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PURPOSE Recent evidence has highlighted the role of hepatocyte growth factor (HGF) as a putative biomarker to predict EGFR inhibitor resistance. This study investigated the impact of plasma HGF levels on EGFR inhibition and the counter effect of MET inhibition in KRAS, NRAS, and BRAF (RAS/RAF) wild-type colorectal cancers (CRCs). METHODS Plasma HGF levels were analyzed with clinical outcomes of patients with metastatic CRC (mCRC) receiving palliative first-line chemotherapy. Then, in vitro experiments were conducted to validate the clinical findings and to establish pre-clinical evidence of MET inhibition by capmatinib. RESULTS A total of 80 patients were included: cetuximab + FOLFIRI (n = 35) and bevacizumab + FOLFIRI (n = 45). Both progression-free survival (PFS) and overall survival (OS) were significantly lesser in the high vs low HGF group: median 11.8 vs. 24.7 months, respectively, for PFS (p = 0.009), and median 21.1 months vs. not reached, respectively, for OS (p = 0.018). The difference was significantly evident in the cetuximab group. In five RAS/RAF wild-type CRC cells, the addition of HGF activated ERK1/2 and AKT via MET phosphorylation, resulting in cetuximab resistance in vitro. In cetuximab-sensitive Caco-2 and SNU-C4 cells, capmatinib overcame cetuximab resistance in the presence of HGF by attenuating HGF-induced MET signaling activation. CONCLUSION Patients with mCRC receiving cetuximab + FOLFIRI who presented with high plasma HGF levels had significantly worse PFS and OS. Cetuximab resistance induced by HGF was mediated by AKT and ERK activation and overcome by MET inhibition in vitro.

We aimed to elucidate the evolutionary trajectories of gallbladder adenocarcinoma (GBAC) using multi-regional and longitudinal tumor samples. Using whole-exome sequencing data, we constructed phylogenetic trees in each patient, and analyzed mutational signatures. A total of 11 patients including 2 rapid autopsy cases were enrolled. The most frequently altered gene in primary tumors was ERBB2 (54.5%), followed by TP53 (45.5%), and FBXW7 (27.3%). Most mutations in frequently altered genes in primary tumors were detectable in concurrent precancerous lesions (biliary intraepithelial neoplasia, BilIN), but some of them were subclonal. Subclonal diversity was common in BilIN (n=4). However, among subclones in BilIN, a certain subclone commonly shrank in concurrent primary tumors. In addition, selected subclones underwent linear and branching evolution, maintaining subclonal diversity. In combined analysis with metastatic tumors (n=11), branching evolution was identified in 9 (81.8%) patients. Of these, 8 patients (88.9%) had a total of 11 subclones expanded at least 7-fold during metastasis. These subclones harbored putative metastasis-driving mutations in tumor suppressor genes such as SMAD4, ROBO1, and DICER1. In mutational signature analysis, 6 mutational signatures were identified: 1, 3, 7, 13, 22, and 24 (cosine similarity >0.9). Signatures 1 (age) and 13 (APOBEC) decreased during metastasis while signatures 22 (aristolochic acid) and 24 (aflatoxin) were relatively highlighted. Subclonal diversity arose early in precancerous lesions and the clonal selection was a common event during malignant transformation in GBAC. However, selected cancer clones continued to evolve and thus maintained subclonal diversity in metastatic tumors.

The blue-green alga Spirulina maxima is a microscopic filamentous cyanobacterium. Spirulina was recently reported to elicit beneficial effects such as reducing cholesterol and inducing weight loss; however, its effects on inflammation are unknown. To determine the effect of S. maxima extract (SME) on innate immunity, we investigated the NLRP3 inflammasome activation, which is a multiprotein scaffolding complex that plays important roles in innate immune responses to many pathogenic infections in macrophages. SME suppressed lipopolysaccharide (LPS)-induced upregulation of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-12, IL-1β, and IL-18 in RAW264.7 cells. In addition, SME attenuated LPS-induced NLRP3 inflammasome activation, and thus pro-IL-1β could not be cleaved to IL-1β by activated caspase-1, which is activated by the NLRP3 inflammasome in RAW264.7 cells. Moreover, SME inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK) in RAW264.7 cells, and attenuated the generation of ERK1 induced-reactive oxygen species (ROS), resulting in decreased expression of NF-κB. These findings suggest that SME suppresses the effects of the NLRP3 inflammasome via regulation of extracellular signal-regulated kinase (ERK). In summary, we demonstrated that SME prevents activation of the NLRP3 inflammasome by inhibiting ERK signaling.

Background: Recent evidence has highlighted the role of hepatocyte growth factor (HGF) as a putative biomarker to predict EGFR inhibitor resistance. This study investigated the impact of plasma HGF levels on EGFR inhibition and the therapeutic implications of MET inhibition in KRAS, NRAS, and BRAF (RAS/RAF) wild-type colorectal cancer (CRC). Methods: Clinical outcomes were analyzed according to the plasma HGF levels in patients with metastatic CRC (mCRC) receiving palliative first-line cetuximab or bevacizumab + FOLFIRI chemotherapy. Then, in vitro experiments were conducted to validate the clinical findings and to establish pre-clinical evidence of MET inhibition in the HGF-high population. Results: From March 2015 to September 2019, a total of 80 patients were enrolled. The median age was 63 (range, 24-85) years. Of these, 45 patients (56.2%) were treated with bevacizumab + FOLFIRI and 35 patients (43.8%) were treated with cetuximab + FOLFIRI. Among patients with evaluable disease (43 patients (95.6%) in the bevacizumab group and 34 patients (97.1%) in the cetuximab group), the objective response rate was 60.0% and 37.8%, respectively (p=0.162). The median serum HGF level was 374.75 (range, 9.43-30,644.44) pg/mL, and the optimal cut-off value of HGF levels was 12.70 pg/mL by the maximal chi-square method. During a median follow-up of 29.3 (range, 1.2-71.3) months, both progression-free survival (PFS) and overall survival (OS) were significantly shorter in the high HGF group compared with the low HGF group: median 11.8 (95% confidence interval [CI], 10.5-13.1) months vs. 24.7 (95% CI, 23.4-25.9) months for PFS (p=0.009), and median 21.1 (95% CI, 17.9-24.3) months vs. not reached for OS (p=0.018). The difference in PFS and OS was statistically significant in the cetuximab group (p=0.003 for PFS and p=0.035 for OS), but not in the bevacizumab group. In five RAS/RAF wild-type CRC cell lines (Caco-2, COLO 320DM, KM12C, SNU-C1, and SNU-C4), the addition of HGF activated ERK1/2 and AKT via MET phosphorylation. In the cytotoxicity assays, Caco-2 and SNU-C4 were relatively sensitive to cetuximab compared with the others and, in the presence of HGF, cetuximab sensitivity was significantly decreased in the two cells. Moreover, capmatinib, a MET inhibitor, abrogated the effect of HGF on common downstream signaling pathways of EGFR and MET and thus the cetuximab resistance was significantly overcome in vitro. Conclusions: Among patients with mCRC receiving cetuximab + FOLFIRI, those with high plasma HGF levels had significantly worse PFS and OS. HGF induced cetuximab resistance by AKT and ERK activation while capmatinib, a MET inhibitor, significantly increased the anti-tumor effects of cetuximab in the presence of HGF in vitro. Our data may provide evidence of future clinical trials of dual EGFR and MET targeting strategies in patients with HGF-high, RAS/RAF wild-type mCRC. Citation Format: Sang-A Kim, Hyejoo Park, Kui-Jin Kim, Ji-Won Kim, Ji Hea Sung, Milang Nam, Ju Hyun Lee, Eun Hee Jung, Koung Jin Suh, Ji Yun Lee, Se Hyun Kim, Jeong-Ok Lee, Jin Won Kim, Yu Jung Kim, Jee Hyun Kim, Soo-Mee Bang, Jong Seok Lee, Keun-Wook Lee. Cetuximab resistance induced by hepatocyte growth factor is overcome by MET inhibition in KRAS, NRAS, and BRAF wild-type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5138.

Background: There is no study so far that analyzed the patterns of clonal evolution from the initiation of carcinogenesis to distant metastasis in patients with gallbladder adenocarcinoma (GBAC). This study aimed to elucidate the evolutionary trajectories of GBAC using multi-regional and longitudinal tumor samples. Methods: Whole-exome sequencing was conducted on tumors and matched normal samples with the coverage of 300x and 200x, respectively. Using PyClone, CITUP, MapScape, and TimeScape, phylogenetic trees were visualized in each patient considering each tumor’s location and timing of acquisition. Mutational signatures were analyzed using Mutalisk. Results: Between 2013 and 2018, a total of 11 patients (male, 5) including 2 rapid autopsy cases were enrolled. The median age was 70 (range, 59-75) years. The dataset consisted of 11 normal samples, 4 biliary intraepithelial neoplasia (BilIN), 11 primary tumors, and 30 metastatic tumors. The most frequently altered gene was ERBB2 (54.5%), followed by TP53 (45.5%) and FBXW7 (27.3%). Of 6 patients with analyzable tumor ploidy, 2 patients (33.3%) had whole genome doubling (WGD) in both primary and metastatic tumors, and 1 patient (16.7%) had WGD not in the primary tumor but in the liver metastasis. In the BilIN analysis (n=4), most mutations in frequently altered genes in GBAC were detectable from the BilIN stage, but some of them were subclonal. In these 4 patients, the fittest subclone in BilIN underwent linear and branching evolution by acquiring additional subclonal mutations and thus expanded in the primary tumor, suggesting a selective sweep phenomenon. In combined analysis with metastatic tumors (n=11), branching and linear evolution was identified in 9 (81.8%) and 2 (18.2%) patients, respectively. Of the 9 patients with branching evolution, eight (88.9%) had a total of 11 subclones expanded at least 7-fold in regional or distant metastasis. These subclones harbored putative metastasis-driving mutations in tumor suppressor genes such as SMAD4, ROBO1, and DICER1. Metastases were polyclonal in all patients. However, metastatic lesions in the same or adjacent organs showed similar clonal compositions and there was evidence of metastasis-to-metastasis spread. In mutational signature analysis, we identified 6 mutational signatures: signatures 1 (age), 3 (DNA double-strand break-repair), 7 (ultraviolet), 13 (APOBEC), 22 (aristolochic acid), and 24 (aflatoxin) (cosine similarity values ≥ 0.9). Among them, signatures 1 and 13 were dominant at the carcinogenesis stage while signatures 22 and 24 were dominant at the metastasis stage. Conclusions: We have constructed evolutionary trajectories of individual patients, highlighting the role of each clone and the dynamics among clones during carcinogenesis and metastasis. This novel approach may help us move forward to precision medicine that enables early detection of carcinogenesis and metastasis. Citation Format: Minsu Kang, Hee Young Na, Soomin Ahn, Ji-Won Kim, Sejoon Lee, Soyeon Ahn, Ju Hyun Lee, Jeonghwan Youk, Haesook T. Kim, Kui-Jin Kim, Koung Jin Suh, Jun Suh Lee, Se Hyun Kim, Jin Won Kim, Yu Jung Kim, Keun-Wook Lee, Yoo-Seok Yoon, Jee Hyun Kim, Jin-Haeng Chung, Ho-Seong Han, Jong Seok Lee. Evolutionary trajectories during carcinogenesis and metastasis in gallbladder adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6083.

Peripheral regulatory T cells (pTregs) are a highly immunosuppressive fraction of CD4+ T cells. We aimed to evaluate the clinical significance of pTregs in patients with gastric cancer and to determine the correlation between pTregs and immune cell infiltration in tumor microenvironment. pTregs status was determined by assessing the pTreg/total T-cell ratio (ratio of Foxp3 Treg-specific demethylated region (TSDR) to CD3G/CD3D demethylation, so-called Cellular Ratio of Immune Tolerance “ImmunoCRIT”) using methylation analyses in 433 patients with gastric cancer who received curative surgery. Among 422 evaluable patients, 230 (54.5%) had high ImmunoCRIT (> 21.0). Patients with high ImmunoCRIT had significantly shorter disease-free survival (DFS) and overall survival (OS) than those with high ImmunoCRIT (p = 0.030, p = 0.008, respectively). In multivariate analysis, high ImmunoCRIT kept a prognostic role for shorter OS (hazard ratio [HR] 1.9, 95% confidence interval [CI] 1.4–2.9; p = 0.005). CD3+ cell density and CD4+ cell density was significantly higher within the tumor in high ImmunoCRIT group than those in low ImmunoCRIT group (CD3+ cell, 202.12/mm2 vs. 172.2/mm2, p = 0.029; CD4+ cell, 56.5/mm2 vs. 43.5/mm2, p = 0.007). In conclusion, the peripheral ImmunoCRIT determined by epigenetic methylation analysis provides prognostic information in resected gastric tumors.

Simple Summary Colorectal cancer (CRC) is the third most common cancer and the second highest cause of cancer related mortality worldwide. Especially, the survival of advanced CRC patients who were failed to achieve durable remission after the anti-angiogenic and anti-epithelial growth factor receptor agents are still poor. The aim of our study was to investigate the anti-tumor activity of the CDK4/6 inhibitor, abemaciclib, as a single agent and to identify an optimal combination agent with abemaciclib in CRC cell lines. We confirmed that abemaciclib monotherapy showed anti-tumor activity and combination therapy with abemaciclib and BYL719 demonstrated synergistic effects in CRC cell lines. Moreover, our study suggested that PIK3CA mutation could be a predictive marker for efficacy of abemaciclib and BYL719 combination therapy. These findings provide novel insight into a possible therapeutic strategy for patients with relapsed and refractory CRC. Abstract Targeting cell cycle regulation in colorectal cancer has not been fully evaluated. We investigated the efficacy of the CDK4/6 inhibitor, abemaciclib, and confirmed a synergistic interaction for PI3K p110α and CDK dual inhibition in colorectal cancer cell lines. Caco-2 and SNU-C4 cell lines were selected to explore the mechanism of action for and resistance to abemaciclib. In vitro and in vivo models were used to validate the anti-tumor activity of abemaciclib monotherapy and BYL719 combination therapy. Abemaciclib monotherapy inhibited cell cycle progression and proliferation in Caco-2 and SNU-C4 cells. CDK2-mediated Rb phosphorylation and AKT phosphorylation appeared to be potential resistance mechanisms to abemaciclib monotherapy. Abemaciclib/BYL719 combination therapy demonstrated synergistic effects regardless of PIK3CA mutation status but showed greater efficacy in the PIK3CA mutated SNU-C4 cell line. Growth inhibition, cell cycle arrest, and migration inhibition were confirmed as mechanisms of action for this combination. In an SNU-C4 mouse xenograft model, abemaciclib/BYL719 combination resulted in tumor growth inhibition and apoptosis with tolerable toxicity. Dual blockade of PI3K p110α and CDK4/6 showed synergistic anti-tumor effects in vivo and in vitro in human colorectal cancer cell lines. This combination could be a promising candidate for the treatment of patients with advanced colorectal cancer.

PIK3CA mutations are frequently observed in various human cancers including gastric cancer (GC). This study was conducted to investigate the anti-tumor effects of alpelisib, a PI3K p110α-specific inhibitor, using preclinical models of GC. In addition, the combined effects of alpelisib and paclitaxel on GC were evaluated. Among the SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1 GC cells, three PIK3CA-mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 phase arrest regardless of PIK3CA mutational status. The alpelisib and paclitaxel combination demonstrated synergistic anti-proliferative effects, preferentially on PIK3CA-mutant cells, resulting in increased DNA damage response and apoptosis. In addition, alpelisib and paclitaxel combination potentiated anti-migratory activity in PIK3CA-mutant cells. Alpelisib partially reversed epithelial–mesenchymal transition markers in PIK3CA-mutant cells. In a xenograft model of MKN1 cells, the alpelisib and paclitaxel combination significantly enhanced anti-tumor activity by decreasing Ki-67 expression and increasing apoptosis. Moreover, this combination tended to prolong the survival of tumor-bearing mice. Our data suggest promising anti-tumor efficacy of alpelisib alone or in combination with paclitaxel in PIK3CA-mutant GC cells.

Obesity results from an imbalance between energy intake and energy expenditure, in which excess fat is stored as triglycerides (TGs) in white adipocytes. Recent studies have explored the anti-obesity effects of certain edible phytochemicals, which suppress TG accumulation and stimulate a brown adipocyte-like phenotype in white adipocytes. Gomisin N (GN) is an important bioactive component of Schisandra chinensis, a woody plant endemic to Asia. GN has antioxidant, anti-inflammatory and hepatoprotective effects in vivo and in vitro. However, the anti-obesity effects of GN in lipid metabolism and adipocyte browning have not yet been investigated. In the present study, we aimed to determine whether GN suppresses lipid accumulation and regulates energy metabolism, potentially via AMP-activated protein kinase (AMPK), in 3T3-L1 adipocytes. Our findings demonstrate that GN inhibited adipogenesis and lipogenesis in adipocyte differentiation. Also, GN not only increased the expression of thermogenic factors, including uncoupling protein 1 (UCP1), but also enhanced fatty acid oxidation (FAO) in 3T3-L1 cells. Therefore, GN may have a therapeutic benefit as a promising natural agent to combat obesity.

Obesity results from an imbalance between energy intake and energy expenditure, in which excess fat is stored as triglycerides (TGs) in white adipocytes. Recent studies have explored the anti-obesity effects of certain edible phytochemicals, which suppress TG accumulation and stimulate a brown adipocyte-like phenotype in white adipocytes. Gomisin N (GN) is an important bioactive component of Schisandra chinensis, a woody plant endemic to Asia. GN has antioxidant, anti-inflammatory and hepatoprotective effects in vivo and in vitro. However, the anti-obesity effects of GN in lipid metabolism and adipocyte browning have not yet been investigated. In the present study, we aimed to determine whether GN suppresses lipid accumulation and regulates energy metabolism, potentially via AMP-activated protein kinase (AMPK), in 3T3-L1 adipocytes. Our findings demonstrate that GN inhibited adipogenesis and lipogenesis in adipocyte differentiation. Also, GN not only increased the expression of thermogenic factors, including uncoupling protein 1 (UCP1), but also enhanced fatty acid oxidation (FAO) in 3T3-L1 cells. Therefore, GN may have a therapeutic benefit as a promising natural agent to combat obesity.

Melanoma has a high propensity to metastasize and exhibits a poor response to classical therapies. Dysregulation of the chemokine receptor gene CXCR4 is associated with melanoma progression, and although n-3 polyunsaturated fatty acids (PUFAs) are known to be beneficial for melanoma prevention, the underlying mechanism of this effect is unclear. Here, we used the n-3 fatty acid desaturase (Fat-1) transgenic mouse model of endogenous n-3 PUFA synthesis to investigate the influence of elevated n-3 PUFA levels in a mouse model of metastatic melanoma. We found that relative to wild-type (WT) mice, Fat-1 mice exhibited fewer pulmonary metastatic colonies and improved inflammatory indices, including reduced serum tumor necrosis factor alpha (TNF-α) levels and pulmonary myeloperoxidase activity. Differential PUFA metabolites in serum were considered a key factor to alter cancer cell travelling to lung, and we found that n-6 PUFAs such as arachidonic acid induced CXCR4 protein expression although n-3 PUFAs such as eicosapentaenoic acid (EPA) decreased CXCR4 levels. In addition, serum levels of the bioactive EPA metabolite, 18-HEPE, were elevated in Fat-1 mice relative to WT mice, and 18-HEPE suppressed CXCR4 expression in B16-F0 cells. Moreover, relative to controls, numbers of pulmonary metastatic colonies were reduced in WT mice receiving intravenous injections either of 18-HEPE or 18-HEPE-pretreated melanoma cells. Our results indicate that 18-HEPE is a potential anticancer metabolite that mediates, at least in part, the preventive effect of n-3 PUFA on melanoma metastasis.

Background: The aim of this study is to elucidate the mechanisms of acquired resistance to pemetrexed in echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer. Methods: We analyzed the sensitivity to pemetrexed and the expression patterns of various proteins after pemetrexed treatment in the cell lines, A549, NCI-H460, NCI-H2228 harboring EML4-ALK variant 3, and NCI-H3122 harboring EML4-ALK variant 1. Pemetrexed-resistant cell lines were also generated through long-term exposure to pemetrexed. Results: The EML4-ALK variant 1 rearranged NCI-H3122 was found to be more sensitive than the other cell lines. Cell cycle analysis after pemetrexed treatment showed that the fraction of cells in the S phase increased in A549, NCI-H460, and NCI-H2228, whereas the fraction in the apoptotic sub-G1 phase increased in NCI-H3122. The pemetrexed-resistant NCI-H3122 cell line showed increased expression of EGFR and HER2 compared to the parent cell line, whereas A549 and NCI-H460 did not show this change. The pan-HER inhibitor afatinib inhibited this alternative signaling pathway, resulting in a superior cytotoxic effect in pemetrexed-resistant NCI-H3122 cell lines compared to that in the parental cells line. Conclusion: The activation of EGFR-HER2 contributes to the acquisition of resistance to pemetrexed in EML4-ALK rearranged non-small cell lung cancer. However, the inhibition of this alternative survival signaling pathway with RNAi against EGFR-HER2 and with afatinib overcomes this resistance.

Our results suggested that thermal stress can lead to activation of hippocampal cell damage and reduction of memory-associated molecules in HT22 cells. These findings also provide a part of molecular rationale for the role of ginsenoside Rg5 as a potent cognitive impairment preventive compound in blocking the initiation of hippocampal damage. Keywords: cognitive impairment, Ginsenoside Rg5, HT22, neuroprotection, thermal stress

Spirulina maxima is a blue-green micro alga that contains abundant amounts of proteins (60-70%), vitamins, chlorophyll a, and C-phycocyanin (C-PC). It has been shown to reduce oxidative stress, and prevent diabetes and non-alcoholic fatty liver disease. However, it is unclear whether Spirulina maxima 70% ethanol extract (SM70EE), chlorophyll a, and C-PC prevent Aβ1-42-induced neurotoxicity in PC12 cells. The aim of this study was to investigate whether SM70EE, chlorophyll a, and C-PC prevent Aβ1-42-induced cell death. SM70EE, chlorophyll a, and C-PC suppressed the Aβ1-42-induced increase in poly-ADP ribose polymerase-1 (PARP-1) cleavage and reduced Aβ1-42-induced decreases in glutathione and its associated factors. The level of brain-derived neurotrophic factor (BDNF), which plays a critical role in neuronal survival and neuroprotection, was increased by SM70EE, chlorophyll a, and C-PC in Aβ1-42-treated cells. SM70EE treatment decreased oxidative stress and cell death in response to Aβ1-42 treatment, while simultaneously suppressing PARP cleavage and increasing the levels of glutathione (GSH) and its associated factors. Moreover, SM70EE lowered the levels of APP and BACE1, two major factors involved in APP processing, and increased BDNF expression during Aβ1-42-induced neurotoxicity in PC12 cells. We suggest that SM70EE prevents cell death caused by Aβ1-42 -induced neurotoxicity via the activation of BDNF signaling.

Background: Pterostilbene is a stilbenoid and major compound and has diverse biological activities, such as antioxidant, anti-cancer, and anti-inflammatory. However, it has not been shown whether pterostilbene affects the mitotic clonal expansion during adipogenesis in 3T3-L1 cells. Purpose: In the present study, we aimed to demonstrate the detailed mechanism of pterostilbene on anti-adipogenesis in 3T3-L1 cells. Methods: Preadipocytes were converted to adipocytes through treatment with MDI (IBMX; 3-isobutyl-1-methylxanthine, DEX; dexamethasone, insulin) in 3T3-L1 cells. Oil Red O staining was performed to measure intracellular lipid accumulation. Western blot analysis was conducted to analyze protein expressions. Results: Our results showed that pterostilbene decreased the lipid accumulation compared to MDI-induced differentiation, using Oil Red O staining. Next, we found that pterostilbene suppressed the expression of C/EBPα, PPARγ, and aP2 as well as the mitotic clonal expansion-associated proteins CHOP10 and C/EBPβ, by western blot analysis. Our results indicated that pterostilbene may repress adipocyte differentiation through the activation of HO-1 expression prior to entering into the mitotic clonal expansion in 3T3-L1 cells. RNA interference was used to determine whether HO-1 acts as a regulator of CHOP10. Conclusion: Our results revealed that pterostilbene induced HO-1 expression which acts as a regulator of CHOP10. Together, we demonstrated that pterostilbene suppresses the initiation of mitotic clonal expansion via up-regulation of HO-1 expression during adipocyte differentiation of 3T3-L1 cells.

Cirsium setidens Nakai is an edible herb. Previously we found that fermented Cirsium setidens Nakai (FCSN) has a large amount of major bioactive compound compared to Cirsium setidens Nakai. In this study, we aimed to examine the anti-obesity effect of FCSN using 3T3-L1 cells in vitro and high calorie diet-induced obese (HDIO) zebrafish model in vivo. Our results demonstrated that FCSN significantly inhibited intracellular lipid accumulation in 3T3-L1 cells. FCSN was shown to reduce the expressions of crucial adipocyte differentiation markers, including PPARγ and aP2. FCSN also decreased the production of ROS due to the up-regulated expressions of SOD1, SOD2, GPx, and catalase. Furthermore, we observed that FCSN also altered the levels of energy metabolism and β-oxidation-associated genes such as AMPK, ACC, and CPT-1. In addition, ATGL, a key lipolysis enzyme, was stimulated while the differentiation of 3T3-L1 was suppressed by FCSN. Strikingly, we found that FCSN dramatically increased both the energy metabolism and β-oxidation associated genes and subsequently prevented the increase of body fat accumulation in high calorie diet-induced obese zebrafish. Taken together, this is the first study that demonstrates that FCSN has the beneficial activity to suppress adipogenesis in 3T3-L1 cells and ameliorate an obese-associated health condition in vivo.

Background: Ginsenoside Rg1 is a class of steroid glycoside and triterpene saponin in Panax ginseng. Many studies suggest that Rg1 suppresses adipocyte differentiation in 3T3-L1. However, the detail molecular mechanism of Rg1 on adipogenesis in 3T3-L1 is still not fully understood. Methods: 3T3-L1 preadipocyte was used to evaluate the effect of Rg1 on adipocyte development in the differentiation in a stage-dependent manner in vitro. Oil Red O staining and Nile red staining were conducted to measure intracellular lipid accumulation and superoxide production, respectively. We analyzed the protein expression using Western blot in vitro. The zebrafish model was used to investigate whether Rg1 suppresses the early stage of fat accumulation in vivo. Results: Rg1 decreased lipid accumulation in early-stage differentiation of 3T3-L1 compared with intermediate and later stages of adipocyte differentiation. Rg1 dramatically increased CAAT/enhancer binding protein (C/EBP) homologous protein-10 (CHOP10) and subsequently reduced the C/EBP b transcriptional activity that prohibited the initiation of adipogenic marker expression as well as triglyceride synthase. Rg1 decreased the expression of extracellular signal-regulated kinase 1/2 and glycogen synthase kinase 3b, which are also essential for stimulating the expression of CEBPb. Rg1 also reduced reactive oxygen species production because of the downregulated protein level of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 4 (NOX4). While Rg1 increased the endogenous antioxidant enzymes, it also dramatically decreased the accumulation of lipid and triglyceride in high fat diet-induced obese zebrafish. Conclusion: We demonstrated that Rg1 suppresses early-stage differentiation via the activation of CHOP10 and attenuates fat accumulation in vivo. These results indicate that Rg1 might have the potential to reduce body fat accumulation in the early stage of obesity.

Fucoxanthin, a pigment from the chloroplasts of marine brown algae, has a number of effects against obesity, diabetes, inflammation and cancer and provides cerebrovascular protection. In this study, we investigated the inhibitory effects of fucoxanthin on lipid accumulation and reactive oxygen species (ROS) production during adipogenesis. Treatment with fucoxanthin suppresses protein levels of the adipogenic transcription factors CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor-γ and of their target protein, fatty acid binding protein 4. Lipogenesis-related enzymes, such as diglyceride acyltransferase 1 and lysophosphatidic acid acyltransferase-θ, were downregulated by fucoxanthin. The ROS-producing enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and the NADPH-generating enzyme glucose-6-phosphate dehydrogenase also decreased following fucoxanthin treatment. The adipokine adiponectin and the ROS-scavenging enzymes superoxide dismutase 2, glutathione reductase and catalase were dose-dependently increased by fucoxanthin. Furthermore, lipolysis-related enzymes and superoxide dismutase 1 were slightly decreased, because of the suppression of lipid-generating factors and the cytosolic enzyme NOX4. To confirm these results, we investigated lipid accumulation and ROS production in zebrafish, where fucoxanthin suppressed lipid and triglyceride accumulation, as well as ROS production. Our data suggest that fucoxanthin inhibits lipid accumulation and ROS production by controlling adipogenic and lipogenic factors and ROS-regulating enzymes. Copyright © 2016 John Wiley & Sons, Ltd.

Ecklonia cava, a brown alga native to the seas of Korea and Japan, contains biologically active polyphenols. Seapolynol (SN) is a polyphenol mixture derived from Ecklonia cava, and dieckol (DK) is a major compound of SN. Both Ecklonia cava-derived polyphenols exert an anti-diabetic effect in C57BL/KsJ-db/db mice. SN and DK supplementation significantly reduced bodyweight, water intake, fasting blood glucose, and plasma insulin levels compared with db/db mice. In addition, C-peptide, a biomarker of insulin secretion, was significantly decreased by SN and DK treatment, whereas plasma adiponectin levels, which are associated with insulin sensitivity, was increased by two-fold compared with the db/db group. Regarding plasma lipid profiles, triacylglycerol, total cholesterol, and low-density lipoprotein (LDL) cholesterol, were effectively reduced in the SN- and DK-treated groups. Moreover, the administration of SN and DK activated the insulin signaling pathway, as indicated by the levels of receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), serine-threonine specific protein kinase (Akt), and glucose transporter type 4 (GLUT4) in diabetic mice. This result showed that SN and DK regulate blood glucose levels by improving insulin sensitivity in the diabetic condition. Our data suggest that SN and DK may be anti-diabetic agents.

Brazilin, is a bioactive compound extracted from Caesalpinia sappan Linn, has been reported the protective effect of the immune system. Particular attention is now devoted to better understanding of the molecular basis of bazilin anti-inflammatory activity. In the present study, we studied the effect of brazilin on the Raw264.7 macrophage cell lines by a nutrigenomics approaches. Raw264.7 cells were treated with braziln, then treated with LPS to cause inflammation. The nuclear transcription κB (NF-κB) promoter activity were analyzed with dual luciferase assay kit. The gene expression and production levels of pro-inflammatory cytokine interleukin (IL)-1β, tumor necrosis factor (TNF)α, and IL-6 were evaluated with semi-quantitative RT-PCR and with ELISA, respectively. We also examined inflammatory signaling, including mitogen-activated protein kinase (MAPK) pathway, iNOS, COX2, and IRAK4. Our findings demonstrated that brazilin down-regulated the expression of IRAK4 protein lead to suppress of c-Jun NH2 terminal kinase (JNK) signaling, and subsequently inactivation of nuclear transcription κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) thus promoting the expression of the downstream target pro-inflammatory cytokines such as IL-1β, TNFα and IL-6 in LPS stimulated Raw264.7 macrophage cell. Thus, brazilin showed anti-inflammatory activity in Raw264.7 macrophage cell targeting IRAK4 mediated signaling pathway.

The medicinal herbal plant has been commonly used for prevention and intervention of disease and health promotions worldwide. Brazilein is a bioactive compound extracted from Caesalpinia sappan Linn. Several studies have showed that brazilein exhibited the immune suppressive effect and anti-oxidative function. However, the molecular targets of brazilein for inflammation prevention have remained elusive. Here, we investigated the mechanism underlying the inhibitory effect of brazilein on LPS-induced inflammatory response in Raw264.7 macrophage cells. We demonstrated that brazilein decreased the expression of IRAK4 protein led to the suppression of MAPK signaling and IKKβ, and subsequent inactivation of NF-κB and COX2 thus promoting the expression of the downstream target pro-inflammatory cytokines such as IL-1β, MCP-1, MIP-2, and IL-6 in LPS-induced Raw264.7 macrophage cells. Moreover, we observed that brazilein reduced the production of nitrite compared to the control in LPS-induced Raw264.7. Thus, we suggest that brazilein might be a useful bioactive compound for the prevention of IRAK-NF-κB pathway associated chronic diseases.

Paclitaxel (PTX) is commonly used to treat urothelial carcinoma (UC) after platinum-based chemotherapy has failed. However, single-agent taxane therapy is not sufficient to inhibit tumor progression and drug resistance in advanced UC. Epithelial-to-mesenchymal transition (EMT) induced by fibroblast growth factor receptor (FGFR)1 signaling has been proposed as a mechanism of PTX resistance, but it is unclear whether this can be overcome by FGFR1 inhibition. The present study investigated whether FGFR1 overexpression contributes to PTX resistance and whether FGFR inhibition can enhance PTX efficacy in UC. The effects of PTX combined with the FGFR inhibitor BGJ398 were evaluated in UC cell lines by flow cytometry, Western blot analysis, cell viability, migration, and colony forming assays, and RNA interference. PTX+BGJ398 induced cell cycle arrest and apoptosis in UC cells with mesenchymal characteristics was accompanied by downregulation of cyclin D1 protein and upregulation of gamma-histone 2A family member X and cleaved poly(ADP-ribose) polymerase. Additionally, PTX+BGJ398 synergistically suppressed UC cell migration and colony formation via regulation of EMT-associated factors, while FGFR1 knockdown enhanced the antitumor effect of PTX. These findings provide a basis for development of effective strategies for overcoming PTX resistance in UC through inhibition of FGFR1 signaling.

Background: Continuous exposure to high temperatures can lead to heat stress. This stress response alters the expression of multiple genes and can contribute to the onset of various diseases. In particular, heat stress induces oxidative stress by increasing the production of reactive oxygen species. The liver is an essential organ that plays a variety of roles, such as detoxification and protein synthesis. Therefore, it is important to protect the liver from oxidative stress caused by heat stress. Korean ginseng has a variety of beneficial biological properties, and our previous studies showed that it provides an effective defense against heat stress. Methods: We investigated the ability of Korean Red Ginseng and Korean black ginseng extracts (JP5 and BG1) to protect against heat stress using a rat model. We then confirmed the active ingredients and mechanism of action using a cell-based model. Results: Heat stress significantly increased gene and protein expression of oxidative stress–related factors such as catalase and SOD2, but treatment with JP5 (Korean Red Ginseng extract) and BG1 (Korean black ginseng extract) abolished this response in both liver tissue and HepG2 cells. In addition, JP5 and BG1 inhibited the expression of inflammatory proteins such as p-NF-κB and tumor necrosis factor alpha-α. In particular, JP5 and BG1 decreased the expression of components of the NLRP3 inflammasome, a key inflammatory signaling factor. Thus, JP5 and BG1 inhibited both oxidative stress and inflammation. Conclusions: JP5 and BG1 protect against oxidative stress and inflammation induced by heat stress and help maintain liver function by preventing liver damage. Keywords: Heat stress, Inflammation, Korean ginseng extracts, Oxidative stress, Sprague–Dawley rats

Background: PIK3CA, encoding p110α subunit of PI3K, is the third most frequently mutated gene in gastric cancer (GC): 9–13% of non-hypermutated tumors and 32% of hypermutated tumors. Thus, PI3K pathway could be a potential therapeutic target in GC. Nevertheless, only few studies have been conducted so far. Materials and Methods: This preclinical study aimed to investigate the anti-tumor effects of alpelisib (Novartis, Basel, Switzerland), a PI3K p110α-specific inhibitor, using in vitro and in vivo models of GC. The combinatorial effects of alpelisib and paclitaxel, a commonly used agent for GC, were also evaluated. Results: Among eight GC cell lines (SNU1, SNU16, SNU484, SNU601, SNU638, SNU668, AGS, and MKN1), three harbored PIK3CA hotspot mutations: E542K in SNU601, E545A and E453K in AGS, and E545K in MKN1, while the others were PIK3CA wild-type. These three PIK3CA mutant cells were predominantly sensitive to alpelisib. Alpelisib monotherapy decreased AKT and S6K1 phosphorylation and induced G0/G1 cell cycle arrest regardless of PIK3CA mutational status. Moreover, in PIK3CA mutant cells, GSK3β and BAD phosphorylation was potently abrogated by alpelisib alone, which was not evident in PIK3CA wild-type cells. Alpelisib in combination with paclitaxel demonstrated synergistic anti-proliferative effects, preferentially in PIK3CA mutant cells, by increasing caspase 3/7 activity and resulting in increased apoptosis. Moreover, the alpelisib and paclitaxel combination more potently inhibited the cell migration in wound healing assays and the expression of epithelial-mesenchymal transition (EMT)-associated proteins including Snail and Twist in PIK3CA mutant cells, compared with PIK3CA wild-type cells. In a mouse xenograft model of MKN1 cells, alpelisib combined with paclitaxel significantly enhanced the anti-tumor activity by decreasing Ki-67 expression and increasing TUNEL expression. Moreover, this combination tended to prolong survival of tumor-bearing mice for 4 weeks of treatment period without resulting in significant change in body weight. Conclusions: Alpelisib alone or in combination with paclitaxel demonstrated promising anti-tumor activity and tolerability in in vitro and in vivo models of PIK3CA mutant GC via inactivating PI3K down-stream molecules, decreasing cell migration, and increasing apoptosis. Our data suggest that this novel combination is worthy of further clinical investigations in patients with PIK3CA mutant GC. Keywords: alpelisib; BYL719; paclitaxel; PIK3CA; gastric cancer; combination; chemotherapy Citation Format: Ji-Won Kim, Kui-Jin Kim, Ji Hea Sung, Koung Jin Suh, Ji Yun Lee, Se Hyun Kim, Jin Won Kim, Jeong-Ok Lee, Yu Jung Kim, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Keun-Wook Lee. Preclinical study of antitumor effect of alpelisib combined with paclitaxel in gastric caner [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C074. doi:10.1158/1535-7163.TARG-19-C074

Compound K, a major metabolite of ginsenosides Rb1, which is produced by human intestinal bacteria after oral administration, is one of the main pharmacologic compounds found in ginseng. In our previous study, we demonstrated that compound K inhibited the production of nitric oxide (NO) and prostaglandin E2 in lipopolysaccharide (LPS)-treated RAW264.7 cells. However, the mechanisms by which compound K may be effective against inflammation remain unknown. In the present study, compound K significantly inhibited LPS-induced NO production by suppression of inducible NO synthase (iNOS) in LPS-treated RAW264.7 cells. Compound K also inhibited LPS-induced cyclooxygenase-2 (COX-2) expression at both the mRNA and protein levels. It effectively suppressed both the release and mRNA expression levels of pro-inflammatory cytokines such as interleukin-1&beta, (IL-1&beta, ) and IL-6. The anti-inflammatory effects of compound K appeared to occur via inhibition of LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibition of NF-&kappa, B translocation from the cytosol to the nucleus by suppressing phosphorylation of inhibitory kappa B-&alpha, (I&kappa, B-&alpha, ). Furthermore, we showed that compound K inhibited LPS-induced NO generation in an experimental zebrafish model. Considering these results, compound K could potentially be developed as a natural anti-inflammatory agent.

Gelidium elegans, a red alga native to the Asia Pacific region, contains biologically active polyphenols. We conducted a molecular biological study of the anti-diabetic effect of Gelidium elegans extract (GEE) in C57BL/KsJ-db/db mice. Mice that had been administered GEE had significantly lower body mass, water consumption, and fasting blood glucose than db/db controls. Moreover, hemoglobin A1c (HbA1c), an indicator of the glycemic status of people with diabetes, was significantly lower in mice that had been administered GEE. We also found that 200 mg/kg/day GEE upregulates the insulin signaling pathway by activating insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K), and increasing the expression of glucose transporter type 4 (GLUT4). In parallel, mitogen-activated protein kinase (MAPK) activity was lower in GEE-treated groups. In summary, these findings indicate that GEE regulates glucose metabolism by activating the insulin signaling pathway and downregulating the MAPK signaling pathway.