Evaluation of the anticancer effects exerted by 5-fluorouracil and heme oxygenase-1 inhibitor hybrids in HTC116 colorectal cancer cells

Figures & data

Figure 1. Chemical structures and HO inhibitory activity in spleen and brain microsomal fractions of tested compounds.

Table 1. Time course of HCT116 cell viability after treatment with 5 µM of the different drugs at different times.

Compound	% of Cell viability ± SD
	24 h	48 h	72 h
Control	100 ± 2.2	100 ± 4.3	100 ± 2
LS6/42	78 ± 7.2	70 ± 5.5	67 ± 6.1
SI1/22	85 ± 4.7	80 ± 3.5	77 ± 5.5
SI1/09	110 ± 6.6	100 ± 5.7	101 ± 4.5
SI1/17	98 ± 7.5	90 ± 3.7	85 ± 5.7
LS4/28	98 ± 8.2	95 ± 5.1	92 ± 5.4
SI1/20	100 ± 9.5	100 ± 4.3	93 ± 8.5

MTT assay was carried out as reported in the methods. Viability was calculated as the percentage of control (HCT116 cells maintained in the presence of vehicle alone) ± SD.

Figure 2. Flow cytometry analysis of the cell cycle phase distribution of HCT116 cells after 72 h incubation with 5 μM SI1/22 or LS6/42. Hypotonic propidium iodide staining was used to evaluate DNA content as described in Methods employing Flowing software.

Figure 3. Effect of 5-FU, HO-1 inhibitors, hybrids, and combo treatment on HCT116 cell viability and morphology. (A) Evaluation of cell viability by MTT assay after 72 h of treatment using different concentrations of the drugs. The results represent the mean ± SD of almost three separate experiments done in triplicate. Significant vs untreated control cells: *p < 0.05; **p < 0.01; ***p < 0.001. (B) Representative images of HCT116 cells after 72 h of treatment with 15 µM of the compounds. Cells were visualised under a light microscope (200× magnification) and the pictures were acquired by IM50 Leica Software (Leika Microsystems, Wetzlar, Germany).

Table 2. Predicted lipophilicity/permeability and experimental antiproliferative effects in HCT116 cells of hybrids.

Compound	Lipophilicity (SK logD)	Permeability (Papp, nm/s)	% of cell death, 72 h [5 μM]
SI1/22	2.97	24.4	23
SI1/17	1.45	20.6	15
SI1/20	2.28	21.8	7

Selected ADME properties were predicted using PreADMET web-based application (http://preadmet.bmdrc.kr).

Table 3. Inhibition of HO-1 enzymatic activity in HCT116 treated cells with LS6/42 and SI1/22.

Compound	picomol/mg prot/1 h	% inhibition
	[5 μM], 72 h
Control	53.5 + 1.118	–
LS6/42	11.42 + 0.903	(78.65%)
SI1/22	17.67 + 1.121	(66.9%)

Data are representative of at least three independent experiments,

Figure 4. Effect of 5-FU, HO-1 inhibitors, hybrids, and combo treatment on oxidative stress. Assessment of ROS levels in HCT116 cells treated with 15 µM of tested drugs for 3 h; the images were acquired under a fluorescence microscope at 200 x magnification.

Figure 5. Analysis of apoptosis and autophagy-related proteins. Representative images and relative densitometric analysis of western blotting against different proteins. HO-1, SOD, pro-caspase-3, p62 (A) and PARP (C) protein expression levels were evaluated after 72 h of treatment, Nrf2 (B) protein after 48 h. The histograms are related to relative intensities over actin. *p < 0.05; **p < 0.01; ***p < 0.001 vs untreated cells (control).

Data availability statement

The data that support this study are available from the corresponding authors upon reasonable request.