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We have explored the effects of 20(S)-protopanaxadiol (aPPD), a naturally derived ginsenoside, against androgen receptor (AR) positive castration resistant prostate cancer (CRPC) xenograft tumors and have examined its interactions with AR. In silico docking studies for aPPD binding to AR, alongside transactivation bioassays and in vivo efficacy studies were carried out in the castration-resistant C4-2 xenograft model. Immunohistochemical (IHC) and Western blot analyses followed by evaluation of AR, apoptotic, cell cycle and proliferative markers in excised tumors was performed. The growth of established CRPC tumors was inhibited by 53% with aPPD and a corresponding decrease in serum PSA was seen compared to controls. The IHC data revealed that Ki-67 was significantly lower for aPPD treated tumors and was associated with elevated p21 and cleaved caspase-3 expression, compared to vehicle treatment. Furthermore, aPPD decreased AR protein expression in xenograft tumors, while significantly upregulating p27 and Bax protein levels. In vitro data supporting this suggests that aPPD binds to and significantly inhibits the N-terminal or the DNA binding domains of AR. The AR androgen binding site docking score for androgen (dihydrotestosterone) was −11.1, while that of aPPD was −7.1. The novel findings described herein indicate aPPD potently inhibits PCa in vivo partly via inhibition of a site on the AR N-terminal domain. This manifested as cell cycle arrest and concurrent induction of apoptosis via an increase in Bax, cleaved-caspase-3, p27 and p21 expression.

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

In spite of possessing desirable anticancer properties, currently, limited clinical success has been achieved with 20(S)-protopanaxadiol (aPPD) and 1,25-dihydroxyvitamin D3 (calcitriol). This study is designed to evaluate if the combination of aPPD with calcitriol can inhibit human prostate cancer xenograft growth by using nuclear receptor signaling. Athymic male nude mice were utilized to establish an androgen-independent human prostate cancer C4-2 cell castration-resistant prostate cancer (CRPC) xenograft model. Mice were treated orally for six weeks with 70 mg/kg aPPD administered once daily or three times per week with 4 µg/kg calcitriol or in combination or only with vehicle control. Contrary to our expectations, calcitriol treatment alone increased C4-2 tumor growth. However, the addition of calcitriol substantially increased aPPD-mediated tumor growth suppression (76% vs. 53%, combination vs. aPPD alone). The combination treatment significantly increased levels of cleaved caspase-3 apoptotic marker compared to vehicle-treated or aPPD-treated C4-2 tumors. The mechanistic elucidations indicate that tumor inhibition by the aPPD and calcitriol combination was accompanied by elevated vitamin D receptor (VDR) protein expression. In silico data suggest that aPPD weakly binds to the native LBD pocket of VDR. Interestingly, the combination of aPPD and calcitriol activated VDR at a significantly higher level than calcitriol alone and this indicates that aPPD may be an allosteric activator of VDR. Overall, aPPD and calcitriol combination significantly inhibited tumor growth in vivo with no acute or chronic toxic effects in the C4-2 xenograft CRPC nude mice. The involvement of VDR and downstream apoptotic pathways are potential mechanistic routes of antitumor effects of this combination.

Androgen receptor (AR) is a key regulatory transcription factor in the development of prostate cancer (PCa). Common treatments for PCa include hormone therapy, radiotherapy and surgery. However, many PCa patients develop resistance towards hormone therapies in later stages of PCa. There is growing evidence suggesting that AR splice variants render PCa treatments that target the LBD, such as enzalutimide, ineffective. Our objective is to develop a drug to treat recurrent and metastatic PCa by targeting the DNA Binding Domain (DBD). Through in silico docking prediction and in vitro bioassays, we have identified a novel small molecule - VPC-14449 (4-(4-(4,5-bromo-1H-imidazol-1-yl)thiazol-2-yl)morpholine) - that may inhibit AR activation through targeting DBD. In order to bring VPC -14449 into the clinic, we conducted preclinical in vivo studies to determine both toxicity and efficacy. CD1 mice were treated with 25, 50, and 100 mg/kg of VPC-14449 I.P. twice daily for four weeks to determine the maximum tolerable dose. During this time, we assessed the signs of physical toxicity, behavioral changes, and gross manifestations of stress. At the end of the experiment, the mice were euthanized and organs were collected for further immunohistochemical analysis. Furthermore, we assessed the in vivo therapeutic effects of VPC-14449 in different nude mouse xenograft models including LNCaP, MR49-F, and C4-2. Body weight, tumor volume (π/6 x length x width x height) and PSA were measured weekly. LNCaP xenograft mice were castrated when PSA level reached 25 ng/ml. Mice were randomly assigned to one of the three treatment groups (VPC-14449 (100mg/kg), enzalutimide (10 mg/kg), or vehicle) when PSA level returned to 25 ng/ml (for LNCaP xenografts) or when the tumor sizes reached 100mm3(for MR49-F and C4-2 xenografts). The mice were treated I.P. twice daily for four weeks before euthanization. Toxicity studies revealed no signs of physical toxicity, behavioral changes, and gross manifestations of stress in animals treated with up to 100 mg/kg dose of VPC-14449. Efficacy studies revealed that VPC-14449 effectively suppresses tumor growth in LNCaP, MR49 and C4-2 xenografts with P value <0.01, <0.05 and < 0.001 respectively, when compared to control. VPC-14449 also significantly reduced PSA levels in LNCaP xenografts (p value <0.01). The results suggest that VPC-14449 is effectively suppressing PCa tumor growth even in androgen insensitive and MDV resistant PCa cell lines in vivo. Citation Format: Mohamed DH Hassona, Leslie G. Chan, Gray R. Meckling, Huifang Li, Eric LeBlanc, Fuqiang Ban, Artem Cherkasov, Paul S. Rennie, Emma Guns. DNA-binding domain as an alternative target site on androgen receptor for prostate cancer therapy: Pre-clinical in vivo study of a novel small molecule for drug development. [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 1727. doi:10.1158/1538-7445.AM2015-1727

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