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

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.

KDM5B regulates the PTEN/PI3K/Akt pathway to increase sorafenib-resistance in hepatocellular carcinoma.

Lysine-specific demethylase 5B (KDM5B) exerts its tumor-promoting functions in numerous malignancies, although the possible mechanisms by which KDM5B promotes cancer aggressiveness in hepatocellular carcinoma (HCC) have been preliminarily explored, the role of this gene in regulating sorafenib-resistance in HCC has not been studied. Thus, the present study was designed to resolve this problem, and our data suggested that KDM5B was significantly upregulated in the HCC tissues collected from patients with sorafenib treatment history. Consistently, continuous low-dose sorafenib administration increased KDM5B expression levels in the sorafenib-resistant HCC cells compared to their sorafenib-sensitive counterparts. Next, by performing the functional experiments, we found that KDM5B positively regulated sorafenib-resistance and cancer stem cell (CSC) properties in HCC cells in vitro and in vivo . Furthermore, upregulation of KDM5B-degraded phosphatase and tensin homolog (PTEN), results in the activation of the downstream oncogenic PI3K/Akt pathway. Subsequently, the rescuing experiments verified that the promoting effects of KDM5B overexpression on chemoresistance and cancer stemness in HCC cells were all abrogated by PI3K (p110) knockdown and PTEN overexpression. Collectively, those data hinted that KDM5B influenced CSC properties and sorafenib-resistance in HCC cells through modulating the PTEN/PI3K/Akt pathway, and KDM5B could be used as a potential target for the treatment of HCC in clinic.

Design and synthesis of N-(1-(6-(substituted phenyl)-pyridazin-3-yl)-piperidine-3-yl)-amine derivatives as JMJD6 inhibitors.

JMJD6 is a member of the JmjC domain-containing family and has been identified as a promising therapeutic target for treating estrogen-induced and triple-negative breast cancer. To develop novel anti-breast cancer agents, we synthesized a class of N-(1-(6-(substituted phenyl)-pyridazine-3-yl)-piperidine-3-yl)-amine derivatives as potential JMJD6 inhibitors. Among them, the anti-cancer compound A29 was an excellent JMJD6 binder (KD = 0.75 ± 0.08 µM). It could upregulate the mRNA and protein levels of p53 and its downstream effectors p21 and PUMA by inhibiting JMJD6. Besides, A29 displayed potent anti-proliferative activities against tested breast cancer cells by the induction of cell apoptosis and cell cycle arrest. Significantly, A29 also promoted a remarkable reduction in tumor growth, with a TGI value of 66.6% (50 mg/kg, i.p.). Taken together, our findings suggest that A29 is a potent JMJD6 inhibitor bearing a new scaffold acting as a promising drug candidate for the treatment of breast cancer.

Inhibition of ALKBH5-mediated m6 A modification of PPARG mRNA alleviates H/R-induced oxidative stress and apoptosis in placenta trophoblast.

The alpha-ketoglutarate-dependent (ALKB) homolog 5 (ALKBH5), an m6 A demethylase, has been reported to be involved in the pathogenesis of preeclampsia (PE), but the exact mechanism requires further investigation. RT-qPCR or Western blotting were used to determine ALKBH5 and peroxisome proliferator-activated receptor gamma (PPARG) expression in placentas from PE patients and normal volunteers, as well as in HTR-8/SVneo cells treated with hypoxia/reoxygenation (H/R). Our results showed that the expression of ALKBH5 was significantly upregulated and PPARG was downregulated in preeclamptic placentas and H/R-treated cells. ALKBH5 interference reduced m6 A levels of PPARG mRNA, and increased PPARG mRNA stability and promoted PPARG translation level. In addition, ALKBH5 silencing increased the cell proliferation, migration, and vimentin protein level, and inhibited cell apoptosis, oxidative stress, and protein levels of endoglin (ENG) and E-cadherin in H/R-treated cells, whereas PPARG interference reversed these effects. Furthermore, PPARG repressed the H3K9me2 levels at activated leukocyte cell adhesion molecule (ALCAM) promoter region by increasing the expression and activity of lysine demethylase 3B (KDM3B). ALCAM inhibition reversed the effects of PPARG overexpression on H/R-treated cell functions. PKF115-584 suppressed the effects of ALKBH5 interference on the behaviors of H/R-treated cells. Finally, inhibition of ALKBH5 alleviates PE-like features in pregnant mice. Inhibition of ALKBH5 promotes KDM3B-mediated ALCAM demethylation by facilitating PPARG mRNA m6 A modification, and further activates the Wnt/ß-catenin pathway, and in turn alleviates PE progression.

JIB-04, a Pan-Inhibitor of Histone Demethylases, Targets Histone-Lysine-Demethylase-Dependent AKT Pathway, Leading to Cell Cycle Arrest and Inhibition of Cancer Stem-Like Cell Properties in Hepatocellular Carcinoma Cells.

JIB-04, a pan-histone lysine demethylase (KDM) inhibitor, targets drug-resistant cells, along with colorectal cancer stem cells (CSCs), which are crucial for cancer recurrence and metastasis. Despite the advances in CSC biology, the effect of JIB-04 on liver CSCs (LCSCs) and the malignancy of hepatocellular carcinoma (HCC) has not been elucidated yet. Here, we showed that JIB-04 targeted KDMs, leading to the growth inhibition and cell cycle arrest of HCC, and abolished the viability of LCSCs. JIB-04 significantly attenuated CSC tumorsphere formation, growth, relapse, migration, and invasion in vitro. Among KDMs, the deficiency of KDM4B, KDM4D, and KDM6B reduced the viability of the tumorspheres, suggesting their roles in the function of LCSCs. RNA sequencing revealed that JIB-04 affected various cancer-related pathways, especially the PI3K/AKT pathway, which is crucial for HCC malignancy and the maintenance of LCSCs. Our results revealed KDM6B-dependent AKT2 expression and the downregulation of E2F-regulated genes via JIB-04-induced inhibition of the AKT2/FOXO3a/p21/RB axis. A ChIP assay demonstrated JIB-04-induced reduction in H3K27me3 at the AKT2 promoter and the enrichment of KDM6B within this promoter. Overall, our results strongly suggest that the inhibitory effect of JIB-04 on HCC malignancy and the maintenance of LCSCs is mediated via targeting the KDM6B-AKT2 pathway, indicating the therapeutic potential of JIB-04.

[Effect of Jumonji domain-containing protein-3 on the proliferation and migration of lung cancer cell line].

For investigating the effect of Jumonji domain-containing protein-3 (JMJD3) on the behavior of lung cancer cell line, A549 proliferation was measured with EDU staining and flow cytometer after JMJD3 expression plasmid and pcDNA3. 1 transfection at 48h. The migration ability of A549 was tested at the same time. The expression of p21 mRNA was measured with RT-PCR. The results showed that JMJD3 transfection increased the EDU positive cells ratio (JMJD3: 40.75% +/- 2.07%, control: 20.97% +/- 1.5%, P < 0.001). G1 phase cell ration also increased after JMJD3 transfection (JMJD3:47. 80% +/- 1.85%, control: 54.60% +/- 0.95%, P = 0.005). The mRNA expression of p21 decreased in JMJD3 group (JMJD3: 35. 89% +/- 3.71%, control: 91.78% +/- 3.74%, P < 0.001). The distances of migration were (0.47 +/- 0.27) cm and (0.96 +/- 0.40) cm after 24h and 48h with JMJD3 tranfection, compared to (0.57 +/- 0.22)cm and (1.08 +/- 0.33)cm in control, respectively (P > 0.05). JMJD3 promoted the proliferation of A549 and decreased the G1 cell numbers, decreased the p21 mRNA, but had no effect on A549 migration.

Overcoming radio-resistance in esophageal squamous cell carcinoma via hypermethylation of PIK3C3 promoter region mediated by KDM5B loss.

Many patients with esophageal squamous cell carcinoma (ESCC) are inoperable because of old age or the advanced stage of the disease; thus radio- and chemotherapy are believed as the standard treatments for these patients. However, due to the radio-resistance of tumor cells that may develop during radiotherapy, results remain unsatisfactory. In this article, the possible relationship between the expression of lysine demethylase 5B (KDM5B) and ESCC radio-resistance is clarified, and the underlying mechanism is evaluated. Using the GSE75241 microarray, we identified KDM5B as a potential oncogene in ESCC. KDM5B was overexpressed in ESCC patients and cells. Inhibition of KDM5B enhanced the H3K4me3 methylation of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) promoter and induced the expression of PIK3C3. Knockdown of KDM5B or overexpression of PIK3C3 in KYSE-150 and TE-10 cells promoted apoptosis, cell cycle arrest, autophagy, and increased sensitivity to radiotherapy. Silencing of PIK3C3 attenuated the promoting effect of sh-KDM5B on the sensitivity of ESCC cells to radiotherapy. The inhibition of sh-KDM5B in radio-resistance of ESCC cells was also reproduced in vivo. Taken together, our findings provide evidence that reduced expression of KDM5B has a critical role in promoting ESCC radio-sensitivity by upregulating PIK3C3, suggesting KDM5B may function as an oncogene in ESCC.

Efficient therapeutic delivery by a novel cell-permeant peptide derived from KDM4A protein for antitumor and antifibrosis.

Cell-penetrating peptide (CPP) based delivery have provided immense potential for the therapeutic applications, however, most of nonhuman originated CPPs carry the risk of possible cytotoxicity and immunogenicity, thus may restricting to be used. Here, we describe a novel human-derived CPP, denoted hPP10, and hPP10 has cell-penetrating properties evaluated by CellPPD web server, as well as In-Vitro and In-Vivo analysis. In vitro studies showed that hPP10-FITC was able to penetrate into various cells including primary cultured cells, likely through an endocytosis pathway. And functionalized macromolecules, such as green fluorescent protein (GFP), tumor-specific apoptosis inducer Apoptin as well as biological active enzyme GCLC (Glutamate-cysteine ligase, catalytic subunit) can be delivered by hPP10 in vitro and in vivo. Collectively, our results suggest that hPP10 provide a novel and versatile tool to deliver exogenous proteins or drugs for clinical applications as well as reprogrammed cell-based therapy.