Albendazole negatively regulates keratinocyte proliferation

effect in a murine model of psoriasis. positive staining. cultured


INTRODUCTION
Psoriasis is one of the most common chronic inflammatory disorders, affecting approximately 2-3% of the population worldwide (1). Well-demarcated, erythematous, scaly plaques, which can involve any part of the skin, but more commonly the extensor surfaces and the scalp, are typical lesions of the disease. Histologically, psoriasis is characterized by keratinocyte hyper-proliferation with parakeratosis and elongation or rete ridges, increased angiogenesis, and dermal infiltration with many inflammatory cells, such as macrophages, dendritic cells, T cells, and neutrophils (2)(3)(4).
The aetiology of psoriasis remains unknown even though accumulating evidence suggests that psoriasis develops in genetically predisposed individuals as a result of the action of various environmental factors, which trigger innate and adaptive immune responses (5,6). As a consequence, many effector cytokines produced in the psoriatic skin [e.g. tumour necrosis factor (TNF), interleukin (IL)-6, IL-17, IL-21, IL-22, and IL-23] stimulate keratinocyte activation/proliferation (7)(8)(9)(10)(11). This sequence of events is accompanied by secretion of chemoattractants, which promote recruitment of other inflammatory cells, such as neutrophils, into the affected skin thereby leading to the amplification of the detrimental inflammatory response (3).
In line with such a hypothesis is the observation that compounds targeting the above cytokines are useful for inducing and maintaining remission in psoriatic patients (12)(13)(14)(15)(16). Unfortunately however, not all the patients respond to these drugs and in some patients treatment must be discontinued due to the development of adverse events or other immune-mediated diseases (17)(18)(19). Thus, the development of novel compounds targeting the key pathogenic events in psoriasis is worth pursuing.
Studies in murine models of psoriasis have also shown that primary defects of keratinocytes could be sufficient to promote epidermal hyper-proliferation and psoriasis-associated immune-Downloaded from https://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20191215/870858/cs-2019-1215.pdf by guest on 02 April 2020 Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20191215 inflammatory reactions, raising the possibility that drugs interfering with keratinocyte function could be useful in the management of psoriatic patients (20)(21)(22).
Since the development of new drugs is a very lengthy and costly process, drug repositioning, defined as identifying novel indications for existing drugs, has recently become a good strategy for overcoming the limitations of traditional methods.
Albendazole is a well-characterized benzimidazole derivative, initially developed as an antihelmintic drug. Albendazole-mediated biologic effects rely on inhibition of tubulin polymerization and blockage of glucose uptake (23)(24)(25). Recent experimental studies have shown that albendazole has also anti-tumoral properties in many epithelial cancers (e.g. hepatocellular carcinoma, cutaneous squamous cell carcinoma, and colorectal carcinoma) mainly depending on its ability to interfere with cancer cell growth (26)(27)(28). Based upon these observations, we hypothesized that albendazole can also regulate keratinocyte proliferation/activation and exert protective effects in psoriasis. The aim of this study was to investigate whether albendazole inhibits psoriatic lesions in an in vivo model of disease and to dissect the mechanisms by which the drug interferes with keratinocyte proliferation.
Downloaded from https://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20191215/870858/cs-2019-1215.pdf by guest on 02 April 2020 Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20191215

Mice studies
C57bl/6 mice (male, 8-10 weeks of age) were purchased from Charles River and hosted in the conventional animal facility at the University of Rome "Tor Vergata". Animal Ethics Committee, according to Italian legislation on animal procedures, approved all the animal experiments (N° 597/2016-PR). All reagents were from Sigma-Aldrich (Milan, Italy) unless specified. To induce a psoriasis-like skin inflammation, shaved mice were treated daily with 62.5 mg of commercially available Aldara cream (Meda, Solda, Sweden) on the back skin for 4 consecutive days. Vaseline cream was used as internal control (vehicle). Methyl 5-propylthio-2benzimidazolecarbamate (albendazole) (30μg/mouse), resuspended in 100 μl of propylene glycol, was daily applied on the shaved back of Aldara-treated mice in the same skin area, starting 12 hours after the Aldara administration. Control mice were daily treated with a similar daily amount of propylene glycol 12 hours after the Aldara treatment. All animals were assessed for the severity of the psoriasis-like skin condition at day 4, using three elements of the Psoriasis Area Severity Index (PASI), assigning a score of 0 -4 (0: none; 1: mild; 2: moderate; 3: severe; 4: very severe) for each of the parameters erythema, scaling and induration. This analysis was performed in at least 4 fields per section of all the skin samples.

Histopathological analysis and immunohistochemistry
Cryosections of mouse skin samples were stained with hematoxylin and eosin (H&E), and epidermal thickness was evaluated by measuring the average interfollicular distance from the basal lamina to the bottom of the stratum corneum (8,29). Mouse sections were also stained with rat anti-ki67 antibody (Dako, Milan, Italy) followed by a secondary antibody anti-rat conjugated to horseradish peroxidase (Dako). Likewise, sections were stained with rabbit anti-CD3 (SP7)   Primer sequences were as follows: mIL-6 forward 5'-AGCCAGAGTCCTTCAGAGAG -3',

Albendazole reduces severity of psoriasis-like lesions and keratinocyte proliferation
To investigate whether albendazole inhibits keratinocyte proliferation, we initially used a well-characterized model of epidermal hyper-proliferation and abnormal keratinocyte differentiation induced in mice by topical application of Aldara cream(30). Our previous timecourse studies showed that mice given Aldara exhibited the more pronounced severe skin pathology at day 4 (31). Therefore, this time point was selected to evaluate the therapeutic effect of albendazole. In such a model, a daily topical application of albendazole, given 12 hours after Aldara treatment, decreased the severity and extent of psoriasis-like lesions in terms of erythema, scaling and skin thickening/induration with significant reduction of PASI score (Fig. 1A). H&E staining showed reduced epidermal thickness, parakeratosis and cellular infiltration in mice treated with albendazole ( Fig. 1B). Similar effects were seen when analysis was performed at day 6 of Aldara treatment (Suppl. Fig 1A). The immunostaining of ki-67, a marker of cell proliferation, revealed less proliferative cells in the epidermal basal stratum of albendazole+Aldara-treated mice as compared to Aldara-treated mice (Fig. 1C). Moreover, albendazole treatment decreased expression of K6A and K16, two differentiation-specific epidermal keratins induced in hyper-proliferative keratinocytes(32, 33) (Fig. 1D). By Western blotting we also showed a reduced expression of cleaved caspase-3 following albendazole treatment clearly indicating the modulatory effect of the drug on keratinocytes is not secondary to induction of apoptosis or cell damage (Suppl. Fig 1B-C).

Albendazole reduces the infiltration of immune cells and production of inflammatory molecules in the skin of Aldara-treated mice
Next, we investigated whether the therapeutic effect of albendazole was paralleled by reduced infiltration of the skin with neutrophils and T cells, as these cells are supposed to amplify pathogenic signals in psoriasis (4,34). Albendazole-treated mice exhibited reduced cutaneous infiltration of CD3-and Ly6G-expressing cells ( Fig. 2A and Suppl. Fig.1D) and this associated with  significant decrease of inflammatory cytokines, such as IL-6, TNF-α, IL-1β, IL-17A, and IL-36(α,β,γ) and reduced expression of chemokines involved in the recruitment of neutrophils and T cells, such as CCL17, CXCL1, CXCL2 and CXCL5 (Fig. 2B).
We also examined whether the in vivo anti-proliferative effect of albendazole on keratinocytes was secondary to inhibition of production of immune cell-derived cytokines, which are known to stimulate keratinocyte growth. To this end, skin explants of Aldara-treated mice were cultured with or without albendazole for a short time (i.e. 6 hours) and the levels of K6A, K16, TNF-α and IL-6 transcripts were then analysed by real-time PCR. Albendazole reduced K6A and K16 RNA transcripts without changing IL-6 and TNF-α RNA expression (Fig. 3).

Albendazole reversibly blocks keratinocyte cell growth
The above findings suggest that albendazole targets primarily keratinocytes. To support this hypothesis, we evaluated the anti-proliferative effect of the compound in HaCaT cells, a nontumorigenic monoclonal highly proliferating human keratinocyte line, which shares some features with psoriatic keratinocytes. In preliminary experiments, we showed that albendazole reduced cell viability at doses ranging from 125 nM to 1 μM (Fig. 4A). Therefore, the subsequent studies were conducted using 30 nM albendazole. HaCaT cells cultured with albendazole exhibited reduced protein expression of K6A and K16, and decreased growth, which was evident at both 24 and 48 hours (Fig. 4B-C). Such effects were fully reversible, because HaCaT cells proliferated regularly after removal of albendazole from the culture (Fig. 4D).

Keratinocytes accumulate in S-phase of the cell cycle following albendazole treatment
In subsequent studies, we assessed the effect of albendazole on cell-cycle progression in Time-course studies showed also that eIF2α phosphorylation was preceded by phosphorylation of PKR (Thr446) (Fig. 6A), while expression of phosphorylated PEKR and GCN2 remained unchanged (data not shown). Down-regulation of PKR expression with a specific siRNA prevented albendazole-mediated eIF2α phosphorylation (Suppl. Fig 2E and Fig. 6B) as well as abrogated the albendazole-mediated inhibitory effect on IL-6, IL-8, IL-1β, and CCL5 expression in TNFα-activated keratinocytes (Fig. 6C-D and Suppl. Fig 2F-G).
To translate these data in vivo, phosphorylation of eIF2α and PKR and expression of CDC25A were evaluated in the skin of Aldara-treated mice receiving or not albendazole.
Albendazole enhanced phosphorylation of both eIF2α and PKR and this was associated with reduction of CDC25A protein expression (Fig. 7).

DISCUSSION
Enhanced keratinocyte proliferation is documented in the psoriatic skin and supposed to make a valid contribution to the pathogenesis of psoriasis (42,43). Therefore, compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients (44). Drug repurposing known also as drug repositioning or drug reprofiling is an approach In conclusion, our findings reveal a novel inhibitory action of albendazole on keratinocyte proliferation, which associates with a therapeutic effect in a murine model of psoriasis.