Dual Proton/Silver-Catalyzed Serial (5 + 2)-Cycloaddition and Nazarov Cyclization of (E)-2-Arylidene-3-hydroxyindanones with Conjugated Eneynes: Synthesis of Indanone-Fused Benzo[cd]azulenes.

A one-pot step-economic tandem process involving (5 + 2)-cycloaddition and Nazarov cyclization reactions has been reported for the facile synthesis of indanone-fused benzo[cd]azulenes from (E)-2-arylidene-3-hydroxyindanones and conjugated eneynes. This highly regio- and stereoselective bisannulation reaction is enabled by dual silver and Brønsted acid catalysis and opens up a new avenue for the construction of important bicyclo[5.3.0]decane skeletons.

Dickkopf-1 drives tumor immune evasion by inducing PD-L1 expression in hepatocellular carcinoma.

Understanding the mechanisms regulating PD-L1 expression in hepatocellular carcinoma (HCC) is important to improve the response rate to PD-1/PD-L1 blockade therapy. Here, we show that DKK1 expression is positively associated with PD-L1 expression and inversely correlated with CD8+ T cell infiltration in human HCC tumor specimens. In a subcutaneous xenograft tumor model, overexpression of DKK1 significantly promotes tumor growth, tumoral PD-L1 expression, but reduces tumoral CD8+ T cell infiltration; whereas knockdown of DKK1 has opposite effects. Moreover, enforced expression of DKK1 dramatically promotes PD-L1 expression, Akt activation, ß-catenin phosphorylation and total protein expression in HCC cells. By contrast, knockdown of DKK1 inhibits all, relative to controls. In addition, CKAP4 depletion, Akt inhibition, or ß-catenin depletion remarkably abrogates DKK1 overexpression-induced transcriptional expression of PD-L1 in HCC cells. Reconstituted expression of the active Akt1 largely increased PD-L1 transcriptional expression in HCC cells. Similarly, expression of WT ß-catenin, but not the phosphorylation-defective ß-catenin S552A mutant, significantly promotes PD-L1 expression. Correlation analysis of human HCC tumor specimens further revealed that DKK1 and PD-L1 expression were positively correlated with p-ß-catenin expression. Together, our findings revealed that DKK1 promotes PD-L1 expression through the activation of Akt/ß-catenin signaling, providing a potential strategy to enhance the clinical efficacy of PD-1/PD-L1 blockade therapy in HCC patients.

HGF/c-MET pathway contributes to cisplatin-mediated PD-L1 expression in hepatocellular carcinoma.

Cisplatin has been reported to promote the expression of programmed cell death ligand-1 (PD-L1) in some cancer cells. However, the underlying mechanism through which PD-L1 is transcriptionally regulated by cisplatin in hepatocellular carcinoma (HCC) cells remains largely unknown. In the present study, we found that the expression of hepatocyte growth factor (HGF), p-Akt, p-ERK, and PD-L1 was increased in cisplatin-treated SNU-368 and SNU-739 cells. HGF stimulation also increased PD-L1 expression in these cells. Moreover, Inhibition of HGF/c-MET, PI3K/Akt, and MEK/ERK signaling pathways can dramatically block cisplatin or HGF-induced PD-L1 expression in SNU-368 and SNU-739 cells. In vivo, combination PHA665752 with cisplatin significantly reduced tumor weight with increased infiltration of CD8+ T cells in the tumor. Taken together, our study suggested that HGF/c-Met axis-induced the activation of PI3K/Akt and MEK/ERK pathways contributes to cisplatin-mediated PD-L1 expression. These findings may provide an alternative avenue for the treatment of HCC.

Corrigendum: PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC.

[This corrects the article DOI: 10.3389/fphar.2020.577108.].

P300-dependent acetylation of histone H3 is required for epidermal growth factor receptor-mediated high-mobility group protein A2 transcription in hepatocellular carcinoma.

High-mobility group protein A2 (HMGA2) is highly expressed in hepatocellular carcinoma (HCC) cells and contributes to tumor metastasis and poor patient survival. However, the molecular mechanism through which HMGA2 is transcriptionally regulated in HCC cells remains largely unclear. Here, we showed that the expression HMGA2 was upregulated in HCC, and that elevated HMGA2 could promote tumor metastasis. Incubation of HCC cells with epidermal growth factor (EGF) could promote the expression of HMGA2 mRNA and protein. Mechanistic studies suggested that EGF can phosphorylate p300 at Ser1834 residue through the PI3K/Akt signaling pathway in HCC cells. Knockdown of p300 can reverse EGF-induced HMGA2 expression and histone H3-K9 acetylation, whereas a phosphorylation-mimic p300 S1834D mutant can stimulate HMGA2 expression as well as H3-K9 acetylation in HCC cells. Furthermore, we identified that p300-mediated H3-K9 acetylation participates in EGF-induced HMGA2 expression in HCC. In addition, the levels of H3-K9 acetylation positively correlated with the expression levels of HMGA2 in a chemically induced HCC model in rats and human HCC specimens.

PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC.

High expression of programmed death-ligand-1 (PD-L1) in hepatocellular carcinoma (HCC) cells usually inhibits the proliferation and functions of T cells, leading to immune suppression in tumor microenvironment. However, very little has been described regarding the mechanism of PD-L1 overexpression in HCC cells. In the present study, we found epidermal growth factor (EGF) stimulation promoted the expression of PD-L1 mRNA and protein in HCC cells. Inhibition of epidermal growth factor receptor (EGFR) could reverse EGF-induced the expression of PD-L1 mRNA and protein. Subsequently, we also observed that the phosphorylation level of Pyruvate kinase isoform M2 (PKM2) at Ser37 site was also increased in response to EGF stimulation. Expression of a phosphorylation-mimic PKM2 S37D mutant stimulated PD-L1 expression as well as H3-Thr11 phosphorylation in HCC cells, while inhibition of PKM2 significantly blocked EGF-induced PD-L1 expression and H3-Thr11 phosphorylation. Furthermore, mutation of Thr11 of histone H3 into alanine abrogated EGF-induced mRNA and protein expression of PD-L1, Chromatin immunoprecipitation (ChIP) assay also suggested that EGF treatment resulted in enhanced H3-Thr11 phosphorylation at the PD-L1 promoter. In a diethylnitrosamine (DEN)-induced rat model of HCC, we found that the expression of phosphorylated EGFR, PKM2 nuclear expression, H3-Thr11 phosphorylation as well as PD-L1 mRNA and protein was higher in the livers than that in normal rat livers. Taken together, our study suggested that PKM2-dependent histone H3-Thr11 phosphorylation was crucial for EGF-induced PD-L1 expression at transcriptional level in HCC. These findings may provide an alternative target for the treatment of hepatocellular carcinoma.

EGF promotes DKK1 transcription in hepatocellular carcinoma by enhancing the phosphorylation and acetylation of histone H3.

The protein Dickkopf-1 (DKK1) is frequently overexpressed at the transcript level in hepatocellular carcinoma (HCC) and promotes metastatic progression through the induction of ß-catenin, a Wnt signaling effector. We investigated how DKK1 expression is induced in HCC and found that activation of the epidermal growth factor receptor (EGFR) promoted parallel MEK-ERK and PI3K-Akt pathway signaling that converged to epigenetically stimulate DKK1 transcription. In HCC cell lines stimulated with EGF, EGFR-activated ERK phosphorylated the kinase PKM2 at Ser37, which promoted its nuclear translocation. Also in these cells, EGFR-activated Akt phosphorylated the acetyltransferase p300 at Ser1834 Subsequently, PKM2 and p300 mediated the phosphorylation and acetylation, respectively, of histone H3 at the DKK1 promoter, which synergistically enhanced DKK1 transcription. The mechanism was supported with mutational analyses in cells and in a chemically induced HCC model in rats. The findings suggest that dual inhibition of the MEK and PI3K pathways might suppress the expression of DKK1 and, consequently, tumor metastasis in patients with HCC.

Wnt5b/Ryk-mediated membrane trafficking of P2X3 receptors contributes to bone cancer pain.

Wnt5b, a member of Wnt family, plays multiple roles in tumor progression and metastasis. However, whether Wnt5b contributes to the sensitization of dorsal root ganglia (DRG) neurons and pathogenesis of bone cancer pain still remains unclear. Here, we found that the protein expression of Wnt5b and its atypical tyrosine protein kinase receptor Ryk was upregulated in ipsilateral DRGs in tumor-bearing mice. Application of Wnt5b evoked an increased discharge frequency in isolated DRG neurons and pain hypersensitivity in naïve mice which were almost completely prevented by anti-Ryk antibody. Moreover, intrathecal injection of anti-Ryk antibody to tumor-bearing mice significantly inhibited bone cancer-induced mechanic allodynia and thermal hyperalgesia. Subsequently, we also demonstrated that application of Wnt5b to cultured DRG neurons could enhance membrane P2X3 receptors and α,ß-meATP-induced currents. Intrathecal injection of calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 or P2X3 receptors antagonist A317491 almost completely abolished Wnt5b-induced mechanical allodynia and thermal hyperalgesia in mice. Meanwhile, pretreatment with anti-Ryk antibody or CaMKII inhibitor KN93 can attenuate bone-cancer induced the upregulation of P2X3 membrane protein as well as pain hypersensitivity. These findings suggested that Wnt5b/Ryk promoted the trafficking of P2X3 receptors to the membrane via the activation of CaMKII in primary sensory neurons, resulting in peripheral sensitization and bone cancer-induced pain. Our results may offer a potential therapeutic strategy for bone cancer pain.

Antihypertensive Activity of Eucommia Ulmoides Oliv: Male Flower Extract in Spontaneously Hypertensive Rats.

Eucommia ulmoides Oliv. is a traditional medical plant in Asia; however, it is still unknown whether Eucommia male flowers have an antihypertensive activity. In this study, we found that the aqueous extract of Eucommia ulmoides Oliv. male flowers can lower the blood pressure of SHR in a dose-dependent manner. Mechanistic studies suggested that the aqueous extract of male flowers can promote the mRNA and protein expressions of ACE2 in the kidney of SHR. ELISA assay showed that the plasma levels of ANG II was decreased, while ANG-(1-7) was increased in SHR treated with the aqueous extract of male flowers. ACE2 inhibitor DX600 can reverse the aqueous extract of Eucommia ulmoides Oliv. male flower-induced downregulation of Ang II and upregulation of Ang-(1-7), as well as the reduction of blood pressure in SHR. Moreover, Ang-(1-7)-Mas receptor antagonist A-779 abolished the antihypertensive effects of the aqueous extract of Eucommia ulmoides Oliv. male flower in SHR. The aqueous extract of Eucommia ulmoides Oliv. male flowers exhibited an antihypertensive action through the activation of ACE2-Ang-(1-7)-Mas signaling pathways in spontaneously hypertensive rats.

FOXC1 up-regulates the expression of toll-like receptors in myocardial ischaemia.

Myocardial ischaemia (MI) remains a major cause of death and disability worldwide. Accumulating evidence suggests a significant role for innate immunity, in which the family of toll-like receptors (TLRs) acts as an essential player. We previously reported and reviewed the changes of Tlr expression in models of MI. However, the underlying mechanisms regulating Tlr expression in MI remain unclear. The present study first screened transcription factors (TFs) that potentially regulate Tlr gene transcription based on in silico analyses followed by experimental verification, using both in vivo and in vitro models. Forkhead box C1 (FOXC1) was identified as a putative TF, which was highly responsive to MI. Next, by focusing on two representative TLR subtypes, an intracellular subtype TLR3 and a cell-surface subtype TLR4, the regulation of FOXC1 on Tlr expression was investigated. The overexpression or knockdown of FoxC1 was observed to up- or down-regulate Tlr3/4 mRNA and protein levels, respectively. A dual-luciferase assay showed that FOXC1 trans-activated Tlr3/4 promoter, and a ChIP assay showed direct binding of FOXC1 to Tlr3/4 promoter. Last, a functional study of FOXC1 was performed, which revealed the pro-inflammatory effects of FOXC1 and its destructive effects on infarct size and heart function in a mouse model of MI. The present study for the first time identified FOXC1 as a novel regulator of Tlr expression and described its function in MI.

Interleukin enhancement binding factor 3 inhibits cardiac hypertrophy by targeting asymmetric dimethylarginine-nitric oxide.

Persistent cardiac hypertrophy eventually leads to deterioration of heart function and changes to normal morphology. Decreased nitric oxide (NO) production plays a critical role in modulating cardiac hypertrophy. Interleukin enhancement binding factor 3 (ILF3), a member of the double-stranded RNA-binding protein family, is known to regulate the transcription and stability of mRNA. Therefore, the major aim of the present study was to determine the role of ILF3 in reduction of NO production in cardiac hypertrophy. Cardiac hypertrophy models of neonatal rat cardiomyocytes (NRCMs) and adult rats were induced by angiotensin II (Ang II) in this study. First, it was found that ILF3 expression, NO production, and nitric oxide synthase (NOS) activity was decreased in cultured cardiomyocytes and adult rats treated with Ang II, compared with NRCMs treated with vehicle and rats treated with saline infusion, respectively. These effects induced by Ang II were significantly exacerbated by specific ILF3 knockdown. Moreover, the level of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS, was increased significantly in the Ang II-induced hypertrophic NRCMs and adult rats. Additionally, decreased protein expression and mRNA level of dimethylarginine dimethylaminohydrolases 1 (DDAH1, which degrades ADMA) were observed. Furthermore, specific ILF3 knockdown further aggravated these effects, but didn't reduce the expression level of NOS isoforms. In conclusion, our data show that ADMA accumulation-mediated decrease in NO production plays an important role in cardiomyocyte remodeling, which may be associated with ILF3-mediated DDAH1 reduction.

Author Correction: Identifying long-term stable refugia for relict plant species in East Asia.

In the original version of this Article, an incorrect sample size was provided for the number of relict species (443 instead of 442) and the number of relict forests (423 instead of 422). These errors have been corrected in both the PDF and HTML versions of the Article.

Identifying long-term stable refugia for relict plant species in East Asia.

Today East Asia harbors many "relict" plant species whose ranges were much larger during the Paleogene-Neogene and earlier. The ecological and climatic conditions suitable for these relict species have not been identified. Here, we map the abundance and distribution patterns of relict species, showing high abundance in the humid subtropical/warm-temperate forest regions. We further use Ecological Niche Modeling to show that these patterns align with maps of climate refugia, and we predict species' chances of persistence given the future climatic changes expected for East Asia. By 2070, potentially suitable areas with high richness of relict species will decrease, although the areas as a whole will probably expand. We identify areas in southwestern China and northern Vietnam as long-term climatically stable refugia likely to preserve ancient lineages, highlighting areas that could be prioritized for conservation of such species.

Potential effects of climate change on geographic distribution of the Tertiary relict tree species Davidia involucrata in China.

This study, using species distribution modeling (involving a new approach that allows for uncertainty), predicts the distribution of climatically suitable areas prevailing during the mid-Holocene, the Last Glacial Maximum (LGM), and at present, and estimates the potential formation of new habitats in 2070 of the endangered and rare Tertiary relict tree Davidia involucrata Baill. The results regarding the mid-Holocene and the LGM demonstrate that south-central and southwestern China have been long-term stable refugia, and that the current distribution is limited to the prehistoric refugia. Given future distribution under six possible climate scenarios, only some parts of the current range of D. involucrata in the mid-high mountains of south-central and southwestern China would be maintained, while some shift west into higher mountains would occur. Our results show that the predicted suitable area offering high probability (0.5‒1) accounts for an average of only 29.2% among the models predicted for the future (2070), making D. involucrata highly vulnerable. We assess and propose priority protected areas in light of climate change. The information provided will also be relevant in planning conservation of other paleoendemic species having ecological traits and distribution ranges comparable to those of D. involucrata.