Potent anti-cancer effects of less polar Curcumin analogues on gastric adenocarcinoma and esophageal squamous cell carcinoma cells

Abstract

Curcumin and its chalcone derivatives inhibit the growth of human cancer cells. It is reported that replacement of two OH groups in curcumin with less polar groups like methoxy increases its anti-proliferative activity. In this study, we explored benzylidine cyclohexanone derivatives with non-polar groups, to see if they possess increased anti-cancer activity. Novel 2,6-bis benzylidine cyclohexanone analogues of curcumin were synthesized, and their inhibitory effects on gastric adenocarcinoma (AGS) and esophageal squamous cell carcinoma (KYSE30) cancer cells were studied using an MTT assay. Cell apoptosis was detected by EB/AO staining, and cell cycle was analyzed by flow cytometry. Real-time PCR was performed for gene expression analysis. All synthesized analogues were cytotoxic toward gastric and esophageal cancer cells and showed lower IC₅₀ values than curcumin. Treatment with 2,6-Bis-(3-methoxy-4-propoxy-benzylidene)-cyclohexanone (BM2) was 17 times more toxic than curcumin after 48 h incubation. All novel compounds were more effective than curcumin in apoptosis induction and cell cycle arrest at G1 phase. These results suggest that less polar analogues of curcumin have potent cytotoxicity in vitro. However, they need to be investigated further, especially with animal tumor models, to confirm their chemotherapeutic activity in vivo.

Figure 1

Synthesized primary aromatic aldehydes.

Figure 2

Synthesized 2,6-bis benzylidene cyclohexanones (4a – 4e also named BM1- BM5).

Figure 3

Inhibitory effect of synthesized compounds on AGS cells assessed with MTT assay at 24 h time point.

Figure 4

Inhibitory effect of synthesized compounds on AGS cells assessed with MTT assay after 48 h time point.

Figure 5

Inhibitory effect of synthesized analogues on AGS cells determined by MTT assay after 72 h time point.

Figure 6

EB/AO staining for detecting apoptotic cells. (A) Control un-treated AGS cells. (B) Representative micrograph of apoptotic cells treated with BM2 that are characterized with fragmented and condensed nuclei of cells. (C) Quantification of the cells with normal, necrotic and apoptotic representation.

Figure 7

Relative expression levels of Bax, cyclin D1, VEGFA, Bcl-2, Caspase 3, c-myc, survivin and RPL38 mRNA in AGS cells. All Ct values were normalized with the homo sapiens ribosomal protein L38 (RPL38) as a housekeeping gene.

Figure 8

Flow cytometric analysis of the cell cycle. Curcumin analogues altered distribution of the AGS cells in cell cycle phases, which indicates that cell cycle arrested at G1 phase.