A small library of chalcones induce liver cancer cell death through Akt phosphorylation inhibition

Abstract

Hepatocellular carcinoma (HCC) ranks as the fifth most common and the second deadliest cancer worldwide. HCC is extremely resistant to the conventional chemotherapeutics. Hence, it is vital to develop new treatment options. Chalcones were previously shown to have anticancer activities in other cancer types. In this study, 11 chalcones along with quercetin, papaverin, catechin, Sorafenib and 5FU were analyzed for their bioactivities on 6 HCC cell lines and on dental pulp stem cells (DPSC) which differentiates into hepatocytes, and is used as a model for untransformed control cells. 3 of the chalcones (1, 9 and 11) were selected for further investigation due to their high cytotoxicity against liver cancer cells and compared to the other clinically established compounds. Chalcones did not show significant bioactivity ([Formula: see text]) on dental pulp stem cells. Cell cycle analysis revealed that these 3 chalcone-molecules induced SubG1/G1 arrest. Akt protein phosphorylation was inhibited by these molecules in PTEN deficient, drug resistant, mesenchymal like Mahlavu cells leading to the activation of p21 and the inhibition of NF[Formula: see text]B-p65 transcription factor. Hence the chalcones induced apoptotic cell death pathway through NF[Formula: see text]B-p65 inhibition. On the other hand, these molecules triggered p21 dependent activation of Rb protein and thereby inhibition of cell cycle and cell growth in liver cancer cells. Involvement of PI3K/Akt pathway hyperactivation was previously described in survival of liver cancer cells as carcinogenic event. Therefore, our results indicated that these chalcones can be considered as candidates for liver cancer therapeutics particularly when PI3K/Akt pathway involved in tumor development.

Figure 1

Structures of the synthesized chalcones.

Figure 2

Real-time cell growth analysis. Human liver cancer cells Huh7 (left panel) and Mahlavu (right panel) were treated with the selected compounds. Cell index measurements were obtained by RT-CES software. DMSO was used as negative control. (A) The growth inhibition values of the compounds were obtained by the normalization with DMSO. Experiments were performed in triplicate. (B) IC₅₀ values calculated from RT-CES experiment data.

Figure 3

Cell death induced by the chalcones. (A) Nuclear staining of the liver cancer cells treated with the chalcones. Human liver cancer cells (Huh7 and Mahlavu) were treated with the IC₅₀ concentrations of the chalcones 1, 9, 11. After 72 h of incubation with the compounds or DMSO control, Hoechst (33258) staining was performed. Images were taken with fluorescent microscope (×40). Due to the strong cytotoxic activities of the chalcones the number of the cells in objective area was much less than DMSO controls. (B) Cell cycle distribution of liver cancer cells. Huh7 and Mahlavu cells were treated for 72 h with IC₅₀ concentrations of the compounds or DMSO control. SubG1 cell cycle arrest was observed upon treatment with the compounds (blue). (C) Investigation of PARP cleavage in Huh7 and Mahlavu cells treated with the selected chalcones for 24 h.

Figure 4

Proteins targeted by the compounds. Human liver cancer cells (Mahlavu) were treated with the IC₅₀ concentrations of the selected compounds or DMSO control for 24 h. (A) Western blot analysis showed that Akt protein is inhibited in liver cancer cells treated with the compounds resulting in alterations of p21 and NFkB proteins. (B–D) Quantification of the results using ImageJ software. (E) Schematic representation of the molecular mechanism of action of the compounds. Blocked signaling is crossed.