J Huazhong Univ Sci Technol［Med Sci］ 31(1):2011 33 Antitumor Activity of Erythromycin on Human Neuroblastoma Cell Line (SH-SY5Y) Yongsheng JIA (贾勇圣), Xiaoyun MA (马晓芸), Xiaoli WEI (魏晓莉), Xin LI (李 昕), Haitao YAN (闫海涛), Xiaoyan LIU (刘晓燕), Jianquan ZHENG (郑建全) # Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2011 Summary: Antitumor effects of erythromycin and the related mechanism were investigated in the pre- sent study. Neuroblastoma cells (SH-SY5Y) were exposed to erythromycin at different concentrations for different durations. Cell proliferation was measured by cell counting, and cell viability was exam- ined by MTT assay. Cell cycle phase distribution and the cytosolic calcium level were detected by flow cytometry. Mitochondrial membrane potential was measured by the JC-1 probe staining and fluorescent microscopy. The expression of an oncogene (c-Myc) and a tumor suppressor [p21 (WAF1/Cip1)] pro- teins was analyzed by using Western blotting. Erythromycin could inhibit the proliferation of SH-SY5Y cells in a concentration- and time-dependent manner. The cell cycle was arrested at S phase. Mitochon- drial membrane potential collapsed and the cytosolic calcium was overloaded in SH-SY5Y cells when treated with erythromycin. The expression of c-Myc protein was down-regulated, while that of p21 (WAF1/Cip1) protein was up-regulated. It was concluded that erythromycin could restrain the proliferation of SH-SY5Y cells. The antitumor mechanism of erythromycin might involve regulating the expression of c-Myc and p21 (WAF1/Cip1) proteins. Key words: erythromycin; c-Myc; p21 (WAF1/Cip1); neuroblastoma Erythromycin is a clinically important broad-spec- trum antibiotic that belongs to the macrolide class. Many esters of erythromycin are well established as agents to treat a variety of respiratory and cutaneous infections, particularly in children [1] . Recently, increasing evidence has revealed that erythromycin exerts a variety of effects that are not related to its antibiotic activity, including an effective aid in the management of aleukemic myeloblas- tic leukemias [2] . Treatment with erythromycin signifi- cantly reversed the resistance of MDR WEHI 164 murine fibrosarcoma cells to the chemotherapeutic drugs by saturating the drug-binding sites on the P-glycoprotein [3] . When given by oral administration, it prolonged the sur- vival time of tumor-bearing mice in both allogeneic and syngeneic mouse systems by two- to three-fold as com- pared with those of vehicle control mice [4] . In some can- cers with HERG (ether-à-go-go related gene) protein preferentially expressed, the antitumor effect of eryth- romycin was considered to be related to inhibiting the HERG channels [5] . However, the molecular and cellular mechanism which underlines the cell proliferation in- hibitory effect of erythromycin remains obscure. Neuroblastoma is the most common extracranial solid cancer in childhood and the most common cancer in infancy [6] . In the present experiment, human neuro- blastoma cells (SH-SY5Y) were used to observe the an- titumor effects of erythromycin. The mechanism was Yongsheng JIA, E-mail: jiays@yahoo.com # Corresponding author, E-mail: zhengjq@bmi.ac.cn explored by observing the effects of erythromycin on cell proliferation, viability, cell cycle phase distribution, and mitochondrial membrane potential. Cancer arises from a stepwise accumulation of genetic changes that liberates neoplastic cells from the homeostatic mechanisms that govern normal cell proliferation [7] . Oncogene and tu- mor-suppressor gene mutations all operate similarly at the physiologic level: they drive the neoplastic process by increasing tumor cell number through the stimulation of cell birth or the inhibition of cell death or cell-cycle arrest [8] . C-Myc gene encodes for a transcription factor that is believed to regulate expression of 15% of all genes [9] through binding on Enhancer Box sequences (E-boxes) and recruiting histone acetyltransferases (HATs). This means that in addition to its role as a clas- sical transcription factor, c-Myc also regulates apoptosis in part through interactions with the p53/Mdm2/Arf sig- naling pathway. Mutation in p53 is commonly observed in patients with relapsed neuroblastoma, contributing to both biology and therapeutic resistance [10] . The cy- clin-dependent kinase inhibitor p21 (WAF1/CIP1) is the major transcriptional target of the tumor suppressor gene, p53; despite this, loss-of-function mutations in p21 do not accumulate in cancer nor do they predispose to can- cer incidence. It was earlier reported that p21 (WAF1/CIP1) expression was required for survival of those differentiating neuroblastoma cells [11] . Recent studies showed that endogenous p21 (WAF1/CIP1) pro- tein in neuroblastoma cells was inactive and may be bound either to a protein complex or in a conformation that precluded its binding to cdk2. The dysfunction of 31(1):33-38,2011 J Huazhong Univ Sci Technol［Med Sci］ DOI 10.1007/s11596-011-0146-4