Suramin augments the antitumor and antimetastatic activity of pentoxifylline in B16F10 melanoma

Authors

  • Pooja Dua,

    1. Gude Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India
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  • Arvind Ingle,

    1. Animal House, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India
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  • Rajiv P. Gude

    Corresponding author
    1. Gude Lab, Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India
    • Cancer Research Institute, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India
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    • Fax: +91-22-27412894/93/92.


Abstract

Rapid tumor growth and metastasis are 2 major problems associated with treatment of malignant melanoma. Therefore, drugs that can intervene these processes are of clinical importance. Pentoxifylline (PTX), a methyl xanthine derivative, has been shown to inhibit B16F10 melanoma tumor growth and metastasis. We hypothesized that suramin when combined with PTX enhances its antineoplastic effects, which we have examined using the B16F10 mouse melanoma model. Suramin in simultaneous or sequential combination potentiated the cytotoxic effects of PTX on B16F10 cells. PTX arrested cells in the G0-G1 phase and suramin augmented the effects. Both the drugs inhibited F10 adhesion to laminin, matrigel and collagen type IV and showed enhanced inhibition in combination The combination also demonstrated significantly higher inhibition in cell motility (p = 0.002) and invasion through matrigel (p = 0.005) as compared to the single agents. Suramin synergized with PTX in its effects on secretion of MMP-9 gelatinase. DBA2/J mice implanted with intradermal B16F10 tumor were used as a model to study tumor growth. Animals were intratumorally treated with 50 mg/kg of PTX, 10 mg/kg of suramin and their combinations. Simultaneous administration of the drugs inhibited tumor growth by 5- to 6-folds. Tumor growth was completely blocked in sequential regimen with regression in some cases. The number and size of metastatic nodules on lung was also reduced significantly by the combination treatment. In conclusion, the novel combination of PTX and suramin has synergistic antitumor and antimetastatic activity in B16F10 melanoma and may be a promising approach in treatment of patients suffering from malignant melanoma. © 2007 Wiley-Liss, Inc.

Malignant melanoma is the deadliest form of skin cancer, and its incidence has shown a 6-fold increase since the past 2 to 3 decades.1 There is no standard treatment for patients with metastatic melanoma. Treatment options include the surgical resection of isolated metastases, therapy with dacarbazine, and immunotherapy.2 However, response rates associated with these measures range between 15 and 20%, and hence there is an urgent need for the development of new therapies. The success of antimetastatic therapy depends on inhibition of diverse stimuli produced by the tumor and its microenvironment while limiting overall toxicity. Thus it has been suggested that a combination of antimetastatic compounds may be more effective than monotherapies.

In our earlier reports we have demonstrated that pentoxifylline (PTX), a methyl xanthine derivative inhibits B16F10 melanoma solid tumor growth and metastasis to lung,3 which is mediated via its inhibitory action on cell adhesion, MMP-9 and -2 secretion,4 and tumor angiogenesis.5 PTX has been used to enhance tumor sensitivity to both radiation6 and alkylating agents7 because of its antioxidant nature and its property to abrogate the G2-M checkpoint, respectively. It has also been reported as a phosphodiesterase inhibitor.8 It inhibits TNFα production9 and NFκβ activation.10 In view of the properties of this drug, it would be advantageous to derive its combination with a compound that can inhibit the outside-in signaling as well, thus completely blocking the growth- promoting cues.

Till date, extensive research has demonstrated that stromal relationships are essential for growth and metastasis of melanoma.11 Melanoma cells interact with their environment not only by direct cell–cell contact, but also through the release of cytokines and growth factors. These by autocrine and paracrine effects enable them to grow autonomously and confer competence to metastasis. The paracine effects mainly include extracellular matrix (ECM) production, modulation of host immune response, vasculogenesis, secretion and activation of proteolytic enzymes, adhesion and motility. Hence, compounds that can block these autocrine and paracrine signaling can have potential clinical application.

One such compound suramin inhibits the binding of growth factors to their receptors present on cell surface, thus inhibiting the outside-in signaling.12 Chemically it is a polysulphonated napthyl urea and has been used long since for the treatment of trypanosomiasis. It is a potent inhibitor of PDGF, IGF, bFGF13, 14 and VEGF,15 which also brings it in focus as an antiangiogenic compound.16 On account of its antiproliferative effects, it has been used for treatment of various malignant cancers. It is in clinical trials for some metastatic cancers such as renal, prostate carcinomas and lymphomas, but has not been considerably successful because of its broad spectrum of toxicity.17, 18 Therefore current studies use suramin in low concentrations as a sensitizer or to reverse b FGF-induced chemoresistance.19 Many preclinical and clinical trials are also being carried out where suramin is being combined with other compounds to reduce its effective concentration and overall toxicity.20, 21

B16F10 melanoma metastasis has shown to be inhibited with suramin.22 It inhibits melanoma cell invasion to brain by inhibiting heparinase activity.23 Considering the aberrant expression of growth factors like b FGF in melanoma cells,24 the efficacy of suramin treatment can be attributed to some extent to its interference in these heparin binding growth factors.

The information regarding the properties of both the compounds suggests that their combined effects could possibly be synergistic in nature, which we have evaluated using the B16F10 melanoma model. Our in vitro studies show that suramin synergizes the antimetastatic effects of PTX. Furthermore, the combination resulted in an enhanced reduction in B16F10 tumor growth and metastasis as compared to PTX alone.

Material and methods

Cell culture

B16F10, a highly metastatic lung selected subline derived from C57/BL6 murine melanoma,25 was purchased from National Centre for Cell Science (Pune, India). The cell line was maintained as a continuous culture in Iscove's minimum Dulbecco's medium (IMDM; GIBCO BRL, MD, USA) supplemented with 10% fetal bovine serum (GIBCO BRL), 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were grown in a humified atmosphere of 5% CO2 and 95% air at 37°C. Media was replenished every third day.

Cytotoxicity assay

B16F10 cells were seeded in 96-well microplates at a density of 5 × 103 cells/well. Cells were allowed to grow and stabilize for 24 hr. They were then treated with serial dilutions of pentoxifylline (1–15 mM) and suramin (0.2–8 mM) for 24 hr to find their IC50 values. Simultaneous (S + P) and sequential (S − P) combination studies were carried out where PTX and suramin were combined in ratios of 4:1, 5:1 and 6:1. We intended to use low concentrations of suramin to keep a check on its toxicity issues and to use it as a sensitizer. Therefore, these ratios were preferred to their equipotent ratio (2:1). The treatment was started with a sequential combination in which cells were exposed to suramin for 24 hr. Thereafter, cells were washed with phosphate buffer saline (PBS) and incubated with PTX for the next 24 hr. At this time, cells were also exposed to single treatments of suramin or PTX and their mixtures in the simultaneous combination, for 24 hr. Each treatment was done in 6-well replicates. Posttreatment cell viability was determined by MTT colorimetric assay. Briefly, MTT reagent (Sigma Aldrich) was added to each well to make a final concentration of 1 mg/ml of media and incubated for 4 hr at 37°C. Formazan crystals were dissolved in 100 μl of DMSO; the optical density was measured in an enzyme-linked immunosorbent assay plate reader (Molecular Devices, Spectra Max 190 with Soft max Pro) at 540 nm with a reference wavelength of 690 nm. Drug effects have been shown as fraction of cell population affected/fraction unaffected (fa/1 − fa).

Flow cytometry analysis of cell cycle distribution

Sub confluent B16F10 cells were treated with 3 mM PTX and 0.6 mM suramin, alone or in combination, for 24 hr. Cells were harvested, washed twice with PBS and fixed in chilled 70% ethanol. After centrifugation, the fixed cell pellet was treated with RNAse at a concentration of 0.5 mg/ml (MBI Fermentas) and finally stained with propidium iodide (50 μg/ml; Sigma Aldrich) for 10 min at room temperature. Ten thousand events were acquired on FACS CALIBUR (Becton-Dickinson) and were analyzed using Modfit software.

Adhesion assay

Adhesion assay was carried out as described.4 Ninety-six well microplate was coated with ECM substrates (50 μl/well): matrigel (10 μg/ml), fibronectin (2.5 μg/ml), collagen type IV (50 μg/ml) and laminin (5 μg/ml). Plates were kept overnight at 4°C for polymerization. Unpolymerized substrates were washed with PBS and the plates were blocked with 2% BSA for 2 hr at 37°C. Subconfluent B16F10 cultures were treated with 3 mM PTX, 0.6 mM surmin alone or in combination for 24 hr. The cells were harvested using saline EDTA, washed and diluted to a final concentration of 3 × 105 cells/ml in IMDM containing 0.1% BSA. Hundred microliters of the cell suspension was added to each substrate-coated well and cell adhesion was seen for15–60 min at 37°C. At the end of incubation, nonadherent cells were removed by giving 2 washes with PBS. The adherent cells were quantified using MTT assay and expressed as a percentage relative of the respective total unwashed cells (adherent as well as nonadherent).

Invasion assay

The cell invasion assay was performed as described previously with some modifications.26 Briefly, 6.5-mm-diameter polycarbonate filters of 8-μm pore size (Millipore) were coated with matrigel at a concentration of 40 μg/insert and kept in a 24-well plate overnight at 37°C. The unpolymerized matrigel was washed with IMDM. Lower chambers were filled with 300 μl of NIH3T3 conditioned media as a chemotactic factor. The cells were pretreated for 24 hr with 3 mM PTX, 0.6 mM suramin and their combination. Inserts were charged with 0.2 million cells in IMDM containing 1% BSA. After 12-hr incubation at 37°C, the suspended media in the lower chamber was collected. Cells from the underside of the insert were harvested and pooled to the collected media. Following centrifugation, the cell pellet was suspended in 30 μl of PBS and counted under a phase contrast microscope (5 fields per sample). The experiment was carried out in duplicate and repeated at least twice.

Condition media preparation and gelatin zymography

To investigate the effect of treatment on secretion of matrix metalloproteinases (MMPs), gelatin zymography was performed for gelatinases MMP-2 and MMP-9 as described.27 Briefly, subconfluent cultures were treated with 3 mM PTX, 0.6 mM suramin alone or in combination for 24 hr. Cells were then washed with PBS and further incubated in serum-free IMDM for 18–20 hr. Conditioned media so obtained was centrifuged to remove the cell debris. Media was dialyzed against PBS and was concentrated 10-fold by lyophilization. The concentrated conditioned media was normalized with respect to cell count and stored at −80°C until further use. To assess the gelatinase activity, samples were run on 10% SDS-PAGE containing 0.1% gelatin (w/v). Gel was washed in developing buffer (50 mM Tris, 100 mM CaCl2, 1 μM ZnCl2, 1% Triton X 100, 0.02% NaN3; pH 7.5) for 1 hr at room temperature and further incubated in the same buffer at 37°C for 48 hr. The gels were stained in Coomassie Brilliant Blue R250 (Sigma Aldrich) and destained in a solution of methanol, water and acetic acid mixture (45:45:10 v/v). Enzymatic activity was visualized as clear zones on a blue background. HT1080 conditioned medium was used as a positive control.

Wound assay

Cells were plated in 35-mm Petri plates. At 60% confluency, they were treated with 3 mM PTX, 0.6 mM suramin and their combination for 24 hr. At the end of incubation, cells were washed with PBS and a wound was induced on the monolayer as described.28 A zero time point, wound was kept as reference plate. Remaining plates were incubated for 12–16 hr in the presence of serum-free IMDM. Subsequently, the plates were fixed and the wound width was measured using PALM ROBO software. Percent wound width was calculated considering initial wound width as 100%. Photographs were taken under phase contrast microscope at 10× magnification.

Animal studies and treatment regimen

Six- to 8-week-old DBA2/J mice obtained from Animal Care Facilities, ACTREC were used for the experiments. The institutional review board of animal experiments reviewed all animal studies.

  • 1Intradermal tumor model: Sex-matched 8-week-old DBA2/J mice were shaved on the ventral side and challenged intradermally with 2 × 105 cells in PBS. Mice were randomly assigned to 5 groups (5 mice/group): (A) control (saline), (B) PTX alone, (C) suramin alone, (D) simultaneous combination, (E) sequential combination.This animal model was used to see the effects of compounds on tumor growth. Post 3 days of implantation, palpable tumors were seen with average tumor volume of 17.69 ± 4.4 mm3. This was considered as Day 1 and treatment was given through intratumoural route in which 50 mg/kg of PTX was given to Group (B), 10 mg/kg of suramin to Group (C) and (E), and mixture of both to Group (D). On Day 2, PTX was given to Group E. This alternative cycle of treatment was repeated till Day 8. Every alternate day tumor volume was measured with the help of a vernier calipers and body weight of mice was taken. The mice were skilled on Day 9. Tumor volume was calculated by the formula:
    equation image
    a is the length and b is the breadth of the tumor implant. Tumor volumes were then converted into relative tumor volume29 (RTV) using the formula:
    equation image
    TV0 is the tumor volume at Day 1 and TVx is the tumor volume at the respective day.
  • 2Experimental metastasis assay: Six- to 8-week-old female DBA/2 mice were injected with 0.1 ml of PBS containing 0.1 million B16F10 cells through intravenous route. Mice were randomly assigned to 5 groups (6 mice/group), as mentioned earlier. The mice were treated from Day 1 onward with 50 mg/kg PTX or 10 mg/kg suramin and their simultaneous and sequential combination through intravenous route in the same fashion as described for the intradermal tumor model. Two cycles of treatment were completed. The mice were killed on Day 20, and the melanotic colonies on the surface of the lung were counted. The lung sections were fixed, stained with hematotoxylin–eosin and were observed under phase contrast microscope. The lung area covered by tumor colonies was measured using Palm Robo software.

Statistical analysis

Median effect analysis

The interaction between PTX and suramin was analyzed using the CalcuSyn software program (Biosoft, Ferguson, MO) to determine whether the combination was antagonistic, additive or synergistic. Data from cell viability assay (MTT) were expressed as the fraction of cell viability inhibited by the individual drugs or the combination versus untreated cells. This program is based upon the Chou-Talalay method,30 which calculates a combination index (CI), and analysis is performed based on the following equation: CI = (D)1/(Dx)1 + (D)2/(Dx)2 + (D)1(D)2/(Dx)1(Dx)2. Here (D)1 and (D)2 are the doses of Drug 1 and Drug 2 that have x effect when used in combination and (Dx)1 and (Dx)2 are the doses of Drug 1 and Drug 2 that have the same x effect when used alone. When CI = 1, this equation indicates additive effects. CI below 1.0 indicates synergism and greater than 1 indicates antagonism. Besides CI values, the software also computes the reduction in the effective concentration of the compounds in combination, which is represented in the form of drug reduction index values (DRI). A DRI value more than 1 indicates presence of synergism.

One-way analysis of variance (ANOVA) with Tukey Multiple comparison test was used to determine significant differences between treatments for the in vitro experiments. In vivo data on tumor growth was analyzed by 1-way ANOVA for repeated measures with Dunnett test. Experimental metastasis results were analyzed with Mann–Whitney U test. p value ≤ 0.05 was regarded as significant. All experiments results are shown as means ± SE.

Results

Synergism in the cytotoxic effects of the combination

The cytotoxic effects of the drugs on B16F10 cells were studied for a period of 24 hr. The half maximal inhibitory concentration (IC50) of PTX found out is 11.65 ± 0.573 mM and of suramin is 5.13 ± 0.213 mM. Combination studies carried out showed that the drugs potentiate each other's effects at lower concentrations. Combination at the ratio of 5:1 gave best results, dose–effect curve of which has been shown. (Fig. 1). Both the simultaneous and sequential combinations showed a drop in IC50 values of the drugs (Table I).

Figure 1.

Cytotoxic effects of the drugs on B16F10 melanoma. PTX, suramin and their combination treatment inhibit B16F10 melanoma cell viability. Cells were treated with serial dilutions of PTX and suramin or their simultaneous and sequential combination for 24 hr. Cell viability was measured using MTT colorimetric assay. Drug effects (fraction of cell population affected/fraction unaffected) are plotted on the Y-axis and drug concentration on the X-axis of dose–response curves. Data presented are the mean of 6-well replicates for each treatment. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Table I. Inhibitory Concentration 50 (IC50) of Suramin and PTX for B16F10 Cells when Given Alone or in combination for a Period of 24 hr
Treatment modalityPTX/suraminS + PS − P
IC50 PTX11.3778.388.59
IC50 suramin5.61171.691.719

The effects of the single treatment and their combination were analyzed using Chaou and Talalay software for median effect analysis, which gave the CI values for all the combinations studied. Drug combinations at lower concentrations were synergistic. With the increase in concentrations, the combination turned out to be additive and further antagonistic in nature as shown in the Table II. The simultaneous and sequential combination of 3 mM PTX and 0.6 mM suramin showed significant cytotoxic effects (p < 0.02 vs. PTX and suramin) and was synergistic in nature (CI 0.78 and 0.687, respectively), and so was chosen for the further studies.

Table II. Combination Index Values of The Combination of PTX and Suramin Studied
Conc. of P:S (mM)S + PS − P
% inhibitedCI% inhibitedCI
01:0.218.70.45190.442
3:0.6300.78330.687
06:1.2401.05838.51.005
10:2.051.91.15654.51.055

Suramin augments the effects of PTX on cell cycle progression

To see whether the synergism in the cytotoxic effects correlates with the effects on the cell cycle, posttreatment cells were analyzed for changes in the cell cycle phase distribution. PTX-treated cells showed G0-G1 arrest (90% as compared to 77% in control), which resulted in decrease in S-phase population. At a concentration of 600 μM, suramin did not show any significant changes in cell cycle profile but potentiated the effects of PTX in combination (Fig. 2). Both sequential and simultaneous treatment of the drugs resulted in further increase in number of cells in the G0-G1 phase (94 and 96%, respectively). In sequential combination, there was a significant increase in the population of cells in sub G1 phase, which contributes to around 10% to the total cell distribution.

Figure 2.

Cell cycle profile of B16F10 cells treated with PTX, suramin and their combinations S + P, S − P. Posttreatment cells were fixed, stained with propium iodide and acquired by flow cytometer. Number of cells in each phase is represented as mean ± SE, and is an average of 3 independent experiments. Three millimolar PTX treatment resulted in a block in G0-G1 phase transition and combining the drug with 0.6 mM suramin potentiated this effect.

Combination results in increased inhibition in cell adhesion to ECM components

Effects of PTX (3 mM) and suramin (600 μM) alone or in combination were studied on cell adhesion to ECM components (Fig. 3). Within 15 min, more than 80% cells got adhered to laminin, matrigel and fibronectin but binding to collagen type IV was lesser as compared to these. PTX inhibited 17.4% adhesion to matrigel as compared to control, whereas suramin had only marginal effects. Simultaneous combination resulted in ∼64% inhibition that was significantly higher than PTX and suramin alone (p = 0.0001), and sequential regimen showed 53% inhibition. Adhesion to laminin was inhibited by both PTX and suramin (59 and 48%, respectively). Both the combinations resulted in a significantly higher inhibition of around 90% (p < 0.0002, vs. PTX and suramin). Inhibition in binding to collagen type IV was maximum as compared to the other substrates. PTX treatment resulted in 77% inhibition and suramin 72%. Simultaneous combination gave 90% inhibition, whereas there was a marginal binding in sequential combination resulting in ∼99% inhibition (p < 0.0004 vs. PTX and suramin). Both the compounds did not show any significant inhibition in binding to fibronectin. However, the binding was seen to comparatively reduce in combination, but the differences were not significant as compared to individual drugs.

Figure 3.

Effects of the treatment on cell adhesion to ECM substrates. Adhesion to the ECM substrates was quantified by MTT assay and is expressed as percent relative to respective unwashed cells. Values are mean of 6-well replicates ± SE and are a representative of 3 independent experiments. ANOVA with Tukey comparison test was used for determining the significance in the differences. Three millimolar PTX and 0.6 mM suramin showed significant inhibition in binding to matrigel, laminin and collagen type IV. (*) Indicates that combination significantly augments the effect of PTX and suramin on F10 adhesion (p < 0.0004).

Effects of the drugs and their combination on F10 invasion through matrigel

Untreated and pretreated F10 cells demonstrated different capacity of invasion through matrigel-coated inserts (Fig. 4). Considering the invasion in untreated cells as 100%, PTX and suramin treatment showed 32.4 and 42% invasion, respectively. Sequential combination resulted in 32% invasion (p = 0.005 vs. suramin). However, simultaneous treatment showed better effects than sequential regimen (17.74%) and turned out to be significantly different to that of suramin (p = 0.0002) and PTX (p = 0.0019).

Figure 4.

Effect of treatment on B16F10 invasion through matrigel. Number of cells invaded has been represented in percent invasion considering invasion in control as 100%. Experiment was carried out in triplicates, and values have been shown as mean ± SE. Three millimolar PTX and 0.6 mM suramin inhibited F10 invasion through matrigel, and the effects were augmented in combination. (#) Indicates significant differences in % invasion of the combinations vs. PTX (p = 0.0019) and (*) vs. suramin (p ≤ 0.005). ANOVA with Tukey multiple comparison tests have been used for the calculation of significance.

Inhibition in secretion of MMP-9 gelatinase

MMPs are key enzymes involved in the process of cancer cell invasion and metastasis. To study the effect of drugs on the secretion and activity of these proteases, gelatin zymography was carried out. As can be seen (Fig. 5), concentrated serum-free media revealed the band of lysis at 105 and 95 kDa that corresponds to the latent and the active forms of MMP-9, respectively. A difference of 13-kDa sequence in the mouse and human MMP-9 protein explains the difference in the position of the MMP-9 bands of B16F10 and HT1080. Densitometry analysis of the gel showed a significant inhibition in the enzymatic activity of MMP-9 with PTX and suramin treatment (23 and 50%, respectively) and the inhibition was enhanced in combination. Sequential treatment resulted in 60% inhibition in the activity (p = 0.001 vs. PTX), whereas simultaneous treatment of the compounds resulted in 75% inhibition, which is significantly higher than that of PTX (p = 0.0001) and suramin (p = 0.016).

Figure 5.

Gelatin zymography of condition media of B16F10 cells treated with 3 mM PTX, 0.6 mM suramin, and their combinations shows inhibition in MMP-9 secretion (a). HT1080 condition media was used as a positive control. Densitometric analysis of the zymogram was done and percentage of relative density was calculated with respect to untreated control (b). (#) Indicates the significant difference in density of PTX vs. simultaneous (p = 0.0001) and sequential combination (p = 0.001). (*) Indicate significant difference between suramin and simultaneous combination (p = 0.016; ANOVA with Tukey multiple comparison test).

Effects of combination of PTX and suramin on cell motility

Wound assay was carried out to determine the effects of 3 mM PTX, 600 μM of suramin and their combination on F10 cell motility. Within 20 hr, more than 85% of the wound width was covered in untreated cells, whereas single treatment of 3 mM PTX and 0.6 mM suramin resulted in around 60% wound closure (p < 0.001 vs. control; Fig. 6). The effects of the treatment were augmented in combination treatment where sequential treatment gave 46% wound coverage, and simultaneous exposure of the drugs resulted in only 34% wound closure (p < 0.002 vs. PTX and suramin).

Figure 6.

B16F10 wound closure. (a) Micrograph of wound widths was taken at 10× magnification. Cells were treated for 24 hr and wounded with a sterile plastic tip. Wound width was measured after 20 hr of incubation. Initial wound width (0 time point) was taken as a reference. PTX, suramin, and their simultaneous and sequential combination inhibit the wound closure. The initial and final wound widths were measured and expressed as percentage of the initial wound width (Fig. 6b). Results are average of triplicate measurements, SE. (*) Indicate significant differences in the wound width 3 mM PTX and 0.6 mM suramin (p < 0.001 vs. control). (#) Represents significant difference in the wound width of the combination (p < 0.002 vs. PTX and suramin; ANOVA with Tukey test).

Combination inhibits intradermal implanted B16F10 melanoma tumor growth

Mice intradermal implanted with B16F10 cells showed tumor latency period of 3 days (Fig. 7). Thereafter (Day 1), treatment was started and tumor volume was measured. Intravenous treatment of 10 mg/kg of suramin resulted in a significant inhibition in tumor growth, with RTV of 5.5 (p = 0.044 vs. control) and PTX 50 mg/kg treatment resulted in RTV of 6.12 as compared to 10.67 of untreated at Day 9. The effects of the single treatment were enhanced when the drugs were combined. Simultaneous treatment of mice with both the compounds resulted in further reduction in tumor growth throughout the schedule of treatment with RTV of 2.48 at Day 9 (p = 0.005 vs. control). In sequential treatment, no tumor growth was observed even after the treatment was discontinued. In contrast there was a regression in the tumor size on Day 9 as compared to that of Day 1 (as shown in the graph 7b), resulting in a RTV of 0.75 (p < 0.002 vs. control). Sequential treatment showed significant differences in tumor volume on Day 9 as compared to that of PTX (p = 0.02) and suramin (p = 0.045). However, the tumor growth throughout the study in the combinations did not differ significantly to that of single agents.

Figure 7.

Effect of treatment on the growth of intradermally implanted B16F10 melanoma tumor. Skin sections of DBA2/J implanted with B16F10 cells shows inhibition in tumor growth and microvascular density on intratumoral treatment with 50 mg/kg PTX, 10 mg/kg suramin and their combination as compared to untreated control (a). Tumor volume was measured each day and is represented by relative tumor volume (ratio of tumor volume on Day x to tumor volume on Day 1) on Y-axis (b). Mean values of RTV (n = 5) with standard deviations are indicated. (*) Indicate significant difference with untreated control (p < 0.04, ANOVA for repeated measures with Dunnett test). (#) Indicate differences in the tumor volume at Day 9 when compared to PTX and suramin (p < 0.05, ANOVA with Tukey test). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Inhibition in experimental metastasis of B16F10 cells to lung

Effects of the compounds when given alone or in combination were studied on experimental metastasis of B16F10 cells to the lung. The number of melanoic F10 colonies on the lung surface was counted, and the lung area covered by the tumor colonies was measured (Fig. 8). Out of 0.1 million cells injected, 440 ± 45 cells showed colonies on the lung. Intravenous treatment of 50 mg/kg of PTX resulted in 45% inhibition in the number of these metastatic colonies (p = 0.028), whereas in case of suramin, 10 mg/kg of the drug inhibited 55% of pulmonary metastasis as compared to untreated control (p = 0.018). In combination regimen, the effects were enhanced to 71% in simultaneous (p = 0.006 vs. control) and 75% in sequential treatment (p = 0.006 vs. control). Both the combinations showed significantly different inhibition with respect to PTX (p = 0.047) but not as compared to suramin. Microscopic evaluation of the histology sections showed that 40% of lung area was covered with the melanoma colonies in untreated control and on treatment with the single agents there was a significant reduction in it. In PTX-treated group only 13.9% of lung area was covered (p < 0.005 vs. control), and with suramin treatment 11.8% area was occupied by melanoma nodules (p < 0.005). In the combination treatment, 2.6–4% of lung area was occupied by the nodules, which is significantly less than the single treatments (p < 0.031).

Figure 8.

Effect of intravenous treatment of 50 mg/kg PTX and 10 mg/kg suramin on B16F10 experimental metastasis to lung (a). Cells were injected through tail vein. After 20 days mice were killed and the pulmonary nodules of B16F10 melanoma were counted and represented as percent nodules considering control as 100% (b, black bar). PTX and suramin inhibited the number of melanotic colonies on the lung (# p < 0.03 vs. control) with additive effects in the combination (*p = 0.006). Both the combinations showed significantly higher inhibition as compared to PTX (p = 0.047) but not as compared to suramin (Mann–Whitney U test). (c) HE-stained sections of the lung show tumor islands. The area of the lung occupied by the colonies in the histology sections was measured microscopically. The treatment reduced colony size and the effects were enhanced on combination (b, grey bar). (@) Indicate significant difference in the values of combinations and the single treatments (p ≤ 0.032; Mann–Whitney U test). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Discussion

One of the major problems in melanoma (besides the rapid primary tumor progression) is an early and widespread metastatic tumor growth. To address this issue, in the present study we have developed a novel combination of suramin with PTX that successfully targets B16F10 melanoma growth and metastasis.

In the first of our studies reported here we investigated the cytotoxic effects of PTX and suramin or a combination of both on B16F10 cell viability. The sequential combination of suramin followed by PTX was chosen than vice versa for all the in vitro and in vivo studies. This is based on the pharmacokinetic profile of both the compounds. Suramin has a very slow clearance, and thus a long half life of ∼40 days.31 In contrast, PTX is cleared off very fast.32 Therefore, suramin administration followed by PTX is more appropriate as it gives a window where both the drugs are present and can interact. Suramin and PTX treatment for 24 hr showed concentration-dependent inhibition in F10 cell viability. Combination of the drugs potentiated each other's effect, thus leading to a drop in the IC50. The analysis of the drug interactions showed that the combination at the lower concentration was synergistic, which turned into additive and further antagonistic with the increase in the concentrations. The potency of the combination treatment was also supported by the DRI values, which were more than 1 for the combinations that were found to be synergistic (data not shown). The combination of 0.6 mM suramin and 3 mM PTX was found to be synergistic in both simultaneous and sequential exposure of the drugs with optimum effects, and thus was chosen for the further in vitro studies. Cell cycle profile of the treated cells indicated that this synergism may be due to an increase in the number of cells getting arrested in the G0-G1 phase, indicating that suramin augments the inhibitory effects of PTX on G0-G1 transition. Sequential combination showed a small sub G1 peak, indicating presence of some cells going into apoptosis. No apoptotic population was observed with simultaneous exposure.

The adhesion of tumor cells to ECM is crucial for metastasis.33, 34 This is followed by ECM degradation, which includes secretion and activation of proteolytic enzymes such as MMPs.35 After gaining an entry in the blood vessels, tumor cells migrate to distant organ with the help of cell protrusions and form metastases. Studies on the antimetastatic effects of the drugs and their combinations were performed, which included a battery of in vitro assays. Effects of the single and combined treatment were seen on B16F10 cell motility, adhesion and invasion.

Collagen type IV is a major component of basement membrane, whereas laminin and fibronectin, besides being present in the basement membrane, also form a part of the interstitial connective tissue.36 Adhesion of cells to laminin, collagen type IV and matrigel was significantly reduced on treatment with PTX and suramin, while no inhibition was observed in adhesion to fibronectin. However in combination, the rate of binding was reduced further with maximum inhibition at a lower time point of 15 min. At this time point, the inhibition was remarkable in case of laminin and collagen type IV. On all the substrates, the inhibition observed with combination treatment was significantly higher as compared to individual drugs.

Suramin treatment significantly inhibited B16F10 invasion through matrigel-coated inserts, which is in agreement with the other studies.23 PTX treatment also resulted in inhibition in F10 invasion. Similar effects of PTX have been seen on macrophage invasion through in vitro cultured peripheral nerves.37 However, no such finding has been earlier reported on any cancerous cell line. These effects were augmented on combining the drugs. The compounds showed effects on cell motility as well. PTX has been found to have contrasting effects on cell motility. It induces motility in normal and pathological human spermatozoa,38 but inhibits polarization and migration of human leukocytes.39 It is evident from our earlier report with 2-hr treatment of PTX3 and also from our present study that PTX has inhibitory effects on F10 motility. The inhibitory effects of suramin on F10 motility can be attributed to its effects on bFGF-induced events.40 Cells treated with both the compounds showed decreased migration as compared to those treated with the individual agents.

MMP-9 expression is high in lung carcinoma and melanoma cells41 and plays a major role in the facilitation of cancer metastasis.42 PTX and suramin treatment inhibited MMP9 gelatinase secretion and activity in F10 cells, and the effect was potentiated with concomitant incubation of the compounds. Simultaneous exposure of the compounds demonstrated superior effects than the sequential exposure in the in vitro functional assays namely invasion, motility and secretion of MMPs. Suramin effects have been reported to be reversible on washout of the drug.43 This might be a reason for the loss of synergism being observed in the in vitro studies on sequential drug interaction.

The effects of the combination treatment were studied in the in vivo system as well. Treatment with 50 mg/kg of PTX and 10 mg/kg of suramin inhibited intradermally implanted B16F10 melanoma tumor growth and experimental metastasis in DBA/2 mice. Suramin dose used in our study is much less than the concentrations used by other researchers.22, 44 Intratumoral treatment of the compound showed inhibition in intradermally implanted tumor growth, and the effects were potentiated in the combination. Moreover, sequential treatment of the compounds showed 25% tumor regression.

Metastasis of F10 cells to lung was also found to be inhibited to a greater extent with the combination regimen. Besides a reduction in the number of melanotic colonies on the lung, there was also a decrease in the lung area occupied by these nodules. This suggests that even if the melanoma cells were successful enough to adhere to the lung endothelium the treatment inhibited their further proliferation, thus restricting their colony size. Considering the side effects and the toxicity problems associated with suramin we chose very low doses of suramin for the in vivo experiments. Though there were no signs of ataxia or weight loss, granular degenerative changes were seen in the histology sections of kidney and liver from the mice treated with suramin. PTX treatment showed no hepatic toxicity. Along with the loss of normal architecture, pyknotic nucleus was seen in liver sections of suramin-treated mice. Less degenerative changes were seen in combinations as compared to suramin, suggesting that PTX may have some protective effects on suramin-induced hepatic toxicity. This is in accordance with some related reports where PTX has been shown to cure liver diseases by downregulating the inflammatory responses and oxidative stress.45, 46 In HE sections of kidney of suramin-treated mice, focal necrosis of tubules were seen but no glomerular toxicity was observed. PTX did not aggravate these changes on combination. No signs of toxicity in pancreas or spleen were observed.

To summarize, our studies demonstrate that suramin and PTX are effective against B16F10 melanoma tumor growth and metastases. The combination was found to be synergistic at low concentrations, which further helps in reducing the effective concentration of suramin, thus minimizing its toxic effects. In vitro studies suggest that simultaneous combination has better effects in inhibiting the early metastatic events namely cell motility, invasion and secretion of MMPs. Whether, the difference between these 2 combination regimens actually exists or is because of the reversible nature of suramin action, is not very clear. Moreover, we were not able to comprehend the same in the in vivo studies, as B16F10 lacks the capacity of spontaneous metastasis and the experimental metastasis model does not account the early steps of metastasis viz invasion and motility. Though our study clearly indicates that both the combinations have antimetastatic effects, it cannot answer which one is better. Nevertheless, it clearly suggests that sequential combination takes an upper hand in inhibiting cell proliferation and tumor growth as compared to simultaneous exposure. Therefore for future implication, sequential exposure is more appropriate. Taking an advantage that there are no major side effects associated with PTX, a modified regimen may be beneficial where suramin is given at alternate days with daily administration of PTX. Hence, PTX and suramin combination treatment for melanoma is indeed very promising and warrants more preclinical and clinical studies.

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