Intermittent fasting modulates human gut microbiota diversity in a phenotype-dependent manner: a systematic review.

Cumulative evidence suggests that intermittent fasting (IF) has beneficial effects on human metabolic health. It has been indicated that its impact on the gut microbiota may mediate these beneficial effects. As a result, we hypothesized that IF may impact the human gut microbiota. A systematic review was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocol using the PubMed, Scopus, and CINAHL databases. We registered our systematic review protocol in PROSPERO under registration number CRD42021270050. Human intervention studies published until April 30, 2023, were included. The quality of the included studies was assessed using National Institutes of Health (NIH) quality assessment study tools for intervention studies. The search in the database returned 166 studies, of which 13 matched all criteria for the final qualitative analysis. The body of evidence suggests that IF modulates human gut microbiota alpha and beta diversity in lean (relatively healthy) and relatively healthy overweight/obese individuals but not in individuals with metabolic syndrome. Furthermore, IF also alters human gut microbiota composition in all phenotypes. Of interest, the gut microbiota taxa or microbial metabolites after an IF intervention are associated with metabolic markers. According to this review, IF influences the diversity and taxonomic levels of the human gut microbiota. Individual metabolic phenotypes may alter the effect of IF on the diversity and taxonomic levels of the gut microbiota.

Nrf2 Regulates the Expression of CYP2D6 by Inhibiting the Activity of Krüppel-Like Factor 9 (KLF9).

AIMS: The aim of the present study is to gain insight into the biology of Parkinson's disease (PD) and cancer to drive translational advances enabling more effective prevention and/or potential treatments. BACKGROUND: The expression of Cytochrome P450 2D6 (CYP2D6) is correlated with various diseases such as PD and cancer; therefore, exploring its regulatory mechanism at transcriptional levels is of interest. NF-E2-related factor 2 (Nrf2) has been known to be responsible for regulating phase II and phase III drug-metabolizing genes. OBJECTIVES: The objectives of this study are to investigate the transcriptional regulation of CYP2D6 by Nrf2 and to analyze its role in PD and cancer. METHODS: Nrf2 was transiently expressed in human hepatoma Hep3B cells, and the expression of CYP2D6 was examined by RT-qPCR. The promoter activity of CYP2D6 and the DNA binding of Nrf2 were examined by luciferase and ChIP assay, respectively. We then investigated the expression and correlation of Nrf2 and CYP2D6 in the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. RESULTS: In the present study, we demonstrated that Nrf2 down-regulated CYP2D6 mRNA expression in hepatoma Hep3B cells. Mechanistically, Nrf2 binds to the antioxidant responsive element (ARE) in the proximity of krüppel- like factor 9 (KLF9)-binding site within the -550/+51 of CYP2D6 promoter. The inhibition and activation of Nrf2 enhanced and suppressed KLF9 effects on CYP2D6 expression, respectively. The expression levels of Nrf2 and CYP2D6 were upregulated and downregulated in the PD patient GEO datasets compared to the healthy control tissues, and Nrf2 was negatively correlated with CYP2D6. In liver cancer patients, decreased CYP2D6 levels were apparent and associated with a lower probability of survival. CONCLUSION: Our work revealed the inhibitory role of Nrf2 in regulating CYP2D6 expression. Moreover, Nrf2- dependent regulation of CYP2D6 can be used as a prognostic factor and therapeutic strategy in PD and liver cancer.

Hydrogen peroxide activates APE1/Ref-1 via NF-κB and Parkin: a role in liver cancer resistance to oxidative stress.

Cancer cells exhibit an altered redox balance and aberrant redox signaling due to genetic, metabolic, and microenvironment-associated reprogramming. Persistently elevated levels of reactive oxygen species (ROS) contribute to many aspects of tumor development and progression. Emerging studies demonstrated the vital role of apurinic/apyrimidinic endonuclease 1 or reduction/oxidation (redox) factor 1(APE1/Ref-1) in the oxidative stress response and survival of cancer cells. APE1/Ref-1 is a multifunctional enzyme involved in the DNA damage response and functions as a redox regulator of transcription factors. We herein demonstrated that basal hydrogen peroxide (H2O2) and APE1/Ref-1 expression levels were markedly higher in cancer cell lines than in non-cancerous cells. Elevated APE1/Ref-1 levels were associated with shorter survival in liver cancer patients. Mechanistically, we showed that H2O2 activated nuclear factor-κB (NF-κB). RelA/p65 inhibited the expression of the E3 ubiquitin ligase Parkin, possibly by interfering with ATF4 activity. Parkin was responsible for the ubiquitination and proteasomal degradation of APE1/Ref-1; therefore, the H2O2-induced suppression of Parkin expression increased APE1/Ref-1 levels. The probability of survival was lower in liver cancer patients with low Parkin and high RelA expression levels. Additionally, Parkin and RelA expression levels negatively and positively correlated with APE1/Ref-1 levels, respectively, in the TCGA liver cancer cohort. We concluded that increases in APE1/Ref-1 via the NF-κB and Parkin pathways are critical for cancer cell survival under oxidative stress. The present results show the potential of the NF-κB-Parkin-APE1/Ref-1 axis as a prognostic factor and therapeutic strategy to eradicate liver cancer.

Nrf2 and Parkin-Hsc70 regulate the expression and protein stability of p62/SQSTM1 under hypoxia.

Solid tumors often contain regions with very low oxygen concentrations or hypoxia resulting from altered metabolism, uncontrolled proliferation, and abnormal tumor blood vessels. Hypoxia leads to resistance to both radio- and chemotherapy and a predisposition to tumor metastases. Under hypoxia, sequestosome 1 (SQSTM1/p62), a multifunctional stress-inducible protein involved in various cellular processes, such as autophagy, is down-regulated. The hypoxic depletion of p62 is mediated by autophagic degradation. We herein demonstrated that hypoxia down-regulated p62 in the hepatoma cell line Hep3B at the transcriptional and post-translational levels. At the transcriptional level, hypoxia down-regulated p62 mRNA by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2). The overexpression of Nrf2 and knockdown of Siah2, a negative regulator of Nrf2 under hypoxia, diminished the effects of hypoxia on p62 mRNA. At the post-translational level, the proteasome inhibitor MG132, but not the lysosomal inhibitors ammonium chloride and bafilomycin, prevented the hypoxic depletion of p62, suggesting the involvement of the proteasome pathway. Under hypoxia, the expression of the E3 ubiquitin ligase Parkin was up-regulated in a hypoxia-inducible factor 1α-dependent manner. Parkin ubiquitinated p62 and led to its proteasomal degradation, ensuring low levels of p62 under hypoxia. We demonstrated that the effects of Parkin on p62 required heat shock cognate 71 kDa protein (Hsc70). We also showed that the overexpression of Nrf2 and knockdown of Parkin or Hsc70 induced the accumulation of p62 and reduced the viability of cells under hypoxia. We concluded that a decrease in p62, which involves regulation at the transcriptional and post-translational levels, is critical for cell survival under hypoxia. The present results show the potential of targeting Nrf2/Parkin-Hsc70-p62 as a novel strategy to eradicate hypoxic solid tumors.

Chlorogenic Acid Activates Nrf2/SKN-1 and Prolongs the Lifespan of Caenorhabditis elegans via the Akt-FOXO3/DAF16a-DDB1 Pathway and Activation of DAF16f.

Chlorogenic acid (CGA) is the most abundant polyphenol in coffee. It has been widely reported to exhibit antioxidant activity by activating nuclear factor erythroid 2-related factor 2 (Nrf2) potentially via the canonical Kelch-like-ECH-associated protein 1 (Keap1)-Nrf2 pathway. We herein demonstrated that the knockdown of WD40 repeat protein 23 (WDR23), but not Keap1, abolished the effects of CGA on the activation of Nrf2. CGA decreased the expression of DDB1, an adaptor for WDR23-Cullin 4A-RING ligase (CRL4AWDR23). FOXO3, a major target for inactivation by the PI3K/Akt pathway, was identified as the transcription factor responsible for the basal and CGA-inhibited expression of the DDB1 gene. CGA blocked FOXO3 binding to importin-7 (IPO7), thereby inhibiting the nuclear accumulation of FOXO3, down-regulating the expression of DDB1, inhibiting the activity of CRL4WDR23, and ultimately increasing that of Nrf2. This pathway was conserved in Caenorhabditis elegans, and CGA extended the lifespan partly through this pathway. We found that in C. elegans, the isoform DAF-16a, but not DAF-16f, regulated the expression levels of ddb-1 mRNA and SKN-1 protein. CGA prolonged the mean lifespan of DAF-16a- and DAF-16f-rescued worms by 24% and 9%, respectively, suggesting that both isoforms involve in lifespan-extending effects of CGA, with DAF-16a being more important than DAF-16f. Based on these results, we established a novel Akt-FOXO3/DAF16a-DDB1 axis that links nutrient sensing and oxidative stress response pathways. Our results also provide a novel molecular mechanism for Nrf2/SKN-1 activation by CGA and the increased lifespan of C. elegans by CGA via this pathway.

Yeast ß-glucan Increases Etoposide Sensitivity in Lung Cancer Cell Line A549 by Suppressing Nuclear Factor Erythroid 2-Related Factor 2 via the Noncanonical Nuclear Factor Kappa B Pathway.

Etoposide is regarded as one of the main standard cytotoxic drugs for lung cancer. However, mutations in Kelch-like ECH-associated protein 1 (Keap1), the main regulator of nuclear factor erythroid 2-related factor 2 (Nrf2), are often detected in lung cancer and lead to chemoresistance. Since the aberrant activation of Nrf2 enhances drug resistance, the suppression of the Nrf2 pathway is a promising therapeutic strategy for lung cancer. We herein used the human lung adenocarcinoma cell line A549 because it harbors a Keap1 loss-of-function mutation. A treatment with ß-glucan, a major component of the fungal cell wall, reduced Nrf2 protein levels; downregulated the expression of cytochrome P450 3A5, UDP glucuronosyltransferase 1A1, and multidrug resistance protein 1; and increased etoposide sensitivity in A549 cells. Furthermore, the ephrin type-A receptor 2 (EphA2) receptor was important for the recognition and biologic activity of ß-glucan in A549 cells. EphA2 signaling includes nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). However, treatment of cells with stattic (STAT3 inhibitor) or SB203580 (p38 MAPK inhibitor) did not diminish the effects of ß-glucan. In contrast, knockdown of v-rel reticuloendotheliosis viral oncogene homolog B (RelB) abolished the effects of ß-glucan, suggesting the involvement of the noncanonical NF-κB pathway. The ß-glucan effects were also attenuated by the knockdown of WD40 Repeat protein 23 (WDR23). The ß-glucan treatment and RelB overexpression induced the expression of Cullin-4A (CUL4A), which increased WDR23 ligase activity and promoted the subsequent depletion of Nrf2. These results revealed a novel property of ß-glucan as a resistance-modifying agent in addition to its widely reported immunomodulatory effects for lung cancer therapy via the EphA2-RelB-CUL4A-Nrf2 axis. SIGNIFICANCE STATEMENT: Chemotherapeutic resistance remains a major obstacle in cancer therapy despite extensive efforts to elucidate the underlying molecular mechanisms and overcome multidrug resistance. The present study revealed a novel resistance-modifying property of ß-glucan, thereby expanding our knowledge on the beneficial roles of ß-glucan and providing an alternative strategy to prevent drug resistance by cancer. The present results provide evidence for the involvement of a novel mode of NF-κB and Nrf2 crosstalk in the drug resistance phenotype.

Sp1 is a substrate of Keap1 and regulates the activity of CRL4AWDR23 ubiquitin ligase toward Nrf2.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical transcription factor that orchestrates cellular responses to oxidative stress. Because the dysregulation of Nrf2 has been implicated in many diseases, precise regulation of its protein level is crucial for maintaining homeostasis. Kelch-like-ECH-associated protein 1 (Keap1) and WD40 repeat protein 23 (WDR23) directly regulate Nrf2 levels via similar but distinct proteasome-dependent pathways. WDR23 forms a part of the WDR23-Cullin 4A-RING ubiquitin ligase complex (CRL4AWDR23), whereas Keap1 serves as a substrate adaptor for the Cullin 3-containing ubiquitin ligase complex. However, the mechanisms underlying crosstalk between these Keap1 and WDR23 pathways for the regulation of Nrf2 levels have not been investigated. Here, we showed that knockdown (KD) of Keap1 upregulated the expression of Cullin4A (CUL4A) in a specificity protein 1 (Sp1)-dependent manner. We also revealed that Sp1 interacted with Keap1, leading to ubiquitination of Sp1. Increases in Sp1 by Keap1 KD triggered Sp1 binding to the fourth Sp1 binding site (Sp1_M4) within the -230/+50 region of the CUL4A gene. We also demonstrated that the overexpression and KD of Sp1 reduced and increased Nrf2 protein levels, respectively. These effects were abrogated by the WDR23 KD, suggesting that Sp1 also regulates Nrf2 levels via the ubiquitin ligase complex CRL4AWDR23. In conclusion, we discovered Sp1 as a novel substrate of Keap1 and provided evidence that Sp1 regulates the expression of CUL4A. We revealed a novel role for Sp1 in mediating crosstalk between two independent regulators of Nrf2 protein levels.

Contribution of DHA diols (19,20-DHDP) produced by cytochrome P450s and soluble epoxide hydrolase to the beneficial effects of DHA supplementation in the brains of rotenone-induced rat models of Parkinson's disease.

Docosahexaenoic acid (DHA) has been shown to have neuroprotective effects in Parkinson's disease, but the underlying mechanism has not been fully elucidated. DHA is metabolized to DHA epoxides (EDPs) and hydroxides by cytochrome P450s (P450s), and EDPs are further hydroxylated to the corresponding diols, dihydroxydocosapentaenoic acids (DHDPs) by soluble epoxide hydrolase (sEH). In the present study, we investigated the roles of these DHA metabolites in the beneficial effects of DHA supplementation on a rotenone-induced rat model of Parkinson's disease. Metabolite analysis by LC-MS revealed that CYP2A1, 2C11, 2C13, 2C23, and 2E1 contributed to the formation of EDPs, and these P450s and sEH were expressed in the rat brain. We found that DHA supplementation in rats improved the motor dysfunction induced by rotenone. In addition, DHA reversed the decrease in tyrosine hydroxylase and the increase in lipid peroxidation generated by rotenone in the striatum. DHA supplementation also induced mRNA expression of antioxidant genes, such as sod1 and catalase, and Nrf2 protein expression in the striatum. However, these effects of DHA supplementation were eliminated by cosupplementation with the sEH inhibitor TPPU. Supplementation with DHA increased the amount of 19,20-DHDP in the rat brain, while the amount of EDPs was not significantly increased. In addition, TPPU suppressed the increase in DHDPs and increased EDPs in the brain. In PC12 cells, 19,20-DHDP increased the mRNA levels of sod1 and catalase along with Nrf2 induction. This study suggests that DHA metabolites-DHDPs generated by P450s and sEH-have an important role in improving rotenone-induced Parkinson's disease.

WDR23 regulates the expression of Nrf2-driven drug-metabolizing enzymes.

Nrf2 plays a central role in the response to xenobiotics and oxidative stress. The activation of Nrf2 induces the expression of drug-metabolizing enzymes (DMEs) and is important for cytoprotection. Keap1 is a widely accepted proteasome-dependent regulator of Nrf2. Keap1 was reported to be absent in Caenorhabditis elegans, and the level of the Nrf2 ortholog SKN-1 was mainly regulated by WDR23. The WDR23 locus is highly conserved from C. elegans to humans. We investigated whether WDR23 regulates Nrf2 activity in mammalian cells, hepatocellular carcinoma cells (Hep3B) and human cervical carcinoma cells (HeLa). We found that WDR23 has two isoforms (1 and 2) and that knockdown of WDR23 was sufficient to stabilize Nrf2 and alter the expression of several DMEs. Keap1 knockdown resulted in higher Nrf2 levels than WDR23 knockdown, and their effects on DMEs differed. These results were consistent with Keap1 being a canonical regulator of Nrf2, and that WDR23 may assist in Nrf2 regulation. We confirmed that WDR23 physically interacted with Nrf2, suggesting that WDR23 directly regulates Nrf2-dependent DMEs. In immunostaining experiments, human WDR23 isoform 1 was localized to the cytoplasm, whereas isoform 2 mainly resided in the nucleus. Taken together, our results suggested WDR23 is a novel regulator of DME expression.

The role of E3 ubiquitin ligase seven in absentia homolog in the innate immune system: An overview.

The innate immune system has been considered as an ancient system and less important than the adaptive immune system. However, the interest in innate immunity has grown significantly in the past few years marked by the identification of Toll-like receptors, a member of pattern recognition receptors (PRRs). The PRRs are crucial for the identification of self- and non-self-antigen and play a role in the initiation of signaling events that activate the effective immune response. These sensor signals through interweaving signaling cascades which result in the production of interferons and cytokines as the effector of immune system. Ubiquitin and ubiquitin-like modifiers (UBLs) actively mediate the rapid and versatile regulatory processes that initiate the activation of the innate immune system cascade. The seven in absentia homolog (SIAH) is a potent RING finger E3 ubiquitin ligase that is known to involve in several stress responses, including hypoxia, oxidative stress, DNA damage stress, and inflammation. In this review, the role of SIAH will be discussed as an E3 ubiquitin ligase on the regulation of innate immune.