Effects of the proapoptotic regulator Bcl-2/adenovirus EIB 19-kDa-interacting protein 3 on the chemosensitivity of human colon cancer cell lines

Abstract

In the clinical setting, drug resistance remains a significant obstacle for successful chemotherapy. Bcl-2/adenovirus EIB 19-kDa-interacting protein 3 (BNIP3) is a proapoptotic member of the Bcl-2 family. To address its potential as a therapeutic target for chemosensitisation, this study investigated the effect of BNIP3 expression on chemosensitivity and reversal of oxaliplatin (L-OHP) resistance in human colon cancer cell lines. A plasmid expressing the BNIP3 gene was transfected into human parental colon cancer cell lines (SW620 and colo320) and L-OHP-resistant colon cancer cell lines (SW620/L-OHP and colo320/L-OHP) using Lipofectamine™ 2000, and the transfection efficiency was determined using fluorescence optics. Western blot analysis identified that SW620/L-OHP and colo320/L-OHP cells expressed lower levels of BNIP3 protein compared with the SW620 and colo320 cells. Transfection with the recombinant BNIP3 plasmid revealed an increase in BNIP3 expression in tumour cells. Following transfection with pDsRed-BNIP3, the chemosensitivity of parental and L-OHP-resistant cell lines to L-OHP was increased (P<0.01), as detected by the Cell Counting Kit-8 (CCK8) assay. Hoechst 33342 staining and flow cytometry revealed that the effects on L-OHP-induced apoptosis were enhanced by the overexpression of BNIP3. Chemosensitisation in human colon cancer cells was observed following treatment with the recombinant BNIP3 plasmid in vitro. The results of this study suggest that BNIP3 is a potential therapeutic target for reversing the resistance of L-OHP-resistant colon cancer cells to L-OHP.

Figure 1.

Fluorescein-labelled pDsRed-N1 or pDsRed-BNIP3-transfected cells were examined using fluorescence optics to determine transfection efficiency after 24 h. The pDsRed-N1 and pDsRed-BNIP3 plasmids were transfected into the four colon cancer cell lines. The overall transfection rates were estimated to be >60% after 24 h of incubation. (A) SW620-N1. (B) SW620-BNIP3. (C) SW620/L-OHP-N1. (D) DW620/L-OHP-BNIP3. (E) colo 320-N1. (F) colo320-BNIP3. (G) colo320/L-OHP-N1. (H) colo320/L-OHP-BNIP3. The images are shown in a single field of view.

Figure 2.

Western blot analysis identified BNIP3 expression in colon cancer cells after 24-h transfection. (A) Lane 1, SW620; lane 2, SW620-N1; lane 3, SW620-BNIP3; lane 4, SW620/L-OHP; lane 5, SW620/L-OHP-N1; lane 6, SW620/L-OHP-BNIP3. (B) Lane 1, colo320; lane 2, colo320-N1; lane 3, colo320-BNIP3; lane 4, colo320/L-OHP; lane 5, colo320/L-OHP-N1; lane 6, colo320/L-OHP-BNIP3. Recombinant human BNIP3 was expressed as 2 bands of approximately 30 and 60 kDa in pDsRed-BNIP3-transfected (lanes 3 and 6) colon cancer cells. Both forms of BNIP3 revealed a visible increase compared with the empty plasmid (pDsRed-N1)-transfected (lanes 1 and 4) or untreated (lanes 2 and 5) colon cancer cells. BNIP3 expression in the L-OHP-resistant colon cancer cell lines (SW620/L-OHP and colo320/L-OHP) was lower compared with that of the parental human colon cancer cell lines (SW620 and colo320). BNIP3, Bcl-2/adenovirus EIB 19-kDa-interacting protein 3; L-OHP, oxaliplatin.

Figure 3.

Survival rates of colon cancer cells treated with various concentrations of L-OHP. (A) Survival rates of SW620 and SW620/L-OHP groups. (B) Survival rates of colo320 and colo320/L-OHP groups. The CCK8 assay revealed that at the same concentration of L-OHP, the surviving fraction of the pDsRed-BNIP3-transfected cells was significantly reduced (P<0.01) compared with pDsRed-N1-transfected cells and the control, whereas that of the pDsRed-N1-transfected cells remained unchanged (P>0.05). L-OHP, oxaliplatin; BNIP3, Bcl-2/adenovirus EIB 19-kDa-interacting protein 3. CCK8, Cell Counting Kit-8.

Figure 4.

Apoptosis of colon cancer cells as detected by Annexin V-FITC/PI. Quadrant 1, PI(+) (cells undergoing necrosis); Quadrant 2, Annexin V-FITC(+) PI(+) (cells in the late period of apoptosis and undergoing secondary necrosis); Quadrant 3, Annexin V-FITC(−) PI(−) (living cells); Quadrant 4, Annexin V-FITC(+) PI(−) (cells in the early period of apoptosis). The total AR was calculated as Quadrant 2 + Quadrant 4. The pDsRed-BNIP3-transfected cells exhibited higher apoptotic rates compared with the pDsRed-N1-transfected cells and the control. Compared with the untransfected cells treated with L-OHP and the pDsRed-BNIP3-transfected cells, the apoptosis regulator in combination with L-OHP significantly increased the apoptosis rates in parental and L-OHP-resistant colon cancer cell lines. FITC, fluorescein isothiocyanate; PI, propidium iodide; BNIP3, Bcl-2/adenovirus EIB 19-kDa-interacting protein 3; L-OHP, oxaliplatin; AR, apoptosis rate.

Figure 5.

(A and B) Morphological features of colon cancer cells undergoing apoptosis, as observed under the fluorescence optics using Hoechst 33342 staining. Typical morphological features of apoptosis, including chromatin condensation and apoptotic bodies, were visualised in pDsRed-BNIP3-transfected cells, compared with the pDsRed-N1-transfected cells and control groups. (C and D) AR of colon cancer cells as detected by Hoechst 33342 staining. pDsRed-BNIP3-transfected cells exhibited higher (^*P<0.01) apoptotic rates than the pDsRed-N1-transfected cells and control groups. Compared with the untransfected cells treated with L-OHP and pDsRed-BNIP3-transfected cells, the apoptosis regulator in combination with L-OHP significantly increased the apoptotic rates (^#P<0.01) in parental and L-OHP-resistant colon cancer cell lines. L-OHP, oxaliplatin; AR, apoptosis rate.