Purpose: To isolate and characterize the antithrombin compounds of Malaysian leeches' saliva collection (LSC) for use as anticoagulant proteins and peptides. Method: Reversed phase -high performance liquid chromatography (RP-HPLC) was used to isolate all proteins from LSC. All isolated proteins were tested for anticoagulant activity by applying two tests, thrombin time test and the inhibition of the amidolytic activity of thrombin on the chromogenic substrate (S-2238). The molecular weights of isolated active compounds were identified using tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE

BACKGROUND Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. Leech saliva contains a large number of peptides and proteins, which have anti-thrombotic, antimicrobial, antitumor and anti-metastatic activities. Currently, leech therapy has an established role as an important tool in microsurgery, reconstructive surgery and salvage of grafted tissues. METHODS Leech saliva extract (LSE) was prepared as an aqueous solution from frozen lyophilized powder. LSE IC50 was determined in-vitro in five prostate cancer cell lines using MTS cell viability assay. In-vivo efficacy of LSE was determined in LNCaP and 22RV-1 in nude mice xenograft models. Mice were injected with 2×106 LNCaP or 22RV-1 cells subcutaneously; the mice were castrated in both studies to resemble castration resistant prostate cancer. After castration, mice were divided into four groups of 6-8 each. Mice were subcutaneously injected with either LSE (5 mg/kg) once a week, LSE (5 mg/kg) twice a week, docetaxel (10 mg/kg) or vehicle twice a week. PSA, tumor volume, and weight were measured weekly in the LNCaP model; and in the 22RV-1 tumor volume and weights were measured twice weekly. After four weeks of treatment, mice were euthanized, tumors and organs were collected for transcriptome and histopathological analysis. RESULTS LSE induced cell death in a panel of prostate cancer cell lines including LNCaP, PC3 and 22RV-1. IC50 values of were determined as 22 μg/ml in both LNCaP and PC3 cells and 53 μg/ml in 22RV-1cells. Furthermore, in vivo studies show that LSE once and twice weekly regimens both caused significant decrease in PSA and tumor volume compared to control. There was no significant difference between the antitumor activity of LSE and docetaxel. Interestingly, once weekly treatment with LSE was associated with significant weight gain (due to good dietary intake) at several time points. Immunohistochemical staining (IHC) showed significant increase in caspase-3 and significant decrease in P21, Ki-67, and PCNA expression in the LSE treated mice compared to the control group. Transcriptome analysis of tumor samples showed that LSE had significant immunomodulatory, anti-inflammatory, along with significant effects on cell-cell adhesion, induction of glutathione transferase and inhibition of certain growth factors. Consequently, these effects led to significant cell cycle arrest, increase in apoptosis and decrease in proliferation. CONCLUSIONS LSE has significant in vitro and in vivo anti-tumor activity with no apparent side effects. This can be attributed, at least partly, to its ability to inhibit cellular proliferation and induce apoptosis through its modulation of immunity, cell-cell adhesion, and inflammation. Citation Format: Amr E. Ammar, Mohamed H. Hassona, Gray R. Meckling, Leslie G. Chan, Mei Y. Chin, Abdulrahman Abdualkader, Mohamed Alaama, Ahmed Merzouk, Abulbashar Helaluddin, Abbas Ghawi, Omer Kucuk, Emma S. Guns. Assessment of the antitumor activity of leech (huridinaria manillensis) saliva extract in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5130. doi:10.1158/1538-7445.AM2015-5130

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271 Background: Ancient traditional physicians from many countries used leeching to treat a wide range of diseases for thousands of years. A large number of peptides and proteins have been identified and characterized in leech saliva extract (LSE), including anti-thrombotic agents, cancer metastasis inhibitors and anti-microbials Currently, leech therapy is established as an important tool in microsurgery and reconstructive operations having demonstrated superior clinical outcomes for the optimal salvage of grafted tissues. Methods: In the current study, we have determined the in vivo efficacy of LSE from (Huridinaria manillensis) on castration resistant LNCaP xenograft mouse model. Mice were divided into three groups of six, mice were subcutaneously injected with either LSE (5 mg/kg), docetaxel (10 mg/kg), or vehicle once a week. PSA and tumor volume were measured weekly. After four weeks of treatment, mice were euthanized, tumors and organs were collected for transcriptome and immunohistochemical (IHC) analysis. Results: There was a significant decrease in the tumor volume and PSA with either docetaxel (10mg/kg) or LSE (5 mg/kg) treated groups compared to the control. While there was no significant difference between the anti-tumor activity of docetaxel (10mg/kg) and LSE (5 mg/kg). IHC showed significant increase in caspase-3 and significant decrease in Ki-67 and PCNA expression in the LSE treated mice compared to the control group. Interestingly, transcriptome analysis of tumor samples showed that LSE modulated cytokine production, monocyte adhesion, steroidogenesis, and P38 MAPK signaling pathways. Conclusions: LSE has significant anti-tumor activity in LNCaP tumor xenograft models with no apparent side effects. This can be attributed, at least partly, to its inhibition of cellular proliferation, induction of apoptosis, modulation of immunity and steroidogenesis.