NMR-based stability evaluation of (E)-1-(3',4'-dimethoxyphenyl)butadiene (DMPBD) from Zingiber cassumunar Roxb. rhizome.

INTRODUCTION: The active compound (E)-1-(3',4'-dimethoxyphenyl)butadiene (DMPBD) isolated from the rhizomes of Zingiber cassumunar Roxb. has potent anti-inflammatory and anticancer activities. Although DMPBD is one of the promising drug candidates for phytomedicine, its limited stability impedes its widespread use. For the development of new drugs, the assessment of their chemical stability is essential, ensuring they maintain their properties within specified limits throughout the period from production until use. OBJECTIVE: In the present study, we aimed to evaluate the stability of DMPBD under various conditions, including different solvents, temperatures, and lighting conditions, to identify the factors affecting stability and optimize the storage and handling conditions. METHODOLOGY: DMPBD samples subjected to the different conditions tested were monitored by quantitative 1H NMR (qHNMR), using an internal standard for the determination of the absolute quantity of DMPBD as a function of time and the changes thereof within 1 month. RESULTS: Significant decomposition of DMPBD was observed in chloroform-d1, whereas its content remained constant in methanol-d4. The content of DMPBD was maintained upon storage at temperatures below 4°C, both as methanolic solution and in the crude extract. Exposure to light had a slight negative impact on its contents. Some degradation products could be identified as resulting from O2-induced cleavage of the diene moiety. CONCLUSIONS: For pharmacological/therapeutic applications, DMPBD should be stored in the form of the crude extract or as a purified material in methanolic solution. Ideally, the storage temperature should be below 4°C and O2 should be excluded.

Pheophorbide a identified in an Eupatorium perfoliatum extract is a novel lymphatic vascular activator.

The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies.

Standardized Extract of Asparagus officinalis Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1ß Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages.

Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1ß. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1ß by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1ß production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.

Cardioprotection by isosteviol derivate JC105: A unique drug property to activate ERK1/2 only when cells are exposed to hypoxia-reoxygenation.

In the present study, we have investigated potential cardioprotective properties of Isosteviol analogue we recently synthesized and named JC105. Treatment of heart embryonic H9c2 cells with JC105 (10 µM) significantly increased survival of cells exposed to hypoxia-reoxygenation. JC105 (10 µM) activated ERK1/2, DRP1 and increased levels of cardioprotective SUR2A in hypoxia-reoxygenation, but did not have any effects on ERK1/2, DRP1 and/or SUR2A in normoxia. U0126 (10 µM) inhibited JC105-mediated phosphorylation of ERK1/2 and DRP1 without affecting AKT or AMPK, which were also not regulated by JC105. Seahorse bioenergetic analysis demonstrated that JC105 (10 µM) did not affect mitochondria at rest, but it counteracted all mitochondrial effects of hypoxia-reoxygenation. Cytoprotection afforded by JC105 was inhibited by U0126 (10 µM). Taken all together, these demonstrate that (a) JC105 protects H9c2 cells against hypoxia-reoxygenation and that (b) this effect is mediated via ERK1/2. The unique property of JC105 is that selectively activates ERK1/2 in cells exposed to stress, but not in cells under non-stress conditions.

Nitroprusside induces melanoma ferroptosis with serum supplementation and prolongs survival under serum depletion or hypoxia.

BACKGROUND: When proliferating tumor cells expand to areas distant from vascular sites, poor diffusion of oxygen and nutrients occur, generating a restrictive hypoxic gradient in which susceptible tumor cells die. The heterogeneous population surviving hypoxia and metabolic starvation include de-differentiated cancer stem cells (CSC), capable of self-renewing tumor-initiating cells (TICs), or those that divide asymmetrically to produce non-tumor-initiating differentiated (NTI-D) cell progeny. Under such restrictive conditions, both populations slowly proliferate, entering quiescence or senescence, when exiting from cell cycle progression. This may drive chemoresistance and tumor recurrence, since most anti-cancer treatments target rapidly proliferating cells. PURPOSE: Since persistent or additional stress may increase NTI-D cells conversion to TICs, we investigated whether nutrient depletion or hypoxia influence expression of tyrosinase, a crucial enzyme for melanin synthesis, and B16 melanoma survival, when exposed to iron-dependent cell death oxidative stress produced by the Fenton reaction, resembling ferroptosis. RESULTS: -a) proliferating B16 melanoma with 10% serum-supplementation (10%S) normoxically express hypoxia inducible factor 1α (HIF1α) but lose tyrosinase, in contrast to those transiently exposed to (SF) serum-free medium, in which both HIF1α and tyrosinase are co-expressed; b) in contrast to the resistance to SNP toxicity in (SF) cells with higher tyrosinase expression, those in (10%S) are killed by iron from nitroprusside/ferricyanide (SNP) irrespective of exogenous H2O2, in a reaction antagonized by the anti-oxidant and MEK inhibitor UO126; c) Moreover, under transient serum depletion, SNP cooperates with hypoxia (1.5% oxygen), prolonging B16 melanoma (SF) survival; d) the hypoxia mimetic CoCl2 inhibits proliferation-associated cyclin A, irrespective of SNP, in (10%S) cells or in transiently serum-depleted (SF) cells. However, only in the latter cells, CoCl2 but not SNP, induce loss of HIF1α and apoptosis-associated PARP cleavage; e) longer term adaptation to survive serum depletion, generates (SS) cells resistant to SNP toxicity, which aerobically co-express HIF1α and tyrosinase. In SS B16 melanoma, exogenous non-toxic 100 µM H2O2 super-induces the ratio of tyrosinase to HIF1α. However, co-treatment of SS-B16 cells with SNP plus exogenous H2O2, partly increases PARP cleavage by reciprocally decreasing tyrosinase expression. SIGNIFICANCE: - These results suggest that a phenotypic plasticity in response to depletion of nutrients and/or oxygen, helps decide whether melanoma cells undergo either death by ferroptosis, or resistance to it, when challenged by the same exogenous oxidative stress (iron ± H2O2).

Ginkgo Biloba Extract Inhibits Metastasis and ERK/Nuclear Factor kappa B (NF-κB) Signaling Pathway in Gastric Cancer.

BACKGROUND Ginkgo biloba extract (EGb761), a standard extract of the Chinese traditional medicine Ginkgo biloba, plays an anti-tumor role in various cancers. However, whether EGb761 is involved in the invasion and metastasis of gastric cancer remains unclear. MATERIAL AND METHODS In the current study, cell viability assay, Western blotting, wound-healing assay, Transwell invasion assay, and orthotopic transplantation model were performed to explore the effects of EGb761 on gastric cancer. RESULTS In vitro, the results showed that EGb761 suppressed the proliferation of gastric cancer cells in a dose-dependent manner. Furthermore, the migration and invasiveness were weakened and the protein levels of p-ERK1/2, NF-kappaB P65, NF-kappaB p-P65, and MMP2 were decreased by EGb761 or U0126 (an inhibitor of ERK signaling pathway) exposure in gastric cancer cells. Moreover, the combined treatment with EGb761 and U0126 significantly inhibited ERK, NF-kappaB signaling pathway, and the expression of MMP2 than those of single drug. In vivo, EGb761 inhibited the tumor growth and hepatic metastasis of gastric cancer in the mouse model. Results of immunohistochemistry indicated that the expression of ERK1/2, NF-kappaB P65 and MMP2 were decreased by EGb761 in the tumor tissues. CONCLUSIONS EGb761 plays a vital role in the suppression of metastasis and ERK/NF-kappaB signaling pathway in gastric cancer.

Anti-Apoptotic Effects of Docosahexaenoic Acid in IL-1ß-Induced Human Chondrosarcoma Cell Death through Involvement of the MAPK Signaling Pathway.

Osteoarthritis (OA) is a degenerative disease characterized by progressive articular cartilage destruction and joint marginal osteophyte formation with different degrees of synovitis. Docosahexaenoic acid (DHA) is an unsaturated fatty acid with anti-inflammatory, antioxidant, and antiapoptotic functions. In this study, the human chondrosarcoma cell line SW1353 was cultured in vitro, and an OA cell model was constructed with inflammatory factor IL-1ß stimulation. After cells were treated with DHA, cell apoptosis was measured. Western blot assay was used to detect protein expression of apoptosis-related factors (Bax, Bcl-2, and cleaved caspase-3) and mitogen-activated protein kinase (MAPK) signaling pathway family members, including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38 MAPK. Our results show that IL-1ß promotes the apoptosis of SW1353 cells, increases the expression of Bax and cleaved caspase-3, and activates the MAPK signaling pathway. In contrast, DHA inhibits the expression of IL-1ß, inhibits IL-1ß-induced cell apoptosis, and has a certain inhibitory effect on the activation of the MAPK signaling pathway. When the MAPK signaling pathway is inhibited by its inhibitors, the effects of DHA on SW1353 cells are weakened. Thus, DHA enhances the apoptosis of SW1353 cells through the MAPK signaling pathway.

CCN2-MAPK-Id-1 loop feedback amplification is involved in maintaining stemness in oxaliplatin-resistant hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. Chemotherapy is an alternative treatment for advanced HCCs, but chemo-resistance prevents cancer therapies from achieving stable and complete responses. Understanding the underlying mechanisms in chemo-resistance is critical to improve the efficacy of HCC. METHODS: The expression levels of Id-1 and CCN2 were detected in large cohorts of HCCs, and functional analyses of Id-1 and CCN2 were performed both in vitro and in vivo. cDNA microarrays were performed to evaluate the alterations of expression profiling of HCC cells with overexpression of CCN2. Finally, the role of downstream signaling of MAPK/Id-1 signaling pathway in oxaliplatin resistance were also explored. RESULTS: The increased expression of Id-1 and CCN2 were closely related to oxaliplatin resistance in HCC. Upregulation of CCN2 and Id-1 was independently associated with shorter survival and increased recurrence in HCC patients, and significantly enhanced oxaliplatin resistance and promoted lung metastasis in vivo, whereas knock-down of their expression significantly reversed the chemo-resistance and inhibited HCC cell stemness. cDNA microarrays and PCR revealed that Id-1 and MAPK pathway were the downstream signaling of CCN2. CCN2 significantly enhanced oxaliplatin resistance by activating the MAPK/Id-1 signaling pathway, and Id-1 could upregulate CCN2 in a positive feedback manner. CONCLUSIONS: CCN2/MAPK/Id-1 loop feedback amplification is involved in oxaliplatin resistance, and the combination of oxaliplatin with inhibitor of CCN2 or MAPK signaling could provide a promising approach to ameliorating oxaliplatin resistance in HCC.

PG102 Upregulates IL-37 through p38, ERK, and Smad3 Pathways in HaCaT Keratinocytes.

IL-37 is an immunomodulatory cytokine that suppresses inflammation in various cell types and disease models. However, its role in keratinocytes has not been clearly understood, and there has been no report on the agents that can increase the expression of IL-37 in keratinocytes. In this study, we investigated the effects of silencing IL37 in HaCaT keratinocytes and the molecular mechanisms involved in the upregulation of IL-37 by PG102, a water-soluble extract from Actinidia arguta. It was found that knockdown of IL37 resulted in the augmented expression of antimicrobial peptides (AMPs) in response to cytokine stimulation. PG102 increased the expression of IL-37 at both mRNA and protein levels presumably by enhancing the phosphorylation of Smad3, ERK, and p38. Indeed, when cells were treated with specific inhibitors for these signaling molecules, the expression level of IL-37 was reduced. PG102 also promoted colocalization of phospho-Smad3 and IL-37. Our results suggest that IL-37 inhibits the expression of AMPs and that PG102 upregulates IL-37 through p38, ERK, and Smad3 pathways in HaCaT cells.

Effects of Electroacupuncture on Expression of D1 Receptor (D1R), Phosphorylation of Extracellular-Regulated Protein Kinase 1/2 (p-ERK1/2), and c-Fos in the Insular Cortex of Ketamine-Addicted Rats.

BACKGROUND The aim of this study was to investigate the effects of electroacupuncture (EA) on expression of the D1 receptor (D1R), phosphorylation of extracellular-regulated protein kinase 1/2 (p-ERK1/2) and c-Fos in the insular cortex (IC) of ketamine-addicted rats. MATERIAL AND METHODS Sprague-Dawley rats were randomly divided into 7 groups: the normal group, the normal saline (NS) group, the ketamine (Ket) group, the U0126+Ket group, the SCH23390+Ket group, the Ket+acupoints EA (EA1) group, and the Ket+ non-acupoints EA (EA2) group. We used immunohistochemistry to detect the expression of D1R, p-ERK1/2, and c-Fos. We also used Nissl staining techniques to study the morphology of IC neurons. RESULTS Our study demonstrated that the ketamine group had sparsely distributed neurons, large intracellular vacuoles, nuclei shift, and unclear nucleolus. The number of Nissl-positive (neuronal) cells in the ketamine group were decreased than in the normal group. Our results also indicated that there was significantly lower expression of D1R, p-ERK1/2, and c-Fos in the IC of the U0126+Ket group, SCH23390+Ket group, and Ket+EA1 group as compared with that of the Ket group. CONCLUSIONS Ketamine addiction induces c-Fos overexpression in the IC by increasing the expression of D1R and p-ERK1/2. Acupoints EA downregulate D1R and p-ERK1/2 by reducing the overexpression of c-Fos.

Fsk and IBMX inhibit proliferation and proapoptotic of glioma stem cells via activation of cAMP signaling pathway.

OBJECTIVE: We aimed to find out the underlying mechanism of forskolin (Fsk) and 3-isobutyl-1-methylxanthine (IBMX) on glioma stem cells (GSCs). METHODS: The expression of cAMP-related protein CREB and pCREB as well as apoptosis-related proteins were detected through Western blot analysis. The level of proliferation and growth rate of human GSCs was measured through thiazolyl blue tetrazolium bromide assay and stem cells forming sphere assay. The apoptosis-related gene expression was measured through reverse transcription-polymerase chain reaction. RESULTS: cAMP signaling pathway was activated in GSCs with Fsk-IBMX administration. Fsk-IBMX could inhibit the proliferation as well as invasion and promote the apoptosis of U87 cells. Besides, U0126 could inhibit MAPK signaling pathway to increase the sensitivity of GSCs to cAMP signaling pathway. As a result, Fsk-IBMX combined with U0126 had more negative effect on GSCs. CONCLUSIONS: The relationship of cAMP and MAPK signaling pathway in GSCs may provide a potential therapeutic strategy in glioma.

Moxibustion eases chronic inflammatory visceral pain through regulating MEK, ERK and CREB in rats.

AIM: To investigate the effects of herb-partitioned moxibustion (HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase (MEK)1, extracellular signal-regulated kinase (ERK)1/2 and cAMP response element binding protein (CREB) in spinal cord of rats with chronic inflammatory visceral pain (CIVP), and to explore the central mechanism of HPM in treating CIVP. METHODS: Male Sprague-Dawley rats were randomized into normal, model, HPM, sham-HPM, MEK-inhibitor and dimethyl sulfoxide (DMSO) groups. The CIVP model was established using an enema mixture of trinitrobenzene sulfonic acid and ethanol. HPM was applied at bilateral Tianshu (ST25) and Qihai (CV6) acupoints in the HPM group, while in the sham-HPM group, moxa cones and herb cakes were only placed on the same points but not ignited. The MEK-inhibitor and DMSO groups received L5-L6 intrathecal injection of U0126 and 30% DMSO, respectively. Abdominal withdrawal reflex (AWR), mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were applied for the assessment of pain behavior. The colonic tissue was observed under an optical microscope after hematoxylin-eosin staining. Expression of phosphor (p)MEK1, pERK1/2 and pCREB in rat spinal cord was detected using Western blotting. The levels of MEK, ERK and CREB mRNA in rat spinal cord were detected using real-time polymerase chain reaction. RESULTS: Compared with the normal group, the AWR scores were increased significantly (P < 0.01) and the MWT and TWL scores were decreased significantly (P < 0.05) in the model, sham-HPM and DMSO groups. Compared with the model group, the AWR scores were decreased significantly (P < 0.01) and the MWT and TWL scores were increased significantly in the HPM and MEK-inhibitor groups (P < 0.05). Compared with the sham-HPM and DMSO groups, the AWR scores were decreased significantly (P < 0.01) and the MWT and TWL scores were increased significantly (P < 0.05) in the HPM and MEK-inhibitor groups. Compared with the normal group, the expression of pMEK1, pERK1/2 and pCREB proteins and the levels of MEK, ERK and CREB mRNA in rat spinal cord were increased significantly in the model, sham-HPM and DMSO groups (P < 0.01 or < 0.05). Compared with the model group, the expression of pMEK1, pERK1/2 and pCREB proteins and the levels of MEK, ERK and CREB mRNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups (P < 0.01 or < 0.05). Compared with the sham-HPM and DMSO groups, expression of pMEK1, pERK1/2 and pCREB proteins and the levels of MEK, ERK and CREB mRNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups (P < 0.01 or < 0.05). CONCLUSION: HPM down-regulates protein phosphorylation of MEK1, ERK1/2 and CREB, and mRNA expression of MEK, ERK and CREB, inhibiting activation of the MEK/ERK/CREB signaling pathway in the spinal cord of CIVP rats, which is possibly a critical central mechanism of the analgesic effect of HPM.

Omega-3 free fatty acids attenuate insulin-promoted breast cancer cell proliferation.

High insulin levels in obese people are considered as a risk factor to induce breast carcinogenesis. And consumption of fish oils which mainly contain omega-3 fatty acids is associated with a reduced risk of breast cancer. However, whether omega-3 free fatty acids (FFAs) modulate insulin signaling pathway to prevent breast cancer is poorly understood. The current study tested the hypothesis that omega-3 FFAs attenuate insulin-induced breast cancer cell proliferation and regulate insulin signaling pathway. We show here that omega-3 FFAs attenuate MCF-7 cell proliferation and Akt and Erk1/2 phosphorylation levels stimulated by insulin. Knockdown Shp2 by siRNA resulted in significantly elevated omega-3 FFAs-activated Akt phosphorylation but failed to change insulin-stimulated Akt and Erk1/2 phosphorylation. And viable cell number was not affected by either downregulation of Shp2 expression or Erk1/2 inhibitor U0126 treatment. These observations indicated that omega-3 FFAs attenuate insulin-promoted breast cancer cell proliferation and insulin-activated Akt phosphorylation.

Anti-Melanogenic Activities of Heracleum moellendorffii via ERK1/2-Mediated MITF Downregulation.

In this study, the anti-melanogenic effects of Heracleum moellendorffii Hance extract (HmHe) and the mechanisms through which it inhibits melanogenesis in melan-a cells were investigated. Mushroom tyrosinase (TYR) activity and melanin content as well as cellular tyrosinase activity were measured in the cells. mRNA and protein expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), TYR-related protein-1 (TYRP-1) and -2 were also examined. The results demonstrate that treatment with HmHe significantly inhibits mushroom tyrosinase activity. Furthermore, HmHe also markedly inhibits melanin production and intracellular tyrosinase activity. By suppressing the expression of TYR, TYRP-1, TYRP-2, and MITF, HmHe treatment antagonized melanin production in melan-a cells. Additionally, HmHe interfered with the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, with reversal of HmHe-induced melanogenesis inhibition after treatment with specific inhibitor U0126. In summary, HmHe can be said to stimulate ERK1/2 phosphorylation and subsequent degradation of MITF, resulting in suppression of melanogenic enzymes and melanin production, possibly due to the presence of polyphenolic compounds.

Water-extracted Perilla frutescens increases endometrial receptivity though leukemia inhibitory factor-dependent expression of integrins.

The leaves and stems of Perilla frutescens var. acuta Kudo (PF) have been used to prevent threatened abortion in traditional medicine in the East Asian countries. Because reduced receptivity of endometrium is a cause of abortion, we analyzed the action of PF on the endometrial receptivity. PF increased the level of leukemia inhibitory factor (LIF), a major cytokine regulating endometrial receptivity, and LIF receptor in human endometrial Ishikawa cells. The PF-induced LIF expression was mediated by c-jun N-terminal kinase (JNK) and p38 pathways. Adhesion between Ishikawa cells and trophoblastic JAr cells stimulated by PF treatment was abolished by knock down of LIF expression or antagonism of LIFR. In addition, the expressions of integrin ß3 and ß5 were increased by PF treatment in Ishikawa cells. The PF-induced expression of integrin ß3 and ß5 was reduced with an LIFR antagonist. Neutralization of both integrins successfully blocked PF-stimulated adhesion of JAr cells and Ishikawa cells. These results suggest that PF enhanced the adhesion between Ishikawa cells and JAr cells by increasing the expression of integrin ß3 and ß5 via an LIF-dependent pathway. Given the importance of endometrial receptivity in successful pregnancy, PF can be a novel and effective candidate for improving pregnancy rate.

Aquaporin 3 Expression Induced by Salvia Miltiorrhiza via ERK1/2 Signal Pathway in the Primary Human Amnion Epithelium Cells from Isolated Oligohydramnios.

Salvia miltiorrhiza is one of the most common Chinese herbal drugs, which is effective to treat oligohydramnios. In this study, the aim was to investigate how Salvia miltiorrhiza regulate aquaporin 3 expression in the human amnion epithelial cells (hAECs) with normal amniotic fluid volume or isolated oligohydramnios, whether via extracellular signal regulated kinase1/2 (ERK1/2) signal transduction pathway or not. Primary hAECs cultures from 120 patients were incubated with Salvia miltiorrhiza or/and ERK1/2 inhibitor-- U0126. Localization of aquaporin 3 was detected by immunohistochemistry and the expression of total ERK1/2, phospho-ERK1/2 (p-ERK1/2) and aquaporin 3 was detected by Western blot. The results were: (1) In hAECs with normal amniotic fluid volume, treatment with 10 µmol/L of U0126 for 6 h resulted in the optimal inhibition of p-ERK1/2 (P<0.05). However, the expression of total ERK1/2 or aquaporin 3 did not significantly change after different concentrations or time of U0126 treatment. Salvia miltiorrhiza significantly up-regulated aquaporin 3 expression, which was not affected by U0126. (2) In hAECs with isolated oligohydramnios, treatment with 5 µmol/L of U0126 for 2 h resulted in the optimal inhibition of p-ERK1/2 and the lowest expression of aquaporin 3 (P<0.05). Moreover, Salvia miltiorrhiza significantly up-regulated aquaporin 3 expression, which was obviously blocked by U0126. These results suggest that Salvia miltiorrhiza may regulate aquaporin 3 expression in hAECs. In addition, in hAECs with isolated oligohydramnios, Salvia miltiorrhiza may regulate the expression of aquaporin 3 via the ERK1/2 signal transduction pathway, which provides a novel thread to the improved treatment for isolated oligohydramnios.

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression.

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins ß3 and ß5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.

Curcumin is a biologically active copper chelator with antitumor activity.

BACKGROUND: Curcumin is a natural product with antitumor activity. The compound targets multiple cell signaling pathways, including cell survival and proliferation, caspase activation and oncogene expression. As a ß-diketone, curcumin also exists as a keto-enol tautomer that chelates transition metal ions with high affinity. PURPOSE: Copper has an integral role in promoting tumor growth and angiogenesis. This study aims to investigate whether curcumin exerts its antitumor activity through copper chelation. METHODS: Copper chelation ability of curcumin was validated by measuring US/VIS spectrum. The antitumor activity and in vivo copper removal ability of curcumin was determined in a murine xenograft model. The effect of curcumin on copper-induced MAPK activation and cell proliferation was determined in cell culture system. RESULTS: Administration of curcumin to tumor-bearing animals resulted in suppression of A549 xenograft growth, an effect that was also observed in animals treated with ammonium tetrathiomolybdate (TM), a metal chelator used for copper storage disorders clinically. The inhibition on tumor growth was associated with reduction of copper concentrations in the serum of treated groups. In cell culture studies, we showed that copper promoted cell proliferation through Erk/MAPK activation. Treatment with curcumin or U0126, a specific MAPK inhibitor, or suppression of cellular uptake of copper by siRNA knockdown of copper transporter protein 1 (CTR1) blocked copper-induced cell proliferation. CONCLUSIONS: This study therefore demonstrates curcumin antitumor effect to its copper chelation capability. These results also implicate copper chelation as a general mechanism for their action of some biologically active polyphenols like flavonoids.

Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

Growth regulating properties of isoprene and isoprenoid-based essential oils.

KEY MESSAGE: Essential oils have growth regulating properties comparable to the well-documented methyl jasmonate and may be involved in localized and/or airborne plant communication. Aromatic plants employ large amounts of resources to produce essential oils. Some essential oils are known to contain compounds with plant growth regulating activities. However, the potential capacity of essential oils as airborne molecules able to modulate plant growth/development has remained uninvestigated. Here, we demonstrate that essential oils from eight taxonomically diverse plants applied in their airborne state inhibited auxin-induced elongation of Pisum sativum hypocotyls and Avena sativa coleoptiles. This response was also observed using five monoterpenes commonly found in essential oils as well as isoprene, the basic building block of terpenes. Upon transfer to ambient conditions, A. sativa coleoptiles resumed elongation, demonstrating an antagonistic relationship rather than toxicity. Inclusion of essential oils, monoterpenes, or isoprene into the headspace of culture vessels induced abnormal cellular growth along hypocotyls of Arabidopsis thaliana. These responses were also elicited by methyl jasmonate (MeJA); however, where methyl jasmonate inhibited root growth essential oils did not. Gene expression studies in A. thaliana also demonstrated differences between the MeJA and isoprenoid responses. This series of experiments clearly demonstrate that essential oils and their isoprenoid components interact with endogenous plant growth regulators when applied directly or as volatile components in the headspace. The similarities between isoprenoid and MeJA responses suggest that they may act in plant defence signalling. While further studies are needed to determine the ecological and evolutionary significance, the results of this study and the specialized anatomy associated with aromatic plants suggest that essential oils may act as airborne signalling molecules.