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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2016 Jul 22;173(17):2633–2644. doi: 10.1111/bph.13543

Antitumour and antiangiogenic activities of [Pt(O,O′‐acac)(γ‐acac)(DMS)] in a xenograft model of human renal cell carcinoma

A Muscella 1,✉, C Vetrugno 2, F Biagioni 3, N Calabriso 4, M T Calierno 3, F Fornai 3,6, S A De Pascali 5, S Marsigliante 7, F P Fanizzi 5
PMCID: PMC4978158  PMID: 27351124

Abstract

Background and Purpose

It is thought that the mechanism of action of anticancer chemotherapeutic agents is mainly due to a direct inhibition of tumour cell proliferation. In tumour specimens, the endothelial cell proliferation rate increases, suggesting that the therapeutic effects of anticancer agents could also be attributed to inhibition of tumour angiogenesis. Hence, we investigated the potential effects of [Pt(O,O′‐acac)(γ‐acac)(DMS)] ([Pt(DMS)]), a new platinum drug for non‐genomic targets, on human renal carcinoma and compared them with those of the well‐established anticancer drug, cisplatin.

Experimental Approach

Tumour growth, tumour cell proliferation and microvessel density were investigated in a xenograft model of renal cell carcinoma, developed by injecting Caki‐1 cells into BALB/c nude mice. The antiangiogenic potential of compounds was also investigated using HUVECs.

Key Results

Treatment of the Caki‐1 cells with cisplatin or [Pt(DMS)] resulted in a dose‐dependent inhibition of cell survival, but the cytotoxicity of [Pt(DMS)] was approximately fivefold greater than that of cisplatin.

[Pt(DMS)] was much more effective than cisplatin at inhibiting tumour growth, proliferation and angiogenesis in vivo, as well as migration, tube formation and MMP1, MMP2 and MMP9 secretion of endothelial cells in vitro. Whereas, cisplatin exerted a greater cytotoxic effect on HUVECs, but did not affect tube formation or the migration of endothelial cells. In addition, treatment of the xenograft mice with [Pt(DMS)] decreased VEGF, MMP1 and MMP2 expressions in tumours.

Conclusions and Implications

The antiangiogenic and antitumour activities of [Pt(DMS)] provide a solid starting point for its validation as a suitable candidate for further pharmacological testing.

Abbreviations

[Pt(DMS)]

[Pt(O,O′‐acac)(γ‐acac)(DMS)]

MMPs

matrix metalloproteases

MTT

3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenol tetrazolium bromide

RCC

renal cell carcinoma

SRB

sulforhodamine B

Tables of Links

TARGETS
Enzymes
MMP1
MMP2
MMP9
LIGANDS
Cisplatin
VEGF
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Introduction

Angiogenesis, the process through which new blood vessels form from pre‐existing vessels, is pivotal in several physiological (reproduction, embryogenesis, wound healing) and pathological processes (diabetic retinopathy, psoriasis, chronic inflammation) (Carmeliet, 2003). Because tumours are unable to increase in size without the creation of new blood vessels (Folkman, 1975), angiogenesis plays a crucial role in the growth of solid cancers and metastases.

Angiogenesis has a pivotal role in the progression of most solid cancers, including those of lung, brain, colon, prostate, breast, bladder and cervix. Enhanced vascular density was also found in the bone marrow of patients with acute myeloid leukaemia or myeloma (Sagar et al., 2006). In addition, renal cell carcinoma (RCC), derived from the renal tubular epithelium, is characterized by hypervascularity and an increase in VEGF (Bard et al., 1986; Dutcher, 2013). RCC is the seventh most common cancer in men and the ninth in women, with over 210 000 cases, resulting in 102 000 deaths per year worldwide (Rini et al., 2009). RCC is resistant to traditional chemotherapy and radiation therapy (Motzer and Russo, 2000). Recently, several drugs have been accepted for RCC therapy: two of these agents inhibit mechanistic target of rapamycin (mTOR), four inhibit the VEGF receptor and hence prevent angiogenesis, and one is an antibody that binds to VEGF and impedes it linking with its receptor. These antiangiogenesis agents displayed clinical benefits (partial responses and stationary disease) in 60–70% of patients, increasing the median survival for responders, thus providing new options for those patients not suitable for or responsive to immunotherapy (Yang et al., 2003; Wilhelm et al., 2004; Motzer et al., 2013, 2006; Escudier et al., 2007; Rini et al., 2011). Obviously, in order to improve the prognosis, new chemotherapeutic molecules are needed.

Our previous studies had shown that [Pt(O,O′‐acac)(γ‐acac)(DMS)] ([Pt(DMS)]), a new platinum drug for non‐genomic targets designed and synthesized by some of us (De Pascali et al., 2009), has antimetastatic responses in vitro, decreasing the synthesis and release of metalloproteases and migration of tumour breast cells (Muscella et al., 2010). This compound has recently gained increasing attention as a potential anticancer agent because of its selectivity for cancer cells, as shown in immortalized cell lines and confirmed in primarily cultured normal and breast cancer cells (Muscella et al., 2007, 2008, 2013, 2011; Vetrugno et al., 2014) and in vivo (Muscella et al., 2014). Remarkably, in a preclinical model based on the s.c. injection of Michigan Cancer Foundation‐7 (MCF‐7) breast cancer cells, [Pt(DMS)] stands out as having higher anticancer activity than cisplatin. In Wistar rats, [Pt(DMS)] also displayed enhanced in vivo pharmacokinetics (PK), biodistribution and tolerability with respect to cisplatin. Noteworthy, in the PK studies, [Pt(DMS)] showed an extended stability in the systemic blood circulation and a reduced nephrotoxicity and hepatotoxicity, the two main target sites of the cytotoxicity of cisplatin (Muscella et al., 2014).

On the basis of these observations, in the present study we investigated whether the antitumour efficacy profile of [Pt(DMS)] in preclinical models could be attributable, in part, to its potential antiangiogenic activity. We therefore evaluated the anticancer effect of [Pt(DMS)] in a xenograft model developed by injection of Caki‐1 cells, derived from the clear cell variant of RCC (Glube et al., 2007). The latter is the most frequent subtype of kidney cancer, occurring in about 70–75% of cases. Clear cell tumours are commonly hypervascular; therefore, this type of cancer is a good model for determining the effectiveness of an antiangiogenesis therapy (Shuch et al., 2015). In addition, the effects of [Pt(DMS)] on angiogenesis were evaluated in vitro, using HUVECs, and compared (both in vivo and in vitro) with those of the well‐established anticancer drug, cisplatin.

Methods

Cell lines and cell culture

HUVECs were harvested and maintained as described previously (Massaro et al., 2010). In particular, human cells were obtained from discarded umbilical veins, and treated anonymously, conforming to the principles outlined in the Declaration of Helsinki (see Cardiovascular Research 1997; 35:2–3).

The authors did not collect the umbilical veins themselves, and the cells were anonymized before use by the authors. As such, approval from the University Ethics Review Board was not necessary (Massaro et al., 2010) (for previous use of this internal rule, and for further details, see Massaro et al., 2015).

Caki‐1 cells were obtained from American Type Culture Collection and were maintained in McCoy's 5A medium at 37°C in 5% CO2. Media were supplemented with 10% FBS, 10 mmol·L−1 HEPES buffer (pH 7.2–7.5), 100 U·mL−1 penicillin and 100 mg·mL−1 streptomycin.

Cell viability assay

Cells at 70–80% confluence were trypsinized (0.25% trypsin with 1 mmol·L−1 EDTA), washed and resuspended in growth medium. Then 100 mL of the cell suspension (105 cells·mL−1) were added to each well of a 96‐well plate. After overnight incubation, cells were treated with specific reagents for different incubation periods.

The SRB (sulforhodamine B) assay was used as an indicator of cell number as described previously (Muscella et al., 2010). In addition, he conversion of MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenol tetrazolium bromide) by cells was also used as an indicator of cell number as previously described (Muscella et al., 2010). The percentage of survival was calculated as the absorbance ratio of treated to untreated cells. Viable cells were also counted by the trypan blue exclusion assay and light microscopy.

The data presented are mean ± SD from eight replicate wells per microtitre plate.

In vivo xenograft experiments

Because animal experiments remain essential to discover improved methods to treat cancer, we developed a xenograft model of RCC, obtained by injection of human Caki‐1 cells.

Thirty‐two nude BALB/c mice (6‐weeks‐old, female, 20 to 30 g body weight) were purchased from Harlan (Carezzana, Italy) and maintained under pathogen‐free conditions. They were given free access to standard food and water, with a 12 h light–dark cycle at a temperature of 22+/−2°C.

Approximately 6 × 106 Caki‐1 cells were injected s.c. into the flank. Animals were monitored daily for general health, and body weights were measured twice weekly. Tumour size was measured with slide callipers, and volumes were calculated as (L × W 2)/2, where L and W are the major and minor diameters respectively. Once tumour volumes reached ~200 mm3, mice were randomly divided into four groups (eight animals per group), in such a manner as to minimize weight and tumour size differences among the groups. After administering a single i.v. injection of saline as a control, or two doses (5 and 10 mg·kg−1) of [Pt(DMS)] or 10 mg·kg−1 cisplatin, the tumour volumes of BALB/c mice were measured every 3 days. The maximum size the tumours were allowed to grow before the mice were killed was 2000 mm3.

The mice were killed after 35 days of treatment, and the tumours were excised. Tumours were divided and either flash frozen in liquid nitrogen, or placed in a paraformaldehyde solution (4%) and 20 h later placed in 70% ethanol until treated with paraffin.

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath & Lilley, 2015).

Immunohistochemical analysis

Sections were deparaffinized, and antigen retrieval was performed with absolute ethanol +5% acetic acid for 15 min at room temperature (RT) [for matrix metalloproteases 1 and 2 (MMP1 and MMP2) antibodies], with pepsin (4 mg·mL−1 in HCl 0.01% at 37°C in humid chamber for 60 min) (for VEGF antibody) and with citrate buffer pH 6.8 for 10 min (for Ki‐67 antibody), soaked in 3% hydrogen peroxide to block endogenous peroxidase activity, then incubated overnight with rabbit anti‐Ki‐67 (1:100; Thermo Scientific, Waltham, MA, USA), with goat anti‐MMP1 antibody (1:50; Santa Cruz Biotechnology, Dallas, TX, USA), with mouse anti‐MMP2 antibody (1:50; Millipore, Temecula, CA, USA), with rabbit anti‐VEGF antibody (1:50; Abcam, Cambridge, UK), and then sections were incubated for 1 h with their respective biotinylated secondary antibodies (1:200; Vector Laboratories, Burlingame, CA, USA). Control staining was performed in the absence of primary antibodies. The sections (MMP1 and Ki‐67) were counterstained with haematoxylin for 6 min. The positivity was carried out with 3,3‐diaminobenzidine tetrachloride (Vector Laboratories) and VEGF with NOVARED (Vector Laboratories).

The number of Ki‐67‐labelled nuclei was automatically counted in five random fields per single animal, using the cellsens Dimension programme (Olympus, Shinjuku, Tokyo, Japan). Counting was performed in a blinded manner, without knowing the assignment of tumours to specific treatment groups. The data are presented as mean ± SD of eight animals per group.

Protein expressions were quantified by image j software (NIH, Bethesda, MD, USA) using five fields, per single animal. Quantification analysis was performed in a blinded manner, and the data are mean ± SD of eight animals per group.

Immunofluorescence analysis

For the analysis of endothelial cells, the sections were treated with rat anti‐mouse CD31conjugated with Alexa Fluor 488 dye (BD Biosciences, San Jose CA, USA). The sections were incubated with for 2 h and cells counted using a microscope.

Microvessel density (MVD) of xenograft tumours was determined by using the method of Weidner et al. (1993). The slides that were stained with anti‐CD31 antibody were scanned at low magnification (×40 and ×100) to identify the five areas with the highest number of discrete microvessels staining for CD31. Then, the number of individual microvessels was counted on a 200× field and a 400× field, by two investigators, blinded to the treatments given to the animals or other pertinent variables. Subsequently, the MVD score was calculated as the mean of the amounts in these five areas. Finally, the data are presented as mean ± SD of eight animals per group.

Endothelial cell tube formation assay

The formation of HUVECs’ capillary‐like structures on a basement membrane matrix was used to investigate the antiangiogenic activity of [Pt(DMS)] and cisplatin. The 24‐well plate was coated with 200 μL matrigel (BD Biosciences) for 30 min at 37°C. HUVECs were seeded on the matrigel (1.5 × 104 cells per well) and cultured in medium containing [Pt(DMS)] or cisplatin (0.1–10 μmol·L−1), for 12 h. Tube formation was photographed, and the tube lengths were quantified by image j software.

Migration assays

Cells were seeded on 24‐well plates at a density of 1.5 × 105 cells per well. At post confluent state, wounds of 1 mm width were created, by scraping the cell monolayer with a sterile pipette tip. Photos, taken at a ×40 magnification, immediately after scraping and 24 h later, documented migration. Cell migration was quantified by measuring the distance between the wound edges before and after injury using the image j software.

Cell migration and invasion assays were also performed by using the QCM™ 24‐well Fluorimetric Cell Migration Kit (Merck Millipore, Darmstadt, Germany) and QCM 24‐well Fluorimetric Cell Invasion Assay Kit (Merck Millipore), respectively, according to the manufacturer's instructions. Both assays exploit a polycarbonate membrane with an 8 mm pore size, which in the invasion assay is coated with a thin layer of ECMatrix™ occluding the membrane pores and physically inhibits the passage of non‐invasive cells. Briefly, HUVECs treated with [Pt(DMS)] were loaded in the upper compartments, while in the lower chambers medium supplemented with 10% FBS was used as the chemoattractant. The plates were incubated for 18 h for the migration and 24 h for the invasion assay. Cells able to migrate through or invade the support were detached from the bottom and then fixed and stained with a fluorescent dye. The fluorescence of the migrated or invaded cells was evaluated using a 480/520 nm filter. Relative variations in the number of migrated and invaded cells were obtained by comparing the mean fluorescence signals of [Pt(DMS)]‐treated samples with those of untreated cells.

MMP gelatin zymography

After treatment, the culture medium was collected, and HUVECs were washed with PBS and harvested by scraping them into 0.1 mmol·L−1 Tris–HCl containing 0.2% Triton X‐100. Both conditioned medium and cell lysates were centrifuged at 450 × g for 5 min at 4°C to remove cells and debris as described previously (Muscella et al., 2010).

Reagents

[Pt(DMS)] was prepared according to previously reported procedures (De Pascali et al., 2009, 2005). Dulbecco's modified Eagle's medium, antibiotics, glutamine and FBS were purchased from Celbio (Pero, MI, Italy). All other reagents were from Sigma (Milan, Italy).

Statistical analysis

The experimenter measuring the tumours and the data analyst were unaware of the treatments given to the animals. Data, presented as mean ± SD, were collected in a blinded fashion and analysed using graphpad prism 5 software (GraphPad Software, La Jolla, CA, USA). Student's unpaired t‐test, the Mann–Whitney U‐test or one‐way ANOVA were used, and when this returned P < 0.05, post hoc analysis using Bonferroni test was performed; we used the Bonferroni‐Dunn post hoc test in the ANOVA after a significant result from the omnibus F‐test. P < 0.05 was accepted as a level of statistical significance. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015).

Results

Cytotoxicity of the drugs

Caki‐1 cells were treated with various concentrations of [Pt(DMS)] or cisplatin, and viable cell number was determined by SRB assay and confirmed by MTT assay 24, 48 h later. Equivalent results were achieved when cell number was directly evaluated by cell counting.

When Caki‐1 cells were incubated with cisplatin or with [Pt(DMS)] at concentrations ranging from 1 to 200 μmol·L−1, a dose‐dependent inhibition of cell survival was seen (Figure 1A). [Pt(DMS)] showed cytotoxicity approximately fivefold greater than that observed for cisplatin (for [Pt(DMS)], IC50 6.92 ± 0.084 μmol·L−1; for cisplatin, IC50 37.35 ± 0.5 μmol·L−1; n = 6) (Figure 1A).

Figure 1.

Figure 1

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Growth inhibitory effect of [Pt(DMS)] and cisplatin in vitro and in a xenograft model of RCC. (A) Caki‐1 cells were treated with or without increasing concentrations of [Pt(DMS)] or cisplatin, and cell viability was monitored by SRB assay over a period of 24 and 48 h. Data are presented as mean ± SD of six‐independent experiments with eight replicates in each and are presented as % of control; values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (B and C) Balb/c nude mice carrying RCC developed by injection of Caki‐1 cells (around 200 mm3) received i.v. [Pt(DMS)] (5 and 10 mg·kg−1) or cisplatin (10 mg·kg−1) (eight mice in each group); tumour volume (B) and body weight of mice (C) were measured every 3 days for 35 days in total. The results are presented as mean ± SD (animals per group n = 8). *P < 0.05, significantly different from saline control; # P < 0.05, significantly different between [Pt(DMS)] and cisplatin. (D) The resected tumours of the xenograft mice were analysed by immunohistochemical staining for Ki‐67. Representative slides of three groups are shown at original magnification, ×200. (E) The proliferative activity was evaluated by Ki‐67 staining. The percentage of positive tumour cells was then calculated using five fields, per single animal. The data are presented as the mean ± SD (animals per group n = 8). Statistically significant differences were determined using the Mann–Whitney U‐test. *P < 0.05, significantly different from saline control; # P < 0.05, significantly different between [Pt(DMS)] and cisplatin. Scale bar = 100 μm.

[Pt(DMS)] prevents cancer growth in a xenograft model of RCC

To assess the anticancer effect of [Pt(DMS)] in vivo, xenograft mice bearing Caki‐1 cells were produced and handled as described in the Methods section.

After tumours had grown to ~200 mm3, the mice were randomized into four groups in order to reduce the differences between the weight and the tumour size among the groups. After administering i.v. (i) saline as a control, or (ii) two doses (5 mg·kg−1 and 10 mg·kg−1) of [Pt(DMS)] or (iii) 10 mg·kg−1 of cisplatin, we measured the volumes of the tumours by a vernier calliper every 3 days for 35 days in total.

Over the course of 5 weeks, the mean cancer volume significantly increased from 198.50 ± 36.27 to 1490.32 ± 163.97 mm3 for the saline group, 753.57 ± 70.71 mm3 for the cisplatin group (10 mg·kg−1), 518.32 ± 57.23 mm3 for the group treated with 5 mg·kg−1 of [Pt(DMS)] and 455.22 ± 58.15 mm3 for the group treated with 10 mg·kg−1 of [Pt(DMS)] (Figure 1B). The results showed that [Pt(DMS)] significantly inhibited tumour growth in a dose‐dependent manner and that 10 mg·kg−1 [Pt(DMS)] was more efficacious than 10 mg·kg−1 cisplatin (Figure 1B).

Interestingly, in the Caki‐1 tumours, a dosage of 10 mg·kg−1 caused complete regression of two tumours, with two of eight animals being tumour free (based on gross observation) when the study was terminated at day 35.

From the beginning of the treatment until the resection of the tumour, no significant decrease of body weight was noticed in Figure 1C. In addition, over the observation time, no damage to health was seen in mice treated with 5 mg·kg−1 and 10 mg·kg−1 [Pt(DMS)], and the overall behaviour was not different between treated and untreated animals.

The expression of human Ki‐67 protein is strictly associated with cell proliferation: to investigate cell proliferation of treated and untreated tumours, immunohistochemical staining for Ki‐67 was performed (Figure 1D). The Ki‐67‐labelling data revealed that cancers from treated mice had reduced proliferation levels with respect to control; furthermore, the proliferation of tumours present in [Pt(DMS)]‐treated animals was significantly lower than that found in tumours of cisplatin‐treated animals (Figure 1E).

Antiangiogenic effects of cisplatin and [Pt(DMS)]

To determine whether the higher inhibitory effect of [Pt(DMS)] on tumour growth is also correlated to tumour angiogenesis suppression, the distribution of the endothelial marker CD31 was assessed. The number of vessels was significantly decreased in all treated groups, relative to the control group (Figure 2A and B), even if [Pt(DMS)] treatment more significantly decreased tumour microvessel when compared with control group or tumours from mice treated with cisplatin (Figure 2B).

Figure 2.

Figure 2

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Effects of [Pt(DMS)] or cisplatin on tumour angiogenesis and viability of HUVEC cells. (A) Following s.c. injection of Caki‐1 cells, mice received i.v. [Pt(DMS)] (10 mg·kg−1) or cisplatin (10 mg·kg−1). The resulting tumours were stained with CD31 antibody, and representative images for each treatment group are shown. Bar graph = 100 μm. (B) Angiogenesis was quantified, and values represent the mean ± SD of at least five results per single animal. Number of vessels per field was measured for mice with tumours treated with saline control (Ctr), (animals per group n = 8). Statistically significant differences were determined using the Mann–Whitney U‐test. *P < 0.05, significantly different from saline control; # P < 0.05, significantly different between [Pt(DMS)] and cisplatin. (C) In vitro cell viability assay was performed using HUVEC cells treated with increasing concentration of [Pt(DMS)] or cisplatin for 24 to 48 h. Data are presented as mean ± SD of six‐independent experiments with eight replicates in each and are presented as % of control. P < 0.0001 by one‐way ANOVA (n = 6); values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.

Inhibition of HUVECs proliferation by cisplatin and [Pt(DMS)]

To better understand the effects of [Pt(DMS)] and cisplatin on endothelial cells, we evaluated their influence on HUVECs in vitro. Exposure of the HUVECs to cisplatin and [Pt(DMS)] at concentrations ranging from 0.1 to 10 μmol·L−1 resulted in a dose‐dependent inhibition of cell survival (Figure 2D). The lowest concentrations of anticancer drugs that significantly decreased cell number were 0,5 μmol·L−1 for cisplatin and 1 μmol·L−1 for [Pt(DMS)] (Figure 2D). Thus, cisplatin was significantly more cytotoxic than [Pt(DMS)] (for cisplatin, IC50 2.35 ± 0.044 μmol·L−1; for [Pt(DMS)], IC50 11.20 ± 1.5 μmol·L−1; after 48 h; n = 6) (Figure 2D).

[Pt(DMS)] inhibited HUVECs tube formation

Tube formation of endothelial cells is one of the critical steps in the formation of new blood vessels (Patan, 2004). To characterize whether [Pt(DMS)] blocked endothelial cell tubulogenesis, we observed its effects on the capillary‐like framework composed of endothelial cells seeded on matrigel. Tubulogenesis was significantly decreased upon treatment with [Pt(DMS)] (Figure 3A and B). Precisely, 1 μmol·L−1, [Pt(DMS)] inhibited three‐dimensional tubular structures by 80%. Cisplatin was inhibitory at a dose of 5 μmol·L−1 for which we cannot exclude a possible cytotoxic effect that may hinder tube development in a non‐specific manner.

Figure 3.

Figure 3

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[Pt(DMS)] inhibits the formation of new vessels. Effect of increasing concentrations of [Pt(DMS)] or cisplatin for 16 h on Matrigel‐induced tube formation of HUVECs in vitro. Tube formation was monitored by an inverted phase contrast microscopy, and representative photographs are presented (A). Branch point relative variations after HUVECs were treated with [Pt(DMS)] (B) or cisplatin (C) were quantified by image analysis. *One‐way ANOVA for [Pt(DMS)]‐treated HUVECs versus controls (n = 6). Scale bar = 100 μm.

Sublethal concentrations of [Pt(DMS)] inhibited HUVECs migration and invasion

Migration plays an important role in angiogenesis, and it is a prerequisite for tumour cell invasion and metastasis. Using the wound closure assay and transwell migration assays, we observed that HUVECs treated with 0.1–1 μmol·L−1 [Pt(DMS)] led to a pronounced reduction in migration rate (Figure 4A and B). HUVECs treated for 24 h with 0.1–1 μM [Pt(DMS)] led to a pronounced decrease in invaded cells (Figure 4C). As shown in Figure 2D, these concentrations of [Pt(DMS)] were sublethal so these effects cannot be attributed to cytotoxicity.

Figure 4.

Figure 4

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[Pt(DMS)] inhibits gelatinase (MMPs) expression and endothelial cell migration. (A) HUVECs were treated or not with the indicated concentrations of [Pt(DMS)], and cell migration after 16 h was measured and was examined using the wound closure assay. Degree of wound closure was assessed by measuring the distance between wound edges in at least eight randomly chosen regions of six different experiments (mean ± SD, n = 6) normalized to 100% wound closure for control cells. Cell migration (B) and invasion (C) were also measured using the transwell invasion assay systems. The relative variations in the migrated and invaded cell number were obtained by comparing the mean fluorescence signal of [Pt(DMS)]‐treated samples with untreated cells. Data are means of five independent experiments with two replica platings each. P < 0.0001 by one‐way ANOVA; Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests. (D) Cells were treated or not with increasing concentrations of [Pt(DMS)] or cisplatin for 24 h; then, conditioned media were subjected to gelatin zymography.

In contrast, upon application of sublethal doses of cisplatin, a subconfluent cell layer formed, inducing cells to migrate not only into the wound but also into the monolayer voids; therefore, potential cisplatin effects could not be quantified. Cisplatin had inhibitory effects only at a dose of 10 μmol·L−1, which can also have cytotoxic effects.

[Pt(DMS)] suppresses the secretion of MMP1, MMP2 and MMP9

Extracellular proteolytic activity is fundamental throughout the course of endothelial cell migration and invasion across the basement membrane and neo angiogenesis. Thus, the secretion levels of MMP1, MMP2 and MMP9, which are the most important proteases in degrading the extracellular matrix, were measured. Gelatin zymography was used to study the effects of [Pt(DMS)] and cisplatin on both MMPs secretion and activation. As shown in Figure 4D, collagen zymography of HUVEC‐conditioned media showed three distinct MMP bands representing MMP1 (40–60 kDa), MMP2 (72 kDa) and MMP9 (95 kDa).

After treatment with different doses of [Pt(DMS)] (0.1 and 5 μmol·L−1) for 24 h, MMP1, MMP2 and MMP9 levels significantly decreased, thus contributing, at least in part, to the migratory ability of HUVECs.

Treatment of HUVECs with 5 and 10 μmol·L−1 of cisplatin caused a reduction in MMP2 and MMP1 activities in conditioned media; conversely, the activity of MMP9 remained unchanged (Figure 4D).

[Pt(DMS)] decreased MMP1, MMP2 and VEGF expression in a mouse xenograft model of RCC

To assess MMP1, MMP2, and VEGF expression in vivo, sections of tissue from the xenograft model of RCC developed by injection of Caki‐1 cells were analysed by use of an immunohistochemical technique. The analysis showed a significant decrease in all proteins considered in animals treated with [Pt(DMS)] and cisplatin compared with control animals; but [Pt(DMS)] was more effective than cisplatin (Figure 5).

Figure 5.

Figure 5

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[Pt(DMS)] decreases MMP1, MMP2 and VEGF protein expressions. Balb/c nude mice carrying breast cancer developed by injection of Caki‐1 cells received i.v. [Pt(DMS)] or cisplatin (10 mg·kg−1). The tumour tissues were removed and cut into 8 μm serial sections and used for histological analysis with specific antibodies. Representative light microscopy images of MMP1, MMP2 and VEGF staining, for each treatment group, are shown. Scale bar = 100 μm. Protein expressions were quantified by image j software using five fields, per single animal. The data are presented as the mean ± SD (animals per group n = 8). Statistically significant differences were determined using the Mann–Whitney U‐test. *P < 0.05, significantly different from saline control; # P < 0.05, significantly different between [Pt(DMS)] and cisplatin.

Discussion

Tumour angiogenesis is extremely important for the progression from a quiescent localized tumour to a developing one with the capacity to metastasize (Folkman, 2006). Blocking tumour‐induced angiogenesis is an attractive strategy for cancer therapy (Liekens et al., 2001). At the moment, several antiangiogenic molecules are used in association with cytotoxic drugs (Cesca et al., 2013) or in maintenance therapy (Johnson et al., 2013). [Pt(DMS)] has a high and selective cytotoxicity for cancer cells, as seen in immortalized cell lines and also in breast cancer cells in primary cultures and in a mouse xenograft model of breast cancer (Muscella et al., 2007, 2008, 2013, 2014, 2011; Vetrugno et al., 2014). In the present study, we concentrated on investigating the antiangiogenic capacity of [Pt(DMS)].

RCC, a vascular cancer resistant to standard chemotherapy, has hence become a model for investigating the efficacy of antian angiogenesis therapy. Because our aimwa s to assess a new therapeutic strategy for advanced metastatic clear cell RCC, for our investigations, we chose the Caki‐1 cell line derived from skin metastasis of clear cell RCC (Koch et al., 1995). In the in vivo tumour‐growth study, both [Pt(DMS)] and an established agent, cisplatin, significantly inhibited the growth of the xenograft tumours. [Pt(DMS)] had a greater activity in vivo and also when directly applied to tumour renal Caki‐1 cell line in vitro. In RCC, Ki‐67 has been shown to be a prognostic factor (Dudderidge et al., 2005): evaluation of the Ki‐67‐labelling substantiated that [Pt(DMS)] diminished cell proliferation more effectively than cisplatin. A likely explanation is that [Pt(DMS)] has extra effects on cancer cells: it exerts tumour growth inhibitory effects through direct cytotoxic effects and through the suppression of neovascularization. A support for this argument comes from our observation that [Pt(DMS)] diminished the MVD in the tumours much more effectively than cisplatin. Inhibition of endothelial cell migration and reduced formation of tube‐like structures in vitro further confirmed the antiangiogenic properties of [Pt(DMS)] observed in vivo.

The proliferation of endothelial cells is thought to be a main occurrence in angiogenesis and is hence a potential target for anticancer therapy (Chen et al., 2008). Thus, we studied the antiproliferative activity of [Pt(DMS)] and cisplatin against HUVECs. The results proved that HUVEC proliferation diminished significantly in a dose‐dependent manner, with cisplatin being more cytotoxic than [Pt(DMS)]. These support our previous results, demonstrating that [Pt(DMS)] is more cytotoxic in cancer than in normal cells (Muscella et al., 2013; Vetrugno et al., 2014). In addition, the inhibition of the proliferation of endothelial cell at the highest dose of [Pt(DMS)] used was not absolute, which could be of great importance for an antiangiogenic agent because cytotoxicity towards endothelial cells would signify damage to blood vessels in non‐cancerous tissue.

The use of cisplatin is linked to vascular toxicity and severe vascular complications (e.g. myocardial infarction and stroke) (Doll et al., 1986; Huddart et al., 2003; Vaughn et al., 2008). Such vascular toxicity is indicated by elevated plasma levels of von Willebrand factor and increased intimae media thickness of the carotid artery (Nuver et al., 2005). Furthermore, cisplatin might induce toxicity towards auditory, renal, peripheral sensory and autonomic nervous systems, likely ascribed to some microvascular damage caused by this drug (Kohn et al., 2002; Kirchmair et al., 2005; Yu et al., 2008). Albeit cisplatin had a good cytotoxicity on HUVECs; it did not influence tube formation, migration and invasion of endothelial cells. The capacity of endothelial cells to differentiate and to migrate out of existing blood vessels (in support of new capillaries organization) in response to specific stimuli is an important part of the angiogenesis process (Tsai et al., 2006). To examine the morphogenic potential of [Pt(DMS)], we used an in vitro assay that allows the visualization of multicellular tube‐like structures on matrigel, which resemble microvascular networks. The results showed that [Pt(DMS)] inhibits microvessel outgrowth, confirming its antiangiogenic properties observed in vivo.

Neovascularization shapes the dissemination of tumour cells, lastly facilitating metastasis. Indeed, the extent of vascularization of a solid tumour correlates to its metastatic potential (Nishida et al., 2006). In this context, we show that [Pt(DMS)] significantly decreased the migration of endothelial cells. This effect was not due to antiproliferative effects of [Pt(DMS)], inasmuch as it was observed using sublethal doses. Noteworthy, [Pt(DMS)] also inhibited angiogenesis at very low doses. This effect was unexpected because hormetic dose‐responses are frequently noted with antiangiogenic agents. Notably, a bell‐shaped dose‐response curve is observed when an agent evokes a stimulatory effect at low doses and an inhibitory effect at higher doses. Agents exhibiting hormetic dose‐response curves include plasminogen activator‐1, 5‐fluorouracil, endostatin, integrin inhibitors, interferon‐α, rapamycin, rosiglitazone, thrombospondin‐1, statins, TGF‐α1 and TGF‐α3 (Reynolds, 2010).

MMPs operate as regulators of the turnover of extracellular matrix by degradation of extracellular matrix proteins (Blavier et al., 2006). The extracellular proteolytic activity is important in the process of endothelial cell migration and invasion, during angiogenesis, and it is also an essential step in tumour invasion and metastasis. Previously, we showed that [Pt(DMS)] prevents the events leading to metastasis via alterations in MMP2 and 9 production, secretion and/or activity (Muscella et al., 2010). We showed here that [Pt(DMS)] down‐regulated the secretion of MMP1, MMP2 and MMP9 by HUVECs. Many studies have shown that MMP9 and MMP2 are required for the mobilization of the sequestered VEGF and the initiation of tumour angiogenesis (Bergers et al., 2000; Kalluri, 2003). Furthermore, VEGF induces MMP1 expression in human endothelial cells, and MMP1‐mediated extracellular matrix degradation in turn stimulates the expression and release of VEGF and angiogenesis (Hicklin and Ellis, 2005). Moreover, VEGF is immunosuppressive, and hence, its inhibition may also increase antitumour immunity (Gabrilovich et al., 1998, 1999; Finke et al., 2008). In the xenograft mouse, [Pt(DMS)] infusion inhibited the expression of both VEGF and MMP‐1 in tumours tissues, breaking this paracrine loop. Our results suggest that the anticancer action of [Pt(DMS)] results from both direct actions on cancer cells and indirect actions through endothelial cells.

Overall, [Pt(DMS)] displayed potent antiangiogenic activity at concentrations that were shown previously to be sublethal in healthy models. The antiangiogenic and antitumour activities in preclinical models were associated with plasma levels of [Pt(DMS)] corresponding to concentrations much higher than those demonstrating significant antiangiogenic activity in vitro and ex vivo (Muscella et al., 2014). Nevertheless, [Pt(DMS)] displayed decreased nephrotoxicity, hepatotoxicity and neurotoxicity, major target sites of cisplatin toxicity (Muscella et al., 2014). The activity demonstrated in the studies reported here is particularly significant, because RCCs currently represent some of the most difficult‐to‐treat tumours by conventional cytotoxic drug therapy. The cancer vascular cells do not generate drug resistance as often as the cancer epithelial cells, and thus, the discovery of a drug targeting angiogenesis is a very promising approach, and further studies are needed to fully investigate the mechanisms of the antiangiogenic effect of [Pt(DMS)].

In summary, these research findings indicate that the antiangiogenic effects of [Pt(DMS)] are due to various interactions at crucial steps in the angiogenic pathway. Both endothelial cell migration and VEGF expression are significant targets of [Pt(DMS)] and might be responsible for its effectiveness as an anticancer drug, which is currently emerging.

Author contributions

A.M., C.V., S.M. and F.P.F. conceived and designed the experiments. C.V., N.C., F.B. and M.T.C. performed the experiments. A.M. analysed the data. F.P.F. contributed reagents/materials/analysis tools. A.M., S.M. and F.P.F. contributed to the writing of the manuscript. S.A.D.P. synthesized and characterized the Pt compound. F.P.F. conceived and supervised the drug design, the synthesis and characterization of Pt compounds.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Acknowledgements

The authors acknowledge Italian Research and Education Ministry MIUR for funding this work within the CAROMICS project RBAP11B2SX_008 CUP: F81J11000340001. This work was also supported by the grant project, the PON (Programma Operativo Nazionale) 254/Ricerca, Potenziamento del ‘Centro Ricerche per la Salute dell'Uomo e dell'Ambiente’ PONa3_00334.

Muscella, A. , Vetrugno, C. , Biagioni, F. , Calabriso, N. , Calierno, M. T. , Fornai, F. , De Pascali, S. A. , Marsigliante, S. , and Fanizzi, F. P. (2016) Antitumour and antiangiogenic activities of [Pt(O,O′‐acac)(γ‐acac)(DMS)] in a xenograft model of human renal cell carcinoma. British Journal of Pharmacology, 173: 2633–2644. doi: 10.1111/bph.13543.

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