Ruangan Lidan decoction inhibits the growth and metastasis of liver cancer by downregulating miR-9-5p and upregulating PDK4.

A growing number of studies have suggested that traditional Chinese medicine (TCM) plays an essential role in the development and occurrence of liver cancer. However, the function of Ruangan Lidan decoction (RLD) in liver cancer are not yet adequately identified and manifested, which attracted our attention. The key genes related to liver cancer and RLD and the upstream miRNAs of PDK4 were obtained based on bioinformatics analysis, followed by verification of the targeting relationship between miR-9-5p and PDK4. Next, Huh7 cells were treated with RLD to detect cell proliferation, colony formation, migration, invasion, and apoptosis by multiple assays with gain- and loss-of-function experiments. Moreover, subcutaneous transplanted tumor model and lung metastasis model of liver cancer in nude mice were established to further verify the functional role of RLD in liver cancer growth and metastasis via miR-9-5p/PDK4 axis. Bioinformatics analysis found that PDK4 and miR-9-5p were related to liver cancer, and PDK4 may be a downstream regulator of RLD. miR-9-5p could target and inhibit PDK4. In vitro cell experiments demonstrated that RLD suppressed liver cancer cell proliferation, invasion and migration, and promoted apoptosis by inhibiting miR-9-5p expression and promoting PDK4 expression. In vivo animal experiments further confirmed that RLD inhibited liver cancer growth and metastasis via upregulation of miR-9-5p-dependent PDK4. RLD downregulated miR-9-5p and upregulated PDK4 to inhibit the proliferation, migration, invasion, and induce apoptosis, thereby suppressing the growth and metastasis of liver cancer, highlighting a potential novel target for treatment of liver cancer.

The efficacy and safety of 125I brachytherapy combined with pre-operative transarterial chemoembolization in patients with locally advanced head and neck cancer.

Objective: To evaluate the safety and effectiveness of Iodine-125 ( 125I) brachytherapy combined with pre-operative transarterial chemoembolization in patients with locally advanced head and neck cancer. Methods: In this study, a total of thirty-seven individuals suffering from locally advanced head and neck cancer were involved. The patients were subjected to transarterial chemoembolization as well as implantation of 125I seeds under the guidance of CT and ultrasonography. Follow-up was conducted for 36 months to study the following parameters: the local control rate, survival rate, and clinical complications. Results: In total, thirty-six patients at the end of three months showed an objective response rate of 69.8% and disease control rate of 93.0%, respectively. The 1, 2, and 3-year cumulative overall survival rate was 89.2%, 73.0%, and 45.9%, respectively. The adverse events of the treatment included infection (n=1, Grade III), radiation brachial plexus injury (n=1, Grade III), leukopenia (n=1, Grade III), cerebrovascular embolism (n=1, Grade IV). Conclusion: The combination of 125I brachytherapy and pre-operative transarterial chemoembolization was safe and effective in patients with locally advanced head and neck cancer.

miR-302a-3p Promotes Radiotherapy Sensitivity of Hepatocellular Carcinoma by Regulating Cell Cycle via MCL1.

Background: The relationship between tumor suppressor gene miR-302a-3p and radiotherapy for hepatocellular carcinoma (HCC) remains unclear. This study intended to illustrate the molecular mechanism how miR-302a-3p regulated radiotherapy sensitivity of HCC. Methods: miR-302a-3p expression in HCC tissues and cells was examined by qRT-PCR. The effect of miR-302a-3p on HCC radiotherapy sensitivity were detected by CCK-8, colony formation, and flow cytometry assays. The expression levels of cell cycle-related proteins were detected by Western blot. The influence of miR-302a-3p on radiotherapy sensitivity of HCC was further investigated via cell cycle inhibitor (Caudatin) treatment. The target gene (MCL1) of miR-302a-3p was obtained by bioinformatics analysis, and their binding relationship was confirmed by RNA-binding protein immunoprecipitation assay. The mechanisms of miR-302a-3p regulating cell cycle and affecting radiotherapy sensitivity of HCC cells through MCL1 were further explored through the rescue experiments. Results: miR-302a-3p expression was remarkably reduced in radiotherapy-resistant tissues and cells of HCC. miR-302a-3p overexpression restored sensitivity of radiotherapy-resistant HCC cells to radiotherapy. Treatment with cell cycle inhibitor Caudatin could reverse suppressive effect of miR-302a-3p downregulation on sensitivity of HCC to radiotherapy. Additionally, miR-302a-3p could restrain MCL1 expression. In vitro cell assays further revealed that miR-302a-3p/MCL1 axis could enhance radiotherapy sensitivity of HCC cells by inducing G0/G1 arrest. Conclusions: miR-302a-3p facilitated radiotherapy sensitivity of HCC cells by regulating cell cycle via MCL1, which provided a new underlying target for radiotherapy resistance of HCC patients.

Efficacy Investigation of TACE Combined with Lenvatinib and Sintilimab in Intermediate-Stage Hepatocellular Carcinoma.

Objective: To evaluate the effectiveness of transarterial chemoembolization (TACE) combined with lenvatinib and sintilimab in treating patients with midstage hepatocellular carcinoma (HCC). Methods: Sixty-two patients with midstage HCC were enrolled in this study. All of them were firstly treated in our hospital between September 1, 2019, and March 1, 2020. According to different treatment regimens, they were divided into the control group (31 cases, TACE group) and the observation group (31 cases, TACE combined with lenvatinib and sintilimab group). Each patient was followed up for at least 30 months to compare the short-term clinical efficacy and survival rate between the two groups. Results: The objective response rate (ORR) and disease control rate (DCR) of the observation group at 3 months were 77.4% and 93.5%, respectively, which were higher than those of the control group (P < 0.05). The 2-year cumulative overall survival rate of the observation group was 64.5%, which was significantly higher than that of the control group (P < 0.05). The survival curve of the disease-free survival rate in the observation group was higher than that in the control group, and the difference was statistically significant (X 2 = 4.313, P < 0.05). Conclusion: TACE combined with lenvatinib and sintilimab in the treatment of Barcelona Clinic Liver Cancer (BCLC) stage B hepatocellular carcinoma can effectively control the tumor progression and prolong the survival time of patients. Those preliminary findings need validation in larger studies, with a prospective design and longer follow-up.

CT-MR Image Fusion for Post-Implant Dosimetry Analysis in Brain Tumor Seed Implantation- a Preliminary Study.

Purpose: To calculate and evaluate postimplant dosimetry (PID) with CT-MR fusion technique after brain tumor brachytherapy and compare the result with CT-based PID. Methods and Materials: 16 brain tumor patients received MR-guided intervention with Iodine-125 (125I) seed implantation entered this preliminary study for PID evaluation. Registration and fusion of CT and MR images of the same patients were performed one day after operation. Seeds identification and targets delineation were carried out on CT, MR, and CT-MR fusion images, each. The number and location of seeds on MR or CT- MR fusion images were compared with those of actually implanted seeds. Clinical target volume (CTV) and dosimetric parameters such as %D90, %V100 and external V100 were measured and calculated. In addition, the correlation of the fusion to CT CTV ratio and other factors were analyzed. Results: The numbers of fusion seeds were not significantly different compared with reference seeds (t =1.76, p >0.05). The difference between reference seeds numbers and truly extracted MR seeds numbers was statistically significant (t =3.91, p <0.05). All dosimetric parameters showed significant differences between the two techniques (p <0.05). The mean CTV delineated on fusion images was 34.3 ± 33.6, smaller than that on CT images. The mean values of external V100, %V100 and %D90 on fusion images were larger than those on CT images. Correlation analysis showed that the fusion-CT V100 ratio was positively and significantly correlated with the fusion-CT volume ratio. Conclusions: This preliminary study indicated that CT-MR fusion-based PID exhibited good accuracy for 125I brain tumor brachytherapy dosimetry when compared to CT-based PID and merits further research to establish best-outcome protocols.

The GPCR-ß-arrestin complex allosterically activates C-Raf by binding its amino terminus.

G protein-coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and ß-arrestins (ßarrs). In a ßarr-dependent signaling pathway, ßarrs link GPCRs to various downstream signaling partners, such as the Raf-mitogen-activated protein kinase extracellular signal-regulated kinase-extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR-ßarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, ßarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR-ßarr1 complexes in vitro. Furthermore, we show that ßarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates ßarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and ßarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between ßarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and ßarr1 bind to the same region on C-Raf. Furthermore, we found that ßarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR-ßarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how ßarrs regulate effector molecules to activate downstream signaling pathways.

Small-Molecule Positive Allosteric Modulators of the ß2-Adrenoceptor Isolated from DNA-Encoded Libraries.

Conventional drug discovery efforts at the ß2-adrenoceptor (ß2AR) have led to the development of ligands that bind almost exclusively to the receptor's hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein-coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first ß2AR small-molecule positive allosteric modulators (PAMs)-compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human ß2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied ß2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and ß-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the ß2AR and potentiates downstream cAMP production and receptor recruitment of ß-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the ß2AR compared with the closely related ß1AR. Structure-activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the ß2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

ß-Arrestin2 mediates progression of murine primary myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm associated with significant morbidity and mortality, for which effective therapies are lacking. ß-Arrestins are multifunctional adaptor proteins involved in developmental signaling pathways. One isoform, ß-arrestin2 (ßarr2), has been implicated in initiation and progression of chronic myeloid leukemia, another myeloproliferative neoplasm closely related to primary myelofibrosis. Accordingly, we investigated the relationship between ßarr2 and primary myelofibrosis. In a murine model of MPLW515L-mutant primary myelofibrosis, mice transplanted with donor ßarr2-knockout (ßarr2-/-) hematopoietic stem cells infected with MPL-mutant retrovirus did not develop myelofibrosis, whereas controls uniformly succumbed to disease. Although transplanted ßarr2-/- cells homed properly to marrow, they did not repopulate long-term due to increased apoptosis and decreased self-renewal of ßarr2-/- cells. In order to assess the effect of acute loss of ßarr2 in established primary myelofibrosis in vivo, we utilized a tamoxifen-induced Cre-conditional ßarr2-knockout mouse. Mice that received Cre (+) donor cells and developed myelofibrosis had significantly improved survival compared with controls. These data indicate that lack of antiapoptotic ßarr2 mediates marrow failure of murine hematopoietic stem cells overexpressing MPLW515L. They also indicate that ßarr2 is necessary for progression of primary myelofibrosis, suggesting that it may serve as a novel therapeutic target in this disease.

GPCR-G Protein-ß-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Changes in conformation at the cytoplasmic ends of the fifth and sixth transmembrane helices of a yeast G protein-coupled receptor in response to ligand binding.

The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor.

Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry.

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ∼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.