Renal cell carcinoma (RCC), also called kidney cancer or renal adenocarcinoma, is highly resistant to current treatments. It has been previously reported that a Kunitz-type inhibitor domain-containing protein, isolated from the salivary glands of the Amblyomma cajennense tick, triggers apoptosis in murine renal adenocarcinoma cells (Renca) by inhibiting the proteasome and endoplasmic reticulum stress. Of note, Amblyomin-X is the corresponding recombinant protein identified in the cDNA library from A. cajennense salivary glands. Herein, using orthotopic kidney tumors in mice, we demonstrate that Amblyomin-X is able to drastically reduce the incidence of lung metastases by inducing cell cycle arrest and apoptosis. The in vitro assays show that Amblyomin-X is capable of reducing the proliferation rate of Renca cells, promoting cell cycle arrest, and down-regulating the expression of crucial proteins (cyclin D1, Ki67 and Pgp) involved in the aggressiveness and resistance of RCC. Regarding non-tumor cells (NIH3T3), Amblyomin-X produced minor effects in the cyclin D1 levels. Interestingly, observing the image assays, the fluorescence-labelled Amblyomin-X was indeed detected in the tumor stroma whereas in healthy animals it was rapidly metabolized and excreted. Taken the findings together, Amblyomin-X can be considered as a potential anti-RCC drug candidate.

During the last two decades, new insights into proteasome function and its role in several human diseases made it a potential therapeutic target. In this context, Amblyomin-X is a Kunitz-type FXa inhibitor similar to endogenous tissue factor pathway inhibitor (TFPI) and is a novel proteasome inhibitor. Herein, we have demonstrated Amblyomin-X cytotoxicity to different tumor cells lines such as pancreatic (Panc1, AsPC1BxPC3) and melanoma (SK-MEL-5 and SK-MEL-28). Of note, Amblyomin-X was not cytotoxic to normal human fibroblast cells. In addition, Amblyomin-X promoted accumulation of ER stress markers (GRP78 and GADD153) in sensitive (SK-MEL-28) and bortezomib-resistant (Mia-PaCa-2) tumor cells. The intracellular calcium concentration [Ca2+]i was slightly modulated in human tumor cells (SK-MEL-28 and Mia-PaCa-2) after 24 h of Amblyomin-X treatment. Furthermore, Amblyomin-X induced mitochondrial dysfunction, cytochrome-c release, PARP cleavage, and activation of caspase cascade in both human tumor (SK-MEL-28 and Mia-PaCa-2) cells. These investigations might help in further understanding of the antitumor properties of Amblyomin-X.

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Amblyomin-X is a Kunitz-type recombinant protein identified from the transcriptome of the salivary glands of the tick Amblyomma cajennense and has anti-coagulant and antitumoral activity. The supposed primary target of this molecule is the proteasome system. Herein, we elucidated intracellular events that are triggered by Amblyomin-X treatment in an attempt to provide new insight into how this serine protease inhibitor, acting on the proteasome, could be comparable with known proteasome inhibitors. The collective results showed aggresome formation after proteasome inhibition that appeared to occur via the non-exclusive ubiquitin pathway. Additionally, Amblyomin-X increased the expression of various chains of the molecular motor dynein in tumor cells, modulated specific ubiquitin linkage signaling and inhibited autophagy activation by modulating mTOR, LC3 and AMBRA1 with probable dynein involvement. Interestingly, one possible role for dynein in the mechanism of action of Amblyomin-X was in the apoptotic response and its crosstalk with autophagy, which involved the factor Bim; however, we observed no changes in the apoptotic response related to dynein in the experiments performed. The characteristics shared among Amblyomin-X and known proteasome inhibitors included NF-κB blockage and nascent polypeptide-dependent aggresome formation. Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules. In investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects. These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition.

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER-and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death. Melanoma is a type of cancer arising from the malignant transformation of melanocytes, pigment producing-cells found predominantly in the basal layer of the epidermis and eyes. Cutaneous melanoma is the most aggressive and treatment-resistant form of skin cancer responsible for the vast majority of skin cancer-related deaths in the Caucasian population 1. The global incidence of melanoma continues to increase at an alarming rate, despite decades of public prevention programs in many countries. Around 232,000 new cases of skin cancer were recorded worldwide in 2012, accounting for 1.6% of all new cases of cancer back then, while over 300,000 new cases of melanoma were diagnosed worldwide in 2018, according to the World Cancer Research Foundation 2,3. In Brazil, 1,547 deaths were recorded in 2013 due to melanoma, with around 5,690 new cases reported back then, while around 6,260 new cases were expected due in 2018, according to the National Cancer Institute (INCA) 4. Cutaneous melanoma usually affects a higher proportion of patients, in the age range 40-60 years. They can be treated by surgical excision when detected in the early stage (0, I, II and resectable III), however, in the later stages (unresectable III, IV and recurrent melanoma) the treatment options are chemotherapy, target therapy (BRAF/ MEK pathway inhibitors), immunotherapy (checkpoint blockade CTLA-4 receptor inhibition, PD-1 ↔ PD-L1 axis inhibition, and interferon-gamma immunotherapy), or a combination of them. Death in most patients is caused by metastatic disease which may have evolved from the primary tumor. Therefore, there is a need for new