A Tick Cysteine Protease Inhibitor RHcyst-1 Exhibits Antitumor Potential

Abstract

Background/Aims: We previously identified a potent and tight-binding inhibitor of cysteine proteases from Rhipicephalus haemaphysaloides, RHcyst-1, which belongs to the cystatin type 1 family. Cathepsins, which are members of the cysteine protease family, participate in various pathological processes, including the initiation and development of cancers. The present study aimed to investigate the antitumor effects of RHcyst-1 and to explore the underlying mechanism of these effects. Methods: Different tumor cells were treated with RHcyst-1 in vitro. Proliferation activity was evaluated using Cell Counting Kit-8, and migration and invasion were determined by wound healing and Transwell® invasion assays. In addition, a mouse tumor therapy model was established by inoculating the left forelimb of mice with B16-F10 cells, and tumor progression was evaluated by assessing tumor volume and survival. Flow cytometry was conducted to evaluate myeloid-derived suppressor cells (MDSCs), CD4⁺, and CD8⁺ T cell levels in PBMCs and spleens. Immunohistochemistry was performed to analyze immune cell infiltration and angiogenesis in the tumors. Results: RHcyst-1 significantly inhibited the proliferation, migration, and invasion of all four different tumor cells in vitro. Additionally, it inhibited tumor growth and improved survival in vivo. A decrease and an increase in MDSCs levels were observed in PBMCs and in the spleen, respectively, after RHcyst-1 application. Conclusions: Tick RHcyst-1 has potential antitumor efficacy, and the observed antitumor activities may be partly attributable to changes in cathepsin expression and MDSCs levels in the PBMCs and spleens. The findings of the present study suggest that RHcyst-1 may have the potential to be utilized in cancer treatment.

Immunosuppressive effects of tick protein RHcyst-1 on murine bone marrow-derived dendritic cells

“…In our previous study, we reported that RHcyst-1 belongs to the type1 cystatin family and is involved in tick physiology [13]. In this study, although RHcyst-1 was not directly detectable in serum from rabbits on which ticks had repeatedly fed (Additional file 1: Figure S1), previous studies have shown that RHcyst-1 has a broad spectrum of mammalian cathepsin-inhibition activities and mediates specific immune responses in tumor-bearing mice [13, 18]. Recently, exosomes have been most extensively studied and tick-derived exosomes were reported to mediate transmission of tick-borne disease [29–32].…”

“…In addition, the safety of RHcyst-1 on the normal cell lines 293T, Vero and CHO has been evaluated in our previous studies. RHcyst-1 treatment did not change the activity of normal cells and did not induce apoptosis in these cell lines [18]. These results show that the suppression of BMDC differentiation by RHcyst-1 is not due to apoptosis or cytotoxicity.…”

Exploring the anticancer activity of five Hyalomma species (Acari: Ixodidae) salivary gland extracts on human colorectal cancer cell line HT-29

“…Our ndings, however, go beyond the prior. A powerful and tightly-binding inhibitor of cysteine proteases, RHcyst-1, belonging to type 1 cystatins, was discovered in Rhipicephalus haemaphysaloides (Acari, Ixodidae) saliva and demonstrated anticancer potential in another work by Wei et al (2018). Four distinct tumor cell types, including the human cervical (HeLa), lung (A549), and ovarian (SKOV-3) cancer cell lines, as well as the murine melanoma B16-F10 cell lines, are strongly inhibited in vitro by RHcyst-1 in terms of proliferation, migration, and invasion.…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part II: Arthropods

“…The investigators attributed their results to the effects of RhCyst-1 in decreasing the number of myeloidderived suppressor cells (MDSCs) from the peripheral blood mononuclear cells (PBMCs). However, the investigators recommended further studies to discover the link between RhCyst-1 and MDSCs regulation from the spleens and PBMCs (60) .…”