In vitro and in vivo anticancer studies of 2'-hydroxy chalcone derivatives exhibit apoptosis in colon cancer cells by HDAC inhibition and cell cycle arrest

Abstract

Considering the therapeutic values of bioflavonoids in colon cancer treatment, six 2'-hydroxy chalcones (C1-C6) were synthesized, characterized and screened for in vitro cytotoxicity on human colon carcinoma (HCT116) and African green monkey kidney epithelial cells (Vero). Only C5 showed selective cytotoxicity against HCT116 cells. Other potent cytotoxic compounds were C1, C2 and C3. Further screening included enzyme inhibition studies on histone deacetylase (HDAC) enzyme where C1 showed lowest IC₅₀ value (105.03 µM). Based on cytotoxicity data C1, C2 and C3 were selected for further in vitro mechanistic studies, namely apoptotic studies (Acridine orange/Ethidium bromide (AO/EB) and Annexin V), cell cycle analysis using propidium iodide (PI) stain and in vivo anticancer efficacy in 1,2-dimethyl hydrazine (DMH) induced colorectal carcinoma in Wistar rats. The compounds induced apoptosis in more than 30 % cells in AO/EB and Annexin V staining. They also showed cell cycle arrest in G₂/M phase with PI staining. They showed a significant reduction in aberrant crypt foci formation and adenocarcinoma count along with a significant (p<0.05) reduction in TNF-α levels as compared to DMH control at 100 mg/kg dose. Thus, it can be concluded that the synthesized 2'-hydroxychalcones were effective against colon adenocarcinoma in in vitro and in vivo studies.

Keywords: DMH; HDAC; apoptosis; cell cycle; chalcones; colon cancer.

Table 1. In vitro cytotoxicity screening in human colon cancer and normal cells

Table 2. Effect of DMH and test compounds on ACF and adenocarcinoma count

Table 3. Effect of various treatments on colon L/W ratio

Table 4. Effect of test compounds on organ index

Figure 1. Synthetic scheme of chalcones.

Synthesis of chalcones was carried out using acetophenone (A) and benzaldehyde (B) where 2ʹ-hydroxy acetophenone was used for the preparation of C1-C4, 2,4-dihydroxy acetophenone for C5 and 2,6-dihydroxy acetophenone for C6. Benzaldehyde used were 4-methyl benzaldehyde for C1, 4-hydroxy benzaldehyde for C2, 4-carboxy benzaldehyde for C3 and C6 and 4-(dimethylamino)benzaldehyde for C4 and C5. The reaction conditions were (a) 20 % w/v potassium hydroxide, ethanol and stirring at room temperature for 12-16 h.

Figure 2. Whole cell HDAC inhibition assay.

The histogram plot represents the IC₅₀ value of whole cell HDAC enzyme inhibition assay carried out in HCT116 cell line. All values are represented as mean ± SEM and the experiments were carried out in triplicate.

Figure 3. Percentage apoptotic cells in HCT116 cell line.

Percentage apoptotic cells in HCT116 cell line determined by AO/EB staining. All values are mean ± SEM of 100 cells. ^ap< 0.05 vs. control.

Figure 4. AO/EB staining in HCT116 cell line.

The images indicate induction of apoptosis in HCT116 cell line by various treatment groups after 48 hrs. (A) Normal control, (B) 5-FU, (C) C1, (D) C2, (E) C3. Arrows indicate condensed nucleus. Magnification 40X.

Figure 5. Determination of apoptosis by Annexin V staining.

Effect of binding of Annexin V on the surface of HCT116 cell after 48 hrs of treatment. Apoptosis was determined as % live, early and late apoptosis/dead cells by MUSE cell analyzer.

Figure 6. Cell cycle analysis in HCT116 cell line.

Effect on the cell cycle of HCT116 cells after 48 hrs of treatment. Histogram showing various phases of the cell cycle where M1 represent G₀/G₁ phase, M2 represent S phase and M3 represent G₂/M phase. (A) Normal control, (B) Doxorubicin, (C) C1, (D) C2, (E) C3

Figure 7. Effect of various treatments on TNF-α levels.

Effect of DMH administration for 21 days on the inflammatory marker TNF-α in colon tissue homogenate. All values are mean ± SEM of 6 animals. ^ap<0.05 vs. Normal control, ^bp<0.05 vs. DMH control, ^cp<0.05 vs. 5-FU.

Figure 8. Effect of test compounds on the histopathology of colon.

Photomicrographs of histological changes in the colon of experimental animals at 400 x magnification. (A) Normal Control, (B) DMH control, (C) 5-FU, (D) C1, (E) C2, (F) C3. The arrows indicate aberrant crypt foci.