Enhanced expression of cyclooxygenase-2 related multi-drug resistance gene in melanoma and osteosarcoma cell lines by TSG-6 secreted from canine adipose-derived mesenchymal stem/stromal cells

Abstract

Background: Multiple drug resistance (MDR) of cancer cells is the main cause of intrinsic or acquired desensitization to chemotherapy in many cancers. A number of studies have found high expression of COX-2 to be a factor for expression of MDR gene in several cancer. Furthermore, adipose tissue derived mesenchymal stem/stromal cells (ADSC) have been found to increase cyclo-oxygenase-2 (COX-2) expression in some tumour cells. The mechanism for this, however, is not yet clear and needs further study.

Objective: The purpose of this study was to determine whether tumour necrosis factor-alpha stimulated gene/protein 6 (TSG-6) secreted from ADSCs is associated with an increase in MDR genes by inducing COX-2 gene expression in melanoma and osteosarcoma cell lines.

Methods: ADSCs were transfected with TSG-6 siRNA or Control RNA respected, and cancer cell line were transfected with COX-2 siRNA or Control RNA respected. Using trans well coculture system, the interactions of ADSCs with tumour cells were investigated.

Results: Increased COX-2 expression was observed in cancer cell co-cultured with ADSCs. Additionally, we identified that COX-2 expression was related to drug resistance genes (P-glycoprotein, multidrug resistance-associated protein). Transfecting canine ADSCs with small interfering RNA, TSG-6 secreted from ADSCs was found to be a major factor in the regulation of COX-2 expression and drug resistance genes in osteosarcoma and melanoma cell lines.

Conclusion: TSG-6 mediated COX-2 up-regulation is a possible mechanism of chemoresistance development induced by ADSCs. These findings provide better understanding about the mechanism associated with ADSC-induced chemoresistance in cancer.

Keywords: adipose tissue-derived stem cell; canine; cyclooxygenase-2; drug resistance gene; melanoma; osteosarcoma; tumour necrosis factor-alpha stimulated gene/protein 6.

FIGURE 1

Canine adipose‐derived stem cells (cADSCs) exhibited characteristics of mesenchymal stem cells. (a) Immune phenotypic analysis of cADSC by flow cytometry. CD29, CD44, CD90 and CD73 were highly expressed and CD 45 and CD34 were low. (Red: stained marker, blue: negative (unstained) control) (b) Osteogenic (Alizarin Red S staining), adipogenic (Oil Red O staining), chondrogenic (Alcian Blue staining) differentiation of cADSCs

FIGURE 2

cADSCs increased COX‐2 expression in cancer cell lines. Co‐culture with cADSCs increased COX‐2 protein levels of (a) canine melanoma (LMeC) and (b) osteosarcoma (D17) cell lines. The COX‐2 protein level increased as the proportion of cADSC increased. Experiments were performed in triplicate and repeated three times with similar results. Results are shown as mean ± SD (*p < .05, **p < .01, ***p < .001 by one‐way ANOVA analysis)

FIGURE 3

Drug resistance gene expression in cancer cell lines increased in the presence of cADSCs, and decreased with interference of COX‐2. COX‐2 protein levels in LMeC (a) and D17 (b) cancer cell lines. mRNA expression levels of COX‐2, MRP1, P‐gp in LMeC (c) and D17 (d) cancer cell lines. COX‐2 and drug resistance gene MRP1, P‐gp expression of cancer cell lines increased significantly with the presence of cADSCs . When COX‐2 was down regulated with COX‐2 targeted siRNA (siCOX‐2) in cancer cell lines, MRP1 and P‐gp expression in LMeC, D17 cells was decreased even when cultured with cADSCs. Experiments were performed in triplicate and repeated three times with similar results. Results are shown as mean ± standard deviation (*p < .05, **p < .01, ***p < .001 by one‐way ANOVA analysis)

FIGURE 4

TSG‐6 secreted from cADSC elevated the expression of COX‐2 in cancer cell lines. mRNA expression levels of COX‐2 in LMeC (a), and D17 (b) cell lines. Protein levels of COX‐2 in LMeC (c) and D17 (d) cell lines. COX‐2 mRNA and protein expression in cancer cell lines decreased significantly when they were co‐cultured with TSG‐6 targeted siRNA (siTSG‐6) transfected cADSCs. Experiments were performed in triplicate and repeated three times with similar results. Results are shown as mean ± SD (*p < .05, **p < .01, ***p < .001 by one‐way ANOVA analysis)

FIGURE 5

TSG‐6 secreted from cADSCs affected drug resistance genes in cancer cell lines. mRNA expression levels of MRP1 and P‐gp in LMeC (a) and D17 (b) cell lines. Experiments were performed in triplicate and repeated three times with similar results. Results are shown as mean ± standard deviation (*p < .05, **p < .01, ***p < .001 by one‐way ANOVA analysis)