ITC-6102RO, a novel B7-H3 antibody-drug conjugate, exhibits potent therapeutic effects against B7-H3 expressing solid tumors.

BACKGROUND: The B7-H3 protein, encoded by the CD276 gene, is a member of the B7 family of proteins and a transmembrane glycoprotein. It is highly expressed in various solid tumors, such as lung and breast cancer, and has been associated with limited expression in normal tissues and poor clinical outcomes across different malignancies. Additionally, B7-H3 plays a crucial role in anticancer immune responses. Antibody-drug conjugates (ADCs) are a promising therapeutic modality, utilizing antibodies targeting tumor antigens to selectively and effectively deliver potent cytotoxic agents to tumors. METHODS: In this study, we demonstrate the potential of a novel B7-H3-targeting ADC, ITC-6102RO, for B7-H3-targeted therapy. ITC-6102RO was developed and conjugated with dHBD, a soluble derivative of pyrrolobenzodiazepine (PBD), using Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linkers with high biostability. We assessed the cytotoxicity and internalization of ITC-6102RO in B7-H3 overexpressing cell lines in vitro and evaluated its anticancer efficacy and mode of action in B7-H3 overexpressing cell-derived and patient-derived xenograft models in vivo. RESULTS: ITC-6102RO inhibited cell viability in B7-H3-positive lung and breast cancer cell lines, inducing cell cycle arrest in the S phase, DNA damage, and apoptosis in vitro. The binding activity and selectivity of ITC-6102RO with B7-H3 were comparable to those of the unconjugated anti-B7-H3 antibody. Furthermore, ITC-6102RO proved effective in B7-H3-positive JIMT-1 subcutaneously xenografted mice and exhibited a potent antitumor effect on B7-H3-positive lung cancer patient-derived xenograft (PDX) models. The mode of action, including S phase arrest and DNA damage induced by dHBD, was confirmed in JIMT-1 tumor tissues. CONCLUSIONS: Our preclinical data indicate that ITC-6102RO is a promising therapeutic agent for B7-H3-targeted therapy. Moreover, we anticipate that OHPAS linkers will serve as a valuable platform for developing novel ADCs targeting a wide range of targets.

Molecular landscape of osimertinib resistance in patients and patient-derived preclinical models.

INTRODUCTION: Osimertinib is a third-generation EGFR tyrosine kinase inhibitor (TKI) that is approved for the use of EGFR-mutant non-small cell lung cancer (NSCLC) patients. In this study, we investigated the acquired resistance mechanisms in NSCLC patients and patient-derived preclinical models. METHODS: Formalin-fixed paraffin-embedded tumor samples and plasma samples from 55 NSCLC patients who were treated with osimertinib were collected at baseline and at progressive disease (PD). Next-generation sequencing was performed in tumor and plasma samples using a 600-gene hybrid capture panel designed by AstraZeneca. Osimertinib-resistant cell lines and patient-derived xenografts and cells were generated and whole exome sequencing and RNA sequencing were performed. In vitro experiments were performed to functionally study the acquired mutations identified. RESULTS: A total of 55 patients and a total of 149 samples (57 tumor samples and 92 plasma samples) were analyzed, and among them 36 patients had matched pre- and post-treatment samples. EGFR C797S (14%) mutation was the most frequent EGFR-dependent mechanism identified in all available progression samples, followed by EGFR G824D (6%), V726M (3%), and V843I (3%). Matched pre- and post-treatment sample analysis revealed in-depth acquired mechanisms of resistance. EGFR C797S was still most frequent (11%) among EGFR-dependent mechanism, while among EGFR-independent mechanisms, PIK3CA, ALK, BRAF, EP300, KRAS, and RAF1 mutations were detected. Among Osimertinib-resistant cell lines and patient-derived models, we noted acquired mutations which were potentially targetable such as NRAS p.Q61K, in which resistance could be overcome with combination of osimertinib and trametinib. A patient-derived xenograft established from osimertinib-resistant patient revealed KRAS p.G12D mutation which could be overcome with combination of osimertinib, trametinib, and buparlisib. CONCLUSION: In this study, we explored the genetic profiles of osimertinib-resistant NSCLC patient samples using targeted deep sequencing. In vitro and in vivo models harboring osimertinib resistance revealed potential novel treatment strategies after osimertinib failure.

SKI-G-801, an AXL kinase inhibitor, blocks metastasis through inducing anti-tumor immune responses and potentiates anti-PD-1 therapy in mouse cancer models.

OBJECTIVES: AXL-mediated activation of aberrant tyrosine kinase drives various oncogenic processes and facilitates an immunosuppressive microenvironment. We evaluated the anti-tumor and anti-metastatic activities of SKI-G-801, a small-molecule inhibitor of AXL, alone and in combination with anti-PD-1 therapy. METHODS: In vitro pAXL inhibition by SKI-G-801 was performed in both human and mouse cancer cell lines. Immunocompetent mouse models of tumor were established to measure anti-metastatic potential of SKI-G-801. Furthermore, SKI-G-801, anti-PD-1 or their combination was administered as an adjuvant or neoadjuvant in the 4T1 tumor model to assess their potential for clinical application. RESULTS: SKI-G-801 robustly inhibited pAXL expression in various cell lines. SKI-G-801 alone or in combination with anti-PD-1 potently inhibited metastasis in B16F10 melanoma, CT26 colon and 4T1 breast models. SKI-G-801 inhibited the growth of B16F10 and 4T1 tumor-bearing mice but not immune-deficient mice. An antibody depletion assay revealed that CD8+ T cells significantly contributed to SKI-G-801-mediated survival. Anti-PD-1 and combination group were observed the increased CD8+Ki67+ and effector T cells and M1 macrophage and decreased M2 macrophage, and granulocytic myeloid-derived suppressor cell (G-MDSC) compared to the control group. The neoadjuvant combination of SKI-G-801 and anti-PD-1 therapy achieved superior survival benefits by inducing more profound T-cell responses in the 4T1 syngeneic mouse model. CONCLUSION: SKI-G-801 significantly suppressed tumor metastasis and growth by enhancing anti-tumor immune responses. Our results suggest that SKI-G-801 has the potential to overcome anti-PD-1 therapy resistance and allow more patients to benefit from anti-PD-1 therapy.

Kinetic Study of Subcritical Water Extraction of Scopoletin, Alizarin, and Rutin from Morinda citrifolia.

Noni fruits (Morinda citrifolia) are a source of phenolic bioactive compounds (scopoletin, alizarin, and rutin), which have antioxidant, antimicrobial, anticancer, and anti-inflammatory activities. In this study, subcritical water was applied to determine the extraction yields and kinetics of phenolic compounds from noni fruits. The scopoletin and alizarin yields increased with the increase in temperature from 100 to 140 °C, while that of rutin increased up to 120 °C and then decreased at 140 °C. The yields of all the compounds rapidly increased from 1 to 2 mL/min and then slightly up to 3 mL/min of water flow rate. The extraction kinetics were assessed using two mathematical models. The two-site kinetic desorption model had a better fit for all experimental conditions throughout the extraction cycle and best described the extraction kinetics of phenolic compounds from noni fruits. The diffusion coefficients of scopoletin and alizarin at 140 °C and 3 mL/min were 3.7- and 16.2-fold higher than those at 100 °C and 1 mL/min, respectively. The activation energies of alizarin were 2.9- to 8.5-fold higher than those of scopoletin at various flow rates. Thus, subcritical water could be an excellent solvent with higher extraction yields and shorter extraction times using an environmentally friendly solvent.

Brief Report: Heterogeneity of Acquired Resistance Mechanisms to Osimertinib and Savolitinib.

INTRODUCTION: MET amplification is a frequently observed mechanism of resistance to osimertinib, and coinhibition strategy of MET and EGFR revealed promising results in recent clinical trials. Nevertheless, acquired resistance mechanisms to combined EGFR and MET inhibition are poorly understood. In this study, we investigated the mechanisms of acquired resistance to osimertinib and savolitinib by using pretreatment and post-treatment tissue analysis. METHODS: Whole-exome sequencing was performed in EGFR-mutant, MET-amplified patients who received osimertinib and savolitinib using tissues obtained both before and after therapy. All patients achieved partial response or durable stable disease to osimertinib and savolitinib before developing acquired resistance. RESULTS: After progression on osimertinib and savolitinib, whole-exome analysis revealed MET-dependent mechanisms of resistance, such as acquired MET p.D1246H mutation, MET p.Y1230C mutation, and MET copy number gain. As for MET-independent mechanisms, development of ERBB2 mutation and amplification and copy number gains in amplifications in CCNE, CCND1, CDK6, and EGFR were observed. Patient 2 harbored an acquired PIK3CA p.H1047R mutation in which resistance could be overcome with combination of PI3K inhibitor and osimertinib in the patient-derived xenograft model. CONCLUSIONS: Our study reveals that acquired resistance to savolitinib plus osimertinib can occur from both MET-dependent and MET-independent mechanisms.

Clinical decision support algorithm based on machine learning to assess the clinical response to anti-programmed death-1 therapy in patients with non-small-cell lung cancer.

OBJECTIVE: Anti-programmed death (PD)-1 therapy confers sustainable clinical benefits for patients with non-small-cell lung cancer (NSCLC), but only some patients respond to the treatment. Various clinical characteristics, including the PD-ligand 1 (PD-L1) level, are related to the anti-PD-1 response; however, none of these can independently serve as predictive biomarkers. Herein, we established a machine learning (ML)-based clinical decision support algorithm to predict the anti-PD-1 response by comprehensively combining the clinical information. MATERIALS AND METHODS: We collected clinical data, including patient characteristics, mutations and laboratory findings, from the electronic medical records of 142 patients with NSCLC treated with anti-PD-1 therapy; these were analysed for the clinical outcome as the discovery set. Nineteen clinically meaningful features were used in supervised ML algorithms, including LightGBM, XGBoost, multilayer neural network, ridge regression and linear discriminant analysis, to predict anti-PD-1 responses. Based on each ML algorithm's prediction performance, the optimal ML was selected and validated in an independent validation set of PD-1 inhibitor-treated patients. RESULTS: Several factors, including PD-L1 expression, tumour burden and neutrophil-to-lymphocyte ratio, could independently predict the anti-PD-1 response in the discovery set. ML platforms based on the LightGBM algorithm using 19 clinical features showed more significant prediction performance (area under the curve [AUC] 0.788) than on individual clinical features and traditional multivariate logistic regression (AUC 0.759). CONCLUSION: Collectively, our LightGBM algorithm offers a clinical decision support model to predict the anti-PD-1 response in patients with NSCLC.

Organic Acid-Catalyzed Subcritical Water Hydrolysis of Immature Citrus unshiu Pomace.

Immature Citrus unshiu pomace (ICUP) was hydrolyzed under organic acid-catalyzed, subcritical water (SW) conditions to produce flavonoid monoglucosides (hesperetin-7-O-glycoside and prunin) and aglycons (hesperetin and naringenin) with high biological activities. The results of single-factor experiments showed that with 8 h of hydrolysis and an increasing citric acid concentration, the yield of flavonoid monoglucosides (hesperetin-7-O-glycoside and prunin) increased from 0 to 7% citric acid. Afterward, the hesperetin-7-O-glycoside yield remained constant (from 7 to 19% citric acid) while the pruning yield decreased with 19% of citric acid, whereas the aglycon yield increased continuously. In response surface methodology analysis, a citric acid concentration and hydrolysis duration of 13.34% and 7.94 h were predicted to produce the highest monoglucoside yield of 15.41 mg/g, while 18.48% citric acid and a 9.65 h hydrolysis duration produced the highest aglycon yield of 10.00 mg/g. The inhibitory activities of the SW hydrolysates against pancreatic lipase (PL) and xanthine oxidase (XO) were greatly affected by citric acid concentration and hydrolysis duration, respectively. PL and α-glucosidase inhibition rates of 88.2% and 62.7%, respectively, were achieved with 18.48% citric acid and an 8 h hydrolysis duration, compared to 72.8% for XO with 16% citric acid and 12 h of hydrolysis. This study confirms the potential of citric acid-catalyzed SW hydrolysis of ICUP for producing flavonoid monoglucosides and aglycons with enhanced enzyme inhibitory activities.

Extraction of flavanones from immature Citrus unshiu pomace: process optimization and antioxidant evaluation.

Dietary guidelines recommend the consumption of flavonoid-rich extracts for several health benefits. Although immature Citrus unshiu pomace (ICUP) contains high levels of flavanone glycosides, many studies have concentrated on the optimization of flavonoid extraction from mature citrus peels. Therefore, we developed an optimized extraction method for hesperidin and narirutin from ICUP, and evaluated their antioxidant activities using ten different assay methods. The extraction conditions for the highest flavonoid yields based on a response surface methodology were 80.3 °C, 58.4% (ethanol concentration), 40 mL/g (solvent/feed), and 30 min, where the hesperidin and narirutin yields were 66.6% and 82.3%, respectively. The number of extractions was also optimized as two extraction steps, where the hesperidin and narirutin yields were 92.1% and 97.2%, respectively. Ethanol was more effective than methanol and acetone. The ethanol extract showed high scavenging activities against reactive oxygen species but relatively low scavenging activities for nitrogen radicals and reactive nitrogen species. The antioxidant activities showed a higher correlation with hesperidin content than narirutin content in the extracts. This study confirms the potential of an optimized method for producing antioxidant-rich extracts for the functional food and nutraceutical industries.

Osimertinib, an Irreversible Next-Generation EGFR Tyrosine Kinase Inhibitor, Exerts Antitumor Activity in Various Preclinical NSCLC Models Harboring the Uncommon EGFR Mutations G719X or L861Q or S768I.

Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI-sensitizing and EGFR T790M-resistance mutations with lower activity against wild-type EGFR and has demonstrated efficacy in non-small cell lung cancer (NSCLC) CNS metastases. The sensitizing mutations, the in-frame deletions in exon 19 and the L858R point mutation in exon 21, represent between 80% and 90% of all EGFR mutations. The remaining 10% to 20% are referred to as uncommon activating mutations and are a diverse group of mutations in exons 18 to 21 within the kinase domain of the EGFR gene. Excluding those found as insertion mutations in exon 20, the uncommon mutations involving codons G719, S768, and L861 are the most prevalent.Although the efficacy of EGFR-TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations, such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series.Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDXs that harbor the G719X mutation, we have evaluated in vitro and in vivo the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of PDX harboring the G719X mutation alone or in combination with L861Q and S768I.Together, these data support clinical testing of osimertinib in patients with uncommon EGFR NSCLC.

Antitumor Activity of Amivantamab (JNJ-61186372), an EGFR-MET Bispecific Antibody, in Diverse Models of EGFR Exon 20 Insertion-Driven NSCLC.

EGFR exon 20 insertion driver mutations (Exon20ins) in non-small cell lung cancer (NSCLC) are insensitive to EGFR tyrosine kinase inhibitors (TKI). Amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR-MET, has shown preclinical activity in TKI-sensitive EGFR-mutated NSCLC models and in an ongoing first-in-human study in patients with advanced NSCLC. However, the activity of amivantamab in Exon20ins-driven tumors has not yet been described. Ba/F3 cells and patient-derived cells/organoids/xenograft models harboring diverse Exon20ins were used to characterize the antitumor mechanism of amivantamab. Amivantamab inhibited proliferation by effectively downmodulating EGFR-MET levels and inducing immune-directed antitumor activity with increased IFNγ secretion in various models. Importantly, in vivo efficacy of amivantamab was superior to cetuximab or poziotinib, an experimental Exon20ins-targeted TKI. Amivantamab produced robust tumor responses in two Exon20ins patients, highlighting the important translational nature of this preclinical work. These findings provide mechanistic insight into the activity of amivantamab and support its continued clinical development in Exon20ins patients, an area of high unmet medical need. SIGNIFICANCE: Currently, there are no approved targeted therapies for EGFR Exon20ins-driven NSCLC. Preclinical data shown here, together with promising clinical activity in an ongoing phase I study, strongly support further clinical investigation of amivantamab in EGFR Exon20ins-driven NSCLC.This article is highlighted in the In This Issue feature, p. 1079.

Semi-Continuous Subcritical Water Extraction of Flavonoids from Citrus unshiu Peel: Their Antioxidant and Enzyme Inhibitory Activities.

We extracted and hydrolyzed bioactive flavonoids from C. unshiu peel using subcritical water (SW) in a semi-continuous mode. The individual flavonoid yields, antioxidant and enzyme inhibitory activities of the SW extracts were analyzed. The extraction yields of hesperidin and narirutin increased with increasing temperature from 145 °C to 165 °C. Hydrothermal hydrolysis products (HHP), such as monoglucosides (hesperetin-7-O-glucoside and prunin) and aglycones (hesperetin and naringenin) were obtained in the SW extracts at temperatures above 160 °C. The sum of hesperidin and its HHP in the SW extracts was strongly correlated with antioxidant activities, whereas the contents of hesperetin and naringenin were strongly correlated with enzyme inhibitory activities. Hesperetin exhibited the highest antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, ferric-reducing antioxidant power, and oxygen radical absorbance capacity), whereas hesperetin-7-O-glucoside exhibited the highest enzyme inhibitory activities (angiotensin-І converting enzyme (ACE) and pancreatic lipase (PL)). Naringenin exhibited the highest enzyme inhibitory activities (xanthine oxidase and α-glucosidase). PMFs (sinensetin, nobiletin, and tangeretin) also exhibited relatively high inhibitory activities against ACE and PL. This study confirms the potential of SW for extracting and hydrolyzing bioactive flavonoids from C. unshiu peel using an environmentally friendly solvent (water) and a shorter extraction time.

Ishophloroglucin A, a Novel Phlorotannin for Standardizing the Anti-α-Glucosidase Activity of Ishige okamurae.

Nutraceutical use of algae requires understanding of the diversity and significance of their active compositions for intended activities. Ishige okamurae (I. okamurae) extract is well-known to possess α-glucosidase inhibitory activity; however, studies are needed to investigate its active composition in order to standardize its α-glucosidase inhibitory activity. In this study, we observed the intensity of the dominant compounds of each I. okamurae extract harvested between 2016 and 2017, and the different potency of each I. okamurae extract against α-glucosidase. By comparing the anti-α-glucosidase ability of the dominant compounds, a novel Ishophloroglucin A with highest α-glucosidase inhibitory activity was identified and suggested for standardization of anti-α-glucosidase activity in I. okamurae extract. Additionally, a validated analytical method for measurement of Ishophloroglucin A for future standardization of I. okamurae extract was established in this study. We suggest using Ishophloroglucin A to standardize anti-α-glucosidase potency of I. okamurae and propose the significance of standardization based on their composition for effective use of algae as marine-derived nutraceuticals.

Diverse Roles of Mitochondria in Immune Responses: Novel Insights Into Immuno-Metabolism.

Lack of immune system cells or impairment in differentiation of immune cells is the basis for many chronic diseases. Metabolic changes could be the root cause for this immune cell impairment. These changes could be a result of altered transcription, cytokine production from surrounding cells, and changes in metabolic pathways. Immunity and mitochondria are interlinked with each other. An important feature of mitochondria is it can regulate activation, differentiation, and survival of immune cells. In addition, it can also release signals such as mitochondrial DNA (mtDNA) and mitochondrial ROS (mtROS) to regulate transcription of immune cells. From current literature, we found that mitochondria can regulate immunity in different ways. First, alterations in metabolic pathways (TCA cycle, oxidative phosphorylation, and FAO) and mitochondria induced transcriptional changes can lead to entirely different outcomes in immune cells. For example, M1 macrophages exhibit a broken TCA cycle and have a pro-inflammatory role. By contrast, M2 macrophages undergo ß-oxidation to produce anti-inflammatory responses. In addition, amino acid metabolism, especially arginine, glutamine, serine, glycine, and tryptophan, is critical for T cell differentiation and macrophage polarization. Second, mitochondria can activate the inflammatory response. For instance, mitochondrial antiviral signaling and NLRP3 can be activated by mitochondria. Third, mitochondrial mass and mobility can be influenced by fission and fusion. Fission and fusion can influence immune functions. Finally, mitochondria are placed near the endoplasmic reticulum (ER) in immune cells. Therefore, mitochondria and ER junction signaling can also influence immune cell metabolism. Mitochondrial machinery such as metabolic pathways, amino acid metabolism, antioxidant systems, mitochondrial dynamics, mtDNA, mitophagy, and mtROS are crucial for immune functions. Here, we have demonstrated how mitochondria coordinate to alter immune responses and how changes in mitochondrial machinery contribute to alterations in immune responses. A better understanding of the molecular components of mitochondria is necessary. This can help in the development of safe and effective immune therapy or prevention of chronic diseases. In this review, we have presented an updated prospective of the mitochondrial machinery that drives various immune responses.

Optimization of Subcritical Water Hydrolysis of Rutin into Isoquercetin and Quercetin.

Maximum production of isoquercetin and quercetin simultaneously from rutin by subcritical water hydrolysis (SWH) was optimized using the response surface methodology. Hydrolysis parameters such as temperature, time, and CO2 pressure were selected as independent variables, and isoquercetin and quercetin yields were selected as dependent variables. The regression models of the yield of isoquercetin and quercetin were valid due to the high F-value and low P-value. Furthermore, the high regression coefficient indicated that the polynomial model equation provides a good approximation of experimental results. In maximum production of isoquercetin from rutin, the hydrolysis temperature was the major factor, and the temperature or time can be lower if the CO2 pressure was increased high enough, thereby preventing the degradation of isoquercetin into quercetin. The yield of quercetin was considerably influenced by temperature instead of time and CO2 pressure. The optimal condition for maximum production of isoquercetin and quercetin simultaneously was temperature of 171.4°C, time of 10.0 min, and CO2 pressure of 11.0 MPa, where the predicted maximum yields of isoquercetin and quercetin were 13.7% and 53.3%, respectively. Hydrolysis temperature, time, and CO2 pressure for maximum production of isoquercetin were lower than those of quercetin. Thermal degradation products such as protocatechuic acid and 2,5-dihydroxyacetophenone were observed due to pyrolysis at high temperature. It was concluded that rutin can be easily converted into isoquercetin and quercetin by SWH under CO2 pressure, and this result can be applied for SWH of rutin-rich foodstuffs.

Interplay between Immune Checkpoint Proteins and Cellular Metabolism.

With the recent successes in immuno-oncology, renewed interest in the role of immune checkpoint modulators, such as the B7 family proteins, has escalated. The immune checkpoint proteins play a crucial role in the regulation of cellular immunity; however, their contribution to other aspects of cancer biology remains unclear. Accumulating evidence indicate that immune checkpoint proteins can regulate metabolic energetics of the tumor, the tumor microenvironment, and the tumor-specific immune response, leading to metabolic reprogramming of both malignant cells and immune cells involved in mounting and sustaining this response. Immune cell metabolism impacts the activation status of immune cells and ultimately the immune response in cancer. Tumor cells may deplete nutrients that immune cells require for optimal generation, expansion, and function. They may also generate toxic metabolites in the microenvironment or induce conserved inhibitory pathways that impair immune function and thus inhibit antitumor responses. In this review, we will discuss how cancer cells with altered expression of immune checkpoint proteins can potently inhibit immune function through the alteration of cellular and microenvironmental metabolism, providing a new perspective on the interplay between these pathways and offering a potential therapeutic intervention strategy in the treatment of malignant disease. Cancer Res; 77(6); 1245-9. ©2017 AACR.

Immunostimulatory Activity of Opuntia ficus-indica var. Saboten Cladodes Fermented by Lactobacillus plantarum and Bacillus subtilis in RAW 264.7 Macrophages.

To increase the functionality of Opuntia ficus-indica var. saboten cladodes, it was fermented by Lactobacillus plantarum and Bacillus subtilis. Eighty percent methanol extracts were investigated for their effects on nitric oxide (NO) production, cytokine secretion, nuclear factor-κB (NF-κB) activity, and mitogen-activated protein kinase (MAPK) phosphorylation in RAW 264.7 cells. Methanol extracts of L. plantarum culture medium (LPCME) and B. subtilis culture medium (BSCME) did not affect lipopolysaccharide (LPS)-induced NO production but, at 500 µg/mL, increased interferon (IFN)-γ-induced NO production by 55.2 and 66.5 µM, respectively, in RAW 264.7 cells. In RAW 264.7 cells not treated with LPS and IFN-γ, LPCME did not affect NO production, but BSCME increased NO production significantly in a dose-dependent manner. In addition, BSCME induced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in RAW 264.7 cells in a dose-dependent manner. BSCME at 500 µg/mL increased TNF-α and IL-1ß mRNA levels by 83.8% and 82.2%, respectively. BSCME increased NF-κB-dependent luciferase activity in a dose-dependent manner; 500 µg/mL BSCME increased activity 9.1-fold compared with the control. BSCME induced the phosphorylation of p38, c-JUN NH2-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) in a dose-dependent manner, but did not affect total ERK levels. In conclusion, BSCME exerted immunostimulatory effects, which were mediated by MAPK phosphorylation and NF-κB activation, resulting in increased TNF-α and IL-1ß gene expression in RAW 264.7 macrophages. Therefore, BSCM shows promise for use as an immunostimulatory therapeutic.

Inhibition of Carbonyl Reductase 1 Safely Improves the Efficacy of Doxorubicin in Breast Cancer Treatment.

AIMS: Doxorubicin (DOX) is a chemotherapeutic drug that is used to treat many cancers, but its use is limited by cardiotoxic side effect. Carbonyl reductase 1 (CBR1) is an NADPH-dependent oxidoreductase that reduces DOX to doxorubicinol (DOXOL), a less potent derivative that is responsible for DOX cardiotoxicity. Thus, we aimed to demonstrate that inhibition of CBR1 enhances the chemotherapeutic efficacy of DOX and attenuates cardiotoxicity. RESULTS: Pharmacological or genetic inhibition of CBR1 improved the anticancer effects of DOX in preclinical models of breast cancer. RNA interference or chemical inhibition of CBR1 improved the anticancer effect of DOX in breast cancer. Moreover, CBR1 overexpression enabled breast cancer cells to obtain chemotherapeutic resistance to DOX treatment. Intriguingly, inhibition of CBR1 decreased DOX-induced cardiotoxicity in animal model. Innovation and Conclusions: Inhibition of CBR1 increases chemotherapeutic efficacy of DOX and reduces cardiotoxicity by blocking DOX reduction to DOXOL. Therefore, we offer preclinical proof-of-concept for a combination strategy to safely leverage the efficacy of doxorubicin by blunting its cardiotoxic effects that limit use of this cytotoxic agent used widely in the oncology clinic. Antioxid. Redox Signal. 26, 70-83.

Enhancement of Phenolic Production and Antioxidant Activity from Buckwheat Leaves by Subcritical Water Extraction.

To enhance the production of phenolic compounds with high antioxidant activity and reduce the level of phototoxic fagopyrin, buckwheat leaves were extracted with subcritical water (SW) at 100~220°C for 10~50 min. The major phenolic compounds were quercetin, gallic acid, and protocatechuic acid. The cumulative amount of individual phenolic compounds increased with increasing extraction temperature from 100°C to 180°C and did not change significantly at 200°C and 220°C. The highest yield of individual phenolic compounds was 1,632.2 µg/g dry sample at 180°C, which was 4.7-fold higher than that (348.4 µg/g dry sample) at 100°C. Total phenolic content and total flavonoid content increased with increasing extraction temperature and decreased with increasing extraction time, and peaked at 41.1 mg gallic acid equivalents/g and 26.9 mg quercetin equivalents/g at 180°C/10 min, respectively. 2,2-Diphenyl-1-picrylhydrazyl free radical scavenging activity and ferric reducing ability of plasma reached 46.4 mg ascorbic acid equivalents/g and 72.3 mmol Fe2+/100 g at 180°C/10 min, respectively. The fagopyrin contents were reduced by 92.5~95.7%. Color values L* and b* decreased, and a* increased with increasing extraction temperature. SW extraction enhanced the yield of phenolic compounds with high antioxidant activity and reduced the fagopyrin content from buckwheat leaves.

Relationship between Chemical Structure and Antimicrobial Activities of Isothiocyanates from Cruciferous Vegetables against Oral Pathogens.

We evaluated the potentials of 10 isothiocyanates (ITCs) from cruciferous vegetables and radish root hydrolysate for inhibiting the growth of oral pathogens, with an emphasis on assessing any structure-function relationship. Structural differences in ITCs impacted their antimicrobial activities against oral pathogens differently. The indolyl ITC (indol-3-carbinol) was the most potent inhibitor of the growth of oral pathogens, followed by aromatic ITCs (benzyl ITC (BITC) and phenylethyl ITC (PEITC)) and aliphatic ITCs (erucin, iberin, and sulforaphene). Sulforaphene, which is similar in structure, but has one double bond, showed higher antimicrobial activity than sulforaphane. Erucin, which has a thiol group, showed higher antimicrobial activity than sulforaphane, which has a sulfinyl group. BITC and iberin with a short chain exhibited higher antimicrobial potential than PEITC and sulforaphane with a longer chain, respectively. ITCs have strong antimicrobial activities and may be useful in the prevention and management of dental caries.

Enhanced Production of Phenolic Compounds from Pumpkin Leaves by Subcritical Water Hydrolysis.

Enhanced production of individual phenolic compounds by subcritical water hydrolysis (SWH) of pumpkin leaves was investigated at various temperatures ranging from 100 to 220°C at 20 min and at various reaction times ranging from 10 to 50 min at 160°C. Caffeic acid, p-coumaric acid, ferulic acid, and gentisic acid were the major phenolic compounds in the hydrolysate of pumpkin leaves. All phenolic compounds except gentisic acid showed the highest yield at 160°C, but gentisic acid showed the highest yield at 180°C. The cumulative amount of individual phenolic compounds gradually increased by 48.1, 52.2, and 78.4 µg/g dry matter at 100°C, 120°C, and 140°C, respectively, and then greatly increased by 1,477.1 µg/g dry matter at 160°C. The yields of caffeic acid and ferulic acid showed peaks at 20 min, while those of cinnamic acid, p-coumaric acid, p-hydroxybenzoic acid, and procatechuic acid showed peaks at 30 min. Antioxidant activities such as 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power values gradually increased with hydrolysis temperature and ranged from 6.77 to 12.42 mg ascorbic acid equivalents/g dry matter and from 4.25 to 8.92 mmol Fe(2+)/100 g dry matter, respectively. Color L* and b* values gradually decreased as hydrolysis temperature increased from 100°C to 140°C. At high temperatures (160°C to 220°C), L* and b* values decreased suddenly. The a* value peaked at 160°C and then decreased as temperature increased from 160°C to 220°C. These results suggest that SWH of pumpkin leaves was strongly influenced by hydrolysis temperature and may enhanced the production of phenolic compounds and antioxidant activities.