Kunitz type protease inhibitor EgKI-1 from the canine tapeworm Echinococcus granulosus as a promising anti-cancer therapeutic

EgKI-1, a member of the Kunitz type protease inhibitor family, is highly expressed by the oncosphere of the canine tapeworm Echinococcus granulosus, the stage that is infectious to humans and ungulates, giving rise to a hydatid cyst localized to the liver and other organs. Larval protoscoleces, which develop within the hydatid cyst, have been shown to possess anti-cancer properties, although the precise molecules involved have not been identified. We show that recombinant EgKI-1 inhibits the growth and migration of a range of human cancers including breast, melanoma and cervical cancer cell lines in a dose-dependent manner in vitro without affecting normal cell growth. Furthermore, EgKI-1 treatment arrested the cancer cell growth by disrupting the cell cycle and induced apoptosis of cancer cells in vitro. An in vivo model of triple negative breast cancer (MDA-MB-231) in BALB/c nude mice showed significant tumor growth reduction in EgKI-1-treated mice compared with controls. These findings indicate that EgKI-1 shows promise for future development as an anti-cancer therapeutic.


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Protein-based therapeutics enable targeted approaches for treating cancer (1). There are many 50 benefits of proteins over small-molecule drugs mainly because of the increased surface area 51 accessing a much wider range of protein targets (2). Protease inhibitors are important as 52 potential cancer therapeutics as proteases are associated with carcinogenesis and cancer 53 progression. Numerous plant protease inhibitors have recently entered human clinical trials 54 (3). Parasites produce a range of protease inhibitors with diverse functions mainly to evade 55 hostile adverse host reactions (4). 56 Several parasites, including the liver flukes, Opisthorchis viverrini and Clonorchis sinensis, 57 and the blood fluke, Schistosoma haematobium, are known risk factors for 58 cholangiocarcinoma and bladder cancer, respectively (5). In contrast, other parasites such as 59 Trypanosoma cruzi (6), Toxoplasma gondii (7) and Echinococcus granulosus (8) produce 60 metabolites with anticancer properties. 61 An increasing number of studies have shown that the presence of neutrophils in tumors, 62 known as tumor associated neutrophils (TAN) correlates with poor prognosis (9), specifically 63 in breast cancers (10). Neutrophils play major roles in tumor initiation, growth and metastasis 64 (11) mainly through the serine protease enzyme neutrophil elastase secreted by active neutrophils. Neutrophil elastase also acts as a chemoattractant for more neutrophils (12). 66 Therefore, potent neutrophil elastase inhibitors have stimulated much interest for 67 development as cancer therapeutics (13). 68 The larval stage of the canine tapeworm (phylum Cestoda) E. granulosus causes 69 echinococcosis (hydatidosis) in humans and ungulates (sheep, goats, cattle etc) when they 70 ingest the parasite eggs containing oncospheres in contaminated food or water (14). The 71 oncospheres hatch and penetrate the intestinal mucosa, enter the blood stream and migrate to 72 the liver or lung. A fluid-filled larva begins to develop from a single oncosphere with 73 subsequent formation of multiple layers, resulting in a metacestode or hydatid cyst (15). 74 Protoscoleces, which develop asexually within the hydatid cyst, have been shown to induce 75 the death of fibrosarcoma cells although the specific molecules involved have not been 76 identified (8). We have shown that EgKI-1, a member of the Kunitz type protease inhibitor 77 family, is highly expressed in oncospheres, is a potent neutrophil elastase and chemotaxis 78 inhibitor (16) and was recently granted an International Patent Publication (17). 79 In this study, recombinant EgKI-1 was expressed in yeast, purified and investigated for 80 potential anti-cancer properties in vitro and in vivo.       Zoom. Images were acquired for each well at 12 hours interval by an in built phase contrast 157 microscope.

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The distance between one side of scratch and the other at each time point was measured using    24-well plates and EgKI-1 or control buffer treatment was carried out on the following day.

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After 24 hours incubation, cells were harvested by trypsinisation and washed with cold PBS.      Real time monitoring using the IncuCyte showed that cells treated with 1 µM EgKI-1 303 eventually lost their typical shape, lost their ability to proliferate with time (Fig 1a), and 304 exhibited dose-dependent growth inhibition compared with the control cells (Fig 1b). 305 Furthermore, the IncuCyte analysis showed that cells continued to grow in the presence of 306 EgKI-1, at a concentration of around 0.5 µM, before starting to die after ~60 hours making it 307 a more sensitive assay than end point staining. and 100% in the controls respectively (Fig 2b). This data indicates that EgKI-1 inhibits 319 cancer cell migration. which can lead to cell membrane collapse and eventually necrosis. There were significant 325 decreases in the intensity of tubulin and total cell area indicating the degradation of the 326 cytoskeleton following treatment with the EgKI-1 protein (Fig 3).     When injected intraperitoneally into mice, the EgKI-1 protein was absorbed into the blood in compared with control mice (Fig 6). Furthermore, expression of Ki67 protein, which is a 366 proliferation marker, was significantly reduced in the EgKI-1-treated tumor tissues indicating 367~80% reduction of tumor proliferation (Fig 6) as suggested by the in vitro data. EgKI-1 is also an important aspect to consider for cancer therapeutic development.

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Further, the apparent cell growth inhibition by EgKI-1 was due to an increase in apoptosis, 14 420 A therapeutic with a molecular size less than 10 kDa allows rapid extravasation from blood 421 vessels and rapid transport to tumor targets resulting in maximal tumor uptake (43).

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However, such molecules have shorter serum half-lives than longer peptides as they are 423 quickly cleared by renal filtration (44). PEGylation, the technique of covalently attaching 424 polyethylene glycol (PEG) to a molecule, which may be a low molecular size protein, 425 enzyme or nanoparticle, has proven to be one of the best methods for the passive targeting of 426 anti-cancer therapeutics (45, 46). Accordingly, this technology will be applied in the future to 427 improve the serum half-life of EgKI-1 if needed.

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Another future consideration will be to monitor the up or down regulation of genes involved 429 in cancer progression and metastasis following EgKI-1 treatment. Further, EgKI-1 treatment 430 can inhibit the cellular activity of different proteases, including trypsin, plasma kallikrein and 431 cathepsins, and matrix metalloproteases as analyzed with P237, P139 and P126 fluorescence 432 substrates (unpublished data). We have also shown that EgKI-1 can induce some anti-433 inflammatory cytokines (unpublished data), which is important as chronic inflammation 434 increases the risk of cancer and a reduction in inflammation helps in cancer therapy (47).

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Therefore immunomodulation effects induced by EgKI-1 may also play a role in cancer 436 growth inhibition in vivo, an area that needs to be explored in the future using 437 immunocompetent mouse models of cancer.  Red fluorescence intensity of tubulin (p<0.0001****) after analysis by Student's t-test.