Ketogenesis attenuated KLF5 disrupts iron homeostasis via LIF to confer oxaliplatin vulnerability in colorectal cancer.

Oxaliplatin has been included as a basal drug in various chemotherapy regimens for colorectal cancer (CRC), a global health concern. However, acquired resistance to oxaliplatin affects the prognosis. This study aimed to determine whether the consumption of a KD increases the sensitivity of CRC cells to oxaliplatin via the inhibition of a classical stem cell marker, Krupple-like factor 5 (KLF5). KLF5 functions as a transcription factor for the leukemia inhibitory factor (LIF) and directly binds to its promoter region. LIF upregulation induces dephosphorylation of metal regulatory transcription factor 1 (MTF1), which is recruited to the promoter area of Ferroportin (FPN1), the only cellular iron exporter. FPN1 upregulation reduces the labile iron pool (LIP) and ferroptosis in CRC cells. KLF5 knockdown inhibits the LIF/MTF1/FPN1 axis and induces iron overload, thereby conferring sensitivity to oxaliplatin to CRC cells. KD mimicked KLF5 silencing and sensitized CRC cells to oxaliplatin via a similar mechanism. Thus, potential correlations were observed among ketogenesis, stemness, and iron homeostasis. This finding can be used to formulate a new strategy for overcoming oxaliplatin resistance in patients with CRC.

Application of circulating tumour DNA in terms of prognosis prediction in Chinese follicular lymphoma patients.

Background: Follicular lymphoma (FL), an indolent non-Hodgkin lymphoma (NHL), is generally incurable. Favourable prognosis and durable remission are crucial for FL patients. The genetic mutation spectrum provides novel biomarkers for determining the prognosis of FL patients, but its detection is easily affected by the collection of tumour tissue biopsies. In this study, we aimed to describe the mutational landscape of FL using circulating tumour DNA (ctDNA) samples and to explore the relationship between mutations and prognostic indicators of clinical outcome in patients with newly diagnosed follicular lymphoma and the prognostic value of such mutations. Methods: A total of 28 patients with newly diagnosed FL were included in this study. A targeted NGS-based 59-gene panel was used to assess the ctDNA mutation profiles. Differences in clinical factors between patients carrying mutations and those without mutations were analysed. We also explored the relationship between gene mutation status, mean VAFs (variant allele frequencies) and clinical factors. The Kaplan‒Meier method was applied to analyse the overall survival (OS) and progression-free survival (PFS) of patients carrying mutations and those without mutations. Results: ctDNA mutations were detectable in 21 (75%) patients. The most commonly mutated genes were CREBBP (54%, 15/28), KMT2D (50%, 14/28), STAT6 (29%, 8/28), CARD11 (18%, 5/28), PCLO (14%, 4/28), EP300 (14%, 4/28), BCL2 (11%, 3/28), and TNFAIP3 (11%, 3/28), with a mutation frequency of >10%. Patients with detectable ctDNA mutation tended to present with advanced Ann Arbor stage (III-IV) (p = 0.009), high FLIPI risk (3-5) (p = 0.023) and severe lymph node involvement (No. of involved areas ≥5) (p = 0.02). In addition, we found that the mean VAF was significantly higher in patients with advanced Ann Arbor stage, high-risk FLIPI, elevated lactate dehydrogenase (LDH: 0-248U/L), advanced pathology grade, bone marrow involvement (BMI) and lymph node involvement. Additionally, KMT2D, EP300, and STAT6 mutations were associated with inferior PFS (p < 0.05). Conclusion: We described the ctDNA mutation landscapes in Chinese patients with newly diagnosed FL and found that ctDNA VAF means reflect tumour burden. Moreover, PFS was shorter in patients with KMT2D, EP300 and STAT6 mutations.

A nomogram model based on pre-treatment and post-treatment MR imaging radiomics signatures: application to predict progression-free survival for nasopharyngeal carcinoma.

BACKGROUND: To establish a novel model using radiomics analysis of pre-treatment and post-treatment magnetic resonance (MR) images for prediction of progression-free survival in the patients with stage II-IVA nasopharyngeal carcinoma (NPC) in South China. METHODS: One hundred and twenty NPC patients who underwent chemoradiotherapy were enrolled (80 in the training cohort and 40 in the validation cohort). Acquiring data and screening features were performed successively. Totally 1133 radiomics features were extracted from the T2-weight images before and after treatment. Least absolute shrinkage and selection operator regression, recursive feature elimination algorithm, random forest, and minimum-redundancy maximum-relevancy (mRMR) method were used for feature selection. Nomogram discrimination and calibration were evaluated. Harrell's concordance index (C-index) and receiver operating characteristic (ROC) analyses were applied to appraise the prognostic performance of nomograms. Survival curves were plotted using Kaplan-Meier method. RESULTS: Integrating independent clinical predictors with pre-treatment and post-treatment radiomics signatures which were calculated in conformity with radiomics features, we established a clinical-and-radiomics nomogram by multivariable Cox regression. Nomogram consisting of 14 pre-treatment and 7 post-treatment selected features has been proved to yield a reliable predictive performance in both training and validation groups. The C-index of clinical-and-radiomics nomogram was 0.953 (all P < 0.05), which was higher than that of clinical (0.861) or radiomics nomograms alone (based on pre-treatment statistics: 0.942; based on post-treatment statistics: 0.944). Moreover, we received Rad-score of pre-treatment named RS1 and post-treatment named RS2 and all were used as independent predictors to divide patients into high-risk and low-risk groups. Kaplan-Meier analysis showed that lower RS1 (less than cutoff value, - 1.488) and RS2 (less than cutoff value, - 0.180) were easier to avoid disease progression (all P < 0.01). It showed clinical benefit with decision curve analysis. CONCLUSIONS: MR-based radiomics measured the burden on primary tumor before treatment and the tumor regression after chemoradiotherapy, and was used to build a model to predict progression-free survival (PFS) in the stage II-IVA NPC patients. It can also help to distinguish high-risk patients from low-risk patients, thus guiding personalized treatment decisions effectively.

Circulating tumor DNA mutation profile is associated with the prognosis and treatment response of Chinese patients with newly diagnosed diffuse large B-cell lymphoma.

Background: Characterization of gene mutation profiles can provide new treatment options for patients with diffuse large B-cell lymphoma (DLBCL). However, this method is challenged by the limited source of tissue specimens, especially those of DLBCL patients at advanced stages. Therefore, in the current study, we aimed to describe the gene mutation landscape of DLBCL using circulating tumor DNA (ctDNA) samples obtained from patients' blood samples, as well as to explore the relationship between ctDNA mutations and the prognosis and treatment response of patients with newly diagnosed DLBCL. Methods: A total of 169 newly diagnosed Chinese DLBCL patients were included in this study, among which 85 patients were divided into a training set and 84 were assigned into a validation set. The mutation profile of a 59-gene panel was analyzed by targeted next generation sequencing (NGS) of the patients' ctDNA samples. Differences in clinical factors between patients with and without ctDNA mutations were analyzed. In addition, we also explored gene mutation frequencies between GCB and non-GCB subtypes, and the relationship between gene mutation status, clinical factors, mean VAF (variant allele frequencies) and the patients' overall survival (OS) and progression-free survival (PFS). Results: ctDNA mutations were detected in 64 (75.3%) patients of the training set and 67 (79.8%) patients of the validation set. The most commonly mutated genes in both sets were PCLO, PIM1, MYD88, TP53, KMT2D, CD79B, HIST1H1E and LRP1B, with mutation frequencies of >10%. Patients with detectable ctDNA mutations trended to present advanced Ann Arbor stages (III-IV), elevated LDH (lactate dehydrogenase) levels, shorter OS and PFS, and a lower complete response (CR) rate to the R-CHOP regimen compared with DLBCL patients without ctDNA mutations. In addition, mean VAF (≥4.94%) and PCLO mutations were associated with poor OS and PFS. Conclusion: We investigated the ctDNA mutation landscape in Chinese patients with newly diagnosed DLBCL and found that ctDNA could reflect tumor burden and patients with detectable ctDNA mutations trended to have shorter OS and PFS and a lower CR rate.

Engagement of gcFKBP5/TRAF2 by spring viremia of carp virus to promote host cell apoptosis for supporting viral replication in grass carp.

Spring viremia of carp virus (SVCV) causes severe morbidity and mortality in grass carp (Ctenopharyngodon idellus) in Europe, America and several Asian countries. We found that FKBP5 (FK506-binding protein 5) is an SVCV infection response factor; however, its role in the innate immune mechanism caused by SVCV infection remains unknown. This study cloned gcFKBP5 (grass carp FKBP5) and made its mimic protein structure for function discussion. We found that gcFKBP5 expression in the primary innate immune organs of grass carp, including intestine, liver and spleen, was highly upregulated by SVCV in 24 h, with a similar result in fish cells by poly(I:C) treatment. gcFKBP overexpression aggravates viral damage to cells and increases viral replication. Furthermore, SVCV engages gcFKBP5 interacting with TRAF2 (tumour necrosis factor receptor-associated factor 2) to promote host cell apoptosis for supporting viral replication. The enhanced viral replication seems not to be due to the repression of IFN and other antiviral factors as expected. For the first time, these data show the pivotal role of gcFKBP5 in the innate immune response of grass carp to SVCV infection.

Microglial Activation of GLP-1R Signaling in Neuropathic Pain Promotes Gene Expression Adaption Involved in Inflammatory Responses.

Background: Neuropathic pain is a common chronic pain, which is related to hypersensitivity to stimulus and greatly affects the quality of life of patients. Maladaptive gene changes and molecular signaling underlie the sensitization of nociceptive pathways. We previously found that the activation of microglial glucagon-like peptide 1 receptor (GLP-1R) could potently relieve formalin-, bone cancer-, peripheral nerve injury-, and diabetes-induced pain hypersensitivity. So far, little is known about how the gene profile changes upon the activation of GLP-1R signaling in the pathophysiology of neuropathic pain. Methods: Spinal nerve ligation (SNL) was performed to induce neuropathic pain in rats. Mechanical allodynia was assessed using von Frey filaments. The expression of IL-10, ß-endorphin, and µ-opioid receptor (MOR) was examined by real-time quantitative polymerase chain reaction (qPCR) and whole-cell recording. Measurements of cellular excitability of the substantia gelatinosa (SG) neurons by whole-cell recording were carried out. R packages of differential gene expression analysis based on the negative binomial distribution (DESeq2) and weighted correlation network analysis (WGCNA) were used to analyze differential gene expression and the correlated modules among GLP-1R clusters in neuropathic pain. Results: The GLP-1R agonist, exenatide, has an antiallodynic effect on neuropathic pain, which could be reversed by intrathecal injections of the microglial inhibitor minocycline. Furthermore, differential gene expression analysis (WGCNA) indicated that intrathecal injections of exenatide could reverse the abnormal expression of 591 genes in the spinal dorsal horn induced by nerve injury. WGCNA revealed 58 modules with a close relationship between the microglial GLP-1R pathway and features of nerve injuries, including pain, ligation, paw withdrawal latency (PWL), and anxiety. The brown module was identified as the highest correlated module, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that inflammatory responses were most correlated with PWL. To further unravel the changes of hyperalgesia-related neuronal electrophysiological activity mediated by microglia GLP-1 receptors, whole-cell recording identified that MOR agonism stimulated a robust outward current in the sham groups compared with the spinal nerve ligation (SNL) groups. This inhibitory effect on the SNL group was more sensitive than that of the sham group after bath application of ß-endorphin. Conclusions: Our results further confirmed that the GLP-1R pathway is involved in alleviating pain hypersensitivity mediated by spinal microglia activation, and inflammatory responses were the most correlated pathway associated with PWL changes in response to exenatide treatment. We found that the identification of gene regulation in response to GLP-1R activation is an effective strategy for identifying new therapeutic targets for neuropathic pain. Investigation for the activation of spinal microglial GLP-1R which might ameliorate inflammatory responses through gene expression and structural changes is providing a potential biomarker in pain management.

Mean apparent diffusion coefficient in a single slice may predict tumor response to whole-brain radiation therapy in non-small-cell lung cancer patients with brain metastases.

OBJECTIVES: This study aimed to access the performance of apparent diffusion coefficient (ADC) as a predictor for treatment response to whole-brain radiotherapy (WBRT) in patients with brain metastases (BMs) from non-small-cell lung cancer (NSCLC). METHODS: A retrospective analysis was conducted of 102 NSCLC patients with BMs who underwent WBRT between 2012 and 2016. Diffusion-weighted MRI were performed pre-WBRT and within 12 weeks after WBRT started. Mean single-plane ADC value of ROIs was evaluated by two radiologists blinded to results of each other. The treatment response rate, intracranial progression-free survival (PFS), and overall survival (OS) were analyzed based on the ADC value and ΔADC respectively. At last, we used COX and logistic regression to do the multivariate analysis. RESULTS: There was good inter-observer agreement of mean ADC value pre-WBRT, post-WBRT, and ΔADC between the 2 radiologists (Pearson correlation 0.915 [pre-WBRT], 0.950 [post-WBRT], 0.937 [ΔADC], p < 0.001, for each one). High mean ADC value were related with better response rate (72.2% vs 37.5%, p = 0.001) and iPFS (7.6 vs 6.4 months, p = 0.031). High ΔADC were related with better response rate (73.6% vs 36.7%, p < 0.001). Multivariate analysis shows that histopathology, BMs number, high ADC value pre-WBRT, and high ΔADC post-WBRT were related to better treatment response of WBRT, and KPS, BMs number, and low ADC value pre-WBRT increased the risk of developing intracranial relapse. CONCLUSIONS: The mean single-plane ADC value pre-WBRT and ΔADC post-WBRT were potential predictor for intracranial tumor response to WBRT in NSCLC patients with brain metastases. KEY POINTS: • ADC value is a potential predictor of intracranial treatment response to WBRT in NSCLC patients with brain metastases. • Higher mean ADC value pre-WBRT and ΔADC post-WBRT of brain metastases were related to better intracranial tumor response. • Prediction of response before WBRT using ADC value can help oncologists to make better therapy plans and avoid missing opportunities for rescue therapy.

Apparent diffusion coefficient for the prediction of tumor response to neoadjuvant chemo-radiotherapy in locally advanced rectal cancer.

BACKGROUND: Patients with locally advanced rectal cancer generally have different response rates to preoperative neoadjuvant chemo-radiotherapy. This study investigated the value of the apparent diffusion coefficient (ADC) as a predictor to forecast the response to neoadjuvant chemo-radiotherapy in patients with locally advanced rectal cancer. METHODS: Ninety-one locally advanced rectal cancer patients who underwent neoadjuvant chemo-radiotherapy between 2015 and 2018 were enrolled. Diffusion-weighted magnetic resonance imaging was performed before treatment and within 4 weeks after the completion of neoadjuvant chemo-radiotherapy. Mean ADC values of regions of interest were evaluated by two radiologists. The tumor response was evaluated according to RESCIST 1.1. The cut-off value for the mean ADC and increasing percentage (ΔADC%) after neoadjuvant chemo-radiotherapy was calculated using the receiver operating characteristic curve. The response rate of pre-ADC and ΔADC% above/below the cut-off values was determined using the chi-square test, respectively. Primary tumor progression-free survival (PFS) was analyzed using the Kaplan-Meier method, based on the pre-ADC and ΔADC% cut-off values. RESULTS: The cut-off value of mean pre-ADC and ΔADC% was 0.94 × 10-3 mm2/s (80.36% sensitivity, 74.29% specificity) and 26.0% (73.21% sensitivity, 77.14% specificity), respectively. Lower mean pre-ADC values were related to a better response rate (83.3% vs 29.7%, P < 0.001) and PFS (26.12 vs 17.70 months, P = 0.004). ΔADC% above the cut-off value was also related to a better response rate (83.7% vs 35.7%, P < 0.001) and PFS (26.93 vs 15.65 months, P = 0.034). CONCLUSIONS: The mean ADC pre-treatment value and ΔADC% were potential predictors for the tumor response in locally advanced rectal cancer patients treated with neoadjuvant chemo-radiotherapy.

GDC-0349 inhibits non-small cell lung cancer cell growth.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related human mortality with a clear need for new therapeutic intervention. GDC-0349 is a potent and selective ATP-competitive mTOR inhibitor. In A549 cells and primary human NSCLC cells, GDC-0349 inhibited cell growth, proliferation, cell cycle progression, migration and invasion, while inducing significant apoptosis activation. Although GDC-0349 blocked Akt-mTORC1/2 activation in NSCLC cells, it also exerted cytotoxicity in Akt1-knockout A549 cells. Furthermore, restoring Akt-mTOR activation by a constitutively-active Akt1 only partially attenuated GDC-0349-induced A549 cell apoptosis, indicating the existence of Akt-mTOR-independent mechanisms. In NSCLC cells GDC-0349 induced sphingosine kinase 1 (SphK1) inhibition, ceramide accumulation, JNK activation and oxidative injury. Conversely, N-acetylcysteine, the JNK inhibitor and sphingosine 1-phosphate alleviated GDC-0349-induced NSCLC cell apoptosis. In vivo, daily oral administration of GDC-0349 potently inhibited NSCLC xenograft growth in mice. Akt-mTOR in-activation, SphK1 inhibition, JNK activation and oxidative stress were detected in NSCLC xenograft tissues with GDC-0349 administration. In summary, GDC-0349 inhibits NSCLC cell growth via Akt-mTOR-dependent and Akt-mTOR-independent mechanisms.

LINC02476 Promotes the Malignant Phenotype of Hepatocellular Carcinoma by Sponging miR-497 and Increasing HMGA2 Expression.

BACKGROUND: Long noncoding RNAs (lncRNAs) can promote hepatocellular carcinoma (HCC) initiation and progression. In this report, we examined the role of lncRNA LINC02476 in HCC. METHODS: The expression levels of different lncRNAs in HCC were explored using the TCGA database and lncRNA LINC02476 was selected for further study. The expression of LINC02476 in HCC tissues was determined by real-time PCR. The clinicopathological characteristics of HCC patients were analyzed relative to the expression of LINC02476. The expression of LINC02476 was downregulated in HCC cells using a lentiviral vector and different assays were performed to study cell growth, proliferation, invasion, apoptosis and the cell cycle. MiR-497 was selected as a miRNA that could interact with LINC02476 which was further tested by RNA immunoprecipitation. HMGA2 was selected as a possible target of miR-497, and their interaction was examined by a luciferase reporter assay. RESULTS: LINC02476 expression was elevated in HCC cell lines and HCC tissues. When LINC02476 was downregulated, the growth and the invasion of HCC cells decreased in vitro and in vivo. LINC02476 negatively regulated the expression of miR-497 by acting as a ceRNA. HMGA2 was directly targeted and inhibited by miR-497. CONCLUSION: The results indicate that LINC02476 functions through the miR-497/HMGA2 axis and that it has a role in the growth and metastasis of HCC cells. Therefore, LINC02476 could be an interesting new molecular target in HCC therapies.

Nuclear receptor co-repressor RIP140 regulates diurnal expression of cytochrome P450 2b10 in mouse liver.

Elucidating the mechanisms for circadian expression of drug-metabolizing enzymes is essential for a better understanding of dosing time-dependent drug metabolism and pharmacokinetics. CYP2B6 (Cyp2b10 in mice) is an important enzyme responsible for metabolism and detoxification of approximately 10% of drugs. Here, we aimed to investigate a potential role of nuclear receptor co-repressor RIP140 in circadian regulation of Cyp2b10 in mice.We first uncovered diurnal rhythmicity in hepatic RIP140 mRNA and protein with peak values at ZT10 (ZT, zeitgeber time). RIP140 ablation up-regulated Cyp2b10 expression and blunted its rhythm in mice and in AML-12 cells. Consistent with a negative regulatory effect, overexpression of RIP140 inhibited Cyp2b10 promoter activity and reduced cellular Cyp2b10 expression.Furthermore, RIP140 suppressed Car- and Pxr-mediated transactivation of Cyp2b10, and the suppressive effects were attenuated when the RIP140 gene was silenced. Chromatin immunoprecipitation assays revealed that recruitment of RIP140 protein to the Cyp2b10 promoter was circadian time-dependent in wild-type mice. More extensive recruitment was observed at ZT10 than at ZT2 consistent with the rhythmic pattern of RIP140 protein. However, the time-dependency of RIP140 recruitment was lost in RIP140-/- mice.Additionally, we identified a D-box and a RORE cis-element in RIP140 promoter. D-box- and RORE-acting clock components such as Dbp, E4bp4, Rev-erbα/ß and Rorα transcriptionally regulated RIP140, potentially accounting for its rhythmic expression.In conclusion, RIP140 regulates diurnal expression of Cyp2b10 in mouse liver through periodical repression of Car- and Pxr-mediated transactivation. This co-regulator-driven mechanism represents a novel source of diurnal rhythmicity in drug-metabolizing enzymes.

CircRNA BIRC6 promotes non-small cell lung cancer cell progression by sponging microRNA-145.

PURPOSE: Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality world-wide. Recently, a number of circular RNAs (circRNAs) has been found to be differentially expressed in human NSCLCs, correlating with clinico-pathological features. As yet, the expression and potential role of circRNA BIRC6 (circBIRC6) in NSCLC have not been studied. METHODS: Expression of circBIRC6 and its target microRNA-145 (miR-145) in human NSCLC cells and tissues was assessed using qRT-PCR. In vitro genetic strategies were used to exogenously alter circBIRC6 and miR-145 expression. Their impact on in vitro and in vivo NSCLC cell behavior was studied. RESULTS: We found that circBIRC6 was upregulated in primary human NSCLC tissues and NSCLC cells, whereas its potential target, miR-145, was downregulated. In A549 NSCLC cells and primary human NSCLC cells, shRNA-induced silencing of circBIRC6 potently inhibited their growth, proliferation, migration and invasion. Conversely, we found that exogenous overexpression of circBIRC6 promoted these characteristics. Using RNA immunoprecipitation (RIP) in A549 cells, we found that Argonaute 2 (Ago2) immunoprecipitated together with both circBIRC6 and miR-145. Additional studies revealed that the miR-145 level increased after circBIRC6 silencing in A549 cells, but decreased after circBIRC6 overexpression. Of note, we found that the circBIRC6 silencing-induced anti-A549 activity could be attenuated by a miR-145 inhibitor. Lastly, we found that circBIRC6 silencing inhibited the growth of NSCLC xenografts in severe combined immunodeficient mice. CONCLUSIONS: From our data we conclude that circBIRC6 overexpression promotes NSCLC cell progression, possibly by sponging miR-145.

Lemairamin, isolated from the Zanthoxylum plants, alleviates pain hypersensitivity via spinal α7 nicotinic acetylcholine receptors.

Lemairamin (also known as wgx-50), is isolated from the pericarps of the Zanthoxylum plants. As an agonist of α7 nicotinic acetylcholine receptors (α7nAChRs), it can reduce neuroinflammation in Alzheimer's disease. This study evaluated its antinociceptive effects in pain hypersensitivity and explored the underlying mechanisms. The data showed that subcutaneous lemairamin injection dose-dependently inhibited formalin-induced tonic pain but not acute nociception in mice and rats, while intrathecal lemairamin injection also dose-dependently produced mechanical antiallodynia in the ipsilateral hindpaws of neuropathic and bone cancer pain rats without affecting mechanical thresholds in the contralateral hindpaws. Multiple bi-daily lemairamin injections for 7 days did not induce mechanical antiallodynic tolerance in neuropathic rats. Moreover, the antinociceptive effects of lemairamin in formalin-induced tonic pain and mechanical antiallodynia in neuropathic pain were suppressed by the α7nAChR antagonist methyllycaconitine. In an α7nAChR antagonist-reversible manner, intrathecal lemairamin also stimulated spinal expression of IL-10 and ß-endorphin, while lemairamin treatment induced IL-10 and ß-endorphin expression in primary spinal microglial cells. In addition, intrathecal injection of a microglial activation inhibitor minocycline, anti-IL-10 antibody, anti-ß-endorphin antiserum or µ-opioid receptor-preferred antagonist naloxone was all able to block lemairamin-induced mechanical antiallodynia in neuropathic pain. These data demonstrated that lemairamin could produce antinociception in pain hypersensitivity through the spinal IL-10/ß-endorphin pathway following α7nAChR activation.

Dual µ-opioid receptor and norepinephrine reuptake mechanisms contribute to dezocine- and tapentadol-induced mechanical antiallodynia in cancer pain.

Dezocine is an opioid analgesic widely used in China, occupying over 45% of the domestic market of opioid analgesics. We have recently demonstrated that dezocine produced mechanical antiallodynia and thermal antihyperalgesia through spinal µ-opioid receptor activation and norepinephrine reuptake inhibition in neuropathic pain. This study further explored the dual µ-opioid receptor and norepinephrine reuptake mechanisms underlying dezocine-induced mechanical antiallodynia in bone cancer pain, compared with tapentadol, the first recognized analgesic in this class. Dezocine and tapentadol, given subcutaneously, exerted profound mechanical antiallodynia in bone cancer pain rats in a dose-dependent manner, yielding similar maximal effects but different potencies: ED50s of 0.6 mg/kg for dezocine and 7.5 mg/kg for tapentadol, respectively. Furthermore, their mechanical antiallodynia was partially blocked by intrathecal injection of the specific µ-opioid receptor antagonist CTAP, but not κ-opioid receptor antagonists GNTI and nor-BNI or δ-opioid receptor antagonist naltrindole. Intrathecal administrations of the specific norepinephrine depletor 6-OHDA (but not the serotonin depletor PCPA) for three consecutive days and single injection of the α-adrenoceptor antagonist phentolamine/α2-adrenoceptor antagonist yohimbine partially blocked dezocine- and tapentadol-induced mechanical antiallodynia. Strikingly, the combination of CTAP and yohimbine nearly completely blocked dezocine- and tapentadol-induced mechanical antiallodynia. Our results illustrate that both dezocine and tapentadol exert mechanical antiallodynia in bone cancer pain through dual mechanisms of µ-opioid receptor activation and norepinephrine reuptake inhibition, and suggest that the µ-opioid receptor and norepinephrine reuptake dual-targeting opioids are effective analgesics in cancer pain.

Role of the CLOCK protein in liver detoxification.

BACKGROUND AND PURPOSE: Whether and how circadian clock proteins regulate drug detoxification are not known. Here, we have assessed the effects of CLOCK (a core circadian clock protein) on drug metabolism and detoxification. EXPERIMENTAL APPROACH: Regulation by CLOCK protein of drug-metabolizing enzymes was assessed using Clock knockout (Clock-/- ) mice and Hepa-1c1c7/AML-12 cells. The relative mRNA and protein levels were determined by qPCR and Western blotting respectively. Toxicity and pharmacokinetic experiments were performed with Clock-/- and wild-type mice after intraperitoneal injection of coumarin or cyclophosphamide. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift, and chromatin immunoprecipitation (ChIP) assays. KEY RESULTS: Clock deletion disrupted hepatic diurnal expressions of a number of drug-metabolizing enzymes in mice. In particular, CYP2A4/5 expressions were markedly down-regulated, whereas CYP2B10 was up-regulated. Positive regulation of Cyp2a4/5 and negative regulation of Cyp2b10 by CLOCK were confirmed in Hepa-1c1c7 and AML-12 cells. Based on a combination of luciferase reporter, mobility shift, and ChIP assays, we found that CLOCK activated Cyp2a4/5 transcription via specific binding to E-box elements in promoter region and repressed Cyp2b10 transcription through REV-ERBα/ß (two target genes of CLOCK and transcriptional repressors of Cyp2b10). Furthermore, Clock ablation sensitized mice to coumarin toxicity by down-regulating CYP2A4/5-mediated metabolism (a detoxification pathway) and to cyclophosphamide toxicity by up-regulating CYP2B10-mediated metabolism (generating the toxic metabolite 4-hydroxycyclophosphamide). CONCLUSION AND IMPLICATIONS: CLOCK protein regulates metabolism by the cytochrome P450 family and drug detoxification. The findings improve our understanding of the crosstalk between circadian clock and drug detoxification.

E4bp4 regulates carboxylesterase 2 enzymes through repression of the nuclear receptor Rev-erbα in mice.

Carboxylesterases (CES) are a family of phase I enzymes that play an important role in xenobiotic clearance and lipid metabolism. Here, we investigate a potential role of E4 promoter-binding protein 4 (E4bp4) in regulation of Ces and CPT-11 (irinotecan, a first-line drug for treating colorectal cancer) pharmacokinetics in mice. Mouse hepatoma Hepa-1c1c7 cells were transfected with Rev-erbα expression plasmid or siRNA targeting E4bp4. The relative mRNA and protein levels of Ces enzymes in the cells or the livers of wild-type and E4bp4-deficient (E4bp4-/-) mice were determined by qPCR and Western blotting, respectively. Transcriptional regulation of Ces by E4bp4/Rev-erbα were investigated using luciferase reporter, mobility shift, and co-immunoprecipitation (Co-IP) assays. Pharmacokinetic studies were performed with wild-type and E4bp4-/- mice after intraperitoneal injection of CPT-11. E4bp4 ablation down-regulated an array of hepatic Ces genes in mice. E4bp4-/- mice also showed reduced Ces-mediated metabolism and elevated systemic exposure of CPT-11, a well-known Ces substrate. Consistently, E4bp4 knockdown reduced the expression of Ces genes (Ces2b, Ces2e and Ces2f) in Hepa-1c1c7 cells. Furthermore, Rev-erbα repressed the transcription of Ces2b, whereas E4bp4 antagonized this repressive action. Co-IP experiment confirmed a direct interaction between E4bp4 and Rev-erbα. Through a combination of promoter analysis and mobility shift assays, we demonstrated that Rev-erbα trans-repressed Ces (Ces2b) through its specific binding to the -767 to-754 bp promoter region. In conclusion, E4bp4 regulates Ces enzymes through inhibition of the transrepression activity of Rev-erbα, thereby impacting the metabolism and pharmacokinetics of Ces substrates.

The milk-derived fusion peptide, ACFP, suppresses the growth of primary human ovarian cancer cells by regulating apoptotic gene expression and signaling pathways.

BACKGROUND: ACFP is an anti-cancer fusion peptide derived from bovine milk protein. This study was to investigate the anti-cancer function and underlying mechanisms of ACFP in ovarian cancer. METHODS: Fresh ovarian tumor tissues were collected from 53 patients who underwent initial debulking surgery, and primary cancer cells were cultured. Normal ovarian surface epithelium cells (NOSECs), isolated from 7 patients who underwent surgery for uterine fibromas, were used as normal control tissue. Anti-viabilities of ACFP were assessed by WST-1 (water-soluble tetrazolium 1), and apoptosis was measured using a flow cytometry-based assay. Gene expression profiles of ovarian cancer cells treated with ACFP were generated by cDNA microarray, and the expression of apoptotic-specific genes, such as bcl-xl, bax, akt, caspase-3, CDC25C and cyclinB1, was assessed by real time PCR and western blot analysis. RESULTS: Treatment with ACFP inhibited the viability and promoted apoptosis of primary ovarian cancer cells but exhibited little or no cytotoxicity toward normal primary ovarian cells. Mechanistically, the anti-cancer effects of ACFP in ovarian cells were shown to occur partially via changes in gene expression and related signal pathways. Gene expression profiling highlighted that ACFP treatment in ovarian cancer cells repressed the expression of bcl-xl, akt, CDC25C and cyclinB1 and promoted the expression of bax and caspase-3 in a time- and dose-dependent manner. CONCLUSIONS: Our results suggest that ACFP may represent a potential therapeutic agent for ovarian cancer that functions by altering the expression and signaling of cancer-related pathways in ovarian cancer cells.

The milk-derived hexapeptide PGPIPN inhibits the invasion and migration of human ovarian cancer cells by regulating the expression of MTA1 and NM23H1 genes.

Some bioactive peptides derived from natural resources or synthesized by rational design have been proved to have very good anticancer effect. We studied the inhibition of PGPIPN, a hexapeptide derived from bovine ß-casein, on the invasion and metastasis of human ovarian cancer cells in vitro and its molecular mechanism. The human ovarian cancer cells studied include the cell line SKOV3 as well as the primary ovarian cancer cells from ovarian tumor tissues of 37 patients at initial debulking surgery, diagnosed as serous ovarian adenocarcinoma. We showed that PGPIPN inhibited the invasion of ovarian cancer cells with Transwell chamber assay, the migration of ovarian cancer cells with cell scratch assay and colony formation of ovarian cancer cells. The expression (mRNAs and proteins) of genes relevant to invasion and metastasis, MTA1, and NM23H1 were analyzed by real-time PCR and western blotting. PGPIPN repressed the expression of MTA1, and promoted NM23H1. The effects of PGPIPN were dose-dependent. Thus, our study suggests that PGPIPN is a potential therapeutic agent for adjuvant therapy of human malignant ovarian tumors.