Growth inhibition and chemo-radiosensitization of esophageal squamous cell carcinoma by survivin-shRNA lentivirus transfection

Abstract

Esophageal cancer is one of the most common types of cancer worldwide, and it has a poor prognosis. Chemo-radiotherapy resistance and cancer relapse are among the most difficult issues in its treatment. Identifying the underlying molecular mechanisms is critical for developing novel therapies. Survivin has been previously suggested to be overexpressed in esophageal cancer cells. The present study identified that down-regulation of survivin sensitized esophageal cancer cells to chemo-radiotherapy. Consistent with previous studies, the present study indicated that survivin was overexpressed in 4 esophageal squamous carcinoma cell lines. Short hairpin RNA delivered by lentivirus successfully knocked down survivin in these cancer cell lines. Consequently, down-regulation of survivin impaired their colony-forming, migratory and invasive capabilities, while the overexpression of survivin in normal human esophagus epithelial cells improved their resistance to cisplatin, paclitaxel and radiation. Survivin knockdown induced apoptosis in esophageal cancer KYSE-150 and ECA-109 cell lines when exposed to the aforementioned chemo-radiotherapy treatments. These results indicate that survivin expression sustains growth in esophageal cancer cells, and confers resistance to chemo-radiotherapy. Targeted survivin ablation may be a promising strategy against esophageal tumor relapse and chemo-radioresistance.

Keywords: chemo-radiosensitization; esophageal cancer; lentivirus; short hairpin RNA; survivin.

Figure 1.

Survivin expression in esophageal cell lines and RNAi knockdown of survivin. (A) Western blot analysis of survivin expression in the normal esophageal HEEC cell line and 4 esophageal squamous carcinoma cell lines. (B) Survivin expression was detected by reverse transcription quantitative polymerase chain reaction at different days following lentivirus delivery of RNAi, svv-lent or control lentivirus vector, cont-lent. (C) Western blot analysis at day 2. RNAi, RNA interference; svv-lent, supernatant virus; control-lent, empty lentiviral vector.

Figure 2.

Survivin knockdown inhibits tumorigenic ability of ESC cells in vitro. Colony formation assay of (A) ECA-109 and (B) KYSE-150 cells infected with survivin shRNA lentivirus or control lentivirus. Cell invasion assay of KYSE-150 and ECA-109 cells infected with survivin or control shRNA lentivirus. (C) The average number of migrated cells in each light microscope field (magnification, ×200). (D) Representative images of migrated cells stained with crystal violet in each group in light microscope (magnification, ×40). Wound healing assay of KYSE-150 and ECA-109 cells infected with survivin or control shRNA lentivirus. (E) The average migrated distance of cells in each group. (F) Representative image to show the distance of migration in the two groups. Results are representative image of at least 3 repeats. *P<0.05 and **P<0.01.

Figure 3.

Overexpression of survivin in normal HEEC cells facilitates chemo-radioresistance. ESC and HEEC cells were treated with various concentration of (A) cisplatin or (B) paclitaxel for 24 h or (C) the indicated dose radiation, and cell viability was measured by MTT experiment. *P<0.05 vs. KYSE-150, ECA-109 or TE-1 cells. (D) Survivin was overexpressed in HEEC cells and cell sensitivity to chemo-radiotherapy was measured. *P<0.05 vs. HEEC cells or HEEC cells infected with empty lentivirus (HEEC-neo). All experiments were repeated at least 3 times and data presented as mean ± standard error of the mean. ESC, esophageal squamous carcinoma; HEEC, Human esophagus epithelial cells; neo, empty plasmid vector.

Figure 4.

Survivin knockdown promotes ESC cells apoptosis and chemo-radiotherapy sensitivity. KYSE150 and ECA-109 cells were pretreated with survivin shRNA lentivirus or negative control, and then a TUNEL assay was performed to evaluate the apoptosis rate of these cells exposure to paclitaxel, cisplatin and radiation. (A) TUNEL assays were performed to assess apoptotic cells infected with svv-lent, cont-lent or untreated, and then svv-lent- and cont-lent-infected cells treated with 1 µg/ml paclitaxel. (B) Western blotting was performed to detect cleaved PARP1 levels following paclitaxel exposure. (C) TUNEL assays were performed to assess apoptotic cells infected with svv-lent, cont-lent or untreated, and then svv-lent- and cont-lent-infected cells treated with 2 µg/ml cisplatin. (D) Western blotting was performed to detect cleaved PARP1 levels following Cisplan exposure. (E) TUNEL assays were performed to assess apoptotic cells infected with svv-lent, cont-lent or untreated, and then svv-lent- and cont-lent-infected cells treated with 3 Gy radiation. (F) Western blotting was performed to detect cleaved PARP1 levels following radiation exposure. All experiments were performed in triplicate and data presented as mean ± standard error of the mean. *P<0.05 with comparisons shown by lines. svv-lent, survivin shRNA lentivirus infected; cont-lent, infected with empty lentivirus; Cisplan, cisplatin; svv-lent+pacl, cells pretreated with survivin shRNA exposed to paclitaxel; PARP1, Poly (adenosine 5′-diphosphate-ribose) polymerase 1; shRNA, short hairpin RNA; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.