Synergistic effects of thalidomide and cisplatin are mediated via the PI3K/AKT and JAK1/STAT3 signaling pathways in cervical cancer

Abstract

Thalidomide (THD) has been found to synergize with cisplatin (DDP) in certain types of cancers; however, their combined use in the treatment of cervical cancer has not been reported to date, at least to the best of our knowledge. Thus, the present study aimed to explore the synergistic effects of THD and DDP and determine their regulatory effects on the phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT) and Janus kinase 1 (JAK1)/signal transducer and activator of transcription 3 (STAT3) pathways in cervical cancer. For this purpose, 0‑160 µM THD and 0‑64 µM DDP monotherapy or in combination were used to treat the HeLa and SiHa cervical cancer cell lines. This was followed by the calculation of the combination index (CI) and 160 µM THD and 16 µM DDP were then used to treat the cells. Relative cell viability and apoptosis, as well as the mRNA and protein levels of PI3K, AKT, JAK1 and STAT3 were evaluated. The results revealed that THD and DDP monotherapy suppressed the viability of the HeLa and SiHa cells in a concentration‑dependent manner. Moreover, THD and DDP treatment exerted a more prominent suppressive effect on the relative viability of HeLa and SiHa cells compared with DDP monotherapy at several concentration settings; further CI calculation revealed that the optimal synergistic concentrations were 160 µM for THD and 16 µM for DDP. Subsequently, combined treatment with THD and DDP suppressed relative cell viability, whereas it promoted cell apoptosis compared with THD or DPP monotherapy; it also inhibited the PI3K/AKT and JAK1/STAT3 signaling pathways compared with DPP or THD monotherapy in both HeLa and SiHa cells. On the whole, the present study demonstrated that THD synergizes with DDP to exert suppressive effects on cervical cancer cell lines. This synergistic action also inactivated the PI3K/AKT and JAK1/STAT3 pathways. Thus, these findings suggest that the combined use of THD and DPP may have potential for use in the treatment of cervical cancer.

Keywords: JAK1/STAT3; PI3K/AKT; cervical cancer; cisplatin; thalidomide.

Figure 1.

Comparison of relative cell viability following treatment with various concentrations of DDP or THD. Comparison of relative cell viability following treatment with various concentrations of (A) DDP or (B) THD treatment in HeLa cells; comparison of relative cell viability following treatment with various concentrations of (C) DDP or (D) THD treatment in SiHa cells. *P<0.05 and **P<0.01 compared with cells treated with 0 µM of DDP or THD. DDP, cisplatin; THD, thalidomide.

Figure 2.

Comparison of relative cell viability by combination treatment involving various concentrations of DDP and THD. Comparison of relative cell viability after treatment with various concentrations of DDP and THD combination treatment in (A) HeLa cells and (B) SiHa cells. *P<0.05 and **P<0.01 compared with cells treated with 0 µM of DDP and THD. DDP, cisplatin; THD, thalidomide.

Figure 3.

Effect of THD plus DDP on HCerEpiC cell viability. *P<0.05 compared with cells without treatment of DDP and THD. DDP, cisplatin; THD, thalidomide.

Figure 4.

Comparison of relative cell viability and apoptosis by THD/DDP monotherapy and their synergistic treatment. Comparison of (A) relative cell viability and (B) apoptosis by THD/DDP monotherapy and their synergistic treatment in HeLa cells; (C) representative images of flow cytometric analysis of apoptosis in HeLa cells. Comparison of (D) relative cell viability and (E) apoptosis by THD/DDP monotherapy and their synergistic treatment in SiHa cells; (F) representative images of flow cytometric analysis of apoptosis in SiHa cells. *P<0.05 and **P<0.01. DDP, cisplatin; THD, thalidomide.

Figure 5.

Comparison of PI3K and AKT expression levels. (A) Comparison of PI3K mRNA expression by THD/DDP monotherapy and their synergistic treatment in HeLa cells. (B) Representative images of PI3K, AKT and p-AKT protein expression detection by western blotting in HeLa cells. Comparison of (C) PI3K and (D) p-AKT protein expression by THD/DDP monotherapy and their synergistic treatment in HeLa cells. (E) Comparison of PI3K mRNA expression by THD/DDP monotherapy and their synergistic treatment in SiHa cells. (F) Representative images of PI3K, AKT and p-AKT protein expression detection by western blotting in SiHa cells. (G) Comparison of PI3K and (H) p-AKT protein expression by THD/DDP monotherapy and their synergistic treatment in SiHa cells. *P<0.05, **P<0.01 and ***P<0.001. PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; THD, thalidomide; DDP, cisplatin; p-, phosphorylated.

Figure 6.

Comparison of JAK1 and STAT3 expression levels. Comparison of (A) JAK1 and (B) STAT3 mRNA expression levels by THD/DDP monotherapy and their synergistic treatment in HeLa cells. (C) Representative images of JAK1 and STAT3 protein expression detection by western blotting in HeLa cells. Comparison of (D) JAK1, (E) STAT3, (F) p-JAK1 and (G) p-STAT3 protein expression levels by THD/DDP monotherapy and their synergistic treatment in HeLa cells. Comparison of (H) JAK1 and (I) STAT3 mRNA expression levels by THD/DDP monotherapy and their synergistic treatment in SiHa cells. (J) Representative images of JAK1 and STAT3 protein expression detection by western blotting in SiHa cells. Comparison of (K) JAK1, (L) STAT3, (M) p-JAK1 and (N) p-STAT3 protein expression levels by THD/DDP monotherapy and their synergistic treatment in SiHa cells. *P<0.05, **P<0.01 and ***P<0.001. JAK1, Janus kinase 1; STAT3, signal transducer and activator of transcription 3; THD, thalidomide; DDP, cisplatin; p-, phosphorylated; NS, not significant.

Figure 7.

Role of PI3K/AKT and JAK1/STAT3 pathways on the regulation of cervical cancer cell viability and apoptosis by THD and DDP synergistic treatment. Comparison of (A) cell viability and (B) apoptosis among groups in HeLa cells. (C) Representative images of apoptosis evaluation among groups in HeLa cells. Comparison of (D) cell viability and (E) apoptosis among groups in SiHa cells. (F) Representative images of apoptosis evaluation among groups in SiHa cells. *P<0.05 and **P<0.01 compared with cells treated with 16 µM of DDP and 160 µM of THD but without treatment of 740 Y-P or RO8191. PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; JAK1, Janus kinase 1; STAT3, signal transducer and activator of transcription 3; THD, thalidomide; DDP, cisplatin.