| Objective: Taxol is a kind of natural medicine with antitumor activity, and has been used for the treatment of refractory ovarian cancer, metastatic breast cancer and uterine tube cancer. More recently, the feasibility study for squamous cell carcinomas of the head and neck (SCCHN) has made great progress. Several clinical trials have demonstrated that Taxol might be a promising agent for advanced SCCHN. Unfortunately, there is still existing multiple drug resistance (MDR) to Taxol. In an attempt to understand the mechanism thoroughly, we conducted the study in which the human laryngeal carcinoma cell strain Hep-2 cell was induced with Taxol to establish a MDR strain, and we characterized the changes between the normal and resistance strain to provide the theoretical support for the reverse of MDR.Methods: Hep-2 cells were exposed in stepwise escalating concentration of Taxol until the resistant cell line was developed. The IC50 and the resistance folds of multiple drug resistance were determined by an ATP assay. The differences of cell cycle distribution, apoptosis, and Rhodamine accumulation between Hep-2 and Hep-2T cells were studied through flow cytometry. Hoechst stain was also used to determine the apoptosis of Hep-2 and Hep-2T cells. The MDR1 and MRP1 genes were detected through real-time quantitative RT-PCR, and the corresponding proteins were detected through western-blotting.Results: A multiple drug resistance cell line-Hep-2T induced by Taxol was effectively developed, whose drug resistance was 104 times that of Hep-2 cells. The drug resistance of Doxorubicin, Gemcitabine, 5-FU, and Cisplatin all increased by 46.78, 1.95, 2.50, 1.05 folds. The percentage of Hep-2T cells in G0/G1 phase was more than that of Hep-2 cells (61.55±3.92% vs.45.83±14.13%, P<0.05), and the percentage in G2/M and S phase were both significantly lower than that of Hep-2 cells (both, P<0.05). The apoptosis of Hep-2 cells was quite greater than that of Hep-2T cells (45.32±6.47% vs. 4.26±1.72%, P<0.01, flow cytometry; 54.47+8.95% vs. 9.84+2.53%, P<0.01, Hoechst staining) after they were exposed to Taxol at IC50 to Hep-2 cells. The copy ratio of MDR1/GAPDH mRNA of Hep-2T cells was 64.2+36.7 times that of Hep-2 cells (P<0.05), while MRP1/GAPDH of Hep-2T cells was only 1.2+0.09 folds more than that of Hep-2 cells (P<0.05). The protein of MDR1/P-gp was greatly over expressed in Hep-2T cells compared with that in Hep-2 cells (P<0.01), so was the same trend for MRP1 (P<0.05), while the elevated expression of MRP1 was lower than that of MDR1/P-gp.Conclusions: When considering the possible methods to reverse MDR of SCCHN, more emphasis should be laid on MDR1/P-gp, and when combined chemotherapy was applied, the non-P-gp substrate chemotherapeutic agents should be considered. At the same time, more attention should be paid to the changes of cell cycle distribution during the drug selection.PART 2Objective: Tumor invasion/metastasis and multidrug resistance (MDR) are the main causes of treatment failure and high mortality in all kinds of cancer patients. Extensive studies have been performed about MDR and invasion, but most have proceeded along separate pathways. Only few documents reported the functional linkage between the two phenotypes, but the detailed mechanism is still unclear. In overall the chemotherapy for advanced SCCHN has been insensitive. One of the main reasons lies at the MDR including the inherently resistant to the drugs and/or the acquisition of resistance during treatment. Moreover SCCHNs are characterized by a marked propensity for local invasion and dissemination to cervical lymph nodes. So investigation of the relationship between MDR and invasion in SCCHN would be more significant. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. VEGF and vascular endothelial growth factor receptor-2 (VEGFR-2) may have an effect on MDR, while little has been reported about VEGF/VEGFR-2 and MDR. In this study, we tried to confirm the phenomena between the MDR and invasion then reveal the detailed mechanism.Methods: Invasion assays were performed using Cell Invasion Assay Kit to detect invasive ability of Hep-2 cell, Hep-2T cell, Hep-2 cell+ VEGF, Hep-2T cell+ VEGF, SU1498+ Hep2-T, MDR1 RNAi + Hep-2T cell, SU1498+ MDR1 RNAi + Hep-2T cell respectively. Realtime Quantitative RT-PCR was applied to detect the expression of VEGFR-2 in Hep-2 and Hep-2T cells, and MDR1 in Hep-2T cells after RNAi.Western-blotting was applied to detect the expression of VEGFR-2 in Hep-2 and Hep-2T cells, and MDR1/P-pg in Hep-2T cells after RNAi.Results: In Hep-2T cells, an increased in vitro invasive ability was observed as compared with the sensitive line Hep-2 cells (P<0.01), and VEGF could increase the vitro invasion both in drug resistant cells and their parental counterparts. 2×2 factorial design ANOVA analysis showed that VEGF and MDR had synergistic enhancing effects on cell invasion (P<0.05). The expression of VEGFR-2 was 1.73±0.24 folds higher in the Hep-2T cells than that in the parental, sensitive cells, as determined by Realtime Quantitative RT-PCR (P<0.05). The expression of VEGFR-2 was 1.17 folds higher in the Hep-2T cells than that in the parental, sensitive cells, as determined by Western-blotting (P<0.05). The invasive ability of Hep-2T cells decreased significantly after VEGFR-2 was blocked by its inhibitor SU1498 (P<0.01). The expression of MDR1 mRNA decreased by 73.5% in Hep-2T cells after RNAi for MDR1(P<0.05). The content of MDR1/P-gp protein also decreased significantly after RNAi (P<0.01). The invasive ability of Hep-2T cells with SU1498 and RNAi was lower than that of Hep-2T cells with either SU1498 or RNAi, so was for Hep-2T cells. 2×2 factorial design ANOVA inferred that there was synergistic decreasing effect of SU1498 and MDR1-knock down on cell invasion (P<0.05).Conclusions: Our results showed that the invasive ability of larynx cancer MDR cells induced by Taxol was higher than that of the parental, sensitive cells. Furthermore, the MDR1/P-gp-mediated and theVEGFR-2-mediated signaling transduction pathway might be involved in the elevated larynx cancer invasion. VEGF could up-regulate the invasive ability through VEGFR-2. Synergistic effect exited between MDR1/P-gp and VEGFR-2 on invasive ability.
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