Salvianolic acid B targets mortalin and inhibits the migration and invasion of hepatocellular carcinoma via the RECK/STAT3 pathway.

BACKGROUND: Tumor migration and invasion is a complex and diverse process that involves the epithelial-mesenchymal transition (EMT) of tumor cells and degradation of the extracellular matrix by matrix metalloproteases (MMPs). Mortalin is an important oncogene. It has been reported to play an important role in tumor migration and invasion through various signaling pathways, but the underlying mechanism is not fully understood. METHODS: Here, we investigated the role of mortalin in the migration of the hepatocellular carcinoma (HCC) cell lines HepG2 and HCCLM3. RESULTS: The overexpression of mortalin in HepG2 cells decreased the protein level of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) and activated the phosphorylation and acetylation of STAT3, thereby up-regulating matrix metalloproteinase 9 (MMP9) and promoting cell migration and invasion. In contrast, in HCCLM3 cells, mortalin knockdown increased the expression of RECK, inhibited the STAT3 pathway and the activity of MMP9, and inhibited cell migration and invasion. Furthermore, we found that salvianolic acid B, a caffeic acid phenethyl ester analog, specifically bound to mortalin and increased the degradation of mortalin proteasomes through ubiquitination, thereby up-regulating RECK, inhibiting STAT3, and finally inhibiting the migration and invasion of HCC cells. CONCLUSION: Our work suggested that mortalin is a potential therapeutic target for hepatocellular carcinoma.

27-Hydroxycholesterol-induced EndMT acts via STAT3 signaling to promote breast cancer cell migration by altering the tumor microenvironment.

Objective: The endothelial to mesenchymal transition (EndMT) plays a major role in cancer metastasis by regulating the complexity of the tumor microenvironment (TME). Here, we investigated whether 27-hydroxycholesterol (27HC) induces EndMT in endothelial cells (ECs). Methods: EndMT markers in the human microvascular endothelial cell-1 (HMEC-1) cell line and human umbilical vein endothelial cells (HUVECs) stimulated with 27HC were evaluated with Western blot. Epithelial to mesenchymal transition (EMT) markers in breast cancer (BC) cells cultured in conditioned medium were investigated with quantitative real time polymerase chain reaction (qRT-PCR). The MMP-2 and MMP-9 mRNA expression and activity were detected with qRT-PCR and gelatin zymography assays, respectively. The effect of activated STAT3 on 27HC-induced EndMT was validated by Western blot, immunofluorescence staining, and cell transfection assays. The migration ability of BC cells was evaluated with Transwell assays. Results: We found that 27HC induced EndMT in HMEC-1 and HUVECs, and 27HC-induced EndMT facilitated EMT and BC cell migration. The 27HC-induced EMT of BC cells also promoted EndMT and HUVEC migration. Investigation of the underlying molecular mechanisms revealed that STAT3 knockdown repressed EndMT in HUVECs as well as migration in BC cells induced with 27HC. In addition, C646 and resveratrol, inhibitors of STAT3 acetylation, repressed the expression of Ac-STAT3, p-STAT3, and EndMT markers in HUVECs exposed to 27HC; these HUVECs in turn attenuated the migration ability of BC cells in 27HC-induced EndMT. Conclusions: Cross-talk between 27HC-induced EndMT and EMT was observed in the TME. Moreover, activation of STAT3 signaling was found to be involved in 27HC-induced EndMT.

Activation of STAT-3 signalling by RECK downregulation via ROS is involved in the 27-hydroxycholesterol-induced invasion in breast cancer cells.

Breast cancer is an important and common tumour among women worldwide. We previously showed that 27-hydroxycholesterol (27HC) promoted the invasion and migration of breast cancer cells and activated signal transducer and activator of transcription 3 (STAT-3) signalling through reactive oxygen species (ROS). However, the regulation of STAT-3 signalling by ROS needs to be further explored. Here, we showed that 27HC caused the accumulation of cellular ROS, which upregulated matrix metalloproteinase 9 (MMP9) and increased the invasive ability of MCF7 and T47D cells. 27HC decreased the protein and mRNA levels of reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) in a time- and dose-dependent manner in MCF7 and T47D cells. RECK downregulation was mediated by 27HC-induced DNA methylation via ROS in MCF7 cells. RECK knockdown increased the activity and mRNA levels of MMP9, and promoted the invasion of MCF7 cells. We also found RECK knockdown upregulated the level of p-STAT-3 in MCF7 cells. Furthermore, overexpression of RECK attenuated 27HC-induced invasion in MCF7 cells. RECK overexpression also inhibited p-STAT-3 upregulation induced by 27HC. Collectively, the results showed that DNA methylation induced by 27HC via ROS downregulated RECK, thereby activating the STAT-3 signalling pathway. RECK could serve as a novel target mediating the effect of 27HC on breast cancer.

Resveratrol inhibits the proliferation of estrogen receptor-positive breast cancer cells by suppressing EZH2 through the modulation of ERK1/2 signaling.

Enhancer of zeste homolog 2 (EZH2) is frequently overexpressed in breast cancer and plays an important role in maintaining the cell proliferative capacity. However, the mechanisms underlying the transcriptional regulation of EZH2 in estrogen receptor (ER)-positive breast cancer cells remain unclear. The antitumor effects of resveratrol have been reported. However, whether EZH2 was involved in these effects needs further exploration. Here, we showed that EZH2 is required for estrogen-induced cell proliferation in ER-positive breast cancer. Exposure to 17ß-estradiol (E2) upregulated EZH2 via ERα signaling, and this effect was blocked by U0126, a MEK inhibiter. Resveratrol inhibited the proliferation and colony formation in ER-positive breast cancer cells and downregulated EZH2 through inhibition of phospho-ERK1/2. These findings indicated that ERK1/2 and ER signaling-mediated EZH2 upregulation is crucial for the proliferation of ER-positive breast cancer cells. The suppression of EZH2 expression by ERK1/2 dephosphorylation is important for the antiproliferative activities of resveratrol against ER-positive breast cancer cells.

Salvianolic acids improve liver lipid metabolism in ovariectomized rats via blocking STAT-3/SREBP1 signaling.

Postmenopausal women, who have reduced circulating estrogen levels, are more prone to develop obesity and related metabolic diseases than premenopausal women. The absence of safe and effective treatments for postmenopausal obesity has changed the focus to natural products as alternative remedies. Total salvianolic acids (TSA) are the major water-soluble ingredients of Danshen. Salvianolic acid (SA) is the major constituent of the TSA. Salvianolic acids, including TSA and SA, are widely used in traditional Chinese medicine. In the present study, ovariectomized rats and LO2 cells were used to study the effects of salvianolic acids on body weight gain and hepatic steatosis. Salvianolic acids reduced ovariectomy (OVX)-induced body weight gain, attenuated the expressions of hepatic lipogenic genes, such as sterol regulatory element binding protein (SREBP)1, fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD)1, and decreased the liver triglyceride (TG) and total cholesterol (TC). For the molecular mechanisms, OVX and high glucose-induced phosphorylation of signal transducer and activator of transcription (STAT)-3 was inhibited by salvianolic acids treatment. In LO2 cells, inhibition of STAT-3 by siRNA attenuated the increased expression of SREBP1 and TG induced by high glucose. Salvianolic acids reduced the upregulation of SREBP1 and TG induced by high glucose in LO2 cells. In conclusion, these findings illustrated that salvianolic acids markedly alleviated the lipid metabolism disorders and protected against the postmenopausal obesity. The underlying mechanism was probably associated with the regulation of STAT-3 signaling.

Blocking of STAT-3/SREBP1-mediated glucose-lipid metabolism is involved in dietary phytoestrogen-inhibited ovariectomized-induced body weight gain in rats.

Postmenopausal women have a decline in circulating estrogen levels and are more prone to obesity and its related metabolic diseases than premenopausal women are. The absence of safe and effective conventional treatments for postmenopausal obesity has changed the focus to natural products as alternative remedies. Here, ovariectomized rats and LO2 cells were used to study the molecular basis of the effect of dietary phytoestrogens on body weight gain and hepatic steatosis. Dietary phytoestrogens can inhibit ovariectomy (OVX)-induced body weight gain, blood glucose concentration, expression of hepatic lipogenic genes, such as sterol regulatory element binding protein (SREBP)1, acetyl-CoA carboxylase (ACC)1, fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD)1, and decrease liver triglyceride (TG) content, but later estradiol withdrawal increased expression of SREBP1. Histological analysis of liver showed that dietary phytoestrogens improved OVX-induced morphological abnormalities. OVX and high glucose-induced phosphorylation of signal transducer and activator of transcription (STAT)-3 were inhibited by phytoestrogens treatment. In LO2 cells, inhibition of STAT-3 by siRNA attenuated the increased TG content and expression of SREBP1 induced by high glucose. Phytoestrogens reduced the upregulation of SREBP1 and TG induced by high glucose in LO2 cells. In conclusion, these findings illustrated that dietary phytoestrogens markedly alleviated the derangement of lipid metabolism. The underlying mechanism is probably associated with regulating STAT-3/SREBP1 signaling.

The Attenuation of 14-3-3ζ is Involved in the Caffeic Acid-Blocked Lipopolysaccharide-Stimulated Inflammatory Response in RAW264.7 Macrophages.

Inflammation plays important roles in the initiation and progress of many diseases. Caffeic acid (CaA) is a naturally occurring hydroxycinnamic acid derivative, which shows hypotoxicity and diverse biological functions, including anti-inflammation. The molecular mechanisms involved in the CaA-inhibited inflammatory response are very complex; generally, the down-regulated phosphorylation of such important transcriptional factors, for example, nuclear factor κB (NF-κB) and signal transducers and activators of transcription-3 (STAT-3), plays an important role. Here, we found that in RAW264.7 macrophage cells, CaA blocked lipopolysaccharide (LPS)-stimulated inflammatory response by attenuating the expression of 14-3-3ζ (a phosphorylated protein regulator). Briefly, the increased expression of 14-3-3ζ was involved in the LPS-induced inflammatory response. CaA blocked the LPS-elevated 14-3-3ζ via attenuating the LPS-induced tumor necrosis factor-α (TNF-α) secretion and via enhancing the 14-3-3ζ ubiquitination. These processes inhibited the LPS-induced activation (phosphorylation) of NF-κB and STAT-3, in turn blocked the transcriptional activation of inducible NO synthase (iNOS), interleukin-6 (IL-6), and TNF-α, and finally attenuated the productions of nitric oxide (NO), IL-6, and TNF-α. By understanding a novel mechanism whereby CaA inhibited the 14-3-3ζ, our study expanded the understanding of the molecular mechanisms involved in the anti-inflammation potential induced by CaA.

The ROS-mediated activation of IL-6/STAT3 signaling pathway is involved in the 27-hydroxycholesterol-induced cellular senescence in nerve cells.

The oxysterol 27-hydroxycholesterol (27HC) is a selective estrogen receptor modulator (SERMs), which like endogenous estrogen 17ß-estradiol (E2) induces the proliferation of ER-positive breast cancer cells in vitro. Interestingly, the observation that 27HC induces adverse effects in neural system, distinguishing it from E2. It has been suggested that high levels of circulating cholesterol increase the entry of 27HC into the brain, which may induce learning and memory impairment. Based on this evidence, 27HC may be associated with neurodegenerative processes and interrupted cholesterol homeostasis in the brain. However, the biological events that participate in this process remain largely elusive. In the present study, we demonstrated that 27HC induced apparent cellular senescence in nerve cells. Senescence-associated ß-galactosidase (SA-ß-Gal) assay revealed that 27HC induced senescence in both BV2 cells and PC12 cells. Furthermore, we demonstrated that 27HC promoted the accumulation of cellular reactive oxygen species (ROS) in nerve cells and subsequently activation of IL-6/STAT3 signaling pathway. Notably, treatment with the ROS scavenger N-acetylcysteine (NAC) markedly blocked 27HC-induced ROS production and activation of IL-6/STAT3 signaling pathway. Either blocking the generation of ROS or inhibition of IL-6/STAT3 both attenuated 27HC-induced cellular senescence. In sum, these findings not only suggested a mechanism whereby 27HC induced cellular senescence in nerve cells, but also helped to recognize the 27HC as a novel harmful factor in neurodegenerative diseases.

The impact of BMI on sperm parameters and the metabolite changes of seminal plasma concomitantly.

The development of male infertility increased rapidly worldwide, which coinciding with the epidemic of obesity. However, the impact of weight abnormalities on sperm quality is still contestable. To assess the correlation between BMI and sperm parameters, we searched relevant articles in PubMed, Embase, Web of science, and Wanfang database published until June 2015 without language restriction. Otherwise, we also recruited some participants who attended fertility clinic as well as some general populations in this report. We performed a systematic review and meta-analysis about BMI and sperm parameters containing total sperm count, concentration, semen volume and sperm motility (overall and progressive). Metabolomic analysis of seminal plasma was performed to explore the mechanism from a new perspective. This study found standardized weighted mean differences (SMD) in sperm parameters (total sperm count, sperm concentration, and semen volume) of abnormal weight groups decreased to different degree compared to normal weight. Dose-response analysis found SMD of sperm count, sperm concentration and semen volume respectively fell 2.4%, 1.3% and 2.0% compared with normal weight for every 5-unit increase in BMI. Metabolomic analysis of seminal plasma showed that spermidine and spermine were likely to play a vital role in the spermatogenesis progress. This systematic review with meta-analysis has confirmed there was a relationship between BMI and sperm quality, suggesting obesity may be a detrimental factor of male infertility.