Natural product myricetin is a pan-KDM4 inhibitor which with poly lactic-co-glycolic acid formulation effectively targets castration-resistant prostate cancer

Abstract

Background: Castration-resistant prostate cancer (CRPC) with sustained androgen receptor (AR) signaling remains a critical clinical challenge, despite androgen depletion therapy. The Jumonji C-containing histone lysine demethylase family 4 (KDM4) members, KDM4A‒KDM4C, serve as critical coactivators of AR to promote tumor growth in prostate cancer and are candidate therapeutic targets to overcome AR mutations/alterations-mediated resistance in CRPC.

Methods: In this study, using a structure-based approach, we identified a natural product, myricetin, able to block the demethylation of histone 3 lysine 9 trimethylation by KDM4 members and evaluated its effects on CRPC. A structure-based screening was employed to search for a natural product that inhibited KDM4B. Inhibition kinetics of myricetin was determined. The cytotoxic effect of myricetin on various prostate cancer cells was evaluated. The combined effect of myricetin with enzalutamide, a second-generation AR inhibitor toward C4-2B, a CRPC cell line, was assessed. To improve bioavailability, myricetin encapsulated by poly lactic-co-glycolic acid (PLGA), the US food and drug administration (FDA)-approved material as drug carriers, was synthesized and its antitumor activity alone or with enzalutamide was evaluated using in vivo C4-2B xenografts.

Results: Myricetin was identified as a potent α-ketoglutarate-type inhibitor that blocks the demethylation activity by KDM4s and significantly reduced the proliferation of both androgen-dependent (LNCaP) and androgen-independent CRPC (CWR22Rv1 and C4-2B). A synergistic cytotoxic effect toward C4-2B was detected for the combination of myricetin and enzalutamide. PLGA-myricetin, enzalutamide, and the combined treatment showed significantly greater antitumor activity than that of the control group in the C4-2B xenograft model. Tumor growth was significantly lower for the combination treatment than for enzalutamide or myricetin treatment alone.

Conclusions: These results suggest that myricetin is a pan-KDM4 inhibitor and exhibited potent cell cytotoxicity toward CRPC cells. Importantly, the combination of PLGA-encapsulated myricetin with enzalutamide is potentially effective for CRPC.

Keywords: Castration-resistant prostate cancer; Enzalutamide; Histone lysine demethylase family 4 (KDM4); Myricetin; Poly lactic-co-glycolic acid (PLGA).

Fig. 1

KDM4A, KDM4B, and KDM4C are overexpressed in PCa. A Expression of KDM4A, KDM4B, and KDM4C in an immortalized prostatic epithelial cell line (RWPE1) and PCa cell lines (LNCaP, C4-2, C4-2B, VCaP, and CWR22Rv1) using Western blotting analysis. Actin served as an internal control. B Expression of KDM4A, KDM4B, and KDM4C in PCa (TCGA) and normal (GTEx) specimens based on the UCSC Xena tool (https://xenabrowser.net). C Analysis of expression of KDM4A, KDM4B, and KDM4C in primary and metastatic PCa tissues based on the Grasso prostate dataset in Oncomine database (https://www.oncomine.org/resource/login.html). Statistical significance was determined using Student's t-test

Fig. 2

Myricetin inhibits the demethylation activity of KDM4A, KDM4B, and KDM4C. A–C Kinetic analyses of myricetin that inhibited the demethylation activity of KDM4A (A), KDM4B (B), and KDM4C C with increasing concentrations of α-KG (kinetics, the first raw; and the corresponding Lineweaver–Burk plot, the second raw), or with increasing concentrations of the H3K9me3 peptide (kinetics, the third raw; and the corresponding Lineweaver–Burk plot, the fourth raw). D Inhibition of H3 demethylation by KDM4A, KDM4B, or KDM4C in the presence of 50 μM myricetin using Western blotting analysis. The reaction mixture containing 10 μM enzyme, 100 μM inhibitor or blank buffer, and 5 μM of H3 in 50 mM HEPES, pH 7.5, 1 mM αKG, 2 mM ascorbate, and 50 μM Fe(II) was incubated at 37 °C for 30 min, followed by western blot analysis. H3 lysine modifications were probed with H3K9me3 and H3K36me3 antisera, respectively. M, myricetin

Fig. 3

Myricetin exhibits cell cytotoxicity against androgen-dependent and -independent PCa cells. A Cells (normal immortalized RWPE-1, androgen-dependent LNCaP, and androgen-independent C4-2B, CWR22Rv1) were treated with different concentrations of myricetin over 3 days as indicated, followed by MTT assay. B H3K9me3 and H3K36me3 levels in myricetin-treated C4-2B cells for 2 days. The H3K9me3, H3K36me3, and H3 signals were detected in cell lysates by western blot analysis. M myricetin

Fig. 4

Effects of myricetin and enzalutamide alone and in combination. C4-2B cells were treated with indicated concentrations of the drugs (M, myricetin; Enza, enzalutamide; M + Enza: the combination of myricetin and enzalutamide). The ratio of Myricetin/enzalutamide is [IC₅₀]_Myricetin/[IC₅₀]_enzalutamide based on the Chou-Talalay method [39]. Cell viability was measured using the MTT assay, the non-linear regression trendline analysis of IC₅₀ values was calculated by Prism7 and the CI values were generated by CompuSyn

Fig. 5

Myricetin impairs tumor growth in C4-2B xenografts. A Comparison of cell viability of C4-2B treated with free-form myricetin (Free M) or PLGA-encapsulated myricetin (PLGA-M). Cells were treated with different concentrations of M or PLGA-M for 24 h, followed by MTT assay. B C4-2B cells (1 × 10⁶) were implanted subcutaneously in the hindlimb of nude mice. PLGA-myricetin formulation (PLGA-M) (intraperitoneal injected, 20 mg/Kg, three times a week), enzalutamide (Enza) (oral gavage feeding, 12.5 mg/Kg, five times a week), and the combined treatment (Enza + PLGA-M) were administered respectively after 14 days implantation. Tumor volumes were measured for up to 3 weeks. Tumor volume was calculated using the formula (length × width × height × 0.52). Statistical significance was evaluated using Student’s t-test. C Representative IHC images of Ki67 and CD31 analysis of xenograft tumor sections from mice treated with vehicle, PLGA-M, Enza, or Enza + PLGA-M. D The proportion of Ki67 + cell was rescored in each field (400 ×) for six random fields in each group of IHC images. CD31 positive vessels in each field (100 ×) were scored for six random fields in each group. Significance was calculated using Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001