Role of ERK-MAPK signaling pathway in pentagastrin-regulated growth of large intestinal carcinoma

Table 1 Primers used for nested reverse transcriptase polymerase chain reaction amplification of cholecystokinin-B receptor, extracellular signal-regulated protein kinase 1/2 and K-ras

Name	Primer sequence	PCR conditions	Size (bp)
CCK-BR	1: 5’TCTCGCGAGCTCTACTTAGGG3’	94 °C, 30 s	185
	2: 5’ACCGACGATGCACGTTGAAG3’	62 °C, 30 s
		72 °C, 30 s
ERK1/2	1: 5’TATTCCCGGGCAAGCACTATTT3’	94 °C, 30 s	243
	2: 5’CGGGCTCATCATTCGGGTCGTA3’	54 °C, 30 s
		72 °C, 30 s
K-ras	1: 5’ACAGTGCAATGAGGGACCAGTA3’	94 °C, 30 s	275
	2: 5’GTATAGAAGGCATCATCAACACC3’	50 °C, 30 s
		72 °C, 30 s
Actin	1: 5’ATGATATCGCCGCGCTCGTCGTC3’	94 °C, 30 s	342
	2: 5’CGCGGTTGGCCTTGGGGTTCAG3’	60 °C, 30 s
		72 °C, 30 s

PCR: Polymerase chain reaction; CCK-BR: Cholecystokinin-B receptor; ERK1/2: Extracellular signal-regulated protein kinase 1/2.

Table 2 Effect of different pentagastrin and proglumide concentrations on the proliferation of HT-29 cells (mean ± SD)

Groups	n	Pentagastrin		Proglumide
		Concentration (mg/L)	OD value	Concentration (mg/L)	OD value
Control group	6	0	0.44 ± 0.00	0	0.44 ± 0.02
Experimental group	6	6.25	0.48 ± 0.03a	8	0.43 ± 0.03
	6	12.5	0.52 ± 0.03a	16	0.44 ± 0.04
	6	25	0.57 ± 0.04a	32	0.43 ± 0.03
	6	50	0.58 ± 0.04a	64	0.45 ± 0.02
	6	100	0.57 ± 0.04a	128	0.45 ± 0.04

^aP < 0.05 vs control group. OD: Optical density.

Table 3 Effect of combined pentagastrin and proglumide on the proliferation of HT-29 cells (mean ± SD)

Groups	n	Pentagastrin + proglumide
		Pentagastrin (mg/L)	Proglumide (mg/L)	OD value
Control group	6	25	0	0.58 ± 0.02
Experimental group	6	25	8	0.54 ± 0.03a
	6	25	16	0.51 ± 0.04a
	6	25	32	0.47 ± 0.04a
	6	25	64	0.47 ± 0.03a
	6	25	128	0.46 ± 0.03a

^aP < 0.05 vs control group. OD: Optical density.

Table 4 Comparison of proliferation index and apoptotic rates between the experimental groups and control group (mean ± SD)

Group	n	PI			AR
		PI (%)	F	P value	AR (%)	F	P value
Control group	5	27.72 ± 5.00			2.53 ± 0.36
Pentagastrin group	5	37.54 ± 5.17a	7.59	0.01	1.91 ± 0.42a	6.50	0.01
Proglumide group	5	27.36 ± 2.68c			2.79 ± 0.45c
Pentagastrin + proglumide group	5	27.31 ± 5.76c			2.41 ± 0.30c

^aP < 0.05 vs control group;

^cP < 0.05 vs pentagastrin group. AR: Apoptosis rate; PI: Proliferation index.

Table 5 Comparison of protein, mRNA and phosphorylation levels of extracellular signal-regulated protein kinase 1/2 and K-ras between the experimental groups (mean ± SD) and control group

Group	n	ERK1/2			K-ras
		mRNA	Protein	p-ERK1/2	mRNA	Protein	p-K-ras
Control group	5	0.76 ± 0.04	0.56 ± 0.05	0.32 ± 0.02	0.54 ± 0.08	0.56 ± 0.04	0.31 ± 0.05
Pentagastrin group	5	0.79 ± 0.05	0.60 ± 0.04	0.43 ± 0.04a	0.59 ± 0.06	0.57 ± 0.04	0.45 ± 0.06a
Proglumide group	5	0.74 ± 0.06	0.55 ± 0.07	0.31 ± 0.02c	0.53 ± 0.05	0.55 ± 0.04	0.27 ± 0.06c
Pentagastrin + proglumide group	5	0.77 ± 0.05	0.58 ± 0.05	0.36 ± 0.01c	0.55 ± 0.06	0.57 ± 0.04	0.35 ± 0.04c

^aP < 0.05 vs control group;

^cP < 0.05 vs pentagastrin group. ERK1/2: Extracellular signal-regulated protein kinase 1/2.