Persistent high-risk human papillomavirus (HPV) infections are among the leading causes of cancer in the world. This infection is mainly associated with cervical cancer as well as other anogenital malignancies and a subgroup of oropharyngeal cancer [1
]. Following the initial infection of the skin or keratinized mucosae, high-risk HPVs can evade the immune system and persist in their infected hosts, and may trigger carcinogenesis [2
]. The lasting inflammatory process rises cellular levels of reactive oxygen and nitrogen species (RONS), which leads to the oxidation of proteins, lipids, and DNA [4
]. Albeit HPV-induced inflammation is a primary source of ERON, HPV proteins, such as E6, E6* and E7, and even E5 indirectly, have been associated with the production and reduction of these species [5
]. HPV16 and HPV18 are the most frequently detected HPV genotypes in malignant lesions [6
]. HPV-induced multiphase carcinogenesis is often associated with chronic inflammation, and experimental studies in HPV transgenic mice suggest that inflammatory phenomena play an essential role in the development of intraepithelial and invasive lesions [9
]. Cyclooxygenase-2 (COX-2), a key mediator of inflammation, is involved in the development of multiple types of cancer, such as colon cancer, making it a useful therapeutic target [10
]. We have previously shown that a specific COX-2 inhibitor, celecoxib, was able to block the progression of HPV16-induced lesions in transgenic mice [14
]. However, oral administration of celecoxib induced significant mortality in HPV16 transgenic mice, indicating that it has unacceptable toxicity in our animal model. In the present work, we chose a different COX-2 inhibitor, parecoxib, to achieve chemopreventive and toxicological studies in vivo using the same animal model. Parecoxib is a water-soluble prodrug of valdecoxib [15
] and is the only selective COX-2 inhibitor that can be administered parenterally, which may contribute to a different toxicological profile compared to other COX-2 inhibitors such as celecoxib [17
Chronic inflammation is associated with factors like reactive species of oxygen and nitrogen, cytokines, chemokines, growth factors, and specific microRNAs, which often contribute to cancer development [19
]. In most epithelial cells, COX-2 is absent or expressed at low levels but is a major contributor to inflammation and is up-regulated in many cancers, [20
], so it is a relevant therapeutic target for cancer prevention and treatment [10
]. Studies in K14-HPV16 mice have demonstrated that COX-2 is overexpressed in skin lesions induced by the HPV16 early genes [20
]. Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of drugs prescribed due to their analgesic, antiplatelet, and anti-inflammatory properties [21
]. NSAIDs are useful in preventing the development of colorectal adenomas [23
]. However, the existing clinical data does not support the use of COX-2 inhibitors for preventing the progression of cervical intraepithelial lesions [24
]. Another difficulty is the potential toxicity of some NSAIDs, as observed with a previous study of celecoxib in K14-HPV16 mice [14
]. Parecoxib, a water-soluble prodrug which is biotransformed in the liver by enzymatic hydrolysis and converted into its active metabolite, valdecoxib, is a selective COX-2 inhibitor used to minimize postoperative pain in noncardiac surgeries. It is also the only selective NSAID marketed that allows parenteral administration [17
K14-HPV16 mice in an FVB/n background express all the early genes of HPV16 under the control of the human cytokeratin 14 promoter. These animals develop multi-step lesions, associated with progressive inflammation, in squamous epithelia such as the skin, similar to HPV-induced lesions in patients [25
]. These animals also show severe systemic inflammation and are particularly susceptible to hepatic damage, as previously demonstrated by our group [28
]. In view of the main role of oxidative stress in triggering and prolonging hepatic damage and the role of inflammation in triggering oxidative stress, we have studied some biomarkers of oxidative stress in liver samples of transgenic (HPV16+/−
), and wild type-mice (HPV16−/−
) treated or not with parecoxib.
In the present study, we made two major sets of observations. The first, concerning the efficacy of parecoxib against HPV16-induced lesions, showed that this drug completely abrogated the onset of epidermal dysplasia in our animal model. Secondly, we observed that parecoxib displayed a favorable toxicological profile concerning several general and liver-specific parameters and the proper functioning of antioxidant defenses.
We observed that HPV+/−
mice treated with parecoxib showed only epidermal hyperplasia that did not progress to dysplasia, while 63.6% of untreated HPV+/−
mice showed epidermal dysplasia. This is of great importance, given that the dysplastic stage precedes the development of squamous cell carcinoma in this model, suggesting that parecoxib may able to block the development of HPV16-induced cancer. A previous study using celecoxib showed similar results [14
]. Another study employing two dietary polyphenols (curcumin and rutin) with some unspecific COX-2 inhibitory activity, also blocked the development of epidermal dysplasia. In this study, they also observed a decrease of COX-2 expression and the decrease of total leukocytes and leukocyte population [20
]. We found, in transgenic animals treated with parecoxib, a decrease of total leukocytes, especially of neutrophils that are involved in inflammation and are attracted to prostaglandin E2, suggesting COX-2 is inhibited by parecoxib. Celecoxib was shown to promote the activation of cytotoxic T lymphocytes present in cutaneous lesions, which may explain its activity against epidermal lesions in this model [14
]. Further studies should address this topic, determining whether parecoxib exerts a similar effect over T cells and if other mechanisms are also involved.
O’Donoghue et al. [29
] employed a 10-fold lower parecoxib dose (0.5 mg/kg/day) intraperitoneally for 15 days against breast cancer mouse xenografts, and observed inhibition of tumor growth and metastasis. An even lower dose of parecoxib (0.22 mg/kg/day) administered for eight weeks has been shown to suppress the growth of esophageal adenocarcinoma xenografts in athymic mice [30
]. Smakman et al. [31
] employed the same dose of parecoxib as we used (5.0 mg/kg/day) for six days after sowing colon cancer cells in the liver and observed a reduced proliferation of cancer cells. In contrast, another study failed to identify antitumor effects of parecoxib at the same dose on Fischer rats with brain tumors [32
]. In our study, 5.0 mg/kg/day was an effective and well-tolerated dose. We only used a single dose of parecoxib, since previous studies have indicated that it is effective and therefore, we could reduce the total number of animals sacrificed.
During the experimental protocol, the animals were monitored for signs of disease and distress that could reflect toxicity. Importantly, no changes in the behavior and physiology of the animals were observed. There were also no significant differences concerning body weight or food consumption, supporting the hypothesis that parecoxib was safe in the present experimental conditions. Transgenic animals had a higher water consumption, as previously reported for this animal model [28
We also observed significant differences concerning the relative mass of internal organs between wild-type and transgenic mice, which had also been reported for these animals. Transgenic animals treated with parecoxib showed near-normal values for the relative masses of internal organs, suggesting that this compound may act systemically to re-establish homeostasis in this setting.
Parecoxib did not induce detectable hepatotoxicity at the biochemical or histological levels. In wild-type animals, parecoxib did not alter the baseline levels of genetic damage. However, in transgenic animals, parecoxib increased the total genetic damage index (GDI) while slightly reducing the GDIFpg. This is likely reflecting the hepatic biotransformation of parecoxib into valdecoxib, and suggests that DNA-repair mechanisms could cope with parecoxib-induced genetic damage, so that it didn’t translate into to biochemical or morphological changes.
Oxidative stress is caused by an imbalance between oxidants–antioxidants that favors the oxidants, leading to the excessive generation of free radicals, particularly reactive oxygen species (ROS), and subsequently to biological damages [33
]. Our results on oxidative stress parameters (GSH:GSSG and LPO) shows that the inflammation caused by HPV16 induces oxidative stress. Therefore, antioxidant system activation is required as can be seen by the increase of SOD and GR when comparing the transgenic mice without treatment (group IV) and its own control (group II). Our data shows that the inflammation caused by HPV16 induces oxidative stress, considering the increased activity of two critical enzymes of the antioxidant system (SOD and GR), which was not sufficient to prevent oxidative damage as demonstrated by the decrease of the GSH:GSSG ratio and increase of lipid peroxidation (TBARS).
Regarding the results of transgenic mice (HPV16) treated with parecoxib (group III) and its control (group IV), it seems that parecoxib leads to a decrease in oxidative stress since lipid peroxidation decreases and the GSH:GSSG ratio increases, although the latter is not statistically significant. The reduction of ROS production, and therefore the lower oxidative stress observed in groups treated with parecoxib will reduce COX-2 expression, leading to the inhibition of prostaglandin synthesis and, subsequently, proinflammatory cytokines which are also responsible for increased free radical production [34
]. For oxidative stress enzymes, SOD has a significant decrease in its activity may be due to the lower generation of its substrate (O2•−
), and GR, GST, and CAT didn’t show any differences. GST activity does not show differences since parecoxib is rapidly converted to valdecoxib, the pharmacologically active substance, by enzymatic hydrolysis in the liver, after that cytochrome P450 system metabolizes it to hydroxyvaldecoxib [16
]. The same type of results were obtained with celecoxib, another COX-2 inhibitor, in Walker-256 tumor-bearing rats, where no differences in GST activity were observed [35
]. The GSH:GSSG ratio, which lowers when oxidative stress increases, is mainly due to a decrease in the amount of GSSG (results not show), which may be related to the GR activity.