JBIC Journal of Biological Inorganic Chemistry

, Volume 22, Issue 6, pp 807–817 | Cite as

In vitro and in vivo anti-tumor effects of selected platinum(IV) and dinuclear platinum(II) complexes against lung cancer cells

  • Milos Arsenijevic
  • Marija Milovanovic
  • Snezana Jovanovic
  • Natalija Arsenijevic
  • Bojana Simovic Markovic
  • Marina Gazdic
  • Vladislav Volarevic
Original Paper

Abstract

In the present study, cytotoxic effects of cisplatin, the most usually used chemotherapeutic agent, were compared with new designed platinum(IV) ([PtCl4(en)] (en = ethylenediamine) and [PtCl4(dach)]) (dach = (±)-trans-1,2-diaminocyclohexane) and platinum(II) complexes ([{trans-Pt(NH3)2Cl}2(μ-pyrazine)](ClO4)2 (Pt1), [{trans-Pt(NH3)2Cl}2(μ-4,4′-bipyridyl)](ClO4)2DMF(Pt2),[{trans-Pt(NH3)2Cl}2(μ-1,2-bis(4pyridyl)ethane)](ClO4)2 (Pt3)), in vitro and in vivo against human and murine lung cancer cells, to determine anti-tumor potential of newly synthesized platinum-based drugs in the therapy of lung cancer. Results obtained by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], Lactate dehydrogenase and Annexin V/Propidium Iodide assays showed that, among all tested complexes, [PtCl4(en)] had the highest cytotoxicity against human and murine lung carcinoma cells in vitro. [PtCl4(en)] showed significantly higher cytotoxicity then cisplatin in all tested concentrations, mainly by inducing apoptosis in lung cancer cells. [PtCl4(en)] was well tolerated in vivo. Clinical signs of [PtCl4(en)]-induced toxicity, such as changes in food, water consumption or body weight, nephrotoxicity or hepatotoxicity was not observed in [PtCl4(en)]-treated mice. [PtCl4(en)] managed to increase presence of CD45+ leukocytes, including F4/80+ macrophages, CD11c+ dendritic cells, CD4+ helper and CD8+ cytotoxic T cells (CTLs) in the lungs, cytotoxic NK, NKT and CTLs in the spleens of tumor bearing mice, resulting with reduction of metastatic lesions in the lungs, indicating its potential to stimulate anti-tumor immune response in vivo. Due to its anti-tumor cytotoxicity, biocompatibility, and potential for stimulation of anti-tumor immune response, [PtCl4(en)] may be a good candidate for further testing in the field of medicinal chemistry.

Keywords

Lung cancer Cytotoxicity Platinum(IV) complexes Platinum(II) complexes 

Introduction

Platinum-based drugs are widely used as anticancer agents with a broad range of anti-tumor activities [1, 2]. Among them, cisplatin is most usually used [3], but has a limited spectrum of activity due to the development of drug resistance [4] and severe side effects [5, 6, 7]. To overcome these defects, many new platinum complexes such as sterically hindered platinum(II), polynuclear platinum(II), and platinum(IV) complexes have been designed and tested over the years with an idea that their different nature of interaction with DNA would enhance anti-tumor activity and attenuate side effects [6, 8, 9, 10]. Recently, we reported that some of platinum(IV) and dinuclear platinum(II) complexes had significantly higher cytotoxic effects against human ovarium carcinoma cells than cisplatin, indicating their potential therapeutic use in the therapy of ovarian cancer [11].

Lung cancer is considered one of the most fatal (the overall ratio of mortality to incidence is 0.87) representing the primary cause of cancer mortality for men worldwide [12, 13, 14]. Although chemotherapeutic agents can improve survival and quality of life of lung cancer patients, the disease still progress after chemotherapy and is usually aggravated by serious side effects. Thus, a lot of pre-clinical and clinical studies are conducted to design the effective chemotherapeutic in lung cancer therapy.

In line with these findings, we explored and reported here in vitro cytotoxic effects of selected platinum(IV) complexes ([PtCl4(en)] (en = ethylenediamine) and [PtCl4(dach)]) (dach = (±)-trans-1,2-diaminocyclohexane) (Fig. 1a, b) and dinuclear platinum(II) complexes [{trans-Pt(NH3)2Cl}2(μ-pyrazine)](ClO4)2(Pt1), [{trans-Pt(NH3)2Cl}2(μ-4,4′-bipyridyl)](ClO4)2 ∙ DMF (Pt2), [{trans-Pt(NH3)2Cl}2(μ-1,2-bis(4-pyridyl)ethane)](ClO4)2(Pt3) (Fig. 1c–e) against human lung carcinoma cell line and tested their effects in vivo using murine model of lung carcinoma.
Fig. 1

Structures of tested complexes. a [PtCl4(en)]; b [PtCl4(dach)]; c ([{trans-Pt(NH3)2Cl}2(μ-pyrazine)](ClO4)2(Pt1); d ([{trans-Pt(NH3)2Cl}2(μ-4,4′-bipyridyl)](ClO4)2DMF(Pt2); e ([{trans-Pt(NH3)2Cl}2(μ-1,2-bis(4pyridyl)ethane)](ClO4)2 (Pt3)

Materials and methods

Chemicals

The compounds ethylenediamine (en) (Merck), (1R, 2R)-1.2-diaminocyclohexane (dach) (Acros Organics), K2PtCl4 (Strem Chemicals), cis-diamminedichloroplatinum(II), cisplatin, cis-[PtCl2(NH3)2] (Aldrich) were used without purification. The trans-diamminedichloroplatinum(II), transplatin, trans-[PtCl2(NH3)2], and ligands such as pyrazine, 4,4′-bipyridyl, 1,2-bis(4-pyridyl)ethane, which were used for synthesis of dinuclear platinum(II) complexes were obtained from Acros Organics. The complexes of platinum(IV), [PtCl4(dach)] [tetrachloro(trans-d,l-1,2-diaminocyclohexane)platinum(IV)] and [PtCl4(en)] [tetrachloro(ethylenediamine)platinum(IV)], were prepared according to the published procedure [15, 16], while the dinuclear platinum(II) complexes, such as [{trans-Pt(NH3)2Cl}2(μ-pyrazine)](ClO4)2(Pt1) (μ-pyrazine-bis[trans-diamminechloroplatinum(II)]-perchlorate), [{trans-Pt(NH3)2Cl}2(μ-4,4′-bipyridyl)](ClO4)2 ∙ DMF (Pt2) (μ-4,4′-bipyridyl-bis[trans-diamminechloroplatinum(II)]-perchlorate), and [{trans-Pt(NH3)2Cl}2(μ-1,2-bis(4-pyridyl)ethane)](ClO4)2(Pt3) (μ-1,2-bis(4-pyridyl)ethane-bis[trans-diamminechloroplatinum(II)]-perchlorate), were prepared as described earlier in the literature [17]. The purity of the all complexes was checked by elemental microanalyses. Anal. Calcd. for [PtCl4(dach)], PtC6N2H14Cl4: H, 3.10; C, 15.97; N, 6.21. Found: H, 3.09; C, 15.93; N, 6.19. Anal. Calcd. for [PtCl4(en)], PtC2N2H8Cl4: H, 2.02; C, 6.04; N, 7.06. Found: H, 2.01; C, 6.05; N, 7.08. Anal. Calcd. for (Pt1), Pt2C4N6H16Cl4O8: H, 2.00; C, 5.94; N, 10.40. Found: H, 1.99; C, 5.95; N, 10.36. Anal. Calcd. for (Pt2), Pt2C13N7H27Cl4O9: H, 2.84; C, 16.31; N, 10.24. Found: H, 2.83; C, 16.34; N, 10.20. Anal. Calcd. for (Pt3), Pt2C12N6H24Cl4O8: H, 2.65; C, 15.80; N, 9.21. Found: H, 2.64; C, 15.83; N, 9.23.

Cell lines

The cell line of human lung carcinoma (A549) was purchased from American Type Culture Collection (ATCC), Manassas, VA, USA. The A549 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS), 100 IU/mL penicillin G and 100 μg/mL streptomycin (Sigma-Aldrich, Munich, Germany), at 37° in a 5% CO2 incubator.

The cell line of murine lung carcinoma (Lewis Lung Cancer 1, LLC1), derived from the lungs of C57BL mice implanted with Lew (Lewis) lung cancer [18], was purchased from American Type Culture Collection (ATCC) (catalog no. CRL-1642™). Cells were routinely grown in suspension in T-25 flask (BD Falcon, USA), in a 5% CO2 incubator with standard conditions [18]. A549 and LLC1 cells in passage 3 were used throughout the experiments.

Cytotoxicity assays

In order to determine cytotoxic activity of selected complexes, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), LDH (Lactate dehydrogenase) and Annexin V/PI assays were used.

MTT assay

A549 and LLC1 cells were diluted with medium to 1 × 106 cells/ml and aliquots (5 × 103 cells/100 μl) were placed in individual wells in 96-multiplates. About 24 h and 48 h later, after the cell adherence, each well received 100 μl of tested complexes, which had been serially diluted two-fold in medium to concentrations ranging from 1000 to 7.8 μM. Cells were incubated at 37 °C in a 5% CO2 incubator for 24 h. After incubation, multiplates were centrifuged, the supernatant was removed, fresh medium and MTT solution (5 mg/ml in PBS) 20 μl were added to each well and the plates were incubated for an additional 4 h. The multiplates were centrifuged, cell-free supernatants were suctioned off, and DMSO (150 μl) and glycine buffer were added to dissolve the crystals. The plates were shaken for 10 min. The optical density of each well was determined at 595 nm using microplate multimode detector Zenyth 3100.

The percentage of cytotoxicity was calculated using the formula: cytotoxicity % = 100-[(TS − BG0) − E/(TS − BG0) × 100], where BG0 is for background of medium alone, TS is for total viability/spontaneous death of untreated target cells, and E is for experimental well [19].

Lactate dehydrogenase (LDH) assay

Cytotoxicity of used complexes was examined by Cytotoxicity Detection Kit (LDH) (Roche Applied Science). Cells were prepared and treated with complexes in the same manner as for MTT assay. Additional wells were prepared as high control cells were treated with Triton X (1%). Cells exposed to medium were used as low controls. After treatment, supernatant (100μL) was transferred to new plate and incubated with an equivalent volume of substrate solution. After incubating the plates for 30 min at RT, 50 μl/well stop solution was added and data were acquired by spectrophotometry at 450 nm. The percentage of dead cells was calculated using the formula [20]:
$$\% {\text{ of dead cells }} = \, \left( {{ \exp }.{\text{ value}} - {\text{low control}}} \right)/\left( {{\text{high control}} - {\text{low control}}} \right) \, \times { 1}00.$$

Annexin V/PI assay

For detection of apoptosis the Annexin V binding capacity of treated cells was examined by flow cytometry using Annexin V FITC Detection Kit (BD Pharmingen, San Jose, CA, USA) according to the manufacturer’s protocol.

After tumor cells reached the subconfluency, medium was replaced with tested complexes eluate diluted 1:1 in complete DMEM (volume, 4 mL). Cells exposed to tested complexes eluate were placed at 37 °C in a 5% CO2 incubator for 24 h. Cultured cells were washed twice with cold phosphate-buffered saline (PBS, Sigma-Aldrich) and resuspended in 1× binding buffer [10x binding buffer: 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2] at a concentration of 1 × 106/mL. Annexin FITC and propidium iodide (PI) were added to the 100 μL of cell suspension and incubated for 15 min at room temperature (25 °C) in the dark. After incubation 400 μL of 1× binding buffer was added to each tube and stained cells were analyzed within 1 h using FACS Calibur (BD, San Jose, USA) and WinMDI software.

Apoptotic cells that lose asymmetry of their membrane phospholipids leave phosphatidylserine behind the outer leaflet of the plasma membrane. Annexin V, a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine, is be used as a sensitive probe for the presence of phosphatidylserine on the cell membrane and hence as a marker of apoptosis. PI is a non-specific DNA intercalating agent, which is excluded by the plasma membrane of living cells, and thus can be used to distinguish necrotic cells from apoptotic and living cells by supravital staining without prior permeabilization. Since Annexin V FITC staining precedes the loss of membrane integrity that accompanies the later stage identified by PI, Annexin V FITC positive and PI negative staining indicates early apoptosis, while viable cells are Annexin V FITC negative and PI negative. Cells that are in late apoptosis or already dead cells are both Annexin V FITC and PI positive [21].

Animals

C57BL/6, 6–8 weeks old male mice, was used. Mice were equalized in weight and randomized in experimental and control groups. All mice were maintained in our animal facilities. Mice were housed in a temperature controlled environment with a 12-h light–dark cycle and were given standard laboratory chow and water ad libitum. All animals received humane care, and all experiments were approved by, and conducted in accord with, the Guidelines of the Animal Ethics Committee of the Faculty of medical sciences of the University of Kragujevac (Kragujevac, Serbia).

Induction of experimental metastasis and in vivo application of platinum complexes

Experimental metastases were induced by intravenous injection of 5 × 104 LLC1 cells [22]. Mice received either [PtCl4(en)] or cisplatin (three times per week, 0.10404 mg/mouse, iv). Mice were killed on 28th day of the experiment, as previously described [22, 23].

Histopathological analysis

All mice were killeded in an atmosphere saturated with diethyl ether (BETA HEM, Belgrade) and organs were isolated for histopathological analysis of metastatic colonies (lungs) or toxicity (livers and kidneys).

The isolated lungs, livers, kidneys were fixed in 10% formalin, embedded in paraffin, and consecutive 4-lm tissue sections mounted on slides. Sections were stained with Hematoxylin and Eosin (H&E) and examined under low-power (100×) light microscopy (Zeiss Axioskop 40, Jena, Germany) equipped digital camera. Metastases were verified by light microscopy (magnification 10× and 40×) as characteristic brown-black pigmented “hot spots” with giant multinucleated cells clearly limited by the surrounding lung tissue. For quantification of metastatic expansion, firstly, the composition of the image was carried out in Adobe Photoshop (Adobe® Photoshop® CS2 Windows® USA). Then, the extent of metastatic deposits within the lung parenchyma was measured using Image J software. Briefly, microscopic images of the entire cross section of the tumor was obtained, at magnification 40×. Next, regions of interest (ROI) confining tumor tissue relative to the background were determined and labeled. Quantification of tumor was estimated selecting the range of labeled area to match the tumor tissue. The measurement results were displayed as a percentage of tumor fields in ROI, as previously described [24].

Isolation of lung-infiltrated immune cells

The lungs were washed with sterile phosphate-buffered saline (PBS) and placed in Petri dishes with DMEM supplemented with 10% FBS. The dissected lung tissue was incubated in medium that contained Collagenase Type IV (0.5 mg/ml) and type IV bovine pancreatic DNAse (Roche Diagnostic; 1 mg/ml) at 37 °C for 45 min. The cells were filtered through a 100-μm nylon cell-strainer into a clean 50-ml conical tube. Then, cells were pelleted by centrifuging 10 min at 300×g, at 10 °C. Red blood cells were depleted with a lysis buffer (0.144 M NH4Cl, 0.0169 M TRIS base, pH 7.4) at 37 °C in a 5% CO2 atmosphere for 5 min [25].

Isolation of splenocytes

The isolation of splenocytes was conducted as previously described [26]. Briefly, the spleens were minced in RPMI 1640 (Sigma-Aldrich, St. Louis, MO, USA) and forced gently through a 40-mm cell-strainer nylon mesh using a sterile syringe plunger and centrifuged at 400 g for 5 min. Pelleted spleen cells were incubated in 2 ml NH4Cl/Tris–Cl (pH 7.2) for 5 min, supplemented with 1 ml FBS, centrifuged at 400 g for 5 min and then resuspended in RPMI 1640 with 10% FCS. Pellet was resuspended in 1 ml of RPMI 1640, total number of cells was determined using trypan blue exclusion on a hemocytometer and cells were used for flow cytometry analysis and for the analysis of cell cytotoxicity.

Flow cytometry analysis

Lung-infiltrated immune cells and splenocytes were analyzed by flow cytometry. Cells were incubated with anti-mouse antibodies CD45, F4/80, CD11c, CD3, CD4, CD8, NK1.1, perforin, CD107, Killer cell lectin-like receptor subfamily G member (KLRG-1) conjugated with fluorescein isothiocyanate [FITC; BD Biosciences, Franklin Lakes, NJ], phycoerythrin [PE; BD Biosciences], peridinin chlorophyll protein [BD Biosciences], or allophycocyanin [APC; BD Biosciences]. Flow cytometric analysis was conducted on a BD Biosciences FACS Calibur and analyzed using the Flowing Software program.

Real-time monitoring of cytotoxicity determined by xCELLigence system

The DP version of the xCELLigence system (Roche) was used in this study for the determination of cytotoxicity of splenocytes against LLC1 cells. The DP version comprises a measurement unit housed within a standard tissue culture incubator with three stations that each takes E16 plates (each E16 plate has 16 wells). 100 µl of complete medium was added to each well and background impedance on the plates was measured on the xCELLigence RTCA DP instrument at 37° and 5% CO2. LLC1 cells were used as target cells for splenocytes. Seeding density of 4 × 104 LLC1 cell/well was considered optimal and used for all assays. Effector to target ratio (E:T ratio) 10:1 was used [27]. LLC1 cells were resuspended in DMEM with 10% FCS at 4 × 105 cells per milliliter. A total of 100 µl tumor cells were added to each well of the E16 plate, which was then placed in the xCELLigence RTCA DP. Splenocytes, isolated from LLC1-treated, LLC1+ cisplatin-treated and LLC1 + [PtCl4(en)]-treated mice, were counted and resuspended at a concentration of 4 × 106 cells per milliliter in DMEM + 10% FCS media. Then 100 µl of splenocytes or media alone was added to the respective wells. The E-plate 16 were placed in the xCELLigence RTCA DP, and impedance measurements were recorded every 15 min for 24 h at 37° and 5% CO2. Splenocytes–mediated death of tumor cells was monitored in real-time and was indicated by a decrease in cell index. Data were analyzed with RTCA Software 1.2 (Acea Biosciences).

Statistics

Data were expressed as the mean ± standard error of the mean (SEM) for each group. Statistical analyses were performed using SPSS 23.0 for Windows software (SPSS Inc., Chicago, IL). The difference was considered significant when p < 0.05.

Results

[PtCl4(en)] showed the highest cytotoxicity against human and murine lung carcinoma cell lines

Cytotoxicity assay showed a dosage and time-dependent correlation between concentration of the tested complexes and cell viability of tumor cells (Fig. 2a). In all tested concentrations, complex [PtCl4(en)] displayed higher cytotoxicity than all other complexes 24 h after treatment (Fig. 2a). The increase in the cytotoxicity of all complexes is observed 48 h after treatment, (Fig. 2b), and similarly as after 24 h, the highest cytotoxicity against A549 cells had [PtCl4(en)], that managed to kill about 50% of tumor cells at the lowest tested concentration (7.8 μM) which is suitable for in vivo application. The most important, [PtCl4(en)] showed significantly higher cytotoxicity then cisplatin in all tested concentrations (Fig. 2b).
Fig. 2

Dose and time-dependent correlation between concentration of the tested complexes and cell viability of tumor cells. a Representative graphs of A549 cell survival after 24-h cell growth in the presence of the investigated complexes and cisplatin, as determined by MTT assay. b Results obtained by MTT assay 48 h after treatment of A549 cells with tested complexes and cisplatin. Mean values from three experiments are shown. Black squares indicates IC50 values

To confirm cytotoxic potential of [PtCl4(en)] against murine lung carcinoma cells and to determine its suitability for in vivo testing in animal model of lung cancer, the cytotoxicity of this complex is further tested against LLC1 cells and compared with anti-tumor potential of cisplatin. Similar as it was observed against A549 cells, [PtCl4(en)] displayed higher cytotoxicity than all other complexes against LLC1, as well (Fig. 3a, b).
Fig. 3

[PtCl4(en)] has higher cytotoxic potential against LLC1 cells than cisplatin in vitro. a Representative graphs of LLC1 cell survival after 24-h cell growth in the presence of the investigated complexes and cisplatin, as determined by MTT assay. Mean values from three experiments are shown. Black squares indicates IC50 values. b Evaluation of cytotoxicity by LDH assay. Mean values from three experiments are shown. cRepresentative dot plots of Annexin V and PI staining

[PtCl4(en)] induced apoptosis of LLC1 cells and has higher cytotoxic potential then cisplatin in vitro

As it was indicated by MTT and LDH tests, similar as cisplatin, [PtCl4(en)] mainly caused apoptosis of LLC1 cells. To quantify cell apoptosis further, LLC1 cells, exposed to [PtCl4(en)] and cisplatin, were stained with Annexin V FITC/PI and analyzed by flow cytometry. The results, shown in Fig. 3c show that majority of LLC1 cells were in late apoptosis 24 h after treatment with [PtCl4(en)] and cisplatin. Importantly, nearly 80% of LLC1 cells that were treated with [PtCl4(en)] were in late apoptosis, while percentage of cisplatin-treated LLC1 cells that were in late-stage apoptosis was about 60%. These results confirmed the results obtained by MTT assay, showing that in vitro [PtCl4(en)] has higher cytotoxic potential then cisplatin against murine lung carcinoma cells, indicating its potential for in vivo application.

[PtCl4(en)] was well tolerated in vivo

Any clinical signs of [PtCl4(en)]-induced toxicity, changes in food or water consumption or body weight (Fig. 4a) were not observed in [PtCl4(en)]-treated mice. Histological examination of kidneys and livers showed that [PtCl4(en)] was neither nephrotoxic nor hepatotoxic (Fig. 4b, c). The structures of renal tubules of [PtCl4(en)]-treated mice showed lower degree of hyperemia, with preserved glomerular structure and intact basement membrane (Fig. 4b). Accordingly, there were no signs of acute or chronic liver failure in the murine livers of [PtCl4(en)]-treated mice (Fig. 4c).
Fig. 4

[PtCl4(en)] managed to reduce metastatic lesions in the lungs by affecting anti-tumor immune response. a Change in body weight. b, c Representative histological images of the renal and hepatic tissue of [PtCl4(en)]-treated mice. d Representative histological analysis of lungs isolated from untreated (a), [PtCl4(en)]-treated (b) and cisplatin-treated tumor bearing mice (c). e Diameter of metastatic fields in the lungs of LLC1-treated mice, as determined by Image J software. f Flow cytometry analysis of the splenocytes. g Cytotoxicity of splenocytes against LLC1 cells, as determined by xCELLigence system. Data are presented as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.001

[PtCl4(en)] managed to reduce metastatic lesions in the lungs by affecting anti-tumor immune response

Although histological analysis of the lungs isolated from [PtCl4(en)]-treated and cisplatin-treated tumor bearing mice showed perivascular infiltration of tumor cells in the lungs, expansion of malignant tissue in these mice was lower when compared to vehicle-treated tumor bearing animals in which lung tissues were almost completely displaced with tumor cells (Fig. 4d, e).

To explore whether [PtCl4(en)]-dependent reduction of metastatic lesions in the lungs are a consequence of its systemic effects of immune cells, cellular make-up of the spleen was analyzed. There were significantly higher percentage of cytotoxic NK1.1+NK cells, CD3+NK1.1+ NKT cells and CD8+ T lymphocytes in the spleens of tumor bearing mice that received [PtCl4(en)] when compared to animals that received vehicle or cisplatin (Fig. 4f).

In line with these findings, the results obtained by xCELLigence system for monitoring real-time cytotoxicity showed that splenocytes isolated from LLC1+[PtCl4(en)]-treated mice were significantly more cytotoxic against LLC1 cells then splenocytes isolated from tumor bearing animals that received only LLC1 cells or cisplatin (Fig. 4f), indicating possible immuno-stimulatory anti-tumor effects of [PtCl4(en)].

[PtCl4(en)] significantly increased infiltration of immune cells in the lungs of LLC1-treated mice

Next, we analyzed cellular make-up of the lungs to determine cellular targets of [PtCl4(en)]-mediated modulation of anti-tumor immune response in LLC1-treated animals. [PtCl4(en)] profoundly increased infiltration of CD45+ leukocytes into the lung parenchyma (p < 0.05; Fig. 5a). As it is shown in Fig. 5b, flow cytometry analysis showed that total number of CD45+F4/80+ macrophages (p < 0.05), CD45+CD11c+ dendritic cells (p < 0.05), CD4+ helper T cells (p < 0.01) and NKT cells (p < 0.01) were significantly higher in the lungs of LLC1-tumor bearing mice that received [PtCl4(en)]. There was no significant increase in lung-infiltrated NK1.1+NK cells (Fig. 5b).
Fig. 5

[PtCl4(en)] significantly increased infiltration of immune cells in the lungs of LLC1-treated mice. Cellular make-up of the lung, determined by flow cytometry, showing: a the total cells number of C45+ leukocytes; b total number of CD45+F4/80+ macrophages; CD5+CD4+ T helper cells; CD45+CD4+NK1.1+NKT cells; CD45+NK1.1+NK cells, and CD45+CD11c+DCs; c total number of CD8+CTLs with representative dot plots; d total number of CD8+perforin+CTLs, CD8+107+CTLs, and CD8+KLRG1+CTLs. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01; ***p < 0.001

Importantly, total number of lung-infiltrated leukocytes, particularly macrophages, T helper lymphocytes and NKT cells were significantly higher in LLC1+[PtCl4(en)]-treated mice when compared to LLC1+cisplatin-treated animals (Fig. 5b) suggesting that [PtCl4(en)] treatment better promoted anti-tumor immune response than cisplatin.

[PtCl4(en)] significantly increased infiltration of CTLs in the lungs of LLC1-treated mice and enhanced expression of perforin and CD107 on their surface

[PtCl4(en)] treatment significantly increased total number of cytotoxic CD8+CTLs (Fig. 5c, p < 0.001) in the lungs of LLC1-treated mice. Moreover, analysis of cytotoxic molecules involved in CTL-mediated anti-tumor immune response (perforin and CD107) showed that [PtCl4(en)] managed to promote influx of perforin+CTLs (p < 0.001) and CD107+CTLs (p < 0.001) in the lungs of tumor bearing animals.

Importantly, total number of cytotoxic, lung-infiltrated perforin+CTLs (p < 0.001) and CD107+CTLs (p < 0.001) were significantly higher, while total number of CTLs expressing inhibitory molecule KLRG1 were profoundly lower (p < 0.05) in LLC1+[PtCl4(en)]-treated mice when compared to LLC1+cisplatin-treated animals (Fig. 5d).

Discussion

Platinum-based drugs are used as cancer chemotherapeutics for the last 40 years [1, 2]. However, drug intrinsic or acquired resistance and nephrotoxicity are the major limitations of the use of platinum-based compounds in cancer therapy [4, 5, 6, 7]. Much effort has been put into the development of new platinum-based anticancer complexes, but none of them has reached worldwide clinical application so far.

In the present study, cytotoxic effects of cisplatin—the most usually used chemotherapeutic— were compared with several newly designed platinum(IV) and platinum(II) complexes, in vitro and in vivo against human and murine lung cancer cells, to determine anti-tumor potential of newly synthesized platinum-based drugs in the therapy of lung cancer.

Among all tested complexes, [PtCl4(en)] had the highest cytotoxicity against human and murine lung carcinoma cells in vitro (Figs. 2, 3). Importantly, [PtCl4(en)] showed significantly higher cytotoxicity than cisplatin in all tested concentrations (Figs. 2, 3), mainly by inducing apoptosis in lung cancer cells. These findings are in line with previously described anti-tumor activity of platinum(IV) complexes that is based on the their activation by reduction to the corresponding platinum(II) analogs [28], followed by aquation and induction of apoptotic cell death. The mechanism of action of cisplatin is based on the intrastrand cross-linking of the cis-Pt(NH3)2 unit to cellular DNA at two neighboring guanine bases [29] and the consequent induction of cellular apoptosis. However, cisplatin, like other platinum(II) complexes, has the ability to non-selectively bind to macromolecules as well, that results with reduced bioavailability, decreased anti-tumor cytotoxicity and increased toxic side effects.

Indeed, the clinical use of platinum(II)-based cytostatics (cisplatin, carboplatin and oxaliplatin) is limited mainly because of their severe side effects (e.g., nephrotoxicity, hepatotoxicity, neurotoxicity, ototoxicity, myelosuppression, emesis, and alopecia) [30, 31, 32, 33, 34]. On the other hand, potential advantage of platinum(IV) complexes compared to mononuclear platinum(II) complexes, is their lower reactivity that decreases the number of toxic side effects and increases the opportunity for their arrival to the target cells. In line with these findings, [PtCl4(en)]— a representative of platinum(IV) complexes— was not only more cytotoxic than cisplatin in vitro (Figs. 2, 3), it was well tolerated in vivo, without any observed side effects (Fig. 4a).

In addition to its ability to directly induce cell death of lung cancer cells, it seems that [PtCl4(en)] can enhance anti-tumor immune response, as well (Figs. 4f, g, 5). [PtCl4(en)] promoted influx of DCs, macrophages, helper CD4+ T cells and CTL in lungs of tumor bearing animals (Fig. 5) and increased presence and cytotoxicity of NK, NKT and CTLs in the spleens of LLC1-treated mice (Fig. 4f, g). In inductive phase of anti-tumor immune response, DCs capture tumor antigens and present them to CD4+ T cells inducing their activation and conversion in effector or memory cells [35, 36, 37]. Effector CD4+ T cells, in effector phase of immune response, migrate into tumors and metastatic lesions where it activates tumor-infiltrated macrophages and promote anti-tumor immunity [38, 39]. Thus, increased number of DCs, T cells and macrophages in the metastatic lungs of LLC1+[PtCl4(en)]-treated mice (Fig. 5) indicates that [PtCl4(en)] treatment enhanced both inductive and effector phase of anti-tumor immune response. Importantly, infiltration of macrophages, DCs, CD4+ T helper cells and CTLs in the lungs was more pronounced in [PtCl4(en)]+LLC1-treated mice then in cisplatin+LLC1-treated animals (Fig. 5).

Cellular make-up of the lungs obtained from LLC1-treated mice revealed reduced number of CD4+ T cells in tumor bearing animals that received cisplatin (Fig. 2b). It is known that cisplatin may suppress proliferation of T cells in mice [40] and to attenuate their capacity to respond to mitogens [41]. In contrast to cisplatin, [PtCl4(en)] increased presence of both CD4+ T helper and CD8+CTLs in the lungs of LLC1-treated mice (Fig. 5b, c). CTLs expand in response to specific tumor antigens in the presence of IL-2 produced by CD4+ T cells [42]. [PtCl4(en)] treatment significantly increased influx of CTLs in metastatic lungs (Fig. 5c) and stimulated expression of anti-tumor molecules (CD107 and perforin) on their surface enhancing their cytotoxicity (Fig. 5d). Additionally, lower number of CTLs that express inhibitory KLRG1 molecule was seen in [PtCl4(en)]+LLC1 mice (Fig. 5d), suggesting that, in comparison with cisplatin, [PtCl4(en)] better promotes development of anti-tumor phenotype of CTLs.

In line with these findings are results obtained from the spleens of tumor bearing animals. As it was shown in Fig. 4f, there were significantly higher percentage of cytotoxic NK cells, NKT cells and CD8+CTLs in the spleens of tumor bearing mice that received [PtCl4(en)] when compared to animals that received vehicle or cisplatin. Moreover, cytotoxicity of splenocytes of LLC1+[PtCl4(en)]-treated mice against LLC1 cells was significantly higher than toxicity of spleen cells obtained from cisplatin+LLC1-treated animals, confirming immuno-stimulatory anti-tumor effects of [PtCl4(en)]. Activated NK, NKT cells and CD8+ T lymphocytes are capable of killing a broad range of cancer cells by detecting danger molecules highly expressed on the surface of cancer cells (NK and NKT cells) or tumor antigens presented in major histocompatibility complex I (CD8+ T cells) and may regulate the innate and adaptive immune responses through the secretion of inflammatory cytokines, such as tumor necrosis factor alpha and IFN-γ [43, 44, 45].

In conclusion, due to its anti-tumor cytotoxicity, biocompatibility and potential for stimulation of anti-tumor immune response, [PtCl4(en)] complex may be a good candidate for further testing in the field of medicinal chemistry.

Notes

Acknowledgements

This work was supported by grants from the Ministry of Education, Science and Technological Development, Republic of Serbia (Projects ON175069, ON175103 and 172011).

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Copyright information

© SBIC 2017

Authors and Affiliations

  • Milos Arsenijevic
    • 1
  • Marija Milovanovic
    • 2
  • Snezana Jovanovic
    • 3
  • Natalija Arsenijevic
    • 4
  • Bojana Simovic Markovic
    • 2
  • Marina Gazdic
    • 5
  • Vladislav Volarevic
    • 2
  1. 1.Department of Thoracic Surgery, Faculty of Medical SciencesUniversity of KragujevacKragujevacSerbia
  2. 2.Department of Microbiology and Immunology, Faculty of Medical Sciences, Center for Molecular Medicine and Stem Cell ResearchUniversity of KragujevacKragujevacSerbia
  3. 3.Department of Chemistry, Faculty of ScienceUniversity of KragujevacKragujevacSerbia
  4. 4.Department for Preventive and Pediatric Dentistry, Faculty of Medical SciencesUniversity of KragujevacKragujevacSerbia
  5. 5.Department of Genetics, Faculty of Medical SciencesUniversity of KragujevacKragujevacSerbia

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