3.1 Th2 cells induce tolerance to cisplatin and co-culture in cervical carcinoma cells in relation to IL-17
We were interested in whether Th2 cells influence the reaction of cervical carcinoma cells to chemotherapeutic drugs or irradiation due to their infiltration of cervical carcinoma tissues and their existence in situ associated to progressive disease [8, 22]. Initially, we tested for cisplatin, which would be the most frequently administered agent in cervical chemotherapy. We cocultured HeLa cells with in vitro-produced Th2 cells in the ratio of 1:20 for overnight. Th2 cells were eliminated and cervical carcinoma cells were challenged with sequential concentrations of cisplatin. The co-culture decreased the reactivity of HeLa cells to induction of cisplatin-induced apoptosis (Fig. 1A), which resulted in an improvement in cell viability. As consistent responses for all the cervical carcinoma cell lines examined, pre-stimulated SiHa and SW756 cells with rhIL-17 exhibited a pronounced growth in cell viability following cisplatin administration at a highest applying cisplatin dose of 6.25 µg/ml (Fig. 1B).
The co-culture declined the reactivity of HeLa-cell to induction of cell viability (Fig. 1A), which resulted in an improvement in cell viability. As consistent responses for all the cervical carcinoma cell lines examined, pre-stimulated SiHa and SW756 cells with human IL-17A exhibited a pronounced growth in cell viability following cisplatin administration at a highest applying cisplatin dose of 6.25 µg/ml (Fig. 1B).
3.2 Th2 cells induce tolerance to irradiation and cisplatin in cervical carcinoma cells
The identical results were gained in exposure experiments where we employed a dose of 6Gy for 48h, which caused the maximum of cell mortality sensitization in the earlier assays (Fig. 2A). Among the cells in all three tested, treatment with 6Gy declined cell survival and pre-stimulating cells with rhIL-17 dramatically enhanced their survival following irradiation (Fig. 2B). As CCRT has been the golden standards for the management of cervical carcinoma >FIGO IIB [2], the combination of cisplatin administration and irradiation was performed to mimic chemo-radiotherapy in vitro. The chemo-radiotherapy significantly depressed the cellular viabilities of the three cell lines (Fig. 2C). Likewise, preconditioning with recombinant IL-17 markedly diminished the cellular reactivity to chemoradiotherapy, which resulted in enhanced cell viability.
3.3 Th2 cells induce tolerance by the IL-17 dependently manner
We were interested in whether Th2 cells affected the responsiveness of cervical carcinoma cells to chemotherapeutic drugs or irradiation. We initially have tested cisplatin, which is the candidate most frequently prescribed in cervical carcinoma chemo. HeLa cells were co-cultured with in vitro Th2 cells at a 1:20 ratio for 24 hours, then Th2 cells were eliminated and treated with cisplatin. The results were that co-culture lessened the reactivity of HeLa cells to cisplatin-induced cell deaths, leading to an improved cell survival rate (Fig.3A).
It was known that therapy with cisplatin on single cell lines SiHa, HeLa or SW756 could lead to a reduction in cell viability [citation needed]. It was known that therapy with cisplatin on single cell lines SiHa, HeLa or SW756 could lead to a reduction in cell viability [citation needed]. Subsequently, as uniform responses for all cancer cell lines tested, pre-stimulation of SiHa, HeLa and SW756 cells with rhIL-17A yielded a pronounced increase in cell viability at the highest application of cisplatin at a dose of 6.25µg/ml (Fig. 3B). We have obtained the identical findings in irradiation assays where we employed a dose of 6Gy, which in previous experiments resulted in the highest cell death sensitization (Fig. 2). The treatment with 6Gy reduced the viability of all three tested cell lines (Figure 3C; dotted bars) and pre-stimulation of cells with rhIL-17 (red bars) dramatically enhanced the post-irradiation increase in cell viability.
We integrated cisplatin therapy with irradiation to mimic chemo-radiotherapy in vitro. Chemo-irradiation administration significantly further decreased cell viability in all three-cell lines (Figure 3D, striped bar). As well, preconditioning with recombinant IL-17 (blue bars) apparently diminished cell reactivity to chemoradiotherapy, resulting in a greater cell viability. Furthermore, Pre-stimulation of SiHa and SW756 cells with CM (pink strips) (Figure 3E) decreased the reactivity to chemoradiotherapy-induced cell death and caused an increase in cell viability. Neutralization of Th17 cells with IL-17 in CM completely abolished Th17-induced resistance (Figure 3E, brown bars). Taken together, our results clearly indicate that IL-17 was the responsible factor for Th2 cells in the chemoresistance of cervical cancer cells.
3.4 The expression of AKT in cervical cancerous cells
To demonstrate the activated AKT signal pathway, the expression of pThr308- and pSer473-AKT was examined by stimulating SiHa and Hela cells with CM arising from Th2 cells in vitro (Fig. 4A and B). The stimulation indicated a pronounced elevation of pThr308- and pSer473- expressed AKT in SiHa and Hela cellular lines tested. AKT included two main different isoforms (AKT1 and 2) (Fig.4C).
3.5 The Th2-induced tolerance of the cervical tumor was AKT dependent
In both AKT1 and AKT2 knockdown markedly reduced IL-17 or CM cell dying tolerance in both cell lines. Under normal conditions, the growing of SiHa and Hela cells could be inhibited by treatment of cisplatin and 6Gy (Control vs Cisplatin+6Gy,**P<0.01). A total reversion was achieved following concomitant elimination of AKT1 and AKT2. While chemotherapy slightly decreased cell viability only slightly, preconditioning with rhIL-17 and CM of Th2 cells entirely recovered their viability (Fig. 5). Together, these results provided proof that the Th2-induced resistances to the combined therapy were determined by the AKT pathways.