Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer

Abstract

Gastric cancer stem-like cells (GCSCs) have been identified to possess the ability of self-renewal and tumor initiation. However, the mechanisms involved remain largely unknown. Here, we isolated and characterized the GCSCs by side population (SP) sorting procedure and cultured sphere cells (SC) from human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, HGC-27 and MKN-28. The sorting and culture assay revealed that SP cells proliferated in an asymmetric division manner. In addition, SP cells exhibited a higher potential of spheroid colony formation and greater drug resistance than non-SP cells (NSP). Moreover, the SC were found with enhanced capabilities of drug resistance in vitro and tumorigenicity in vivo. Sox2 mRNA and protein was highly and significantly overexpressed in the SP cells and SC. Importantly, downregulation of Sox2 with siRNA obviously reduced spheroid colony formation and doxorubicin efflux, as well as increased apoptosis rate in sphere cells in vitro and suppressed tumorigenicity in vivo. These results suggest that both SP cells and cultured SC enrich with GCSCs and that Sox2 plays a pivotal role in sustaining stem cell properties and might be a potential target for gastric cancer therapy.

Keywords: CD44; Sox2; chemoresistance; gastric cancer stem-like cells; side population.

Fig. 1. The side population (SP) analysis and CD44 expression of human gastric cancer cell lines.

A: Representative SP analysis of five gastric cancer cell lines, the flow cytometry gate for SP cells was defined by the diminished area in the presence of 100 µmol/L verapamil. V: verapamil. B: Expression of CD44 in five human gastric cancer cell lines. SGC-7901, BGC-823 and MGC-803 cells expressed significantly higher CD44 levels than HGC-27 and MKN-28. Data was expressed as mean±SEM, **P < 0.01.

Fig. 2. Asymmetric cell division-like proliferation of the SP cells.

SP and NSP cells of SGC-7901 and BGC-823 cells were sorted and cultured independently in RPMI 1640 for 7 d and 14 d; and then evaluated for the appearance of SP cells. The ratios of SP fraction in cultured SGC-7901 and BGC-823 SP cells decreased from 30.6%, 33.5% to 7.56%, 4.56%, respectively, suggesting that SP cells regenerated both NSP and SP cells. However, NSP cells only regenerated NSP cells. SP: side population; NSP: non-side population.

Fig. 3. The SP cells and SC exhibit more stem cell like characteristics than NSP cells and AC.

A: Potential of spheroid colony formation in SGC-7901 and BGC-823 cells was measured and SP cells generated more colonies than NSP cells. B: The tumorigenicity of SC and AC in SGC-7901 and BGC-823 cells was determined by xenograft assay in nude mice. SC formed more and larger xenografts than AC. C: SP cells and SC treatment with 0.1 µg/ml doxorubicin or cisplatin for 24 h induced lower apoptosis rate than NSP cells and AC. Data was expressed as mean±SEM, *P < 0.05, **P < 0.01. AC: adherent cells, SC: sphere cells, SP: side population, NSP: non-SP.

Fig. 4. ABC transporters and stem cell markers expressed in gastric SP cells and SC.

Q- RT-PCR (A, B) and Western blotting assays (C) were used to detect the expression of ABC transporters and stem cell markers in SP, NSP cells, AC and SC. Data was expressed as mean±SEM, *P < 0.05, **P < 0.01.

Fig. 5. Knockdown of Sox2 decreased GCSCs of GC cells.

SGC-7901-SC and BGC- 823-SC were transfected with Sox2 siRNA (si-Sox2) or negative control siRNA (si-con) for 48 h. A: Western blotting was used to examine the expression of Sox2. Knockdown of Sox2 decreased the ability of doxorubicin efflux (B), increased apoptosis rate (C) and resulted in the low sphere formation (D) and tumorigenicity of SC (E). The doxorubicin efflux assay was done to show the activity of ABC transporters by the intracellular doxorubicin fluorescence difference between the verapamil-treated cells and the untreated cells. GC: Gastric cancer, AC: adherent cells, SC: sphere cells, SP: side population, NSP: non-SP. Data was expressed as mean±SEM, *P < 0.05, **P < 0.01.