Anti-neoplastic efficacy of Haimiding on gastric carcinoma and its mechanisms

Table 1 Cytotoxicity of HMD to human tumor cell lines (MTT assay)

Drug	IC50(mg/ml)
	KB	BGC-823	A549	MCF-7	A2780	Bel-7420	HCT-8	Eca-109
HMD	83.89	25.28	292.41	34.14	16.84	36.57	28.29	12.78
5-Fu	0.12	0.06	0.19	0.10	0.08	0.09	0.08	0.04
HMD without 5-Fu	428.58	274.90	685.70	365.00	124.65	295.60	187.58	100.42

Table 2 Effect of HMD on formation of colonies in culture of BGC-823, E_ca-109 and HCT-8 cell lines (n = 8)

Drug	Percentage of colony formation (mean ± SD)
	Dose (mg/ml)	BGC-823	HCT-8	Eca-109
HMD	10.00	72.18 ± 7.29c	83.18 ± 9.01	61.58 ± 4.12a
HMD	20.00	50.21 ± 5.18d	59.45 ± 6.78c	22.43 ± 3.65d
HMD	40.00	28.43 ± 3.42d	30.15 ± 4.20d	0
5-Fu	0.04	61.18 ± 4.27c	68.70 ± 5.15a	69.27 ± 5.48a
HMD without 5-Fu	19.63	90.51 ± 7.42	82.15 ± 7.92	78.29 ± 6.25a
Blank control		98.27 ± 6.58	95.68 ± 9.12	97.14 ± 8.26c

^aP < 0.05, ^bP < 0.01 vs blank control;

^cP < 0.05,

^dP < 0.01 vs HMD without 5-Fu.

Table 3 Effect of HMD on tumor weight of FC mice

Drug	Dose (g/kg)	Route of administration	No. of animals	Average weight of tumor (g)	Rate of inhibition (%)
HMD	27.05	ip	30	0.42 ± 0.21bd	76.92
HMD	13.53	ip	30	0.58 ± 0.30bc	68.13
HMD	47.63	ip	30	0.88 ± 0.34a	51.65
HMD
Without 5-Fu	13.50	ip	30	1.21 ± 0.45	33.52
5-Fu	0.03	ip	30	0.89 ± 0.40	51.10
Normal saline		ip	30	1.82 ± 0.43

^aP < 0.05,

^bP < 0.01 vs HMD without 5-Fu;

^cP < 0.05,

^dP < 0.01 vs 5-Fu.

Table 4 Effect of HMD on tumor weight of S₁₈₀ bearing mice

Drug	Dose (g/kg)	Route	No. of animals	Average weight of tumor (g)	Rate of inhibition (%)
HMD	27.05	ip	30	0.28 ± 0.10bc	73.33
HMD	13.53	ip	30	0.40 ± 0.15a	61.91
HMD	6.76	ip	30	0.49 ± 0.31a	53.33
HMD
Without 5-Fu	13.50	ip	28	0.79 ± 0.33	24.76
5-Fu	0.03	ip	28	0.54 ± 0.21	48.57
Normal saline		ip	29	1.05 ± 0.32d

^aP < 0.05,

^bP < 0.01 vs HMD without 5-Fu;

^cP < 0.05,

^dP < 0.01 vs 5-Fu.

Table 5 HMD at doses of 27.05 g/kg·d, 13.53 g/kg·d and 6.76 g/kg·d prolonged life span of H₂₂- bearing mice

Drug	Dose (g/kg)	Route	No.of animals	Life-span(d)	Rate of life prolongation (%)
HMD	27.05	iv	27	21.1 ± 1.6ac	74.4
HMD	13.53	iv	28	18.6 ± 2.9ac	53.7
HMD	6.76	iv	30	17.1 ± 3.2	41.3
HMD
Without 5-Fu	13.50	iv	30	15.8 ± 2.5	30.6
5-Fu	0.03	iv	29	14.6 ± 2.7	20.7
Normal saline		iv	30	12.1 ± 3.1

^aP < 0.05, ^bP < 0.01 vs HMD without 5-Fu;

^cP < 0.05, ^dP < 0.01 vs 5-Fu.

Table 6 Effect of HMD on cell cycle of human gastric carcinoma

Group	Dose(µg/ml)	No. of cases	G0/G1 (%)	G2/M (%)	S (%)
Blank	Media only	10	22.20 ± 5.06	4.09 ± 1.27	73.56 ± 12.10
5-Fu	50	10	44.81 ± 10.25	0.03 ± 0.01	55.13 ± 7.28b
HMD	50	10	22.63 ± 3.34	11.01 ± 3.56	63.38 ± 7.31a
HMD	100	10	26.70 ± 7.99	19.33 ± 4.67	52.83 ± 10.03b
HMD	200	10	12.65 ± 2.43	31.36 ± 7.06	54.27 ± 8.33b

^aP < 0.05 vs blank control;

^bP < 0.01 vs blank control.

Table 7 Effect of HMD on cell apoptosis of human gastric carcinoma

Group	Dose (μg/ml)	No. of cases	APO (%)
Blank control	Media only	10	0.15 ± 0.04
5-Fu	50	10	0.17 ± 0.06
HMD	50	10	1.16 ± 0.23b
HMD	100	10	1.14 ± 0.20b
HMD	200	10	1.72 ± 0.41b

^bP < 0.01 vs blank control.

Table 8 Changes of intracellular [Ca²⁺]i in human gastric car-cinoma cells after treated with HMD for 12 h

Group	Concentration of drug (µg/ml)	No. of cells	[Ca2+]i (FI)
A	Same vol. of medium	20	320.26 ± 147.99
B	50	22	386.00 ± 163.85b
C1	200	25	1 367.56 ± 133.98d
C2	100	28	916.78 ± 151.46d
C3	50	26	724.26 ± 151.33d
D	100+20	24	776.36 ± 144.10f

^bP < 0.01 vs negative control;

^dP < 0.001 vs negative control;

^fP < 0.01 vs medium dose group and negative control.

Table 9 Changes of intracellular [Ca²⁺]i in human gastric car-cinoma cells after treated with HMD for 24 h

Group	Concentration (µg/ml)	No. of cells	[Ca2+]i (FI)
A	Same vol. of medium	22	323.50 ± 141.35
B	50	25	382.67 ± 135.02b
C1	200	23	824.63 ± 155.65d
C2	100	23	672.65 ± 165.16d
C3	50	27	532.46 ± 146.76d
D	100 + 20	26	579.16 ± 147.20f

^bP < 0.01,

^dP < 0.001 vs negative control;

^fP < 0.01 vs medium dose group and negative control, respectively.

Table 10 Changes of intracellular [Ca²⁺]i in human gastric car-cinoma cells after treated with HMD for 36 h

Group	Concentration of drug (µg/ml)	No. of cells	[Ca2+]i (FI)
A	Same vol of medium	30	339.97 ± 150.19
B	50	23	399.83 ± 159.52b
C1	200	25	833.84 ± 133.10d
C2	100	26	684.31 ± 148.45d
C3	50	23	519.00 ± 170.92d
D	100 + 20	32	583.67 ± 144.80f

^bP < 0.01,

^dP < 0.001 vs negative control;

^fP < 0.01 vs medium dose group and negative control group separately.

Table 11 Changes of intracellular [Ca²⁺]i in human gastric carcinoma cells after treated with HMD for 48 h

Group	Concentration of drug given (µg/ml)	No.of cells	[Ca2+]i (FI)
A	Same vol. of mediun	22	328.45 ± 135.15
B	50	36	380.42 ± 151.46b
C1	200	33	1 119.55 ± 193.94d
C2	100	28	787.00 ± 178.19d
C3	50	21	547.29 ± 142.25d
D	100 + 20	26	693.38 ± 168.15f

^bP < 0.01,

^dP < 0.001 vs negative control;

^fP < 0.01 vs separately medium dose group and negative control group.

Table 12 Changes of [Ca²⁺]i in human gastric carcinoma cells after treated with HMD for different times

Different durations of treatment (h)	No. of cells	[Ca2+]i (FI)
12	28	916.78 ± 151.46
24	23	672.65 ± 165.16
36	26	684.31 ± 148.41
48	28	787.00 ± 178.19