P38 Signal Transduction Pathways Research Articles

Abstract 576: The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for Phase 1a clinical trials in USA and is currently under clinical evaluation. RNA-seq whole-genome transcriptome analysis revealed that QN-302 selectively targets G-quadruplexes in cancer-related genes, notably by forming stable G-quadruplex complexes with appropriate promoter sequences. This results in the transcriptional down regulation of affected genes, which map to for example, the hedgehog, axon guidance, WNT/β-catenin and P38 signal transduction pathways. In many instances individual expression changes are modest, though statistically significant: cumulatively, they affect whole pathways. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their genes products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Cellular transcriptome data on these eleven genes is presented here, which together with earlier reports on them, supports the designation of QN-302 as a pan-G-quadruplex agent targeting a group of major genes involved in PDAC maintenance and metastasis. Data from a xenograft model for pancreatic cancer is also available for four of these genes, which concurs with the cellular data. We suggest that the identification of these 11 genes may also provide a basis for biomarker selection and possible patient stratification based on transcriptome data. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 576.

CLL Cell-Derived Exosomes Impair Immune Function and Normal Hematopoiesis in CLL

CLL is characterized by immune and hematopoietic abnormalities such as T-cell dysfunction, hypogammaglobulinemia, increased levels of inflammatory cytokines, aberrant monocyte polarity, anemia, neutropenia, and low-grade bone marrow fibrosis. As a result, CLL patients become susceptible to increased rates of infection and/or second neoplasms. Exosomes are nanovesicles that participate in bidirectional communication between neoplastic cells and their microenvironment. Using electron microscopy, we found that CLL cells produce and release CD19 +/CD5 +exosomes, and in agreement with other investigators, we detected high levels of CLL cell-derived exosomes in the plasma of patients with CLL. By using bead-assisted flow cytometry, we found that at least 50% of circulating extracellular vesicles are CLL cell-derived exosomes, identified as CD63 +/CD41a - particles co-expressing CD19 +/CD5 +, whereas only a small proportion of CLL patients' plasma-derived exosomes expressed T-cell or endothelial cell surface markers. Furthermore, we found a positive correlation between levels of CLL cell-derived exosomes in the plasma of patients with CLL and the patients' peripheral white blood cell counts, indicating that CLL cells are the primary source of circulating exosomes. To investigate the effects of CLL cell-derived exosomes, we conducted an analysis of CLL cells and CLL cell-derived exosomes from a cohort of 78 previously untreated patients. To identify the molecular profiles of CLL cell-derived exosomes, we analyzed their RNA transcripts using RNA-seq and qRT-PCR. Our data revealed that CLL cell-derived exosomes expressed significant levels of oncogenic micro-RNAs. Additionally, we identified 1052 differentially expressed genes, and enrichment analysis further unveiled the significant upregulation of gene clusters associated with p53, NF-κB, MAPK, mTOR, and interleukin signal transduction pathways, consistent with their neoplastic cell origin. The distinctive transcriptional profile of CLL cell-derived exosomes led us to explore their effects on normal cell populations. We labeled ultra-centrifuged CLL cell-derived exosomes with fluorescent markers and incubated them with various cell types in culture conditions conducive to their physiological activity. Using confocal microscopy and flow cytometry, we observed significant uptake of CLL cell-derived exosomes by normal monocytes, fibrocytes, B cells, T cells, and CLL cells. Furthermore, incubation with CLL exosomes resulted in notable alterations in normal CD14 + monocyte polarity and subset-specific shifts in the expression of inflammatory cytokines. Remarkably, CLL cell-derived exosomes induced overexpression of the T-cell makers CD5 and CD8 in normal CD19 + cells, suggesting that CLL exosomes may promote immune-evasive reprogramming of normal B cells. Moreover, when normal T cells were incubated with CLL cell-derived exosomes, CyTOF analysis using 46 markers revealed elevated levels of immune checkpoints, including PD1, CTLA4, and LAG3, in both CD4 + and CD8 + subsets, indicating that exosomes derived from CLL cells induce T-cell dysfunction. Given their profound effect on immune cells, we wondered whether CLL exosomes affect hematopoietic cells. We previously found that normal monocyte derived fibrocytes stimulate hematopoietic colony proliferation. In sharp contrast to control fibrocytes, fibrocytes preincubated with CLL exosomes and CLL cell-derived exosomes inhibited normal BFU-E and CFU-GM colony formation, suggesting that CLL exosomes might contribute to CLL patients' anemia or neutropenia. Wondering whether CLL cell-derived exosomes affect the viability of other cell types, we found that they induced a dose-dependent apoptosis of CD19 + B cells, CLL cells but not normal T cells. In summary, CLL cell-derived exosomes exert a profound effect on gene and cell surface protein expression in immune and tissue-remodeling cells, leading to functional modifications, alteration of inflammatory cytokine levels, and suppression of normal hematopoiesis. The impact of CLL exosomes may vary across different cell types; however, as the leukemic cell mass increases, exosome levels escalate, invariably affecting CLL cell environment. The identification of exosomes with distinct molecular signatures holds promising potential for novel therapeutic interventions in future treatment of CLL.

Pharmacological Effects of Cisplatin Combination with Natural Products in Cancer Chemotherapy.

Cisplatin and other platinum-based drugs, such as carboplatin, ormaplatin, and oxaliplatin, have been widely used to treat a multitude of human cancers. However, a considerable proportion of patients often relapse due to drug resistance and/or toxicity to multiple organs including the liver, kidneys, gastrointestinal tract, and the cardiovascular, hematologic, and nervous systems. In this study, we sought to provide a comprehensive review of the current state of the science highlighting the use of cisplatin in cancer therapy, with a special emphasis on its molecular mechanisms of action, and treatment modalities including the combination therapy with natural products. Hence, we searched the literature using various scientific databases., such as MEDLINE, PubMed, Google Scholar, and relevant sources, to collect and review relevant publications on cisplatin, natural products, combination therapy, uses in cancer treatment, modes of action, and therapeutic strategies. Our search results revealed that new strategic approaches for cancer treatment, including the combination therapy of cisplatin and natural products, have been evaluated with some degree of success. Scientific evidence from both in vitro and in vivo studies demonstrates that many medicinal plants contain bioactive compounds that are promising candidates for the treatment of human diseases, and therefore represent an excellent source for drug discovery. In preclinical studies, it has been demonstrated that natural products not only enhance the therapeutic activity of cisplatin but also attenuate its chemotherapy-induced toxicity. Many experimental studies have also reported that natural products exert their therapeutic action by triggering apoptosis through modulation of mitogen-activated protein kinase (MAPK) and p53 signal transduction pathways and enhancement of cisplatin chemosensitivity. Furthermore, natural products protect against cisplatin-induced organ toxicity by modulating several gene transcription factors and inducing cell death through apoptosis and/or necrosis. In addition, formulations of cisplatin with polymeric, lipid, inorganic, and carbon-based nano-drug delivery systems have been found to delay drug release, prolong half-life, and reduce systemic toxicity while other formulations, such as nanocapsules, nanogels, and hydrogels, have been reported to enhance cell penetration, target cancer cells, and inhibit tumor progression.

Adiponectin inhibits lipoplysaccharide-induced inflammation and promotes osteogenesis in hPDLCs.

Periodontal diseases are infections of the structures that surround and support the teeth; they are characterized by local inflammation and alveolar bone loss. Most treatments focus on only one aspect, inhibiting inflammation, or promoting osteoblasts. We set out to develop a new method that would intervene in the two aspects simultaneously. Adiponectin (APN), secreted by adipocytes, inhibits the inflammatory response and promotes osteogenesis. However, its role in human periodontal ligament cells (hPDLCs) is unclear. Therefore, we aim to investigate whether APN could suppress lipopolysaccharide (LPS)-induced inflammation and promote osteogenesis in hPDLCs. In the present study, we stimulated hPDLCs with LPS in the presence or absence of APN. Real-time PCR and Western blotting results demonstrated that APN partially inhibited the activation of the classical nuclear factor κ-B (NF-κB) pathway. These results were confirmed by a change of expressions of NF-κB downstream inflammatory genes, such as decreased cyclooxygenase (COX)-2 and tumor necrosis factor α (TNF-α), along with increased interleukin (IL)-10. As for the role of APN in osteogenesis, Alizarin Red S staining showed that APN treatment induced more calcium deposition nodules than controls. We also found that APN enhanced the expression of osteoblast-related genes (osteopontin (OPN), collagen 1, osteocalcin, alkaline phosphatase, runt-related transcription factor 2 (RUNX2), and bone morphogenetic protein 2) in hPDLCs via the APPL1 (the adaptor protein containing PH domain, PTB domain, and leucine zipper motif 1)/p38 signal transduction pathway. Therefore, APN inhibits LPS-induced inflammation and promotes osteogenesis in hPDLCs and may have potential therapeutic value in treating periodontitis by inhibiting the inflammatory lesions and contributing to bone tissue regeneration.

AUNIP Expression Is Correlated With Immune Infiltration and Is a Candidate Diagnostic and Prognostic Biomarker for Hepatocellular Carcinoma and Lung Adenocarcinoma.

AUNIP, a novel prognostic biomarker, has been shown to be associated with stromal and immune scores in oral squamous cell carcinoma (OSCC). Nonetheless, its role in other cancer types was unclear. In this study, AUNIP expression was increased in hepatocellular carcinoma (HCC) and lung adenocarcinoma (LUAD) according to data from The Cancer Genome Atlas (TCGA) database, Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB), and Gene Expression Omnibus (GEO) database (GSE45436, GSE102079, GSE10072, GSE31210, and GSE43458). Further, according to copy number variation analysis, AUNIP up-regulation may be associated with copy number variation. Immunohistochemistry showed AUNIP expression was higher in HCC and LUAD compared with the normal tissues. Receiver operating characteristic (ROC) curve analysis demonstrated that AUNIP is a candidate diagnostic biomarker for HCC and LUAD. Next, TCGA, International Cancer Genome Consortium (ICGC), and GEO (GSE31210 and GSE50081) data showed that increased AUNIP expression clearly predicted poor overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) in HCC and LUAD. Additionally, multivariate Cox regression analysis involving various clinical factors showed that AUNIP is an independent prognostic biomarker for HCC and LUAD. Next, the role of AUNIP in HCC and LUAD was explored via a co-expression analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and a gene set variation analysis (GSVA). HCC and LUAD exhibited almost identical enrichment results. More specifically, high AUNIP expression was associated with DNA replication, cell cycle, oocyte meiosis, homologous recombination, mismatch repair, the p53 signal transduction pathway, and progesterone-mediated oocyte maturation. Lastly, the Tumor Immune Estimation Resource (TIMER) tool was used to determine the correlations of AUNIP expression with tumor immune infiltration. AUNIP expression was positively correlated with the infiltration degree of B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells in HCC. However, AUNIP expression was negatively correlated with the infiltration degree of B cells, CD4+ T cells, and macrophages in LUAD. In addition, AUNIP expression was correlated with immune infiltration in various other tumors. In conclusion, AUNIP, which is associated with tumor immune infiltration, is a candidate diagnostic and prognostic biomarker for HCC and LUAD.

Paclitaxel induces apoptosis through the TAK1-JNK activation pathway.

Paclitaxel (PTX) has previously been used to treat tumours of various tissue origins, such as lung, breast, ovarian, prostate cancers and leukemia. PTX‐induced apoptosis is associated with p38 mitogen‐activated protein kinase (p38 MAPK), extracellular signal‐regulated kinase (ERK), nuclear factor‐kappa B (NF‐κB) and c‐Jun N‐terminal kinase or stress‐activated protein kinase (JNK/ SAPK) pathways. Transforming growth factor‐beta‐activated kinase 1 (TAK1) and TAK1‐binding protein 1 (TAB1) play an important role in cell apoptosis through the p38, ERK, NF‐κB and JNK signal transduction pathways. To investigate the role of TAK1 in PTX‐induced cell apoptosis, we treated HEK293 and 8305C cells with 0–20 µm PTX for 6, 12 or 24 h. To investigate whether TAK1 can cooperate with PTX for cancer treatment, we transfected cells with TAK1, TAB1 or control plasmid and treated them with PTX (3–10 µm) for 9–24 h. Apoptosis rates were analysed by flow cytometry (Annexin V/PI). Endogenous TAK1 and TAB1, caspase‐7 cleavage, poly ADP‐ribose polymerase (PARP) cleavage, Bcl‐xL level, phospho‐p44/42, phospho‐JNK and phospho‐p38 were detected by western blot. We show that in HEK293 and 8305C cells, PTX enhanced the endogenous TAK1/TAB1 level and induced cell apoptosis in a dose‐ and time‐dependent manner. Upon TAK1 overexpression in HEK293 cells treated with PTX, apoptosis rate, JNK phosphorylation and PARP cleavage increased contrary to heat‐shocked or untreated cells. CRISPR editing of the tak1 gene upon PTX treatment resulted in lower phospho‐JNK and PARP cleavage levels than in cells transfected with the control or the TAK1‐ or TAB1 + TAK1‐containing plasmids. TAK1‐K63A could not induce JNK phosphorylation or PARP cleavage. We conclude that PTX induces HEK293 and 8305C cell apoptosis through the TAK1–JNK activation pathway, potentially highlighting TAK1’s role in chemosensitivity.

P38 Signal Transduction Pathway Has More Cofactors on Apoptosis of SGC-7901 Gastric Cancer Cells Induced by Combination of Rutin and Oxaliplatin.

Currently, gastric cancer treatment is mainly based on first-line intervention with oxaliplatin (OXA) after surgical resection, but the application of OXA has been limited due to the toxic side effects caused by the cumulative dose. The toxicity of OXA mainly includes hepatotoxicity, nephrotoxicity, and ototoxicity, and there is an urgent clinical need to find alternatives that are less toxic and more effective. Rutin (RT) is a natural flavonoid with many biological activities. Studies have found that RT inhibits tumor cell growth and enhances their sensitivity toward certain drugs. As the underlying impact of RT on gastric cancer and its molecular mechanism remain poorly understood, we performed a series of experiments to determine whether RT has the effect of treating gastric cancer, and whether it can cooperate with OXA to treat gastric cancer and its related mechanisms. In the present study, we founded that RT suppressed cell viability, inhibited cell proliferation by causing G0/G1 arrest, and induced apoptosis in SGC 7901 cells. And RT can play as an antitumor agent together with OXA. The mechanism of RT-induced apoptosis may be associated with the activation of the p38/Caspase signal pathway. These results demonstrated the potential of RT as a promising therapeutic compound to treat gastric cancer. At the same time, RT can synergize with OXA to reduce the dose of OXA and reduce the toxicity.

Expression of gastrin in colon cancer and its effect on human colon cancer cell proliferation and P38 signal transduction pathway

Expression of gastrin in colon cancer and its effect on human colon cancer cell proliferation and P38 signal transduction pathway

Inactivation of Cyclic AMP Response Element Transcription Caused by Constitutive p38 Activation Is Mediated by Hyperphosphorylation-Dependent CRTC2 Nucleocytoplasmic Transport.

The p38 signal transduction pathway can be activated transiently or constitutively, depending on the contexts in which the activation occurs. However, the biological consequence of constitutive activation of p38 is largely unknown. After screening 300 transcriptional cofactors, we identified CRTC2 as a downstream substrate of constitutively activated p38. Constitutive, rather than transient, activation of p38 led to hyperphosphorylation of CRTC2, resulting in CRTC2 cytosolic relocation and subsequent inactivation of cyclic AMP response element (CRE)-mediated transcription. Interestingly, the cytosolic translocation of CRTC2 depended on phosphorylation accumulation at multiple sites (≥11 phosphoserine/phosphothreonine residues) but not on specific sites. The hyperphosphorylation-driven nucleocytoplasmic transport of CRTC2 may not be a rare case of nuclear export of proteins, as we also observed that constitutively activated p38 promoted FOS nuclear export in a hyperphosphorylation-dependent manner. Collectively, our study uncovered a previously unknown mechanism of inactivation of selected transcription, which results from hyperphosphorylation-driven nucleocytoplasmic transport of cofactors or transcription factors mediated by constitutively active kinase.

Screening of cell cycle-related genes regulated by KIAA0101 in gastric cancer

To screen the genes related to cell cycle under regulation by KIAA0101 in gastric cancer. RT-PCR was used to detect the expression level of KIAA0101 gene in gastric cancer tissue and paired adjacent tissues. GO function enrichment analysis and KEGG pathway enrichment analysis were carried out using DAVID database. KEGG was used to map the pathways and the corresponding genes were analyzed. The list of genes associated with the KIAA0101 expression pattern was imported into TCGA cBioPortal to analyze the relationship between the interacting genes and generate a genetic topology map. The candidate genes were screened by RT-PCR. The expression level of KIAA0101 mRNA was significantly higher in cancer tissues than in paired adjacent tissues (1.104 ± 0.379 vs 0.421 ± 0.172; P=0.0179). The system screened genes related with KIAA0101 from 478 tissues by pooled analysis of the expression intensity of all the gene probes. GO function analysis showed that the differential genes were mainly enriched in protein phosphorylation, RNA processing, cell cycle, DNA metabolism, protein transport, acetylation, apoptosis, proteolysis, and redox. The changes in the expression level of KIAA0101 mainly affect the gastric cancer-related pathways including cell cycle, spliceosome, DNA replication, and p53 signal transduction pathway. KEGG pathway maps and gene topology maps showed that the genes related to KIAA0101 (such as BUB1B, MAD2L1, CDC45, CDK1, CCNE1 and CCNB2) were also related to cell cycle. RT-PCR results confirmed significant increments of the expression levels of BUB1B, MAD2L, CDK1, CCNE1, and CCNB2 mRNA in gastric cancer tissues as compared with the paired adjacent gastric tissues (P < 0.05), but CDC45 mRNA did not show significant differential expression in gastric cancer tissues (P > 0.05). KIAA0101 may affect cell cycle by regulating the expression of BUB1B, MAD2L1, CDK1, CCNE1 and CCNB2, and this finding may provide evidence for understanding how KIAA0101 affects cell cycle and for screening of tumor markers and selection of drug targets.

Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Down-Regulation of AQP4 Expression via p38 MAPK Signaling in Temozolomide-Induced Glioma Cells Growth Inhibition and Invasion Impairment.

Glioma is the most common and lethal central nervous system tumors. Temozolomide (TMZ) is an effective drug for malignant glioma, however, the intracellular and molecular mechanisms behind this anti-cancer effect have yet to be fully understood. The aim of the present study was to determine whether TMZ inhibits proliferation, invasion of glioma cells in vitro and whether these effects can be mediated through modulation of aquaporin 4 (AQP4) and phosphorylation of the MAPK pathway. The viability of U87 and U251 human glioma cells was evaluated using MTT assay. The cell cycle distribution was detected with flow cytometry. Migration ability and invasion ability were tested by scratch assays and transwell assays, respectively. The levels of AQP4 and MAPK were measured using immunoblot analyses. Our results showed that TMZ inhibited proliferation, migration and invasion, and induced G2/M arrest in U87 and U251 glioma cell lines. These changes were associated with a decrease in the levels of AQP4 expression as well as activation phosphorylated level of p38. Treatment with a p38 chemical activator (anisomycin) resulted in similar effects as TMZ treatment on glioma cells. And p38 chemical inhibitor (SB203580) could block these effects in glioma treated with TMZ, suggesting a direct up-regulation of the p38 signaling pathway. Therefore, we identified that TMZ might have therapeutic potential for controlling proliferation, invasion of malignant glioma by inhibiting AQP4 expression through activation of p38 signal transduction pathway. J. Cell. Biochem. 118: 4905-4913, 2017. © 2017 Wiley Periodicals, Inc.

MicroRNA‑146a promotes the proliferation of rat vascular smooth muscle cells by downregulating p53 signaling.

The present study aimed to detect and verify gene expression profile differences for microRNA (miR)‑146a and its role in the proliferation of vascular smooth muscle cells (VSMCs). Artificially synthesized miR‑146a mimics, miR‑146 inhibitor, scramble‑miRNA or PBS was transfected into cultured primary rat VSMCs invitro. Reverse transcription‑quantitative polymerase chain reaction confirmed that the miR‑146a expression level was significantly decreased in VSMCs treated with miR‑146a inhibitor (P<0.01). Cell Counting Kit‑8 was used to determine the proliferation ability, which demonstrated that proliferation was significantly decreased in VSMCs treated with miR‑146a inhibitor (P<0.01). Microarray expression profiling analysis revealed that the p53 signal pathway was upregulated in VSMCs treated with the miR‑146a inhibitor. Compared with untransfected VSMCs, the mRNA and protein expression levels of caspase‑3 and phosphatase and tensin homolog (PTEN) in p53 signal transduction pathway did not exhibit a significant difference (P>0.05); however, the mRNA and protein expression levels of p53 were significantly decreased in cells transfected with miR‑146a mimics and increased in miR‑146a inhibitor transfected cells (both P<0.01). The mRNA and protein expression levels of cyclin D1 significantly increased in miR‑146a mimics transfected cells and decreased in cells transfected with the miR‑146a inhibitor (both P<0.05). The present data indicated that miR‑146a may promote the proliferation of rat VSMCs by downregulating p53 and upregulating cyclin D1 expression.

Suppression of inflammation by the rhizome of Anemarrhena asphodeloides via regulation of nuclear factor-κB and p38 signal transduction pathways in macrophages.

The rhizome of Anemarrhena asphodeloides Bunge (A. asphodeloides) has been used as a traditional East Asian medicine for the treatment of various types of inflammatory disease. However, to the best of our knowledge, there have been no systemic studies regarding the molecular mechanisms of action of the A. asphodeloides rhizome anti-inflammatory effects. The aim of the present study was to elucidate the anti-inflammatory effects and underlying mechanism of action of ethanol extracts of the rhizome of A. asphodeloides (EAA) in murine macrophages. Non-cytotoxic concentrations of EAA (10-100 µg/ml) significantly decreased the production of NO and interleukin (IL)-6 in lipopolysaccharide (LPS)-stimulated macrophages, while the production of tumor necrosis factor-α was not regulated by EAA. EAA-mediated reduction of nitric oxide (NO) was due to reduced expression levels of inducible NO synthase (iNOS). Furthermore, protein expression levels of LPS-induced cyclooxygenase-2, another inflammatory enzyme, were alleviated in the presence of EAA. EAA-mediated reduction of those proinflammatory mediators was due to inhibition of nuclear factor-κB (NF-κB) and activator protein 1 transcriptional activities followed by the stabilization of inhibitor of κ Bα and inhibition of p38, respectively. These results indicate that EAA suppresses LPS-induced inflammatory responses by negatively regulating p38 and NF-κB, indicating that EAA is a candidate treatment for alleviating inflammation.

3-MCPD 1-Palmitate Induced Tubular Cell Apoptosis In Vivo via JNK/p53 Pathways.

Fatty acid esters of 3-chloro-1, 2-propanediol (3-MCPD esters) are a group of processing induced food contaminants with nephrotoxicity but the molecular mechanism(s) remains unclear. This study investigated whether and how the JNK/p53 pathway may play a role in the nephrotoxic effect of 3-MCPD esters using 3-MCPD 1-palmitate (MPE) as a probe compound in Sprague Dawley rats. Microarray analysis of the kidney from the Sprague Dawley rats treated with MPE, using Gene Ontology categories and KEGG pathways, revealed that MPE altered mRNA expressions of the genes involved in the mitogen-activated protein kinase (JNK and ERK), p53, and apoptotic signal transduction pathways. The changes in the mRNA expressions were confirmed by qRT-PCR and Western blot analyses and were consistent with the induction of tubular cell apoptosis as determined by histopathological, TUNEL, and immunohistochemistry analyses in the kidneys of the Sprague Dawley rats. Additionally, p53 knockout attenuated the apoptosis, and the apoptosis-related protein bax expression and cleaved caspase-3 activation induced by MPE in the p53 knockout C57BL/6 mice, whereas JNK inhibitor SP600125 but not ERK inhibitor U0126 inhibited MPE-induced apoptosis, supporting the conclusion that JNK/p53 might play a critical role in the tubular cell apoptosis induced by MPE and other 3-MCPD fatty acid esters.

Role of p38 Signaling Pathway in Pentagastrin-Regulated Cell Proliferation of Colorectal Carcinoma Cell Line HT-29

Objective: To investigate the effects and mechanisms of p38 signaling pathway in pentagastrin-regulated cell proliferation of colorectal carcinoma cell line HT-29. Methods: HT-29 cell line of colorectal carcinoma was in vitro incubated and divided into the control group, pentagastrin group, proglumide group, and pentagastrin + proglumide group. MTT reduction assay was performed to detect the proliferation status of HT-29 cell line and determine the optimal dosage of pentagastrin and proglumide. Annexin V-fluorescein isothiocyanate flow cytometry was used to detect the proliferation index (PI) and apoptosis rate (AR) of HT-29 cells. Reverse transcriptase polymerase chain reaction was performed to detect the mRNA expression of the pentagastrin receptor/cholecystokinin-B receptor (CCK-BR) and p38. The protein and phosphorylation levels of p38 were estimated by western blotting. Results: RT-PCR detection showed that CCK-BR mRNA was expressed in the HT-29 cell line. Pentagatrin improved HT-29 cell proliferation in dosage of 6.25 - 100 mg/L, and the optimal dosage of pentagastrin was 25.0 mg/L. Proglumide had no significant effect on the proliferation of HT-29 cells, but significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the dosage of proglumide was 8.0 - 128.0 mg/L, and the optimal dosage was 32.0 mg/L. The AR in the pentagastrin group was significantly lower than that in the control group and in the pentagastrin + proglumide group. The PI in the pentagastrin group was significantly higher than that in the control group and in the pentagastrin + proglumide group. P38 phosphorylation level in the pentagastrin group was significantly lower than that in the control group, and in the pentagastrin + proglumide group. There were no significant differences in the mRNA and protein expression of p38 in the control, pentagastrin, proglumide and pentagastrin + proglumide groups. Conclusion: Pentagastrin can improve proliferation of the CRC cell line HT-29 and inhibit apoptosis via the p38 signal transduction pathway. This mechanism may be associated with suppressed p38 protein phosphorylation level due to inhibition of proglumide, a gastrin receptor antagonist.

Naringin inhibits the invasion and migration of human glioblastoma cell via downregulation of MMP-2 and MMP-9 expression and inactivation of p38 signaling pathway.

Gliomas are the most common and malignant primary brain tumors. They are associated with a poor prognosis despite the availability of multiple therapeutic options. Naringin, a common dietary flavonoid abundantly present in fruits and vegetables, is believed to possess strong anti-proliferative and anti-cancer properties. However, there are no reports describing its effects on the invasion and migration of glioblastoma cell lines. Our results showed that the treatment of U251 glioma cell lines with different concentrations of naringin inhibited the invasion and migration of these cells. In addition, we revealed a decrease in the levels of matrix metalloproteinases (MMP-2) and (MMP-9) expression as well as proteinase activity in U251 glioma cells. In contrast, the expression of tissue inhibitor of metalloproteinases (TIMP-1) and (TIMP-2) was increased. Furthermore, naringin treatment decreased significantly the phosphorylated level of p38. Combined treatment with a p38 inhibitor (SB203580) resulted in the synergistic reduction of MMP-2 and MMP-9 expressions correlated with an increase of TIMP-1 and TIMP-2 expressions and the anti-invasive properties. However, p38 chemical activator (anisomycin) could block these effects produced by naringin, suggesting a direct downregulation of the p38 signaling pathway. These data suggest that naringin may have therapeutic potential for controlling invasiveness of malignant gliomas by inhibiting of p38 signal transduction pathways.

Changes in gene expression in SIRT3 knockout liver cells

The sirtuin (SIRT) gene family is reported to regulate critical intracellular processes from aging to cellular metabolism and repair. SIRT3 knockout (SIRT3-/-) mice develop receptor positive mammary tumors starting at 13 months and SIRT3 expression is decreased in human breast cancer samples as well as several other diseases. It is established that carcinogenesis results from the accumulation of multiple aberrant genetic events including the activation of oncogenes and inactivation of tumor-suppressor genes. To determine the potential early genomic events that may play a role in the tumor-permissive phenotype observed in SIRT3-/- cells, we compared gene expression profile in SIRT3-/- and wild-type (SIRT3+/+) mouse livers. Differences between the expression profiles of genes important in the p53 and apoptosis pathway and signal transduction pathways, as well as genes involved with insulin and cholesterol metabolism, were determined. These results demonstrate that the expression of several oncogenes including Cdkn1, Myc, and Nos2 are increased. In contrast, several genes shown to be downregulated in human breast cancer including Btg2, Egr-1, Fos, Jun, Gadd4, and Wnt1 had decreased expression. The current work demonstrates that the loss of function of SIRT3 results in a cellular environment permissive for carcinogenesis and is characterized by altered metabolism.

The Roles of MAPKs in Rabbit Nucleus Pulposus Cell Apoptosis Induced by High Osmolality

Earlier work has suggested that the p38 MAPK, JNK1/2 and ERK1/2 signal pathway existed in nucleus pulposus cells and the cell growth, differentiation and apoptosis were regulated by them. Because osmotic fluctuations are inevitable in the physicochemical environment of intervertebral disc cells, high osmolality could activate p38 MAPK, JNK1/2 and ERK1/2 signal pathway. The effects of high osmolality on the catabolic program and proliferation of nucleus pulposus cells are still not clear. To explore the possible roles of MAPKs in rabbit nucleus pulposus cell apoptosis induced by high osmolality. Rabbit nucleus pulposus cells were cultured and divided into different group at random. The cells were pretreated with inhibitor for p38 MAPK, JNK1/2 and ERK1/2 signal pathway respectively. In next step, the cells were cultured in different osmolality environment for different time at 37°C in 5% carbon dioxide incubator. After treatments, ratio of apoptosis was measured by flow cytometry, and western blotting was performed to quantify the expression of the activated forms of p38 MAPK, JNK1/2 and ERK1/2. Furthermore, immunofluorescence analysis with confocal microscopy was performed to confirm the hyperosmolality effects on activation of p38 MAPK, JNK1/2 and ERK1/2 signal pathways in nucleus pulposus cells. Our results show that in 500 and 600 mOsm/kg medium, rabbit nucleus pulposus cell apoptosis increased, and a persistent phosphorylation of p38 MAPK, JNK1/2 and ERK1/2 proteins were observed. In the same condition, the apoptotic cells death remarkably decreased when the p38 MAPK and JNK1/2 signal pathways were blocked by their inhibitors SB203580, SP600125 repectively. On the other side, the apoptotic cells death rate reraised greatly when the ERK1/2 signal pathways were blocked by its inhibitor PD98059. High osmolality activated p38MAPK, JNK1/2 and ERK1/2 in rabbit nucleus pulposus cell, and the activated p38 MAPK and JNK1/2 induced cell apoptosis, on the contrary, the activated ERK1/2 made the cell survived.

Expression profiling and functional analysis of hsa-miR-125b and its target genes in drug-resistant cell line of human gastric cancer

The expression of hsa-miR-125b is significantly downregulated in the fluorouracil-resistant cell line of human gastric cancer (BGC823/Fu). In order to investigate the role of hsa-miR-125b in the drug-resistance acquisition process of human gastric cancer, we performed a series of analysis on the sequence characteristics, species conservation, target genes, function annotation and signal transduction pathway enrichment of hsa-miR-125b using a combined bioinformatic approach such as miRbase, TargetScan6.2, PicTar, miRanda, Gene Ontology(GO) and KEGG. The results showed that the sequence of miR-125b is highly conserved in multiple species. A total of 79 target genes related to transcription regulation, protein binding, enzyme activity (P<0.001) were predicted by bioinformatics software. These genes involved in many biological processes including cell cycle, cell proliferation, cell apoptosis and cell responses to cytokine, drug responses and DNA damage (P<0.001). And these target genes mainly belong to MAPK, Wnt and p53 signal transduction pathways (P<0.01). The results revealed that hsa-miR-125b may regulate multiple biological processes and signal transduction pathways, and drug-resistant occurrence is associated with cell proliferation, cell apoptosis, cell cycle and signaling pathways including MAPK, Wnt and p53. We suggest that hsa-miR-125b may affect chemosensitivity by regulating target genes involved in the above processes and these target genes might be reliable candidates for exploring the role of hsa-miR-125b in tumor chemoresistance.