Pilot Study Of The Erythromycin's Neuroblastoma Proliferation Inhibiting Effect And Its Mechanism

Erythromycin (EM) is produced from Streptomyces erythraeus. EM is a macrolide antibiotic which has an antimicrobial spectrum similar to or slightly wider than that of penicillin, and is often used for people that have an allergy to penicillins. It has been widely used as effective antibiotics against Gram-positive organisms.As reported previously, EM can inhibit the ether-à-go-go (eag) related gene-encoded potassium current in a concentration dependent manner, and can inhibit the proliferation of of tumor cells in which HERG protein preferentially expressed. There is accumulating evidence that herg gene and HERG protein are overexpressed in many types of human cancers. Overexpression of HERG contributes to the progression, metastasis, apopotosis, angiopoiesis, cell differentiation and chemotherapy sensitivity of a variety of tumors. Our previous study demonstrated that inhibition of herg expression effectively depressed human neuroblastoma growth both in vitro and in vivo. However, much research has been done, little is konown about the molecular and cellular mechanism of EM's cell proliferation inhibiting effect. Therefore, the aim of our study is to clarify the possible mechanism of EM's cell proliferation inhibiting effect at molecular and cellular level. SH-SY5Y cells were used in which HERG protein highly expressed to investigate EM's neuroblastoma inhibiting effect and its mechanism. Our study mainly focuses on two aspects below:1. The influence of EM on cell proliferation, apoptosis, autophagy and cell cycle regulation;2. The influence of EM on the expression of oncoprotein and tumor suppressor.MethodsHuman neuroblastoma cells of the line SH-SY5Y, Cell growth curve assay and MTT were used to evaluate the effect of erythromycin on SH-SY5Y cell proliferation and survival. Annexin V-FITC/PI, PI staining and flow cytometry were used to evaluate the effect of erythromycin on SH-SY5Y cells apoptosis and cell cycle regulation; SH-SY5Y cell autophagy were observed under fluorescence microscope and flow cytometry with cellular LC3 immunofluorescence staining technique; Cytosolic calcium level of cells after long-term erythromycin exposure was measured using a calcium probe fluo-3AM by flow cytometry. siRNA was used to knock down HERG protein expression; Western blotting was used to detect the protein expression of c-Myc, p21(WAF1/Cip1), LC3-Ⅱ, HERG; RT-PCR was used to detect the mRNA expression of herg and c-myc.Results1. The treatment of SH-SY5Y cells with EM result in a concentration-dependent and time-dependent inhibition in cell proliferation. The inhibition rate of cell proliferation was 13.50%, 24.75%, 37.25% and 90.75% respectively which were treated with EM for 72 h in a concentration of 62.5, 125, 250 and 500μM. The inhibition rate of cell proliferation was 4%, 26%, 37%, 53% respectively which were treated with 250μM EM for 24 h, 48 h, 72 h and 96 h.2. Viability of SH-SY5Y cells was inhibited significantly upon treatment with EM in a concentration-dependent and time-dependent manner. The survival rate was 77.28±0.01%, 59.71±0.02% and 44.27±0.01% respectively which treated with 500μM EM for 24, 48 and 72 h, and IC50 was 205.01μM for 24 h, 187.29μM for 48 h, 187.97μM for 72 h.3. Exposure of SH-SY5Y cells to EM for increasing time intervals produced a time dependent S phase cell cycle arrest. For example, after incubation with 500μM EM for 24 h, 48 h and 72 h, the percentage of cells in the S phase was 3.5±1.56%, 6.12±0.26 % and 14.95±1.63 % , respectively, showing significant difference compared with the control group 3.66±0.49, 5.66%±0.11% and 7.05±1.02%.4. Some SH-SY5Y cells became round, detacted, followed by extensive membrane budding after exposure to EM for 48 h or 72 h. Treament of SH-SY5Y cells with EM for 72 h remarkably increased the percentage of cells with sub-diploid DNA content. After treatment with 500μM EM for 24 h, 48 h and 72 h, the percentage of cells with sub-diploid DNA content increased from 0.02±0.01% to 0.04±0.05%, 0.81±0.03%, 21.57±1.22%, respectively.5. Flow cytometric quantification of apoptotic SH-SY5Y cells stained with annexin V-FITC/PI showed that 500μM EM treatment increase the percentage of apoptotic cells to 2.81±0.43%, 5.3±0.04%, 10.10±0.40%, respectively at 24 h, 48 h and 72 h.6. After treatment with EM for 72 h, the cytosolic calcium level was 2.04-folds higher in SH-SY5Y cells compared with control.7. Western blotting analysis of LC3 protein after incubation with 500μM EM for 72 h. Densitometry detects 6-folds higher LC3-II/LC3-I ratios in 500μM EM treated cells, compared with control, which indicate SH-SY5Y cells autophagy was induced by EM.8. When SH-SY5Y cells were treated with 62.5-500μM EM for 72 h, a concentration-dependent decrease of c-Myc expression was observed. 500μM EM treatment after 72 h caused a time-dependent decrease of c-Myc expression.9. There is no expression of p21 (WAF1/Cip1) in normal SH-SY5Y cells and control. When SH-SY5Y cells were treated with 62.5-500μM EM for 48 h, a concentration-dependent increase of p21 (WAF1/Cip1) expression was detected.10. C-Myc mRNA and protein expression decreased after HERG protrein was knocked down in SH-SY5Y cells.ConclusionEM can inhibit SH-SY5Y cells proliferation in a concentration-dependent and time-dependent manner; furthermore, EM may also induce cell cycle arrest at S phase in SH-SY5Y cells, which may be attributable to the down-regulation of c-Myc. Our data also provide evidence for the induction of apoptotic cell death of EM, which may be attributable to the up-regulation of p21 (WAF1/Cip1), cytosolic calcium overloading. HERG potassium channel inhibition by EM may be underline the c-Myc protein down-regulation. SH-SY5Y cells autophagy was also induced by EM. The results suggest that EM, which can be administered p.o., may ultimately prove useful as a potential therapeutic agent against human neuroblastoma.