Rhein enhances the cytotoxicity of effector lymphocytes in colon cancer under hypoxic conditions

Abstract

The immunosuppressive tumor microenvironment limits the application of adoptive immunotherapy for solid tumors. Hypoxia is closely associated with the formation of the immunosuppressive tumor microenvironment. Hypoxia-inducible factor-1 (HIF-1) is an oxygen-sensitive transcriptional activator that drives the transcription of several immunosuppressive molecules. In addition, previous studies confirmed that rhein downregulated the expression of HIF-1α, a subunit of HIF-1, in pancreatic cancer cells. The present study established correlations between mRNA expression levels of HIF-1α and six immunosuppressive molecules in colorectal cancer (CRC) tissue samples. This study examined the effect of rhein on the expression levels of HIF-1α and six immunosuppressive molecules in CRC cell lines under hypoxic conditions by western blot analysis and reverse transcription-quantitative polymerase chain reaction. This study demonstrated that rhein downregulated the expression of HIF-1α and immunosuppressive molecules in CRC cells under hypoxic conditions. In addition, the present study analyzed the cytotoxicity of peripheral blood lymphocytes in vitro using a non-toxic cytotoxicity assay. This study demonstrated that in vitro, rhein enhanced the cytotoxicity of effector lymphocytes toward tumor cells under hypoxic conditions, and therefore rhein may be used in combination with effector lymphocytes for the treatment of CRC.

Keywords: colorectal cancer; effector lymphocyte; hypoxia; hypoxia-inducible factor-1; immunosuppressive tumor microenvironment; rhein.

Figure 1.

HIF-1α mRNA expression correlates with immunosuppressive gene expression in colorectal cancer tissue. The expression levels of HIF-1α and six genes with known immunosuppressive functions were determined by reverse transcription-quantitative polymerase chain reaction from 12 colorectal cancer tissue samples. The Pearson's R coefficient was used to measure the correlation between the mRNA expression level of HIF-1α and the six immunosuppressive molecules. HIF-1α, hypoxia-inducible factor 1 subunit α; PD-L1, programmed cell death 1 ligand 1; COX-2, cyclooxygenase-2; IL-10, interleukin 10; VEGF, vascular endothelial growth factor; TGF-1β, transforming growth factor β-1.

Figure 2.

Rhein cytotoxicity in CRC cell lines and PBLs. (A) MTT assay was used to examine cell viability in four CRC cell lines following treatment with various concentrations of rhein for 48 h. *P<0.05 vs. untreated cells. (B) Cell counting kit-8 assay was used to examine cell viability in PBL following treatment with rhein for 48 h. Data are presented as the mean ± standard deviation and normalized to untreated control. CRC, colorectal cancer, PBL, peripheral blood lymphocyte; HT29, HCT116, Colo205 and SW620, human CRC cell lines.

Figure 3.

Rhein downregulates the expression of HIF-1α and six immunosuppressive molecules. (A) The protein expression levels of HIF-1α were determined by western blot analysis in HT29 cells cultured under conditions of normoxia, hypoxia, hypoxia + 25 µM rhein, and hypoxia + 50 µM rhein for 16 h. (B) The mRNA expression levels of PD-L1, VEGF, COX-2, galectin-1, IL-10 and TGF-β1 were determined by reverse transcription-quantitative polymerase chain reaction in HT29 cells treated as above. The mRNA expression levels were normalized to that of cells under normoxia. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 and **P<0.01 as indicated. RQ, relative quantification; CRC, colorectal cancer; HIF-1α hypoxia-inducible factor 1 subunit α; PD-L1, programmed cell death 1 ligand 1; COX-2, cyclooxygenase-2; IL-10, interleukin 10; VEGF, vascular endothelial growth factor; TGF-β1, transforming growth factor-β1; HT29, human CRC cell line.

Figure 4.

Cytotoxicity of PBLs toward HT29-CD3scfv cells. (A) Schematic representation of the lentiviral vector construct encoding membrane-bound anti-CD3scfv expression cassette. (B) The protein expression levels of CD3scfv in HT29-CD3scfv and HT29-control cells were determined by western blot analysis. (C) The cellular localization of CD3scfv in HT29-CD3scfv and HT29-control cells was detected by confocal microscopy (magnification, ×400). (D) The proportion of cells expressing membrane-bound anti-CD3scfv was determined by fluorescence-activated cell sorting analysis. (E) HT29-CD3scfv and HT29-control cells were co-cultured with PBLs at different E:T cell ratios, respectively for 16 h. CytoTox96^® Non-Radioactive Cytotoxicity assay was used to determine cell lysis of HT29-CD3scfv and HT29-control cells. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 vs. HT29-control cells at the corresponding E:T ratio. HT29-control, untreated HT29 cells; HT29-CD3scfv, HT29 cells stably expressing mCD3scfv; HT29, human CRC cell line; PBL, peripheral blood lymphocyte; CRC, colorectal cancer; HA, hemagglutinin; E:T, effector-to-target cell ratio.

Figure 5.

Rhein enhances the cytotoxicity of PBLs in hypoxia. HT29-CD3scfv cells were cultured under conditions of normoxia, hypoxia and hypoxia + 50 µM rhein for 8 h. Cells were subsequently co-cultured with PBLs at different E:T cell ratios for 16 h. HT29-CD3scfv cell lysis was examined by CytoTox96^® Non-Radioactive Cytotoxicity assay. The data are presented as the mean ± standard deviation from three independent experiments. *P<0.05 and **P<0.01 as indicated. PBLs, peripheral blood lymphocytes; HT29-CD3scfv, HT29 cells stably expressing mCD3scfv; E:T, effector-to-target cell ratio.