Evaluation of Novel 3-Hydroxyflavone Analogues as HDAC Inhibitors against Colorectal Cancer

Abstract

Alteration of epigenetic enzymes is associated with the pathophysiology of colon cancer with an overexpression of histone deacetylase 8 (HDAC8) enzyme in this tissue. Numerous reports suggest that targeting HDAC8 is a viable strategy for developing new anticancer drugs. Flavonols provide a rich source of molecules that are effective against cancer; however, their clinical use is limited. The present study investigated the potential of quercetin and synthetic 3-hydroxyflavone analogues to inhibit HDAC8 enzyme and evaluated their anticancer property. Synthesis of the analogues was carried out, and cytotoxicity was determined using MTT assay. Nonspecific and specific HDAC enzyme inhibition assays were performed followed by the expression studies of target proteins. Induction of apoptosis was studied through annexin V and caspase 3/7 activation assay. Furthermore, the analogues were assessed against in vivo colorectal cancer. Among the synthesized analogues, QMJ-2 and QMJ-5 were cytotoxic against HCT116 cells with an IC₅₀ value of 68 ± 2.3 and 27.4 ± 1.8 µM, respectively. They inhibited HDAC enzyme in HCT116 cells at an IC₅₀ value of 181.7 ± 22.04 and 70.2 ± 4.3 µM, respectively, and inhibited human HDAC8 and 1 enzyme at an IC₅₀ value of <50 µM with QMJ-5 having greater specificity towards HDAC8. A reduction in the expression of HDAC8 and an increase in acetyl H3K9 expression were observed with the synthesized analogues. Both QMJ-2 and QMJ-5 treatment induced apoptosis through the activation of caspase 3/7 evident from 55.70% and 83.55% apoptotic cells, respectively. In vivo studies revealed a significant decrease in colon weight to length ratio in QMJ-2 and QMJ-5 treatment groups compared to DMH control. Furthermore, a reduction in aberrant crypt foci formation was observed in the treatment groups. The present study demonstrated the potential of novel 3-hydroxyflavone analogues as HDAC8 inhibitors with anticancer property against colorectal cancer.

Figure 1

Synthesis of 3-hydroxyflavone analogues was carried out by the reaction of various substituted acetophenones with substituted benzaldehyde using 40% KOH and methanol to form chalcones (1a–f). The chalcones were cyclized using 10% KOH, methanol, and 15% H₂O₂ to produce 3-hydroxyflavones (QMJ-1–6).

Figure 2

Effect of various compounds incubated for 24 h on HDAC8, acetyl histone H3K9, and p21^Waf1/Cip1 protein expression in HCT116 cells. All values are expressed as mean ± SEM of three experiments. ^ap < 0.05 vs. normal control (NC).

Figure 3

Apoptotic profile after treatment with various compounds in HCT116 cells was determined as % live,% early, and late apoptosis and % dead cells by MUSE cell analyzer: (a) normal control; (b) normal control; (c) SAHA (5 µM); (d) quercetin (100 µM); (e) QMJ-2 (100 µM); (f) QMJ-5 (50 µM).

Figure 4

Apoptosis induction through the activation of caspase 3/7 after treatment with various compounds in HCT116 cells was determined as % live, % early, and late apoptosis and % dead cells by the MUSE cell analyzer. (a) Normal control; (b) normal control; (c) SAHA (5 μM); (d) quercetin (100 μM); (e) QMJ-2 (100 μM); (f) QMJ-5 (50 μM).

Figure 5

Effect of various compounds on different phases of HCT116 cells was estimated using flow cytometer. Histogram plots display the different phases of the cell cycle and the percentage of cells in each phase. (a) Normal control; (b) SAHA (5 μM); (c) quercetin (100 μM); (d) QMJ-2 (100 μM); (e) QMJ-5 (50 μM).

Figure 6

The bar graphs represent colon weight/colon length ratio. All values are expressed as mean ± SEM of six animals. ^ap < 0.05 vs. normal control; ^bp < 0.05 vs. DMH control.

Figure 7

The bar graphs represent the effect of various treatments on the major organ index. All values are expressed as mean ± SEM of six animals. ^ap < 0.05 vs. normal control; ^bp < 0.05 vs. DMH control. (a) Kidney index; (b) spleen index; (c) liver index.

Figure 8

Histopathological changes of colon tissues from different treatment groups at 40x magnification. Arrows indicate aberrant crypt foci. (a) Normal control; (b) DMH control; (c) 5-FU; (d) quercetin; (e) QMJ-2; (f) QMJ-5.