JI017 Induces Cell Autophagy and Apoptosis via Elevated Levels of Reactive Oxygen Species in Human Lung Cancer Cells.

Lung cancer is one of the most common malignant tumors and a leading cause of cancer-related death in the worldwide. Various anticancer drugs, such as cisplatin and pemetrexed, have been developed for lung cancer treatment but due their drug resistance and side effects, novel treatments need to be developed. In this study, the efficacy of the natural drug JI017, which is known to have few side effects, was tested in lung cancer cells. JI017 inhibited A549, H460, and H1299 cell proliferation. JI017 induced apoptosis, regulated apoptotic molecules, and inhibited colony formation. Additionally, JI017 increased intracellular ROS generation. JI017 downregulated PI3K, AKT, and mTOR expression. JI017 increased the cytosolic accumulation of LC3. We found that JI017 promoted apoptosis through ROS-induced autophagy. Additionally, the xenograft tumor size was smaller in JI017-treated mice. We found that JI017 treatment increased MDA concentrations, decreased Ki-67 protein levels, and increased cleaved caspase-3 and LC3 levels in vivo. JI017 decreased cell proliferation and increased apoptosis by inducing autophagy signaling in H460 and H1299 lung cancer cells. Targeting JI017 and autophagy signaling could be useful in lung cancer treatment.

Reduced HIF-1α Stability Induced by 6-Gingerol Inhibits Lung Cancer Growth through the Induction of Cell Death.

Lung cancer (LC) is the leading global cause of cancer-related death, and metastasis is a great challenge in LC therapy. Additionally, solid cancer, including lung, prostate, and colon cancer, are characterized by hypoxia. A low-oxygen state is facilitated by the oncogene pathway, which correlates with a poor cancer prognosis. Thus, we need to understand the related mechanisms in solid tumors to improve and develop new anticancer strategies. The experiments herein describe an anticancer mechanism in which heat shock protein 90 (HSP90) stabilizes HIF-1α, a master transcription factor of oxygen homeostasis that has been implicated in the survival, proliferation and malignant progression of cancers. We demonstrate the efficacy of 6-gingerol and the molecular mechanism by which 6-gingerol inhibits LC metastasis in different oxygen environments. Our results showed that cell proliferation was inhibited after 6-gingerol treatment. Additionally, HIF-1α, a transcriptional regulator, was found to be recruited to the hypoxia response element (HRE) of target genes to induce the transcription of a series of target genes, including MMP-9, vimentin and snail. Interestingly, we found that 6-gingerol treatment suppressed activation of the transcription factor HIF-1α by downregulating HSP90 under both normoxic and hypoxic conditions. Furthermore, an experiment in an in vivo xenograft model revealed decreased tumor growth after 6-gingerol treatment. Both in vitro and in vivo analyses showed the inhibition of metastasis through HIF-1α/HSP90 after 6-gingerol treatment. In summary, our study demonstrates that 6-gingerol suppresses proliferation and blocks the nuclear translocation of HIF-1α and activation of the EMT pathway. These data suggest that 6-gingerol is a candidate antimetastatic treatment for LC.

Ru(II)-Catalyzed C-H Hydroxyalkylation and Mitsunobu Cyclization of N-Aryl Phthalazinones.

Ruthenium(II)-catalyzed C(sp2)-H functionalization of N-aryl phthalazinones with a range of aldehydes and activated ketone is described. Initial formation of hydroxyalkylated phthalazinones and subsequent Mitsunobu cyclization provided facile access to biologically relevant indazolophthalazinones. The utility of this method is highlighted by synthetic transformations into a series of potentially bioactive scaffolds.

Ruthenium(II)-Catalyzed Site-Selective Hydroxymethylation of Indolines with Paraformaldehyde.

The facile synthesis of hydroxymethylated indole derivatives is crucial for their further development as pharmaceutical compounds and other synthetic purposes. Herein, we describe the ruthenium(II)-catalyzed hydroxymethylation of indolines and other N-heterocycles using paraformaldehyde as an abundant C1 feedstock. A wide substrate scope range and high levels of site selectivity as well as functional group tolerance were observed.

Atf3 induction is a therapeutic target for obesity and metabolic diseases.

Activating transcription factor 3 (Atf3) has been previously demonstrated to impact obesity and metabolism. However, a metabolic role of Atf3 in mice remains debatable. We investigated the role of Atf3 in mice and further investigated Atf3 expression as a therapeutic target for obesity and metabolic diseases. Atf3 knockout (KO) mice fed with a high fat diet (HFD) aggravated weight gain and impaired glucose metabolism compared to littermate control wild type (WT) mice. Atf3 KO aged mice fed with a chow diet (CD) for longer than 10 months also displayed increased body weight and fat mass compared to WT aged mice. We also assessed requirements of Atf3 in a phytochemical mediated anti-obese effect. Effect of sulfuretin, a previously known phytochemical Atf3 inducer, in counteracting weight gain and improving glucose tolerance was almost completely abolished in the absence of Atf3, indicating that Atf3 induction can be a molecular target for preventing obesity and metabolic diseases. We further identified other Atf3 small molecule inducers that exhibit inhibitory effects on lipid accumulation in adipocytes. These data highlight the role of Atf3 in obesity and further suggest the use of chemical Atf3 inducers for prevention of obesity and metabolic diseases.

Prdm4 induction by the small molecule butein promotes white adipose tissue browning.

Increasing the thermogenic activity of adipocytes holds promise as an approach to combating human obesity and related metabolic diseases. We identified induction of mouse PR domain containing 4 (Prdm4) by the small molecule butein as a means to induce expression of uncoupling protein 1 (Ucp1), increase energy expenditure, and stimulate the generation of thermogenic adipocytes. This study highlights a Prdm4-dependent pathway, modulated by small molecules, that stimulates browning of white adipose tissue.

The prevention of 2,4-dinitrochlorobenzene-induced inflammation in atopic dermatitis-like skin lesions in BALB/c mice by Jawoongo.

BACKGROUND: Jawoongo is an herbal mixture used in traditional medicine to treat skin diseases. This study aimed to investigate whether Jawoongo ameliorates Atopic dermatitis (AD)-like pathology in mice and to understand its underlying cellular mechanisms. METHODS: AD was induced by 2, 4-Dinitrocholrlbenzene (DNCB) in BALB/c mice. Treatment with Jawoongo was assessed to study the effect of Jawoongo on AD in mice. Histological Analysis, blood analysis, RT-PCR, western blot analysis, ELISA assay and cell viability assay were performed to verify the inhibitory effect of Jawoongo on AD in mice. RESULTS: We found that application of Jawoongo in an ointment form on AD-like skin lesions on DNCB-exposed BALB/c mice reduced skin thickness and ameliorated skin infiltration with inflammatory cells, mast cells and CD4+ cells. The ointment also reduced the mRNA levels of IL-2, IL-4, IL-13 and TNF-α in the sensitized skin. Leukocyte counts and the levels of IgE, IL-6, IL-10 and IL-12 were decreased in the blood of the DNCB-treated mice. Furthermore, studies on cultured cells demonstrated that Jawoongo exhibits anti-inflammatory activities, including the suppression of proinflammatory cytokine expression, nitric oxide (NO) production, and inflammation-associated molecule levels in numerous types of agonist-stimulated innate immune cell, including human mast cells (HMC-1), murine macrophage RAW264.7 cells, and splenocytes isolated from mice. CONCLUSION: These findings indicate that Jawoongo alleviates DNCB-induced AD-like symptoms via the modulation of several inflammatory responses, indicating that Jawoongo might be a useful drug for the treatment of AD.

Sulfuretin has therapeutic activity against acquired lymphedema by reducing adipogenesis.

Acquired lymphedema is a pathological condition associated with lymphatic dysfunction caused by surgical treatments for cancer. Although global estimates of the prevalence of acquired lymphedema have been rising, there are currently no effective therapeutics available. Since adipose tissue accumulation is a clinical hallmark of lymphedema, we hypothesized that regulation of adipogenesis in lymphedematous tissue could be used as a therapeutic intervention against lymphedema. Toward this, we investigated the possibility of anti-adipogenic 30% ethanol Rhus verniciflua Stokes (RVS) extract as a potential lymphedema treatment. Oral administration of RVS extract ameliorated volumetric symptoms of lymphedema in a mouse model. RVS administration also reduced adipose tissue accumulation in lymphedematous tissue and downregulated expression of adipocyte markers, including Pparγ and Fabp4. Sulfuretin was identified as a major bioactive compound in the 30% ethanol RVS extract in liquid chromatography-mass spectrometry analysis. Similar to the activities of RVS, sulfuretin inhibited adipocyte differentiation in 3T3-L1 preadipocytes. Moreover, treatment with sulfuretin on lymphedema-induced mice reduced lymphedema volume, decreased the expression of adipogenic markers, but induced the expression of markers associated with lymphangiogenesis. Taken together, our data raise the possibility that sulfuretin might be used in therapeutic interventions against acquired lymphedema.

Effects of Angelicae dahuricae Radix on 2, 4-Dinitrochlorobenzene-Induced Atopic Dermatitis-Like Skin Lesions in mice model.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory, chronically relapsing, and intensively pruritic skin disease that affect 10-30% of the global population. Angelicae dahuricae Radix (ADR) has been reported to be anti-inflammatory in Korean Medicine. In the present study, we investigated whether ADR suppresses the progression of AD in animal model. METHODS: AD was induced by 2, 4-Dinitrochlorobenzene (DNCB). ADR was orally administered to mice to study the effect of ADR on AD. Histological Analysis, immunohistochemistry, blood analysis, RT-PCR, and ELISA assay were performed. RESULTS: ADR significantly suppressed AD-like symptoms in BALB/c mice: ADR decreased skin thickness and spleen weight of mice. ADR reduced infiltration of mast cells, inflammatory cells and CD4+ cells into mouse skin. ADR lowered the number of WBCs in the blood of mice. ADR reduced the levels of IgE, IL-6, IL-10 and IL-12 in mice serum. ADR down-regulated mRNA expression of IL-4, IL-6 and TNF-α in mouse skin tissue. CONCLUSION: Our present study clearly indicates that ADR suppresses the progression of AD induced by DNCB in BALB/c mice. This suggests that ADR might be a useful drug for the treatment of AD.

Tonggyu-tang, a traditional Korean medicine, suppresses pro-inflammatory cytokine production through inhibition of MAPK and NF-κB activation in human mast cells and keratinocytes.

BACKGROUND: Allergic diseases including allergic rhinitis, asthma, and atopic dermatitis are increasing worldwide. Common medications used to treat these inflammatory disorders are anti-histamines and corticosteroids, but they have their own limitations such as short duration and severe side effects. Thus, interest in complementary and alternative medicine is continually growing. Here, we investigate the anti-inflammatory mechanisms of Tonggyu-tang (TGT), a traditional Korean medicine that has been used to treat patients with allergic nasal disorders. METHODS: We measured mRNA expressions and production of pro-inflammatory cytokines such as interleukin (IL)-4, IL-6, IL-8 and tumor necrosis factor alpha (TNF-α) by RT-PCR and ELISA assays in HMC-1 (human mast cell line-1) and HaCaT cells, immortalized human keratinocytes. Moreover, we evaluated the effect of TGT on two major inflammation-related pathways, mitogen activated protein kinase (MAPK) and NF-κB signaling pathway in these two cells. RESULTS: Our results revealed that that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-α in the agonist-treated HMC-1 and HaCaT cells. We also found that TGT suppressed MAPK signaling pathway including extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK) as well as NF-κB pathway, which are known to regulate inflammatory cytokine expression. CONCLUSION: Taken together, our results demonstrate that TGT inhibits expression of pro-inflammatory cytokines by suppressing MAPK and NF-kB pathway in both mast cells and keratinocytes, suggesting the potential use of TGT in treating allergic inflammatory diseases.

Anti-allergic effects of So-Cheong-Ryong-Tang in ovalbumin-induced allergic rhinitis model.

Allergic rhinitis (AR) is an allergic inflammation of the nasal airways. The Korean herbal medicine, So-Cheong-Ryong-Tang (SCRT) has been typically used for the treatment of AR for hundreds of years. In the present study, we investigated whether SCRT suppresses the progression of AR in animal model. AR was induced by ovalbumin (OVA). Treatment with SCRT was assessed to study the effect of SCRT on AR in mice. Histological analysis, multiplex cytokine assay, blood analysis, cell viability assay, RT-PCR and Elisa assay were performed to verify inhibitory effect of SCRT on AR. SCRT reduced infiltration of inflammatory cells into nasal cavity. SCRT reduced infiltration of mast cells into nasal mucosa. SCRT reduced the levels of cytokines (IL-4 and LIF) in the serum. SCRT reduced the levels of leukocytes in the blood. SCRT decreased cell viability of HMC-1 cells and splenocyte. SCRT suppressed IL-4 level in HMC-1 cells and splenocyte cells in a dose-dependent manner. SCRT suppressed IL-6 level and TNF-α level in splenocyte. SCRT suppresses the progression of AR induced by OVA. SCRT might be a useful drug for the treatment of AR.

Cucurbitacin D induces cell cycle arrest and apoptosis by inhibiting STAT3 and NF-κB signaling in doxorubicin-resistant human breast carcinoma (MCF7/ADR) cells.

Breast cancer is the most common cancer for women and is a major cause of mortality in women. Doxorubicin is a generally used chemotherapy drug for breast cancer. However, multidrug resistance of breast cancer interferes with the chemotherapy. We examined whether cucurbitacin D affects doxorubicin resistance of MCF7/ADR breast cancer cells. Cell viability was measured by MTT assay. Levels of p-STAT3, p-NF-κB, IκB, and caspases were measured by Western blot analysis. Nuclear staining of Stat3 and NF-κB was measured by immunocytochemistry. STAT3 and NF-κB transcriptional activity was detected by STAT3 and NF-κB luciferase reporter gene assays. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by cucurbitacin D was measured by Annexin V-FITC/propidium iodide assay. More than 90% of MCF7/ADR cells lived upon treatment with doxorubicin for 24 h. However, upon treatment with cucurbitacin D, cell death was more than 60%. Co-administration of cucurbitacin D and doxorubicin induced apoptosis, and G2/M cell cycle arrest, and inhibited upregulated Stat3 by doxorubicin on MCF7/ADR cells. Additionally, cucurbitacin D led to an increase in the IκBα level in the cytosol and a decrease in the p-NF-κB level in the nucleus. Finally, cucurbitacin D inhibited translocation of Stat3 and NF-κB and decreased transcriptional activity in the nucleus. Cucurbitacin D decreases cell proliferation and induces apoptosis by inhibiting Stat3 and NF-κB signaling in doxorubicin-resistant breast cancer cells. Cucurbitacin D could be used as a useful compound to treat adriamycin-resistant patients.

Sulfuretin induces osteoblast differentiation through activation of TGF-ß signaling.

The identification and examination of potential determinants controlling the progression of cell fate toward osteoblasts can be intriguing subjects. In this study, the effects of sulfuretin, a major compound isolated from Rhus verniciflua Stokes, on osteoblast differentiation were investigated. Treatments of sulfuretin induced alkaline phosphatase (ALP) activity in mesenchymal C3H10T1/2 cells and mineralization in preosteoblast MC3T3-E1 cells. Pro-osteogenic effects of sulfuretin were consistently observed in freshly isolated primary bone marrow cells. In mechanical studies, sulfuretin specifically induced expression of TGF-ß target genes, such as SMAD7 and PAI-1, but not other signaling pathway-related genes. Similar to the results of gene expression analysis, reporter assays further demonstrated TGF-ß-specific induction by sulfuretin. Furthermore, disruption of TGF-ß signaling using treatment with TGF-ß-specific inhibitor, SB-431542, and introduction of SMAD2/3 small interfering RNA impaired the effects of sulfuretin in inducing ALP activity and expression of ALP mRNA. Together, these data indicate that the pro-osteogenic effects of sulfuretin are mediated through activation of TGF-ß signaling, further supporting the potential of sulfuretin in the prevention of bone-related diseases such as bone fracture and osteoporosis.

Read-through compound 13 restores dystrophin expression and improves muscle function in the mdx mouse model for Duchenne muscular dystrophy.

Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders. Two such read-through compounds, RTC13 and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations in the ATM and the dystrophin genes. We have now tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD). Direct intramuscular injection of compound RTC14 did not result in significant read-through activity in vivo and demonstrated the levels of dystrophin protein similar to those detected using gentamicin. In contrast, significant higher amounts of dystrophin were detected after intramuscular injection of RTC13. When administered systemically, RTC13 was shown to partially restore dystrophin protein in different muscle groups, including diaphragm and heart, and improved muscle function. An increase in muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase levels. These studies establish the therapeutic potential of RTC13 in vivo and advance this newly identified compound into preclinical application for DMD.

A new series of small molecular weight compounds induce read through of all three types of nonsense mutations in the ATM gene.

Chemical-induced read through of premature stop codons might be exploited as a potential treatment strategy for genetic disorders caused by nonsense mutations. Despite the promise of this approach, only a few read-through compounds (RTCs) have been discovered to date. These include aminoglycosides (e.g., gentamicin and G418) and nonaminoglycosides (e.g., PTC124 and RTC13). The therapeutic benefits of these RTCs remain to be determined. In an effort to find new RTCs, we screened an additional ~36,000 small molecular weight compounds using a high-throughput screening (HTS) assay that we had previously developed and identified two novel RTCs, GJ071, and GJ072. The activity of these two compounds was confirmed in cells derived from ataxia telangiectasia (A-T) patients with three different types of nonsense mutation in the ATM gene. Both compounds showed activity comparable to stop codons (TGA, TAG, and TAA) PTC124 and RTC13. Early structure-activity relationship studies generated eight active analogs of GJ072. Most of those analogs were effective on all three stop codons. GJ071 and GJ072, and some of the GJ072 analogs, appeared to be well tolerated by A-T cells. We also identified another two active RTCs in the primary screen, RTC204 and RTC219, which share a key structural feature with GJ072 and its analogs.

Effects of Hyeonggaeyeongyo-tang in ovalbumin-induced allergic rhinitis model.

Allergic rhinitis (AR) is an allergic inflammation of the nasal airways. The prevalence of AR is increasing worldwide. We investigated whether Hyeonggaeyeongyo-tang (HYT) is effective to suppress the progression of AR induced by ovalbumin (OVA). Male BALB/c mice were used for this study. Allergic rhinitis was induced by OVA. Treatment with HYT was assessed to study the effect of HYT on allergic rhinitis in mice. Histological analysis, immunohistochemistry, multiplex cytokine assay, blood analysis, and cell viability assay were performed to verify inhibitory effect of HYT on allergic rhinitis. HYT did not show any toxicity maintaining body weight. Food intake was steady without variation in mice. HYT reduced infiltration of inflammatory cells and mast cells into nasal cavity. HYT reduced the levels of cytokines and leukocytes in the blood. HYT decreased the splenocyte cell viability. Antihistamines and steroids are the most common medications used to treat allergic rhinitis. However, long-term use of drug generates resistance or side effects requiring the development of new drug. Our present study clearly demonstrates that HYT suppresses the progression of allergic rhinitis induced by OVA. This suggests that HYT might be a useful drug for the treatment of allergic rhinitis.

Oral administration of herbal mixture extract inhibits 2,4-dinitrochlorobenzene-induced atopic dermatitis in BALB/c mice.

CP001 is four traditional herbal medicine mixtures with anti-inflammatory properties. In this study, we investigated the effect of oral administration of CP001 ethanol extract on the 2,4-dinitrochlorobenzene- (DNCB-) induced AD mouse models. For that purpose, we observed the effects of oral administration of CP001 on skin inflammatory cell infiltration, skin mast cells, production of serum IgE, and expression of Th2 cytokine mRNA in the AD skin lesions of DNCB treated BALB/c mice. Histological analyses demonstrated that CP001 decreased dermis and epidermis thickening as well as dermal infiltration induced by inflammatory cells. In addition, CP001 decreased mast cell infiltration in count as well as dermal infiltration induced by inflammatory cells. In the skin lesions, mRNA expression of interleukin- (IL-) 4 and IL-13 was inhibited by CP001. CP001 also reduced the production of IgE level in mouse plasma. In addition, we investigated the effect of CP001 on the inflammatory allergic reaction using human mast cells (HMC-1). In HMC-1, cytokine production and mRNA levels of IL-4, IL-13, IL-6, and IL-8 were suppressed by CP001. Taken together, our results showed that oral administration of CP001 exerts beneficial effects in AD symptoms, suggesting that CP001 might be a useful candidate for the treatment of AD.

Butein derivatives prevent obesity and improve insulin resistance through the induction of energy expenditure in high-fat diet-fed obese mice.

Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3 s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3 s at 5 mg/kg prevented weight gain by 13.6 % in high-fat diet-fed mice without any toxicological observation. In addition, compound 3 s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T1/2), and blood exposure ratio of i.p to i.v were significantly improved in compound 3 s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3 s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3 s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation.

Silibinin inhibits the production of pro-inflammatory cytokines through inhibition of NF-κB signaling pathway in HMC-1 human mast cells.

BACKGROUND: Silibinin is the major active molecule of silymarin, the mixture of flavonolignans extracted from Cirsium japonicum. It has been used for the treatment of hepatitis and inflammation-related diseases. In the present study, the effects of silibinin on allergic inflammation and its signaling were investigated in the induced human mast cells. METHODS: Cell growth inhibition induced by silibinin was measured by MTS assay. Histamine release was measured by enzyme immunoassay. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) secreted protein levels and mRNA levels were measured by the ELISA assay and RT-PCR, respectively. The NF-κB promoter activity was examined by a luciferase assay. RESULTS: Silibinin suppressed the growth of HMC-1 cells and also reduced the production and mRNA expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-8. Moreover, silibinin inhibited the nuclear translocation of nuclear factor (NF)-κB through inhibition of the phosphorylation of IκBα and suppressed NF-κB transcriptional activity in stimulated HMC-1 cells. CONCLUSIONS: Taken together, these results indicate that silibinin inhibits the production of pro-inflammatory cytokines through inhibition of NF-κB signaling pathway in HMC-1 human mast cells, suggesting that silibinin could be used for the treatment of mast cell-derived allergic inflammatory diseases.

Hydroxynaphthoic acids identified in a high throughput screening potently ameliorate endoplasmic reticulum stress as novel chemical chaperones.

Folding of newly synthesized protein occurs in endoplasmic reticulum (ER) and is assisted by chaperone molecules. In ER stress conditions, misfolded proteins are enriched in a lumen of ER perturbing its normal function, which triggers cellular self-defense mechanism, the unfolded protein response (UPR). It was reported that tunicamycin-induced ER stress can be modulated with high concentration of chemicals such as 4-phenylbutyric acid and salicylate. In search of assay systems to identify such compounds, we have developed a cell-based reporter assay where renilla luciferase activity is driven by glucose-regulated protein 78 (GRP78) promoter. Using our reporter assay, we have screened chemical libraries and found that hydroxynaphthoic acids, especially 1-, 3-, and 6-hydroxy-2-naphthoic acids, potently decrease the ER stress signal, showing an order of magnitude better activity than salicylate. UPR markers such as GRP78, C/EBP homology protein (CHOP) and phosphorylated protein kinase RNA-activated (PKR)-like ER kinase (PERK) were significantly down-regulated with hydroxynaphthoic acids in western blot. Among the analogues, 1-hydroxy-2-naphthoic acid was the most potent in down-regulating those UPR markers. Further, both phosphorylated inositol-requiring enzyme 1α (IRE1α) and spliced form of X-box binding protein 1 (XBP1) were decreased in the protein and the mRNA level, implying both PERK and IRE1α branches in UPR mechanism are controlled with hydroxynaphthoic acids. Taken together, it was suggested that hydroxynaphthoic acids exert their ER stress-reducing activity prior to the UPR activation as chemical chaperones do. In summary, we report a cell-based assay system for the screening of ER stress-reducing compounds and hydroxynaphthoic acids as novel series of chemical chaperones.