Natural stilbenes have gained significant attention in the scientific community owing to their potential anticancer effects against prostate cancer. We recently reported that Gnetin C, a resveratrol (Res) dimer, demonstrated more potent inhibition of metastasis-associated protein 1/v-ets avian erythroblastosis virus E26 oncogene homolog 2 (MTA1/ETS2) axis in prostate cancer cell lines than other stilbenes. In this study, we investigated in vivo antitumor effects of Gnetin C in two doses (50 and 25 mg/kg, i.p.) using PC3M-Luc subcutaneous xenografts and compared these to Res and pterostilbene (Pter). We found that while vehicle-treated mice revealed rapid tumor progression, compounds-treated mice showed noticeable delay in tumor growth. Gnetin C in 50 mg/kg dose demonstrated the most potent tumor inhibitory effects. Gnetin C in 25 mg/kg dose exhibited tumor inhibitory effects comparable with Pter in 50 mg/kg dose. Consistent with the effective antitumor effects, Gnetin C-treated tumors showed reduced mitotic activity and angiogenesis and a significant increase in apoptosis compared to all the other groups. The data suggest that Gnetin C is more potent in slowing tumor progression in prostate cancer xenografts than Res or Pter. Taken together, we demonstrated, for the first time, that Gnetin C is a lead compound among stilbenes for effectively blocking prostate cancer progression in vivo.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

Overexpression of chromatin modifier protein, metastasis-associated protein 1 (MTA1) in prostate cancer contributes to tumor aggressiveness and metastasis. MTA1 ChiP-Seq analysis identified downstream targets that are transcriptionally regulated by MTA1 and suggested a link between MTA1 and ETS2. The effects of ETS2 in cancer are context-dependent and both oncogenic and tumor suppressive functions have been described. We have shown that there is a positive correlation between MTA1 and ETS2 using loss of function studies and various preclinical models of prostate cancer. Our conclusion that MTA1 functions as a co-activator for regulating ETS2 expression in prostate cancer leading to promotion of prostate cancer progression, prompted us to look for pharmacological inhibitors of MTA1/ETS2 axis. In our previous studies, we reported inhibition of MTA1 by resveratrol and its potent analog pterostilbene in vitro and in vivo. We have demonstrated that pterostilbene treatment blocks the progression of PIN and adenocarcinoma in xenografts and transgenic mouse models by inhibiting MTA1 expression and signaling. Here, we found that Gnetin C, double-resveratrol, has more potent inhibition of MTA1/ETS2 axis than other stilbenes. Gnetin C, a resveratrol dimer, is found abundantly in melinjo plant widely cultivated in Southeast Asia; its seeds and fruits are common ingredients in Indonesian culinary. As resveratrol and pterostilbene, Gnetin C has been reported to possess anti-inflammatory and anticancer activity, however it has not been considered as chemopreventive agent in prostate cancer. Using two prostate cancer cell lines, namely DU145 and PC3M cells, we found that Gnetin C shows significant inhibitory effect on cell proliferation, more potent effects in colony formation and wound healing assays than resveratrol or pterostilbene. Importantly, Gnetin C specifically and strongly downregulates MTA1 and ETS2 expression in prostate cancer cells. Taken together, our findings implicate the potential of Gnetin C in MTA1/ETS2-mediated chemoprevention and therapy in prostate cancer. Citation Format: Urvi Kolhatkar, Kshiti Dholakia, Gabriela Sikorska, Avinash Kumar, Luis A. Martinez, Anait S. Levenson. Gnetin C as a candidate for targeted chemopreventive and therapeutic measures in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 253.

In our previous studies, we reported MTA1-targeted chemopreventive and therapeutic properties of resveratrol (Res) and its potent analog pterostilbene (Pter) in prostate cancer (PCa). We recently reported that Gnetin C, a Res- dimer, has more potent inhibition of MTA1/ETS2 axis in PCa than other stilbenes. Gnetin C is found abundantly in melinjo (Gnetum gnemon) plant widely cultivated in Southeast Asia; its seeds and fruits are common ingredients in Indonesian culinary. As Res and Pter, Gnetin C has been reported to possess anti-inflammatory, antioxidant and anticancer activity, however it has not been considered as a chemopreventive agent in PCa. Using two PCa cell lines, namely DU145 and PC3M cells, we demonstrated that Gnetin C shows significant inhibitory effects on cell proliferation and viability, more potent effects in colony formation and wound healing assays than Res or Pter. We next studied in vivo antitumor effects of Gnetin C in two doses (50 and 25 mg/kg, i.p., 5 days/week) using PC3M-Luc subcutaneous xenografts and compared to Res and Pter effects (50 mg/kg, i.p., 5 days/week). 5-6 week-old male nude mice were randomized into 5 treatment groups: 1) Vehicle; 2) Res50; 3) Pter50; 4) Gnetin C50; and 5) Gnetin C25. Weekly bioluminescent imaging was used to monitor tumor development. After 21 days of treatment with corresponding compounds, mice were sacrificed and tumor tissues were analyzed. Results demonstrated that while vehicle-treated mice showed rapid tumor progression, compounds-treated mice showed noticeable delay in tumor growth, especially in Gnetin C-treated groups. Gnetin C in 50 mg/kg dose demonstrated the most potent tumor inhibitory effects. Gnetin C in 25 mg/kg dose exhibited tumor inhibitory effects comparable with Pter in 50 mg/kg dose. Consistent with the effective antitumor effects, Gnetin C-treated tumors showed reduced mitotic activity (Ki67) compared to all the other groups. Accordingly, cleaved caspase 3 staining revealed a large increase in apoptosis in tumors from Gnetin C-treated mice. Additionally, microvessel area as assessed by CD31 staining was significantly decreased in Gnetin C-treated tumors compared to Res, Pter or vehicle-treated mice. Together, this data suggest that Gnetin C is more potent in slowing tumor progression in PCa xenografts than Res or Pter. Steady state serum levels and tissue distribution of compounds also were analyzed. Taken together, we demonstrated, for the first time, that Gnetin C is a lead compound among stilbenes for effectively blocking PCa progression. In conclusion, our findings implicate the potential of Gnetin C in PCa chemoprevention and therapy. Citation Format: Ketaki Gadkari, Rutu Hemani, Urvi Kolhatkar, Gisella Campanelli, Qing Cai, Avinash Kumar, Anait S. Levenson. Does dimer resveratrol offer twice the benefits in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3.