The Curcumin Analog EF24 Targets NF-κB and miRNA-21, and Has Potent Anticancer Activity In Vitro and In Vivo

<div><p>EF24 is a curcumin analog that has improved anticancer activity over curcumin, but its therapeutic potential and mechanism of action is unknown, which is important to address as curcumin targets multiple signaling pathways. EF24 inhibits the NF-κB but not the JAK-STAT signaling pathway in DU145 human prostate cancer cells and B16 murine melanoma cells. EF24 induces apoptosis in these cells apparently by inhibiting miR-21 expression, and also enhances the expression of several miR-21 target genes, PTEN and PDCD4. EF24 treatment significantly suppressed the growth of DU145 prostate cancer xenografts in immunocompromised mice and resulted in tumor regression. EF24 enhanced the expression of the miR-21 target PTEN in DU145 tumor tissue, but suppressed the expression of markers of proliferating cells (cyclin D1 and Ki67). In syngeneic mice injected with B16 cells, EF24 treatment inhibited the formation of lung metastasis, prolonged animal survival, inhibited miR-21 expression and increased the expression of miR-21 target genes. Expression profiling of miRNAs regulated by EF24 <i>in vitro</i> and <i>in vivo</i> showed that the antitumor activity of EF24 reflected the enhanced expression of potential tumor suppressor miRNAs as well as the suppressed expression of oncogenic miRNAs, including miR-21. Taken together, our data suggest that EF24 is a potent anticancer agent and selectively targets NF-κB signaling and miRNA expression, indicating that EF24 has significant potential as a therapeutic agent in various cancers.</p></div

The effects of EF24 and miR-21 KD on apoptosis <i>in vitro</i>.

<p>EV and miR-21KD DU145 cells were treated with EF24 (A) at varying concentrations for 24 hr or (B) at 5 µM for varying times, and the extent of PARP cleavage (cPARP) as a measure of apoptosis was determined by immunoblotting with anti-PARP. EV and miR-21KD B16 cells were treated with EF24 (C) at 5 µM for varying times or (D) at varying concentrations for 48 hr, and the fraction of annexin V positive cells was determined by flow cytometry.</p

The effects of EF24 on activation of the p65 subunit of NF-κB in DU145 cells <i>in vitro</i>.

<p>DU145 cells plated on 8-well chamber slides were treated with EF24 (5 µM) or vehicle (control) for 24 hr prior to stimulation with human IFNα (1000 IU/ml) for 30 min. Cells were fixed with 4% paraformaldehyde and methanol, and permeabilized with 1% Triton X100. After blocking with 5% goat serum, slides were stained for p65 and mounted using Vectashield medium with DAPI, and images were captured on a Zeiss LSM700 laser scanning confocal microscope.</p

The effects of EF24 on miRNA expression <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Total RNA was prepared from DU145 cells treated with vehicle and EF24 (5 µM, 24 hr), or tumor tissue from DU145 xenografts of control and EF24-treated mice (200 µg/kg body weight) at autopsy, and miRNA expression profiling was conducted on the nCounter Analysis System using the human V1 miRNA assay kit. (B) Venn diagram analysis of the genes upregulated or downregulated by EF24 treatment in cells and tumor tissue. (C) PCR validation of the genes upregulated or downregulated by EF24 treatment in both cells and tumor tissue (n = 3), and expressed as relative gene expression (EF24-treated/control).</p

The effects of EF24 on STAT and NF-κB activation <i>in vitro</i>.

<p>DU145 (A,B) and B16 (D,E) cells were treated with EF24 (5 µM) or vehicle (control) for 24 hr prior to stimulation with IFN (1000 IU/ml) for the indicated times. Whole cell extracts were prepared and immunoblotted for (A,D) IκB and actin, or (B,E) phosph-STAT3 and STAT3. (C,F) Nuclear extracts were prepared from IFN-stimulated cells (1000 IU/ml for 30 min) DU145 (C) or B16 (F) cells pretreated for 24 hr with EF24 (5 µM) or vehicle (control) and subjected to EMSA with oligonucleotide probes for NF-κB or SIE. A 50-fold excess of unlabeled probe (cold) was used to show the specificity of binding. Representative results from at least three experiments are shown.</p

Primers used for mRNA Expression Analysis.

<p>Primers used for mRNA Expression Analysis.</p

Forward Primers Used for miRNA Expression Analysis.

<p>Forward Primers Used for miRNA Expression Analysis.</p

The effects of EF24 miR-21 target protein expression in DU145 and B16 cells <i>in vitro</i>.

<p>DU145 and B16 cells were treated with 5 µM EF24. At 24 hr after EF24 treatment cells were lysed, and the expression of miR-21 target proteins was determined by immunoblotting.</p

EF24 treatment inhibits the tumorigenic potential of B16 melanoma cells <i>in vivo</i>.

<p>Mice were injected with 10⁶ B16 cells into the tail vein and after 1 week were injected daily with EF24 (200 µg/kg body weight). (A) Bioluminescent imaging of mice injected with luciferin performed at the indicated days, and (B) Kaplan-Meier analysis of survival data (n = 8). (C) RNA was extracted from tumor tissue at necropsy and subjected to gene expression analysis by qPCR as indicated (n = 3). (C) Lungs of control and EF24 treated mice at necropsy (17 days post-tail vein injection).</p

The effects of EF24 on the expression of key target genes in prostate cancer tumor xenografts <i>in vivo</i>.

<p>At the end of treatment (∼8 weeks) the control and EF24-treated mice (200 µg/kg body weight) injected with DU145 cells were sacrificed, and the tumors were removed, sectioned with a cryostat and immunostained for (A) PTEN, (B) Cyclin D1, or (C) Ki67. Sections were imaged on a laser scanning confocal microscope.</p