**1. Introduction**

Echinochrome A has a chemical structure of 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone, which exhibits cardioprotective activity and reduces the myocardial ischemia/reperfusion injury via its antioxidant effect and enhancement of mitochondria biogenesis [1–3]. Echinochrome A has a number of structural analogues and together they comprise the class of spinochrome pigments of sea urchins. Biological effects of spinochromes were investigated mainly on crude extracts [4] and there is not so much information on the activity of individual pigments, particularly regarding cardioprotective ability. Spinochrome D (SpD) is one of six main spinochromes and it is biosynthesized by many sea urchin species (Figure 1A) [5]. SpD is a side product of echinochrome A isolation from the flat sea urchin *Scaphechinus mirabilis*, which is utilized for the preparation of the active substance

of the antioxidant and the cardioprotective drug *Histochrome*® [6]. Nevertheless, the content of SpD in sea urchins is usually pretty low (0.001–0.003% of dry weight), but recently by Balaneva et al. was developed a simple and effective synthesis scheme with the yield of SpD in 58% [7]. SpD might be assumed to inherit the cardioprotective ability of echinochrome A, but the detailed mechanism has been unknown.

**Figure 1.** Spinochrome D (SpD) protected cardiac cells from the cytotoxicity of doxorubicin. (**A**) Chemical structure of SpD (MW, 238.15044); (**B**) AC16 human cardiomyocytes were treated with 0–200 μM SpD for 24 h and cell viability was measured using a Cell Counting Kit-8 reagent (CCK-8) assay. SpD did not affect the cell viability of cardiomyocytes. \* *p* < 0.05 compared with control; (**C**) Treatment with SpD (10 μM, 24 h) attenuated the cardiotoxicity of doxorubicin (0.1 μM) in AC16 cells.

Doxorubicin is an anthracycline that was firstly extracted from *Streptomyces peucetius* and it has been routinely used for the treatment of several cancers, including breast, lung, gastric, ovarian, non-Hodgkin's, and Hodgkin's lymphoma [8,9]. There are several proposed anti-cancer mechanisms of doxorubicin, including intercalation into DNA and generation of reactive oxygen species (ROS). The inhibition of topoisomerase II has been known to induce apoptosis in cells [10–12].

Unfortunately, cumulative dose of doxorubicin over 550 mg/m2 body surface area has been known to develop cardiomyopathy [13,14]. The exact mechanism of cardiomyopathy is still controversial but iron-related free radical formation and mitochondrial disruption have been considered to be the main causes [15]. There have been trials to overcome the doxorubicin's cardiac toxicity by reducing its oxygen radical formation to achieve important accomplishment [16–18].

In the present study, we demonstrated that SpD has a cardio-protective effect against the cardiac toxicity of doxorubicin without interfering the cytotoxicity to cancer cell lines. We used an integration of 1H-NMR based metabolomics and mass-spectrometry based proteomics to specify molecular pathways that are affected by SpD treatment in human cardiomyocytes. We also measured changes of the mitochondrial membrane potential and mitochondrial calcium in SpD treated human cardiomyocytes.

#### **2. Results**

#### *2.1. SpD Protected AC16 Cells against Doxorubicin Cytotoxicity*

Human cardiomyocyte AC16 cells were treated with SpD at 1–200 μM (Figure 1B). We found no harmful effects on cell viability. Identical concentrations of SpD were tested on rat cardiomyocyte H9c2 cells and also they showed no effect on cell viability (Figure S1). The cell viability of AC16 cells decreased after 0.1 μM doxorubicin treatment for 24 h, and an exposure duration of 48 h resulted in cell death for the majority of cells (data not shown). However, adding 10 μM SpD treatment in addition to doxorubicin for 24 h led to 80–90% AC16 cell survival (Figure 1C).
