Ⅰ.Introduction
Salivary gland cancer is relatively uncommon type of cancer, comprising about 3~5% of all head and neck cancer 1). The most of them are mucoepidermoid carcinoma (MEC) which is diagnosed about 30~40% of patients with salivary gland cancer2). Current standard treatment strategy for MEC is surgical resection, but the patients with high risk of recurrence and poor prognosis are considered adjunctive radiotherapy3,4). To date, the efficacy of chemotherapy using doxorubicin, paclitaxel and 5-fluorouracil is hindered by their low activity in MEC5). In addition, limited establishment of cell lines by low frequency of MEC has disturbed to develop anti-cancer drugs with higher therapeutic effect. Thus, exploring effective anti-cancer drug and experimental models is urgent to optimize therapeutic approaches for patients with MEC.
Cisplatin is the first FDA-approved platinum-containing anti-cancer drug against various cancers. It efficiently interferes with DNA repair mechanism and causes DNA damage by crosslinking with the purine bases on DNA, which ultimately triggers cells to die6). Even though cisplatin exerts modest activity in the treatment of advanced salivary gland cancer, the therapeutic effect of cisplatin is the most effective in monotherapy or in combination therapy7). In practice, new therapeutic strategies with cisplatin were constantly developed to enhance their efficiency in head and neck cancer8,9), indicating that cisplatin may be still first anti-cancer drug for chemotherapy. However, attractive biomarker and its underlying mechanism of cisplatin in MEC were not well established.
In this study, we demonstrated that pro-apoptotic effect of cisplatin by regulating Bax activation in human MEC cells, suggesting that cisplatin may serve as a potential anti-cancer drug for human MEC.
Ⅱ.Materials and Methods
Cell culture and drug treatment
Human mucoepidermoid carcinoma (MC3 and YD15) cells were kindly given by Prof. Jun-Zheng Wu (Forth Military Medical University, Xi’an, China) and Prof. Jin Kim (Yonsei University, Seoul, Korea), respectively. MC3 and YD15 cells were cultured in DMEM and RPMI1640 containing 10% FBS and antibiotics. Both cells were incubated at 37°C in 5% CO2 atmosphere. Cisplatin (Sigma, St. Louis, MO, USA) was dissolved in DMSO, aliquoted, and stored at -20°C. All experiments were prepared in cells cultured at 50% confluence, each cells were treated with various concentration of Cisplatin for 48hr.
Trypan blue exclusion assay
MC3 and YD15 cells were seeded on 6well plates, and then both cells were treated with cisplatin for 48hr. The cell viability was determined with trypan blue solution (Gibco, Paisley, UK). Detached cells were stained with trypan blue (0.4%), and then viable cells were counted using a hemocytometer.
Western blot analysis
Protein lysates extracted with lysis buffer were prepared to measure the protein concentration using DC Protein Assay Kit (BIO-RAD Laboratories, Madison, WI, USA). After normalization, equal amounts of protein were separated by SDS-PAGE gel and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST at RT for 2hr, and maintained with primary antibodies and corresponding HRP-conjugated secondary antibodies diluted with 2% skim milk. Antibodies against cleaved PARP, Bak, Bad, Bim, Bcl-xL and Bcl-2 were supplied from Cell Signaling Technology, Inc., (Charlottesville, VA, USA). Bax (6A7) antibody was obtained from BD Pharmingen™ (San Jose, CA, USA) and actin antibody was from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA). The immunoreactive bands were visualized by ImageQuant™ LAS 500 (GE Healthcare Life Sciences, Piscataway, NJ, USA).
4'-6-diamidino-2-phenylindole (DAPI) staining
Nuclear morphological changes of apoptotic cells were assessed by DAPI (Sigma-Aldrich, Louis, MO, USA) staining. Briefly, detached cells were fixed in 100% methanol at RT for 10 min, deposited on slides, and stained with DAPI solution (2 μg/ml). Chromatin condensation or nuclear fragmentation of apoptotic cells were observed under a fluorescence microscopy.
Live/dead assay
The cytotoxicity of Cisplatin was determined using Live/Dead & Viability/Cytotoxicity assay (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. Briefly, cells were stained with 2 μM Calcein-AM and 4 μM Ethidium homodimer-1, and then incubated for 30 min at RT. Cells were analyzed under a fluorescence microscope equipped with the appropriate excitation and emission filters.
Immunocytochemistry
Cells were seeded on 4well plates, and then treated with higher concentration of cisplatin for 48hr. Cells were fixed and permeabilized using a Cytofix/Cytoperm Solution (BD Bioscience, San Diego, CA, USA) for 1hr at 4°C. Cells were blocked with 1% BSA for 1hr at RT, and maintained overnight at 4°C with Bax (6A7) antibody and followed by incubation with FITC-conjugated secondary antibody for 1hr at RT. Cells were observed by fluorescence microscope equipped with appropriate filters for FITC and DAPI fluorescence dye.
Statistical analysis
Student’s t-test was used to determine the significance of differences between the control and treatment groups; p values of < 0.05(*), 0.01(**), and 0.001(***) were considered significant.
Ⅲ.Results
Cisplatin suppresses cell growth and induces apoptosis in human MEC cells.
To identify the anti-proliferative functions of cisplatin in human MEC cells, cisplatin-treated MC3 and YD15 cells were investigated by trypan blue exclusion assay. The treatment of cells to various concentrations of cisplatin for 48hr inhibited cell growth (Figure 1A). We then clarified whether anti-proliferative function of cisplatin in both cells was involved in apoptosis. As shown in Figure 1B, treatment of cisplatin led to marked induction of cleaved PARP in a concentration-dependent manner. These results indicate that cisplatin-induced apoptosis may contribute to growth inhibition of MC3 and YD15 cells.
Qualitative analysis of Cisplatin-induced apo ptosis indicates potential pro-apoptotic activi ty in human MEC cells.
To further confirm the apoptotic effect of cisplatin, we analyzed morphological change of nuclei by DAPI staining. The results showed that treatment of cells to cisplatin enhanced nuclear fragmentation and condensation compared to the DMSO treatment group (Figure 2A). Cisplatin-induced apoptosis was also qualitatively analyzed by live/dead assay which differentially labels live (green) and dead (red) cells with two fluorescent dyes. As presented in Figure 2B, cisplatin significantly decreased green population and increased red population indicating the induction of apoptosis. These results clearly showed that cisplatin is an effective apoptotic inducer in human MEC cells.
Cisplatin-induced apoptosis is associated wit h Bax activation.
In response to apoptotic stimuli, pro-apoptotic Bcl-2 family protein Bax is activated by its conformational change, translocation into mitochondria and oligomerization10). Therefore, we next investigated whether Cisplatin could regulate Bax activation to induce apoptosis. The results showed that treatment of cells to cisplatin markedly increased the expression of active Bax using specific antibody against N-terminus of active Bax (Figure 3A). Immunofluorescence staining also showed that higher concentration of cisplatin increased the expression levels of active Bax compared with the DMSO treatment group (Figure 3B). By contrast, cisplatin had no significant effect on other Bcl-2 family proteins such as Bak, Bad, Bim, Bcl-xL and Bcl-2 (Figure 4). These results suggest that Bax activation may contribute to cisplatin-induced apoptosis.
Ⅳ.Discussion
Developing an effective therapeutic strategy for patients with MEC was severely limited because of low frequency of MEC, which may impede to establish experimental models for MEC. However, several groups have made an effort to establish cell lines from primary tumors in spite of their low success rate11-13). In this study, we provide a baseline data for developing an effective treatment modality against MEC using two MEC cell lines which were established by other group.
Previous studies reported that the existence of cancer stem cells within MEC cell lines and resistance to chemotherapy may be associated with treatment failure of patients with MEC14,15). Hence, new approaches for sensitizing MEC cells to chemotherapy have been attempted 4,16). We also reported that several natural compounds had potential abilites to inhibit cell growth and induced apoptosis in MEC cells17,18). In addition, we demonstrated the underlying molecular mechanism that is associated with STAT3 inhibitor-induced apoptosis in MEC cells and nude mice xenograft models without any systemic toxicity19). Here, we found that cisplatin has anti-cancer properties including growth inhibition and apoptosis induction against MEC cells, indicating that the treatment of cisplatin may be still considered as first anti-cancer drug for patients with MEC, even though it has moderate activity.
Bcl-2 family proteins have critical roles in mitochondriamediated apoptosis, which regulate the balance of pro-survival and pro-apoptosis proteins20). In particular, pro-apoptotic Bax acts as final executioner upon the mitochondria-mediated apoptotic signal. Bax is conformational changed, translocated into mitochondria and oligomerized when triggered by apoptotic stimuli, thereby inducing release of apoptogenic factor from mitochondria like cytochrome c10). Thus, we investigated the potential effect of cisplatin on Bax expression in MEC cells, and found that cisplatin significantly increased Bax activation using specific antibody that can detect their N-terminal domain. This specific antibody is limited by the N-terminal domain in the inactive Bax10), implying that cisplatin is sufficient to induce apoptosis by activation of Bax in MEC cells.
In conclusion, we demonstrated that Cisplatin suppresses cell growth of MEC and induces apoptosis by regulating Bax activation. These findings provide a basic molecular mechanism of cisplatin for treatment of human MEC.
