Plumbagin Enhances the Anticancer Effects of PF Chemotherapy via Downregulation of the PI3K/AKT/mTOR/p70S6K Pathway in Human Tongue Squamous Cell Carcinoma

Abstract

Cisplatin plus 5-fluorouracil (PF) is used as the standard neoadjuvant chemotherapy (also called preoperative chemotherapy) in the treatment of tongue squamous cell carcinoma (TSCC). Although PF chemotherapy reduces the distant metastasis of TSCC, the five-year survival rate has not significantly improved. In recent years, components considered in traditional Chinese medicine have been researched as adjuvant drugs for radiotherapy and chemotherapy. Plumbagin (PB) is a quinone component isolated from Plumbago zeylanica L. Notably, PB demonstrates numerous anticancer properties. In order to examine the chemosensitization effect of PB on PF and its associated mechanisms, in vitro experiments using TSCC Cal27 and cisplatin (CDDP)-resistant Cal27/CDDP cells were carried out in the present study, and the results were subsequently verified using nude mice xenografts. Results of the present study demonstrated that PB enhanced the anticancer effects of PF on the proliferation, migration, and invasion of Cal27 and Cal27/CDDP cells. Cell cycle assays demonstrated that both Cal27 and Cal27/CDDP cells were arrested in the S phase following the combined treatment of PF and PB. Moreover, the PF and PB combination group induced higher levels of apoptosis in Cal27 and Cal27/CDDP cells compared with the group treated with PF alone. In addition, the results of the present study demonstrated that combined PB and PF inhibited the PI3K/AKT/mTOR/p70S6K pathway in TSCC cells. Moreover, the weight and volumes of tumors in nude mice were reduced following treatment with a combination of PF and PB. Results of the present study also demonstrated that the expression levels of Ki67 were markedly reduced in the combined treatment group compared with the group treated with PF alone. In summary, the results of the present study demonstrated that PB enhanced the PF sensitivity of TSCC through induction of S-phase arrest and apoptosis via the PI3K/AKT/mTOR/p70S6K pathway.

Figure 1

Viability of TSCC cells after various treatments. (a-c) Inhibition of the proliferation of Cal27 and Cal27/CDDP cells following treatment with PB, 5-Fu, and CDDP. (d) A combination of PB and PF exerted synergistic cytotoxic effects in Cal27 and Cal27/CDDP cells. TSCC, tongue squamous cell carcinoma; PB, plumbagin; 5-Fu, 5-fluorouracil; and PF, cisplatin plus 5-fluorouracil.

Figure 2

Combined treatment of PB and PF inhibited the proliferation and colony formation of TSCC cells. (a) Results of the Edu assay demonstrated the inhibited proliferation of Cal27 and Cal27/CDDP cells following the treatment with PB and/or PF. (b-c) Histogram of Edu fluorescence in Cal27 and Cal27/CDDP cells. (d) Colony formation of Cal27 and Cal27/CDDP cells following the treatment with PB and/or PF. (e-f) Histogram of numbers of colonies of Cal27 and Cal27/CDDP cells. Independent biological experiments were repeated three times. ^∗∗P < 0.01, ^∗∗∗P < 0.001 and ^∗∗∗∗P < 0.0001. PB, plumbagin; PF, cisplatin plus 5-fluorouracil; and TSCC, tongue squamous cell carcinoma.

Figure 3

Combined treatment of PB and PF inhibited the migration and invasion of TSCC cells. (a) Results of the transwell assay demonstrated the invasion of Cal27 and Cal27/CDDP cells following treatment with PB and/or PF. (b) Histogram of invading Cal27 and Cal27/CDDP cells. (c) Results of the scratch test demonstrated the migration of Cal27 and Cal27/CDDP cells following treatment with PB and/or PF. (d) Histogram of migration rate of Cal27 and Cal27/CDDP cells. Independent biological experiments were repeated three times (magnification, ×50; scale bar, 200 μm). ^∗∗P < 0.01, ^∗∗∗P < 0.001. PB, plumbagin; PF, cisplatin plus 5-fluorouracil; and TSCC, tongue squamous cell carcinoma.

Figure 4

Combined PB and PF induced S phase arrest of TSCC cells. (a) A representative cell cycle in Cal27 and Cal27/CDDP cells following the treatment with PB and/or PF. (b-c) A histogram of cell cycle distribution in Cal27 and Cal27/CDDP cells. PB, plumbagin; PF, cisplatin plus 5-fluorouracil; and TSCC, tongue squamous cell carcinoma.

Figure 5

PB enhanced the proapoptotic effects of PF in TSCC cells. (a) Results of the flow cytometry assay demonstrated apoptosis in Cal27 and Cal27/CDDP cells following the treatment with PB and/or PF. (b-c) A histogram of the levels of apoptosis in Cal27 and Cal27/CDDP cells. (d) Representative blots of the proapoptotic protein Bax and Bad and antiapoptotic protein Bcl-2 and Bcl-xL in Cal27 and Cal27/CDDP cells after the treatment of PB and/or PF. (e) Ratio of Bax/Bcl-2 in Cal27 and Cal27/CDDP cells. (f-g) Histogram of the expression levels of Bcl-xL and Bad. Independent biological experiments were repeated three times, and statistical significance is denoted by ^∗P <  0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 and ^∗∗∗∗P < 0.0001. PB, plumbagin; PF, cisplatin plus 5-fluorouracil; and TSCC, tongue squamous cell carcinoma.

Figure 6

Combined PB and PF inhibited the PI3K/AKT/mTOR/p70S6K pathway in TSCC cells. (a) Representative blots demonstrating the expression of proteins involved in the PI3K/AKT/mTOR pathway in Cal27 and Cal27/CDDP cells following treatment with PB and/or PF. (b–e) A histogram demonstrating the expression level of PI3K, p-AKT/AKT, p-mTOR/mTOR, and p-p70S6K/p70S6K. Independent biological experiments were repeated three times. ^∗P <  0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001. PB, plumbagin; PF, cisplatin plus 5-fluorouracil; TSCC, tongue squamous cell carcinoma; and p-, phosphorylated.

Figure 7

Combined treatment with PB and PF inhibited TSCC xenograft tumor growth in vivo. (a-b) Cal27/CDDP-derived xenograft models via subcutaneous injection into BALB/c nude mice. (c) Tumor weights in the PB + PF group were significantly lower than those in the control group. (d) Tumor volumes in the PB + PF group were significantly lower than those in the control group. (e) No significant changes in body weight were observed in the PB + PF group compared with the control group. (f) Ki67 expression in the PB + PF group was significantly lower than that in the control group. (g) H and E staining verified that the PB + PF group did not induce toxicity in major organs (n = 6/group). PB, plumbagin; PF, cisplatin plus 5-fluorouracil; and TSCC, tongue squamous cell carcinoma.