Anti-cancer Research on Arnebiae Radix-derived Naphthoquinone in Recent Five Years

Background: In recent years, many naphthoquinone compounds with anticancer activity have been identified in Arnebiae Radix, and some of them have the potential to be developed into anticancer drugs. Objective: This article aimed to provide a comprehensive overview of the anticancer effects of naphthoquinone compounds through a detailed review of literature and Chinese patents, and discuss their potential to be developed as anticancer drugs for clinical application. Methods: Research papers were collected through the databases of PubMed, Cnki and SciDirect using keyword searches “naphthoquinone compounds” and “anticancer”. The keywords of “shikonin” and “shikonin derivatives” were also used in PubMed, Cnki and SciDirect databases to collect research articles. The Chinese patents were collected using the Cnki patent database. Results: Naphthoquinone compounds have been found to possess anti-cancer activity, and their modes of action are associated with inducing apoptosis, inhibiting cancer cell proliferation, promoting autophagy in cancer cells, anti-cancer angiogenesis and inhibition of cell adhesion, invasion and metastasis, inhibiting glycolysis and inhibiting DNA topoisomerase activity. Conclusion: Most of the naphthoquinone compounds show effective anti-cancer activity in vitro. The structure modification of naphthoquinone aims to develop anti-cancer drugs with high efficacy and low toxicity.

Shikonin Derivatives from Onsoma visianii Decrease Expression of Phosphorylated STAT3 in Leukemia Cells and Exert Antitumor Activity

Antitumor effects of shikonins on chronic lymphocytic leukemia (CLL) and B-cell prolymphocytic leukemia (B-PLL) are mostly unexplored. The antitumor activity of shikonins, isolated from Onosma visianii Clem (Boraginaceae), in BCL1, mouse CLL cells and JVM-13, human B-PLL cells was explored in this study. The cytotoxicity of shikonin derivatives was measured by an MTT test. Cell death, proliferation, cell cycle, and expression of molecules that control these processes were analyzed by flow cytometry. Expression of STAT3-regulated genes was analyzed by real-time q-RT-PCR (Quantitative Real-Time Polymerase Chain Reaction). The antitumor effects of shikonin derivatives in vivo were analyzed, using flow cytometry, by detection of leukemia cells in the peripheral blood and spleens of mice intravenously injected with BCL1 cells. The two most potent derivatives, isobutyrylshikonin (IBS) and α-methylbutyrylshikonin (MBS), induced cell cycle disturbances and apoptosis, inhibited proliferation, and decreased expression of phospho-STAT3 and downstream-regulated molecules in BCL1 and JVM-13 cells. IBS and MBS decreased the percentage of leukemia cells in vivo. The link between the decrease in phosphorylated STAT3 by MBS and IBS and BCL1 cell death was confirmed by detection of enhanced cell death after addition of AG490, an inhibitor of Jak2 kinase. It seems that IBS and MBS, by decreasing STAT3 phosphorylation, trigger apoptosis, inhibit cell proliferation, and attenuate leukemia cell stemness.

Synthesis and Pharmacological In Vitro Investigations of Novel Shikonin Derivatives with a Special Focus on Cyclopropane Bearing Derivatives

Melanoma is the deadliest form of skin cancer and accounts for about three quarters of all skin cancer deaths. Especially at an advanced stage, its treatment is challenging, and survival rates are very low. In previous studies, we showed that the constituents of the roots of Onosma paniculata as well as a synthetic derivative of the most active constituent showed promising results in metastatic melanoma cell lines. In the current study, we address the question whether we can generate further derivatives with optimized activity by synthesis. Therefore, we prepared 31, mainly novel shikonin derivatives and screened them in different melanoma cell lines (WM9, WM164, and MUG-Mel2 cells) using the XTT viability assay. We identified (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopropyl-2-oxoacetate as a novel derivative with even higher activity. Furthermore, pharmacological investigations including the ApoToxGloTM Triplex assay, LDH assay, and cell cycle measurements revealed that this compound induced apoptosis and reduced cells in the G1 phase accompanied by an increase of cells in the G2/M phase. Moreover, it showed hardly any effects on the cell membrane integrity. However, it also exhibited cytotoxicity against non-tumorigenic cells. Nevertheless, in summary, we could show that shikonin derivatives might be promising drug leads in the treatment of melanoma.

Alcohol Dehydrogenase Activity Converts 3″-Hydroxy-geranylhydroquinone to an Aldehyde Intermediate for Shikonin and Benzoquinone Derivatives in Lithospermum erythrorhizon

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2′–C3′ double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.

Acetylshikonin Sensitizes Hepatocellular Carcinoma Cells to Apoptosis through ROS-Mediated Caspase Activation

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown strong and explicit cancer cell-selectivity, which results in little toxicity toward normal tissues, and has been recognized as a potential, relatively safe anticancer agent. However, several cancers are resistant to the apoptosis induced by TRAIL. A recent study found that shikonin b (alkannin, 5,8-dihydroxy-2-[(1S)-1-hydroxy-4-methylpent-3-en-1-yl]naphthalene-1,4-dione) might induce apoptosis in TRAIL-resistant cholangiocarcinoma cells through reactive oxygen species (ROS)-mediated caspases activation. However, the strong cytotoxic activity has limited its potential as an anticancer drug. Thus, the current study intends to discover novel shikonin derivatives which can sensitize the liver cancer cell to TRAIL-induced apoptosis while exhibiting little toxicity toward the normal hepatic cell. The trypan blue exclusion assay, western blot assay, 4′,6-diamidino-2-phenylindole (DAPI) staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as well as the ‘comet’ assay, were used to study the underlying mechanisms of cell death and to search for any mechanisms of an enhancement of TRAIL-mediated apoptosis in the presence of ASH. Herein, we demonstrated that non-cytotoxic doses of acetylshikonin (ASH), one of the shikonin derivatives, in combination with TRAIL, could promote apoptosis in HepG2 cells. Further studies showed that application of ASH in a non-cytotoxic dose (2.5 μM) could increase intracellular ROS production and induce DNA damage, which might trigger a cell intrinsic apoptosis pathway in the TRAIL-resistant HepG2 cell. Combination treatment with a non-cytotoxic dose of ASH and TRAIL activated caspase and increased the cleavage of PARP-1 in the HepG2 cell. However, when intracellular ROS production was suppressed by N-acetyl-l-cysteine (NAC), the synergistic effects of ASH and TRAIL on hepatocellular carcinoma (HCC) cell apoptosis was abolished. Furthermore, NAC could alleviate p53 and the p53 upregulated modulator of apoptosis (PUMA) expression induced by TRAIL and ASH. Small (or short) interfering RNA (siRNA) targeting PUMA or p53 significantly reversed ASH-mediated sensitization to TRAIL-induced apoptosis. In addition, Bax gene deficiency also abolished ASH-induced TRAIL sensitization. An orthotopical HCC implantation mice model further confirmed that co-treated ASH overcomes TRAIL resistance in HCC cells without exhibiting potent toxicity in vivo. In conclusion, the above data suggested that ROS could induce DNA damage and activating p53/PUMA/Bax signaling, and thus, this resulted in the permeabilization of mitochondrial outer membrane and activating caspases as well as sensitizing the HCC cell to apoptosis induced by TRAIL and ASH treatment.

Synthesis of Novel Shikonin Derivatives and Pharmacological Effects of Cyclopropylacetylshikonin on Melanoma Cells

Despite much research in the last centuries, treatment of malignant melanoma is still challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate. Therefore, the discovery of novel drug leads is an important goal. In a previous study, we have isolated several shikonin derivatives from the roots of Onosma paniculata Bureau & Franchet (Boraginaceae) which evolved as promising anticancer candidates. β,β-Dimethylacrylshikonin (1) was the most cytotoxic derivative and exhibited strong tumor growth inhibitory activity, in particular, towards melanoma cells. In this study, we synthesized eighteen novel shikonin derivatives in order to obtain compounds which exhibit a higher cytotoxicity than 1. We investigated their cytotoxic potential against various melanoma cell lines and juvenile skin fibroblasts. The most active compound was (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropylacetate (cyclopropylacetylshikonin) (6). It revealed significant stronger tumor growth inhibitory activity towards two melanoma cell lines derived from metastatic lesions (WM164 and MUG-Mel2). Further investigations have shown that 6 induced apoptosis caspase-dependently, increased the protein levels of cleaved PARP, and led to double-stranded DNA breaks as shown by phosphorylation of H2AX. Cell membrane damage and cell cycle arrest were not observed.