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Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis.

Yin SY, Jian FY, Chen YH, Chien SC, Hsieh MC, Hsiao PW, Lee WH, Kuo YH, Yang NS - Nat Commun (2016)

Bottom Line: Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression.Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel.Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

View Article: PubMed Central - PubMed

Affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression. Here we investigate the role of IL-25, an endogenous anticancer factor secreted from TAFs, in suppression of mouse 4T1 mammary tumour metastasis. We show that a synthetic dihydrobenzofuran lignan (Q2-3), the dimerization product of plant caffeic acid methyl ester, suppresses 4T1 metastasis by increasing fibroblastic IL-25 activity. The secretion of IL-25 from treated human or mouse fibroblasts is enhanced in vitro, and this activity confers a strong suppressive effect on growth activity of test carcinoma cells. Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel. Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

No MeSH data available.


Related in: MedlinePlus

IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells.(a) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). (b) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. (c) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (two-tailed Student's t-test). (d) Western blot analysis of IL-17RA and IL-17RB (IL-25R) expression in MDA-MB-231 and MCF-10A cells. Knock down of human IL-25R (both 56 and 33 kDa molecules) expression in MDA-MB-231 cells was performed with three designed siRNAi treatments. Neg, negative control RNAi. (e) Western blot analysis of the cleavage of caspases 8 and 3 in MDA-MB-231 cells. MDA-MB-231 cells were treated by control-, Neg RNAi- or IL-25R RNAi, for 48 h. Some cells in each test group were cultivated with recombinant human IL-25 (200 ng ml−1), WI38-CM, or WI38-CM with the immunodepletion of IL-25, for another 24 h. White arrowheads, cleaved protein. Black arrowheads, uncleaved protein. β-Actin served as an internal control. Similar results were obtained from three independent experiments.
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f4: IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells.(a) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). (b) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. (c) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (two-tailed Student's t-test). (d) Western blot analysis of IL-17RA and IL-17RB (IL-25R) expression in MDA-MB-231 and MCF-10A cells. Knock down of human IL-25R (both 56 and 33 kDa molecules) expression in MDA-MB-231 cells was performed with three designed siRNAi treatments. Neg, negative control RNAi. (e) Western blot analysis of the cleavage of caspases 8 and 3 in MDA-MB-231 cells. MDA-MB-231 cells were treated by control-, Neg RNAi- or IL-25R RNAi, for 48 h. Some cells in each test group were cultivated with recombinant human IL-25 (200 ng ml−1), WI38-CM, or WI38-CM with the immunodepletion of IL-25, for another 24 h. White arrowheads, cleaved protein. Black arrowheads, uncleaved protein. β-Actin served as an internal control. Similar results were obtained from three independent experiments.

Mentions: To characterize and analyse the possible suppressive effect of fibroblast-secreted IL-25 on growth activity of mammary tumour cells, the levels of secreted IL-25 protein in conditioned media of test mouse and human fibroblasts were collected and compared by using an anti-IL-25 antibody-mediated immunoprecipitation approach. Before immunoprecipitation, aliquot samples of conditioned medium from Q2-3-treated fibroblasts, including 3T3 fibroblast-conditioned media (3T3-CM) and WI38 fibroblast-conditioned media (WI38-CM), were immunodepleted for IL-25. In this test, anti-rabbit IgG antibody (isotype control) was used as a negative control for immunodepletion. The quantity of IL-25 in each conditioned medium and the efficiency of immunodepletion for IL-25 were then assessed by immunobloting analysis (Fig. 4a). In comparison, the levels of secreted IL-25 in the Q2-3-treated fibroblast-conditioned media were found to be significantly higher than that in the untreated fibroblast-conditioned media (Fig. 4a). Importantly, this result also demonstrates that most secreted IL-25 (∼90%) in both human and mouse Q2-3-treated fibroblast-conditioned media could be immunodepleted by using anti-IL-25 antibody (Fig. 4a). Only a very small fraction (4 to 9% decrease) of nonspecific protein binding was detected for the isotype control antibody, showing high specificity and efficiency of the used anti-IL-25 antibody. The conditioned media immunodepleted for IL-25 from cultivation of mouse and human fibroblasts were also employed to culture 4T1 and MDA-MB-231 tumour cells, respectively. In this test, fresh conditioned media were applied every 24 h for 5 days. 4T1 cells cultured with 3T3-CM showed much higher growth activity than cells cultured with fresh medium only (Fig. 4b). This result is consistent with that obtained from MDA-MB-231 cells (Fig. 4c) and taken together these data suggest that fibroblasts can release important cellular and molecular factors for tumour cell expansion. In addition, exogenously added IL-25 protein (100 ng ml−1 for human and mouse IL-25 recombinant protein) decreased the growth activity of test 4T1 and MDA-MB-231 cells. In agreement, 4T1 cells cultured with Q2-3-treated 3T3-CM (Fig. 4b) or MDA-MB-231 cells cultured with Q2-3-treated WI38-CM (Fig. 4c) also showed relatively decreased cell growth activity, as compared with IL-25 protein-treated tumour cells. In contrast, depletion of IL-25 attenuated the growth-suppressive effect of Q2-3-treated fibroblasts CM on mouse (Fig. 4b) and human (Fig. 4c) mammary tumour cells. This attenuation was not observed by the use of isotype IgG antibody. Taken together, our results suggest that the Q2-3-induced IL-25 secretion from fibroblasts plays a critical role in suppressing the growth activity of metastatic mammary tumour cells.


Induction of IL-25 secretion from tumour-associated fibroblasts suppresses mammary tumour metastasis.

Yin SY, Jian FY, Chen YH, Chien SC, Hsieh MC, Hsiao PW, Lee WH, Kuo YH, Yang NS - Nat Commun (2016)

IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells.(a) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). (b) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. (c) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (two-tailed Student's t-test). (d) Western blot analysis of IL-17RA and IL-17RB (IL-25R) expression in MDA-MB-231 and MCF-10A cells. Knock down of human IL-25R (both 56 and 33 kDa molecules) expression in MDA-MB-231 cells was performed with three designed siRNAi treatments. Neg, negative control RNAi. (e) Western blot analysis of the cleavage of caspases 8 and 3 in MDA-MB-231 cells. MDA-MB-231 cells were treated by control-, Neg RNAi- or IL-25R RNAi, for 48 h. Some cells in each test group were cultivated with recombinant human IL-25 (200 ng ml−1), WI38-CM, or WI38-CM with the immunodepletion of IL-25, for another 24 h. White arrowheads, cleaved protein. Black arrowheads, uncleaved protein. β-Actin served as an internal control. Similar results were obtained from three independent experiments.
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f4: IL-25 secreted by Q2-3-treated fibroblasts suppresses the growth of mammary tumour cells.(a) Western blot analysis of the IL-25 secretion activity of mouse (3T3) and human (WI38) fibroblasts in response to Q2-3 treatment. Different fibroblast-conditioned media (CM), including 3T3-CM and WI38-CM, were collected from the 3D cultures and were stained with coomassie blue, revealing that the total protein level in tested CM was normalized. Aliquots of 3T3-CM and WI38-CM were immunodepleted for IL-25. Rabbit IgG (isotype control) was used as a negative control. Amounts of IL-25 (relative staining intensity) were further normalized with the value detected for Q2-3-treated 3T3-CM (blue) or Q2-3-treated WI38-CM samples (purple). (b) Reduction in cytotoxicity of 3T3-CM on 4T1 cells after immunodepletion of IL-25. The control (fresh) medium, 3T3-CM, Q2-3-treated 3T3-CM, Q2-3-treated 3T3-CM with added IL-25 protein, Q2-3-treated 3T3-CM with the immunodepletion of IL-25 and Q2-3-treated 3T3-CM with control IgG-mediated immunodepletion, were compared for their suppressive effect on growth of 4T1 cells. (c) Reduction in cytotoxicity of WI38-CM on MDA-MB-231 after immunodepletion of IL-25. Similarly, WI38-CM with added IL-25 protein, WI38-CM with the immunodepletion of IL-25, or Q2-3-treated WI38-CM with control IgG-mediated immunodepletion, were compared for their effect on growth of MDA-MB-231 cells. The growth activity of 4T1 cells or MDA-MB-231 cells was determined using MTT assay at 5 days post cultivation, and was normalized to the control group (with fresh medium only). Data are reported as mean±s.d. (n=3). *P<0.05; **P<0.01; ***P<0.001 (two-tailed Student's t-test). (d) Western blot analysis of IL-17RA and IL-17RB (IL-25R) expression in MDA-MB-231 and MCF-10A cells. Knock down of human IL-25R (both 56 and 33 kDa molecules) expression in MDA-MB-231 cells was performed with three designed siRNAi treatments. Neg, negative control RNAi. (e) Western blot analysis of the cleavage of caspases 8 and 3 in MDA-MB-231 cells. MDA-MB-231 cells were treated by control-, Neg RNAi- or IL-25R RNAi, for 48 h. Some cells in each test group were cultivated with recombinant human IL-25 (200 ng ml−1), WI38-CM, or WI38-CM with the immunodepletion of IL-25, for another 24 h. White arrowheads, cleaved protein. Black arrowheads, uncleaved protein. β-Actin served as an internal control. Similar results were obtained from three independent experiments.
Mentions: To characterize and analyse the possible suppressive effect of fibroblast-secreted IL-25 on growth activity of mammary tumour cells, the levels of secreted IL-25 protein in conditioned media of test mouse and human fibroblasts were collected and compared by using an anti-IL-25 antibody-mediated immunoprecipitation approach. Before immunoprecipitation, aliquot samples of conditioned medium from Q2-3-treated fibroblasts, including 3T3 fibroblast-conditioned media (3T3-CM) and WI38 fibroblast-conditioned media (WI38-CM), were immunodepleted for IL-25. In this test, anti-rabbit IgG antibody (isotype control) was used as a negative control for immunodepletion. The quantity of IL-25 in each conditioned medium and the efficiency of immunodepletion for IL-25 were then assessed by immunobloting analysis (Fig. 4a). In comparison, the levels of secreted IL-25 in the Q2-3-treated fibroblast-conditioned media were found to be significantly higher than that in the untreated fibroblast-conditioned media (Fig. 4a). Importantly, this result also demonstrates that most secreted IL-25 (∼90%) in both human and mouse Q2-3-treated fibroblast-conditioned media could be immunodepleted by using anti-IL-25 antibody (Fig. 4a). Only a very small fraction (4 to 9% decrease) of nonspecific protein binding was detected for the isotype control antibody, showing high specificity and efficiency of the used anti-IL-25 antibody. The conditioned media immunodepleted for IL-25 from cultivation of mouse and human fibroblasts were also employed to culture 4T1 and MDA-MB-231 tumour cells, respectively. In this test, fresh conditioned media were applied every 24 h for 5 days. 4T1 cells cultured with 3T3-CM showed much higher growth activity than cells cultured with fresh medium only (Fig. 4b). This result is consistent with that obtained from MDA-MB-231 cells (Fig. 4c) and taken together these data suggest that fibroblasts can release important cellular and molecular factors for tumour cell expansion. In addition, exogenously added IL-25 protein (100 ng ml−1 for human and mouse IL-25 recombinant protein) decreased the growth activity of test 4T1 and MDA-MB-231 cells. In agreement, 4T1 cells cultured with Q2-3-treated 3T3-CM (Fig. 4b) or MDA-MB-231 cells cultured with Q2-3-treated WI38-CM (Fig. 4c) also showed relatively decreased cell growth activity, as compared with IL-25 protein-treated tumour cells. In contrast, depletion of IL-25 attenuated the growth-suppressive effect of Q2-3-treated fibroblasts CM on mouse (Fig. 4b) and human (Fig. 4c) mammary tumour cells. This attenuation was not observed by the use of isotype IgG antibody. Taken together, our results suggest that the Q2-3-induced IL-25 secretion from fibroblasts plays a critical role in suppressing the growth activity of metastatic mammary tumour cells.

Bottom Line: Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression.Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel.Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

View Article: PubMed Central - PubMed

Affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan.

ABSTRACT
Tumour-associated fibroblasts (TAFs), as a functionally supportive microenvironment, play an essential role in tumour progression. Here we investigate the role of IL-25, an endogenous anticancer factor secreted from TAFs, in suppression of mouse 4T1 mammary tumour metastasis. We show that a synthetic dihydrobenzofuran lignan (Q2-3), the dimerization product of plant caffeic acid methyl ester, suppresses 4T1 metastasis by increasing fibroblastic IL-25 activity. The secretion of IL-25 from treated human or mouse fibroblasts is enhanced in vitro, and this activity confers a strong suppressive effect on growth activity of test carcinoma cells. Subsequent in vivo experiments showed that the anti-metastatic effects of Q2-3 on 4T1 and human MDA-MD-231 tumour cells are additive when employed in combination with the clinically used drug, docetaxel. Altogether, our findings reveal that the release of IL-25 from TAFs may serve as a check point for control of mammary tumour metastasis and that phytochemical Q2-3 can efficiently promote such anticancer activities.

No MeSH data available.


Related in: MedlinePlus