Staurosporine was first isolated by Omura, S. et al. in 1977 from a Streptomyces strain [20]. Until present, several authors have confirmed its pharmacological activities like hypotensive, antiprotozoal, anticoagulant and antifungal agent [21, 22]. In a previous work, we have proved the amoebicidal activity of Staurosporine against various Acanthamoeba strains [23]. The approach was based on molecule isolation and bio-guided fractionation from a Streptomyces sanyensis extract. In addition, we have confirmed that the present indolocarbazole could induce program cell death in Acanthamoeba castellanii Neff [23, 24]. Yet, to confirm the obtained results and to indicate the mechanism of action, we opt to study the effect of the present molecules on the proteomic profile of Acanthamoeba and to confirm its amoebicidal activity using and ex vivo approach.
The main objectives of the present work were first to confirm the amoebicidal effect of Staurosporine on this clinical strain of Acanthamoeba castellanii following protein expression in the early stage of treatment. Second to establish a new protocol to study the effect on amoebicidal drug on an amoebic keratitis murine ex vivo model, which turned out to be efficient since data similar to those observed and described in the cornea of hamster (Mesocricetus auratus) were obtained.
In our previous work, we highlighted the morphological alterations induced by the Staurosporine on Acanthamoeba in the early stage of treatment. To corroborate the effect of the present drug on the cytoskeleton of Acanthamoeba, a specific staining of actin and tubulin was done. After 30 min of incubation with the present drug, we observed a dramatic alteration of the cell cytoskeleton; actin staining revealed the formation of long elongation. Various reports confirmed the present findings, Hedberg et al., (1990) have reported the alteration of the cytoskeleton of several cell lines, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts, by the Staurosporine [25]. While Xie et al., (2017) have observed the formation of filaments in the fungal pathogen Candida albicans [26]. In fact, they have related the Staurosporine-induced filament to a defect in septin ring formation implicating cell cycles kinases as potential Staurosporine targets [26]. As for the microtubules network, we observed that the present drug induces alteration in its rearrangement as a disorganisation network with the presence of concentrated points. All those events could be a result of a cell dismantling upon program cell death [27].
The proteomic analysis of cells treated with the Staurosporine (IC50) for 24 hours revealed that various membrane protein kinases were significantly downregulated, including dual specificity protein kinase shkB and Serine/threonine-protein kinase. Those proteins have been described as regulators of chemotaxis and phagocytosis processes in Acanthamoeba. Inhibiting those proteins could reduce the pathogenicity and growth of Acanthamoeba. Along with this effect, we notice the inhibition of cysteine protease implicated in the tissue invasion as well as in the encystation pathway [28].
In treated cells, almost 16% of the total identified proteins were overexpressed. Among those proteins, we have observed the upregulation of Profilin. This molecule is implicated in the regulation of actin polymerization and affects the structure of the cytoskeleton. Various reports have confirmed that at lower concentrations this protein enhances the actin polymerization while at high concentrations would prevent it and trigger the autophagy via the mTOR pathway [29, 30]. Among the most upregulated proteins, we found proteins involved in cell survival under oxidative stress.
Although, we have confirmed in vitro the effect of Staurosporine on Acanthamoeba, these results are still insufficient to scale it up in vivo. For this reason, one of the main objectives of the present study was to establish an ex vivo model to study the effect of drug therapy on AK infection. Omaña-Molina et al., (2004) established an ex vivo model to study the cytopathic effect of Acanthamoeba castellanii and A. polyphaga on hamster corneas [11]. In this study, the ex vivo assay revealed that trophozoites of Acanthamoeba castellanii infected mouse corneas, showing that the amoebas invaded corneal epithelium penetrating the junctions of the epithelial cells without lysis process, only phagocyting the detached cells. This type of invasion suggest that Acanthamoeba infections are contact-dependent. In this sense, Omaña-Molina et al., (2013) demonstrated that A. castellanii and A. polyphaga invasion and disruption of corneal epithelium in the hamster model is performed by the penetration of the amoebae through cell junctions either by the action of proteases and/or a mechanical effect exerted by trophozoites, suggesting that the contact-dependent activity is an important pathogenic mechanism of these strains of Acanthamoeba [12]. Moreover, in a previous study Omaña-Molina et al., (2010) co-cultivated A. castellanii trophozoites with human corneas and reported mechanisms of pathogenicity of amoebic infections were very similar to the previous study using a hamster model and our findings using a mouse model, which validate these animal models for the study of the pathogenesis of AK [11, 13, 31].
Currently, the agents normally recommended to treat AK need to be administered for a prolonged period, which often result in severe ocular surface toxicity [4, 32–35]. In this study, mouse corneas cultured alone with Staurosporine revelated that this compound causes no adverse effect on corneal epithelium compared to other drugs, maintaining the corneal tissues intact. Importantly, it has been shown that Staurosporine with only 30 min pre-treatment affects the adhesion of the amoebae to the corneal epithelium and prevents the trophozoites from invading the corneal tissue. However, a 30 min pre-treatment did not completely eliminate the amoebae population, possibly due to insufficient time for pre-treatment of the trophozoites. The corneal epithelium was intact in the infected mouse corneas treated simultaneously and 30 min after infection. Staurosporine eliminated the entire amoebic population, preventing adhesion and infection of amoebae to the epithelium. Therefore, this study demonstrated that Staurosporine could be used to develop a new line of eye drops for the treatment of superficial AK or early stages of amoebic infection.
Nevertheless, our study has limitations. For example, in patients with AK in the chronic stage of the disease, where the infection is located at deeper levels of the corneal epithelium, further studies are needed to determine whether the effect of Staurosporine could be observed deeper in the corneal tissue and completely eliminate the amoebic infection.