-018-2135-7

The cytotoxic and antitumour activities of five organotin complexes (1–5) with o-hydroxy-benzoic or p-hydroxy-benzoic acids were evaluated in a series of in vitro and in vivo experiments. All complexes exhibited strong cytotoxic activity against all cancer cells lines, whereas complexes 1, 2 and 4 induced apoptosis at significantly lower doses than complexes 3 and 5. Human cancer cells treated with increasing concentrations of complexes 1, 2 and 4 gradually lost their ability to form colonies. Only complexes 1 and 2 inhibited colony formation efficiency in rat leiomyosarcoma cells. Histopathology of liver and kidney showed mild damage and lung oedema after a single injection of 10 mg/kg body wt of complex 1 to Wistar rats. At higher doses (100 mg/kg body wt) brain stem oedema was observed. Daily administration of tumour-bearing Wistar rats (leiomyosarcoma) with 1 mg/kg body wt of complex 1 until death reduced the mean tumour growth rate by more than 3× fold and prolonged mean survival time by 120%. These findings indicate that the organotin complexes with ortho- or para-hydroxy-benzoic acids possess potent cytotoxic and antitumour activity and they could be used as potential chemotherapeutic agents.

Full text of DOI:10.1007/s00044-018-2135-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2135-7
ORIGINAL RESEARCH
Cytotoxic effect, antitumour activity and toxicity of organotin
derivatives with ortho- or para-hydroxy-benzoic acids
Maria S. Agiorgiti1 Angelos Evangelou1 Patra Vezyraki1 Sotiris K. Hadjikakou2 Vasiliki Kalfakakou1
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Ioannis Tsanaktsidis1 Anna Batistatou3 John Zelovitis1 Yannis V. Simos
Vasilios Ragos4
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Spyridon Karkabounas1 Dimitrios Peschos1
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Received: 7 September 2017 / Accepted: 13 January 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
The cytotoxic and antitumour activities of five organotin complexes (1–5) with o-hydroxy-benzoic or p-hydroxy-benzoic
acids were evaluated in a series of in vitro and in vivo experiments. All complexes exhibited strong cytotoxic activity against
all cancer cells lines, whereas complexes 1, 2 and 4 induced apoptosis at significantly lower doses than complexes 3 and 5.
Human cancer cells treated with increasing concentrations of complexes 1, 2 and 4 gradually lost their ability to form
colonies. Only complexes 1 and 2 inhibited colony formation efficiency in rat leiomyosarcoma cells. Histopathology of liver
and kidney showed mild damage and lung oedema after a single injection of 10 mg/kg body wt of complex 1 to Wistar rats.
At higher doses (100 mg/kg body wt) brain stem oedema was observed. Daily administration of tumour-bearing Wistar rats
(leiomyosarcoma) with 1 mg/kg body wt of complex 1 until death reduced the mean tumour growth rate by more than 3×
fold and prolonged mean survival time by 120%. These findings indicate that the organotin complexes with ortho- or para-
hydroxy-benzoic acids possess potent cytotoxic and antitumour activity and they could be used as potential
chemotherapeutic agents.
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Keywords Organotin complexes Hydroxy benzoic acid Cytotoxicity Anticancer In vivo Toxicity
Introduction
tumour resistance (Koberle et al. 2010) lead to the devel-
opment of platinum and non-platinum metal complexes
with similar anticancer activity (Garbutcheon-Singh et al.
2011). Especial interest was invested on organotin deriva-
tives characterised by potent cytotoxic and anticancer
activity (Arjmand et al. 2014; Amir et al. 2014; Muhammad
and Guo 2014). Organotin derivatives can cause direct
DNA damage (Liu et al. 2006) acting through different
mechanisms than cisplatin, e.g., the rise of cytosolic Ca2+
which leads the cells to apoptosis (Aw et al. 1990) or the
inhibition of essential macromolecules synthesis and the
mitochondrial metabolism (Alama et al. 2009). Moreover,
the cytotoxicity exerted by some organotin complexes is
correlated with the inhibition of peroxidation of linoleic
acid induced by lipoxygenase (Abdellah et al. 2009).
Organotin derivatives show potent cytotoxic properties
that can be even 200 times higher of cisplatin (Verginadis
et al. 2011). Ttriphenyltin(VI) complexes of non-steroidal
anti-inflammatory drugs were found to be highly cytotoxic
against the cancer cell lines MCF-7 (breast), T-24 (urinary
bladder) and A549 (lung) (Dokorou et al. 2011), while tri-
phenyltin 2-(triphenyltinmercapto) nicotinate exhibited an
Cisplatin is one of the most potent anticancer agent used
alone or in combination with chemotherapy agents to treat
various human tumours (Miller et al. 2010). However, the
serious side effects (mainly nephrotoxicity) exerted by cis-
platin (Miller et al. 2010) in conjunction with the increased
* Yannis V. Simos
simosyannis@gmail.com
* Dimitrios Peschos
dpeschos@cc.uoi.gr
1
Department of Experimental Physiology, Faculty of Medicine,
University of Ioannina, 45110 Ioannina, Greece
2
Department of Inorganic and Analytical Chemistry, Faculty of
Chemistry, University of Ioannina, 45110 Ioannina, Greece
3
Department of Pathology, Faculty of Medicine, University of
Ioannina, 45110 Ioannina, Greece
4
Clinic of Maxillofacial Surgery, Medical Department, University
of Ioannina, 45110 Ioannina, Greece

Medicinal Chemistry Research
IC50 as low as 5.5 nM against leiomyosarcoma (LMS) cells
(Verginadis et al. 2011). Moreover, organotin(IV) deriva-
tives of L-cysteine show cytotoxic activity against six
human cancer cells lines: leukaemia (HL60), liver
(Bel7402), stomach (BGC823), cervix epithelial (HeLa),
nasopharyngeal (KB) and lung (PG) (Chasapis et al. 2004).
Hence, these compounds exert cytotoxic activity against
cisplatin-sensitive and cisplatin-resistant cell lines. In vivo
experiments in tumour bearing animals (LMS) with orga-
notin metal complexes prolonged the mean survival time
(MST) of the animals (Verginadis et al. 2011; Metsios et al.
2012) and inhibited tumour growth (Verginadis et al. 2011)
with minor side effects (mainly in the kidney) (Verginadis
et al. 2011; Metsios et al. 2012) or liver damage (Metsios
et al. 2012).
(2009) (Table 1). The molecular diagram of complexes 1
and 2 is presented in Fig. 2.
Cell proliferation assay
Cell viability was evaluated using MTT (3-(4, 5-dimethyl-
thiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Can-
cer cells (LMS, HeLa, MCF-7 and U2-OS) and normal cells
(MRC-5) were cultured at a density of 3 × 103 and 5 × 103,
respectively, in 96-well plates for 24 h. There were then
washed with PBS and a solution containing different con-
centrations of complexes 1–5 or cisplatin (diluted in
DMSO) was added. After incubation for another 48 h, 50 μl
of MTT were added in each well from a stock solution (5
mg/ml) and the cells were incubated for another 4 h at 37 °
C. The formed formazan crystals were solubilized with 50
μl of DMSO under gentle shaking and absorbance was
determined at 570 nm (subtract background absorbance
measured at 690 nm) using a microplate spectrophotometer
(Multiskan Spectrum, Therno Fisher Scientific, Waltham,
USA). Data were expressed as a percentage of control
measured in the absence of complexes (DMSO control). All
experiments were performed in triplicate.
The aim of this study was to examine the cytotoxic
effects of organotin compounds with ortho- and para-
hydroxybenzoic acid on malignant cell lines and to evaluate
their toxic effects as well as their antitumour properties in a
series of in vitro and in vivo experiments.
Materials and methods
Cell lines and culture conditions
Colony formation efficiency assay
Human cell lines MCF-7 (breast adenocarcinoma - ATCC®,
HTB-22™), HeLa (cervical cancer—ATCC®, CCL-2™),
U2-OS (human osteosarcoma—ATCC®, HTB-96™) and
MRC-5 (normal foetal lung fibroblasts - ATCC®, CCL-
171™) as well as cells (LMS) isolated from tumours his-
tological identified as LMS in Wistar rats (Avdikos et al.
2007) were cultured in Dulbecco’s modified Eagle’s med-
ium supplemented with 10% FBS, 1% penicillin/strepto-
mycine and 0.5% L-glutamine (1% L-glutamine for MRC-5
cells). Cells were maintained in culture dishes at 37 °C in a
5% CO2 and 95% air atmosphere incubator.
LMS, HeLa, MCF-7 and U2-OS cells were seeded into six-
well plates at a density of 6 × 104 cells per well and incu-
bated for 24 h. Cells were then washed with PBS, treated
with media containing various concentrations of complexes
1–5 in DMSO and incubated for 48 h. LMS cells were also
treated with cisplatin (0.5, 1 and 2.5 μΜ). Supernatants and
cells were collected and counted using Neubauer cytometer
and 5 × 102 cells were seeded onto six-well plates. After
7 days, dishes were fixed with methanol–acetic acid 3:1 at
−20 °C for 10 min, dried and stained with Giemsa dye 1:10
for 30 min. The dishes were then washed, dried and the
colonies were counted.
Synthesis of organotin compounds 1–5
Quantification of apoptosis
The Organotin(IV) complexes 1–5 were synthesised
according to the method described earlier (Abdellah et al.
2009). Thus, a suspension of the ligand (2-hydroxybenzoic
acid or 4-hydroxybenzoic acid) in distilled water was trea-
ted with an equimolar amount of KOH, and a clear solution
was immediately formed. A methanolic solution of di-
organotin(IV) or tri-organotin(IV) chloride was then added
to the above solution. The white precipitation formed is
filtered off, washed with water and it dried in vacuo over
silica gel. The reaction route for the preparation is shown in
Fig. 1. The analytical and spectroscopic data (m.p., ele-
mental analysis, FT-IR, 1H-NMR and ESI-MS) of 1–5 are
identical to those reported in reference Abdellah et al.
LMS cells were seeded into six-well plates at a density of
6 × 104 cells per well and incubated for 24 h before the
experiment. Cells were washed with PBS, treated with
media containing various concentrations of complexes 1–5
diluted in DMSO and cisplatin (IC50 value) and incubated
for 48 h. Supernatants and cells were collected, centrifuged
and cell pellets were suspended in calcium buffer 1× at a
rate 105 cells/100 μl. Cells were stained with 5 μl of
Annexin V-fluorescein isothiocynate and 4 μl of propidium
iodide in a dark room for 15 min. DNA content was
determined on a FACScan flow cytometer (Partec ML,
Partec GmbH, Germany).

Medicinal Chemistry Research
Fig. 1 Reaction route for the preparation of 1–5 (Abdellah et al. 2009)
Table 1 Characteristic vibration bands (cm−1) and 119Sn Mossbauer spectroscopic data for complexes 1–5 (Abdellah et al. 2009)
Compound
1
2
3
4
5
ν (OH)
ν (CH)
3467
2817–2705
1631, 1388
580, 536
446
3450
2928–2869
1628, 1419
562, 530
435
3450
3447
3063
1636, 1356
600, 533
420
3439
2957–2364
1633, 1352
564, 538
445
2958–2922
1613, 1351
606, 509
457
Infrared
Raman
ν as, ν (COO)
sy
ν (Sn–C)
ν (Sn–C)
ν (Sn–O)
ν (Sn–C)
δ (mm s−1
–
–
–
–
410
–
–
–
–
520
)
1.22
1.31
1.34
1.24
1.44
Δ (mm s−1
)
3.09
3.53
2.93
2.48
3.18
C–Sn–C (°) angles
130
143
Area (%)
56
29
100
–
100
–
100
–
δ (mm s−1
Δ (mm s−1
)
1.33
1.56
)
3.65
3.68
–
–
–
C–Sn–C (°) angles
148
149
Area (%)
44
71
–
–
–

Medicinal Chemistry Research
Fig. 2 Molecular diagram with
atomic numbering of a [Me2Sn
(o-BZA)2] (1) and b [(n-Bu)2Sn
(o-BZA)2] (2)
DNA fragmentation assay
The animals survived were sacrificed by ether on day 15.
The internal organs were removed en bloc and fixed in 10%
buffered formalin. Tissue sections of formalin-fixed,
DNA fragmentation was estimated as described by Vergi-
nadis et al. (Verginadis et al. 2011). Briefly, LMS cells were
treated with complexes 1–5 for 48 h. Supernatants and cells
were collected and incubated with lysis buffer. Fragmented
DNA was digested with RNAse and Proteinase K and
precipitated with isopropanol. Finally, DNA fragments were
fractionated by 1.2% agarose gel and were visualised by
ethidium bromide under ultraviolet light.
paraffin-embedded
tumours
were
stained
with
haematoxylin–eosin. The sections were examined under
light microscope.
Subacute toxicity
Subacute toxicity was studied in 30 female Wistar rats that
were divided into three groups (10 animals per group), and
received i.p. daily dosages of solvent (0.5 ml), 0.5 and 1 mg/
kg body wt of complex 1 for 4 weeks. Animals were
monitored for their behaviour once per day. Body weight
was recorded every 2 days and was used as an index of
toxicity, while the percentage change in body wt was cal-
culated as previously. All animals were sacrificed by ether
at the end of experiment. Immediate autopsy, fixation of the
organs in 10% buffered formalin and a histopathological
examination were performed as described previously (Ver-
ginadis et al. 2011).
Animals and treatment
Female Wistar rats, aged 3–4 months and weighting 242
13 g, were reared at controlled room temperature (20 2 °
C), with periodical lighting (12 h light/12 h dark). Standard
Wistar rat diet and water ad libitum were used in all the
experiments. Experiments on animals were handled with
human care in accordance with the European Union direc-
tive for the care and the use of laboratory animals (EEC
Directive 2010/63/EU) and according to the permission
number 20EEP02.
Antitumour activity in vivo
Acute toxicity
Twenty-nine female Wistar rats were inoculated with 3 ×
106 LMS cells/1 ml Hank’s balanced salt solution on the
upper scapula after anaesthetisation (mixture of anaesthesia
was composed of ketamine 10% (Imalgene), midazolame
40% (Roche) and saline solution 0.9, 50% to a total volume
of 0.5 ml per animal). After the appearance of a palpable
tumour mass (smaller than 1.5 cm diameter), the inoculated
animals were divided into two groups: the control group
(CG, consisted of 15 animals) and the treatment group (TG,
consisted of 14 animals). All animals in TG Group were
treated daily till death with 1 mg/kg body wt of complex 1
(diluted in 0.5 ml PBS/DMSO 7:1), while the animals of the
CG received daily 0.5 ml PBS/DMSO(7:1). The animals
were observed for their behaviour once a day. After the
animals’ death, an autopsy, fixation of the organs in 10%
Forty female Wistar rats were divided into four groups (ten
animals per group) and injected intra-peritoneally (i.p.)
with a single dose of 1, 10 and 100 mg/kg body wt of
complex 1 in order to achieve a logarithmic increase of the
dose. Control group was injected with a similar volume of
the solvent (1 ml of PBS/DMSO (7:1)). All animals were
starved for 3–4 h before treatment. Twenty minutes after
the administration, the rats were allowed to access freely
food and water. The animals were observed for their
behaviour twice a day. Body weight (body wt) was recor-
ded every 2 days and used as an index of toxicity. The
percentage change in body wt was calculated as: Body wt
(%) = [(mean body wt on day D − mean body wt on day
1)/mean body wt on day 1] × 100, where D = day of the
experiment.
formaldehyde and
a
histological examination were

Medicinal Chemistry Research
performed. For both groups, the MST of the animals,
tumour weight inhibition (TWI%), mean tumour weight and
mean tumour growth rate (MTGR) were calculated. The
tumour growth rate for each animal was calculated by the
following equation: Tumour growth rate (g/d) = tumour
weight (g)/survival time (days), while TWI% was calculated
as: TWI% = [(mean control tumour weight-mean treatment
tumour weight)/ mean control tumour weight] × 100.
Furthermore, the cytotoxic activities of complexes 1–5
and cisplatin was studied in LMS cells. Complex 1 pre-
sented the lower IC50 (31 nM) followed by complex 2 (43
nM) and 4 (45 nM). The IC50 values for complexes 3 and 5
were higher than the other 3 complexes but still extremely
lower compared to cisplatin. Trypan blue assay was pre-
viously performed by Abdellah et al. (2009) in order to
determine the cell membrane integrity. The complexes have
high activity against LMS cells while complex 4 was the
most active one. Except from complex 5 the rest of the
complexes exhibited no inhibitory activity on growth pro-
liferation of MRC-5 cells (IC50 concentration of complexes
against LMS cells) (Table 3).
Statistical analysis
The data are expressed as mean S.D., and the difference
between two groups was evaluated using Student’s t-test
and two-way analysis of variance (SPSS version 17.0,
Statistical Package for the social Sciences software, SPSS,
Chicago, USA). A probability level of p < 0.05 was used to
establish significance.
Colony formation efficiency after treatment
The cells were tested for their ability to form colonies after
drug treatment compared to untreated cells. Complexes 1
and 4 presented a dose-dependent inhibition of MCF-7,
HeLa and U2-OS cells colony formation. All human cancer
cell lines treated with dosages higher than 20 nM of com-
plex 2 lost their ability to form colonies (Fig. 3)
Results
Cytotoxicity
After treatment with complexes 1 and 2, LMS cells
gradually lost their ability to form colonies. Complex 2 at
the highest dose (100 nM) completely abolished the for-
mation of colonies while LMS cells resisted treatment with
complex 4 and were able to form colonies even at the
highest concentration of the complex (Fig. 4a). Complexes
3 and 5 failed to inhibit colony formation at concentrations
lower than 100 nM (results not shown). On the other hand,
cisplatin also exerted a dose-dependent inhibition of colony
formation of LMS cells but at significantly higher doses
than complexes 1, 2 and 4 (Fig. 4b).
Complexes 1–5 were tested for their in vitro cytotoxic
activity against all cell lines with the MTT assay. The IC50
values for cell growth proliferation after 48 h of incubation
were calculated. Cisplatin was used as the reference treat-
ment. Moreover, the survival of MRC-5 cells, at the IC50
values of the complexes against the LMS cells, was also
calculated.
All complexes showed potent cytotoxic activity against
the three human cells lines ranging from 108 to 724 nM for
the MCF-7 cells, 21 to 295 nM for the HeLa cells and 97 to
820 nM for the U2-OS cells. The IC50 value of complex 2
was 304 and 211 times lower than the corresponding value
of cisplatin for the MCF-7 and U2-OS cells, respectively
while the IC50 value of complex 4 was 264 times lower for
the HeLa cells. Among the tested compounds, complexes 3
and 5 presented the weaker cytotoxic activity (Table 2).
Flow cytometry and DNA fragmentation
Flow cytometry assay was used to quantify apoptotic or
necrotic death in LMS cells treated with compounds 1–5 or
Table 3 Cytotoxic activity of complexes 1–5 and cisplatin against
LMS cells after 48-h incubation (IC50 values) and survival of MRC-5
cells
Table 2 IC50 values for cell viability calculated for complexes 1–5 and
cisplatin against MCF-7 (breast cancer), HeLa (cervical cancer) and
U2-OS cells (osteosarcoma)
IC50 (nM)
Complex
LMS
MTT
MRC-5
Trypan Bluea % Survival (LMS
IC50 value)
IC50 (nM)
Compound
MCF-7
HeLa
U2-OS
1
31 1.6
>2000
150
97.5 0.15
104.1 0.2
98.1 0.1
94.5 0.2
78.4 0.2
1
142 4.3
108 2.6
724 5.4
121 3.7
325 2.3
32850 2390
84 3.2
130 2.3
97 2.1
2
43 1.5
2
37 1.2
3
738 2.4
45 1.8
150
3
295 3.8
21 1.1
820 5.7
258 3.2
535 4.6
20510 1250
4
5–10
30–40
5000
4
5
134 2.8
5480 530
5
120 2.9
5540 680
Cisplatin
Cisplatin
aAbdellah et al. (2009)

Medicinal Chemistry Research
Fig. 3 Formation of colonies for a MCF-7, b HeLa and c U2-OS cells after treatment with the complexes 1, 2 and 4. Different letters indicate the
significant differences (p < 0.05) among the current and the exact previous (lower) dose
i.p. administration of complex 1, while 40% of the
animals that received 10 mg/kg body wt died within 24 h.
None of the animals from the other two groups
(control and 1 mg/kg body wt) died during the experiment
(Table 4).
All animals that received 1 mg/kg body wt of complex 1
did not show any signs of toxicity. The absence of toxicity
was verified by the histologic examination. Animals injec-
ted with 10 mg/kg body wt of complex 1 showed reduced
mobility and difficulty in breathing during the first 24 h.
Animals presented intent seizures right after the adminis-
tration of the complex 1 at the highest dose (100 mg/kg
Fig. 4 Formation of colonies for LMS cells after treatment with a the
complexes 1, 2 and 4 and b cisplatin. Different letters indicate the
body wt). The main organs targeted were liver, kidneys and
lungs. The diseased animals showed cerebral oedema as
well (Fig. 6).
significant differences (p < 0.05) among the current and the exact
previous (lower) dose
the IC50 of cisplatin (5.5 μΜ). Compared to untreated LMS
cells that showed a total of 6.2 1.3 and 1.7 0.5%
apoptotic and necrotic cell death, respectively, the cells
treated with complex 1 at 50 nM showed 22.4 1.8%
apoptosis and 2.1 0.2% necrosis. At higher concentrations
(100 nM), the cells showed 21.1 2.8% apoptosis and 1.6
0.6% necrosis. As for complexes 2 and 4, cells showed
33.8 2.3 and 13.7 2.6 apoptosis and 1.8 0.1 and 2.2
1.4% necrosis after treatment with 50 nM of the complexes,
respectively and 38.7 2.2 and 28.9 1.6% apoptosis and
7.5 0.9 and 2.8 1.2% necrosis after treatment with 100
nM. Complexes 3 and 5 induced apoptosis of LMS cells at
higher concentrations (>300 nM) (results not shown).
Apoptosis induced by cisplatin (IC50 value) was estimated
at 35.0 3.0% and necrosis at 3.3 1.6% (Fig. 5a, b).
The characteristic laddering pattern observed at the
electrophoresis gel after treatment of LMS cells with 150
and 100 nM of complexes 1 and 2 and 4, respectively,
indicated that cells underwent apoptosis triggered by the
DNA damage and fragmentation (Fig. 5c).
Subacute toxicity
None of the animals presented any signs of toxicity. Mild
kidney damage was only observed at animals treated daily
with the highest dose (1 mg/kg body wt of complex 1)
(results not shown).
Antitumour activity
Daily administration of tumour bearing Wistar rats with 1
mg/kg with complex 1 until death prolonged the MST
(120% increase) and reduced the MTGR by more than 3×
fold and with minimal side effects (Table 5). Furthermore
tumour weight inhibition was calculated as TWI% =
60.2%. Long terms survival was noted for LTS = 1/15. This
animal belonged in TG group and was excluded from the
above calculations.
It is worth mentioning that total tumour regression
was observed in one animal treated with complex 1. The
histopathological examination showed an abscess cavity at
the site of the inoculation. This animal was the only one
with an observed lung metastasis, which was resistant to
treatment.
Acute toxicity
Ninety percent of the animals at the highest dosage
group (100 mg/kg body wt) died within an hour after the

Medicinal Chemistry Research
Fig. 5 Quantification of apoptotic and necrotic cells after treatment with 50 and 100 nM of complex 1, 2 and 4 and cisplatin (CP, 5.5 μΜ) for 48 h.
*Statistically significant from untreated cells, p < 0.05. Data are presented as mean SD
Fig. 6 Acute toxicity report. Healthy samples from the control group: a
liver, b lung, c kidney and d brain stem and representative histological
images from animals that received 100 mg/kg body wt of complex 1: e
liver, f lung, g kidney and h brain stem. Tissue sections were stained
with Hematoxylin–eosin. Liver and lung sections were magnified by
×200 while kidney and brain stem sections by ×400
Table 4 Toxic effects (acute toxicity) induced by complex 1
Group
Kidney
Liver
Lung
Brain stem
ΔBody wt (%)
Death (%)
Control
(−)
(−)
(−)
(−)
(+)
(+)
(−)
(−)
(−)
(+)
+2.01 0.10
+1.37 0.32
−2.40 0.23
−0.10 0.00
0
1 mg/kg body wt
10 mg/kg body wt
100 mg/kg body wt
(−)
(−)
0
Oedema
(+)
Oedema
(+)
40
90
Body wt (%) = [(mean body wt on day D − mean body wt on day 1)/mean body wt on day 1] × 100, where D = day of the experiment
(−) absent, (+) mild histological alterations
Discussion
with the corresponding IC50 value of the complex being
significantly lower than that of cisplatin (41 nM versus 25
μM) (Verginadis et al. 2011). Di-n-butyltin(IV) compounds
related to salicylic acid were found to be 4–10×-fold more
effective than cisplatin against A204, MCF-7 and T24
human tumour cells (Ross et al. 1991). This is in accordance
with our results where all complexes exerted higher cyto-
toxic activity than cisplatin against MCF-7, HeLa, U2-OS
and LMS cells.
Ever since cisplatin was introduced in cancer therapy, many
non-conventional platinum compounds and non-platinum
metal complexes were designed and tested. Among them,
organotin derivatives have shown potent cytotoxic and
antitumour properties (Arjmand et al. 2014).
Verginadis et al. showed that a triorganotin complex with
2-mercapto-nicotinic acid inhibits LMS cell proliferation

Medicinal Chemistry Research
Table 5 Mean survival time and mean tumour growth rate of tumour
bearing Wistar rats
metabolic acidosis (Matsuno-Yagi and Hatefi 1993). This is
a very likely explanation considering that all animals pre-
sented Kussmaul type of breath before death. Similar find-
ings were reported after i.p. administration of triphenyltin 2-
(triphenyltinmercapto) nicotinate with kidney damage being
more severe reaching total tubular necrosis (Verginadis
et al. 2011). A simple i.p. administration of the complex
at lower doses (10 mg/kg BW) showed toxic effects and
caused death to the 40% of the animals within 24 h.
Repeated administration of the complex at very low doses
(1 mg/kg/day) for 30 days caused only slight kidney
damage. The same dosage was used to study the antitumour
effects of the complex.
Control group
Treatment group
(N = 15)
(N = 14)
MST (days)
20.7 5.5
108 25
45.1 5.9*
72 9*
Mean tumour
weight (g)
MTGR (g/day)
5.2 0.2
1.6 0.1*
MST mean survival time, MTGR mean tumour growth rate
*Statistically significant from the control group, p < 0.05. Data are
presented as mean SD
Flow cytometry revealed that complexes 1, 2 and 4
caused LMS cell death through apoptosis and inhibited
human cancer cells and LMS cell’s ability to form colonies.
Moreover, electrophoresis of the LMS cell’s DNA showed
that apoptosis was triggered by the DNA damage and
fragmentation after incubation with complexes 1, 2 and 4.
Such DNA damage is shown for several organotin deriva-
tives for instance tributiltin (Liu et al. 2006). The inability
of complexes 3 and 5 to induce apoptosis at similar, low
doses as complexes 1, 2, and 4 a well as to inhibit colony
formation could be possibly attributed to an alternative
apoptotic mechanism and specifically to the rise of intra-
cellular Ca2+ that would lead the cell to apoptosis (Nakatsu
et al. 2007; Reader et al. 1999; Viviani et al. 1995) but
would also allow the cells to develop after the complex was
removed from the culture media. This is only a hypothesis
that would need additional experiments to be verified
although the relative lower cytotoxic activity of complexes
3 and 5 compared to 1, 2, and 4 suggest that they should be
excluded from further research. Apoptosis seems to be the
main type of cell death caused by many organotin deriva-
tives even though the path that leads to apoptosis may differ
between different complexes (Nakatsu et al. 2007; Grondin
et al. 2007; Tada-Oikawa et al. 2008). Diorganotin(IV) and
triorganotin(IV) complexes of meso-tetra(4-sulfonatophe-
nyl) porphine induce apoptosis in A375 human melanoma
cells via both death receptor-mediated and mitochondrial
apoptotic pathways (Amir et al. 2014), while triphenyl tin
benzimidazolethiol induces mitochondrial-mediated apop-
tosis in human cervical cancer cells (Hoti et al. 2003). On
the other hand, trimethyl tin seems to cause both apoptotic
and necrotic death in human hepatoma G2 cells (Cai et al.
2009).
Tumour-bearing animals treated with complex 1 showed
prolongation of survival time and tumour growth inhibition.
Other organotin derivatives have higher TWI% values
(Verginadis et al. 2011), while oral administration of trie-
thyltin(IV)-lupinylsulfide hydrochloride showed as high as
90% tumour volume inhibition (Muhammad and Guo
2014). Not all organotin complexes are active in vivo.
Organotin complexes with salicylic acid derivatives are
inactive against L1210 leukaemia in mice (Gielen et al.
1993), while triphenyltin 5-sulfosalicylate and triphenyltin
5-aminosalicylate offer no protection against murine colon
carcinoma colon 26 (Gielen 1995). Both treated and
untreated animals showed slight liver damage and minor
lung oedema, which could be attributed to the tumour, but
only treated animals showed kidney damage, as expected by
the subacute toxicity results. It is noteworthy to mention
that one of the treated animals showed total tumour
regression at the site of the inoculation. Unfortunately, this
animal was the only one presenting lung metastasis. The
lung metastasis presented to this animal was resistant to
treatment and showed no signs of necrosis during the his-
tological evaluation.
Conclusions
In this study, we evaluated the cytotoxic and antitumour
properties of five organotin complexes (1–5) with o-
hydroxy-benzoic or p-hydroxy-benzoic acids. Amongst
them, complex 1 presented the highest cytotoxic activity
against LMS cells (IC50 = 31 1.6 nM), complex 2 against
MCF-7 and U2-OS cells (IC50 = 108 2.6 nM and 97
2.1 nM, respectively) and complex 4 against HeLa cells
(IC50 = 21 1.1 nM). The induction of apoptosis was
identified as the mechanism of LMS cell death. Complexes
1 and 2 showed a higher inhibitory effect on LMS colony
formation efficiency compared to complex 4. The following
acute toxicity study revealed a mildly toxic effect of com-
plex 1 at a dose of 10 mg/kg body weight in the liver,
kidney and lungs of adult Wistar rats. Tumour-bearing
Organotin derivatives are known to cause neurotoxicity
and bile duct damage (Boyer 1989; Winship 1988). Com-
plex 1 caused the death of 90% of the animals after a single
dose of 100 mg/kg within an hour of the i.p. administration.
Since the main pathological findings (brain stem and lung
oedema and mild liver and kidney damage) were not cap-
able to cause the animal’s death, a possible cause was

Medicinal Chemistry Research
Wistar rats administrated daily with 1 mg/kg body weight of
complex 1 lived longer than the control rats (45.1 versus
20.7 days) probably due to the deceleration of tumour
growth rate (1.6 versus 5.2 g/day) caused by the complex.
In short, our results demonstrated that complex 1 exerted
potent cytotoxic activity, tumour growth retardation and
prolongation of MST in tumour-bearing (LMS) Wistar rats.
Thus, it could be used as a potential candidate for cancer
treatment at primary site of the tumour. Further investiga-
tion is needed to confirm the antitumour properties of the
other organotin complexes.
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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