Abstract 5130: Assessment of the antitumor activity of leech (huridinaria manillensis) saliva extract in prostate cancer
Authors: Ammar Amr E. , Hassona Mohamed H. , Meckling Gray R. , Chan Leslie G. , Chin Mei Y. , Abdualkader Abdulrahman , Alaama Mohamed , Merzouk Ahmed , Helaluddin Abulbashar , Ghawi Abbas , Kucuk Omer , Guns Emma S. 
Published: August 2015

Abstract
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