Indole phytoalexin derivatives induce mitochondrial-mediated apoptosis in human colorectal carcinoma cells

Table 1 Flow cytometry staining

Analysis	Staining solution	Manufacturer
Caspase activation	Caspase-3-PE	BD Biosciences Pharmingen, San Diego, CA, United States
	Cleaved Caspase-9-FITC	BIOSS Antibodies, Woburn, MA, United States
Cytochrome c release	Cytochrome c Antibody (6H2) FITC Conjugate	Invitrogen, Carlsbad, CA, United States
Cleavage of PARP	Cleaved-PARP (Asp214) XP® Rabbit mAb (PE Conjugate)	Cell Signaling Technology, Danvers, MA, United States
Mitochondrial membrane potential	TMRE (tetramethylrhodamine ethyl ester perchlorate) Final concentration 0.1 µmol/L	Sigma-Aldrich, St. Louis, Missouri, United States
Protein analysis	Phospho-Bcl-2 (Ser70) Rabbit mAb Alexa Fluor® 488 Conjugate	Cell Signaling Technology®, Danvers, MA, United States
	Phospho-Bad (Ser112) Rabbit mAb (PE Conjugate)

Table 2 List of Western blot antibodies

Name	Mr (kDA)	Origin	Manufacturer
Primary antibodies
PARP	116 + 89	Rabbit	Cell Signaling Technology®, Danvers, Massachusetts, United States
β-actin	45	Mouse
p38 MAPK	43	Rabbit
NF-κB p65	65	Rabbit
NF-κB p105/p50	105/50	Rabbit
Secondary antibodies
Anti-rabbit IgG HRP-Linked	-	Goat	Cell Signaling Technology®, Danvers, Massachusetts, United States
Anti-mouse IgG/HRP	-	Goat	Dako, Glostrup, Denmark

Table 3 IC₅₀ (μmol/L) of tested compounds in different cell lines after 72 h incubation

Compound	Cancer Cell Lines
	CaCo-2	HCT116	3T3
K-452	> 100	> 100	> 100
K-453	67.39 ± 0.78	32.22 ± 1.14	60.55 ± 3.35
K-455	> 100	> 100	> 100
K-459	> 100	> 100	> 100
K-462	> 100	> 100	> 100
K-463	74.18 ± 0.001	33.04 ± 2.67	61.13 ± 2.25
K-465	35.66 ± 6.49	33.74 ± 2.78	34.94 ± 2.01
K-466	> 100	> 100	> 100
K-467	> 100	> 100	> 100
K-471	> 100	> 100	> 100

Data are presented as the mean ± SD of three independent experimental determinations performed in triplicate.

Table 4 Flow cytometric analysis of cell cycle distribution in HCT116 cells treated with compound K-453 (in %)

Treatment	Time (h)	sub-G1	G0/G1	S	G2/M
Control	24	1.30 ± 0.07	48.38 ± 0.81	23.28 ± 0.53	27.05 ± 0.35
	48	1.75 ± 0.22	52.1 ± 2.12	22.88 ± 0.08	23.30 ± 2.26
	72	1.64 ± 0.09	48.57 ± 0.52	24.85 ± 0.64	24.95 ± 0.21
K-453	24	19.7 ± 2.26b	62.97 ± 0.37b	7.05 ± 1.35b	10.29 ± 1.29b
	48	48.9 ± 7.21c	35.58 ± 3.22a	9.31 ± 2.54b	6.22 ± 1.45b
	72	55.11 ± 14.13c	34.11 ± 9.89a	5.71 ± 1.73b	5.07 ± 2.52b

The results are presented from three independent experiments as the mean ± SD; significantly different,

^aP < 0.05,

^bP < 0.01,

^cP < 0.001 vs untreated cells (control); sub-G1 fraction of cells identified as apoptotic population.

Table 5 Annexin V/PI flow cytometry analysis of apoptosis occurrence in HCT116 cells after K-453 treatment (in %)

Treatment	Time (h)	An-/PI-	An+/PI-	An+/PI+
Control	24	89.8 ± 1.41	4.46 ± 0.35	1.82 ± 1.03
	48	92.45 ± 0.07	2.38 ± 0.45	1.71 ± 0.25
	72	94.55 ± 0.92	2.13 ± 0.59	1.51 ± 0.31
K-453	24	66.35 ± 7.0b	23.4 ± 3.25b	6.38 ± 6.84
	48	37.85 ± 9.12c	34.75 ± 4.88c	11.43 ± 7.88a
	72	36.05 ± 14.07c	30.25 ± 11.1c	10.2 ± 3.39a

The results are presented from 3 independent experiments. Living cells are presented as An^-/PI^- events, cells in a stage of early apoptosis are presented as An⁺/PI^-, late apoptotic and necrotic cells as An⁺/PI⁺. Significantly different,

^aP < 0.05,

^bP < 0.01,

^cP < 0.001 vs untreated cells (control).