PUMA mediates the combinational therapy of 5-FU and NVP-BEZ235 in colon cancer

Abstract

Colon cancer is the third most common cancer in humans which has a high mortality rate, and 5-Fluorouracil (5-FU) is one of the most widely used drugs in colon cancer therapy. However, acquired chemoresistance is becoming the major challenges for patients, and the molecular mechanism underlying the development of 5-FU resistance is still poorly understood. In this study, a newly designed therapy in combination with 5-FU and NVP-BEZ235 in colon cancer cells (HCT-116 and RKO) was established, to investigate the mechanism of 5-FU resistance and optimize drug therapy to improve outcome for patients. Our results show 5-FU induced cell apoptosis through p53/PUMA pathway, with aberrant Akt activation, which may well explain the mechanism of 5-FU resistance. NVP-BEZ235 effectively up-regulated PUMA expression, mainly through inactivation of PI3K/Akt and activation of FOXO3a, leading to cell apoptosis even in the p53-/- HCT-116 cells. Combination treatment of 5-FU and NVP-BEZ235 further increased cell apoptosis in a PUMA/Bax dependent manner. Moreover, significantly enhanced anti-tumor effects were observed in combination treatment in vivo. Together, these results demonstrated that the combination treatment of 5-FU and NVP-BEZ235 caused PUMA-dependent tumor suppression both in vitro and in vivo, which may promise a more effective strategy for colon cancer therapy.

Keywords: Akt; NVP-BEZ235; PUMA; colon cancer; p53.

Figure 1. Efffects of 5-FU, NVP-BEZ235 or their combination on cell viability and apoptosis

A–D. Cells viability was analyzed using Cell Counting Kit-8 at 0, 3, 6, 12 and 24 hours after (A) 50, 100, 200, or 400 uM 5-FU treatment, (B) 100, 200, 400, or 800 nM NVP-BEZ235 treatment, (C) the combination treatment of 200 uM 5-FU and 400 nM NVP-BEZ235 in HCT-116 cells, (D) Cell viability was detected in RKO cells after 24 hours combination treatment. E. HCT-116 cells apoptosis was analyzed using FACS technique at 12 hours after the treatment of 200 uM 5-FU, or 400 nM NVP-BEZ235, or their combination. F. The percentage of apoptotic cells were used to calculate. Data represent the mean ± S.D. of four independent experiments.

Figure 2. 5-FU induces cell apoptosis through p53/PUMA pathway

A–C and E–G. The expression of p53, PUMA, cleaved PARP and P-Akt(S473) were detected by western blotting in different conditions. (A) and (E) HCT-116 cells were treated with various doses of 5-FU for 24 hours. (B) and (F) HCT-116 cells were treated with 200 uM 5-FU, then harvested at different time points after stimulation. (C) and (G) Wild-type, p53^−/− or PUMA^−/− HCT-116 cells were treated with 200 uM 5-FU for 12 hours. (D) Hoechst 33342 morphological examination of apoptosis in wild-type, p53^−/− or PUMA^−/− HCT-116 cells. Cells were treated with 200 uM 5-FU and incubated for 12 hours, then stained with Hochest 333342, visualized under a Nikon fluorescent microscope (60 ×). Similar results were obtained from three independent experiments.

Figure 3. Cell apoptosis induced by NVP-BEZ235 is dependent on Akt/mTOR/PUMA pathway, not on p53

A–C. Western blotting showing the expression of P-Akt, p53, PUMA and cleaved PARP after (A) various doses of NVP-BEZ235 for 24 hours (B) 400 nM NVP-BEZ235 for different time stimulation in HCT-116 cells (C) 400 nM NVP-BEZ235 for 12 hours in wild-type, p53^−/− or PUMA^−/− HCT-116 cells. (D) Hoechst 33342 morphological examination of apoptosis in wild-type, p53^−/− or PUMA^−/− HCT-116 cells after the treatment of 400 nM NVP-BEZ235 for 12 hours. Similar results were obtained from three independent experiments.

Figure 4. The effect of NVP-BEZ235, NVP-BBD130, Wortmannin, Rapamycin or Akt inhibitor VIII on PUMA expression and apoptosis

A–C. The expression of P-Akt(S473), PUMA, P-FOXO3a, cleaved PARP and Caspase3 were detected after the treatment of 400 nM NVP-BEZ235, 400 nM NVP-BBD130, 250 nM Wortmannin, 50 nM Rapamycin or 5 uM Akt inhibitor VIII in (A) HCT-116 cells, (B) RKO cells. (C) p53^−/− HCT-116 cells. (D) FOXO3a and PUMA were detected in p53^−/− HCT-116 cells after the treatment of NVP-BEZ235, with FOXO3a deletion or not. Similar results were obtained from three independent experiments.

Figure 5. Combination treatment induced PUMA-dependent apoptosis

A–C. Western blotting showing the expression of p53, P-Akt(S473), PUMA, cleaved PARP and Caspase3 after the treatment of 200 uM 5-FU, 400 nM NVP-BEZ235 or their combination for (A) 12 hours in HCT-116 cells, (B) 12 hours in RKO cells, (C) 24, 48 or 72 hours in HCT-116 cells. (D and G). Colony formation of HCT-116 cells. Cells were treated with different drugs for 24 hours, followed with crystal violet staining of attached cells at 14 days. (D) 200 uM 5-FU, 400 nM NVP-BEZ235, or their combination in wild-type HCT-116 cells, (G) combination treatment in wild-type, p53^−/− or PUMA^−/− HCT-116 cells. E. and H. Hoechst 33342 morphological examination of apoptosis in HCT-116 cells, (E) treated with 200 uM 5-FU, 400 nM NVP-BEZ235, or their combination for 12 hours in wild-type HCT-116 cells, (H) combination treatment for 12 hours in wild-type, p53^−/− or PUMA^−/− HCT-116 cells. F. The expression of p53, P-Akt, PUMA and cleaved PARP were detected in wild-type, p53^−/− or PUMA^−/− HCT-116 cells. Similar results were obtained from three independent experiments.

Figure 6. PUMA/Bax pathway is required for combination treatment induced apoptosis

A. The interaction between PUMA and Bax was detected by Co-IP. Co-immunoprecipitation with an anti-PUMA antibody was used to pull down PUMA, western blotting for Bax shows the amount of Bax binding to PUMA. B. PUMA and activated Bax were detected in wide-type or PUMA^−/− HCT-116 cells after combination treatment. C. Transfecting PUMA into PUMA^−/− HCT-116 cells or not, western blot to check the expression of PUMA, activated Bax and cleaved PARP. D. P-Akt(S473), p53, PUMA, cleaved PARP and Caspase3 were detected in wild-type or Bax^−/− HCT-116 cells after combination treatment for 24 or 48 hours. E. Cell viability was analyzed by CCK-8 in wild-type or Bax^−/− HCT-116 cells after combination treatment for 48 hours. F. Hoechst 33342 morphological examination of apoptosis in in wild-type or Bax^−/− HCT-116 cells after combination treatment for 12 hours. Similar results were obtained from three independent experiments.

Figure 7. The antitumor effect of combination treatment in vivo

A–C. Nude mice were injected s.c. with 1 × 10⁶ HCT-116 cells. Once the tumor was measurable, mice were treated daily with 5-FU at 25 mg/kg by i.p. injection, or 40 mg/kg NVP-BEZ235 by oral gavage, or their combination for 15 consecutive days. (A) Representative tumors at the end of the experiment, (B) tumors weight, (C) tumor volume at indicated time points after treatment was calculated. Data represent the mean ± S.D. of four independent experiments. D. Paraffin-embedded sections of control or treated tumor tissues from mice were analyzed by P-Akt(S473), ki67 and active Caspase-3 IHC staining. E. and F. Quantitative analysis of ki67 and active Caspase-3 staining corresponding to the images in D. Data represent the mean ± S.D. of four independent experiments.

Figure 8. Schematic representation of 5-FU and NVP-BEZ235 induced apoptotic pathway