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. 2016 Mar 29;7(13):16840-54.
doi: 10.18632/oncotarget.7614.

Thymic stromal lymphopoietin (TSLP) inhibits human colon tumor growth by promoting apoptosis of tumor cells

Wenjie Yue  1   2 , Yuli Lin  1 , Xuguang Yang  1 , Bingji Li  1 , Jie Liu  1   2 , Rui He  1   3
Affiliations

Thymic stromal lymphopoietin (TSLP) inhibits human colon tumor growth by promoting apoptosis of tumor cells

Wenjie Yue et al. Oncotarget. .

Abstract

Thymic stromal lymphopoietin (TSLP) has recently been suggested in several epithelial cancers, either pro-tumor or anti-tumor. However, the role of TSLP in colon cancer remains unknown. We here found significantly decreased TSLP levels in tumor tissues compared with tumor-surrounding tissues of patients with colon cancer and TSLP levels negatively correlated with the clinical staging score of colon cancer. TSLPR, the receptor of TSLP, was expressed in all three colon cancer cell lines investigated and colon tumor tissues. The addition of TSLP significantly enhanced apoptosis of colon cancer cells in a TSLPR-dependent manner. Interestingly, TSLP selectively induced the apoptosis of colon cancer cells, but not normal colonic epithelial cells. Furthermore, we demonstrated that TSLP induced JNK and p38 activation and initiated apoptosis mainly through the extrinsic pathway, as caspase-8 inhibitor significantly reversed the apoptosis-promoting effect of TSLP. Finally, using a xenograft mouse model, we demonstrated that peritumoral administration of TSLP greatly reduced tumor growth accompanied with extensive tumor apoptotic response, which was abolished by tumor cell-specific knockdown of TSLPR. Collectively, our study reveals a novel anti-tumor effect of TSLP via direct promotion of the apoptosis of colon cancer cells, and suggests that TSLP could be of value in treating colon cancer.

Keywords: TSLP; TSLPR; apoptosis; caspase3; colon cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST

There is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Figures

Figure 1
Figure 1. Decreased tumoral TSLP expression in human colon cancer
A. TSLP expression in colon adenomas and normal mucosa by using two sets of public data from the GEO database (NCBI GEO: GSE8671) and TCGA cancer genome. B and C. TSLP mRNA expression was examined by quantitative real-time RT-PCR (qRT-PCR) analysis (B) and TSLP protein levels were measured by ELISA (C) in tumor and tumor-surrounding tissues from surgical specimens of 40 colon cancer patients. D. Representative photomicrographs of immunohistochemical (IHC)-staining of TSLP in tumor and tumor-surrounding tissues (100X magnification) and quantitiative analysis of the intensity of TSLP staining. *P<0.05. E. The analysis of TSLP mRNA and protein levels in tumor tissues from colon cancer patients according to the criteria of Duke's classification. F. Representative photomicrographs of IHC-staining of TSLP in tumor tissues from colon cancer patients in stage A and stage D (100X magnification). Individual tissue samples are represented as dots in a,b,c.*P<0.05 **P<0.01.
Figure 2
Figure 2. TSLPR expression in colon cancer cells
A. TSLPR mRNA expression was examined by conventional RT-PCR analysis in colon cancer cell lines, SW1116, SW480 and DLD-1. PBMCs were used as a positive control. B and C. TSLPR expression in colon cancer cell lines was detected by immunostaining (100X magnification, inserted: 200X magnification) (B) and was analyzed by flow cytometry (C). D and E. qRT-PCR analysis of TSLPR mRNA expression (D) and representative photomicrographs of IHC-staining of TSLPR (100X magnification) (E) in tumor and tumor-surrounding tissues, respectively. Individual tissue samples are represented as dots in d. NS=no significant.
Figure 3
Figure 3. Exogenous TSLP treatment promotes apoptosis of colon cancer cells
A. Representative data of flow cytometric analysis of Annexin V-FITC/PI double-staining apoptotic cells in three colon cancer cell lines treated with TSLP at indicated concentrations for 48 h. B. The percentages of apoptotic cells in TSLP-treated colon cancer cells. Columns and error bars are representatives of mean±SEM of triplicate in one experiment. Similar results were obtained in three independent experiments. **P<0.01 versus medium group. C. Western blotting analysis of protein levels of total and cleaved caspase3, t-PARP, and cleaved-PARP in TSLP-treated colon cancer cells at indicated concentrations for 48 h. β-actin was used as the control. D. TSLPR was specifically knocked down by siRNA in all three colon cancer cells. The average percentages of Annexin V-FITC+ apoptotic cells in TSLP-treated colon cancer cells were determined by flow cytometric analysis. *P<0.05 NS=no significant.
Figure 4
Figure 4. TSLP preferentially promotes the apoptosis of primary colon cancer cells
EpCAM+ cells were from FACS sorted dissociated tumor tissues (as primary colon cancer cells) or tumor-surrounding tissues (as non-transformed colonic epithelial cells) from two patients with colon cancer. A. Representative data of flow cytometric analysis of Annexin V-FITC/PI double-staining apoptotic cells and the percentages of AnnexinV+ apoptotic cells in primary colon cancer cells and non-transformed colonic epithelial cells treated with or without TSLP. Columns and error bars are representatives of mean±SEM of triplicate in one experiment. Similar results were obtained in two independent experiments. B. Western blotting analysis of protein levels of total and cleaved caspase-3. β-actin was used as the control.
Figure 5
Figure 5. TSLP activates both extrinsic and intrinsic apoptosis pathway, and the apoptosis-promoting effect of TSLP is largely dependent on caspase-8-initiated extrinsic pathway
A and B. Western blotting analysis of protein levels of total and phosphorylated MARKs (JNK and p38) (A) as well as total and cleaved caspase-8 and caspase-9 (B) in TSLP-treated colon cancer cells at indicated concentrations for 48 h. β-actin was used as the control. C. The percentages of Annexin V-FITC+ apoptotic cells in TSLP-treated colon cancer cells with or without caspase-8 inhibitor. Columns and error bars are representatives of mean±SEM of triplicate in one experiment. Similar results were obtained in three independent experiments. D. Western blotting analysis of protein levels of total and cleaved caspase-3 in TSLP-treated colon cancer cells with or without caspase-8 inhibitor. E. Western blotting analysis of protein levels of Bid, Bax, Bak, and Bad in TSLP-treated colon cancer cells at indicated concentrations for 48 h with or without caspase8 inhibitor. β-actin was used as the control. F. The alteration in mitochondrial membrane potential was determined by flow cytometric analysis of JC-1 staining of TSLP-treated colon cancer cells at indicated concentrations for 48 h. JC-1 is a type of fluorescence probe, which exists as JC-aggregates emitting red fluorescence detected at FL-2 channel when cells are alive and as JC-monomers emitting green fluorescence detected at FL-1 channel when early apoptosis occurs. Columns and error bars are representatives of mean±SEM of triplicate in one experiment. Similar results were obtained in three independent experiments. **P<0.01 versus medium group.
Figure 6
Figure 6. Administration of exogenous TSLP inhibits tumor growth in a xenograft mouse model of human colon cancer
SW1116 and TSLPRkd-SW1116 cells were subcutaneously injected, respectively, into nude mice at day 0. A. Experimental procedure used to administer TSLP to nude mice bearing colon tumors. PBS-0.1%BSA was used as control. B. Tumor-bearing mice and tumors excised from each group at day 21. C. Tumor volume and tumor weight at day 21 were measured. **p<0.01 n=7. Data are presented as mean±SEM. D and E. Tumor sections from each group were stained with H&E to show necrotic area (D) and with TUNEL kit to show the apoptotic cells (E). F. Western blotting analysis of protein levels of total and cleaved caspase-3 in tumor tissue lysates.
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