**5. Conclusions**

In conclusion, the results of this study showed that ISL e ffectively inhibited triple-negative MDA-MB-231 breast cancer cells through the activation of apoptotic cell death and cause p62 accumulation activated autophagy mediated apoptosis cell death progressions. In the mouse breast cancer tumor xenograft model, the preventive e ffect of ISL on antitumor growth was demonstrated (Figure 7). Therefore, supplementation with ISL may be an e ffective anticancer approach for use in clinical trials.

**Figure 7.** Schematic representation of the inhibitory e ffect of ISL on the proliferation of triple-negative breast cancer MDA-MB-231 cells through p62 accumulation and activation of apoptotic cell death programs. Blue arrow: decreased; Red arrow: increased.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/3/228/s1, **Figure S1**: ISL and bafilomycin A1 (BAF) treatment induced the expression of autophagy-associated proteins and caused cell toxicity. MDA-MB-231 cells were seeded in 96-well plates (3000 cells per well). Cell were pretreated with BAF for 3 h, and combined with ISL for 48 h. At the end of incubation, (**a**) cell viability was measured by MTT assay. MDA-MB-231 cells were pretreated with BAF for 3 h, and combined with ISL for 48 h. The expression of (**b**) p62 and (**c**) Beclin1 protein were analyzed using western blotting. Data were represented as means ± SD (*n* = 3). a, *p* < 0.05, compared with control group. b, *p* < 0.05, compared with ISL group. **Figure S2**: Effects of ISL on the expression and secretion of VEGF. MDA-MB-231 tumor cells (5 × 106 cells per mouse) were implanted and mice were treated with ISL for 25 days. At the end of experiment, (**a**) tumor tissues were isolated and performed immunohistochemistry to analyze VEGF protein level. Images were photographed at 200× magnification. (**b**) Serum VEGF levels were measured using ELISA. Data represent as means ± SEM (*n* = 5 each group). \*\* *p* < 0.01 compared with the control group. **Figure S3**: E ffects of ISL on the capillary-like tube formation. ISL inhibited capillary-like tube formation of mouse endothelial cells SVEC4-10 cells in matrigel. Images were photographed at 100× magnification.

**Author Contributions:** Conceptualization, P.-H.L., T.-M.S., H.-Y.C., T.-C.H., and S.-M.H.; experimentation, P.-H.L., T.-M.S., H.-Y.C., Y.-F.C., T.-H.W., K.-L.W., and S.-M.H.; data analysis and figure preparation, P.-H.L., T.-M.S., H.-Y.C., and S.-M.H.; methodology and resources Y.-F.C., C.-K.S., S.-C.L., and Y.-H.H.; writing—original draft preparation, P.-H.L., Y.-F.C., and S.-M.H.; writing—review and editing, P.-H.L. and S.-M.H.; editing and approval of the final version of the manuscript, S.-M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Science and Technology (MOST), Taiwan, Republic of China, under gran<sup>t</sup> numbers MOST103-2313-B-038-003-MY3, MOST106-2320-B-038-064-MY3, MOST106-2811-B-038-033-, MOST 108-2314-B-039-009-MY3 and MOST 108-2314-B-468-001- and the Council of Agriculture, Taiwan, China, under gran<sup>t</sup> numbers 106AS-16.4.1-ST-a4 and 107AS-13.4.1-ST-a6 and China Medical University, under gran<sup>t</sup> numbers CMU108-MF-25.

**Conflicts of Interest:** The authors declare no conflict of interest.
