Inhibition of FOXO1­mediated autophagy promotes paclitaxel­induced apoptosis of MDA­MB­231 cells.

Triple­negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and it often becomes resistant to paclitaxel (PTX) therapy. Autophagy plays an important cytoprotective role in PTX­induced tumor cell death, and targeting autophagy has been promising for improving the efficacy of tumor chemotherapy in recent years. The aim of the present study was to clarify the mechanism of PTX inducing autophagy in TNBC cells to provide a potential clinical chemotherapy strategy of PTX for TNBC. The present study reported that PTX induced both apoptosis and autophagy in MDA­MB­231 cells and that inhibition of autophagy promoted apoptotic cell death. Furthermore, it was found that forkhead box transcription factor O1 (FOXO1) enhanced PTX­induced autophagy through a transcriptional activation pattern in MDA­MB­231 cells, which was associated with the downstream target genes autophagy related 5, class III phosphoinositide 3­kinase vacuolar protein sorting 34, autophagy related 4B cysteine peptidase, beclin 1 and microtubule associated protein 1 light chain 3ß. Knocking down FOXO1 attenuated the survival of MDA­MB­231 cells in response to PTX treatment. These findings may be beneficial for improving the treatment efficacy of PTX and to develop autophagic targeted therapy for TNBC.

Endogenous leptin promotes autophagy in EBSS-induced PFCs.

Leptin is an important adipokine and plays a vital role in animals. However, the role of leptin in the autophagic response of pig fibroblast cells (PFCs) has not been fully elucidated. In this study, we investigated the relationship between leptin and autophagy as well as underlying molecular basis. We found that PFCs treated with EBSS could secrete leptin, and the leptin concentration in the supernatant of leptin transgenic PFCs was higher than that of WT PFCs. We found an increase in LC3-II protein level and a decrease in p62 protein level in treated leptin transgenic PFCs compared with treated WT PFCs. Meanwhile, we observed an increase of autophagosomes by transmission electron microscopy and an enhancement of the accumulation of LC3 puncta in the cytoplasm of treated leptin transgenic PFCs, and these effects were further augmented by Baf A1 treatment. Furthermore, we detected the expression levels of 7 autophagy signaling pathway genes and 17 autophagy-related (ATG) genes by q-PCR. We found that between the two types of EBSS-treated cells 3 genes expression pattern were significantly different among the 7 autophagy signaling pathway genes and 8 genes expression pattern were significantly differernt among the ATG genes. These results indicated that leptin may promote autophagy and involving the downregulation of FOXO1 and LMNA genes via an unknown pathway which causes the upregulation of the 4 genes and the downregulation of 4 genes.

The molecular basis for p53 inhibition of autophagy in porcine fibroblast cells.

BACKGROUND: Autophagy regulation involves an intricate network that can degrade and recycle cytosolic components in autophagosomes when cells are subject to various stress signals. p53 plays a dual role of induction or inhibition in the regulation of autophagy. Recently, pigs have been considered an excellent large animal model for their many anatomical and physiological similarities to humans. Here, we investigated the relationship between p53 and autophagy, as well as the underling molecular basis, in porcine fibroblast cells (PFCs). METHODS: Autophagy was induced by Earle's balanced salt solution (EBSS) in p53-/- and p53wt PFCs. The autophagy response was assessed by immunoblotting, transmission electron microscopy (TEM) and fluorescent staining. The molecular basis for p53 regulation of autophagy was analyzed by qPCR. RESULTS: We found that the increased expression of LC3-II and the decreased expression of P62 occurred earlier in p53-/- PFCs than in p53wt PFCs, the relative autophagic flux of p53-/- PFCs was stronger than that of p53wt PFCs in a time-dependent manner. Meanwhile, we observed a visualized increase of autophagosomes in p53-/- PFCs. Moreover, we found greater accumulation of LC3 punctate and acidic vesicular organelle (AVOs) in the cytoplasm of p53-/- PFCs than in that of p53wt PFCs, and these effects were further augmented by Baf A1 treatment. Furthermore, we detected the expression of 6 autophagy signaling pathway-related genes and 14 autophagy-related (ATG) genes by qPCR. We found that the expression patterns of the 6 genes had significant differences in both groups of treated PFCs. These results demonstrated that p53 negatively regulated autophagy and involving the downregulation of LMNA gene by p53 via an unknown pathway, which causes the upregulation of the LC3, ULK1, ATG4B, ATG16L1 and ATG9A genes and the downregulation of the P62 gene. CONCLUSIONS: p53-/- PFCs responded to autophagy earlier than p53wt PFCs, which implied that p53 might inhibit autophagy. The expression patterns of autophagy signaling pathway-related genes and ATG genes were most different between p53-/- and p53wt PFCs. Our study will provide a new biological model and contribute to further study of the molecular basis for p53 and autophagy.

Low-Dose Paclitaxel Inhibits Tumor Cell Growth by Regulating Glutaminolysis in Colorectal Carcinoma Cells.

Paclitaxel (PTX) is a natural alkaloid isolated from the bark of a tree, Taxus brevifolia, and is currently used to treat a variety of tumors. Recently, it has been found that low-dose PTX is a promising treatment for some cancers, presenting few side effects. However, antitumor mechanisms of low-dose PTX (<1 nM) have rarely been illuminated. Here we report a new antitumor mechanism of low-dose PTX in colorectal carcinoma cells. We treated colorectal carcinoma HCT116 cells with PTX at 0.1 and 0.3 nM for 0, 1, 2, or 3 days, and found that low-dose PTX inhibits cell growth without altering cell morphology and cell cycle. There was a significant decrease of pH in culture media with 0.3 nM PTX for 3 days. Also, lactate production was significantly increased in a dose- and time-dependent manner. Furthermore, expression of glutaminolysis-related genes GLS, SLC7A11 and SLC1A5 were significantly decreased in the colorectal carcinoma cells treated with low-dose PTX. Meanwhile, protein expression levels of p53 and p21 increased significantly in colorectal carcinoma cells so treated. In summary, low-dose PTX down-regulated glutaminolysis-related genes and increased their lactate production, resulting in decreased pH of tumor microenvironments and inhibition of tumor cell growth. Up-regulation of p53 and p21 in colorectal carcinoma cells treated with low-dose PTX also contributed to inhibition of tumor cell growth.