Regulatory role of Echinochrome A in cancer-associated fibroblast-mediated lung cancer cell migration.

Echinochrome A (Ech A), a marine biosubstance isolated from sea urchins, is a strong antioxidant, and its clinical form, histochrome, is being used to treat several diseases, such as ophthalmic, cardiovascular, and metabolic diseases. Cancer-associated fibroblasts (CAFs) are a component of the tumor stroma and induce phenotypes related to tumor malignancy, including epithelial-mesenchymal transition (EMT) and cancer stemness, through reciprocal interactions with cancer cells. Here, we investigated whether Ech A modulates the properties of CAFs and alleviates CAF-induced lung cancer cell migration. First, we observed that the expression levels of CAF markers, Vimentin and fibroblast-activating protein (FAP), were decreased in Ech A-treated CAF-like MRC5 cells. The mRNA transcriptome analysis revealed that in MRC5 cells, the expression of genes associated with cell migration was largely modulated after Ech A treatment. In particular, the expression and secretion of cytokine and chemokine, such as IL6 and CCL2, stimulating cancer cell metastasis was reduced through the inactivation of STAT3 and Akt in MRC5 cells treated with Ech A compared to untreated MRC5 cells. Moreover, while conditioned medium from MRC5 cells enhanced the migration of non-small cell lung cancer cells, conditioned medium from MRC5 cells treated with Ech A suppressed cancer cell migration. In conclusion, we suggest that Ech A might be a potent adjuvant that increases the efficacy of cancer treatments to mitigate lung cancer progression.

Physicochemical characterization and phase II metabolic profiling of echinochrome A, a bioactive constituent from sea urchin, and its physiologically based pharmacokinetic modeling in rats and humans.

Echinochrome A, a natural naphthoquinone pigment found in sea urchins, is increasingly being investigated for its nutritional and therapeutic value associated with antioxidant, anticancer, antiviral, antidiabetic, and cardioprotective activities. Although several studies have demonstrated the biological effects and therapeutic potential of echinochrome A, little is known regarding its biopharmaceutical behaviors. Here, we aimed to investigate the physicochemical properties and metabolic profiles of echinochrome A and establish a physiologically-based pharmacokinetic (PBPK) model as a useful tool to support its clinical applications. We found that the lipophilicity, color variability, ultraviolet/visible spectrometry, and stability of echinochrome A were markedly affected by pH conditions. Moreover, metabolic and pharmacokinetic profiling studies demonstrated that echinochrome A is eliminated primarily by hepatic metabolism and that four possible metabolites, i.e., two glucuronidated and two methylated conjugates, are formed in rat and human liver preparations. A whole-body PBPK model incorporating the newly identified hepatic phase II metabolic process was constructed and optimized with respect to chemical-specific parameters. Furthermore, model simulations suggested that echinochrome A could exhibit linear disposition profiles without systemic and local tissue accumulation in clinical settings. Our proposed PBPK model of echinochrome A could be a valuable tool for predicting drug interactions in previously unexplored scenarios and for optimizing dosage regimens and drug formulations.

Effect of Echinochrome A on Submandibular Gland Dysfunction in Ovariectomized Rats.

Post-menopausal dry mouth or xerostomia is caused by reduced salivary secretion. This study aimed to investigate the efficacy of echinochrome A (Ech A) in alleviating submandibular gland dysfunctions in ovariectomized rats that mimic menopause. Female rats that were eight-weeks-old were randomly divided into SHAM-6, -12; OVX-6, -12; and ECH-6, -12 groups (consisting of 6- and 12-weeks post-sham-operated, ovariectomized, and Ech A-treated ovariectomized rats, respectively). The ECH groups had lower body weight than OVX but similar food intake and estradiol or estrogen receptor ß expression. However, the ECH groups had lower mRNA expression of sterol-regulatory element binding protein-1c (Srebp-1c), acetyl-CoA carboxylase (Acc), fatty acid synthase (Fasn), cluster of differentiation 36 (Cd36), and lipid vacuole deposition than OVX mice. Moreover, reactive oxygen species (ROS), malondialdehyde (MDA), and iron accumulation were lower in the ECH than in the OVX groups. Fibrosis markers, transforming growth factor ß (Tgf-ßI and Tgf-ßII mRNA) increased in the OVX than SHAM groups but decreased in the ECH groups. Aquaporin (Aqp-1 and Aqp-5 mRNA) and mucin expressions were downregulated in the OVX groups but improved with Ech A. In addition, Ech A prevented post-menopausal salivary gland dysfunction by inhibiting lipogenesis and ferroptosis. These findings suggest Ech A as an effective remedy for treating menopausal dry mouth.

Echinochrome A Reverses Kidney Abnormality and Reduces Blood Pressure in a Rat Model of Preeclampsia.

We aimed to observe the effects of Echinochrome A (Ech A) on systemic changes using a rat model of preeclampsia. The results showed that an infusion of angiotensin II (Ang II) through an osmotic pump (1 µg/kg/min) on GD 8 increased systolic and diastolic blood pressures and reduced fetal weight and placental weight. The diameters of the glomeruli were expended and glomeruli capillaries were diminished. No change was observed in the heart and liver in the Ang II group, but epithelial structures were disrupted in the uterus. Ech A treatment on GD 14 (100 µg/µL) through the jugular vein reduced systolic and diastolic blood pressures and reversed glomerulus alterations, but the fetal or placental parameters were unaffected. Ech A only partly reversed the effect on the uterus. The mRNA expression of TNF-α was increased and IL-10 and VEGF were reduced in the uterus of the Ang II group, while Ech A restored these changes. A similar trend was observed in the kidney, liver, and heart of this group. Furthermore, Bcl-2 was reduced and Bcl-2/Bax ratios were significantly reduced in the kidney and heart of the Ang II group, while Ech A reversed these changes. We suggest that Ech A modulates inflammation and apoptosis in key systemic organs in Ang II-induced rat preeclampsia and preserves kidney and uterus structures and reduces blood pressure.

Echinochrome A Inhibits Melanogenesis in B16F10 Cells by Downregulating CREB Signaling.

Excessive increase in melanin pigment in the skin can be caused by a variety of environmental factors, including UV radiation, and can result in spots, freckles, and skin cancer. Therefore, it is important to develop functional whitening cosmetic reagents that regulate melanogenesis. In this study, we investigated the effects of echinochrome A (Ech A) on melanogenesis in the B16F10 murine melanoma cell line. We triggered B16F10 cells using α-MSH under Ech A treatment to observe melanin synthesis and analyze expression changes in melanogenesis-related enzymes (tyrosinase, tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2)) at the mRNA and protein levels. Furthermore, we measured expression changes in the microphthalmia-associated transcription factor (MITF), CREB, and pCREB proteins. Melanin synthesis in the cells stimulated by α-MSH was significantly reduced by Ech A. The expression of the tyrosinase, TYRP1, and TYRP2 mRNA and proteins was significantly decreased by Ech A, as was that of the MITF, CREB, and pCREB proteins. These results show that Ech A suppresses melanin synthesis by regulating melanogenesis-related enzymes through the CREB signaling pathway and suggest the potential of Ech A as a functional agent to prevent pigmentation and promote skin whitening.

Multifaceted Clinical Effects of Echinochrome.

The marine drug histochrome is a special natural antioxidant. The active substance of the drug is echinochrome A (Ech A, 7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone), the most abundant quinonoid pigment in sea urchins. The medicine is clinically used in cardiology and ophthalmology based on the unique properties of Ech A, which simultaneously block various links of free radical reactions. In the last decade, numerous studies have demonstrated the effectiveness of histochrome in various disease models without adverse effects. Here, we review the data on the various clinical effects and modes of action of Ech A in ophthalmic, cardiovascular, cerebrovascular, inflammatory, metabolic, and malignant diseases.

Antiviral Potential of Sea Urchin Aminated Spinochromes against Herpes Simplex Virus Type 1.

Herpes simplex virus type 1 (HSV-1) is one of the most prevalent pathogens worldwide requiring the search for new candidates for the creation of antiherpetic drugs. The ability of sea urchin spinochromes-echinochrome A (EchA) and its aminated analogues, echinamines A (EamA) and B (EamB)-to inhibit different stages of HSV-1 infection in Vero cells and to reduce the virus-induced production of reactive oxygen species (ROS) was studied. We found that spinochromes exhibited maximum antiviral activity when HSV-1 was pretreated with these compounds, which indicated the direct effect of spinochromes on HSV-1 particles. EamB and EamA both showed the highest virucidal activity by inhibiting the HSV-1 plaque formation, with a selectivity index (SI) of 80.6 and 50.3, respectively, and a reduction in HSV-1 attachment to cells (SI of 8.5 and 5.8, respectively). EamA and EamB considerably suppressed the early induction of ROS due to the virus infection. The ability of the tested compounds to directly bind to the surface glycoprotein, gD, of HSV-1 was established in silico. The dock score of EchA, EamA, and EamB was -4.75, -5.09, and -5.19 kcal/mol, respectively, which correlated with the SI of the virucidal action of these compounds and explained their ability to suppress the attachment and penetration of the virus into the cells.

PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line.

Polyhydroxylated naphthoquinones (PHNQs), known as spinochromes that can be extracted from sea urchins, are bioactive compounds reported to have medicinal properties and antioxidant activity. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay showed that pure echinochrome A exhibited a cytotoxic effect on Saos-2 cells in a dose-dependent manner within the test concentration range (15.625-65.5 µg/mL). The PHNQ extract from New Zealand sea urchin Evechinus chloroticus did not induce any cytotoxicity within the same concentration range after 21 days of incubation. Adding calcium chloride (CaCl2) with echinochrome A increased the number of viable cells, but when CaCl2 was added with the PHNQs, cell viability decreased. The effect of PHNQs extracted on mineralized nodule formation in Saos-2 cells was investigated using xylenol orange and von Kossa staining methods. Echinochrome A decreased the mineralized nodule formation significantly (p < 0.05), while nodule formation was not affected in the PHNQ treatment group. A significant (p < 0.05) increase in mineralization was observed in the presence of PHNQs (62.5 µg/mL) supplemented with 1.5 mM CaCl2. In conclusion, the results indicate that PHNQs have the potential to improve the formation of bone mineral phase in vitro, and future research in an animal model is warranted.

The Protective Effect of Echinochrome A on Extracellular Matrix of Vocal Folds in Ovariectomized Rats.

Here, we investigated the effects of sex hormones on extracellular matrix (ECM)-related gene expression in the vocal fold lamina propria of ovariectomized (after ovary removal) rats and verified whether echinochrome A (ECH) exerts any therapeutic effects on ECM reconstitution after estrogen deficiency in ovariectomized rats. Sprague-Dawley female rats (9 weeks old) were acclimatized for a week and randomly divided into three groups (n = 15 each group) as follows: group I (sham-operated rats, SHAM), group II (ovariectomized rats, OVX), group III (ovariectomized rats treated with ECH, OVX + ECH). Rats from the OVX + ECH group were intraperitoneally injected with ECH at 10 mg/kg thrice a week after surgery for 6 weeks. And rats were sacrificed 6 weeks after ovariectomy. Estradiol levels decreased in OVX group compared with the SHAM group. ECH treatment had no effect on the levels of estradiol and expression of estrogen receptor ß (ERß). The evaluation of ECM components showed no significant changes in elastin and hyaluronic acid levels between the different groups. Collagen I and III levels were lower in OVX group than in SHAM group but increased in OVX + ECH group. The mRNA levels of matrix metalloproteinase (MMP)-1, -2, -8, and -9 were significantly higher in the OVX group than in the SHAM group, but decreased in the OVX + ECH group. Thus, changes were observed in ECM-related genes in the OVX group upon estradiol deficiency that were ameliorated by ECH administration. Thus, the vocal fold is an estradiol-sensitive target organ and ECH may have protective effects on the ECM of vocal folds in ovariectomized rats.

Echinochrome A Promotes Ex Vivo Expansion of Peripheral Blood-Derived CD34+ Cells, Potentially through Downregulation of ROS Production and Activation of the Src-Lyn-p110δ Pathway.

Intracellular reactive oxygen species (ROS) play an important role in the proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). HSPCs are difficult to be expanded ex vivo while maintaining their stemness when they are exposed to oxidative damage after being released from the bone marrow. There have been efforts to overcome this limitation by using various cytokine cocktails and antioxidants. In this study, we investigated the effects of echinochrome A (Ech A)-a well-established and non-toxic antioxidant-on the ex vivo expansion of HSPCs by analyzing a CD34+ cell population and their biological functions. We observed that Ech A-induced suppression of ROS generation and p38-MAPK/JNK phosphorylation causes increased expansion of CD34+ cells. Moreover, p38-MAPK/JNK inhibitors SB203580 and SP600125 promoted ex vivo expansion of CD34+ cells. We also demonstrated that the activation of Lyn kinase and p110δ is a novel mechanism for Ech A to enhance ex vivo expansion of CD34+ cells. Ech A upregulated phospho-Src, phospho-Lyn, and p110δ expression. Furthermore, the Ech A-induced ex vivo expansion of CD34+ cells was inhibited by pretreatment with the Src family inhibitor PP1 and p110δ inhibitor CAL-101; PP1 blocked p110δ upregulation and PI3K/Akt activation, whereas CAL-101 and PI3K/Akt pathway inhibitor LY294002 did not block Src/Lyn activation. These results suggest that Ech A initially induces Src/Lyn activation, upregulates p110δ expression, and finally activates the PI3K/Akt pathway. CD34+ cells expanded in the presence of Ech A produced equal or more hematopoietic colony-forming cells than unexpanded CD34+ cells. In conclusion, Ech A promotes the ex vivo expansion of CD34+ cells through Src/Lyn-mediated p110δ expression, suppression of ROS generation, and p38-MAPK/JNK activation. Hence, Ech A is a potential candidate modality for the ex vivo, and possibly in vivo, expansion of CD34+ cells.

Therapeutic Cell Protective Role of Histochrome under Oxidative Stress in Human Cardiac Progenitor Cells.

Cardiac progenitor cells (CPCs) are resident stem cells present in a small portion of ischemic hearts and function in repairing the damaged heart tissue. Intense oxidative stress impairs cell metabolism thereby decreasing cell viability. Protecting CPCs from undergoing cellular apoptosis during oxidative stress is crucial in optimizing CPC-based therapy. Histochrome (sodium salt of echinochrome A-a common sea urchin pigment) is an antioxidant drug that has been clinically used as a pharmacologic agent for ischemia/reperfusion injury in Russia. However, the mechanistic effect of histochrome on CPCs has never been reported. We investigated the protective effect of histochrome pretreatment on human CPCs (hCPCs) against hydrogen peroxide (H2O2)-induced oxidative stress. Annexin V/7-aminoactinomycin D (7-AAD) assay revealed that histochrome-treated CPCs showed significant protective effects against H2O2-induced cell death. The anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and Bcl-xL were significantly upregulated, whereas the pro-apoptotic proteins BCL2-associated X (Bax), H2O2-induced cleaved caspase-3, and the DNA damage marker, phosphorylated histone (γH2A.X) foci, were significantly downregulated upon histochrome treatment of hCPCs in vitro. Further, prolonged incubation with histochrome alleviated the replicative cellular senescence of hCPCs. In conclusion, we report the protective effect of histochrome against oxidative stress and present the use of a potent and bio-safe cell priming agent as a potential therapeutic strategy in patient-derived hCPCs to treat heart disease.

Spinochrome D Attenuates Doxorubicin-Induced Cardiomyocyte Death via Improving Glutathione Metabolism and Attenuating Oxidative Stress.

Doxorubicin, an anthracycline from Streptomyces peucetius, exhibits antitumor activity against various cancers. However, doxorubicin is cardiotoxic at cumulative doses, causing increases in intracellular reactive oxygen species in the heart. Spinochrome D (SpD) has a structure of 2,3,5,6,8-pentahydroxy-1,4-naphthoquinone and is a structural analogue of well-known sea urchin pigment echinochrome A. We previously reported that echinochrome A is cardioprotective against doxorubicin toxicity. In the present study, we assessed the cardioprotective effects of SpD against doxorubicin and determined the underlying mechanism. ¹H-NMR-based metabolomics and mass spectrometry-based proteomics were utilized to characterize the metabolites and proteins induced by SpD in a human cardiomyocyte cell line (AC16) and human breast cancer cell line (MCF-7). Multivariate analyses identified 12 discriminating metabolites (variable importance in projection > 1.0) and 1814 proteins from SpD-treated AC16 cells. Proteomics and metabolomics analyses showed that glutathione metabolism was significantly influenced by SpD treatment in AC16 cells. SpD treatment increased ATP production and the oxygen consumption rate in D-galactose-treated AC16 cells. SpD protected AC16 cells from doxorubicin cytotoxicity, but it did not affect the anticancer properties. With SpD treatment, the mitochondrial membrane potential and mitochondrial calcium localization were significantly different between cardiomyocytes and cancer cell lines. Our findings suggest that SpD could be cardioprotective against the cytotoxicity of doxorubicin.