SFN inhibited ESCC but activated Akt/mTOR pathway
ECa109 and EC9706 cells were treated with 0, 1, 10, 20, 50 and 100 µM of SFN, the results of cell viability measured by MTT were shown in Fig. 1a, SFN inhibited the proliferation of ECa109 and EC9706 cells in a dose- and time-dependent manner, with IC50 values of 31.58 ± 1.50 and 14.95 ± 1.72 µM in ECa109 cells and 38.78 ± 1.59, 22.79 ± 1.36 µM in EC9706 cells at 48 and 72 h, respectively. The anti-proliferative effect of SFN was further demonstrated by the clone formation assay. As shown in Fig. 1b, SFN inhibited cell colony formation, with lower cell colony numbers (89 ± 26 and 67 ± 17 for ECa109 and 101 ± 21 and 87 ± 19 for EC9706 at 5 and 10 µM, respectively) than that in the control group (176 ± 32 for ECa109 and 202 ± 23 for EC9706) (P < 0.05). Apoptosis of ECa109 and EC9706 cells assayed by a flow cytometer was shown in Fig. 1c, the cell apoptosis rates were 6.93 ± 0.37%, 26.60 ± 0.73% and 36.63 ± 1.02% in ECa109 cells, and 5.00 ± 0.73%, 19.20 ± 0.57% and 29.37 ± 1.23% in EC9706 cells treated with 0, 10, and 20 µM of SFN, respectively, for 48 h, which had a statistical difference between the control group and the experimental group (P < 0.01), indicating that SFN induced apoptosis of ESCC cells. In addition, expression of proteins in apoptotic and Akt/mTOR pathway was detected by Western blot after ECa109 and EC9706 cells were treated with 20 µM of SFN for 0, 3, 6, 12 and 24 h, respectively, and the results showed that SFN promoted the expression of Cleaved-caspase 9, but not Bcl-2 and Bax (Fig. 2a). However, we found SFN activated Akt/mTOR pathway by promoting the phosphorylation of Akt (Ser473) and p70S6K (Thr389) (Fig. 2b). The results suggested that SFN inhibited ESCC through activating apoptotic pathways, while the activating effects of SFN on Akt/mTOR pathway might impair the anti-tumor efficiency of SFN.
Pp242 enhanced inhibitory effects of SFN on ESCC by inhibiting activation of SFN on Akt/mTOR pathway
Considering the activation of SFN on Akt/mTOR pathway, we speculated the combination of Akt/mTOR pathway inhibitors and SFN might have better anti-tumor effects. Therefore, cell proliferation was investigated after ESCC cells were treated with SFN ( 0, 10, 20 µM) combined with Akt/mTOR inhibitors MK2206 ( 0, 5, 10, 15 and 20 µM), PP242 ( 0, 0.5, 1, 5 and 10 µM) and RAD001(0, 5, 10, 20 and 30 µM) for 48 h, respectively, and the combination efficiency was judged according to the CI value. As shown in Fig. 3 and Table 1, CI > 1 in both ECa109 and EC9706 cells treated with SFN combined with RAD001; when cells were treated with SFN combined with MK2206, CI > 1 in ECa109 cells and CI < 1 in EC9706 cells; while CI < 1 in the two cell line cells treated with SFN combined with PP242, indicating that SFN combined with PP242 had better inhibiting efficiency on ESCC than combined with MK2206 or RAD001 (P < 0.001 or P < 0.01).
Table 1 CI of SFN combined with AKT/mTOR inhibitor on ESCC cells
|
CI ECa109
|
CI EC9706
|
|
SFN(μmol/L)
|
SFN (μmol/L)
|
|
10
|
20
|
10
|
20
|
PP242 (μmol/L)
|
|
|
|
|
0.5
|
0.83 ± 0.09
|
0.69 ± 0.26
|
0.14 ± 0.13
|
0.17 ± 0.09
|
1
|
0.72 ± 0.53
|
0.63 ± 0.25
|
0.17 ± 0.01
|
0.19 ± 0.02
|
5
|
0.56± 0.14
|
0.40 ± 0.02
|
0.24 ± 0.07
|
0.32 ± 0.11
|
10
|
0.45 ± 0.33
|
0.31 ± 0.09
|
0.43 ± 0.41
|
0.45 ± 0.11
|
MK2206 (μmol/L)
|
|
|
|
|
5
|
1.39 ± 0.21
|
1.92 ± 0.23
|
0.42 ± 0.33
|
0.43 ± 0.11
|
10
|
1.06 ± 0.48
|
0.93 ± 0.47
|
0.66 ± 0.01
|
0.54 ± 0.24
|
15
|
1.03 ± 0.38
|
0.89 ± 0.27
|
0.77 ± 0.23
|
0.74 ± 0.20
|
20
|
0.83 ± 0.36
|
0.72 ± 0.30
|
0.90 ± 0.40
|
0.82 ± 0.50
|
RAD001 (μmol/L)
|
|
|
|
|
5
|
1.80 ± 0.95
|
0.96 ± 0.29
|
1.25 ± 0.90
|
1.78 ± 0.35
|
10
|
1.27 ± 0.24
|
0.69 ± 0.30
|
1.16 ± 0.79
|
1.67 ± 0.10
|
20
|
1.12 ± 0.87
|
0.61 ± 0.46
|
1.17 ± 0.38
|
1.60 ± 0.10
|
30
|
0.58 ± 0.46
|
0.48 ± 0.09
|
0.94 ± 0.40
|
1.27 ± 0.11
|
To further demonstrate the combined effect of SFN and PP242, colony formation, migration, cell cycle and apoptosis of ECa109 and EC9706 cells treated with SFN and PP242 alone or combined were investigated. Compared to the control group, SFN and PP242 inhibited the formation of colonies (P < 0.05), while the combination of SFN and PP242 had stronger inhibition effects than that of the single-drug group (Fig. 4a, P < 0.05). Also, SFN and PP242 inhibited the migration of cells and retarded cells in the G2 phase, especially when they were combined (Fig. 4b, c). PP242 had no obvious effects at the current concentration, while enhanced the apoptosis-inducing and JC-1 monomer-promoting effects of SFN on ESCC cells (Fig. 4d, e). The results above indicated that SFN combined with PP242 had better anti-tumor effects on ESCC.
To explore the molecular mechanism that PP242 combined with SFN inhibited ESCC, ECa109 and EC9706 cells were treated with 20 µM of SFN and 4 µM of PP242 alone or combined for 24 h, and expression of proteins in the Akt/mTOR pathway was shown as Fig. 4f. Compared to the corresponding control group, SFN promoted but PP242 inhibited the expression of p-p70S6K (Thr389), p-Akt (Ser473) and p-PRAS40, while their expression was still inhibited when SFN combined with PP242 in ECa109 and EC9706 cells, indicated that PP242 blocked the activation of SFN on proteins of Akt/mTOR pathway in ESCC (P < 0.001 or P < 0.01, P < 0.05).
SFN combined with PP242 had stronger inhibiting effects on xenografts of ESCC
As shown in Fig. 5a, b, as the treatment time prolonging, the growth of tumors treated with SFN or PP242 was slower and the tumor volume was smaller than that in the control group, especially the combined group. Compared to the control group, apoptosis-related features such as cell shrinkage and nuclear merging, as well as more brown positive cells were observed in experimental groups, especially in the SFN + PP242 group (Fig. 5c, P < 0.001 or P < 0.01). The tumor inhibition rate in SFN + PP242 group is 56.51%, which was obviously higher than SFN (20.55%) or PP242 (39.97%) group, and the relative tumor growth rate (T/C%) in SFN + PP242 group was < 40% (Table 2). These results above indicated SFN and PP242 alone could inhibit the growth of xenografts in nude mice, while the combination of them had more significant efficiency. Results of protein expression were consistent with that in vitro, SFN stimulated expression of p-Akt (Ser473), p-p70S6K (Thr389) and p-PRAS40 (Thr246), and inhibited expression of p-Rictor (Thr1135), while PP242 weakened the activation of SFN on them (Fig. 6d).
Table 2
Efficiency of drugs on nude mice
Group
|
RTV
|
T/C (%)
|
Tumor weight (g)
|
Tumor inhibitory rate (%)
|
Control
|
12.61
|
-
|
1.42 ± 0.13
|
-
|
SFN
|
7.84
|
62.19
|
1.13 ± 0.11
|
20.55
|
PP242
|
7.03
|
55.78
|
0.85 ± 0.05
|
39.97
|
S + P
|
4.10
|
32.55*
|
0.62 ± 0.04
|
56.51*
|
Note: Relative tumor volume (RTV) = Vt / V0, in them V0 was tumor volume at the beginning of treatment, and Vt was tumor volume at every treatment). T/C% = TRTV/CRTV×100%, in them TRTV was the mean of RTV in experiment group during the treatment, and CRTV was the mean of RTV in the control group during the treatment). T/C% ≤ 40% and P < 0.05 were considered as effective treatment, T/C % > 40% was considered as invalid treatment. |
The toxicity of drugs was primarily evaluated according to the body weight, H&E staining of live and kidney, blood routine, the indicators of liver and kidney function, as well as the organ coefficients of nude mice, and the results showed the data above had no obvious difference in each group (Fig. 6e, f, Table. 3–5), indicating that SFN and PP242 had no obvious toxicity at the current dose.