Genbao Shao

Upregulation of autophagy by hypoxia-inducible factor-1α promotes EMT and metastatic ability of CD133+ pancreatic cancer stem-like cells during intermittent hypoxia

Epithelial-to-mesenchymal transition (EMT) facilitates the escape of pancreatic cancer cells from the primary tumor site, which is a key early event in metastasis. In the present study, we examined if intermittent hypoxia facilitates the invasiveness of human pancreatic cancer cell lines (Panc-1 and BxPC-3) by Transwell assay. We used western blotting and flow cytometry analysis to quantify stem-like cells in the migratory cells during intermittent hypoxia in the human pancreatic cancer cells. Under normoxia or intermittent hypoxia, the expression of autophagy-related proteins (LC3-II and Beclin), hypoxia-inducible factor-1α (HIF-1α) and EMT-related markers (E-cadherin, Vimentin and N-cadherin) was examined by western blotting. siRNA and the autophagic inhibitor were used to access the role of HIF-1α and autophagy in promoting metastasis and EMT. Under intermittent hypoxia, pancreatic cancer cells demonstrated enhanced invasive ability and enriched stem-like cells. The migratory cells displayed stem-like cell characteristics and elevated the expression of LC3-II and Beclin-1, HIF-1α, E-cadherin, Vimentin and N-cadherin under intermittent hypoxia conditions. Moreover, enhanced autophagy was induced by the elevated level of HIF-1α. The metastatic ability and EMT of pancreatic cancer stem cells was associated with HIF-1α and autophagy. This novel finding may indicate the specific role of HIF-1α and autophagy in promoting the metastatic ability of pancreatic cancer stem cells. Additionally, it emphasizes the importance of developing therapeutic strategies targeting cancer stem cells and autophagy to reduce metastasis.

Economical and green synthesis of bagasse- derived fluorescent carbon dots for biomedical applications

Carbon quantum dots (CDs) are promising nanomaterials in biomedical, photocatalytical and photoelectronic applications. However, determining how to explore an ideal precursor for a renewable carbon resource is still an interesting challenge. Here, for the first time, we report that renewable wastes of bagasse as a new precursor were prepared for fluorescent CDs by a hydrothermal carbonization (HTC) process. The characterization results show that such bagasse-derived CDs are monodispersed, contain quasi spherical particles with a diameter of about 1.8 nm and exhibit favorable photoluminescence properties, super-high photostability and good dispersibility in water. Most importantly, bagasse-derived CDs have good biocompatibility and can be easily and quickly internalized by living cancer cells; they can also be used for multicolour biolabeling and bioimaging in cancer cells. It is suggested that bagasse-derived CDs might have potential applications in biomedical and photoelectronic fields.

Role of histone methylation in zygotic genome activation in the preimplantation mouse embryo

Numerous previous studies demonstrated that gene expression was influenced by histone modifications. However, little information is available about the relation of histone methylation with embryonic gene expression. Here, we examine the significance of histone H3 dimethyl-lysine 4 (H3K4me2) during mouse zygotic genome activation (ZGA) by inhibiting demethylation with the specific histone H3 lysine 4 demethylase inhibitor bisguanidine 1c (1c). A 1c treatment of one-cell embryos did not significantly affect the level of eIF-4C transcripts but did affect Oct4 levels by the two-cell stage. Furthermore, 1c treatment significantly inhibited cleavage of the embryos to the four-cell stage (from 82.7% to 18.2%), and the inhibitory effect was identified to be irreversible. These results suggest that histone methylation may be closely correlated with the formation of a transcriptionally repressive state during ZGA and that the repressive state actually dictates the appropriate pattern of gene expression required for further development.

Dynamic patterns of histone H3 lysine 4 methyltransferases and demethylases during mouse preimplantation development

Extensive and dynamic chromatin remodeling occurs after fertilization, including DNA methylation and histone modifications. These changes underlie the transition from gametic to embryonic chromatin and are thought to facilitate early embryonic development. Histone H3 lysine 4 methylation (H3K4me) is an important epigenetic mechanism that associates with gene-specific activation and functions in development. However, dynamic regulation of H3K4me during early embryonic development remains unclear. Herein, the authors examined the dynamic changes of H3K4me and its key regulators (Ash1l, Ash2l, Kmt2a, Kmt2b, Kmt2c, Setd1a, Setd7, Kdm1a, Kdm1b, Kdm5a, Kdm5b, Kdm5c, and Kdm5d) in mouse oocytes and preimplantation embryos. An increase in levels of H3K4me2 and me3 was observed at the one- to two-cell stages (P < 0.05), corresponding to the period of embryonic genome activation (EGA). Subsequently, the H3K4me2 level dramatically decreased at the four-cell stage and remained at low level until the blastocyst stage (P < 0.05), whereas the H3K4me3 level transiently decreased in the four-cell embryos but steadily increased to the peak in the blastocysts (P < 0.05). The high level of H3K4me2 during the EGA was coinciding with a peak expression of its methyltransferase, ASH2L, which may stabilize this methylation level during this period. Correspondingly, a concomitant decrease in levels of its demethylases, KDM5B and KDM1A, was observed. H3K4me3 was correlated to the expression of its methyltransferase (KMT2B) and demethylase (KDM5A). Thus, these enzymes may function for the EGA and the first lineage segregation in preimplantation mouse embryos.

Effect of trychostatin A treatment on gene expression in cloned mouse embryos

Histone deacetylation occurs upon the transfer of somatic nuclei into enucleated oocytes, but its role in reprogramming somatic chromatin to the totipotent state is unknown. To investigate the importance of histone deacetylation in reprogramming, we constructed embryos by electrofusing breast cancer cells with enucleated mouse oocytes. The reconstructed embryos were then cultured before and/or after activation for 6h in the presence of trychostatin A (TSA), a potent inhibitor of histone deacetylase. Total RNA was isolated from these TSA-treated and untreated embryos and real-time reverse transcription PCR was conducted to monitor transcription of ErbB2, Muc1, eIF-4C, MuERV-L, and c-mos genes. The nuclear-cytoplasmic interaction inhibited typical expression of ErbB2 and Muc1 in the somatic cells. Moreover, the inhibition of histone deacetylation prior to activation did not increase the levels of eIF-4C, MuERV-L, and c-mos expression in the nuclear transfer (NT) embryos (P>0.05), whereas additional treatment with 100nM TSA beyond the activation point improved expression of these genes (P<0.05). Trychostatin A treatment also improved the development rates of NT embryos at the 2-cell, 4-cell, and blastocyst stages (78.6% vs. 90.2%, 45.2% vs. 68.9%, and 16.7% vs. 30.3%, respectively, P<0.05). We hypothesized that the reprogramming of gene expression in NT embryos is independent of somatic histone deacetylation, and that hyperacetylation may have a positive effect on NT embryo development.

Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

ABSTRACT The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.

Autophagy contributes to ING4-induced glioma cell death

Previous studies suggest that ING4, a novel member of ING (inhibitor of growth) family, can inhibit brain tumor growth. However, whether autophagy is involved in ING4-induced cell death still remains unknown. In this study, we found that in addition to apoptosis, autophagy also contributed to cell death induced by ING4. Autophagy levels were elevated following the exposure to Ad-ING4, including enhanced fluorescence intensity of monodansylcadervarine (MDC), a specific in vivo marker for autophagic vacuoles, and increased expression levels of the LC3-II and Beclin-1, wheras the autophagic levels were attenuated following the pretreatment of 3-MA, the inhibitor of autophagy, which significantly decreased the Ad-ING4-induced cell death compared with caspase inhibitor zVAD. Furthermore, ING4 also induced mitochondrial dysfunction, such as mitophagy, collapse of mitochondrial membrane potential and the intracellular ROS, which indicated that mitochondria might be associated with the process of autophagic cell death of glioma cells. Finally, the relationship among Bax, Bcl-2, Beclin-1 and caspase family proteins levels were analyzed in glioma cells U251MG and LN229 infected with Ad-ING4 or Ad-lacZ. It is suggested that both autophagy and apoptosis could contribute to ING4-induced glioma cell death, and mitochondria might play an important role in this process. Our findings reveal novel aspects of the autophagy in glioma cells that underlie the cytotoxic action of ING4, possibly providing new insights in the development of combinatorial therapies for gliomas.

Lysine-specific demethylase 1 mediates epidermal growth factor signaling to promote cell migration in ovarian cancer cells

Epigenetic abnormalities play a vital role in the progression of ovarian cancer. Lysine-specific demethylase 1 (LSD1/KDM1A) acts as an epigenetic regulator and is overexpressed in ovarian tumors. However, the upstream regulator of LSD1 expression in this cancer remains elusive. Here, we show that epidermal growth factor (EGF) signaling upregulates LSD1 protein levels in SKOV3 and HO8910 ovarian cancer cells overexpressing both LSD1 and the EGF receptor. This effect is correlated with a decrease in the dimethylation of H3K4, a major substrate of LSD1, in an LSD1-dependent manner. We also show that inhibition of PI3K/AKT, but not MEK, abolishes the EGF-induced upregulation of LSD1 and cell migration, indicating that the PI3K/PDK1/AKT pathway mediates the EGF-induced expression of LSD1 and cell migration. Significantly, LSD1 knockdown or inhibition of LSD1 activity impairs both intrinsic and EGF-induced cell migration in SKOV3 and HO8910 cells. These results highlight a novel mechanism regulating LSD1 expression and identify LSD1 as a promising therapeutic target for treating metastatic ovarian cancer driven by EGF signaling.

Effects of hyperoxia on transdifferentiation of primary cultured typeII alveolar epithelial cells from premature rats

Hyperoxia exposure is a significant risk factor for the impaired alveolarization characteristic of bronchopulmonary dysplasia. Type II alveolar epithelial cells (AECIIs) may serve as “alveolar stem cells” to transdifferentiate into type I alveolar epithelial cells (AECIs). Here, we show that hyperoxia is capable of inducing transdifferentiation of AECIIs in premature rats in vitro. Hyperoxia-induced transdifferentiation was characterized by typical morphological changes, inhibition of cellular proliferation, decline in expression rate of Ki67, accumulation of cells in the G1 phase of the cell cycle, increased expression of AECI-specific protein aquaporin 5, and decreased expression of AECII-associated protein surfactant protein C. These results suggest that hyperoxia may induce transdifferentiation of AECIIs into AECIs and the transdifferentiation may be responsible for repairing early lung injury.