Research ArticleExperimental Studies

Combination Chemoprevention with Diclofenac, Calcipotriol and Difluoromethylornithine Inhibits Development of Non-melanoma Skin Cancer in Mice

HANS-CHRISTIAN POMMERGAARD, JAKOB BURCHARTH, JACOB ROSENBERG and HANS RASKOV
Anticancer Research August 2013, 33 (8) 3033-3039;

Abstract

Background/Aim: With increasing incidence of non-melanoma skin cancer (NMSC), focus on chemoprevention of this disease is growing. The aim of this study was to evaluate topical combination therapies as chemoprevention of UV radiation-induced tumors in a mouse model. Materials and Methods: A total of 160 SKH-1 mice were randomized to one placebo group and four chemoprevention groups (diclofenac plus difluoromethylornithine; diclofenac plus calcipotriol; difluoromethylornithine plus calcitriol; and diclofenac plus difluoromethylornithine plus calcipotriol). The mice received UVB radiation for 20 weeks followed by 17 weeks with topical application of chemoprevention. The number of mice with tumors, number of tumors per group and tumor area size were compared using a linear regression model. Results: Chemoprevention with diclonefac plus calcipotriol and diclonefac plus difluoromethylornithine had a significant inhibiting effect on the number of tumors per group and the area of tumors. Moreover, diclonefac plus difluoromethylornithine had a significant inhibiting effect on the number of mice with tumors. Conclusion: Potentially, non-melanoma skin cancer in humans may be prevented with these agents with few adverse effects. Therefore, clinical studies are needed to determine their therapeutic/preventive effect and possible adverse effects.

Non-melanoma skin cancer (NMSC) is the most common form of skin cancer in humans (1). NMSC is often treatable by surgery, and fortunately has a low tendency to metastasize, however local recurrence after complete surgical resection may occur (2). The incidence of NMSC has doubled over the past 18 years, which makes this disease an increasing clinical problem with substantial patient morbidity and treatment-related costs (3, 4).

A clear relationship has been found between the degree of sun light exposure and the incidence of skin cancer (1, 5), which is caused by UV-light induced DNA damage (6-8). Preventive measures typically focus on risk reducing behavior, such as sun protection and limiting sun exposure. However, in the high-risk population, e.g. immunosuppressed patients (5) and patients with precursor lesions such as actinic keratoses (AK) (9), additional prevention may be needed. Chemoprevention agents act to prevent carcinogenic progression to NMSC (10) and among these are diclofenac [a non-steriod anti-inflammatory drug (NSAID)], calcipotriol (vitamin D-3 analog) and difluoromethylornithine (DFMO). Since few adverse effects are tolerated when preventing disease, topical instead of systemic administration seems alluring. These three agents applied topically as monotherapy chemoprevention failed to show beneficial effects in a mouse model (11), however combinations of these drugs have not been evaluated.

The aim of this study was to evaluate the chemopreventive potential of topically-applied combinations of diclonefac, calcipotriol and DFMO, in a mouse model of UVB-induced skin tumors.

Materials and Methods

Animals and environment. The study used 160 female SKH-1 hairless mice (6-7 weeks of age) from Charles River Breeding Laboratories (Köln, Germany) caged together in numbers of 10 in standard polypropylene cages, at a constant room temperature of 18-22°C. Cages were housed in Scantainers with HEPA filters (Class EU10, withholding 98.5% of all particles >0.3 mm). Bedding materiel (Lignocel, Hahn & Co., Faserstoffwerk, Bredenbeck-Kronsburg, Germany) was changed twice a week in a laminar air flow unit. A 12-h automated light/dark cycle was maintained. Diet (Altromin 1324, Altromin Denmark, Chr. Pedersen A/S, 4100 Ringsted, Denmark) was sterilized and administered ad libitum. The mice had a pathogen-free status and were exclusively handled by educated personnel under veterinary supervision. Daily records concerning animal welfare were made and clear humane end-points were followed.

Induction of skin cancer. This study used an existing model of UVB-light induced skin tumors in SKH-1 hairless mice (12). The UV-treated hairless mouse model produces skin lesions resembling AK and squamous cell carcinoma observed in humans, which are associated with repeated exposure to UV-light. Each cage, with all interior materials removed, containing 10 mice, was placed under an FS72T12-UVB-HO sunlamp (National Biological Corporation, Twinsburg, OH, USA) and were exposed to UVB-irradiation for one hour twice a week, altogether for 20 weeks. The UV lamps were calibrated with a radiometer/photometer and the mice were exposed to a UV irradiation dose corresponding to 30 mJ/cm2. This dose of UV irradiation has been used in earlier models (12,13) and corresponds to the dose for development of human skin cancer (14). All animals were observed for their wellbeing during and after each UV treatment.

Preparation of agents. Mixtures of the topically applied agents were made according to the following recipes: NSAID (diclofenac sodium, 3% Solaraze gel; Bioglan Pharma, Malmö, Sweden), vitamin D-3 analog (calcipotriol 50 μg/g, Daivonex cream; Leo Pharma, Ballerup, Denmark), DFMO (eflornithine hydrochloride, 13.9% Vaniqa cream; Bristol-Myers Squibb, Lyngby, Denmark), placebo (Matas skin lotion, Denmark). For combination therapy with two active agents, one part of each active agent and one part of skin lotion (placebo) was used. For the combination therapy with three active agents, one part of each active agent was used. All the resulting mixtures were diluted five times with distilled water (1:4 mixture:water). This made the active agents 15-times diluted (1/15 of the original concentration). The decision to dilute the original drugs was made on the basis of an earlier animal study, in which mice were treated topically with undiluted Solaraze gel (15). In that study the animals had high mortality due to ulcers and severe gastrointestinal bleeding. In the present study, a similar degree of dilution was made of all active substances to make comparison between the original products possible.

Randomization and chemopreventive treatment. After UV treatment, mice without tumors were randomly divided into five groups, four chemoprevention groups (diclofenac plus DFMO; diclofenac plus calcipotriol; DFMO plus calcitriol; and diclofenac plus DFMO plus calcipotriol) and one placebo group (skin lotion). The placebo group used in this study was the same as the one used in an earlier study (11). The mice were treated with test mixtures once a day, five days a week, for a total of 17 weeks. The test mixtures were applied topically on the dorsal surface of the mice. Ten microliters were applied by a pipette after which the mixture was rubbed onto the skin. This corresponded to the following doses of each active substance in the treatments: 100 μg/week for diclofenac (30 mg/g undiluted), 0.166 μg/week for calcitriol (50 μg/g undiluted), and 463.3 μg/week for difluoromethylornithine (DFMO) (139 mg/g undiluted).

Once a week, all mice were examined for tumors, and the body weight was noted. The number of mice with tumors in each group, as well as the number of tumors on each mouse was recorded. Tumor diameter was measured with a calibrated optical magnifying device. Cysts were not recorded. The UV-induced tumors were easily distinguishable from cysts, since cysts were yellowish and tumors were reddish due to vascularization. The experiment was ended after 17 weeks of treatment.

From each group, two randomly chosen samples of tumors were histologically evaluated. Tissues with pathological findings were fixed in 4% buffered formaldehyde, embedded in paraffin and stained with hematoxylin and eosin prior to histopathological evaluation.

Outcome measures were number of mice with tumors, total number of tumors, and total area of tumors in each group.

Data analysis and permissions. Sample size was based on the assumption that 80% of the mice in the placebo group developed one or more tumors due to the UV irradiation (14). We chose a minimum relevant difference (MIREDIF) of 50%, which led to a group size of 25 mice. This means that it was expected that treatment would reduce the tumor occurrence from 80% to 40%. Based on this calculation, a group size of 32 was chosen in order to allow drop-outs due to death of animals in the groups.

A linear regression model was used to evaluate the outcomes. These linear correlations were assumed on the basis of R2 coefficients exceeding 0.90, as well as outcome vs. time plots. For the total number of tumors and total tumor area an exponential tendency was seen in the plots which is why a logarithmic transformation of the data was used. Chi2-test was used to evaluate distribution of deaths between the groups at the end of the experiment. p-Values <0.05 were considered significant. SPSS version 19 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.

The study was approved by the Danish Council of Animal Experiments before initiation (license number: 220801-087).

Results

Survival. One out of the 32 animals in each of the groups died, except for the diclofenac plus DFMO plus calcipotriol group, where all animals survived. Survival was equally distributed between the groups (p=1.000, Chi2-test).

Histological evaluation. The randomly-selected tumors from the groups consisted of planocellular papillomas with hyperkeratosis, dysplasia and acute/chronic inflammation of the underlying stroma and invasive carcinomas.

Number of tumors per group. Diclofenac plus calcipotriol, diclofenac plus DFMO and diclofenac plus DFMO plus calcipotriol seemed to have a tumor-reducing effect (Figure 1). However, using a linear regression model (log-transformed data), we only found significant difference for two of the treatment combinations (diclofenac plus calcipotriol vs. placebo p=0.005; diclofenac plus DFMO vs. placebo p=0.001); the difference between diclofenac plus DFMO plus calcipotriol and placebo was non-significant.

Area of tumors. Diclofenac plus calcipotriol, diclofenac plus DFMO and diclofenac plus DFMO plus calcipotriol seemed to have a reducing effect on the area of tumors (Figure 2). However, a significant reduction was only seen in two of the chemoprevention groups (diclofenac plus calcipotriol vs. placebo, p<0.001; diclofenac plus DFMO vs. placebo, p<0.001; linear regression on logtransformed data).

Figure 1.
Figure 1.

Number of tumors in each group after initiation of treatment.

Number of mice with tumors per group. As with number of tumors per group and area of tumors, it seemed that all diclofenac-containing mixtures reduced the number of mice with tumors (Figure 3). However, this reduction was only significant for the diclofenac plus DFMO group based on linear regression (diclofenac plus DFMO vs. placebo, p<0.001).

Weight. The weight gain was significantly smaller in the groups treated with diclofenac plus calcipotriol (p=0.018) and diclofenac plus DFMO plus calcipotriol (p=0.002) compared with placebo (linear regression model) (Figure 4). Weight gain in the remaining groups was not significantly different from that of the placebo group.

Discussion

We found that preventive treatment with diclofenac plus calcipotriol and diclofenac plus DFMO reduced the number of tumors, as well as the total tumor area, compared with placebo. Moreover, diclofenac plus DFMO reduced the number of mice with tumors. Therapy with diclofenac plus calcipotriol and diclofenac plus DFMO plus calcipotriol reduced weight gain in the treated mice. As seen in Figures 1, 2 and 3, the combination therapy of diclofenac plus DFMO plus calcipotriol seemed to have an effect on all of the three above mentioned parameters. However, this effect was not significant when using a linear regression model. Based on the figures and the regression model, the combination therapy of DFMO plus calcipotriol seemed to have no effect. Thus, the common denominator of the significant treatment combinations was the inclusion of diclofenac. Histological evaluation of the tumors showed pre-malignant lesions, e.g. AK and planocellular carcinomas, which may be found in patients with chronic sun exposure (16).

In a previous publication from our group using the same experimental setup, we found no tumor inhibiting properties of these drugs when used as topical monotherapy (11). However, the results in the present study with topically-administered combination therapy of the same drugs seem very promising. The reduced weight gain for the groups treated with diclofenac plus calcipotriol and diclofenac plus DFMO plus calcipotriol was not reflected in an increased mortality for these groups, hence this phenomenon is not regarded as a result of toxicity of these drug combinations.

The mechanisms of action of NSAID, DFMO and vitamin D-3 in inhibiting tumorigenesis share similarities. Regarding NSAID, the cyclooxygenase-2 (COX-2) enzyme has been suggested to play a role as a promoter of skin neoplasia, where increased COX-2 activity leads to increased prostaglandin E-2 levels. Such elevated levels of prostaglandin E-2 have been correlated to promotion of tumors and metastases (17-19). NSAIDs inhibit COX-2, whereby tumor development may be suppressed. It has been shown that UVB induces the expression of COX-2 in epidermis and that COX-2 concentrations are high in NMSC (17, 20, 21). On the other hand, inhibition of COX-1 and/or COX-2 may suppress tumorgenesis by keratinocyte differentiation as shown in a previous experimental study (21). This is supported by a clinical randomized controlled trial (RCT) demonstrating that oral celecoxib reduced the number of NMSC (22), and a clinical study suggesting that long-term use of NSAIDs may reduce the risk of skin melanoma in humans (23). Furthermore, a large cohort study evaluating 172,057 individuals concluded that the use of non-aspirin NSAID had protective effects against colon, rectal, stomach and ovarian cancer (24).

Figure 2.
Figure 2.

Area of tumors in each group after initiation of treatment.

Figure 3.
Figure 3.

Number of mice with tumors in each group after initiation of treatment.

Figure 4.
Figure 4.

Weight change in each group after initiation of treatment.

Vitamin D is known to have antiproliferative and pro-apoptotic properties and studies have suggested that high levels of vitamin D may protect against different types of cancers (25, 26). Experimental studies have found a tumor-inhibiting effect of vitamin D on NMSC (27) and a clinical study has suggested an association between high levels of vitamin D and reduced risk of NMSC (28).

DFMO inhibits the enzyme ornithine decarboxylase (ODC), which is essential for cellular proliferation and differentiation. This enzyme is induced by UV radiation whereby increased activity may lead to tumor formation (29). Experimental studies found that DFMO can inhibit tumor growth (30-32). In a RCT in 48 patients with AK, topical treatment with DFMO reduced the number of AK lesions and subsequent evaluation of skin biopsies found a reduced p53 protein expression, which may help to explain the effect (33, 34).

Besides the drugs evaluated in the present study, others have also been studied. Among these, retinols (35, 36) and photodynamic therapy (PDT) (37) have shown promising results. However, these treatments may be associated with significant adverse effects and patient discomfort (38). In these cases, topical administration of diclofenac plus calcipotriol or diclofenac plus DFMO could be of interest. If combination treatment with diclofenac is as efficient as indicated by our results, this treatment may have a role in chemoprevention of high-risk patients as an alternative to retinols and PDT. Given the expected negligible side-effects of topically-administered diclofenac plus calcipotriol and diclofenac plus DFMO, these drugs may potentially also be used in normal-risk individuals with a high level of sun exposure. However, before routine clinical use, the effect should be tested in human RCTs with special focus on potential adverse effects of diclofenac, such as cardiovascular events and gastrointestinal bleeding (39, 40).

The mouse model was designed to resemble humans at high risk of NMSC, e.g. patients with precursor lesions such as AK and normal individuals heavily exposed to sun early in life (5, 18). Therefore, the chemoprevention was not initiated until after 20 weeks of UV radiation. In a model resembling healthy individuals, where the chemoprevention was started at the same time as UV radiation, the beneficial effects may be even greater.

In conclusion, we found that diclofenac plus calcipotriol and diclofenac plus DFMO had tumor reducing effects in a mouse model of UVB-induced tumors. Potentially, these treatments may offer fewer and less severe adverse effects compared with existing drugs such as retinols and PDT. Thus, clinical studies are warranted to determine the effect and possible adverse effects of these combination therapies in humans.

Acknowledgements

The experiment was conducted by Pipeline Biotech A/S, Roevedvej 1,8380 Trige, Denmark. No funding was used for this study.

Footnotes

  • Conflicts of Interest

    The Authors declare that they have no conflict of interests or financial interest associated with the study.

  • Received May 9, 2013.
  • Revision received May 29, 2013.
  • Accepted June 3, 2013.

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