Investigating the efficacy and mechanisms of Jinfu'an decoction in treating non-small cell lung cancer using network pharmacology and in vitro and in vivo experiments.

ETHNOPHARMACOLOGICAL RELEVANCE: Jinfu'an Decoction (JFAD) is a traditional Chinese decoction used in lung cancer treatment to improve patient quality of life and survival. Previous research has established that JFAD has a significant therapeutic effect on non-small cell lung cancer (NSCLC), although the underlying molecular mechanisms have not been largely underexplored. AIM OF THE STUDY: We used network pharmacology to identify the putative active ingredients of JFAD and conducted experimental studies to determine the potential molecular mechanism of JFAD in NSCLC treatment. MATERIALS AND METHODS: The herbal components in JFAD-containing serum were identified by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS), and targets associated with the anti-lung cancer metastasis effects of JFAD were retrieved from various databases. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Next, the protein-protein interactions network and the "JFAD-Chemical Component-Target-KEGG Pathway" network were constructed. The network pharmacology findings were confirmed by in vitro and in vivo experiments. In vitro experiments were conducted to assess cell viability by CCK8 assay, cell cycle analysis by propidium iodide (PI) assay, and migration and invasion ability of cells by the transwell assay. In vivo experiments were performed to assess the efficacy of JFAD on the tumor by observing the growth of transplanted tumor models in nude mice and evaluated by in vivo bioluminescence imaging. Moreover, we assessed the effect of JFAD on the PI3K/Akt signaling pathway and proteins of Lumican, p120ctn, and specific RhoGTP enzyme family members (RhoA, Rac1, and RhoC) by Western Blot and immunohistochemistry. RESULTS: 32 herbal components were identified in the JFAD-containing serum, which potentially acted on 229 targets related to lung cancer metastasis. Network pharmacology results suggested that JFAD may treat lung cancer metastasis by targeting the PI3K/Akt pathway via regulating multiple core targets. Our experiments showed that JFAD suppressed the proliferation of A549 cells in vitro, induced cell cycle arrest, and reduced the migration and invasion ability of A549 cells. Our in vivo study revealed that JFAD inhibited tumor growth in a nude mouse model. Additionally, we found that JFAD could downregulate the expression of the PI3K/Akt pathway and affect the expression of Lumican, p120ctn, and specific RhoGTPase family members. CONCLUSIONS: In conclusion, through network pharmacology, we have unveiled the underlying mechanisms that link the various components, targets, and pathways influenced by JFAD in the context of lung cancer metastasis. Our experimental results suggest that the oncostatic effects of JFAD may be achieved by upregulating the expression of Lumican/p120ctn and downregulating the levels of specific RhoGTPase family members, which in turn block the PI3K/Akt signaling pathway.

Repurposing cancer drugs identifies kenpaullone which ameliorates pathologic pain in preclinical models via normalization of inhibitory neurotransmission.

Inhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expression­enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.

An LTR Retrotransposon-Derived Long Noncoding RNA lncMER52A Promotes Hepatocellular Carcinoma Progression by Binding p120-Catenin.

Long terminal repeat (LTR) retrotransposons are a major class of transposable elements, accounting for 8.67% of the human genome. LTRs can serve as regulatory sequences and drive transcription of tissue or cancer-specific transcripts. However, the role of these LTR-activated transcripts, especially long non-coding RNAs (lncRNA), in cancer development remains largely unexplored. Here, we identified a novel lncRNA derived from MER52A retrotransposons (lncMER52A) that was exclusively expressed in hepatocellular carcinoma (HCC). HCC patients with higher lncMER52A had advanced TNM stage, less differentiated tumors, and shorter overall survival. LncMER52A promoted invasion and metastasis of HCC cells in vitro and in vivo. Mechanistically, lncMER52A stabilized p120-catenin and triggered the activation of Rho GTPase downstream of p120-catenin. Furthermore, we found that chromatin accessibility was crucial for the expression of lncMER52A. In addition, YY1 transcription factor bound to the cryptic MER52A LTR promoter and drove lncMER52A transcription in HCC. In conclusion, we identified an LTR-activated lncMER52A, which promoted the progression of HCC cells via stabilizing p120-catenin and activating p120-ctn/Rac1/Cdc42 axis. LncMER52A could serve as biomarker and therapeutic target for patients with HCC. SIGNIFICANCE: A novel long noncoding RNA lncMER52 modulates cell migration and invasion via posttranslational control of p120-catenin protein stability. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/5/976/F1.large.jpg.

Jinfu'an Decoction Inhibits Invasion and Metastasis in Human Lung Cancer Cells (H1650) via p120ctn-Mediated Induction and Kaiso.

BACKGROUND Previous research showed that Jin-Fu-An decoction has a significant effect on lung cancer. However, it remains unclear whether p120ctn and its transcription factor Kaiso play a role in lung cancer cell proliferation, adhesion, migration, and metastasis. MATERIAL AND METHODS Proliferation inhibition was detected by CCK-8 assay. The migration and invasion were detected using Transwell assay. The location and expression of p120ctn and Kaiso were monitored by immunofluorescence staining. The expression changes of p120ctn, its isoform 1A, its S288 phosphorylation, and Kaiso were measured by Western blot assay. RESULTS The lung cancer cell line H1650 administered Jin-Fu-An decoction had significantly reduced the growth in dose-dependent and time-dependent manners. Migration and metastasis were significantly inhibited by application of Jin-Fu-An decoction in a dose-dependent manner. Additionally, Jin-Fu-An decoction decreased the expressions of p120ctn, its isoform 1A, and its S288 phosphorylation, but the protein level of Kaiso was elevated. CONCLUSIONS Jin-Fu-An decoction inhibits the proliferation, adhesion, migration, and metastasis though down-regulation of p120ctn or its isoform 1A expression, mediating the up-regulation of Kaiso. The underlying mechanism of Jin-Fu-An decoction might involve targeting the lower expression of p120ctn S288 phosphorylation, which suggests that Jin-Fu-An decoction may be a potential therapeutic measure as prevention and control of recurrence and metastasis of lung cancer.

p120-Catenin Is Required for Dietary Calcium Suppression of Oral Carcinogenesis in Mice.

Previous studies have shown that dietary calcium suppresses oral carcinogenesis, but the mechanism is unclear. p120-catenin (p120) is a cytoplasmic protein closely associated with E-cadherin to form the E-cadherin-ß-catenin complex and may function as a tumor suppressor in the oral epithelium. To determine whether p120 is involved in the mechanism by which dietary calcium suppresses oral carcinogenesis, The normal, low, or high calcium diet was fed control mice (designated as floxed p120 mice) or mice in which p120 was specifically deleted in the oral squamous epithelium during the adult stage (designated as p120cKO mice). All mice were exposed to a low dose of oral cancer carcinogen 4-nitroquinoline 1-oxide and rates of oral squamous cell carcinoma (OSCC) and proliferation and differentiation in the cancerous and non-cancerous oral epithelium of these mice were examined. The results showed that the low calcium diet increased rates of OSCC and proliferation of the non-cancerous oral epithelium and decreased differentiation of the non-cancerous oral epithelium, but had no effect on cancerous oral epithelium. In contrast, the high calcium diet had opposite effects. However, the effect of the dietary calcium on the rates of OSCC, proliferation, and differentiation of the non-cancerous epithelium were not seen in p120cKO mice. Based on these results, we conclude that p120 is required for dietary calcium suppression of oral carcinogenesis and oral epithelial proliferation and dietary calcium induction of oral epithelial differentiation. J. Cell. Physiol. 232: 1360-1367, 2017. © 2016 Wiley Periodicals, Inc.

Continuous association of cadherin with beta-catenin requires the non-receptor tyrosine-kinase Fer.

The function of Type 1, classic cadherins depends on their association with the actin cytoskeleton, a connection mediated by alpha- and beta-catenin. The phosphorylation state of beta-catenin is crucial for its association with cadherin and thus the association of cadherin with the cytoskeleton. We now show that the phosphorylation of beta-catenin is regulated by the combined activities of the tyrosine kinase Fer and the tyrosine phosphatase PTP1B. Fer phosphorylates PTP1B at tyrosine 152, regulating its binding to cadherin and the continuous dephosphorylation of beta-catenin at tyrosine 654. Fer interacts with cadherin indirectly, through p120ctn. We have mapped the interaction domains of Fer and p120ctn and peptides corresponding to these sequences release Fer from p120ctn in vitro and in live cells, resulting in loss of cadherin-associated PTP1B, an increase in the pool of tyrosine phosphorylated beta-catenin and loss of cadherin adhesion function. The effect of the peptides is lost when a beta-catenin mutant with a substitution at tyrosine 654 is introduced into cells. Thus, Fer phosphorylates PTP1B at tyrosine 152 enabling it to bind to the cytoplasmic domain of cadherin, where it maintains beta-catenin in a dephosphorylated state. Cultured fibroblasts from mouse embryos targeted with a kinase-inactivating ferD743R mutation have lost cadherin-associated PTP1B and beta-catenin, as well as localization of cadherin and beta-catenin in areas of cell-cell contacts. Expression of wild-type Fer or culture in epidermal growth factor restores the cadherin complex and localization at cell-cell contacts.

Characterization of cadherin-24, a novel alternatively spliced type II cadherin.

Cadherins comprise a superfamily of calcium-dependent cell-cell adhesion molecules. Within the superfamily are six subfamilies including type I and type II cadherins. Both type I and type II cadherins are composed of five extracellular repeat domains with conserved calcium-binding motifs, a single pass transmembrane domain, and a highly conserved cytoplasmic domain that interacts with beta-catenin and p120 catenin. In this study, we describe a novel cadherin, cadherin-24. It is a type II cadherin with a 781-codon open reading frame, which encodes a type II cadherin protein complete with five extracellular repeats containing calcium-binding motifs, a transmembrane domain, and a conserved cytoplasmic domain. Cadherin-24 has the unusual feature of being alternatively spliced in extracellular repeat 4. This alternative exon encodes 38 in-frame amino acids, resulting in an 819-amino-acid protein. Sequence analysis suggests the presence of beta-catenin and p120 catenin-binding sequences, and immunoprecipitation experiments confirm the ability of both forms of the novel cadherin to associate with alpha-catenin, beta-catenin, and p120 catenin. In addition, aggregation assays show that both forms of cadherin-24 mediate strong cell-cell adhesion.

Expression analysis of delta-catenin and prostate-specific membrane antigen: their potential as diagnostic markers for prostate cancer.

The current approach to prostate cancer diagnosis has major limitations including the inability of prostate-specific antigen (PSA) assays to accurately differentiate between prostate cancer and benign prostate hyperplasia (BPH) and the imprecision of transrectal ultrasound (TRUS) biopsy sampling. We have employed cDNA microarray screening to compare gene expression patterns in BPH and tumour samples to identify expression markers that may be useful in discriminating between these conditions. Screening of 3 individual cDNA arrays identified 8 genes with expression 3-fold greater in 6 tumour tissues than in 1 nontumour sample and 1 BPH sample. Real-time PCR was used to confirm the overexpression of these 8 genes and 12 genes selected from the literature against a panel of 17 tumours and 11 BPH samples. Two genes, delta-catenin (delta-catenin; CTNND2) and prostate-specific membrane antigen (PSMA; FOLH1), were significantly overexpressed in prostate cancer compared to BPH. Prostate epithelial cells stained positively for delta-catenin and PSMA in our prostate cancer tissues, whereas the majority of our BPH tissues were negative for both markers. Thus we have identified delta-catenin (not previously associated with prostatic adenocarcinoma) and confirmed the potential of PSMA as potential candidates for the diagnosis and management of prostate cancer.

Isolation and characterization of XKaiso, a transcriptional repressor that associates with the catenin Xp120(ctn) in Xenopus laevis.

The Armadillo family of catenin proteins function in multiple capacities including cadherin-mediated cell-cell adhesion and nuclear signaling. The newest catenin, p120(ctn), differs from the classical catenins and binds to the membrane-proximal domain of cadherins. Recently, a novel transcription factor Kaiso was found to interact with p120(ctn), suggesting that p120(ctn) also possesses a nuclear function. We isolated the Xenopus homolog of Kaiso, XKaiso, from a Xenopus stage 17 cDNA library. XKaiso contains an amino-terminal BTB/POZ domain and three carboxyl-terminal zinc fingers. The XKaiso transcript was present maternally and expressed throughout early embryonic development. XKaiso's spatial expression was defined via in situ hybridization and was found localized to the brain, eye, ear, branchial arches, and spinal cord. Co-immunoprecipitation of Xenopus p120(ctn) and XKaiso demonstrated their mutual association, whereas related experiments employing differentially epitope-tagged XKaiso constructs suggest that XKaiso additionally self-associates. Finally, reporter assays employing a chimera of XKaiso fused to the GAL4 DNA binding domain indicate that XKaiso is a transcriptional repressor. These data suggest that XKaiso functions throughout development and that its repressor functions may be most apparent in the context of neural tissues. The significance of the XKaiso-p120(ctn) interaction has yet to be determined, but it may include transducing information from cadherin-mediated cell-cell contacts to transcriptional processes within the nucleus.

Detection of differentially expressed genes in human colon carcinoma cells treated with a selective COX-2 inhibitor.

Numerous reports suggest that use of nonsteroidal anti-inflammatory drugs (NSAIDs) decrease mortality from colorectal cancer. To better understand all of the mechanisms underlying this effect, the global pattern of gene expression in colon carcinoma cells following treatment with NS-398, a selective cyclo-oxygenase-2 inhibitor was evaluated. We utilized suppression subtractive hybridization combined with differential screening to identify genes whose expression was affected following treatment. Among the subtracted cDNA fragments confirmed as differentially expressed, there were two which are known to be involved in the regulation of cell adhesion (human FAT and proto-cadherin-7). We identified two other genes whose levels were decreased and these are known to be involved in the regulation of cell proliferation (cyclin K and p-100). We identified additional genes which are involved in different signaling pathways which regulate programmed cell death (Dynamin 2, Pdcd4 and LIP.1). These results provide evidence that some of the effects of NS-398 on carcinoma cells may be due to modulation of genes which regulate programmed cell death, cell proliferation and cell-cell communication. Additional studies are underway to determine the biological function of the novel genes that were identified.