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Abstract Cyclic guanosine-adenosine monophosphate synthase (cGAS) is a key cytosolic DNA sensor that plays a pivotal role in the innate immune response. Although a decade of research on the cGAS has advanced our understanding of inflammasome formation, cytokine production, and signaling pathways, the role of cGAS in the nucleus remains unclear. In this study, we found that the nuclear localization of endogenous and stably expressed cGAS differed from transiently expressed cGAS, which mainly localized in the cytosol. In the nucleus, cGAS is tightly bound to chromatin DNA. The chromatin DNA binding of cGAS was dependent on RSK2. Our molecular mechanism study indicated that the N-lobe of RSK2 harboring 1–323 interacted with the NTase domain of cGAS harboring residues 213–330. This interaction increased RSK2-induced cGAS phosphorylation at Ser120 and Thr130, resulting in the tightly binding of cGAS to chromatin. Importantly, epidermal growth factor (EGF)-induced cell transformation and anchorage-independent colony growth showed an increase in growth factors, such as EGF or bFGF, in cGAS stable expression compared to mock expression. Notably, the cGAS-S120A/T130A mutant abolished the increasing effect of cell transformation of JB6 Cl41 cells and colony growth of SK-MEL-2 malignant melanoma cells. The results suggested that cGAS’s chromatin DNA binding, which is indispensable to RSK2-dependent phosphorylation of cGAS at Ser120/Thr130, provides the first clue to how cGAS may participate in chromatin remodeling in the nucleus.

Abstract Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4’s transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

Abstract Accumulating evidence demonstrates that the activity regulation of ELK3, a member of the E26 transformation-specific oncogene family, is critical to regulating cell proliferation, migration, and survival in human cancers. However, the molecular mechanisms of how ELK3 induces chemoresistance in prostate cancer (PCa) have not been elucidated. In this study, we found that SPOP and ELK3 are an interacting partner. The interaction between SPOP and ELK3 resulted in increased ELK3 ubiquitination and destruction, assisted by checkpoint kinase-mediated ELK3 phosphorylation. Notably, the modulation of SPOP-mediated ELK3 protein stability affected the c-Fos-induced cell proliferation and invasion of PCa cells. The clinical involvement of the SPOP-ELK3 axis in PCa development was confirmed by an immunohistochemical assay on 123 PCa tissues, with an inverse correlation between increased ELK3 and decreased SPOP being present in ~80% of the specimens. This observation was supported by immunohistochemistry analysis using a SPOP-mutant PCa specimen. Finally, docetaxel treatment induced cell death by activating checkpoint kinase- and SPOP-mediated ELK3 degradation, while SPOP-depleted or SPOP-mutated PCa cells showed cell death resistance. Notably, this observation was correlated with the protein levels of ELK3. Taken together, our study reveals the precise mechanism of SPOP-mediated degradation of ELK3 and provides evidence that SPOP mutations contribute to docetaxel resistance in PCa.

Abstract Cancer cells often exhibit resistance to apoptotic cell death, but they may be vulnerable to other types of cell death. Elucidating additional mechanisms that govern cancer cell death is crucial for developing new therapies. Our research identified cyclic AMP-responsive element-binding protein 3 (CREB3) as a crucial regulator and initiator of a unique cell death mechanism known as karyoptosis. This process is characterized by nuclear shrinkage, deformation, and the loss of nuclear components following nuclear membrane rupture. We found that the N-terminal domain (aa 1-230) of full-length CREB3 (CREB3-FL), which is anchored to the nuclear inner membrane (INM), interacts with lamins and chromatin DNA. This interaction maintains a balance between the outward force exerted by tightly packed DNA and the inward constraining force, thereby preserving INM integrity. Under endoplasmic reticulum (ER) stress, aberrant cleavage of CREB3-FL at the INM leads to abnormal accumulation of the cleaved form of CREB3 (CREB3-CF). This accumulation disrupts the attachment of CREB3-FL to the INM, resulting in sudden rupture of the nuclear membrane and the onset of karyoptosis. Proteomic studies revealed that CREB3-CF overexpression induces a DNA damage response akin to that caused by UVB irradiation, which is associated with cellular senescence in cancer cells. These findings demonstrated that the dysregulation of CREB3-FL cleavage is a key factor in karyoptotic cell death. Consequently, these findings suggest new therapeutic strategies in cancer treatment that exploit the process of karyoptosis.

Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-Ⅰ to LC3-Ⅱ and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

The extracts of Corydalis heterocarpa, a salt-tolerant plant, exhibit diverse physiological properties, including anti-inflammatory, anticancer, and antiadipogenic effects. However, the anti-aging effects of C. heterocarpa extract (CHE) on human skin cells have not yet been investigated. In the present study, we determined that CHE inhibited senescence-associated β-galactosidase (SA-β-gal)-stained senescent human dermal fibroblasts (HDFs). Furthermore, CHE markedly suppressed the expression of major regulatory proteins involved in senescence, including p53, p21, and caveolin-1. Interestingly, CHE promoted autophagic flux, as confirmed by the formation of microtubule-associated protein 1 light chain 3B (LC3B) puncta and lysosomal activity. Notably, using RNA sequencing (RNA-seq), we showed that CHE selectively regulated the gene expression of leucine-rich repeat and sterile alpha motif-containing 1 (LRSAM1), an important regulator of autophagy. The adenosine-monophosphate activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway, which is essential for autophagy regulation, was also modulated by CHE. LRSAM1 depletion not only inhibited LC3B expression but also decreased the autophagy flux induced by CHE. Moreover, the knockdown of LRSAM1 suppressed the reversal of CHE-induced senescence in old HDFs. Collectively, our study has revealed the rejuvenating effects and molecular mechanisms of CHE, suggesting that CHE may be a promising anti-aging agent.

The inflammasome is a molecular platform that is created in the cytosolic compartment to mediate the host immunological response to cellular injury and infection. Caspase-1 may be activated by the inflammasome, which leads to the generation of the inflammatory cytokines interleukin-1β (IL-1β) and IL-18 and the beginning of pyroptosis, which is a type of proinflammatory cell death. Scientists have identified a number of different inflammasomes in the last 2 decades. The NLRP3 inflammasome has been studied the most, and its activity may be triggered by a broad range of different inducers. However, activation of the NLRP3 inflammasome in a manner that is not properly controlled is also a factor in the etiology of many human illnesses. Accumulating evidence indicates that the NLRP3 inflammasome plays a significant role in the innate and adaptive immune systems and the development of various arthritic illnesses, such as rheumatoid arthritis, ankylosing spondylitis, and gout. The present review provides a concise summary of the biological properties of the NLRP3 inflammasome and presents the fundamental processes behind its activation and control. We discuss the role of the inflammasome in the pathogenesis of arthritic diseases, such as rheumatoid arthritis, ankylosing spondylitis, and gout, and the potential of newly developed therapies that specifically target the inflammasome or its products for the treatment of inflammatory diseases, with a particular emphasis on treatment and clinical application.

Cancer: Significance of stability of regulatory protein The activity of STAT2, a protein stimulated by molecular signalling systems to activate selected genes in ways that can lead to cancer, is regulated by factors controlling its rate of degradation. Yong-Yeon Cho and colleagues at The Catholic University of Korea in South Korea review the role of STAT2 in links between molecular signals of the immune response and the onset of cancer. They focus on the significance of factors that regulate the stability of STAT2. One key factor appears to be the molecular mechanisms controlling the degradation of STAT2 by cellular structures called proteasomes. These structures break down proteins as part of routine cell maintenance. Deeper understanding of the stimulation, action and degradation of STAT2 will assist efforts to treat the many cancers in which STAT2 activity is involved.

Abstract Tumor microenvironment favors tumor cells to promote their growth and metastasis such as migration, invasion, and angiogenesis. IL-1β, one of the inflammatory cytokines released from myeloid cells in tumor microenvironment, plays an important role in development and progress of tumor. The activation of inflammasome is a critical step to secrete mature IL-1β through stepwise reactions to activate capspase-1. Therefore, we investigated whether the inhibition of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in macrophages regulated the metastatic potential of tumor cells. NLRP3 inflammasome was activated by ATP in bone marrow-derived primary mouse macrophages. The metastatic potential of mouse melanoma cell line (B16F10) was determined by migration and invasion assays with transwell system. ATP-treated wild-type macrophages increased the migration and invasion of melanoma cells. However, NLRP3- or caspase-1-knockout macrophages exhibited greatly diminished ability to promote the migration and invasion of melanoma cells. In addition, treatment with celastrol, an inhibitor of NLRP3 inflammasome, reduced the potency of macrophages to stimulate migration and invasion of melanoma cells. The results demonstrate that inhibition of the NLRP3 inflammasome in macrophages by genetic deficiency or a pharmacological inhibitor is linked to suppression of the metastatic potential of tumor cells. The results would provide a novel anti-cancer strategy to modulate tumor microenvironment by suppressing NLRP3 inflammasome and consequently reducing IL-1β production.

Inflammation is a host protection mechanism that eliminates invasive pathogens from the body. However, chronic inflammation, which occurs repeatedly and continuously over a long period, can directly damage tissues and cause various inflammatory and autoimmune diseases. Pattern recognition receptors (PRRs) respond to exogenous infectious agents called pathogen-associated molecular patterns and endogenous danger signals called danger-associated molecular patterns. Among PRRs, recent advancements in studies of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome have established its significant contribution to the pathology of various inflammatory diseases, including metabolic disorders, immune diseases, cardiovascular diseases, and cancer. The regulation of NLRP3 activation is now considered to be important for the development of potential therapeutic strategies. To this end, there is a need to elucidate the regulatory mechanism of NLRP3 inflammasome activation by multiple signaling pathways, post-translational modifications, and cellular organelles. In this review, we discuss the intracellular signaling events, post-translational modifications, small molecules, and phytochemicals participating in the regulation of NLRP3 inflammasome activation. Understanding how intracellular events and small molecule inhibitors regulate NLRP3 inflammasome activation will provide crucial information for elucidating the associated host defense mechanism and the development of efficient therapeutic strategies for chronic diseases.

The toxicokinetics of β-amanitin, a toxic bicyclic octapeptide present abundantly in Amanitaceae mushrooms, was evaluated in mice after intravenous (iv) and oral administration. The area under plasma concentration curves (AUC) following iv injection increased in proportion to doses of 0.2, 0.4, and 0.8 mg/kg. β-amanitin disappeared rapidly from plasma with a half-life of 18.3–33.6 min, and 52.3% of the iv dose was recovered as a parent form. After oral administration, the AUC again increased in proportion with doses of 2, 5, and 10 mg/kg. Absolute bioavailability was 7.3–9.4%, which resulted in 72.4% of fecal recovery from orally administered β-amanitin. Tissue-to-plasma AUC ratios of orally administered β-amanitin were the highest in the intestine and stomach. It also readily distributed to kidney > spleen > lung > liver ≈ heart. Distribution to intestines, kidneys, and the liver is in agreement with previously reported target organs after acute amatoxin poisoning. In addition, β-amanitin weakly or negligibly inhibited major cytochrome P450 and 5′-diphospho-glucuronosyltransferase activities in human liver microsomes and suppressed drug transport functions in mammalian cells that overexpress transporters, suggesting the remote drug interaction potentials caused by β-amanitin exposure.

Mitochondrial targeting is a promising approach for solving current issues in clinical application of chemotherapy and diagnosis of several disorders. Here, we discuss direct conjugation of mitochondrial-targeting moieties to anticancer drugs, antioxidants and sensor molecules. Among them, the most widely applied mitochondrial targeting moiety is triphenylphosphonium (TPP), which is a delocalized cationic lipid that readily accumulates and penetrates through the mitochondrial membrane due to the highly negative mitochondrial membrane potential. Other moieties, including short peptides, dequalinium, guanidine, rhodamine, and F16, are also known to be promising mitochondrial targeting agents. Direct conjugation of mitochondrial targeting moieties to anticancer drugs, antioxidants and sensors results in increased cytotoxicity, anti-oxidizing activity and sensing activity, respectively, compared with their non-targeting counterparts, especially in drug-resistant cells. Although many mitochondria-targeted anticancer drug conjugates have been investigated in vitro and in vivo, further clinical studies are still needed. On the other hand, several mitochondria-targeting antioxidants have been analyzed in clinical phases I, II and III trials, and one conjugate has been approved for treating eye disease in Russia. There are numerous ongoing studies of mitochondria-targeted sensors. KEY WORDS: Anticancer agents, Antioxidants, Direct conjugation, Mitochondria-targeting, Sensing agents

Inflammation and immunity are linked to the onset and development of obesity and metabolic disorders. Pattern recognition receptors (PRRs) are key regulators of inflammation and immunity in response to infection and stress, and they have critical roles in metainflammation. In this study, we investigated whether RIG-I (retinoic acid-inducible gene I)-like receptors were involved in the regulation of obesity-induced metabolic stress in RIG-I knockout (KO) mice fed a high-fat diet (HFD). RIG-I KO mice fed an HFD for 12 weeks showed greater body weight gain, higher fat composition, lower lean body mass, and higher epididymal white adipose tissue (eWAT) weight than WT mice fed HFD. In contrast, body weight gain, fat, and lean mass compositions, and eWAT weight of MDA5 (melanoma differentiation-associated protein 5) KO mice fed HFD were similar to those of WT mice fed a normal diet. RIG-I KO mice fed HFD exhibited more severely impaired glucose tolerance and higher HOMA-IR values than WT mice fed HFD. IFN-β expression induced by ER stress inducers, tunicamycin and thapsigargin, was abolished in RIG-I-deficient hepatocytes and macrophages, showing that RIG-I is required for ER stress-induced IFN-β expression. Our results show that RIG-I deficiency promotes obesity and insulin resistance induced by a high-fat diet, presenting a novel role of RIG-I in the development of obesity and metabolic disorders.

Oxidized phospholipids are well known to play physiological and pathological roles in regulating cellular homeostasis and disease progression. However, their role in cancer metastasis has not been entirely understood. In this study, effects of oxidized phosphatidylcholines such as 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) on epithelial-mesenchymal transition (EMT) and autophagy were determined in cancer cells by immunoblotting and confocal analysis. Metastasis was analyzed by a scratch wound assay and a transwell migration/invasion assay. The concentrations of POVPC and 1-palmitoyl-2-glutaroyl-sn-glycero-phosphocholine (PGPC) in tumor tissues obtained from patients were measured by LC-MS/MS analysis. POVPC induced EMT, resulting in increase of migration and invasion of human hepatocellular carcinoma cells (HepG2) and human breast cancer cells (MCF7). POVPC induced autophagic flux through AMPK-mTOR pathway. Pharmacological inhibition or siRNA knockdown of autophagy decreased migration and invasion of POVPC-treated HepG2 and MCF7 cells. POVPC and PGPC levels were greatly increased at stage II of patient-derived intrahepatic cholangiocarcinoma tissues. PGPC levels were higher in malignant breast tumor tissues than in adjacent nontumor tissues. The results show that oxidized phosphatidylcholines increase metastatic potential of cancer cells by promoting EMT, mediated through autophagy. These suggest the positive regulatory role of oxidized phospholipids accumulated in tumor microenvironment in the regulation of tumorigenesis and metastasis.

Gout is a type of inflammatory arthritis caused by the deposition of monosodium uric acid (MSU) crystals in tissues. The etiology of gout is directly linked to the NLRP3 inflammasome, since MSU crystals are NLRP3 inflammasome activators. Therefore, we decided to search for a small-molecule inhibitor of the NLRP3 inflammasome for the prevention of gout inflammation. We found that loganin suppressed MSU crystals-induced caspase-1 (p20) and interleukin (IL)-1β production and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) specks formation in mouse primary macrophages, showing its ability to inhibit the NLRP3 inflammasome. In an air pouch inflammation model, oral administration of loganin to mice prevented MSU crystals-induced production of mature IL-1β and IL-18 in air pouch exudates, resulting in decreased neutrophil recruitment. Furthermore, oral administration of loganin suppressed MSU crystals-induced gout inflammation in a mouse foot gout model, which was accompanied by the inhibition of the NLRP3 inflammasome. Loganin blocked de novo synthesis of mitochondrial DNA in air pouches and foot tissues injected with MSU crystals. Consistently, loganin prevented MSU crystals-induced mitochondrial damage in macrophages, as it increased mitochondrial membrane potential and decreased the amount of mitochondrial reactive oxygen species. These data demonstrate that loganin suppresses NLRP3 inflammasome activation by inhibiting mitochondrial stress. These results suggest a novel pharmacological strategy to prevent gout inflammation by blocking NLRP3 inflammasome activation and mitochondrial dysfunction.

Eudesmin, fargesin, epimagnolin A, magnolin, and yangambin are tetrahydrofurofuranoid lignans with various pharmacological activities found in Magnoliae Flos. The inhibition potencies of eudesmin, fargesin, epimagnolin A, magnolin, and yangambin on six major human uridine 5′-diphospho-glucuronosyltransferase (UGT) activities in human liver microsomes were evaluated using liquid chromatography–tandem mass spectrometry and cocktail substrates. Eudesmin, fargesin, epimagnolin A, magnolin, and yangambin inhibited UGT1A1 and UGT1A3 activities, but showed negligible inhibition of UGT1A4, UGT16, UGT1A9, and UGT2B7 activities at 200 μM in pooled human liver microsomes. Moreover, eudesmin, fargesin, epimagnolin A, magnolin, and yangambin noncompetitively inhibited UGT1A1-catalyzed SN38 glucuronidation with Ki values of 25.7, 25.3, 3.6, 26.0, and 17.1 μM, respectively, based on kinetic analysis of UGT1A1 inhibition in pooled human liver microsomes. Conversely, the aforementioned tetrahydrofurofuranoid lignans competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation with 39.8, 24.3, 15.1, 37.6, and 66.8 μM, respectively in pooled human liver microsomes. These in vitro results suggest the necessity of evaluating whether the five tetrahydrofurofuranoid lignans can cause drug–drug interactions with UGT1A1 and UGT1A3 substrates in vivo.

Indazole carboxamide synthetic cannabinoid, AB-PINACA, has been placed into Schedule I of the Controlled Substances Act by the US Drug Enforcement Administration since 2015. Despite the possibility of AB-PINACA exposure in drug abusers, the interactions between AB-PINACA and drug-metabolizing enzymes and transporters that play crucial roles in the pharmacokinetics and efficacy of various substrate drugs have not been investigated. This study was performed to investigate the inhibitory effects of AB-PINACA on eight clinically important human major cytochrome P450s (CYPs) and six uridine 5′-diphospho-glucuronosyltransferases (UGT) in human liver microsomes and the activities of six solute carrier transporters and two efflux transporters in transporter-overexpressing cells. AB-PINACA reversibly inhibited the metabolic activities of CYP2C8 (Ki, 16.9 µM), CYP2C9 (Ki, 6.7 µM), and CYP2C19 (Ki, 16.1 µM) and the transport activity of OAT3 (Ki, 8.3 µM). It exhibited time-dependent inhibition on CYP3A4 (Ki, 17.6 µM; kinact, 0.04047 min−1). Other metabolizing enzymes and transporters such as CYP1A2, CYP2A6, CYP2B6, CYP2D6, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, OAT1, OATP1B1, OATP1B3, OCT1, OCT2, P-glycoprotein, and BCRP, exhibited only weak interactions with AB-PINACA. These data suggest that AB-PINACA can cause drug-drug interactions with CYP3A4 substrates but that the significance of drug interactions between AB-PINACA and CYP2C8, CYP2C9, CYP2C19, or OAT3 substrates should be interpreted carefully.

The tight binding of pDNA with a cationic polymer is the crucial requirement that prevents DNA degradation from undesired DNase attack to safely deliver the pDNA to its target site. However, cationic polymer-mediated strong gene holding limits pDNA dissociation from the gene complex, resulting in a reduction in transfection efficiency. In this study, to control the decomplexation rate of pDNA from the gene complex in a hard-to-transfect cell or an easy-to-transfect cell, either α-poly(l-lysine) (APL) or ε-poly(l-lysine) (EPL) was incorporated into branched polyethylenimine (bPEI)-based nanocomplexes (NCs). Compared to bPEI/pDNA NCs, the addition of APL or EPL formed smaller bPEI-APL/pDNA NCs with similar zeta potentials or larger bPEI-EPL/pDNA NCs with reduced zeta potentials, respectively, due to the different characteristics of the primary amines in the two poly(l-lysine)s (PLs). Interestingly, although both bPEI-APL/pDNA NCs and bPEI-EPL/pDNA NCs showed similar pDNA compactness to bPEI/pDNA NCs, the addition of APL or EPL resulted in slower or faster pDNA release, respectively, from the bPEI-PL/pDNA NCs than from the bPEI/pDNA NCs. bPEI-EPL/pDNA NCs with a decomplexation enhancer (i.e., EPL) improved the transfection efficiency (TE) in both a hard-to-transfect HepG2 cell and an easy-to-transfect HEK293 cell. However, although a decomplexation inhibitor (i.e., APL) reduced the TE of bPEI-APL/pDNA NCs in both cells, the degree of reduction in the TE could be compensated by PL-mediated enhanced nuclear delivery, particularly in HepG2 cells but not HEK293 cells, because both PLs facilitate nuclear localization of the gene complex per its cellular uptake. In conclusion, a decomplexation rate controller could be a potential factor to establish a high TE and design clinically available gene complex systems.

Doxorubicin, an anthracycline antitumor antibiotic, acts as a cancer treatment by interfering with the function of DNA. Herein, liquid chromatography-tandem mass spectrometry was for the first time developed and validated for the simultaneous determination of doxorubicin and its major metabolites doxorubicinol, doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone in mouse plasma. The liquid−liquid extraction of a 10 μL mouse plasma sample with chloroform:methanol (4:1, v/v) and use of the selected reaction monitoring mode led to less matrix effect and better sensitivity. The lower limits of quantification levels were 0.5 ng/mL for doxorubicin, 0.1 ng/mL for doxorubicinol, and 0.01 ng/mL for doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone. The standard curves were linear over the range of 0.5−200 ng/mL for doxorubicin; 0.1−200 ng/mL for doxorubicinol; and 0.01−50 ng/mL for doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone in mouse plasma. The intra and inter-day relative standard deviation and relative errors for doxorubicin and its four metabolites at four quality control concentrations were 0.9−13.6% and −13.0% to 14.9%, respectively. This method was successfully applied to the pharmacokinetic study of doxorubicin and its metabolites after intravenous administration of doxorubicin at a dose of 1.3 mg/kg to female BALB/c nude mice.

Mertansine, a tubulin inhibitor, is used as the cytotoxic component of antibody−drug conjugates (ADCs) for cancer therapy. The effects of mertansine on uridine 5′-diphospho-glucuronosyltransferase (UGT) activities in human liver microsomes and its effects on the mRNA expression of cytochrome P450s (CYPs) and UGTs in human hepatocytes were evaluated to assess the potential for drug−drug interactions (DDIs). Mertansine potently inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-β-glucuronidation, and UGT1A4-catalyzed trifluoperazine N-β-d-glucuronidation, with Ki values of 13.5 µM, 4.3 µM, and 21.2 µM, respectively, but no inhibition of UGT1A6, UGT1A9, and UGT2B7 enzyme activities was observed in human liver microsomes. A 48 h treatment of mertansine (1.25−2500 nM) in human hepatocytes resulted in the dose-dependent suppression of mRNA levels of CYP1A2, CYP2B6, CYP3A4, CYP2C8, CYP2C9, CYP2C19, UGT1A1, and UGT1A9, with IC50 values of 93.7 109.1, 36.8 18.3, 160.6 167.4, 32.1 14.9, 578.4 452.0, 539.5 233.4, 856.7 781.9, and 54.1 29.1 nM, respectively, and decreased the activities of CYP1A2-mediated phenacetin O-deethylase, CYP2B6-mediated bupropion hydroxylase, and CYP3A4-mediated midazolam 1-hydroxylase. These in vitro DDI potentials of mertansine with CYP1A2, CYP2B6, CYP2C8/9/19, CYP3A4, UGT1A1, and UGT1A9 substrates suggest that it is necessary to carefully characterize the DDI potentials of ADC candidates with mertansine as a payload in the clinic.

The mitochondrion is an important intracellular organelle for drug targeting due to its key roles and functions in cellular proliferation and death. In the last few decades, several studies have revealed mitochondrial functions, attracting the focus of many researchers to work in this field over nuclear targeting. Mitochondrial targeting was initiated in 1995 with a triphenylphosphonium-thiobutyl conjugate as an antioxidant agent. The major driving force for mitochondrial targeting in cancer cells is the higher mitochondrial membrane potential compared with that of the cytosol, which allows some molecules to selectively target mitochondria. In this review, we discuss mitochondria-targeting ligand-conjugated anticancer agents and their in vitro and in vivo behaviors. In addition, we describe a mitochondria-targeting nanocarrier system for anticancer drug delivery. As previously reported, several agents have been known to have mitochondrial targeting potential; however, they are not sufficient for direct application for cancer therapy. Thus, many studies have focused on direct conjugation of targeting ligands to therapeutic agents to improve their efficacy. There are many variables for optimal mitochondria-targeted agent development, such as choosing a correct targeting ligand and linker. However, using the nanocarrier system could solve some issues related to solubility and selectivity. Thus, this review focuses on mitochondria-targeting drug conjugates and mitochondria-targeted nanocarrier systems for anticancer agent delivery.

Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability.

Embryonic stem (ES) cells are pluripotent cells with the capacity for unlimited self-renewal or differentiation. Inhibition of MAPK pathways enhances mouse ES cell pluripotency characteristics. Compared to wildtype ES cells, jnk2−/− ES cells displayed a much higher growth rate. To determine whether JNKs are required for stem cell self-renewal or differentiation, we performed a phosphorylation kinase array assay to compare mouse ES cells under LIF+ or LIF− culture conditions. The data showed that activation of JNKs was induced by LIF withdrawal. We also found that JNK1 or 2 phosphorylated Klf4 at threonines 224 and 225. Activation of JNK signaling and phosphorylation of Klf4 inhibited Klf4 transcription and transactivation activity. Importantly, jnk1−/− and jnk2−/− murine embryonic fibroblasts (MEFs) exhibited a significantly greater potency in the ability to increase the number of iPS colonies compared with jnk wildtype MEFs. Overall, our results demonstrated that JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

APINACA (known as AKB48, N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide), an indazole carboxamide synthetic cannabinoid, has been used worldwide as a new psychoactive substance. Drug abusers take various drugs concomitantly, and therefore, it is necessary to characterize the potential of APINACA-induced drug−drug interactions due to the modulation of drug-metabolizing enzymes and transporters. In this study, the inhibitory effects of APINACA on eight major human cytochrome P450s (CYPs) and six uridine 5′-diphospho-glucuronosyltransferases (UGTs) in human liver microsomes, as well as on the transport activities of six solute carrier transporters and two efflux transporters in transporter-overexpressed cells, were investigated. APINACA exhibited time-dependent inhibition of CYP3A4-mediated midazolam 1′-hydroxylation (Ki, 4.5 µM; kinact, 0.04686 min−1) and noncompetitive inhibition of UGT1A9-mediated mycophenolic acid glucuronidation (Ki, 5.9 µM). APINACA did not significantly inhibit the CYPs 1A2, 2A6, 2B6, 2C8/9/19, or 2D6 or the UGTs 1A1, 1A3, 1A4, 1A6, or 2B7 at concentrations up to 100 µM. APINACA did not significantly inhibit the transport activities of organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, OATP1B3, organic cation transporter (OCT)1, OCT2, P-glycoprotein, or breast cancer resistance protein at concentrations up to 250 μM. These data suggest that APINACA can cause drug interactions in the clinic via the inhibition of CYP3A4 or UGT1A9 activities.

Catalposide, an active component of Veronica species such as Catalpa ovata and Pseudolysimachion lingifolium, exhibits anti-inflammatory, antinociceptic, anti-oxidant, hepatoprotective, and cytostatic activities. We characterized the in vitro metabolic pathways of catalposide to predict its pharmacokinetics. Catalposide was metabolized to catalposide sulfate (M1), 4-hydroxybenzoic acid (M2), 4-hydroxybenzoic acid glucuronide (M3), and catalposide glucuronide (M4) by human hepatocytes, liver S9 fractions, and intestinal microsomes. M1 formation from catalposide was catalyzed by sulfotransferases (SULTs) 1C4, SULT1A1*1, SULT1A1*2, and SULT1E1. Catalposide glucuronidation to M4 was catalyzed by gastrointestine-specific UDP-glucuronosyltransferases (UGTs) 1A8 and UGT1A10; M4 was not detected after incubation of catalposide with human liver preparations. Hydrolysis of catalposide to M2 was catalyzed by carboxylesterases (CESs) 1 and 2, and M2 was further metabolized to M3 by UGT1A6 and UGT1A9 enzymes. Catalposide was also metabolized in extrahepatic tissues; genetic polymorphisms of the carboxylesterase (CES), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) enzymes responsible for catalposide metabolism may cause inter-individual variability in terms of catalposide pharmacokinetics.

Gout is a chronic inflammatory disease evoked by the deposition of monosodium urate (MSU) crystals in joint tissues. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is responsible for the gout inflammatory symptoms induced by MSU crystals. We investigated whether epigallocatechin-3-gallate (EGCG) suppresses the activation of the NLRP3 inflammasome, thereby effectively preventing gouty inflammation. EGCG blocked MSU crystal-induced production of caspase-1(p10) and interleukin-1β in primary mouse macrophages, indicating its suppressive effect on the NLRP3 inflammasome. In an acute gout mouse model, oral administration of EGCG to mice effectively alleviated gout inflammatory symptoms in mouse foot tissue injected with MSU crystals. The in vivo suppressive effects of EGCG correlated well with the suppression of the NLRP3 inflammasome in mouse foot tissue. EGCG inhibited the de novo synthesis of mitochondrial DNA as well as the production of reactive oxygen species in primary mouse macrophages, contributing to the suppression of the NLRP3 inflammasome. These results show that EGCG suppresses the activation of the NLRP3 inflammasome in macrophages via the blockade of mitochondrial DNA synthesis, contributing to the prevention of gouty inflammation. The inhibitory effects of EGCG on the NLRP3 inflammasome make EGCG a promising therapeutic option for NLRP3-dependent diseases such as gout.

25B-NBF, 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine, is a new psychoactive substance classified as a phenethylamine. It is a potent agonist of the 5-hydroxytryptamine receptor, but little is known about its metabolism and elimination properties since it was discovered. To aid 25B-NBF abuse screening, the metabolic characteristics of 25B-NBF were investigated in human hepatocytes and human cDNA-expressed cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes using liquid chromatography⁻high resolution mass spectrometry. At a hepatic extraction ratio of 0.80, 25B-NBF was extensively metabolized into 33 metabolites via hydroxylation, O-demethylation, bis-O-demethylation, N-debenzylation, glucuronidation, sulfation, and acetylation after incubation with pooled human hepatocytes. The metabolism of 25B-NBF was catalyzed by CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and UGT2B7 enzymes. Based on these results, it is necessary to develop a bioanalytical method for the determination of not only 25B-NBF but also its metabolites in biological samples for the screening of 25B-NBF abuse.

Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the "stemness" of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, including intracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16(INK4a), p21 and phosphorylated p53 (Ser15). Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers.

Melanosomes are specific organelles dedicated to melanin synthesis and accumulation in melanocytes. Autophagy is suggestively involved in melanosome degradation, although the potential underlying molecular mechanisms remain elusive. In selective autophagy, autophagy receptors and E3-ligases are the key factors conferring cargo selectivity. In B16F10 cells, β-mangostin efficiently induced melanosome degradation without affecting other organelles such as mitochondria, peroxisomes, and the endoplasmic reticulum. Among various autophagy receptors, optineurin (OPTN) contributes TANK-binding kinase 1 (TBK1)-dependently to melanosome degradation and its knockdown inhibited β-mangostin-mediated melanosome degradation. OPTN translocation to melanosomes was dependent on its ubiquitin-binding domain. Moreover, OPTN-mediated TBK1 activation and subsequent TBK1-mediated S187 OPTN phosphorylation were essential for melanosome degradation. β-mangostin increased K63-linked melanosome ubiquitination. Finally, the E3-ligase RCHY1 knockdown inhibited the melanosome ubiquitination required for OPTN- and TBK1-phosphorylation as well as melanosome degradation. This study suggests that melanophagy, melanosome-selective autophagy, contributes to melanosome degradation, and OPTN and RCHY1 are an essential autophagy receptor and a E3-ligase, respectively, conferring cargo selectivity in melanophagy. [ABSTRACT FROM AUTHOR]