Induction of the human CDC45 gene promoter activity by natural compound trans­resveratrol.

GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to trans­resveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'­upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RT­PCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'­upstream 556­bp of the CDC45 gene was cloned and inserted into a multi­cloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutation­introduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.

Supplementation of Nicotinic Acid and Its Derivatives Up-Regulates Cellular NAD+ Level Rather than Nicotinamide Derivatives in Cultured Normal Human Epidermal Keratinocytes.

Nicotinamide adenine dinucleotide (NAD+) plays a pivotal role in various physiological processes within mammalian cells, including energy metabolism, redox homeostasis, and genetic regulation. In the majority of mammalian cellular contexts, NAD+ biosynthesis primarily relies on vitamin B3, including nicotinamide (NAM) and nicotinic acid (NA). The concept of NAD+ augmentation therapy has recently emerged as a promising strategy to mitigate aging-associated phenomena, termed rejuvenation. Despite the involvement of diverse enzymatic cascades in NAD+ biosynthesis, certain cellular environments exhibit deficiencies in specific enzymes, suggesting cell type-dependent variability in optimal NAD+ precursor selection. However, the optimization of NAD+ precursors for topical formulations has received scant attention thus far. In the present investigation, we sought to delineate the most efficacious precursor for augmenting NAD+ levels in human skin keratinocytes. Remarkably, NA supplementation led to a significant 1.3-fold elevation in intracellular NAD+ levels, even in the presence of nicotinamide phosphoribosyltransferase inhibition by FK866. Additionally, NA mononucleotide demonstrated a 1.5-fold increase (but not significant) in NAD+ levels following 100 µM application. Conversely, NAM and its derivatives failed to elicit a NAD+ response in keratinocytes. Notably, NA supplementation elicited up-regulation of mitochondrial superoxide dismutase (SOD2) and sirtuin 3 (SIRT3), indicative of its beneficial impact on mitochondrial function. Furthermore, NA mitigated rotenone-induced mitochondrial reactive oxygen species (ROS) accumulation. Collectively, these findings advocate for the potential utility of NA in topical applications aimed at skin rejuvenation.

Niacin restriction with NAMPT-inhibition is synthetic lethal to neuroendocrine carcinoma.

Nicotinamide phosphoribosyltransferase (NAMPT) plays a major role in NAD biosynthesis in many cancers and is an attractive potential cancer target. However, factors dictating therapeutic efficacy of NAMPT inhibitors (NAMPTi) are unclear. We report that neuroendocrine phenotypes predict lung and prostate carcinoma vulnerability to NAMPTi, and that NAMPTi therapy against those cancers is enhanced by dietary modification. Neuroendocrine differentiation of tumor cells is associated with down-regulation of genes relevant to quinolinate phosphoribosyltransferase-dependent de novo NAD synthesis, promoting NAMPTi susceptibility in vitro. We also report that circulating nicotinic acid riboside (NAR), a non-canonical niacin absent in culture media, antagonizes NAMPTi efficacy as it fuels NAMPT-independent but nicotinamide riboside kinase 1-dependent NAD synthesis in tumors. In mouse transplantation models, depleting blood NAR by nutritional or genetic manipulations is synthetic lethal to tumors when combined with NAMPTi. Our findings provide a rationale for simultaneous targeting of NAR metabolism and NAMPT therapeutically in neuroendocrine carcinoma.

UVC-Protective Activity of Lemongrass Among 12 Fat-soluble Herbal Extracts: Rapid Decay Due to Cytotoxicity.

BACKGROUND/AIM: The COVID-19 pandemic led to the rapid spread of the use of ultraviolet C (UVC) sterilizers in many public facilities. Considering the harmful effects of prolonged exposure to UVC, manufacturing of safe skin care products is an important countermeasure. In continuation of our recent study of water-soluble herbal extracts, the present study aimed at searching for anti-UVC components from fat-soluble herbal extracts. MATERIALS AND METHODS: Human dermal fibroblast and melanoma cells were exposed to UVC (1.193 W/m2) for 3 min. Viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell-cycle analysis was performed using a cell sorter. UVC-protective activity was quantified by the selective index (SI), i.e., the ratio of the 50% cytotoxic concentration for unirradiated cells to the concentration that restored viability of UVC-treated cells by 50%. RESULTS: Only lemongrass extract, among 12 fat-soluble herbal extracts, showed significant anti-UVC activity, comparable to that of lignified materials and tannins, but exceeding that of N-acetyl-L-cysteine and resveratrol. Lemongrass extract was highly cytotoxic, producing a subG1 cell population. During prolonged incubation in culture medium, the anti-UVC activity of lemongrass extract, sodium ascorbate and vanillic acid declined with an approximate half-life of <0.7, 5.4-21.6, and 27.8-87.0 h, respectively. CONCLUSION: Removal of cytotoxic principle(s) from lemongrass extract is crucial to producing long-lasting UVC-protective effects.

A Comparative Study of Tumor-Specificity and Neurotoxicity between 3-Styrylchromones and Anti-Cancer Drugs.

Background. Many anti-cancer drugs used in clinical practice cause adverse events such as oral mucositis, neurotoxicity, and extravascular leakage. We have reported that two 3-styrylchromone derivatives, 7-methoxy-3-[(1E)-2-phenylethenyl]-4H-1-benzopyran-4-one (Compound A) and 3-[(1E)-2-(4-hydroxyphenyl)ethenyl]-7-methoxy-4H-1-benzopyran-4-one (Compound B), showed the highest tumor-specificity against human oral squamous cell carcinoma (OSCC) cell lines among 291 related compounds. After confirming their superiority by comparing their tumor specificity with newly synthesized 65 derivatives, we investigated the neurotoxicity of these compounds in comparison with four popular anti-cancer drugs. Methods: Tumor-specificity (TSM, TSE, TSN) was evaluated as the ratio of mean CC50 for human normal oral mesenchymal (gingival fibroblast, pulp cell), oral epithelial cells (gingival epithelial progenitor), and neuronal cells (PC-12, SH-SY5Y, LY-PPB6, differentiated PC-12) to OSCC cells (Ca9-22, HSC-2), respectively. Results: Compounds A and B showed one order of magnitude higher TSM than newly synthesized derivatives, confirming its prominent tumor-specificity. Docetaxel showed one order of magnitude higher TSM, but two orders of magnitude lower TSE than Compounds A and B. Compounds A and B showed higher TSM, TSE, and TSN values than doxorubicin, 5-FU, and cisplatin, damaging OSCC cells at concentrations that do not affect the viability of normal epithelial and neuronal cells. QSAR prediction based on the Tox21 database suggested that Compounds A and B may inhibit the signaling pathway of estrogen-related receptors.

Potentiation of Anticancer Activity of G2/M Blockers by Mild Hyperthermia.

BACKGROUND/AIM: Hyperthermia (HT), combined with chemotherapy, has been used to treat various types of cancer. This study aimed to investigate the HT-sensitivity of malignant and non-malignant cells, and then evaluate the combination effect of docetaxel (DTX) and a newly synthesized chromone derivative (compound A) with HT. MATERIALS AND METHODS: The number of viable cells was determined using the MTT method. Cell cycle distribution was analyzed using a cell sorter, and DNA fragmentation pattern was detected using agarose gel electrophoresis. RESULTS: Among 12 cultured cells, oral squamous cell carcinoma (OSCC), especially Ca9-22 cells, and myelogenous leukemia cells showed higher sensitivity to HT than lung carcinoma and glioblastoma cell lines, while normal oral cells were the most resistant. Cytotoxicity of DTX on Ca9-22 cells was maximum at 41-42°C and 45~60 min exposure to HT. DXT, compound A, and HT induced G2/M arrest of Ca-22 cells. Mild HT enhanced the DTX- and compound A-induced subG1 arrest, in a synergistic fashion. CONCLUSION: The combination G2/M blockers and mild-HT can potentially be used for the treatment of OSCC.

Comprehensive Study of Anti-UVC Activity and Cytotoxicity of Hot-water Soluble Herb Extracts.

BACKGROUND/AIM: COVID-19 pandemic caused the rapid dissemination of ultraviolet C (UVC) sterilization apparatuses. Prolonged exposure to UVC, however, may exert harmful effects on the human body. The aim of the present study was to comprehensively investigate the anti-UVC activity of a total of 108 hot-water soluble herb extracts, using human dermal fibroblast and melanoma cell lines, for the future development of skin care products. MATERIALS AND METHODS: Exposure time to UVC was set to 3 min, and cell viability was determined using the MTT assay. Anti-UVC activity was determined using the selective index (SI), a ratio of 50% cytotoxic concentration for unirradiated cells to 50% effective concentration that restored half of the UVC-induced decrease of viability. RESULTS: Dermal fibroblasts at any population doubling level were more resistant to UVC irradiation than melanoma cells. Both 49 herb extracts recommended by Japan Medical Herb Association (JAMHA) and 59 additional herb extracts showed comparable anti-UVC activity. SI values of selected herbs (Butterbur, Cloves, Curry Tree, Evening Primrose, Rooibos, Stevia, Willow) were several-fold lower than those of vitamin C and vanillin. Their potent anti-UVC activity was maintained for at least 6 h post irradiation, but declined thereafter to the basal level, possibly due to cytotoxic ingredients. CONCLUSION: UVC sensitivity may be related to the growth potential of target cells. Removal of cytotoxic ingredients of herb extracts may further potentiate and prolong their anti-UVC activity.

Prominent Anti-UVC Activity of Lignin Degradation Products.

BACKGROUND/AIM: The rapid spread of COVID-19 resulted in the revision of the value of ultraviolet C (UVC) sterilization in working spaces. This study aimed at re-evaluating the anti-UVC activity of four groups of natural products against human melanoma COLO679 and human normal dermal fibroblast (HDFa) cells, based on chemotherapeutic index. MATERIALS AND METHODS: Various cell lines were exposed to UVC for 3 min in the presence of increasing concentrations of test compounds and viable cell numbers were determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The anti-UVC activity was quantified by the ratio of the 50% cytotoxic concentration (determined without irradiation) to the 50% effective concentration (which abolished by 50% the UVC-induced loss of viability). Apoptosis was quantified as the subG1 population proportion following cell-cycle analysis. RESULTS: Among four groups of major natural products, six phenylpropanoids showed the highest anti-UVC activity, followed by the lignified products and alkaline products that contain lignin and its degradation products. On the other hand, tannins and flavonoids showed lower activity due to their higher cytotoxicity. UVC-sensitive COLO679 cells lack dectin-1 protein expression. CONCLUSION: These data suggest the prominent anti-UVC activity of lignin degradation products, and the possible involvement of dectin-1 expression in UVC-sensitivity.

Passion fruit seed extract protects beta-amyloid-induced neuronal cell death in a differentiated human neuroblastoma SH-SY5Y cell model.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with accompanying perceptive disorder. We previously reported that decreasing levels of brain-derived neurotrophic factor (BDNF) promoted beta-amyloid (Aß)-induced neuronal cell death in neuron-like differentiated SH-SY5Y (ndSH-SY5Y) human neuroblastoma cells in an AD mimic cell model. We investigated the neuroprotective effects of passion fruit seed extract (PFSE) and one of the main stilbene compounds, piceatannol, in an AD cell model using ndSH-SY5Y cells. Both PFSE and piceatannol were found to protect Aß-induced neurite fragmentation in the cell model (protection efficacy; 34% in PFSE and 36% in piceatannol). In addition, both PFSE and piceatannol suppress Aß-induced neuronal cell death in the cell model (inhibitory effect; 27% in PFSE and 32% in piceatannol). Our study is the first to report that piceatannol-rich PFSE can repress Aß-induced neuronal cell death by protecting against neurite fragmentation in the AD human cell model. These findings suggest that piceatannol-rich PFSE can be considered a potentially neuroprotective functional food for both prevention and treatment of AD.

A Dual Anti-Inflammatory and Anti-Proliferative 3-Styrylchromone Derivative Synergistically Enhances the Anti-Cancer Effects of DNA-Damaging Agents on Colon Cancer Cells by Targeting HMGB1-RAGE-ERK1/2 Signaling.

The current anti-cancer treatments are not enough to eradicate tumors, and therefore, new modalities and strategies are still needed. Most tumors generate an inflammatory tumor microenvironment (TME) and maintain the niche for their development. Because of the critical role of inflammation via high-mobility group box 1 (HMGB1)-receptor for advanced glycation end-products (RAGE) signaling pathway in the TME, a novel compound possessing both anti-cancer and anti-inflammatory activities by suppressing the HMGB1-RAGE axis provides an effective strategy for cancer treatment. A recent work of our group found that some anti-cancer 3-styrylchromones have weak anti-inflammatory activities via the suppression of this axis. In this direction, we searched such anti-cancer molecules possessing potent anti-inflammatory activities and discovered 7-methoxy-3-hydroxy-styrylchromone (C6) having dual suppressive activities. Mechanism-of-action studies revealed that C6 inhibited the increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) under the stimulation of HMGB1-RAGE signaling and thereby suppressed cytokine production in macrophage-like RAW264.7 cells. On the other hand, in colorectal cancer HCT116 cells, C6 inhibited the activation of ERK1/2, cyclin-dependent kinase 1, and AKT, down-regulated the protein level of XIAP, and up-regulated pro-apoptotic Bax and caspase-3/7 expression. These alterations are suggested to be involved in the C6-induced suppression of cell cycle/proliferation and initiation of apoptosis in the cancer cells. More importantly, in cancer cells, the treatment of C6 potentiates the anti-cancer effects of DNA-damaging agents. Thus, C6 may be a promising lead for the generation of a novel class of cancer therapeutics.

Hinokitiol-induced decreases of tyrosinase and microphthalmia-associated transcription factor are mediated by the endoplasmic reticulum-associated degradation pathway in human melanoma cells.

Tyrosinase (TYR) is a key enzyme for melanin production. We previously showed that hinokitiol, a naturally occurring seven-membered ring terpenoid, potently inhibits human TYR activity. Interestingly, hinokitiol was recently reported to decrease expression of TYR and microphthalmia-associated transcription factor (MITF), which is a main transcription factor of the TYR gene, in murine melanoma cells. However, the mechanisms by which hinokitiol decreases the intracellular levels of TYR and MITF have not been fully elucidated. Here, we investigated the underlying mechanisms of the decreases using cultured human melanoma cells. As a result, hinokitiol treatment decreased TYR protein level in a time- and dose-dependent manner in G361 human melanoma cells, while MITF protein level was decreased only at higher concentrations after 3 days treatment. Notably, the mRNA levels of TYR and MITF were slightly increased by hinokitiol treatment. Therefore, we focused on the degradation of TYR and MITF in endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Importantly, co-treatment of ERAD inhibitor with hinokitiol restored the protein levels of TYR and MITF to approximately 30% and 20% of total those in untreated control cells, respectively. Hinokitiol affected the ER homeostasis as well as degradation of TYR and MITF in two human melanoma cell lines, G361 and HT-144, but the changes of ER-stress markers under the hinokitiol treatment were different in the two human melanoma cell lines. Taken together, these observations indicate that hinokitiol may induce ER stress and trigger the degradation of unfolded newly synthesizing TYR and MITF via the ERAD pathway.

GLO 1 and PKCλ Regulate ALDH1-positive Breast Cancer Stem Cell Survival.

BACKGROUND/AIM: We examined the inhibitory effects of both glyoxalase 1 (GLO 1) and protein kinase C (PKC)λ in aldehyde dehydrogenase 1 (ALDH1)-positive breast cancer stem cells (CSCs). MATERIALS AND METHODS: Breast cancer genomics datasets (TCGA, n=593; METABRIC, n=1904) were downloaded and statistically analyzed. The effects of GLO 1 and PKCλ on trypan blue staining and tumor-sphere formation by ALDH1high cells derived from triple negative breast cancer (TNBC) and basal-like breast cancer were examined. RESULTS: GLO 1high, PKCλhigh, and ALDH1A3high tumors were enriched in stage I/II/III/IV samples, associated with the HER2 and TNBC subtypes according to receptor status, and associated with the HER2-enriched and basal-like subtypes according to PAM50. Inhibition of either GLO 1 (TLSC702) or PKCλ (ANF) suppressed tumor-sphere formation and enhanced death in ALDH1high cells. TLSC702 also effectively inhibited tumor-sphere formation and induced death in PKCλ knockout ALDH1high cells. CONCLUSION: GLO 1 and PKCλ are important for the survival of ALDH1-positive breast CSCs, and may represent potential therapeutic targets for the treatment of ALDH1-positive breast CSCs.

Trimebutine suppresses Toll-like receptor 2/4/7/8/9 signaling pathways in macrophages.

Because of the critical roles of Toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE) in the pathophysiology of various acute and chronic inflammatory diseases, continuous efforts have been made to discover novel therapeutic inhibitors of TLRs and RAGE to treat inflammatory disorders. A recent study by our group has demonstrated that trimebutine, a spasmolytic drug, suppresses the high mobility group box 1‒RAGE signaling that is associated with triggering proinflammatory signaling pathways in macrophages. Our present work showed that trimebutine suppresses interleukin-6 (IL-6) production in lipopolysaccharide (LPS, a stimulant of TLR4)-stimulated macrophages of RAGE-knockout mice. In addition, trimebutine suppresses the LPS-induced production of various proinflammatory cytokines and chemokines in mouse macrophage-like RAW264.7 cells. Importantly, trimebutine suppresses IL-6 production induced by TLR2-and TLR7/8/9 stimulants. Furthermore, trimebutine greatly reduces mortality in a mouse model of LPS-induced sepsis. Studies exploring the action mechanism of trimebutine revealed that it inhibits the LPS-induced activation of IL-1 receptor-associated kinase 1 (IRAK1), and the subsequent activations of extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor-κB (NF-κB). These findings suggest that trimebutine exerts anti-inflammatory effects on TLR signaling by downregulating IRAK1‒ERK1/2‒JNK pathway and NF-κB activity, thereby indicating the therapeutic potential of trimebutine in inflammatory diseases. Therefore, trimebutine can be a novel anti-inflammatory drug-repositioning candidate and may provide an important scaffold for designing more effective dual anti-inflammatory drugs that target TLR/RAGE signaling.

Human microRNA hsa-miR-15b-5p targets the RNA template component of the RNA-dependent RNA polymerase structure in severe acute respiratory syndrome coronavirus 2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of the coronavirus disease in 2019. RNA-dependent RNA polymerase (RdRp) plays an essential role in RNA replication and transcription in SARS-CoV-2. In this study, we focused on the RNA template component of viral RdRp structure and analyzed human microRNAs (miRNAs) targeting specific sequences in this RNA. By examining miRNA databases and using an in vitro RNA-RNA interaction assay, we observed hsa-miR-15b-5p interacting with the RNA component of viral RdRp. Our findings provide evidence that hsa-miR15b-5p may suppresses viral infection and proliferation by targeting the RNA template component of SARS-CoV-2 RdRp.

ß-Thujaplicin Enhances TRAIL-Induced Apoptosis via the Dual Effects of XIAP Inhibition and Degradation in NCI-H460 Human Lung Cancer Cells.

Background: ß-thujaplicin, a natural tropolone derivative, has anticancer effects on various cancer cells via apoptosis. However, the apoptosis regulatory proteins involved in this process have yet to be revealed. Methods: Trypan blue staining, a WST-8 assay, and a caspase-3/7 activity assay were used to investigate whether ß-thujaplicin sensitizes cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Additionally, western blotting was performed to clarify the effects of ß-thujaplicin on X-linked inhibitor of apoptosis protein (XIAP) in NCI-H460 cells and a fluorescence polarization binding assay was used to evaluate the binding-inhibitory activity of ß-thujaplicin against XIAP-BIR3. Results: ß- and γ-thujaplicins decreased the viability of NCI-H460 cells in a dose-dependent manner; they also sensitized the cells to TRAIL-induced cell growth inhibition and apoptosis. ß-thujaplicin significantly potentiated the apoptosis induction effect of TRAIL on NCI-H460 cells, which was accompanied by enhanced caspase-3/7 activity. Interestingly, ß-thujaplicin treatment in NCI-H460 cells decreased XIAP levels. Furthermore, ß-thujaplicin was able to bind XIAP-BIR3 at the Smac binding site. Conclusions: These findings indicate that ß-thujaplicin could enhance TRAIL-induced apoptosis in NCI-H460 cells via XIAP inhibition and degradation. Thus, the tropolone scaffold may be useful for designing novel nonpeptidic small-molecule inhibitors of XIAP and developing new types of anticancer drugs.

A 3-styrylchromone converted from trimebutine 3D pharmacophore possesses dual suppressive effects on RAGE and TLR4 signaling pathways.

Receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) are potential therapeutic targets in the treatment of acute and chronic inflammatory diseases. We previously reported that trimebutine, a spasmolytic drug, suppresses RAGE pro-inflammatory signaling pathway in macrophages. The aim of this study was to convert trimebutine to a new small molecule using in silico 3D pharmacophore similarity search, and dissect the mechanistic anti-inflammatory basis. Of note, a unique 3-styrylchromone (3SC), 7-methoxy-3-trimethoxy-SC (7M3TMSC), converted from trimebutine 3D pharmacophore potently suppressed both high mobility group box 1-RAGE and lipopolysaccharide-TLR4 signaling pathways in macrophage-like RAW264.7 cells. More importantly, 7M3TMSC inhibited the phosphorylation of extracellular signaling-regulated kinase 1 and 2 (ERK1/2) and downregulated the production of cytokines, such as interleukin-6. Furthermore, 3D pharmacophore-activity relationship analyses revealed that the hydrogen bond acceptors of the trimethoxy groups in a 3-styryl moiety and the 7-methoxy-group in a chromone moiety in this compound are significant in the dual anti-inflammatory activity. Thus, 7M3TMSC may provide an important scaffold for the development of a new type of anti-inflammatory dual effective drugs targeting RAGE/TLR4-ERK1/2 signaling.

Glyoxalase 1 and protein kinase Cλ as potential therapeutic targets for late-stage breast cancer.

Cancer cells upregulate the expression levels of glycolytic enzymes in order to reach the increased glycolysis required. One such upregulated glycolytic enzyme is glyoxalase 1 (GLO 1), which catalyzes the conversion of toxic methylglyoxal to nontoxic S-D-lactoylglutathione. Protein kinase Cλ (PKCλ) is also upregulated in various types of cancer and is involved in cancer progression. In the present study, the association between enhanced glycolysis and PKCλ in breast cancer was investigated. In human breast cancer, high GLO 1 expression was associated with high PKCλ expression at the protein (P<0.01) and mRNA levels (P<0.01). Furthermore, Wilcoxon and Cox regression model analysis revealed that patients with stage III-IV tumors with high GLO 1 and PKCλ expression had poor overall survival compared with patients expressing lower levels of these genes [P=0.040 (Gehan-Breslow generalized Wilcoxon test) and P=0.031 (hazard ratio, 2.36; 95% confidence interval, 1.08-5.16), respectively]. Treatment of MDA-MB-157 and MDA-MB-468 human basal-like breast cancer cells with TLSC702 (a GLO 1 inhibitor) and/or aurothiomalate (a PKCλ inhibitor) reduced both cell viability and tumor-sphere formation. These results suggested that GLO 1 and PKCλ were cooperatively involved in cancer progression and contributed to a poor prognosis in breast cancer. In conclusion, GLO 1 and PKCλ serve as potentially effective therapeutic targets for treatment of late-stage human breast cancer.

A Unique Anti-Cancer 3-Styrylchromone Suppresses Inflammatory Response via HMGB1-RAGE Signaling.

Background: High mobility group box 1 (HMGB1)-receptor for advanced glycation endo-products (RAGE) axis serves as a key player in linking inflammation and carcinogenesis. Recently, papaverine was revealed to suppress the HMGB1-RAGE inflammatory signaling pathway and cancer cell proliferation. Therefore, a dual suppressor targeting this axis is expected to become a new type of therapeutic agent to treat cancer. Methods: Papaverine 3D pharmacophore mimetic compounds were selected by the LigandScout software from our in-house, anti-cancer chemical library and assessed for their anti-inflammatory activities by a HMGB1-RAGE-mediated interleukin-6 production assay using macrophage-like RAW264.7 cells. Molecular-biological analyses, such as Western blotting, were performed to clarify the mechanism of action. Results: A unique 6-methoxy-3-hydroxy-styrylchromone was found to possess potent anti-inflammatory and anti-cancer activities via the suppression of the HMGB1-RAGE-extracellular signal-regulated kinase 1/2 signaling pathway. Furthermore, the 3D pharmacophore-activity relationship analyses revealed that the hydroxyl group at the C4' position of the benzene ring in a 3-styryl moiety was significant in its dual suppressive effects. Conclusions: These findings indicated that this compound may provide a valuable scaffold for the development of a new type of anti-cancer drug possessing anti-inflammatory activity and as a tool for understanding the link between inflammation and carcinogenesis.

Addition of hydrophobic side chains improve the apoptosis inducibility of the human glyoxalase I inhibitor, TLSC702.

Glyoxalase I (GLO I) is a known therapeutic target in cancer. Even though TLSC702, a GLO I inhibitor that we discovered, induces apoptosis in tumor cells, exceptionally higher doses are required compared with those needed to inhibit GLO I activity in vitro. In this work, structure-activity optimization studies were conducted on four sections of the TLSC702 molecule to determine the partial structural features necessary for the inhibition of GLO I. Herein, we found that the carboxy group in TLSC702 was critical for binding with the divalent zinc at the active site of GLO I. In contrast, the side chain substituents in the meta- and para- positions of the benzene ring had little influence on the in vitro inhibition of GLO I. The CLogP values of the TLSC702 derivatives showed a positive correlation with the antiproliferative effects on NCI-H522 cells. Thus, two derivatives of TLSC702, which displayed either high or low lipophilicity due to the types of substituents at the phenyl position, were selected. Even though both derivatives showed comparable inhibitory effects as that of their parent compound, the derivative with the high CLogP value was distinctly more antiproliferative than TLSC702. In contrast, the derivative with the low CLogP value did not decrease cell viability in NCI-H522 and HL-60 cells. These findings suggested that structural improvements, such as the addition of hydrophobic moieties to the phenyl group, enhanced the ability of TLSC702 to induce apoptosis by increasing cell membrane permeability.

Antinociceptive activity of the novel RAGE inhibitor, papaverine, in a mouse model of chronic inflammatory pain.

Extracellular high-mobility group box 1 (HMGB1) is known to mediate the inflammatory response through pattern recognition receptors, including the receptor for advanced glycation end products (RAGE) or the toll-like receptors (TLRs). The aim of the present study was to investigate whether papaverine, a novel RAGE inhibitor, could suppress inflammatory pain in mice after several time points, which was induced by the injection of complete Freund's adjuvant (CFA). We also investigated the influence of redox modulation during a state of chronic inflammatory pain. Although papaverine did not suppress CFA-induced mechanical allodynia on Day 7, papaverine significantly suppressed CFA-induced mechanical allodynia on Days 14 and 28. In contrast, the radical scavenger N-tert-Butyl-α-phenylnitrone (PBN) suppressed mechanical allodynia in mice on Days 7 and 14, but not on Day 28. We demonstrated that the RAGE inhibitor improves mechanical allodynia in chronic inflammatory conditions. Moreover, we also found that high levels of reactive oxygen species (ROS) contributed to the early phase of CFA-induced mechanical allodynia. Precisely, lower ROS levels contributed to the inflammatory pain response via the all-thiol HMGB1/RAGE signaling pathway during the chronic state. These findings led us to propose that ROS levels modulate RAGE and/or TLR4-mediated inflammatory allodynia by regulating the concentrations of disulfide HMGB1 or all-thiol HMGB1.