Cyclin D1 overexpression enhances chemosensitivity to TPF chemotherapeutic agents via the caspase-3 pathway in oral cancer

Abstract

Induction chemotherapy has been previously demonstrated to downgrade locally advanced or aggressive cancers and increase the likelihood of primary lesion eradication. Based on our previous phase 3 trial on TPF (docetaxel, cisplatin and fluorouracil) induction chemotherapy in patients with oral squamous cell carcinoma (OSCC), in which short-term prognostic and predictive values of cyclin D1 expression were reported, the present study aimed to determine the long-term predictive value of cyclin D1 expression in the same patients with OSCC who were eligible to receive TPF induction chemotherapy. In addition, the present study investigated the potential association between cyclin D1 expression and chemosensitivity to TPF agents during OSCC cell intervention, and the underlying apoptotic mechanism of action. In total, 232 patients with locally advanced OSCC from our previous trial with a median follow-up of 5 years were included for survival analysis using the Kaplan-Meier method and the log-rank test in the present study, where cyclin D1 expression in their tissues was detected by immunohistochemistry. Cyclin D1 knockdown, cytotoxicity assays assessing the efficacy of the TPF chemotherapeutic agents and measurements of caspase-3 and PARP activity in HB96, CAL27 and HN30 cell lines were performed. Patients with OSCC in the low cyclin D1 expression group exhibited significantly superior long-term clinical outcomes compared with those in patients in the high cyclin D1 expression group [overall survival (OS), P=0.001; disease-free survival, P=0.003; local recurrence-free survival, P=0.004; distant metastasis-free survival (DMFS), P=0.001]. Furthermore, patients with stage clinical nodal stage 2 (cN2) OSCC in the high cyclin D1 expression group benefitted from TPF induction chemotherapy (OS, P=0.024; DMFS, P=0.024), whilst patients with cN2 OSCC in the low cyclin D1 expression group did not benefit from this chemotherapy. Overexpression of cyclin D1 expression was found to enhance chemosensitivity to TPF chemotherapeutic agents in OSCC by mediating caspase-3-dependent apoptosis. Based on these findings, TPF induction chemotherapy can benefit patients with cN2 OSCC and high cyclin D1 expression in terms of long-term survival from compared with standard treatment. In addition, OSCC cell lines overexpressing cyclin D1 are more sensitive to TPF chemotherapeutic agents in a caspase-3-dependent manner (clinical trial. no. NCT01542931; February 2012).

Keywords: apoptosis; chemosensitivity; cisplatin; cyclin D1; docetaxel; fluorouracil; induction chemotherapy; oral squamous cell carcinoma.

Figure 1.

Survival analysis of patients with oral squamous cell carcinoma as a function of tumor cyclin D1 expression. Patients with low cyclin D1 expression exhibited superior (A) overall, (B) disease-free, (C) locoregional recurrence-free and (D) distant metastasis-free survival rates compared with those of patients with high cyclin D1 expression.

Figure 2.

Survival comparison between patients with cN2 OSCC with low and high cyclin D1 expression in the experimental and control groups. Patients with cN2 OSCC and high cyclin D1 expression in the experimental group exhibited superior survival rates compared with those in patients in the control group with respect to (A) overall and (B) distant metastasis-free survival. Patients with cN2 OSCC and low cyclin D1 expression did not benefit from TPF induction chemotherapy with respect to (C) overall and (D) distant metastasis-free survival. OSCC, oral squamous cell carcinoma.

Figure 3.

Association between cyclin D1 expression and sensitivity to chemotherapeutic agents docetaxel, cisplatin and 5-FU. After (A) HB96 and (B) CAL27 cells were transfected with siRNA1 and siRNA2 against cyclin D1, cell viability was measured following treatment with a series of concentrations of docetaxel, cisplatin and 5-FU for 72 h. After (C) CAL27 and (D) HN30 cells were transfected with cyclin D1 overexpression vectors, cell viability was measured following treatment with a series of concentrations of docetaxel, cisplatin and 5-FU for 72 h. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. 5-FU, 5-fluorouracil; siRNA, small interfering RNA; NC, negative control.

Figure 4.

Association between cyclin D1 expression and cleaved caspase-3 and PARP levels following treatment with chemotherapeutic agents docetaxel, cisplatin and 5-FU. After (A) CAL27 and (B) HB96 cells were transfected with siRNA1 and siRNA2 against cyclin D1, cleaved caspase-3 and PARP protein levels were measured after treatment with 5 nM docetaxel, 2 µg/ml cisplatin or 2 µg/ml 5-FU for 72 h. After (C) CAL27 and (D) HN30 cells were transfected with the cyclin D1 overexpression vector, cleaved caspase-3 and PARP protein levels were measured following treatment with 5 nM docetaxel, 2 µg/ml cisplatin or 2 µg/ml 5-FU for 72 h. 5-FU, 5-fluorouracil; siRNA, small interfering RNA; NC, negative control; PARP, poly (ADP-ribose) polymerase.