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Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon- γ -induced malignant transformation of primary bovine mammary epithelial cells

View Article: PubMed Central - PubMed

ABSTRACT

Autophagy has been linked to the regulation of both the prevention and progression of cancer. IFN-γ has been shown to induce autophagy in multiple cell lines in vitro. However, whether IFN-γ can induce autophagy and whether autophagy promotes malignant transformation in healthy lactating bovine mammary epithelial cells (BMECs) remain unclear. Here, we provide the first evidence of the correlation between IFN-γ treatment, autophagy and malignant transformation and of the mechanism underlying IFN-γ-induced autophagy and subsequent malignant transformation in primary BMECs. IFN-γ levels were significantly increased in cattle that received normal long-term dietary corn straw (CS) roughage supplementation. In addition, an increase in autophagy was clearly observed in the BMECs from the mammary tissue of cows expressing high levels of IFN-γ. In vitro, autophagy was clearly induced in primary BMECs by IFN-γ within 24 h. This induced autophagy could subsequently promote dramatic primary BMEC transformation. Furthermore, we found that IFN-γ promoted arginine depletion, activated the general control nonderepressible-2 kinase (GCN2) signalling pathway and resulted in an increase in autophagic flux and the amount of autophagy in BMECs. Overall, our findings are the first to demonstrate that arginine depletion and kinase GCN2 expression mediate IFN-γ-induced autophagy that may promote malignant progression and that immunometabolism, autophagy and cancer are strongly correlated. These results suggest new directions and paths for preventing and treating breast cancer in relation to diet.

No MeSH data available.


Related in: MedlinePlus

Autophagy is required for IFN-γ-induced BMEC transformation. (a) The expression levels of LC3-II in BMECs treated with IFN-γ (0 or 10 ng/ml) for up to 8 weeks were determined by western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01. (b) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. Cells (1250) were grown on soft agar, and colonies were monitored after 3 weeks. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05, **P<0.01. (c) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. The expression levels of LC3-II were analysed using western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01.
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fig5: Autophagy is required for IFN-γ-induced BMEC transformation. (a) The expression levels of LC3-II in BMECs treated with IFN-γ (0 or 10 ng/ml) for up to 8 weeks were determined by western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01. (b) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. Cells (1250) were grown on soft agar, and colonies were monitored after 3 weeks. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05, **P<0.01. (c) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. The expression levels of LC3-II were analysed using western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01.

Mentions: To determine the role of IFN-γ-induced autophagy in malignant transformation, we first determined whether autophagic activity was altered during IFN-γ-induced BMEC transformation. The cells were exposed to 10 ng/ml of sodium-IFN-γ for 8 weeks, as described in the Materials and Methods section. We first monitored autophagic activity by evaluating LC3-II expression. As shown in Figure 5a, IFN-γ treatment increased the expression level of LC3-II. After LC3-II upregulation reached a peak at 72 h, it decreased but remained at an elevated level, compared with the control, for 2–8 weeks in response to subchronic IFN-γ exposure. Cell transformation was determined by anchorage-independent growth in soft agar. After 8 weeks of treatment, IFN-γ induced more cell transformation than was observed in the control cells (Figure 5b). The cells were then either left untreated, treated with 3-methyladenine (3-MA) to inhibit autophagic flux, or treated with rapamycin to induce autophagy. The results demonstrated that 3-MA, an inhibitor of autophagy, reduced the IFN-γ-induced anchorage-independent cell growth on soft agar (Figure 5b). However, rapamycin produced the opposite effect, increasing IFN-γ-induced cell transformation (Figure 5b). Western blot analysis further showed that the level of LC3-II expression induced by IFN-γ was reduced in 3-MA-treated cells but was increased in rapamycin-treated cells compared with the levels observed in cells treated only with IFN-γ (Figure 5c). These data indicate that the transformation of BMECs are completely consistent with the intensity of autophagy. Taken together, these results indicate that the initiation of cell transformation was closely related to the autophagy induced by accumulated exposure to IFN-γ.


Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon- γ -induced malignant transformation of primary bovine mammary epithelial cells
Autophagy is required for IFN-γ-induced BMEC transformation. (a) The expression levels of LC3-II in BMECs treated with IFN-γ (0 or 10 ng/ml) for up to 8 weeks were determined by western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01. (b) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. Cells (1250) were grown on soft agar, and colonies were monitored after 3 weeks. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05, **P<0.01. (c) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. The expression levels of LC3-II were analysed using western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01.
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fig5: Autophagy is required for IFN-γ-induced BMEC transformation. (a) The expression levels of LC3-II in BMECs treated with IFN-γ (0 or 10 ng/ml) for up to 8 weeks were determined by western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01. (b) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. Cells (1250) were grown on soft agar, and colonies were monitored after 3 weeks. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05, **P<0.01. (c) BMECs were exposed to IFN-γ (10 ng/ml), 3-MA (1 mM) or rapamycin (Rap; 10 nM) alone or in combination. The expression levels of LC3-II were analysed using western blot analysis with specific antibodies as described in the Materials and Methods section. The data represent the mean±S.E.M. of three independent experiments. Error bars are ±S.E.M. One-way ANOVA; *P<0.05; **P<0.01.
Mentions: To determine the role of IFN-γ-induced autophagy in malignant transformation, we first determined whether autophagic activity was altered during IFN-γ-induced BMEC transformation. The cells were exposed to 10 ng/ml of sodium-IFN-γ for 8 weeks, as described in the Materials and Methods section. We first monitored autophagic activity by evaluating LC3-II expression. As shown in Figure 5a, IFN-γ treatment increased the expression level of LC3-II. After LC3-II upregulation reached a peak at 72 h, it decreased but remained at an elevated level, compared with the control, for 2–8 weeks in response to subchronic IFN-γ exposure. Cell transformation was determined by anchorage-independent growth in soft agar. After 8 weeks of treatment, IFN-γ induced more cell transformation than was observed in the control cells (Figure 5b). The cells were then either left untreated, treated with 3-methyladenine (3-MA) to inhibit autophagic flux, or treated with rapamycin to induce autophagy. The results demonstrated that 3-MA, an inhibitor of autophagy, reduced the IFN-γ-induced anchorage-independent cell growth on soft agar (Figure 5b). However, rapamycin produced the opposite effect, increasing IFN-γ-induced cell transformation (Figure 5b). Western blot analysis further showed that the level of LC3-II expression induced by IFN-γ was reduced in 3-MA-treated cells but was increased in rapamycin-treated cells compared with the levels observed in cells treated only with IFN-γ (Figure 5c). These data indicate that the transformation of BMECs are completely consistent with the intensity of autophagy. Taken together, these results indicate that the initiation of cell transformation was closely related to the autophagy induced by accumulated exposure to IFN-γ.

View Article: PubMed Central - PubMed

ABSTRACT

Autophagy has been linked to the regulation of both the prevention and progression of cancer. IFN-&gamma; has been shown to induce autophagy in multiple cell lines in vitro. However, whether IFN-&gamma; can induce autophagy and whether autophagy promotes malignant transformation in healthy lactating bovine mammary epithelial cells (BMECs) remain unclear. Here, we provide the first evidence of the correlation between IFN-&gamma; treatment, autophagy and malignant transformation and of the mechanism underlying IFN-&gamma;-induced autophagy and subsequent malignant transformation in primary BMECs. IFN-&gamma; levels were significantly increased in cattle that received normal long-term dietary corn straw (CS) roughage supplementation. In addition, an increase in autophagy was clearly observed in the BMECs from the mammary tissue of cows expressing high levels of IFN-&gamma;. In vitro, autophagy was clearly induced in primary BMECs by IFN-&gamma; within 24&thinsp;h. This induced autophagy could subsequently promote dramatic primary BMEC transformation. Furthermore, we found that IFN-&gamma; promoted arginine depletion, activated the general control nonderepressible-2 kinase (GCN2) signalling pathway and resulted in an increase in autophagic flux and the amount of autophagy in BMECs. Overall, our findings are the first to demonstrate that arginine depletion and kinase GCN2 expression mediate IFN-&gamma;-induced autophagy that may promote malignant progression and that immunometabolism, autophagy and cancer are strongly correlated. These results suggest new directions and paths for preventing and treating breast cancer in relation to diet.

No MeSH data available.


Related in: MedlinePlus