Oral squamous cell carcinoma (OSCC) metastasis is characterized by distant metastasis and local recurrence. Combined chemotherapy with cisplatin and 5-fluorouracil is routinely used to treat patients with OSCC, and the combined use of gefitinib with cytotoxic drugs has been reported to enhance the sensitivity of cancer cells in vitro . However, the development of drug resistance because of prolonged chemotherapy is inevitable, leading to a poor prognosis. Therefore, understanding alterations in signaling pathways and gene expression is crucial for overcoming the development of drug resistance. However, the altered characterization of Ca2+ signaling in drug-resistant OSCC cells remains unclear. In this study, we investigated alterations in intracellular Ca2+ ([Ca2+]i) mobilization upon the development of gefitinib resistance in human tongue squamous carcinoma cell line (HSC)-3 and HSC-4 using ratiometric analysis. This study demonstrated the presence of altered epidermal growth factor- and purinergic agonist-mediated [Ca2+]i mobilization in gefitinib-resistant OSCC cells. Moreover, Ca2+ content in the endoplasmic reticulum, store-operated calcium entry, and lysosomal Ca2+ release through the transient receptor potential mucolipin 1, were confirmed to be significantly reduced upon the development of apoptosis resistance. Consistent with [Ca2+]i mobilization, we identified modified expression levels of Ca2+ signaling-related genes in gefitinib-resistant cells. Taken together, we propose that the regulation of [Ca2+]i mobilization and related gene expression can be a new strategy to overcome drug resistance in patients with cancer.

Introduction
Materials and Methods
    1. Cell culture and reagents
    2. Establishment of gefitinib-resistant cells
    3. Western blot assay
    4. Cell proliferation assay
    5. Measurement of [Ca2+]i
Results
    1. Establishment of gefitinib-resistant sub-cell line ofHSC-3 and HSC-4
    2. EGF- and ATP-mediated [Ca2+]i mobilization isdecreased in gefitinib-resistant cells
    3. Endoplasmic reticulum Ca2+ content and storeoperatedCa2+ entry is reduced in gefitinibresistantcells
    4. Lysosomal Ca2+ release through TRPML1 isdecreased in gefitinib-resistant cells
Discussion
References

• Mi Seong Kim(Department of Oral Physiology, Institute of Biomaterial-Implant, School of Dentistry, Wonkwang University/Wonkwang Dental Research Institute, School of Dentistry, Wonkwang University)
• Min Seuk Kim(Department of Oral Physiology, Institute of Biomaterial-Implant, School of Dentistry, Wonkwang University) Correspondence to