Radiol Oncol 2017; 51(3): 295-306. doi:10.1515/raon-2017-0034
295
research article
In vitro and in vivo evaluation of 
electrochemotherapy with trans-platinum 
analogue trans-[PtCl2(3-Hmpy)2] 
Simona Kranjc1, Maja Cemazar1,2, Gregor Sersa1,3, Janez Scancar4, Sabina Grabner5
1 Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia
2 University of Primorska, Faculty of Health Sciences, Izola, Slovenia
3 Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia
4 Department of Environmental Sciences, Jozef Stefan Institute, Ljubljana, Slovenia
5 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
Radiol Oncol 2017; 51(3): 295-306.
Received 20 July 2017 
Accepted 4 August 2017
Correspondence to: Simona Kranjc, Ph.D., Department of Experimental Oncology, Institute of Oncology Ljubljana,  
Zaloška 2, SI-1000 Ljubljana, Slovenia. Phone/Fax: +386 1 5879 434; E-mail: skranjc@onko-i.si
Disclosure: No potential conflicts of interest were disclosed.
Background. Cisplatin is used in cancer therapy, but its side effects and acquired resistance to cisplatin have led 
to the synthesis and evaluation of new platinum compounds. Recently, the synthesized platinum compound trans-
[PtCl2(3-Hmpy)2] (3-Hmpy = 3-hydroxymethylpyridine) (compound 2) showed a considerable cytotoxic and antitu-
mour effectiveness. To improve compound 2 cytotoxicity in vitro and antitumour effectiveness in vivo, electroporation 
was used as drug delivery approach to increase membrane permeability (electrochemotherapy). 
Materials and methods. In vitro, survival of sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensi-
tive, TBLCl2Pt resistant and SA-1 moderately sensitive) was determined using a clonogenic assay after treatment with 
compound 2 or cisplatin electrochemotherapy. In vivo, the antitumour effectiveness of electrochemotherapy with 
compound 2 or cisplatin was evaluated using a tumour growth delay assay. In addition, platinum in the serum, tumours 
and platinum bound to the DNA in the cells were performed using inductively coupled plasma mass spectrometry.
Results. In vitro, cell survival after treatment with compound 2 electrochemotherapy was significantly decreased 
in all tested sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensitive, TBLCl2Pt resistant and SA-1 
moderately sensitive). However, this effect was less pronounced compared to cisplatin. Interestingly, the enhancement 
factor (5-fold) of compound 2 cytotoxicity was equal in cisplatin-sensitive TBLCl2 and cisplatin-resistant TBLCl2Pt cells. In 
vivo, the growth delay of subcutaneous tumours after treatment with compound 2 electrochemotherapy was lower 
compared to cisplatin. The highest antitumour effectiveness after cisplatin or compound 2 electrochemotherapy was 
obtained in TBLCl2 tumours, resulting in 67% and 11% of tumour cures, respectively. Compound 2 induced significantly 
smaller loss of animal body weight compared to cisplatin. Furthermore, platinum amounts in tumours after compound 
2 or cisplatin electrochemotherapy were approximately 2-fold higher compared to the drug treatment only, and the 
same increase of platinum bound to DNA was observed. 
Conclusions. The obtained results in vitro and in vivo suggest compound 2 as a potential antitumour agent in elec-
trochemotherapy.
Key words: platinum analogue; cisplatin; 3-Hmpy; electroporation; electrochemotherapy; mouse sarcoma
Introduction
Cisplatin is used for the treatment of variable 
types of cancers (testicular, bladder, ovarian, en-
dometrial, cervical, lung, head and neck), and 
primarily systemically used as single drug or in 
combination with other drugs and/or in combina-
tion with radiation therapy, immunotherapy or 
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue296
other targeted therapies.1-3 Despite many efforts 
in adapting an appropriate therapy schedule with 
cisplatin, the treatment of patients with cisplatin 
induces side effects, and cancer cells can acquire 
resistance to cisplatin through different mecha-
nisms. Among these mechanisms, the inactivation 
of cisplatin via thiol-containing molecules, such as 
glutathione and metallothionein, increased the re-
pair of cisplatin-DNA adducts, enhanced tolerance 
to cisplatin-DNA adducts, resulting in the failure 
of apoptotic pathways and reduction of platinum 
accumulation through decreased drug uptake or 
increased drug efflux.4-8 Therefore, overcoming 
side effects and acquired resistance of cells to cis-
platin during the treatment of patients with cis-
platin still remains the main challenge in studies 
of platinum analogues. Modification of platinum 
compounds with variable ligands (iminoethers, 
aliphatic amines, amine piperazine and pyridine) 
in the trans position of ligands resulted in variable 
cytotoxic effects.9-17 Pyridine ligands with hydroxyl 
groups, which participate in the formation of stable 
complex between DNA and platinum compounds, 
were demonstrated as promising ligands in activat-
ing platinum (II) compound cytotoxicity.9,11,14,18-20 
Recently, we reported that two 3-hydroxymethyl-
pyridine (3-Hmpy) ligand in the trans position in 
the platinum (II) complex increased cytotoxicity in 
vitro and antitumour effectiveness in vivo.21 
The effectiveness of chemotherapeutic drugs re-
lies on their uptake into tumour cells. For poorly 
permeable drugs, higher doses are needed for ef-
fective cytotoxicity to tumour cells, consequently 
producing higher side effects. Cisplatin is a poorly 
permeable drug; therefore, many approaches have 
been examined to increase its uptake, i.e., using na-
noparticles, magnetic particles, liposomes, and tu-
mour-specific antibodies and chemically or physi-
cally inducing pores in the cell membrane.16,22-23 
Among physical approaches, electroporation, 
inducing pores in the cell membrane via electric 
pulses, could be used.24 Electroporation increases 
the cytotoxicity up to 80-fold in cisplatin-sensitive 
and cisplatin-resistant tumour models, in vitro and 
in vivo.25-29 The treatment of tumour nodules with 
the local application of electric pulses after sys-
temic or local administration of drug is referred 
to as electrochemotherapy. Electrochemotherapy 
with cisplatin is currently used for the treatment of 
variable cutaneous and subcutaneous tumours in 
human and veterinary clinics, showing a local ef-
fectiveness of up to 80% of local tumour control.30-33 
The antitumour effectiveness of electrochemother-
apy primarily relies on increased drug uptake and 
platinum binding to DNA.25,29,34 However, some ef-
fects might be attributed to immune system modu-
lation and changes in tumour blood flow.27,35-36 
Thus far, electroporation has been used to in-
crease antitumour effectiveness of platinum (II) 
analogue 3P-SK containing squarato (skv = 3,4-di-
oxocyclobut-1-ene-1,2-diolate) and 3-Hmpy li-
gands (3P-SK, [Pt(3-Hmpy)2(skv)]). Indeed, 3P-SK 
electrochemotherapy treatment had a profound 
antitumour effectiveness in mouse MCA mam-
mary carcinoma, demonstrating 3P-SK as the first 
biologically active squarato compound with two 
3-Hmpy ligands in vivo.19
The promising cytotoxic and antitumour effec-
tiveness of compound 2 from a previous study21 
encouraged us to assess the electroporation, as 
drug delivery method, for compound 2 in in vitro 
and in vivo tumour models with different intrinsic 
sensitivities to cisplatin. In vitro, clonogenic assays 
were performed to demonstrate the cytotoxicity of 
compound 2. The cytotoxicity of compound 2 alone 
or in combination with electroporation was inves-
tigated in sarcoma cisplatin-sensitive TBLCl2 cells 
and the cisplatin-resistant subclone TBLCl2Pt, and 
in sarcoma SA-1 cells with moderate sensitivity to 
cisplatin, based on in vitro data.25,37 Furthermore, 
the antitumour effectiveness of compound 2 elec-
trochemotherapy using tumour growth delay as-
say was determined in the same sarcoma tumour 
models in vivo. In addition, the animal body weight 
loss was monitored to estimate the influence of the 
intratumoural administration of compound 2 and 
local application of electric pulses to the tumour on 
animal wellbeing. To clarify the underlying mech-
anism of antitumour effectiveness of compound 2 
electrochemotherapy, the amount of platinum in 
serum and the platinum uptake into tumours, and 
platinum binding to DNA were measured.
Materials and methods
Drugs
Cisplatin (CDDP, Cysplatyl, Aventis Laboratory, 
Paris, France) was dissolved in sterile water at 
concentration of 10 mM and frozen in aliquots at 
-20˚C until further use. The compound 2 (trans-
[PtCl2(3-Hmpy)2] was synthesized at the Faculty of 
Chemistry and Chemical Technology, University 
of Ljubljana (Ljubljana, Slovenia)21 and dissolved 
in N,N-dimethyl-formamide (DMF, Sigma-Aldrich 
Co. LCC, St. Louis, MO) at a concentration of 10 
mM and frozen in aliquots at -20°C until further 
use. 
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue 297
In the in vitro experiments, concentrations of 
CDDP and compound 2 ranging from 1.7 µM to 
1333 µM were prepared in a sterile electroporation 
buffer (EP buffer, 125 mM sucrose, 10 mM K2HPO4, 
2.5 mM KH2PO4, and 2 mM MgCl2 x 6 H2O) imme-
diately prior to the experiments. 
In the in vivo experiments, fresh solutions of CDDP 
and compound 2 were prepared in sterile NaCl solu-
tion (0.9%) at a final concentration of 13.3 mmol/kg.
Tumour cell lines 
Mouse SA-1 sarcoma cells (Jackson Laboratory, 
Bar Harbor, ME, USA), mouse TBLCl2 sarcoma 
cells and the cisplatin-resistant subclone TBLCl2Pt 
(generously provided by J. Belehradek of the 
Institute Gustave Roussy, Villejuif, France)38 were 
used for the in vitro experiments. The SA-1, TBLCl2 
and TBLCl2Pt cells were cultured in a humidi-
fied incubator at 37°C and 5% CO2 in advanced 
minimum essential media (AMEM, Gibco, Life 
Technologies Corporation, Grand Island, NY, 
USA), supplemented with 5% of foetal bovine se-
rum (FBS, Gibco). Two days after sub-culturing, 
the cells in the exponential phase of growth were 
obtained and used for the cytotoxicity experiments 
or for the induction of subcutaneous tumours.
Animals and tumour models
Animal experiments were conducted in accord-
ance with the Veterinary Administration of the 
Republic of Slovenia (permission No: 34401-
10/2009/6). Inbreed A/J and C57Bl/6 mice, females 
10-12 weeks old, were purchased from the Institute 
of Pathology, Medical Faculty, University of 
Ljubljana (Ljubljana, Slovenia) and acclimated to 
the facility for 10 days. The mice were maintained 
under specific pathogen-free conditions, at a con-
stant room temperature in a 12-hour day/night 
light cycle. Food and water were provided ad libi-
tum. Subcutaneous tumours were induced via the 
injection of 100 µL of cell suspension in the right 
shaved flank of mice; injection of SA-1 sarcoma 
tumour cell suspension (5×106 cells/mL; obtained 
from ascites of the donor animal) in A/J mice; injec-
tion of TBLCl2 sarcoma (25×106 cells/mL; obtained 
in vitro) and TBLCl2Pt cell suspension (40×106 cells/
mL; obtained in vitro) in C57Bl/6 mice.
Cytotoxicity of electrochemotherapy in vitro
In the experiments, variable groups were used: 
control (untreated cells), CDDP or compound 
2 (5 minutes incubation of cells with drug), elec-
troporation (EP, cells exposed to application of 
electric pulses), electrochemotherapy (ECT, cells 
exposed to combination of EP and drug, CDDP 
or compound 2). Briefly, cell suspensions of SA-1, 
TBLCl2 and TBLCl2Pt sarcoma cells in EP buffer 
at concentration 2.2×107 cells/mL were prepared via 
the trypsinization (Trypsin, Gibco) of two day-old 
monolayers. The cytotoxicity of treatments was de-
termined using a clonogenic assay. In the EP group, 
5 µL of EP buffer was added to 45 µL (1×106) of cell 
suspension and subsequently placed between two 
parallel stainless-steel plate electrodes at a 2-mm 
distance and thereafter exposed to 8 electric pulses 
(electric field over distance ratio of 1300 V/cm, du-
ration time of each pulse 100 µs, at frequency of 
1 Hz; generated at Jouan GHT 1287, St. Herblain, 
France).26,27 In CDDP or compound 2 groups, 45 µL 
(1×106) of cell suspension was diluted with 5 µL of 
drug solution at a ten times higher final concentra-
tion (1.7 µM-1333 µM). In the ECT group, 45 µL 
(1×106) of cell suspension was diluted with 5 µL of 
drug solution at a ten times higher final concentra-
tion (1.7 µM-1333 µM) and immediately exposed 
to electric pulses. Regardless of the treatment, the 
cells were incubated for 5 minutes at room tem-
perature (22°C) in an ultra-low attachment 24-well 
plate. Subsequently, the cells (50 µL) were diluted 
in 1 mL of fresh medium and a variable number of 
cells were seeded onto a Petri dish (diameter = 6 
cm; 200 to 800 cells/Petri dish) for clonogenic assay. 
In 7-14 days, viable clonogenic cells formed colo-
nies that were stained with crystal violet (0.005%, 
Sigma, St. Louis, USA), and only the colonies con-
taining at least 50 cells were counted. The experi-
ment was repeated three times, and each group 
comprised three replicates.
Electrochemotherapy of tumours in vivo
The antitumour effectiveness of electrochemo-
therapy was evaluated using a tumour growth 
delay assay. When subcutaneous tumours grew to 
a volume of 40 mm3 in approximately 7-14 days, 
the animals were divided randomly in experimen-
tal groups: control (untreated tumours), CDDP or 
compound 2 group (intratumoural injection of 50 
µL drug solution, CDDP or compound 2), EP (lo-
cal application of 8 electric pulses in two perpen-
dicular directions (4+4), with electric field over dis-
tance ratio of 1300 V/cm, 100 µs long, 1 Hz, using 
two plate parallel stainless-steel electrodes 7 mm 
apart placed percutaneously at the opposite mar-
gins of tumour; generated by Jouan GHT 1287, 
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue298
St. Herblain, France) and combined treatment, 
electrochemotherapy, i.e., 60 seconds after intra-
tumoural injection of 50 µL drug solution electric 
pulses were applied. Each experimental group 
comprised 6 to 12 animals. The antitumour effec-
tiveness of therapies was followed by measuring 
three orthogonal tumour diameters (a, b, c) every 
other day after therapy using a Vernier calliper and 
the tumour volume was subsequently calculated 
using the formula a×b×c×π/6. The tumour dou-
bling time for each experimental group based on, 
tumour growth delay (the tumour doubling time 
for each experimental group subtracted from the 
tumour growth delay of control group), was de-
termined as the endpoint of antitumour effective-
ness of electrochemotherapy with cisplatin or com-
pound 2. Growth of treated tumours was followed 
up to a volume of 350 mm3 or in the case of com-
pletely regressed tumours, up to 100 days after the 
treatment (complete response, CR). At these times, 
the animals were humanely sacrificed via cervical 
dislocation. Potential side effects of therapies were 
determined after weighing the animals and assess-
ing behaviour and locomotion.
Platinum determination in tumour and 
serum
Measurements of platinum in the serum, tumours 
and platinum bound to the DNA in the cells were 
performed using inductively coupled plasma mass 
spectrometry (ICP-MS, Agilent Technologies, mod-
el 7700x, Tokyo, Japan), which monitored the 195Pt 
isotope. To determine platinum accumulation in 
the serum at different time points after treatment 
(3 minutes, 1 hour, 8 and 24 hours), the blood was 
collected using a glass capillary from intraorbital 
sinus (8 samples per group) and coagulated at 
room temperature for two hours. Subsequently, 
the blood was centrifuged at 3000 rpm for 10 
minutes, and the serum was collected and stored 
at -20°C. Furthermore, the total volume of serum 
samples was digested in 1 mL of 1:1 mixture of 65% 
nitric acid (MERCK KgaA, Darmstadt, Germany) 
and 30% hydrogen peroxide (MERCK KgaA, 
Darmstadt, Germany) at 90°C for 48 hours. The 
obtained clear solutions were diluted with Milli-Q 
water and analysed using ICP-MS. 
To obtain tumour samples for platinum determi-
nation, the animals were humanely sacrificed im-
mediately after the blood was collected. Tumours 
were excised (8 samples per group), removed from 
the overlying skin, weighed and stored at -20°C. 
Subsequently, the tumours were digested in 2 mL 
of 1:1 mixture of 65% nitric and 30% hydrogen per-
oxide) at 90°C for 48 hours, diluted with Milli-Q 
water and analysed using ICP-MS.
For measurements of platinum binding to the 
DNA in the tumour cells, the tumours were ob-
tained as described above. However, the tumours 
were weighed, immediately mechanically de-
graded and washed with 3 mL of freshly prepared 
PBS. Collected samples were filtered through a 
cell strainer with a pore size of 40 µm (Corning 
Incorporated, Life Sciences, Durham, USA) and cen-
trifuged at 1500 rpm for 10 minutes. The collected 
cells were used for the fast DNA isolation using a 
salting-out protocol. First, the cells were lysed with 
lysis buffer (10 mM Tris-HCl, 1 mM EDTA, and 1% 
SDS) containing proteinase K (0.2 µg/mL) at 55°C 
and constant shaking for 30 minutes. The proteins 
in the cooled samples were precipitated using 120 
µL of 4 M NaCl and shaking for 15 seconds. The 
precipitated proteins were two times centrifuged 
at 13000 rpm for 6 minutes. DNA was precipitated 
from the obtained supernatant using 1 mL of etha-
nol (70%) for 2 minutes by gentle mixing of tube 
and centrifugation at 13000 rpm for 2 minutes. The 
precipitated DNA was washed with an additional 
1 mL of ethanol (70%) and centrifuged at 13000 
rpm for 2 minutes. The pellet of DNA was dried, 
dissolved in 100 µL of distilled water. The concen-
tration of DNA in the samples was spectrophoto-
metrically determined at 260 nm (Epoch, Take3, Bio 
Tek Germany). Finally, the samples of DNA were 
digested using the same procedure described above 
and the platinum binding to DNA was determined 
in the diluted samples using ICP-MS. 
Statistical analysis
Statistical analysis was performed using SigmaPlot 
software (Systat Software, Chicago, IL, USA). The 
Shapiro-Wilk test was used to determine the nor-
mality of data distribution. The differences between 
mean values of experimental groups were tested 
using the t-test or one-way ANOVA, followed by 
the Holm-Sidak test for multiple comparisons. 
Values of p < 0.05 were considered significant.
Results and discussion
Recently, discovered biological activity of trans 
platinum complexes with planar aromatic N-donor 
base21, encouraged us to examine compound 2 with 
electroporation (electrochemotherapy) as a drug 
delivery approach in tumour cells with different 
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Kranjc S et al. / Electrochemotherapy with trans-platinum analogue 299
intrinsic sensitivities to cisplatin in vitro and tu-
mour models in vivo. In vitro, compound 2 electro-
chemotherapy was effective in cisplatin-sensitive 
and cisplatin-resistant cells. However, its cytotoxic 
effect (at the IC50 level) was less evident compared 
to cisplatin electrochemotherapy, with up to 4-fold 
in cisplatin-sensitive TBLCl2 cells, up to 1.6-fold in 
moderately cisplatin-sensitive SA-1 cells and up to 
15-fold in cisplatin-resistant TBLCl2Pt cells. Similar 
to in vitro data, growth of tumours with variable in-
trinsic sensitivity to cisplatin (sensitive, moderate-
ly sensitive and resistant) after compound 2 elec-
trochemotherapy was less delayed (up to 9.7 days) 
compared to cisplatin electrochemotherapy. As 
expected, the most evident antitumour effective-
ness of compound 2 and cisplatin electrochemo-
therapy was obtained in cisplatin-sensitive TBLCl2 
tumours, resulting in 11% and 67% of cured tu-
mours, respectively. However, compound 2 elec-
trochemotherapy induced less animal body weight 
loss compared to cisplatin electrochemotherapy 
and considering its antitumour effectiveness could 
be potentially used in further investigation.
Electrochemotherapy with cisplatin or 
compound 2 efficiently decreased cell 
survival, in vitro
Cytotoxicity of platinum compound with 3-hy-
droxymethilpyridine ligands in cis and trans posi-
tion has previously been demonstrated in various 
human and mouse tumour cells.19-21 First, in mouse 
B16 melanoma cells, cis platinum (II) compound 
with acyclovir (acv, (9-[(2-hydroxyetoxy)methyl]
guanine)) and 3-Hmpy ([Pt([(acv-N7)2(3-Hmpy)2]
(NO3)2) was 1600-fold less cytotoxic compared to 
cisplatin. Furthermore, an increase of cytotoxicity 
in cisplatin-sensitive and cisplatin-resistant human 
tumour cell lines was obtained using another plati-
num (II) compound 3P-SK containing 3-Hmpy and 
squarato ligands.19 However, the enhancement of 
cytotoxicity was less pronounced compared to cis-
platin, particularly in cisplatin-sensitive cells, with 
a 42-fold enhancement in MCF7 mammary carci-
noma, and a 10-fold enhancement in T24 bladder 
carcinoma and IGROV ovarian carcinoma cells. The 
compound 3P-SK was least effective in cisplatin-
resistant IGROV/RDDP ovarian carcinoma cells, 
showing only a 3-fold increase in IC50 value com-
pared to cisplatin.19 Further synthesis of platinum 
analogues with 3-hmpy ligands led to the discovery 
of biological active platinum (II) compound with 
trans geometry possessing two 3-Hmpy ligands, 
compound 2. Recently, compound 2 was dem-
onstrated as equally cytotoxic in various human 
cisplatin-sensitive T24 bladder carcinoma cells and 
IGROV 1 ovarian carcinoma cells, and in cisplatin-
resistant IGROV 1/RDDP ovarian carcinoma cells 
compared to cisplatin.21 In the present study, the 
cytotoxicity of compound 2 using clonogenic as-
say was determined in another pair of cisplatin-
sensitive and cisplatin-resistant cells, TBLCl2 and 
TBLCl2Pt, respectively. Similarly, equal sensitivity 
of cisplatin-sensitive TBLCl2 and cisplatin-resistant 
TBLCl2Pt cells was observed, suggesting that com-
pound 2 partially overcomes the mechanisms of re-
sistance to cisplatin (Table 1, Figure 1). Thus, com-
pound 2 could be potentially used in treatment to 
cisplatin resistant tumours alone or combined with 
other treatments, such as irradiation, in combina-
tion with other chemotherapeutics.
Knowing that cell membrane is the main bar-
rier for drug import in cells, electroporation as 
the drug delivery method (electrochemotherapy) 
TABLE 1. IC50 values after electrochemotherapy with cisplatin or compound 2 in various mouse tumour cell lines 
SA-1 TBLCl2 TBLCl2Pt
Group IC50 (μM) EF IC50 (μM) EF IC50 (μM) EF
CDDP 276.7‡ 180.0‡ 926.6‡
Compound 2 500.0* 220.0* 1066.6*
ECT CDDP 28.3♦ 9.8 8.0♦ 22.5 12.0♦ 77.2
ECT Compound 2 80.0 6.3 41.0 5.4 206.7 5.2
IC50 = dose of drug which reduced cell survival to 50%; EF = enhancement factor for electroporation of cells, calculated on the bases of IC50 of electroporated and non-
electroporated cells; ‡p (< 0.05) statistically significant difference compared to treatment with cisplatin electrochemotherapy (ECT CDDP); *p (< 0.05) significant difference 
compared to treatment with compound 2 electrochemotherapy (ECT Compound 2); ♦p (< 0.05) statistically difference compared to treatment with compound 2 
electrochemotherapy.
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Kranjc S et al. / Electrochemotherapy with trans-platinum analogue300
could be used for increasing the amount of com-
pound 2 in the cell, thereby achieving the increased 
cytotoxic effectiveness of compound 2. The cyto-
toxicity of cisplatin or compound 2 electrochemo-
therapy was evaluated using a clonogenic assay 
in cell lines with different intrinsic sensitivities 
to cisplatin (sensitive TBLCl2, moderate sensitive 
SA-1 and resistant TBLCl2Pt cells) to obtain reli-
able results for the cytotoxic effect of compound 2 
compared to cisplatin. Electrochemotherapy using 
either drug significantly decreased IC50 value in all 
tested cell lines. The most potentiated cytotoxic ef-
fect of cisplatin electrochemotherapy was observed 
in cisplatin-resistant TBLCl2Pt cells, resulting in a 
77-fold decrease in IC50 value, as previously dem-
onstrated (79-fold).25 However, in moderately cis-
platin-sensitive SA-1 cells and cisplatin-sensitive 
TBLCl2 cells, the reduction in IC50 value was less 
pronounced in cells exposed to cisplatin electro-
chemotherapy, and the determined decrease in 
IC50, up to 23-fold (Table 1), was in the range of 
previously published data (10.5-fold in SA-1 and 
24-fold in TBLCl2 cells).25,37 Using electroporation 
to increase compound 2 uptake into the cells im-
proved cytotoxicity, resulting in an enhancement 
factor of 6.3-fold in SA-1 cells, 5.4-fold in TBLCl2 
and 5.2-fold in TBLCl2Pt cells. However, the po-
tentiation in the cytotoxic effect of compound 2 
electrochemotherapy was significantly lower com-
pared to cisplatin electrochemotherapy, but reflect-
ing a similar enhancement factor in cisplatin-resist-
ant TBLCl2Pt and cisplatin-sensitive TBLCl2 cells 
(Table 1, Figure 1), compound 2 still represents a 
promising drug for further testing. 
Since electroporation of cells with cisplatin ren-
ders cisplatin-resistant TBLCl2Pt cells to equal lev-
el of sensitivity as cisplatin-sensitive cells, and IC50 
values were statistically non-significant, consistent 
with the data obtained in a previous study.25 These 
data indicate increased membrane permeability 
as the predominant mechanism for the improved 
cytotoxicity of cisplatin. In fact, the same amount 
of platinum in both, cisplatin-sensitive TBLCl2 or 
cisplatin-resistant TBLCl2Pt cells induced equal 
cell killing.25 Furthermore, the platinum content 
measured after electrochemotherapy with cisplatin 
or short 5 minutes incubation with cisplatin alone 
was comparable in both, cisplatin-sensitive TBLCl2 
and cisplatin-resistant TBLCl2Pt cells.25 In con-
trast, significant higher IC50 value determined after 
compound 2 electrochemotherapy (5-fold higher) 
in cisplatin-resistant TBLCl2Pt cells compared 
to cisplatin-sensitive TBLCl2 cells suggests the 
involvement of other mechanisms besides mem-
brane permeability, such as transport mechanisms 
(influx or efflux of drug), increased level of thiol 
molecules in cells, enhanced DNA repair, tolerance 
to DNA damage and cell death decrease.4-6 The 
function of compound 2 differs from that of cispl-
atin. Compound 2 induces severe conformational 
changes in plasmid DNA, suggesting that once in 
the cell, compound 2 likely induces few reparable 
or irreparable DNA damages, which are reflected 
in delayed apoptosis.21
Concentration (µM)
2 3.5 6 20 35 60 200 350 60010 100
S
ur
vi
vi
ng
 fr
ac
tio
n
0.0005
0.0007
0.002
0.005
0.007
0.02
0.05
0.07
0.2
0.5
0.7
0.001
0.01
0.1
1
CDDP
Compound  2
ECT CDDP 
ECT Compound 2 
Concentration (µM)
2 3.5 6 20 35 60 200 350 60010 100 1000
S
ur
vi
vi
ng
 fr
ac
tio
n
0.0005
0.0007
0.002
0.005
0.007
0.02
0.05
0.07
0.2
0.5
0.7
0.001
0.01
0.1
1
CDDP
Compound 2  
ECT CDDP 
ECT Compound 2 
Concentration (µM)
2 3.5 6 20 35 60 200 350 60010 100 1000
S
ur
vi
vi
ng
 fr
ac
tio
n
0.0005
0.0007
0.002
0.005
0.007
0.02
0.05
0.07
0.2
0.5
0.7
0.001
0.01
0.1
1
CDDP
Compound 2
ECT CDDP
ECT Compound 2
SA-1 TBLCl2 TBLCl2Pt
*
*
*
*
*
*
*
*
* ***
*
*
*
*
*
*
**
*
*
*
*
*
**
A B C
FIGURE 1. Survival of SA-1 sarcoma (A), TBLCl2 sarcoma (B) and TBLCl2Pt sarcoma cells (C) after treatment with cisplatin (CDDP) or compound 2 or 
cisplatin electrochemotherapy (ECT CDDP) or compound 2 electrochemotherapy (ECT Compound 2), determined using a clonogenic assay. Data are 
presented as the arithmetic mean and standard error of the mean (AM±SE) of triplicates pooled from three independent experiments. The survival of 
cells treated with electrochemotherapy was normalized to the survival of cells treated with electric pulses alone. The survival of SA-1, TBLCl2 and TBLCl2Pt 
cells treated with electric pulses alone was 0.96 ± 0.05, 0.93 ± 0.09 and 0.92 ± 0.10, respectively.
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue 301
Electrochemotherapy with cisplatin or 
compound 2 efficiently delayed tumour 
growth in vivo
Until recently, few trans-Pt(II) complexes were 
evaluated in vivo. The analogues of transplatin 
(trans-[PtCl2(NH3)2]) with one NH3 exchanged with 
pyridine or 4-methylpyridine had no effect in the 
S180 sarcoma tumour model.39 In P388 leukaemia 
an evident antitumour effectiveness was obtained 
with compound having one iminoether instead of 
an amine ligand.40 However, the replacement of 
two amine ligands with dimethylamine and iso-
propylamine failed to improve the antitumour ac-
tivity in human CH ovarian carcinoma, reflecting 
its extracellular inactivation after binding to plas-
ma proteins.41 These results indicate the nature of 
the ligand as an important player in the biological 
activation of trans-Pt(II) compounds. Recently, we 
showed the promising biological activity of trans-
Pt compound 2 with both amino groups replaced 
with pyridine derivate containing hydroxyl group, 
3-Hmpy. Interestingly, a comparable antitumour 
effectiveness of treatment with triple intravenous-
ly administered compound 2 in sarcoma SA-1 tu-
mours to cisplatin was obtained.21
To evaluate compound 2 as a potential chemo-
therapeutic drug in combination with electropora-
tion, for the induction of subcutaneous tumours, 
the same mouse sarcoma tumour cell lines as 
used in vitro (moderately cisplatin-sensitive SA-
1 cells, cisplatin-sensitive TBLCl2 and -resistant 
TBLCl2Pt cells) were selected. Since in vitro data 
have shown sarcoma cells as resistant to com-
pound 2 (high IC50), the purpose of the research 
was to evaluate whether the intrinsic resistance 
of these cells is reflected in tumours in vivo. The 
route (intratumoural or intravenous) of drug ad-
ministration plays an important role in drug dis-
tribution and toxicity to normal tissues. In some 
cases, electrochemotherapy with intratumoural 
injection of cisplatin showed evident antitumour 
potential over systemic administration, reflect-
ing a higher concentration of the drug obtained 
in tumours and lower concentration in normal 
tissues during electroporation.34,42 Furthermore, 
compound 2 pharmacologically behaves differ-
ently compared to cisplatin, and more platinum in 
serum and tumour was observed.21 According to 
these observations, in the present study, the sin-
gle intratumoural administration of drug was used 
to potentially induce less side effects of the treat-
ment and achieve the highest drug concentration 
in the tumours at the time of electroporation. In 
general, the antitumour effectiveness of treatment 
with compound 2 alone was less pronounced com-
pared to cisplatin in all tumour models with in-
trinsically different sensitivity to cisplatin (Table 2, 
Figure 2). Treatment of moderately cisplatin-sensi-
tive SA-1 tumours with cisplatin alone resulted in 
a significant delay of tumour growth compared to 
compound 2. Regarding the route of drug admin-
istration in SA-1 tumours, triple intravenous21 or 
single intratumoural injection resulted in compa-
rable antitumour effectiveness of cisplatin alone. 
However, the delay of tumour growth after triple 
intravenous administration of compound 221 was 
more pronounced (up to 2.2 days) compared to 
single intratumoural administration of compound 
2 in the present study. Furthermore, antitumour 
TABLE 2. Antitumour effectiveness of electrochemotherapy with cisplatin or compound 2 in mouse sarcoma tumours; SA-1, TBLCl2 and TBLCl2Pt
SA-1 TBLCl2 TBLCl2Pt
Group n DT (days)(AM±SE)
GD 
(days) CR (n, %) n
DT (days)
(AM±SE)
GD 
(days) CR (n, %) n
DT (days)
(AM±SE)
GD 
(days) CR (n, %)
Control 10 1.7±0.2 0 8 2.8±0.3 0 6 3.1±0.2 0
EP 10 3.1±0.3 1.4 0 8 5.1±1.0 2.3 0 6 4.2±0.9 1.1 0
CDDP 10 5.2±1.0‡ 3.5 0 8 5.3±0.7‡ 2.5 0 6 3.4±0.5‡ 0.3 0
Compound 2 12 2.3±0.3* 0.6 0 9 3.5±0.5* 0.7 0 6 3.2±0.3* 0.1 0
ECT CDDP 12 15.8±2.2
*
14.1 0 9 11.6±1.0 8.8 6, 66.6 6 7.6±0.9
* 4.5 0
ECT Compound 2 12 5.1±0.4 3.4 0 9 9.4±1.8 6.6 1, 11.1 6 5.1±0.6 2.0 0
EP = application of electric pulses; CDDP = cisplatin, intratumoural injection at a dose of 13.3 mmol/kg; Compound 2- intratumoural injection at a dose 13.3 mmol/kg; ECT = 
electrochemotherapy, application of EP at 1 minute after intratumoural injection of CDDP or compound 2; DT = tumour doubling time; GD = tumour growth delay; CR = complete 
response; AM = mean; SE = standard error of the mean; ‡p (< 0.05) significant difference compared to treatment with CDDP electrochemotherapy; *p (< 0.05) significant 
difference compared to treatment with compound 2 electrochemotherapy.
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue302
effectiveness was less evident in cisplatin-sensi-
tive TBLCl2 and cisplatin-resistant TBLCl2Pt as in 
moderately cisplatin-sensitive SA-1 tumours, in-
dicating different intrinsic features of the tumour 
models examined, particularly the mechanisms 
of resistance to cisplatin. Several efforts to over-
come the mechanisms of cisplatin resistance, i.e., 
using approaches to decrease glutathione level, 
affect DNA repair, activate signal transduction 
pathways leading to cell death and increase cispl-
atin accumulation, have been used.5,17,23,25,43-46 The 
predominant mechanism of cisplatin resistance in 
TBLCl2Pt cells was suggested as the membrane 
restriction of cisplatin uptake.25 Thus, electropo-
ration could be used to overcome the resistance 
to cisplatin and potentiate antitumour effective-
ness of cisplatin, as previously demonstrated in 
variable tumour models.19,25,28,34,37,42 Application of 
electric pulses to the tumours alone insignificantly 
delayed tumour growth in all tested tumour mod-
els and the data are consistent with previous stud-
ies.25,28,38 However, combined treatment of intratu-
moural injection either of cisplatin or compound 2 
shortly before application of electric pulses, signif-
icantly potentiated (up to 3-fold) antitumour effec-
tiveness in all tumour models, but less prominent 
effects were observed with compound 2. As ex-
pected, most pronounced antitumour effectiveness 
after single electrochemotherapy treatment was 
obtained in cisplatin-sensitive TBLCl2 tumours, 
resulting in 67% tumour cures after cisplatin elec-
trochemotherapy and 11% tumour cures after 
compound 2 electrochemotherapy. Notably, cis-
platin or compound 2 alone did not delay growth 
of cisplatin-resistant TBLCl2Pt tumours, but elec-
troporation potentiated antitumour effectiveness 
of either of the drugs used, resulting in prolonged 
tumour 2.2-fold and 1.6-fold growth delay, re-
spectively (Table 2, Figure 2). These data suggest 
that compound 2 electrochemotherapy as an ap-
propriate alternative therapy in cisplatin-resistant 
tumours or tumours with acquired cisplatin-re-
sistance. Studies using electroporation to improve 
the antitumour effectiveness of newly synthesized 
chemotherapeutic are scarce. Among the reported 
studies, two ruthenium (III) compounds, KP418 
((imH)[trans-RuCl4(im)2], im=imidazole) and 
KP1339 (Na[trans-RuCl4(in)2], in=indazole), ad-
ministered intravenously, were used in combina-
tion with electroporation.47-48 However, significant 
antitumour effectiveness was only obtained with 
KP1339 electrochemotherapy in SA-1 tumours, 
Time (days)
0 2 4 6 8 10 12 14 16 18 20
A
ni
m
al
 b
od
y 
w
ei
gh
t (
%
)
92.5
95.0
97.5
102.5
105.0
107.5
0.0
100.0
Control
EP 
CDDP
Compound 2
ECT CDDP
ECT Compound 2
*
Time (days)
0 2 4 6 8 10 12 14 16 18 20
Tu
m
ou
r v
ol
um
e 
(m
m
3 )
20
30
50
70
200
300
500
100
Control
EP
CDDP 
Compound 2
ECT CDDP 
ECT Compound 2
Time (days)
0 2 4 6 8 10 12 14 16
Tu
m
ou
r v
ol
um
e 
(m
m
3 )
20
30
50
70
200
300
500
100
Control
EP
CDDP 
Compound 2
ECT CDDP 
ECT compound 2 
Time (days)
0 2 4 6 8 10 12 14 16
A
ni
m
al
 b
od
y 
w
ei
gh
t (
%
)
92.5
95.0
97.5
102.5
105.0
107.5
0.0
100.0
Control
EP 
CDDP
Compound 2
ECT CDDP
ECT Compound 2
Time (days)
0 2 4 6 8 10 12 14 16 18 20
A
ni
m
al
 b
od
y 
w
ei
gh
t (
%
)
92.5
95.0
97.5
102.5
105.0
107.5
0.0
100.0
110.0
Control
EP
CDDP 
Compound 2
ECT CDDP 
ECT Compound 2 
Time (days)
0 2 4 6 8 10 12 14 16 18 20 22
Tu
m
ou
r v
ol
um
e 
(m
m
3 )
20
30
60
70
200
300
600
100
Control
EP
CDDP 
Compound 2
ECT CDDP 
ECT Compound 2
SA-1 TBLCl2 TBLCl2Pt
*
*
A B C
FIGURE 2. Tumour growth and animal body weight change after cisplatin or compound 2 electrochemotherapy in mouse sarcoma tumours; SA-1, 
TBLCl2 and TBLCl2Pt. Mice (6-12 per group) were treated with intratumoural injection of cisplatin (CDDP, 13.3 mmol/kg) or compound 2 (13.3 mmol/kg) 
or with local application of electric pulses at 1 minute after drug injection (ECT CDDP; ECT Compound 2; 8 pulses, 1300 V/cm, 100 µs, 1 Hz). Data are 
presented as the arithmetic mean and standard error of the mean (AM±SE) of tumour volumes.*p (< 0.05) significant difference compared to treatment 
with cisplatin electrochemotherapy
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue 303
while electrochemotherapy with KP418 has no sig-
nificant effect on tumour growth delay in SA-1 and 
in B16F1 melanoma tumours. Although, the type 
of administration of KP1339 was different (intrave-
nously), at equimolar doses, its antitumour effec-
tiveness was comparable to the effect of compound 
2. To our knowledge, only one synthesized plati-
num (II) compound with 3-Hmpy ligands, 3P-SK, 
was used in combination with electroporation in 
recent study of our research group.19 In that study, 
similar to the results of the present study, the anti-
tumour effectiveness of local application of electric 
pulses after intratumoural administration of 3P-SK 
was less pronounced compared to cisplatin. Still, 
3P-SK electrochemotherapy significantly reduced 
growth of mouse MCA mammary carcinoma tu-
mours and cured 14% of tumours.19 Overall, con-
sidering the 3-fold potentiation in antitumour ef-
fectiveness of compound 2 electrochemotherapy 
compared to the 2-fold potentiation obtained after 
3P-SK electrochemotherapy19, compound 2 may 
potentially be more biologically active. Moreover, 
the potentiation level in antitumour effectiveness 
of compound 2 electrochemotherapy compared 
to cisplatin electrochemotherapy, varied between 
1.6- and 3-fold, suggesting that different transport 
mechanisms and intracellular mechanisms of ac-
tion between compound 2 and cisplatin could be 
involved in tumour responses (Table 2, Figure 2). 
In addition, animal body weight, behaviour and 
locomotion as indicators of animal wellbeing were 
monitored after cisplatin or compound 2 electro-
chemotherapy. Single treatment with intratumour-
al administration of compound 2 and application 
of electric pulses induced significantly less body 
weight loss compared to cisplatin. The animals 
showed a loss of body weight up to 6 days after 
treatment (approximately 6%) and recovered there-
after (Figure 2). These results are consistent with 
those of a previous study, showing that animals 
bearing SA-1 sarcoma lost significantly less body 
weight after triple intravenous administration of 
compound 2 compared to cisplatin.21 However, 
the body weight loss obtained after single intratu-
moural administration of compound 2 compared 
to triple intravenous administration of compound 
2 was less pronounced, indicating that the intra-
tumoural administration of compound 2 was less 
toxic. Since electrochemotherapy with compound 2 
demonstrates fewer side effects on animal wellbe-
ing and despite less pronounced antitumour effec-
tiveness compared to cisplatin, further studies are 
warranted, particularly for testing the repetitive 
treatment of tumours with electrochemotherapy. 
Electrochemotherapy with cisplatin or 
compound 2 increases platinum uptake 
in tumour and amount of platinum 
bound to DNA
To clarify whether the antitumour effectiveness of 
electrochemotherapy either with compound 2 or 
cisplatin was consistent with increased uptake of 
the drug in the cells and partially clarify the phar-
macology of the drugs used, the amount of plati-
num was measured in tumours, serum and bound 
to the DNA. Previous studies have determined an 
increase in platinum accumulation after application 
of electric pulses in various mouse tumours (sar-
coma (SA-1, LPB) and carcinoma (EAT) tumours), 
which consequently improve the antitumour effec-
tiveness of cisplatin.27,29,34 To prevent the washout 
of drug from the tumour and ensure the highest 
concentration in SA-1 tumours, electric pulses were 
delivered at 1 minute after intratumoural injection, 
as previously optimized in an EAT tumour mod-
el.34 Measurement of platinum amount using ICP-
MS in the present study demonstrated electropora-
tion as an effective delivery method for compound 
2, transplatin analogue, and cisplatin. An approxi-
mately significant 2-fold increase of platinum in tu-
mours was achieved, resulting in pronounced anti-
tumour effectiveness (Figure 3). Initial level of plat-
inum amount in tumours treated with compound 
2 alone or compound 2 electrochemotherapy was 
1.3-fold higher compared to cisplatin. In addition, 
the wash out of platinum from the tumours treat-
ed with cisplatin or compound 2 alone was up to 
1.5-fold quicker compared to tumours treated with 
electrochemotherapy using either of the drugs. 
The initial platinum amount in the tumours at one 
hour after the treatment with drug alone or with 
electrochemotherapy was reduced up to 48% and 
up to 18%, respectively, indicating the binding of 
tested drug to the proteins in serum, in extracel-
lular tumour matrix and transport into the cell. In 
fact, the aromatic ring of pyridine and hydroxyl 
groups participates in non-covalent interactions 
between platinum compound and DNA.9, 11, 14, 18-20 
Furthermore, triple intravenous administration of 
compound 2 compared to cisplatin resulted in sig-
nificant higher platinum amount in tumours and 
serum, 4-fold and 40-fold, respectively.21 Similarly, 
significantly higher concentration (up to 39-fold) of 
platinum concentration in serum of animals treat-
ed either with compound 2 alone or compound 2 
electrochemotherapy compared to cisplatin corre-
lated with more platinum in tumours (Figure 3). 
Altogether, compound 2 exhibits a different phar-
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue304
macology compared to cisplatin, which could affect 
tumour responses. Compared to the pharmacology 
of cisplatin, other mechanisms could be involved 
in tumour responses to electrochemotherapy. In 
particular, the drug distribution in tumours de-
pends on tumour vascularization and the content 
of tumour extracellular matrix, which plays an im-
portant role before application of electric pulses. 
Vascular disruption, reduced oxygenation and in-
duced immune responses greatly contribute to tu-
mour responses.35-36,49-50 
DNA is considered the main intracellular target 
of cisplatin and its analogues. Hence, to clarify that 
antitumour effectiveness of electrochemotherapy 
primarily depends on increased drug uptake, the 
amount of platinum bound to DNA was evaluated 
as an indicator that compound 2 escaped bind-
ing to intracellular thiol proteins and reached 
DNA (Figure 3). Indeed, in tumours treated with 
electrochemotherapy compared to the drug ad-
ministration only, approximately 2-fold higher 
platinum binding to DNA was achieved, corre-
lated with pronounced antitumour effectiveness 
(Figure 3). Similarly, the higher level of platinum 
in tumours and serum in animals treated with 
compound 2 electrochemotherapy compared to 
cisplatin was reflected in a significantly (approxi-
mately 3-fold) higher amount of platinum bound 
to DNA. However, the antitumour effectiveness 
was less evident. Overall, a 4-fold increase of 
platinum bound to DNA was obtained in tumours 
treated with compound 2 compared to cisplatin. 
In vitro, despite the enhanced accumulation of a 
trans-platinum (II) compounds, either with amine, 
oxine or piperidine ligand, in tumour cells, the 
cytotoxicity remained comparable or lower than 
that of cisplatin.51-53 Thus, the increased accumula-
tion and binding capacity to DNA obviously are 
not crucial for cytotoxic potential of compounds, 
suggesting the importance of the mode DNA in-
teraction and DNA repair. Thus, depending on the 
ligand in trans platinum compound a monofunc-
tional and bifunctional adducts can be formed with 
DNA.9-10,54-56 Recently, we demonstrated the forma-
tion of severe conformational changes in plasmid 
DNA after treatment with compound 2, and con-
sistent with the findings using other transplati-
num analogues9,54,56, the formation of bifunctional 
DNA crosslinks was suggested. Additionally, the 
effectiveness of DNA repair mechanisms may also 
render compound 2 less cytotoxic compared to cis-
platin. Moreover, the route of drug administration 
to some extent affected the level of platinum bound 
to the DNA. Approximately 14% more platinum 
bound to DNA was obtained after intratumoural 
compared to the intravenous administration of ei-
ther compound 2 or cisplatin.21 Thus, as 80% of cis-
platin in blood circulation is bound to proteins, pri-
marily albumin, this difference could be expected, 
suggesting less free cisplatin molecules reached tu-
mour nodule.57-58 Taken altogether, the pharmacol-
ogy of compound 2 seems to differ from cisplatin, 
A B CTime (h)
0 2 4 6 8 10 12 14 16 18 20 22 24
P
t c
on
te
nt
 in
 tu
m
or
  (
μg
/g
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
CDDP 
ECT CDDP
Compound 2
ECT Compound 2 
Time (h)
0 2 4 6 8 10 12 14 16 18 20 22 24
P
t b
ou
nd
 to
 th
e 
D
N
A
  (
pg
/ μ
g)
0
5
10
15
20
25
30
CDDP 
ECT CDDP
Compound 2
ECT Compound 2 
Time (h)
0 2 4 6 8 10 12 14 16 18 20 22 24
P
t c
on
ce
nt
ra
tio
n 
 ( μ
g/
m
l)
1
3
5
7
9
11
13
0
2
4
6
8
10
12
CDDP
ECT CDDP
Compound 2
ECT Compound 2
SA-1 (tumor) SA-1 (serum) SA-1 (bound to DNA)a.) b.) c.)
*
*
*
*
*
*
*
*
*
*
*
*
**
*
*
*
*
*
FIGURE 3. Platinum amount in sarcoma SA-1 tumours (A), platinum amount in the serum (B) and platinum amount bound to DNA in the cells isolated 
from SA-1 tumours (C). Animals (8 per group) were treated with intratumoural injection of cisplatin (CDDP, 13.3 mM) alone or compound 2 (13.3 mM) 
alone or with local application of electric pulses 1 minute after intratumoural drug injection (ECT CDDP; ECT Compound 2; 8 pulses, 1300 V/cm, 100 µs, 
1 Hz). The data are presented as the arithmetic mean and standard error of the mean (AM±SE) obtained from 8 samples. *p (< 0.05) under (A) and (C) 
statistically significant difference compared to corresponding drug treatment only; *p (< 0.05) under (B) statistically significant difference compared to 
cisplatin or cisplatin electrochemotherapy.
Radiol Oncol 2017; 51(3): 295-306.
Kranjc S et al. / Electrochemotherapy with trans-platinum analogue 305
involving binding to the proteins in blood circula-
tion, in tumour extracellular matrix and accumula-
tion in cells. The accumulation of cisplatin in the 
cell is not fully understood. Cisplatin could enter 
in cells through passive diffusion and facilitated 
or active transport mediated through membrane 
transporters (the Copper transporter 1 (CTR1) 
and 2 (CTR2); the P-type copper-transporting 
ATPases ATP7A and ATP7B, multidrug and toxin 
extrusion transporters).7 Currently, little is known 
about the accumulation of transplatin or its ana-
logues. Active transport was demonstrated to play 
an important role in accumulation, independent 
of transporter CTR1 and ATP, depending on pro-
tein ATP7B and CTR2.6,8 The details of compound 
2 transport mechanisms and its interaction with 
biological molecules and antitumour effectiveness 
need further investigation. 
Conclusions
In summary, compound 2, a trans-Pt(II) analogue 
with two 3-Hmpy ligands, shows potential for 
electrochemotherapy treatment in vitro and in vivo. 
Electroporation increased compound 2 cytotoxicity 
up to 6-fold in vitro and antitumour effectiveness 
up to 3-fold in vivo, but it had less evident effects 
compared to cisplatin. The underlying mechanism 
of antitumour effectiveness of electrochemother-
apy could be increased drug uptake, and an ap-
proximately 2 times higher amount of platinum 
in the tumours was reflected to the same extent in 
higher amount of Pt bound on its target of action to 
the DNA. Based on the compound 2 electrochemo-
therapy cytotoxicity and antitumour effectiveness 
in cisplatin-resistant tumour model TBLCl2Pt, this 
molecule could potentially be used in the treat-
ment of tumours with intrinsic or acquired resist-
ance to cisplatin. To improve the antitumour ef-
fectiveness of compound 2 electrochemotherapy, 
multiple treatments should be tested in the future. 
Furthermore, compound 2 could be used in combi-
nation with other chemotherapeutics, affecting dif-
ferent targets, immunotherapy, vascular-targeted 
therapy or irradiation; however, further studies are 
warranted.
Acknowledgements
The authors gratefully acknowledge the research 
grants from the Slovenian Research Agency (P3-
0003 and P1-0134).
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