ECL reagent - 探討水溶性大蒜萃取物S-allylcysteine對非小細胞肺癌細胞株的影響; The effect of garlic water-soluble compou

1:1, reagent A: reagent B

Antibodies

Cell signaling (Danvers, MA, U.S.A.)

p-Akt (1:1000) and NFκB (1:1000)

R&D (Minneapolis, MN, U.S.A.)

p-mTOR (1:2000).

Jackson ImmunoReserch (West Grove, PA, U.S.A.)

Goat anti-mouse IgG

Goat anti-rabbit IgG

Procedure

1. Protein samples are quantified to 100 μg protein and added with 6x loading dye and heated at 90 ℃ for 5 minutes then put on ice immediately before loading.

2. Protein samples are separated in 10% SDS polyacrylamide gel electrophoresis with 6x loading dye and running at 80 V in upper gel and 100 V in lower gel with running buffer.

3. Protein samples were transferred to nitrocellulose membranes with 400 mA for 4 hours in transfer buffer.

4. The membranes are washed with TTBS for 5 minutes then blocked with TTBS contain 5 % non-fat dried milk for 1 hour.

5. The membranes are incubated at 4 ℃ overnight with specific primary antibody diluted in TTBS contain 5 % non-fat dried milk.

6. Before exposing to secondary antibody, the membranes are washed with TTBS 5 minutes repeat for 3 times then incubate at room temperature for at least 1 hour with specific secondary antibody diluted in TTBS

contained 5 % non-fat dried milk.

7. The membranes are washed with TTBS 5 minutes repeat for 3 times then added ECL reagent to detect protein level expression by using LAS 4000 system to take the pictures of the membranes.

8. The stripping buffer was used when the membranes needed to be deprobed. Before reprobed the next primary antibody, the membranes were washed with stripping buffer 5 minutes repeat for 3 times and then washed with TTBS 5 minutes repeat for 3 times.

Results

SAC suppressed growth of A549 human lung cancer cell xenograft in

BALB/c nu/nu nude mice

To evaluate the effect of SAC on tumor growth, tumor xenografts were used by implanting A549 cells subcutaneously into the left thigh of mice.

After xenograft implantation, the tumor size was observed by Non-invasive in vivo imaging system (IVIS) for 2 weeks. However, the tumor formation was not similar after A549 xenograft implantation in each mouse. Total of 3 mice were not observed the tumor expression. Therefore were not continued the following experiment (Fig.1). The mice were equally and randomly divided into three groups (N=9 for each group) according to their tumor size.

The tumor group and health control group was fed by oral gavage with PBS.

The low SAC group and high SAC group were fed by oral gavage with 240 mg/kg BW/mice/day and 480 mg/kg BW/mice/day diluted in PBS. During the period of SAC treatment, the diet intake for the first week was lower in high SAC group when compared to other groups. However, the diet intake became normal from the second weeks in high SAC group (Fig.2). During the period of SAC treatment, the initial and final body weights showed no significantly differences in each group when compared to the control group (Fig.3).

The tumor volume was substantially smaller than the tumor group mice after SAC oral gavage from 5 to 7 weeks (Fig.4). During SAC treatment, we used Non-invasive in vivo imaging system (IVIS) to observe tumor size

every week. Before SAC treatment, the tumor size was similar. Interestingly, the tumor size was smaller than the tumor group mice after SAC oral gavage since the first week (Fig.5). The mean weight of tumors in SAC treatment group was also significantly lower than the tumor group (Fig.6). Therefore, SAC treatment markedly decreased tumor weight and volume when compared to the tumor group. These results indicated that SAC can reduce tumor formation in BALB/c nu/nu nude mice.

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Figure 1. The tumor formation after A549 xenograft implantation for 1 and 2 weeks.

(a) The tumor size expressed after A549 xenograft implantation for 1 week. (b) The tumor size expressed after A549 xenograft implantation for 2 weeks.

b

0 1 2 3 4 5 6 7 8

1 2 3 4 5 6 7

Period of SAC treatment (weeks)

diet (g) / mice / day

Control Tumor

SAC 240 mg/kg BW SAC 480 mg/kg BW

Figure 2. The average dietary intake during SAC treatment.

15 16 17 18 19 20 21

0 1 2 3 4 5 6 7

Preiod of SAC treatment (weeks)

Mice average BW (g)

Control Tumor

SAC 240 mg/kg BW SAC 480 mg/kg BW

Figure 3. The average body weight during SAC treatment.

Values are expressed as mean ± SEM.

0 50 100 150 200 250

5 6 7

Peroid during SAC treatment (weeks) Tumor volume (mm3 )

Tumor

SAC 240 mg/kg BW SAC 480 mg/kg BW

Figure 4. The tumor volume during SAC treatment.

One way ANOVA was used to compare the differences. Values are expressed as mean ± SEM. ＊: Statistically significant differences compared with tumor group (P<0.05).

*

treatment. (a) The tumor size before SAC treatment. (b) After 1 week (c) After 2 weeks (d) After 3 weeks (e) After 4 weeks (f) After 5 weeks (g) After 6 weeks (h) After 7 weeks of SAC treatment.

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