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PeerJ ◽  
2022 ◽  
Vol 10 ◽  
pp. e12750
Supharada Tessiri ◽  
Anchalee Techasen ◽  
Sarinya Kongpetch ◽  
Achira Namjan ◽  
Watcharin Loilome ◽  

Background Genetic alterations in ARID1A were detected at a high frequency in cholangiocarcinoma (CCA). Growing evidence indicates that the loss of ARID1A expression leads to activation of the PI3K/AKT pathway and increasing sensitivity of ARID1A-deficient cells for treatment with the PI3K/AKT inhibitor. Therefore, we investigated the association between genetic alterations of ARID1A and the PI3K/AKT pathway and evaluated the effect of AKT inhibition on ARID1A-deficient CCA cells. Methods Alterations of ARID1A, PI3K/AKT pathway-related genes, clinicopathological data and overall survival of 795 CCA patients were retrieved from cBio Cancer Genomics Portal (cBioPortal) databases. The association between genetic alterations and clinical data were analyzed. The effect of the AKT inhibitor (MK-2206) on ARID1A-deficient CCA cell lines and stable ARID1A-knockdown cell lines was investigated. Cell viability, apoptosis, and expression of AKT signaling were analyzed using an MTT assay, flow cytometry, and Western blots, respectively. Results The analysis of a total of 795 CCA samples revealed that ARID1A alterations significantly co-occurred with mutations of EPHA2 (p < 0.001), PIK3CA (p = 0.047), and LAMA1 (p = 0.024). Among the EPHA2 mutant CCA tumors, 82% of EPHA2 mutant tumors co-occurred with ARID1A truncating mutations. CCA tumors with ARID1A and EPHA2 mutations correlated with better survival compared to tumors with ARID1A mutations alone. We detected that 30% of patients with PIK3CA driver missense mutations harbored ARID1A-truncated mutations and 60% of LAMA1-mutated CCA co-occurred with truncating mutations of ARID1A. Interestingly, ARID1A-deficient CCA cell lines and ARID1A-knockdown CCA cells led to increased sensitivity to treatment with MK-2206 compared to the control. Treatment with MK-2206 induced apoptosis in ARID1A-knockdown KKU-213A and HUCCT1 cell lines and decreased the expression of pAKTS473 and mTOR. Conclusion These findings suggest a dependency of ARID1A-deficient CCA tumors with the activation of the PI3K/AKT-pathway, and that they may be more vulnerable to selective AKT pathway inhibitors which can be used therapeutically.

2022 ◽  
Vol 2022 ◽  
pp. 1-11
Yujie Xing ◽  
Shuo Pan ◽  
Ling Zhu ◽  
Qianwei Cui ◽  
Zhiguo Tang ◽  

Objective. The objective of this study was to investigate the involved mechanisms of advanced glycation end product- (AGE-) exacerbated atherosclerosis (AS). Methods. Toll-like receptor 4 (TLR4) inhibitor was administrated to type 2 diabetes mellitus (T2DM) AS rats. Atherosclerotic plaque, M1 macrophage infiltration, and VSMCs phenotypes were evaluated. AGE-exposed primary macrophages were treated with specific siRNAs knocking down receptor for AGEs (RAGE) and TLR4. Phenotypes of M1 macrophage and VSMCs were identified by fluorescent stains. Contact and noncontact coculture models were established. VSMCs and macrophages were cocultured in these models. ELISA was used to detect inflammatory cytokine concentrations. Relative mRNA expression levels were determined by real-time PCR. Relative protein expression and phosphorylation levels were evaluated by Western blots assays. Results. TLR4 inhibitor treatment significantly reduced arterial stenosis, infiltration of M1 polarized macrophages, and contractile-to-synthetic phenotype conversion of VSMCs in DM AS animals. RAGE and TLR4 silencing dramatically reduced AGE-induced macrophage M1 polarization, inflammatory cytokine secretion, and RAGE/TLR4/forkhead box protein C2 (FOXC2)/signaling which inhibited delta-like ligand 4 (Dll4) expression in macrophages. AGE-treated macrophages induced VSMC phenotypic conversion via activating Notch pathway in a contact coculture model rather than a noncontact model. The VSMC phenotypic conversion induction capability of macrophages was attenuated by RAGE and TLR4 silencing. Conclusions. AGEs induced activation of RAGE/TLR4/FOXC2 signaling, which featured macrophage with Dll4 high expression during M1 polarization. These macrophages promoted contractile-synthetic phenotypic conversion of VSMCs through the Dll4/Notch pathway after direct cell-to-cell contacts.

2022 ◽  
Vol 22 (1) ◽  
Guangtao Zhang ◽  
Zhaoyan Li ◽  
Jiahuan Dong ◽  
Weili Zhou ◽  
Zhanxia Zhang ◽  

Abstract Background Epithelial-to-mesenchymal transition (EMT) is a pivotal cellular phenomenon involved in tumour metastasis and progression. In gastric cancer (GC), EMT is the main reason for recurrence and metastasis in postoperative patients. Acacetin exhibits various biological activities. However, the inhibitory effect of acacetin on EMT in GC is still unknown. Herein, we explored the possible mechanism of acacetin on EMT in GC in vitro and in vivo. Methods In vitro, MKN45 and MGC803 cells were treated with acacetin, after which cell viability was detected by CCK-8 assays, cell migration and invasion were detected by using Transwell and wound healing assays, and protein expression was analysed by western blots and immunofluorescence staining. In vivo, a peritoneal metastasis model of MKN45 GC cells was used to investigate the effects of acacetin. Results Acacetin inhibited the proliferation, invasion and migration of MKN45 and MGC803 human GC cells by regulating the expression of EMT-related proteins. In TGF-β1-induced EMT models, acacetin reversed the morphological changes from epithelial to mesenchymal cells, and invasion and migration were limited by regulating EMT. In addition, acacetin suppressed the activation of PI3K/Akt signalling and decreased the phosphorylation levels of TGF-β1-treated GC cells. The in vivo experiments demonstrated that acacetin delayed the development of peritoneal metastasis of GC in nude mice. Liver metastasis was restricted by altering the expression of EMT-related proteins. Conclusion Our study showed that the invasion, metastasis and TGF-β1-induced EMT of GC are inhibited by acacetin, and the mechanism may involve the suppression of the PI3K/Akt/Snail signalling pathway. Therefore, acacetin is a potential therapeutic reagent for the treatment of GC patients with recurrence and metastasis.

Endocrines ◽  
2022 ◽  
Vol 3 (1) ◽  
pp. 16-28
Vanessa Moisan ◽  
Catherine Brousseau ◽  
Jacques J. Tremblay

Members of the pre-B-cell leukemia transcription factor (PBX) family of homeoproteins are mainly known for their involvement in hematopoietic cell differentiation and in the development of leukemia. The four PBX proteins, PBX1, PBX2, PBX3 and PBX4, belong to the three amino acid loop extension (TALE) superfamily of homeoproteins which are important transcriptional cofactors in several developmental processes involving homeobox (HOX) factors. Mutations in the human PBX1 gene are responsible for cases of gonadal dysgenesis with absence of male sex differentiation while Pbx1 inactivation in the mouse causes a failure in Leydig cell differentiation and function. However, no data is available regarding the expression profile of this transcription factor in the testis. To fill this knowledge gap, we have characterized PBX1 expression during mouse testicular development. Real time PCRs and Western blots confirmed the presence Pbx1 mRNA and PBX1 protein in different Leydig and Sertoli cell lines. The cellular localization of the PBX1 protein was determined by immunohistochemistry and immunofluorescence on mouse testis sections at different embryonic and postnatal developmental stages. PBX1 was detected in interstitial cells and in peritubular myoid cells from embryonic life until puberty. Most interstitial cells expressing PBX1 do not express the Leydig cell marker CYP17A1, indicating that they are not differentiated and steroidogenically active Leydig cells. In adults, PBX1 was mainly detected in Sertoli cells. The presence of PBX1 in different somatic cell populations during testicular development further supports a direct role for this transcription factor in testis cell differentiation and in male reproductive function.

Baorui Xing ◽  
Nana Feng ◽  
Juan Zhang ◽  
Yunmei Li ◽  
Xiuxiu Hou ◽  

Whether pinocembrin (PCN) could be utilized to alleviate hip fracture-induced pain is investigated in this research. Aged rats with hip fractures were treated with vehicle or 80 mg/kg/day PCN from week 3 to week 4. Then hind paw mechanical allodynia, unweighting, warmth, and thickness were measured. The microglia and astrocytes activation and proliferation markers in the spinal dorsal horn were detected with real-time PCR and immunofluorescence staining. The relative expression of substance P and its receptor, tachykinin receptor 1 (Tacr1), were detected with enzyme-linked immunosorbent assay (ELISA) and Western blots. The antinociceptive effect of Tacr1 inhibitor LY303870 was also testified. PCN alleviated hip fracture-induced hind paw nociceptive (allodynia and unweighting) and vascular changes (warmth and thickness) in aged rats with diminished microglia and astrocytes activation and proliferation in the spinal dorsal horn. Up-regulated substance P and Tacr1 were induced after hip fracture, which could be reversed by PCN treatment. Furthermore, LY303870 treatment partially reversed both spinal nociceptive sensitization and vascular changes after hip fracture. Substance P signaling contributes to the nociceptive and vascular changes observed in the hip fracture, which could be alleviated by PCN.

2022 ◽  
Vol 12 ◽  
Sungyub Lee ◽  
Minsun Kim ◽  
Sooyeon Hong ◽  
Eom Ji Kim ◽  
Jae-Hyun Kim ◽  

Postmenopausal osteoporosis is caused by an imbalance between osteoclasts and osteoblasts and causes severe bone loss. Osteoporotic medicines are classified into bone resorption inhibitors and bone formation promoters according to the mechanism of action. Long-term use of bisphosphonate and selective estrogen receptor modulators (SERMs) can cause severe side effects in postmenopausal osteoporosis patients. Therefore, it is important to find alternative natural products that reduce osteoclast activity and increase osteoblast formation. Sparganii Rhizoma (SR) is the dried tuberous rhizome of Sparganium stoloniferum Buchanan-Hamilton and is called “samreung” in Korea. However, to date, the effect of SR on osteoclast differentiation and the ovariectomized (OVX)-induced bone loss model has not been reported. In vitro, tartrate-resistant acid phosphatase (TRAP) staining, western blots, RT-PCR and other methods were used to examine the effect of SR on osteoclast differentiation and osteoblasts. In vivo, we confirmed the effect of SR in a model of OVX-induced postmenopausal osteoporosis. SR inhibited osteoclast differentiation and decreased the expression of TNF receptor-associated factor 6 (TRAF6), nuclear factor of activated T cells 1 (NFATc1) and c-Fos pathway. In addition, SR stimulates osteoblast differentiation and increased protein expression of the bone morphogenetic protein 2 (BMP-2)/SMAD signaling pathway. Moreover, SR protected against bone loss in OVX-induced rats. Our results appear to advance our knowledge of SR and successfully demonstrate its potential role as a osteoclastogenesis-inhibiting and osteogenesis-promoting herbal medicine for the treatment of postmenopausal osteoporosis.

2022 ◽  
Vol 44 (1) ◽  
pp. 222-232
Wen-Jung Chen ◽  
Wen-Wei Sung ◽  
Chia-Ying Yu ◽  
Yu-Ze Luan ◽  
Ya-Chuan Chang ◽  

Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC.

2021 ◽  
Vol 23 (1) ◽  
pp. 219
Qing Ye ◽  
Brianne Falatovich ◽  
Salvi Singh ◽  
Alexey V. Ivanov ◽  
Timothy D. Eubank ◽  

There is an unmet clinical need to identify patients with early-stage non-small cell lung cancer (NSCLC) who are likely to develop recurrence and to predict their therapeutic responses. Our previous study developed a qRT-PCR-based seven-gene microfluidic assay to predict the recurrence risk and the clinical benefits of chemotherapy. This study showed it was feasible to apply this seven-gene panel in RNA sequencing profiles of The Cancer Genome Atlas (TCGA) NSCLC patients (n = 923) in randomly partitioned feasibility-training and validation sets (p < 0.05, Kaplan–Meier analysis). Using Boolean implication networks, DNA copy number variation-mediated transcriptional regulatory network of the seven-gene signature was identified in multiple NSCLC cohorts (n = 371). The multi-omics network genes, including PD-L1, were significantly correlated with immune infiltration and drug response to 10 commonly used drugs for treating NSCLC. ZNF71 protein expression was positively correlated with epithelial markers and was negatively correlated with mesenchymal markers in NSCLC cell lines in Western blots. PI3K was identified as a relevant pathway of proliferation networks involving ZNF71 and its isoforms formulated with CRISPR-Cas9 and RNA interference (RNAi) profiles. Based on the gene expression of the multi-omics network, repositioning drugs were identified for NSCLC treatment.

Marine Drugs ◽  
2021 ◽  
Vol 20 (1) ◽  
pp. 28
D. P. Nagahawatta ◽  
Hyun-Soo Kim ◽  
Young-Heun Jee ◽  
Thilina U. Jayawardena ◽  
Ginnae Ahn ◽  

Sargassum horneri is an invasive brown seaweed that grows along the shallow coastal areas of the Korean peninsula, which are potentially harmful to fisheries and natural habitats in the areas where it is accumulated. Therefore, the author attempted to evaluate the anti-inflammatory mechanism of Sargachromenol isolated from S. horneri against particulate matter (PM)-stimulated RAW 264.7 macrophages. PM is a potent inducer of respiratory diseases such as lung dysfunctions and cancers. In the present study, the anti-inflammatory properties of Sargachromenol were validated using enzyme-linked immunosorbent assay (ELISA), Western blots, and RT-qPCR experiments. According to the results, Sargachromenol significantly downregulated the PM-induced proinflammatory cytokines, Prostaglandin E2 (PGE2), and Nitric Oxide (NO) secretion via blocking downstream activation of Toll-like receptor (TLR)-mediated nuclear factor kappa B (NF-κB) and MAPKs phosphorylation. Thus, Sargachromenol is a potential candidate for innovation in various fields including pharmaceuticals, cosmeceuticals, and functional food.

Nutrients ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 66
Xingyue Yang ◽  
Wenyan Sun ◽  
Qian Wu ◽  
Hongyan Lin ◽  
Zhixing Lu ◽  

Use of folic acid (FA) during early pregnancy protects against birth defects. However, excess FA has shown gender-specific neurodevelopmental toxicity. Previously, we fed the mice with 2.5 times the recommended amount of FA one week prior to mating and during the pregnancy and lactation periods, and detected the activated expression of Fos and related genes in the brains of weaning male offspring, as well as behavioral abnormalities in the adults. Here, we studied whether female offspring were affected by the same dosage of FA. An open field test, three-chamber social approach and social novelty test, an elevated plus-maze, rotarod test and the Morris water maze task were used to evaluate their behaviors. RNA sequencing was performed to identify differentially expressed genes in the brains. Quantitative real time-PCR (qRT-PCR) and Western blots were applied to verify the changes in gene expression. We found increased anxiety and impaired exploratory behavior, motor coordination and spatial memory in FA-exposed females. The brain transcriptome revealed 36 up-regulated and 79 down-regulated genes in their brains at weaning. The increase of Tlr1; Sult1a1; Tph2; Acacb; Etnppl; Angptl4 and Apold1, as well as a decrease of Ppara mRNA were confirmed by qRT-PCR. Among these genes; the mRNA levels of Etnppl; Angptl4andApold1 were increased in the both FA-exposed female and male brains. The elevation of Sult1a1 protein was confirmed by Western blots. Our data suggest that excess FA alteres brain gene expression and behaviors in female offspring, of which certain genes show apparent gender specificity.

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