Comparison of effects of P-coumaric acid and coumarin on colorectal cancer cell line by inducing apoptosis and autophagy

Abstract

Objective: Autophagy, as a cellular pathway involved in removing damaged proteins and organelles, performs a vital function in the homeostasis and fate of cells. Natural compounds of coumarin (CO) are found in a variety of herbs. Due to their many medicinal properties, including antitumor and anti-proliferative activity, they are involved in apoptosis and autophagy processes. This investigation desired to analyze the apoptotic and autophagic effects of p-coumaric acid (PCA) and CO on HT-29 cells cultured in fibrin hydrogel.

Materials and methods: Cell viability and apoptotic and autophagic changes were evaluated by MTT assay, Acridine Orange, 4',6-diamidino-2-phenylindole (DAPI), and monodansylcadaverine (MDC) staining. The expression Bax, Bad, Bcl2, Lc3, Beclin-1, P53 and Atg5 was respectively measured by qRT-PCR and Western blotting.

Results: CO (IC50=25 μM) and PCA (IC50=150 μM) had a dose- and time-dependent cytotoxic effect in HT-29 cells. So, the cytotoxic effects of CO were significantly higher than PCA and these differences were also evident in cell morphology investigations. The data illustrated a high expression of pro-apoptotic and pro-autophagic genes and a declined expression of anti-apoptotic and anti-autophagic genes.

Conclusion: CO (that was more potent) and p-coumaric acid-induced autophagy via PI3K/Akt/mTOR and AMPK/mTOR signaling on HT-29 cells.

Keywords: Autophagy; Cancer phytotherapy; Coumarin; Signaling pathway.

Figure 1

Scanning microscopy results obtained from morphology analysis of fibrin hydrogel scaffold and HT-29 cells cultured after 72 hr. A: cell-free fibrin hydrogel scaffold with the magnification of 1.00 KX. B: HT-29 cells cultured on fibrin hydrogel scaffold with the magnification of 1.00 KX - These images show the desired properties and appropriate porosity of the scaffold

Figure 2

Cell viability charts using MTT assay. A: Effects of different concentrations of coumarin on the viability rate of HT-29 cells on days 1,3, and 5 using MTT assay and determination of 25 μM concentration as IC50 after 24 hr. B: Effects of different concentrations of p-coumaric acid on the viability rate of HT-29 cells on days 1, 3, and 5 using MTT assay and determination of 150 μM concentration as IC50 after 4 hr (n=3). *Significant difference with the control group at p<0.05. Dissimilar letters indicate significant differences

Figure 3

. Morphological study using Giemsa staining of HT-29 cells by invert microscope A: HT-29 control cells B: HT-29 treated cells with IC50 concentration of coumarin after 24 hr C: Treated HT-29 cells with IC50 concentration of p-coumaric acid after 24 hours D: Normal HT-29 cells with Giemsa E: HT-29 cells treated with IC50 concentration of coumarin after 24 hr with Giemsa F: HT-29 cells treated with IC50 concentration of p-coumaric acid after 24 hr by Giemsa staining. In coumarin-treated and p-coumaric acid-treated cells showed a decrease in cell volume and number compaction compared to the control group, confirming cell death

Figure 4

Morphological characterization of the nucleus using DAPI staining in HT-29 cells cultured on fibrin hydrogel scaffold in control group. A: untreated or control, B: treated with 25 μM coumarin, C: treated with 150 μM p-coumaric acid

Figure 5

Apoptosis study using acridine orange/ethidium bromide staining of HT-29 cells cultured in fibrin hydrogel scaffold by magnification (20 X) by fluorescent microscopy. A: HT-29 control cells B: HT-29 cells treated with IC50 concentration of coumarin after 24 hr C: HT-29 cells treated with IC50 concentration of p-coumaric acid after 24 hr. Orange-colored cells confirm cell apoptosis and green-colored cells indicate live cells in the samples

Figure 6

Autophagy study using MDC staining in HT-29 cells cultured in fibrin hydrogel scaffold by fluorescence microscopy. A: MDC staining in control group of HT-29 B: DAPI staining in control group of HT-29 C: MDC staining of cells treated with coumarin at IC50 concentration 24 hr after treatment D: DAPI staining of cells treated with coumarin at IC50 concentration 24 hr after treatment E: MDC staining of cells treated with p-coumaric acid at IC50 concentration 24 hr after treatment F: DAPI staining of cells treated with p-coumaric acid at IC50 concentration 24 hr after treatment

Figure 7

Gene expression alterations in HT-29 cells treated with coumarin and p-coumaric acid. Increased expression of Bad, Bax, P53, Lc3 genes and decreased expression of Bcl2 gene in HT-29 cells treated with coumarin and p-coumaric acid A: after 24 hr and B: after 48 hr, confirmed apoptosis and increased expression of Beclin-1 and Atg5 genes, approved autophagy. These changes were significant compared to the control group. *** Indicates significant difference with the control group at p<0.001, ** Indicates significant difference with the control group at level (p≤ 0.01), * Significant difference with the control group at level (p≤ 0.01). All experiments were conducted in triplicate, and GAPDH was used as the internal normalizer for gene expression analysis. Also, the control group is considered as the reference with a value of 1.

Figure 8 A

Determination of the effects of IC50 concentrations of coumarin and p-coumaric acid on the induction of autophagy and identification of mTOR, AMPK, AKT, Beclin-1, Atg5, and Lc3 proteins by Western blotting in HT-29 treated cells compared to control cells. As can be seen, the expression of P-mTOR and P-AKT was significantly decreased in the treated samples and the expression of P-AMPK, Beclin-1, Atg5, and Lc3 in the treated samples was increased compared to the control. GAPDH was also used as a loading control