RU 09. Upregulating CCL2 protects bladder cancer cells from apoptosis upon challenging with DS - P21

Introduction and Objective: Chemokine C-C motif ligand 2 (CCL2) has long been thought to be chemotactic factor that attracts monocytes and basophils. Here we show that CCL2 expressed in bladder cancer cells has anti-apoptosis property, and CCL2 expression is upregulated upon challenging with ds- P21, an synthesized double strand RNA corresponding to the -322 upstream of P21 gene promoter.

Methods: Epithelium and cancer cell lines SV-UVC-1, T24, UM-UC-3, 5637 and LNCaP (ATCC) were cultured in regular RPMI containing 10% FBS. The mRNA and protein expression of CCL2 in cell line were investigated by real-time PCR and western- blot. Time course of CCL2 upregulation upon challenging with ds-P21 were investigated by cDNA microarray. The apoptosis was assayed by Annexin V flowcytometry. Viability and proliferation assay were performed by WST-1 method. AKT pathway, ERK pathway and apoptosis pathway related proteins were assayed by western-blot. The mechanism for CCL2 upregulation was investigated by NF-kB inhibitor assay.

Results: The mRNA and protein expression of CCL2 were upregulated in T24, UM-UC-3, 5637 compared with SVUVC- 1. Challenging T24 cells with ds-P21 induce apoptosis, with CCL2 expression steadily increased. Knock down CCL2 expression with siCCL2 in T24 cells also induce apoptosis and growth inhibition, with suppressed AKT and ERK pathways, decreased expression of BCL-xL and increased cleavage of PARP. Apoptosis and upregulation of CCL2 upon ds-P21 challenge were inhibited by pretreatment T24 cells with NF-kB inhibitor.

Conclusions: CCL2 is an anti-apoptosis factor in T24 cells. The anti-apoptosis property was attributed to the suppressed AKT and ERK pathways, decreased expression of BCL-xL and increased cleavage of PARP. The mechanism of CCL2 upregulation is NF-kB related.