The Antitumor Effects of Icaritin Against Breast Cancer is Related to Estrogen Receptors.

OBJECTIVE: We aim to investigate the anticancer effects and mechanisms of icaritin against breast cancer. MATERIALS AND METHODS: Both estrogen receptor (ER) positive breast cancer cells MCF- 7 and ER-negative MDA-MB-231 cells were employed. We examined the effects of icaritin on the proliferation and migration by wound healing assay and transwell assay. Cell apoptosis and cell cycle of MCF-7 and MDA-MB-231 cells were analyzed using Flow cytometry. Cell autophagy of MCF-7 and MDA-MB-231 cells was assessed by western blotting, acridine orange staining and confocal microscopy. We also detected the expression of apoptosis-related genes by western blotting. In addition, an autophagy inhibitor was used to investigate whether cytoprotective autophagy was induced. Meanwhile, an ER inhibitor was utilized to explore whether ER was involved in autophagy. RESULTS: Icaritin inhibited the proliferation and migration, and induced cell cycle arrest of both MDA-MB-231 and MCF-7 cells. Icaritin significantly induced apoptosis of MDA-MB- 231 cells by activating caspase-3. And icaritin stimulated autophagy in MCF-7 cells, as evidenced by increased LC3II/LC3I, enhanced p62 degradation, the accumulation of endogenous LC3 puncta formation, and the increased autophagy flux. Icaritin induced autophagy through upregulating the phosphorylation of AMPK and ULK1. Chloroquine, an autophagy inhibitor, increased icaritin-induced apoptosis and proliferation inhibition of MCF-7 cells. Meanwhile, tamoxifen, an ER inhibitor, reversed icaritin-induced autophagy and proliferation inhibition of MCF-7 cells. CONCLUSION: Our study demonstrated that the antitumor effects of icaritin against breast cancer are related to ER, which suggested that the status of ER should be considered in the clinical application of icaritin.

Cloning short DNA into plasmids by one-step PCR.

BACKGROUND: Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one-step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10-15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo. METHODS: The pEGFP-N1-HA plasmid was constructed by one-step PCR and transformation. Cells were transfected with pEGFP-N1-HA and pEGFP-N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA-GFP fusion protein was detected by confocal microscopy. RESULTS: The pEGFP-N1-HA plasmid was successfully constructed and HA expression in cells. CONCLUSIONS: Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction. KEY POINTS: Significant findings of the study A method to clone short DNA into plasmids was found. What this study adds Our study provides a flexible and economical option to clone short DNA into plasmids.