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Echinochrome A (EchA), a marine bioactive pigment isolated from various sea urchin species, is the active agent of the clinically approved drug Histochrome®. EchA is currently only available in the form of an isotonic solution of its di- and tri-sodium salts due to its poor water solubility and sensitivity to oxidation. Electrospun polymeric nanofibers have lately emerged as promising drug carriers capable of improving the dissolution and bioavailability of drugs with limited water solubility. In the current study, EchA isolated from sea urchins of the genus Diadema collected at the island of Kastellorizo was incorporated in electrospun micro-/nanofibrous matrices composed of polycaprolactone and polyvinylpyrrolidone in various combinations. The physicochemical properties of the micro-/nanofibers were characterized using SEM, FT-IR, TGA and DSC analyses. The fabricated matrices exhibited variable dissolution/release profiles of EchA, as evidenced in in vitro experiments using gastrointestinal-like fluids (pH 1.2, 4.5 and 6.8). Ex vivo permeability studies using the EchA-loaded micro-/nanofibrous matrices showed an increased permeation of EchA across the duodenum barrier. The results of our study clearly show that electrospun polymeric micro-/nanofibers represent promising carriers for the development of new pharmaceutical formulations with controlled release, as well as increased stability and solubility of EchA, suitable for oral administration, while offering the potential for targeted delivery.

Echinochrome A (EchA) is a natural bioproduct extracted from sea urchins, and is an active component of the clinical drug, Histochrome®. EchA has antioxidant, anti-inflammatory, and antimicrobial effects. However, its effects on diabetic nephropathy (DN) remain poorly understood. In the present study, seven-week-old diabetic and obese db/db mice were injected with Histochrome (0.3 mL/kg/day; EchA equivalent of 3 mg/kg/day) intraperitoneally for 12 weeks, while db/db control mice and wild-type (WT) mice received an equal amount of sterile 0.9% saline. EchA improved glucose tolerance and reduced blood urea nitrogen (BUN) and serum creatinine levels but did not affect body weight. In addition, EchA decreased renal malondialdehyde (MDA) and lipid hydroperoxide levels, and increased ATP production. Histologically, EchA treatment ameliorated renal fibrosis. Mechanistically, EchA suppressed oxidative stress and fibrosis by inhibiting protein kinase C-iota (PKCι)/p38 mitogen-activated protein kinase (MAPK), downregulating p53 and c-Jun phosphorylation, attenuating NADPH oxidase 4 (NOX4), and transforming growth factor-beta 1 (TGFβ1) signaling. Moreover, EchA enhanced AMPK phosphorylation and nuclear factor erythroid-2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) signaling, improving mitochondrial function and antioxidant activity. Collectively, these findings demonstrate that EchA prevents DN by inhibiting PKCι/p38 MAPK and upregulating the AMPKα/NRF2/HO-1 signaling pathways in db/db mice, and may provide a therapeutic option for DN.

Echinochrome A (Ech A), a naphthoquinoid pigment from sea urchins, is known to have anti-inflammatory and analgesic effects that have been suggested to be mediated by antioxidant activity and intracellular signaling modulation. In addition to these mechanisms, the ion channels in keratinocytes, immune cells, and nociceptive neurons may be the target for the pharmacological effects. Here, using the patch clamp technique, we investigated the effects of Ech A on the Ca2+-permeable TRPV3, TRPV1 and Orai1 channels and the two-pore domain K+ (K2P) channels (TREK/TRAAK, TASK-1, and TRESK) overexpressed in HEK 293 cells. Ech A inhibited both the TRPV3 and Orai1 currents, with IC50 levels of 2.1 and 2.4 μM, respectively. The capsaicin-activated TRPV1 current was slightly augmented by Ech A. Ech A alone did not change the amplitude of the TREK-2 current (ITREK2), but pretreatments with Ech A markedly facilitated ITREK2 activation by 2-APB, arachidonic acid (AA), and acidic extracellular pH (pHe). Similar facilitation effects of Ech A on TREK-1 and TRAAK were observed when they were stimulated with 2-APB and AA, respectively. On the contrary, Ech A did not affect the TRESK and TASK-1 currents. Interestingly, the ITREK2 maximally activated by the combined application of 2-APB and Ech A was not inhibited by norfluoxetine but was still completely inhibited by ruthenium red. The selective loss of sensitivity to norfluoxetine suggested an altered molecular conformation of TREK-2 by Ech A. We conclude that the Ech A-induced inhibition of the Ca2+-permeable cation channels and the facilitation of the TREK/TRAAK K2P channels may underlie the analgesic and anti-inflammatory effects of Ech A.

Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS− level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS− in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.

The diverse therapeutic feasibility of the sea urchin-derived naphthoquinone pigment, Echinochrome A (Ech A), has been studied. Simple and noninvasive administration routes should be explored, to obtain the feasibility. Although the therapeutic potential has been proven through several preclinical studies, the biosafety of orally administered Ech A and its direct influence on intestinal cells have not been evaluated. To estimate the bioavailability of Ech A as an oral administration drug, small intestinal and colonic epithelial organoids were developed from mice and humans. The morphology and cellular composition of intestinal organoids were evaluated after Ech A treatment. Ech A treatment significantly increased the expression of LGR5 (~2.38-fold change, p = 0.009) and MUC2 (~1.85-fold change, p = 0.08). Notably, in the presence of oxidative stress, Ech A attenuated oxidative stress up to 1.8-fold (p = 0.04), with a restored gene expression of LGR5 (~4.11-fold change, p = 0.0004), as well as an increased expression of Ly6a (~3.51-fold change, p = 0.005) and CLU (~2.5-fold change, p = 0.01), markers of revival stem cells. In conclusion, Ech A is harmless to intestinal tissues; rather, it promotes the maintenance and regeneration of the intestinal epithelium, suggesting possible beneficial effects on the intestine when used as an oral medication.

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.

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 β 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 β (Tgf-βI and Tgf-βII 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.

Endothelial–mesenchymal transition (EndMT) is a process by which endothelial cells (ECs) transition into mesenchymal cells (e.g., myofibroblasts and smooth muscle cells) and induce fibrosis of cells/tissues, due to ischemic conditions in the heart. Previously, we reported that echinochrome A (EchA) derived from sea urchin shells can modulate cardiovascular disease by promoting anti-inflammatory and antioxidant activity; however, the mechanism underlying these effects was unclear. We investigated the role of EchA in the EndMT process by treating human umbilical vein ECs (HUVECs) with TGF-β2 and IL-1β, and confirmed the regulation of cell migration, inflammatory, oxidative responses and mitochondrial dysfunction. Moreover, we developed an EndMT-induced myocardial infarction (MI) model to investigate the effect of EchA in vivo. After EchA was administered once a day for a total of 3 days, the histological and functional improvement of the myocardium was investigated to confirm the control of the EndMT. We concluded that EchA negatively regulates early or inflammation-related EndMT and reduces the myofibroblast proportion and fibrosis area, meaning that it may be a potential therapy for cardiac regeneration or cardioprotection from scar formation and cardiac fibrosis due to tissue granulation. Our findings encourage the study of marine bioactive compounds for the discovery of new therapeutics for recovering ischemic cardiac injuries.

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.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which skin barrier dysfunction leads to dryness, pruritus, and erythematous lesions. AD is triggered by immune imbalance and oxidative stress. Echinochrome A (Ech A), a natural pigment isolated from sea urchins, exerts antioxidant and beneficial effects in various inflammatory disease models. In the present study, we tested whether Ech A treatment alleviated AD-like skin lesions. We examined the anti-inflammatory effect of Ech A on 2,4-dinitrochlorobenzene (DNCB)-induced AD-like lesions in an NC/Nga mouse model. AD-like skin symptoms were induced by treatment with 1% DNCB for 1 week and 0.4% DNCB for 5 weeks in NC/Nga mice. The results showed that Ech A alleviated AD clinical symptoms, such as edema, erythema, and dryness. Treatment with Ech A induced the recovery of epidermis skin lesions as observed histologically. Tewameter® and Corneometer® measurements indicated that Ech A treatment reduced transepidermal water loss and improved stratum corneum hydration, respectively. Ech A treatment also inhibited inflammatory-response-induced mast cell infiltration in AD-like skin lesions and suppressed the expression of proinflammatory cytokines, such as interferon-γ, interleukin-4, and interleukin-13. Collectively, these results suggest that Ech A may be beneficial for treating AD owing to its anti-inflammatory effects.

Echinochrome A (Ech A, 7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone) has been known to exhibit anti-oxidative and anti-inflammatory effects. However, no study has been carried out on the efficacy of Ech A against skin photoaging; this process is largely mediated by oxidative stress. Six-week-old male SKH-1 hairless mice (n = 36) were divided into five groups. Except for a group that were not treated (n = 4), all mice underwent ultraviolet-B (UVB) exposure for 8 weeks while applying phosphate-buffered saline or Ech A through intraperitoneal injection. UVB impaired skin barrier function, showing increased transepidermal water loss and decreased stratum corneum hydration. UVB induced dermal collagen degeneration and mast cell infiltration. Ech A injection was found to significantly lower transepidermal water loss while attenuating tissue inflammatory changes and collagen degeneration compared to the control. Furthermore, Ech A was found to decrease the relative expression of matrix metalloproteinase, tryptase, and chymase. Taken together, these results suggest that Ech A protects against UVB-induced photoaging in both functional and histologic aspects, causing a lowering of collagen degradation and inflammatory cell infiltration.

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.

The high-performance liquid chromatography method coupled with diode array and mass spectrometric detector (HPLC-DAD-MS) method for quinonoid pigment identification and quantification in sea urchin samples was developed and validated. The composition and quantitative ratio of the quinonoid pigments of the shells of 16 species of sea urchins, collected in the temperate (Sea of Japan) and tropical (South-China Sea) climatic zones of the Pacific Ocean over several years, were studied. The compositions of the quinonoid pigments of sea urchins Maretia planulata, Scaphechinus griseus, Laganum decagonale and Phyllacanthus imperialis were studied for the first time. A study of the composition of the quinonoid pigments of the coelomic fluid of ten species of sea urchins was conducted. The composition of quinonoid pigments of Echinarachnius parma jelly-like egg membrane, of Scaphechinus mirabilis developing embryos and pluteus, was reported for the first time. In the case of Scaphechinus mirabilis, we have shown that the compositions of pigment granules of the shell epidermis, coelomic fluid, egg membrane, developing embryos and pluteus are different, which should enable a fuller understanding of the functions of pigments at different stages of life.

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.

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.

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 β (ERβ). 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.

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.

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.

The aim of this study was to examine the in vitro antioxidant and antiviral activities of echinochrome A and echinochrome-based antioxidant composition against tick-borne encephalitis virus (TBEV) and herpes simplex virus type 1 (HSV-1). The antioxidant composition, which is a mixture of echinochrome A, ascorbic acid, and α-tocopherol (5:5:1), showed higher antioxidant and antiviral effects than echinochrome A. We suppose that echinochrome A and its composition can both directly affect virus particles and indirectly enhance antioxidant defense mechanisms in the hosting cell. The obtained results allow considering the echinochrome A and the composition of antioxidants on its basis as the promising agents with the both antioxidant and antiviral activities.

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.

Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca2+. To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 μM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 μM of Ech A with an IC50 for PKCι activity of 107 μM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low KD of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.