Notch1 signaling modulates hypoxia-induced multidrug resistance in human laryngeal cancer cells

Laryngeal carcinoma is one of the common malignant tumors of the head and neck. Multidrug resistance (MDR) remains a critical problem in the chemotherapy of patients with laryngeal cancer. This study aims to clarify the role and mechanisms of Notch1 signaling in MDR induced by hypoxia in laryngeal cancer cells. Laryngeal carcinoma cells were cultured under normoxia or hypoxia. Notch1 expression was inhibited by small interfering RNA (siRNA). The mRNA expression of Notch1, Hes1, Hey1, MDR1 and survivin was analyzed by real-time PCR. The protein expression of Notch1, the Notch1 intracellular domain (N1ICD), MDR1/P-gp and survivin was analyzed by Western blotting. Current research has shown that hypoxia can upregulate Notch1 expression and Notch1 signaling activity. Furthermore, suppression of Notch1 expression effectively downregulated Notch1 signaling activity and the expression of the MDR and survivin genes in laryngeal cancer cells under hypoxic conditions (P < 0.05). The Cell Counting Kit-8 (CCK-8) assay results confirmed that the sensitivity of hypoxic laryngeal cancer cells to a variety of drugs could be upregulated by suppressing Notch1 expression (P < 0.05). Additionally, flow cytometry (FCM) showed that suppression of Notch1 expression significantly increased drug-induced apoptosis and intracellular rhodamine 123 (Rh123) accumulation in hypoxic laryngeal carcinoma cells (P < 0.05). Notch1 signalling could be regarded as a pivotal regulator of hypoxia-induced MDR in laryngeal cancer cells through the regulation of survivin-mediated apoptosis resistance and MDR1/P-gp-mediated drug transport.


Introduction
Laryngeal carcinoma is one of the common malignant tumors of the head and neck. Concurrent chemoradiation has been considered as the primary treatment for locally advanced laryngeal cancer. However, multidrug resistance (MDR) remains a critical problem in the chemotherapy of patients with laryngeal cancer. Unfortunately, the regulatory mechanisms related to MDR in laryngeal carcinoma remain unclear.
Cell cytotoxicity assay. A CCK-8 assay was to assess the sensitivity of neoplastic cells to adriamycin, paclitaxel, cisplatin, 5-FU and gemcitabine. Cells were seeded in 96-well culture plates (5 × 10 3 cells/well). After 12 h, the cells were treated with a certain dose of chemotherapeutic drugs and cultured for another 48 h under hypoxia or normoxia.
As mentioned in a previous study [4], the drug concentration that led to a 50% reduction in the cell number (IC 50 ), was calculated.
FCM assay was used to analyze the accumulation of Rh123 in Hep-2 and AMC-HN-8 cells as described previously [18]. A FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the we previously found that Notch1 expression in laryngeal cancer tissues was evidently higher than that in laryngeal normal tissues and was related to lymph node metastasis and clinical stage [11], suggesting that Notch1 signaling might play a pivotal role in regulation the malignant progression of laryngeal cancer. Recently, a number of studies have confirmed that Notch1 signaling is involved in regulating MDR of various neoplasic cells [12][13][14]. Furthermore, several studies have indicated that Notch1 expression has a positive correlation with cisplatin [15,16] and paclitaxel [16] resistance in head and neck squamous cell carcinoma. The above findings suggest that Notch1 signaling may be involved in regulating MDR in laryngeal cancer cells in the hypoxic microenvironment. Up to now, there have been no relevant literature reports.
In the current study, we were to investigate the regulatory role of Notch1 signaling in hypoxia-induced MDR in laryngeal cancer cells and clarify its possible molecular mechanisms.

Materials and methods
Cell lines and cell culture.
Laryngeal carcinoma cell lines Hep-2 and AMC-HN-8 were gained from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. Neoplastic cells were cultured in DMEM (Gibco Corporation, USA) supplemented with 1% penicillin/streptomycin (Invitrogen) and 10% fetal bovine serum (Hyclone, USA). For normoxic conditions, cells were placed in an incubator at 37 °C in an atmosphere of 21% O 2 , 74% N 2 and 5% CO 2 . For hypoxic conditions, cells were placed in a hypoxic incubator (NuaireTM US autoflow CO 2 water jacketed incubator) at 37 °C containing 1% O 2 , 94% N 2 and 5% CO 2 .
Real-time PCR analysis.
Suppression of Notch1 expression inhibited multidrug resistance in laryngeal carcinoma cells under hypoxia.
The drug sensitivity of the Notch1-siRNA group with that of the control groups was compared by a CCK-8 assay in our study. The results showed that the sensitivity of hypoxic Hep-2 and AMC-HN-8 cells to a variety of drugs was obviously enhanced by inhibition of Notch1 expression (P < 0.05) (Supplementary Tables 1 and 2).
Suppression of Notch1 expression inhibited the mRNA expression of MDR1 and survivin in hypoxic laryngeal cancer cells.
Real-time PCR analysis showed that the MDR1 and survivin mRNA expression levels in the Notch1-siRNA group were obviously lower than those in the control groups (P < 0.05) (Fig. 3 A-C). In addition, Western blot analysis showed that the MDR1/P-gp and survivin protein expression levels in the Notch1-siRNA group were lower than those in the control groups (P < 0.05) ( Fig. 3D and E). The above data indicated that MDR1 and survivin expression in hypoxic laryngeal cancer cells was downregulated by inhibition of Notch1 expression.
Suppression of Notch1 expression increased drug accumulation in hypoxic laryngeal cancer cells.
The FCM results showed that the percentage of Rh123positive Hep-2 cells in the Notch1-siRNA group was evidently higher than that in the control groups (89.48 ± 1.97% vs. 70.39 ± 1.66% and 70.63 ± 0.71%; P < 0.05) (Fig. 4 A). In addition, the percentage of Rh123-positive AMC-HN-8 cells in the Notch1-siRNA group was higher than that in the control groups (92.35 ± 2.13% vs. 73.12 ± 3.10% and 72.84 ± 2.24%; P < 0.05) (Fig. 4B). The above data revealed that suppression of Notch1 expression could enhance drug accumulation in hypoxic laryngeal cancer cells.
Suppression of Notch1 expression enhanced druginduced apoptosis in hypoxic laryngeal cancer cells.
Annexin-V/PI staining assay showed that the apoptosis rate of Hep-2 or AMC-HN-8 cells induced by cisplatin in the Notch1-siRNA group was obviously higher than that in the control groups (P < 0.05) (Fig. 4 C, 4D). Likewise, cell suspension with an excitation wavelength of 488 nm. Then, Cell-Quest™ software (BD Biosciences) was used to analyze the experimental data.
Cell apoptosis analysis. Hep-2 (3 × 10 5 cells/well) and AMC-HN-8 (4 × 10 5 cells/ well) cells were plated in six-well plates and cultured overnight at 37˚C. Then, the cells were cultured under hypoxic or normoxic conditions for 12 h after the culture medium was refreshed. Paclitaxel or cisplatin was added to each well to a concentration of 5.0 × 10 − 9 M or 2.5 × 10 − 9 M, respectively. After that, the cells were cultured for another 48 h. As in our previous research, the apoptosis index (AI) of cells was assessed by FCM and Annexin-V-FITC/propidium iodide (PI) staining method [4]. Finally, the apoptosis rate was measured as the average fluorescence intensity.

Statistical analysis
Quantitative variables were compared by Student's t test with SPSS 20.0. P values of less than 0.05 were regarded as statistically significant.

Results
Hypoxia upregulated Notch1 expression and Notch1 signaling activity in laryngeal carcinoma cells.
Laryngeal cancer cells were cultured under normoxic or hypoxic conditions for 12, 24, 48 h. Real-time PCR analysis showed that hypoxia obviously induced the mRNA expression of Notch1, Hes1, and Hey1 in neoplastic cells (P < 0.05) ( Fig. 1 A-C). Hes1 and Hey1 are downstream target genes of Notch signaling and are usually used to reflect Notch signaling activity. Similarly, Western blot assay showed that Notch1 and N1ICD expression in laryngeal cancer cells was upregulated with exposure to hypoxia (P < 0.05) ( Fig. 1D and E). N1ICD can be considered the active component of the Notch1 protein. Thus, the above data indicated that hypoxia could upregulate Notch1 expression and Notch1 signaling activity.
Suppression of Notch1 expression downregulated Notch1 signaling activity in hypoxic laryngeal carcinoma cells.
Real-time PCR analysis showed that the mRNA expression levels of Notch1, Hes1 and Hey1 in the Notch1-siRNA group were evidently lower than those in the control groups (P < 0.05) (Fig. 1 A-C). In addition, Western blot analysis revealed that the Notch1 and N1ICD protein expression levels in the Notch1-siRNA group were lower than those in the control groups (P < 0.05) (Fig. 2D and E). The above data demonstrated that suppression of Notch1 expression can downregulate Notch1 signaling activity in hypoxic laryngeal cancer cells. Up to date, a number of studies in other cancers have shown that Notch1 signaling is involved in regulating MDR in various neoplastic cells [12][13][14]. Furthermore, the studies of Zuping Zhang et al. [16] and Feng Gu et al. [15] demonstrated that Notch1 expression was positively correlated with chemoresistance of head and neck carcinoma. Then, the present work showed that the sensitivity of hypoxic laryngeal cancer cells to a variety of chemotherapeutic drugs was obviously enhanced by suppressing Notch1 signaling activity. That is, Notch1 signaling might play a significant role in mediating hypoxia-induced MDR in laryngeal cancer cells.
MDR1/P-gp, a crucial drug transporter, affects the regulation of intracellular drug concentrations. MDR1/P-gp has been confirmed as an important regulator of MDR in laryngeal cancer cells [19,20]. Furthermore, our previous work has suggested that MDR1/P-gp could serve a significant role in regulating hypoxia-induced MDR in laryngeal carcinoma cells through cellular drug effluxing mechanism [21]. Recently, Jiayuan Huang et al. [22] indicated that Notch-1 signaling may play a role in regulating chemoresistance in lung adenocarcinoma by mediating MDR1 expression. Likewise, our present work has elucidated that suppression of Notch1 expression could downregulate MDR1 expression in hypoxic laryngeal carcinoma cells and reduce the drug efflux ability of neoplastic cells. Consequently, these findings suggest that Notch1 signaling might participate in the regulation of MDR1/P-gp-mediated drug transport in hypoxic laryngeal cancer cells.
Survivin belongs to the inhibitor of apoptosis family and participates in the regulation of apoptosis in laryngeal cancer cells [23,24]. Besides, a study of Himani Sharma et al. [25] has indicated that survivin participates in the regulation of drug sensitivity in head and neck squamous cell carcinoma cells, including Hep-2 cells. Recently, our research has already confirmed that survivin might play a regulatory role in hypoxia-induced MDR in laryngeal carcinoma cells by regulating apoptosis resistance [26]. Moreover, several studies have identified that Notch-1 signaling might regulate survivin expression in basal breast cancer cells [27] and lung cancer cells [28]. In this series of studies, our work confirmed that suppression of Notch1 expression can downregulate survivin expression in hypoxic laryngeal carcinoma cells and enhance drug-induced apoptosis in neoplastic cells. Accordingly, these findings indicate that Notch1 signaling might be involved in the regulation of survivin-mediated apoptosis resistance in hypoxic laryngeal cancer cells.
In summary, the current research indicates that Notch1 signaling may play an important role in regulating hypoxia-induced MDR in laryngeal cancer cells by regulating survivin-mediated apoptosis resistance and the apoptosis rate of Hep-2 or AMC-HN-8 cells induced by paclitaxel in the Notch1-siRNA group was also higher than that in the control groups (P < 0.05) (Fig. 4E F). The above data demonstrated that suppression of Notch1 expression enhanced chemotherapeutic drug-induced apoptosis in hypoxic laryngeal cancer cells.

Discussion
Notch signaling is a crucial signal transduction pathway for the regulation of biological behaviors of neoplasic cells under hypoxia [5]. Previously, the findings of Meng-Yuan Dai et al. [10] and our work [11] have demonstrated that high expression of Notch1 in laryngeal cancer tissues was associated with lymph node metastasis. Furthermore, current research exhibited that hypoxia could enhance Notch1 expression and the activity of Notch1 signaling in laryngeal cancer cells. The above results suggested that in the hypoxic microenvironment of laryngeal cancer tissue, Notch1   Hes1 (B) and Hey1 (C) in Hep-2 and AMC-HN-8 cells under hypoxia. Western blot analysis estimated the expression of Notch1 and N1ICD protein in Hep-2 (D) and AMC-HN-8 (E) cells under hypoxia. *P < 0.05, versus the control groups analysis and interpretation, manuscript drafting and revising, final approval, and accountability for all aspects of the work; Dan Xu, study execution, data acquisition, analysis and interpretation, manuscript drafting and revising, final approval, and accountability for all aspects of the work; Penghui Chen, study execution, data acquisition, analysis and interpretation; Jin Xie, study design, data analysis and interpretation, manuscript revising, final approval, and accountability for all aspects of the work.
Funding This work was supported by grants from the National Natural