Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC. AIM OF THE STUDY: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated. MATERIALS AND METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1ß and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot. RESULTS: After administration of BEPD-containing serum, the levels of IL-1ß, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins. CONCLUSIONS: BEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

p63, a key regulator of Ago2, links to the microRNA-144 cluster.

As a key component of the RNA-induced silencing complex (RISC), Argonaute2 (Ago2) exhibits a dual function regulatory role in tumor progression. However, the mechanistic basis of differential regulation remains elusive. p63 is a homolog of the tumor suppressor p53. p63 isoforms play a critical role in tumorigenesis and metastasis. Herein, we show that p63 isoforms physically interact with and stabilize Ago2. Expression of p63 isoforms increases the levels of Ago2 protein, while depletion of p63 isoforms by shRNA decreases Ago2 protein levels. p63 strongly guides Ago2 dual functions in vitro and in vivo. Ectopic expression of the miR-144/451 cluster increases p63 protein levels; TAp63 transactivates the miR-144/451 cluster, forming a positive feedback loop. Notably, miR-144 activates p63 by directly targeting Itch, an E3 ligase of p63. Ectopic expression of miR-144 induces apoptosis in H1299 cells. miR-144 enhances TAp63 tumor suppressor function and inhibits cell invasion. Our findings uncover a novel function of p63 linking the miRNA-144 cluster and the Ago2 pathway. FACTS AND QUESTIONS: Identification of Ago2 as a p63 target. Ago2 exhibits a dual function regulatory role in tumor progression; however, the molecular mechanism of Ago2 regulation remains unknown. p63 strongly guides Ago2 dual functions in vitro and in vivo. Unraveling a novel function of p63 links the miRNA-144 cluster and the Ago2 pathway.

Hsp70 acts as a fine-switch that controls E3 ligase CHIP-mediated TAp63 and ΔNp63 ubiquitination and degradation.

The major clinical problem in human cancer is metastasis. Metastases are the cause of 90% of human cancer deaths. TAp63 is a critical suppressor of tumorigenesis and metastasis. ΔNp63 acts as a dominant-negative inhibitor to block the function of p53 and TAp63. Although several ubiquitin E3 ligases have been reported to regulate p63 stability, the mechanism of p63 regulation remains partially understood. Herein, we show that CHIP, an E3 ligase with a U-box domain, physically interacts with p63 and promotes p63 degradation. Notably, Hsp70 depletion by siRNA stabilizes TAp63 in H1299 cells and destabilizes ΔNp63 in SCC9 cells. Loss of Hsp70 results in a reduction in the TAp63-CHIP interaction in H1299 cells and an increase in the interaction between ΔNp63 and CHIP in SCC9 cells. Our results reveal that Hsp70 acts as a molecular switch to control CHIP-mediated ubiquitination and degradation of p63 isoforms. Furthermore, regulation of p63 by the Hsp70-CHIP axis contributes to the migration and invasion of tumor cells. Hence, our findings demonstrate that Hsp70 is a crucial regulator of CHIP-mediated ubiquitination and degradation of p63 isoforms and identify a new pathway for maintaining TAp63 or ΔNp63 stability in cancers.

Plasmodium infection inhibits tumor angiogenesis through effects on tumor-associated macrophages in a murine implanted hepatoma model.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death in China. The lack of an effective treatment for this disease results in a high recurrence rate in patients who undergo radical tumor resection, and the 5-year survival rate of these patients remains low. Our previous studies demonstrated that Plasmodium infection provides a potent antitumor effect by inducing innate and adaptive immunity in a murine Lewis lung carcinoma (LLC) model. METHODS: This study aimed to investigate the inhibitory effect of Plasmodium infection on hepatocellular carcinoma in mice, and various techniques for gene expression analysis were used to identify possible signal regulation mechanisms. RESULTS: We found that Plasmodium infection efficiently inhibited tumor progression and prolonged survival in tumor-bearing mice, which served as a murine implanted hepatoma model. The inhibition of tumor progression by Plasmodium infection was related to suppression of tumor angiogenesis within the tumor tissue and decreased infiltration of tumor-associated macrophages (TAMs). Further study demonstrated that matrix metalloprotease 9 (MMP-9) produced by TAMs contributed to tumor angiogenesis in the tumor tissue and that the parasite-induced reduction in MMP-9 expression in TAMs resulted in the suppression of tumor angiogenesis. A mechanistic study revealed that the Plasmodium-derived hemozoin (HZ) that accumulated in TAMs inhibited IGF-1 signaling through the PI3-K and MAPK signaling pathways and thereby decreased the expression of MMP-9 in TAMs. CONCLUSIONS: Our study suggests that this novel approach of inhibiting tumor angiogenesis by Plasmodium infection is of high importance for the development of new therapies for cancer patients. Video abstract.

MicroRNA-498 promotes proliferation and migration by targeting the tumor suppressor PTEN in breast cancer cells.

Triple negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis and high mortality rate. The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays an important role in cell proliferation and cell migration by negatively regulating the PI3K/Akt pathway. PTEN is downregulated by microRNAs in multiple cancers. However, few microRNAs have been reported to directly target PTEN in TNBC. In this study, microRNAs predicted to target PTEN were screened by immunoblotting and luciferase reporter assays. Expression levels of microRNA-498 (miR-498) were measured by TaqMan microRNA assays. We performed clonogenic, cell cycle and scratch wound assays to examine the oncogenic role of miR-498. We demonstrated that miR-498 directly targeted the 3'untranslated region of PTEN mRNA and reduced PTEN protein levels in TNBC cells. Compared with the non-tumorigenic breast epithelial cell line MCF-10A, TNBC cell lines overexpressed miR-498. Moreover, miR-498 promoted cell proliferation and cell cycle progression in TNBC cells in a PTEN-dependent manner. Suppressing miR-498 overexpression impaired the oncogenic effects of miR-498 on cell proliferation and cell migration. This study identified a novel microRNA (miR-498) overexpressed in TNBC cells and its oncogenic role in suppressing PTEN. These results provide new insight into the downregulation of PTEN and indicate a potential therapeutic target for treating TNBC.

MicroRNA-1301 suppresses tumor cell migration and invasion by targeting the p53/UBE4B pathway in multiple human cancer cells.

The p53 protein plays a critical role in preventing tumor development. Although numerous factors have been shown to directly or indirectly regulate p53, the mechanism of how microRNAs (miRNAs) modulate p53 remains unclear. Here, we identified miR-1301, a microRNA that regulates the activity and function of p53, by directly targeting the ubiquitination factor E4B (UBE4B), an E3 and E4 ubiquitin ligase. Notably, ectopic expression of miR-1301 inhibits dissemination and metastasis of tumor cells in a p53-dependent manner. Depletion of miR-1301 downregulates p53 function. Our results reveal that there is an inverse correlation between miR-1301 and UBE4B expression and p53 status in prostate cancer. Furthermore, low miR-1301 expression is often associated with incomplete methylation of its gene in human prostate tumors. Together, our results provide the first report indicating that miR-1301 functions as a tumor suppressor that inhibits tumor cell migration and invasion in multiple human cancer cells by regulating the UBE4B-p53 pathway.

The Regulation of Tumor Suppressor p63 by the Ubiquitin-Proteasome System.

The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is capable of inducing apoptosis, cell cycle arrest, or senescence. p63 has at least six isoforms, which can be divided into two major groups: the TAp63 variants that contain the N-terminal transactivation domain and the ΔNp63 variants that lack the N-terminal transactivation domain. The TAp63 variants are generally considered to be tumor suppressors involved in activating apoptosis and suppressing metastasis. ΔNp63 variants cannot induce apoptosis but can act as dominant negative inhibitors to block the function of TAp53, TAp73, and TAp63. p63 is rarely mutated in human tumors and is predominately regulated at the post-translational level by phosphorylation and ubiquitination. This review focuses primarily on regulation of p63 by the ubiquitin E-3 ligase family of enzymes via ubiquitination and proteasome-mediated degradation, and introduces a new key regulator of the p63 protein.

Transition of tumor-associated macrophages from MHC class II(hi) to MHC class II(low) mediates tumor progression in mice.

BACKGROUND: Tumor-associated macrophages (TAMs) are the most abundant immune cells within the tumor stroma and play a crucial role in tumor development. Although clinical investigations indicate that high levels of macrophage (MΦ) infiltration into tumors are associated with a poor prognosis, the exact role played by TAMs during tumor development remains unclear. The present study aimed to investigate dynamic changes in TAM major histocompatibility complex (MHC) class II expression levels and to assess the effects of these changes on tumor progression. RESULTS: Significant inhibition of tumor growth in the murine hepatocellular carcinoma Hepa1-6 model was closely associated with partial TAM depletion. Strikingly, two distinct TAM subsets were found to coexist within the tumor microenvironment during Hepa1-6 tumor development. An MHC class II(hi) TAM population appeared during the early phase of tumor development and was associated with tumor suppression; however, an MHC class II(low) TAM population became increasingly predominant as the tumor progressed. CONCLUSIONS: Tumor progression was positively correlated with increasing infiltration of the tumor tissues by MHC class II(low) TAMs. Thus, targeting the transition of MΦ may be a novel strategy for drug development and immunotherapy.