Abstract
Colorectal cancer stem cells (CSCs) have the potential for self-renewal, proliferation, and differentiation. And LGR5 is a stem cell marker gene of colorectal cancer. Curcumin can suppress oncogenicity of many cancer cells, yet the effect and mechanism of curcumin in LGR5(+) colorectal cancer stem cells (CSCs) have not been studied. In this study, we studied the effect of curcumin on LGR5(+) colorectal CSCs using the experiments of tumorsphere formation, cell viability and cell apoptosis. Then autophagy analysis, RNA-Seq, and real-time PCR were used to identify the mechanism responsible for the inhibition of LGR5(+) colorectal CSCs. Our results showed that curcumin inhibited tumorsphere formation, decreased cell viability in a dose-dependent manner, and also promoted apoptosis of LGR5(+) colorectal CSCs. Next, we found curcumin induced autophagy of LGR5(+) colorectal CSCs. When LGR5(+) colorectal CSCs were co-treated with curcumin and the autophagy inhibitor (hydroxychloroquine), curcumin-induced cell proliferation inhibition decreased. In addition, we also found that curcumin inhibited the extracellular matrix (ECM)-receptor interaction pathway via the downregulation of the following genes: GP1BB, COL9A3, COMP, AGRN, ITGB4, LAMA5, COL2A1, ITGB6, ITGA1, and TNC. Further, these genes were transcriptionally regulated by TFAP2A, and the high expression of TFAP2A was associated with poor prognosis in colorectal cancer. In conclusion, curcumin suppressed LGR5(+) colorectal CSCs, potentially by inducing autophagy and repressing the oncogenic TFAP2A-mediated ECM pathway.Graphic abstract
Highlights
Materials and methodsColorectal cancer (CRC) is the world’s fourth most deadly cancer, and the second and third most common cancer diagnosed in women and men, respectively [1]
Colorectal cancer (CRC) is the world’s fourth most deadly cancer, and evidence shows that rare cancer stem cells (CSCs) have the potential for self-renewal, proliferation, and differentiation in CRC [26]
LGR5 was originally isolated from colon cancer cells, where 56% LGR5(+) cells were observed in colorectal CSCs [27]
Summary
Colorectal cancer (CRC) is the world’s fourth most deadly cancer, and the second and third most common cancer diagnosed in women and men, respectively [1]. After treated with curcumin (5 μM) for 24 h, the LGR5(+) colorectal CSCs were suspended in 100 μL binding buffer, and stained with 5 μL APC-conjugated Annexin V and 5 μL propidium iodide for 15 min at room temperature in the dark. The LGR5(+) colorectal CSCs were transfected with AdmCherry-GFP-LC3B (2.51 × 1010 PFU/mL), and treated with different concentrations of curcumin (1 μM and 5 μM) or rapamycin (100 nM) for 48 h. To confirm the effect of curcumininduced autophagy on curcumin-mediated cell inhibition of LGR5(+) colorectal CSCs, cells were co-treated and incubated with curcumin (5 μM) and HCQ (10 μM); sphere formation and cell viability were analyzed. Total RNA of the LGR5(+) colorectal CSCs treated with and without curcumin (5 μM), were extracted using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The following values were considered statistically significant: *p < 0.05, **p < 0.01, ***p < 0.001
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