Suppression of esophageal tumor growth and chemoresistance by directly targeting the PI3K/AKT pathway

Abstract

Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide. Novel therapeutic intervention is urgently needed for this deadly disease. The functional role of PI3K/AKT pathway in esophageal cancer is little known. In this study, our results from 49 pairs of human esophageal tumor and normal specimens demonstrated that AKT was constitutively active in the majority (75.5%) of esophageal tumors compared with corresponding normal tissues. Inhibition of the PI3K/AKT pathway with specific inhibitors, wortmannin and LY294002, significantly reduced Bcl-xL expression, induced caspase-3-dependent apoptosis, and repressed cell proliferation and tumor growth in vitro and in vivo without obvious toxic effects. Moreover, significantly higher expression level of p-AKT was observed in fluorouracil (5-FU)-resistant esophageal cancer cells. Inactivation of PI3K/AKT pathway markedly increased the sensitivity and even reversed acquired resistance of esophageal cancer cells to chemotherapeutic drugs in vitro. More importantly, the resistance of tumor xenografts derived from esophageal cancer cells with acquired 5-FU resistance to chemotherapeutic drugs was significantly abrogated by wortmannin treatment in animals. In summary, our data support PI3K/AKT as a valid therapeutic target and strongly suggest that PI3K/AKT inhibitors used in conjunction with conventional chemotherapy may be a potentially useful therapeutic strategy in treating esophageal cancer patients.

Conflict of interest statement

Conflict of interest statement

None.

Figures

Figure 1. Constitutive activation of PI3K/AKT signaling pathway in esophageal cancer

(A) Expression levels of p-AKT and total AKT were determined in 49 pairs of esophageal tumor and matched normal tissues by Western blot, and results of 6 representative esophageal tumor tissues (T) and their matched normal tissues (N) were shown. Actin was included as loading control. (B) p-AKT/total AKT ratio in 49 tumor tissues relative to matched normal esophageal tissues. Higher ratio of p-AKT to total AKT was found in 75.5% (37 of 49) of human primary esophageal cancer, compared with their corresponding normal tissues. (C) Comparison of p-AKT/total AKT ratios between tumor tissues and normal tissues. The boxes contain the values between 25th and 75th percentiles of the 49 cases, and the whiskers extend to the highest and lowest values. The lines across the boxes indicate the median values, and the white diamonds inside the boxes represent the mean values.

Figure 2. Effects of wortmannin and LY294002 on PI3K/AKT pathway and expressions of apoptosis-associated proteins

Four esophageal cancer cell lines were treated with different concentrations of wortmannin (A) or LY294002 (B) respectively for 48 h, and cell lysates were collected for Western blot analysis of p-AKT, AKT, p-GSK3β, GSK3β, Bcl-xL, Bax, caspase-3, and cleaved caspase-3. (C) Comparison of sub-G1 population percentage by flow cytometry in the esophageal cancer cells treated with wortmannin (40 μM), LY294002 (40 μM), Z-DEVD-FMK (50 μM) alone, or a combination of Z-DEVD-FMK and wortmannin or LY294002. Bars, SD; *** P < 0.001 compared with DMSO-treated cells.

Figure 3. Effects of PI3K/AKT inhibitors on esophageal cancer cell proliferation

MTT assay was used to determine the effects of different concentrations of wortmannin (A) and LY294002 (B) on viability of esophageal cancer cells lines KYSE150, HKESC-1, KYSE270, and T.Tn. Bars, SD; * P < 0.05; **, P < 0.01; ***, P < 0.001 compared with DMSO-treated cells.

Figure 4. Effects of PI3K/AKT inhibitors on colony-formation ability of esophageal cancer cells

Colony-formation assay showed that exposure of the four esophageal cancer cell lines to wortmannin (A) and LY294002 (B) decreased colony-formation ability in a dose-dependent manner. Bars, SD; * P < 0.05; **, P < 0.01; ***, P < 0.001 compared with DMSO-treated cells.

Figure 5. Effects of wortmannin on suppressing growth of human esophageal tumor xenografts in nude mice

(A) Human esophageal cancer cells KYSE150 (left panel) and KYSE270 (right panel) were injected subcutaneously into the flanks of nude mice (n = 6 per group). Treatment of the mice with 0.6 mg/kg wortmannin twice a week significantly delayed the growth of the tumor xenografts. Note that the treatment had a better response in KSYE270-derived xenografts. (B) Western blot analysis indicated reduced expression levels of p-AKT, p-GSK3β, and increased cleaved caspase-3 in the tumors of wortmannin-treated mice, compared with the DMSO-treated group. (C, D) Comparison of body weight (C) and histological examination of lung, liver and kidney specimens (D) between wortmannin-treated and vehicle-treated animals showed no toxic effects. Bars, SD; *** P < 0.001 compared with DMSO-treated mice.

Figure 6. Effects of PI3K/AKT inhibitors on sensitivity of esophageal cancer cells to conventional chemotherapeutic drug in vitro and in vivo

(A) Esophageal cancer cells were treated with wortmannin (5 μM), LY294002 (5 μM), 5-FU (2.5 μM), DDP (10 μM), wortmannin plus 5-FU, wortmannin plus DDP, LY294002 plus 5-FU, or LY294002 plus DDP, and cell viability and colony-formation ability measured by MTT (A) and colony-formation assays (B). (C) Nude mice xenografted with human esophageal cancer cells KYSE270 were treated with wortmannin (0.3 mg/kg), 5-FU (20 mg/kg), DDP (2 mg/kg), a combination of wortmannin and 5-FU, or a combination of wortmannin and DDP twice weekly (n = 6 per group). Bars, SD; *** P < 0.001 compared with 5-FU- or DDP-treated cells or mice.

Figure 7. Effects of PI3K/AKT inhibition on reversing resistance of FR cells to 5-FU

(A) Western blot analysis of expression levels of p-AKT and AKT in FR and corresponding parental cells. The bands were quantified using ImageJ. (B) Comparison of the viability of FR esophageal cancer cells treated with 5-FU (40 μM for KYSE150FR and 10 μM for KYSE410FR), wortmannin (5 μM), LY294002 (5 μM) alone, or a combination of 5-FU and wortmannin or LY294002. (C-D) Parental KYSE150 and KYSE410 cells were treated with 5-FU, whereas the respective FR cells were treated with 5-FU, wortmannin, LY294002 alone, or the combination of 5-FU and wortmannin or LY294002 for 48 h. The drugs were used at the same concentrations as in (B). The cells were collected for cell cycle analysis of sub-G1 population percentage using flow cytometry (C), and for Western blot analysis of caspase-3 and cleaved caspase-3 (D), respectively. (E) Nude mice bearing KYSE410FR-derived tumor xenografts were treated with wortmannin (0.3 mg/kg), 5-FU (20 mg/kg), or a combination of wortmannin and 5-FU twice weekly for three weeks (n = 6 per group). Bars, SD; * P < 0.05; **, P < 0.01; ***, P < 0.001.