Synthesis, biological function and evaluation of Shikonin in cancer therapy

Fitoterapia 134 (2019) 329–339

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Review

Synthesis, biological function and evaluation of Shikonin in cancer therapy Fangfang Wang a b c

a,b

, Xinsheng Yao

c,⁎

a,b

a,b,⁎⁎

T

, Youwei Zhang , Jinshan Tang

Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drug Research, Jinan University, Guangzhou 510632, China Department of Pharmacology, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Shikonin Review Anti-cancer Chemical synthesis Biogenic synthesis

Shikonin is a natural compound isolated from herbs and traditional medicines that have been used in a number of countries to treat various illnesses including inflammation, virus infection and cancer for centuries. Recent studies have shed light on the molecular mechanisms underlying these biological activities of Shikonin. Here we review the latest advances in our understanding of this compound class in the anti-cancer regimen. We focus on signaling pathways and cellular targets involved in the anticancer activity of Shikonin. We also briefly discuss approaches in evaluating the in vivo bioactivity and drug delivery of Shikonin in the anti-cancer treatment. Subsequently, we highlight recently developed strategies in the chemical and biogenic synthesis of Shikonin and summarize the structure-activity relationship studies of Shikonin. We anticipate that these lines of information would facilitate the functional identification and future clinical development of Shikonin and its derivatives in the combat against cancer.

1. Introduction Shikonin, a natural red naphthoquinone compound, is a major component of Zicao (purple gromwell), the dried root of Lithospermum erythrorhizon Sieb. et Zucc, Arnebia euchroma (Royle) Johnst, or Arnebia guttata Bunge. Zicao is a commonly used herbal medicine in China and other countries. It had been demonstrated that Zicao extracts possess multiple pharmacological activities including anti-inflammation, antioxidative stress, anti-virus, anti-bacteria and anti-cancer [1–6]. In addition, scattering reports showed beneficial effects of Shikonin on many other pathologies or disorders, such as inhibiting hypertrophic scar formation [7–9], reducing hepatic fibrosis [10], inhibiting pulmonary hypertension [11,12], protecting hypoxia and reoxygenation damage in heart [13] and brain [1,14], protecting β-amyloid-induced neurotoxicity [15], improving spinal cord injury [16], etc.

The biological function of Shikonin had been previously summarized in several reviews [1–3,17]. However, the broad spectrum of activities somehow diluted the focus of these reviews, and there was the lack of understanding of the detailed molecular mechanisms underlying these functions of Shikonin. The purpose of this review is to summarize recent advances in our understanding of the mechanisms by which Shikonin elicit its anti-cancer effect, one of the most widely investigated activities of Shikonin. Specifically, we survey the signaling pathways, the cellular targets, and in vivo bioactivity of Shikonin and its analogs. Further, we review recently developed strategies in the chemical and biogenic synthesis of Shikonin and its derivatives with the purpose of increasing the biological efficacy of this compound class. These lines of information will assist us in the design and the development of Shikonin derivatives that hold clinical potential in the future.

Abbreviations: 5' adenosine monophosphate-activated protein kinase, AMPK; acetoacetyl-CoA thiolase, ACTH; ceric ammonium nitrate, CAN; c-Jun N-terminal kinase, JNK; 4-coumaroyl-CoA ligase, 4CL; Diisobutylaluminum hydride, DIBALH; DNA methyltransferase 1, DNMT1; epithelial-to-mesenchymal transition, EMT; endoplasmic reticulum, ER; extracellular signal-regulated kinase ½, ERK1/2; G-protein coupled receptors, GPCRs; hypoxia-inducible factor 1 alpha, HIF1α; c-Jun Nterminal kinase, JNK); mitogen-activated protein kinase, MAPK; Chloromethyl methyl ether, MOMCl; natural killer, NK; phosphatidylinositol-4,5-triphosphate, PIP2; phosphatidylinositol-3,4,5-triphosphate, PIP3; phosphatidylinositol-4,5-bisphosphate 3-kinase, PI3K; pyruvate kinase isoenzyme M2, PKM2; receptor interacting protein 1/3, RIP1/3; signal transducer and activator of transcription 3, STAT3; cycline dependent kinase inhibitor 2A, p16; phosphatase and tensin homolog, PTEN; Poly ethylene glycol, PEG; Pyridinium dichromate, PDC; poly lactide-co-glycolides, PLGA; Pyridine sulfur trioxide, Py-SO3; reactive oxygen species, ROS; spleen tyrosine kinase, Syk; Tetra-n-butylammonium fluoride, TBAF; Tert-butyl-diphenylsilyl chloride, TBDPSCl; Tert-butyldimethylsilyl chloride, TBSCl; Tetrahydrofuran, THF; Titanium tetraisopropoxide, Ti[OCH(CH3)2]4; Trimethyl sulfoxonium iodide, TMSOI ⁎ Corresponding author at: 2109 Adelbert Road, Cleveland, OH 44106, USA. ⁎⁎ Corresponding author at: W601 Huangpu Ave., Guangzhou 510632, China. E-mail addresses: [email protected] (Y. Zhang), [email protected] (J. Tang). https://doi.org/10.1016/j.fitote.2019.03.005 Received 29 January 2019; Received in revised form 4 March 2019; Accepted 7 March 2019 Available online 08 March 2019 0367-326X/ © 2019 Published by Elsevier B.V.

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Table 2 In vivo bioactivity for the anti-cancer effect of Shikonin.

Fig. 1. Chemical structures of Shikonin (1) and its enantiomer, Alkannin (2) Table 1 Representative IC50 of Shikonin (1) and Alkanin (2) in different cell lines. Cancer cell lines

IC50 (μM)

References

Shikonin

Alkannin

Breast cancer MCF-7 MDA-MB-231 MDA-MB-468

2.88 ± 0.2a 6.15 ± 0.25a 3.61 ± 0.34

2.47 ± 0.3

Lung cancer SPC-A1 H1299 A549

18.1 1.88 ± 0.15 1.8

Colon cancer HT-29 HCT-15 HCT-116

16.7 3.03 ± 0.12a 1.84 ± 0.18

Hepatic carcinoma BEL-7402 Hep G2

54.5 0.42 ± 0.08

Leukemia K562 HL60

0.7 ± 0.1 1.03

Skin cancer A875 B16-F10

1.06 ± 0.06a 17.9 ± 0.90

Prostate cancer DU145

16.0 ± 0.7

Cervical cancer HeLa

4.60 ± 0.81

[28,30,34]

Head and neck cancer CNE

0.7

[24]

Gastric cancer MGC-803

1.51 ± 0.01

[32]

Liposarcoma cancer SW872-s

0.76

[31,36]

Glioblastoma U87MG U87MG.ΔEGFR

4.11 ± 0.18 2.14 ± 0.07

> 100 > 100

Normal cell lines Human skin fibroblasts (HSF) Mouse fibroblast L-929 LO2 MCF-10A

1.2 ± 0.1 3.2 4.74 ± 1.31 23.5 ± 3.01

1.65 ± 0.1

a

[22,29] [24,27,32,33] [30]

A549 HeLa EC109 PANC1 SW480 HCT116 Namalwa C6 143B MDA-MB-231 OCUM-2MD3 CNE-2Z MB-49

1-10 mg/kg 0.5-2 mg/kg 30 mg/kg 2.5-5 mg/kg 3-6 mg/kg 2-10 mg/kg 4 mg/kg 2 mg/kg 2 mg/kg 1-4 mg/kg 0.8 mg/kg 0.5-1 mg/kg 2 mg/kg

[37–39] [40] [41] [42] [43] [44] [45] [46] [47] [21] [48] [49] [50]

2.2.1. Cellular localization-dependent biological activities A large number of studies have solidified the idea that Shikonin inhibits cancer through targeting multiple aspects of this devastating disease, including the inhibition of cell growth, migration and invasion, and the induction of cell death. Shikonin was also reported to trigger autophagy activation [39,51,52]. However, this activated autophagy did not seem to protect cells from Shikonin-induced death [39,52]; therefore, it may represent just a cellular response to the stress elicited by Shikonin treatment [50,53,54]. Despite being confirmed by numerous in vitro and in vivo results, the molecular mechanisms including the signaling pathways and the cellular targets in the anti-cancer activity of Shikonin appear to be complex and remained elusive. One interesting observation is that the activity of Shikonin may depend on its ability to locate within unique cellular organelles or interact with specific cellular proteins. When accumulated in mitochondria, Shikonin induced the breakdown of mitochondria membrane potential [55,56], leading to mitochondrial apoptotic cell death in various cancer cell lines [43,57–59]. When located in the endoplasmic reticulum (ER), Shikonin increases the ER stress followed by apoptotic cell death [57,60]. In addition to the induction of apoptosis, Shikonin could cause necrosis-dependent cancer cell death (so-called necroptosis) through increased expression of the necrosis factor - receptor interacting protein 1/3 (RIP1/3) [42,46,49,61–63] followed by the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) [39].

[20,22–24,27] [20] [31,34] [35]

18.2 ± 1.7

References

2.2. Anti-cancer mechanisms

[29] [34] 1.4 ± 0.2

Dose

Shikonin and Alkannin did not obviously affect their biological activities (Table 1). For instance, both compounds showed similar anti-inflammatory activities [3,19]. Similarly, the anticancer effects of Shikonin and Alkannin were very similar in the majority of cases. Therefore, attention has been mainly focused on the in vitro and in vivo anticancer activity of Shikonin. Importantly, this anti-cancer activity has been confirmed by a variety of in vivo bioactivity models (Table 2).

[20–27] [21,24,28] [21] [29] [30] [31]

1.11

Xenograft cell lines

[23,26]

[22–24,27] [24] [28,34] [21]

2.2.2. Signaling pathways and cellular targets of Shikonin 2.2.2.1. The PKM2-dependent tumor glycolysis. The cellular targets by which Shikonin inhibit cancer cell growth and/or induce cancer cell death seem to be complicated and are often in a cellular context dependent manner (Table 2). Although the growth inhibitory effect of Shikonin was observed in a wide range of human tumors, myelogenous leukemia cells appear to be particularly sensitive to Shikonin with an IC50 at sub-μM concentration [64]. Using a pull down assay, pyruvate kinase (PK) muscle isoenzyme M2 (PKM2) was identified to be a cellular protein target that bound to Shikonin [65]. PKM2 is a ratelimiting enzyme that controls the last step of glycolysis [66]. Tumors, especially those solid ones, are known to utilize glycolysis to generate energy for tumor metabolism and growth, which is called the Warburg effect [67]. PKM2 is often overexpressed/elevated in tumors,

Average number from different studies.

2. Anti-cancer activities 2.1. In vivo bioactivity Shikonin (1) and its enantiomer, Alkannin (2) (Fig. 1), are two important 1,4-naphthoquinones isolated from the roots of Lithospermum erythrohizon in Asia and Alkanna tinctoria in Europe, respectively. These compounds and their derivatives display fascinating biological activities, including growth inhibition of a wide range of human cancer cells (Table 1 for IC50) [1,18]. It appears that the absolute configuration of 330

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suggesting a critical role of this gene in tumor metabolism and growth [68,69]. Binding with Shikonin inhibits the enzymatic activity of PKM2 and therefore suppresses the aerobic glycolysis and growth of human breast, cervical and esophageal cancers [41,46,65], as well as mouse Lewis lung carcinoma and B16 melanomas [70]. In addition, the inhibition of PKM2 by Shikonin overcame the resistance of EGFR wild type non-small cell lung cancers to gefitinib [41], providing a novel approach in treating this subtype of lung cancer. In addition to the direct binding effect, Shikonin might also indirectly inhibit PKM2 through RIP1/3 [46,71]. Nonetheless, these studies suggest an important role of PKM2 and the affiliated glycolysis in the inhibitory effect of Shikonin on cancer growth.

by the phosphatase and tensin homolog (PTEN) terminates the growth signaling [105]. Hence, the PI3K/AKT/mTOR/PTEN pathway plays a critical role in cell growth, nutrient sensing and metabolism. It is probably the most frequently mutated signaling pathway in human cancers with mutations occurring on both tumor drivers and tumor suppressors in this pathway [105]. Consistently, this is also one of the major targets of Shikonin in showing its anticancer effect. For instance, Shikonin inhibited the activation of PI3K/AKT in a variety of cancers [41,45,87,106,107]. On the other hand, Shikonin upregulated PTEN to counteract the growth signal elicited by PI3K/AKT [107–109]. Under certain circumstances, Shikonin could both inhibit PI3K/AKT and induce PTEN, potentiating the growth inhibitory effect [107].

2.2.2.2. The RAS/RAF/MEK/ERK pathway. The MAPK signaling is an evolutionally conserved pathway that regulates virtually every aspects of cellular function such as growth, proliferation, differentiation, apoptosis, motility, etc [72]. Canonically, the MAPK family includes the extracellular signal-regulated kinase ½ (ERK1/2), the c-Jun Nterminal kinase (JNK1/2/3), p38 and ERK5 [73]. Cells respond to growth stimuli by activating the receptor tyrosine kinases on cell membrane. Subsequently, the RAS/RAF/MEK/ERK pathway will be activated to transduce the signal to the nucleus, which induces transcription of genes required for cell growth and differentiation [74]. Hence, it is not surprising that Shikonin was often reported to inhibit the RAS/RAF/MEK/ERK signaling, serving as an important mechanism to inhibit the growth of cancer cells, as well as normal cells during inflammation. For instance, Shikonin inhibited the growth of acute leukemia cells [75], human epidermal keratinocytes [76], mouse lung tissues [77] or mouse adipocytes [78] by inhibiting ERK. However, given that fact that ERK activation is also involved in the induction of apoptotic cell death [79], there are almost equal number of reports showing that Shikonin induced ERK activation [60,80–82]. Thus, care should be given when determining the role of the ERK signaling in the anticancer effect of Shikonin, which may depend on the cell line used, drug concentration and treatment duration, etc.

2.2.2.5. c-Myc. A critical downstream target of the above-mentioned signaling pathways (JNK, MAPK and PI3K/AKT) is the proto-oncogene c-Myc [110,111]. c-Myc encodes a transcription factor that is critical for cell proliferation, differentiation and metabolism [112,113]. Overexpression or amplification of c-Myc is frequently observed in human cancers and indicates poor patient outcome [113–115]. In cell growth control, c-Myc controls the cell cycle entry into the S phase by regulating the transcription of S phase cyclins and cyclin-dependent kinases [112,116]. Therefore, inhibition of c-Myc will induce cell cycle arrest in the G0/G1 phase. Shikonin and its derivatives had been reported to inhibit the expression of c-Myc at concentrations much lower than known Myc inhibitors such as 10074-G5 and 10058-F4 in U937 leukemia cells [75]. Such inhibition of c-Myc was also observed in Burkitt’s lymphoma [45] and acute promyelocytic leukemia cells [58], through which Shikonin suppressed cancer growth. Further, the combined inhibition of PI3K/AKT and c-Myc by Shikonin not only suppressed the cell growth, but also induced cell death of Burkitt’s lymphoma [45]. 2.2.2.6. ROS generation. As a naphthoquinone derivative, Shikonin and its analogs have been shown to undergo redox cycling in isolated mitochondria [117], resulting in the generation of ROS. Therefore, in addition to directly targeting cellular proteins, it is tempting to speculate that at least some of the functional activities, such as apoptosis induction, of Shikonin might be indirectly related to ROS production by Shikonin [63,82,86,91,118,119]. Cellular senescence in lung adenocarcinoma induced by Shikonin was also suggested to be through ROS production [37]. However, given the toxic effect of ROS to normal tissues and organs, further investigation is warranted to determine both the kinetics and the spectrum of ROS production by Shikonin. These lines of information will prove to be critical in identifying a suitable treatment (i.e., concentration and duration) of Shikonin that displays a therapeutic effect while minimizing its toxicity to normal tissues through ROS generation.

2.2.2.3. The JNK pathway. As a MAPK family member, JNK senses both extracellular and intracellular signals [83,84]. JNK has been thought to mainly regulate stress-induced apoptosis [83,84], although transient activation of JNK may promote cell growth [84,85]. Shikonin treatment triggered robust activation of apoptosis through the activation of JNK with simultaneous inhibition of the ERK signaling in BCR/ABL-positive chronic myelogenous leukemia cells [86]. This apoptosis-inducing effect of Shikonin was also reported in cancer cells originated from gallbladder, breast, stomach, liver and acute promyelocytic leukemia [58,75,87–90]. A critical mediator involved in the activation of JNK by Shikonin is reactive oxygen species (ROS), and the cell death induction effect of Shikonin may depend on the presence of wild type P53 at least in breast cancer cells [88]. However, P53 mutant thyroid cancer cells showed stronger sensitivity than P53 wild type cells to Shikonin [91]. Therefore, whether the status of the TP53 gene affects the therapy response of Shikonin remains unclear, and further studies are needed to thoroughly address this issue.

2.2.2.7. Additional targets. Shikonin was also reported to affect other cellular targets. Shikonin inhibits STAT3 [21,38,120], a highly activated transcription factor in tumors [121], and therefore inhibiting expression of tumor-promoting factors. Shikonin inhibits expression of hypoxia-inducible factor 1α (HIF1α) [58,75,86], a critical transcription factor that regulates gene expression under low oxygen levels [122], leading to the inhibition of protein translation, tumor growth and angiogenesis [28,41,44]. Shikonin activated SIRT2 to inhibit the growth and metastasis of colorectal SW480 cancer cells [123]. In addition, Shikonin upregulated p16INK4A, p73 [123] or P53 [37,75,86,88,125,126] to suppress cell growth and/or induce cell death. Interestingly, Shikonin could induce P21 expression in a P53independent manner [37], which then leads to cell growth inhibition or even senescence. Additionally, Shikonin inhibited DNMT1 [108], topoisomerases [127–129] and glycolysis enzymes [130], which at least partially contributed to the documented anti-cancer activity of Shikonin (Table 3).

2.2.2.4. The PI3K/AKT/mTOR/PTEN pathway. In addition to the RAS/ RAF/MEK/ERK pathway, receptor tyrosine kinases also activate the phosphatidylinositol 3-kinase (PI3K), which converts phosphatidylinositol-4,5-triphosphate (PIP2) to phosphatidylinositol3,4,5-triphosphate (PIP3) through which the AKT/PKB kinase is recruited to the cellular membrane [92–95]. AKT will be phosphorylated at multiple residues by PDK1 [96,97], mTORC2 or PKC [98–101]. These phosphorylation events induce fully activation of AKT, which then phosphorylates a number of downstream targets, such as FOXOs, GSK3, TSC2, IKK, etc., to regulate gene expression, protein synthesis, and signaling transduction for cell survival and growth [102–104]. On the other hand, the removal of the phosphate of PI3P 331

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Poly-peptide-derived nano-gel specifically delivered Shikonin to sarcomas, which significantly suppressed sarcoma growth in mice [47]. Liposome-coated Shikonin greatly suppressed the growth of MDA-MB231 cancer cells and increased the uptake of the drug into cancers in mice [145]. By encapsulating in Poly (ethylene glycol) (PEG) and PLGA poly (lactide-co-glycolides) derived nanoparticles with surface coated with lactoferrin to improve the blood brain barrier crossing, Shikonin was specifically delivered to the brain to treat glioblastoma in mice [146]. Shikonin entrapped in a degradable thermo-sensitive nanomicelle increased the drug uptake in MCF7 breast cancer xenografts in BALC/c nude mice and displayed strong anti-tumor effect [147]. These studies provide the foundation for engineering deliverable Shikonin specifically into tumors in future.

Table 3 Anti-cancer signaling and major targets of Shikonin. Signaling pathways

Molecular targets

References

Glycolysis Apoptosis Necrosis

PKM2 MMP RIP1/3

[41,46,65] [55,56] [42,46,49,61–63]

MAPK ↓ HIF1α ↓ DNMT1 ↓ c-Myc ↓ JNK ↓ PI3K/AKT ↓ STAT3 ↓ Topo ↓ p16INK4A ↑ PTEN ↑

[58,75,86] [28,41,44] [108] [58,75] [89,90] [41,45,87,106] [21,38] [127–129] [124] [107–109]

Growth

3. Chemical synthesis A bottleneck in studying the anti-cancer function of Shikonin is to acquire a large quantity of the compound or its derivatives. Therefore, chemical and/or biological synthesis and structure-activity relationship studies of Shikonin are crucial. The total synthesis of Shikonin (1) and Alkannin (2) was first developed by Terada in 1983 [148], and previous work had been reviewed by Papageorgiou et al in 1999 [3] and Wang et al in 2012 [149]. Here we will focus on recent advances in the acquisition of key intermediates of Shikonin, and those established methods in the chemical synthesis of Shikonin will only be briefly mentioned. Further, we will emphasize the structure-activity relationship of this compound. Although the chemical structure of Shikonin (1)/Alkannin (2) appears simple (Fig. 1), the total synthesis of these compounds proves to be challenging due largely to the instability of Shikonin to Brønsted and Lewis acids, light, and oxygen [3,149]. Meanwhile, the naphthoquinone core of Shikonin has high affinity for both silica gel and alumina, making chromatographic purification of Shikonin difficult. Therefore, a critical step in the total synthesis of Shikonin is to develop approaches to effectively protect the naphthoquinone core. Up to present, two strategies have been developed to protect the naphthoquinone core, i.e., methylation and bis-methyleneacetalation of Shikonin. Since Hauser annulation and Stobbe condensation are widely used for the construction of the naphthoquinone core (Scheme 1), the main intermediates for the total synthesis of Shikonin (1)/Alkannin (2) include 1,4,5,8-tetramethoxynaphthalene (3), 1,8:4,5-bis(methylenedioxy)naphthalene (4), 5,8-dimethoxynaphthalen-1-ol (5), and 4,7-dimethoxy-cyanophthalide (6) (Fig. 2). Among them, the intermediate 3 remains the most successful synthon for the total synthesis of Shikonin (1)/Alkannin (2). Another key issue in the total synthesis of Shikonin (1)/Alkannin (2) is the introduction of the stereo specific hydroxyl group on the side chain. Several strategies have been developed to accomplish this goal, such as stereoselective reduction of the carbonyl group, asymmetric alkylation, chiral resolution of the key intermediate, etc. Finally, deprotection is also challenging as it usually leads to low-yield, making the synthesis almost impractical. The deprotection protocol developed by Terada included cascade oxidation of ceric ammonium nitrate (CAN) and AgO/HNO3, which has been widely employed for methylated

2.2.3. Cancer cell migration and metastasis In addition to the regulation of cell growth and/or death, Shikonin also demonstrated activities in inhibiting cancer cell migration, invasion and metastasis in a wide range of tumors including bladder cancer [50], adenoid cystic cancer [131], prostate cancer [132], hepatocarcinoma [133], medullary thyroid carcinoma [134], and glioblastomas [135]. At least for tyrosine kinase inhibitor resistant EGFR mutated lung cancer cells, inhibition of cancer cell migration by Shikonin greatly enhanced the efficacy of PI3K/AKT inhibitors [54]. Mechanistically, Shikonin inhibited the epithelial-to-mesenchymal transition (EMT) through modulating expression or activity of critical EMT mediators including Snail, c-Met and matrix metallopeptidase (MMP-2/9) [53,54]. Shikonin suppressed expression of MMP2/9 in adenoid cystic cancer cells [131], prostate cancer cells [132], glioblastomas [106,135] and bladder cancer cells [50], through which Shikonin inhibited the migration and invasion of these cancer cells. These findings also suggest MMP-2/9 as a key molecular target underlying the inhibitory effect of Shikonin on EMT. In osteosarcoma cancer cells, Shikonin inhibits cancer invasion through suppressing MMP13 [136]. Inhibition of cancer migration and metastasis demonstrates the capability of Shikonin to suppress a wide range of biological and pathological aspects of cancers. 2.2.4. Combination therapy In addition to functioning as a single agent, Shikonin and its derivatives have been extensively studied in combination with other therapies to treat cancer, especially with chemotherapeutic agents such as cisplatin [50,137,138], temozolomide [135], doxorubicin [45,139], adriamycin [140,141], taxol [142], and gemcitabine [42]. Shikonin also enhanced the effects of both the first generation (gefitinib) and the second generation (afatinib) EGFR inhibitors on non-small cell lung cancers [38,143], as well as erlotinib on glioblastomas [36]. Recent findings suggest that Shikonin could also enhance the immune response of natural killer cells towards colon cancers [144]. An intriguing extension of these research studies would be to determine how exactly Shikonin enhanced the effect of these targeted therapies. Collectively, these findings strongly support the development of Shikonin as a potent anti-cancer agent. 2.3. Drug delivery approaches Structurally, Shikonin is lipophilic, has short half-life and displays poor bioavailability [1]. These disadvantages restrict Shikonin as an effective anti-cancer agent. Therefore, a number of approaches had been developed to actively increase the solubility and the stability of Shikonin in vitro and in vivo, such as mixing with pharmaceutically safe proteins, encapsulating with ethylcellulose, gelatin, biodegradable or biocompatible materials [1].

Scheme 1. Retrosynthetic analysis for Shikonin (1) and Alkannin (2), modified from [150]. 332

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13 with AgO in THF almost exclusively afforded Shikonin (1/2). In addition, Couladouros et al applied a Hauser-type annulation of cyanophthalide (6) with enone intermediate to construct the aromatic system of Shikonin [150]. Asymmetric chemical synthesis is always challenging and also very important for the bioactivity of chiral compounds. Asymmetric synthesis of Shikonin has been developed by stereoselective reduction of the carbonyl group, asymmetric alkylation, and chiral resolution of the key intermediate, etc, which has been reviewed previously [149]. Strategies include the asymmetric reduction of the carbonyl group via (S)- / (R)Corey′s catalyst, Ru(II)-catalyzed asymmetric hydrogenation by C2symmetric planar chiral ruthenocene phosphinooxazoline ligand [RuCl2(PPh3)3 [155], and chiral resolution of the key intermediate [156]. In addition, Zhang et al [157] reported a new asymmetric synthesis of Shikonin (1) and Alkannin (2) from 5,8-dimethoxynaphthalen-1-ol (5) (Scheme 4). The chiral center was established through alkylation of D-isopropylideneglyceraldehyde (15). Treatment of the intermediate 5 with EtMgBr, followed by aldehyde 15, then subjected to ultrasonic wave at 0 °C, led to the syn addition product 16 in high diastereoisomeric excess (d.e.) of 91%. On the other hand, treatment of 5 with titanium tetraisopropoxide (Ti[OCH(CH3)2]4) followed by aldehyde 15 gave rise to the anti product 17 in 90% d.e.. Subsequently, the key intermediates 18 and 19 were synthesized according to previous procedures [158]. Elimination of tert-alchols with SOCl2 and pyridine, followed by alkaline hydrolysis with NaOH and subsequent acidification with HCl led to Shikonin (1) and Alkannin (2) from 19 and 18, respectively.

Fig. 2. Key intermediates used for the total synthesis of Shikonin (1) and Alkannin (2).

protective groups [148]. The electrooxide deprotection protocol developed by Nicolaou was used for synthesizing bismethyleneacetal derivatives [151]. 3.1. Total synthesis strategies of Shikonin Since Terada et al firstly synthesized Shikonin from 1,4,5,8-tetramethoxynaphthalene (3) in 1983 [148], many strategies have been reported for total synthesis of Shikonin based on this key intermediate [3,149]. In 1998, Nicolaou et al developed a second new protecting strategy for concise synthesis of Shikonin from 1,8:4,5-bis(methylenedioxy)naphthalene (4) [151]. In the latter method, the deprotection was accomplished by mild anodic oxidation in the final step. From then on, a large number of studies have been reported on the total synthesis of Shikonin using different intermediates [149,152]. Apart from 1,4,5,8-tetramethoxynaphthalene (3) and 1,8:4,5-bis (methylenedioxy) naphthalene (4), 5,8-dimethoxynaphthalen-1-ol (5) (Fig. 2) and its derivatives were also important intermediates for Shikonin synthesis. Yang et al [153] applied a Stobbe reaction to construct the naphthalene skeleton (9) from 2,5-dimethoxy-benzaldehyde (7) and diethyl succinate (8) (Scheme 2). After protection of the phenol hydroxyl group of intermediate 9 with chloromethyl methyl ether (MOMCl), the ester group was reduced with LiAlH4. The resultant hydroxyl group was oxidized with pyridinium dichromate (PDC) to afford naphthoaldehyde 10, which was subjected to Babier-type reaction with bromopropane/Zn. Protection of the hydroxyl group with tert-butyldimethylsilyl chloride (TBSCl) produced the intermediate 11. Cleavage of the terminal double bond of 11 followed by oxidation with NaIO4 produced aldehyde 12. Wittig Reaction of aldehyde 12 with ylide of 2iodopropane followed by removal of the protective group using HCl in iPrOH and tetrahydrofuran (THF) produced the intermediate 13. Oxidation of 13 with AgO/HNO3 led to Shikonin. Later on, Lu et al [154] further modified this method to synthesize Shikonin from 5,8-dimethoxynaphthalen-1-ol derivative 9 (Scheme 3), in which 9 was reduced with LiAlH4 followed by oxidation with pyridine sulfur trioxide (Py-SO3) to generate aldehyde 14. Epoxidation of aldehyde 14 with trimethyl sulfoxonium iodide (TMSOI) followed by reaction with 2,2dimethylvinyl magnesium bromide afforded naphthol 13. Oxidation of

3.2. Modification of Shikonin in the anti-cancer activity Structural modifications of Shikonin have mainly focused on the hydroxyl groups of the naphthazarin ring and the side chain (Fig. 3). Previous investigation showed that many derivatives with acyl side chain isolated from natural sources exhibited strong bioactivity [3]. Hence, a series of acyl derivatives of 1′-OH of Shikonin was synthesized and their anti-cancer effects were evaluated (Fig. 3). Ahn et al [129] reported that Shikonin analogues bearing an acyl group of short chain length (C2-C6) exerted stronger inhibitory effect on DNA topoisomerase I than those with long chain length (C7-C20). Rao et al [159] prepared novel β-hydroxyisovaleryl Shikonin analogues with oxygen-containing substituents at 1′-OH and found that analogues with ether substitution displayed the most potent anti-tumor activity selectively toward multidrug resistant prostate cancer cell DU-145. They further designed and synthesized sulfur-containing acylshikonin derivatives and found that introduction of a thioether group at the 1′-OH is associated with increased cytotoxicity [32]. Glycosylation is another efficient approach in natural product modification as it usually increases the compound solubility. Su et al [20] introduced an acylglycosyl group to 1′-OH of Shikonin, allowing

Scheme 2. Total synthesis of Shikonin (1) and Alkannin (2) [153]. Reagents and conditions: a) I: NaH, toluene. II: Ac2O, AcONa, reflux. III: CH3COCH3, HCl, reflux. b) CH3OCH2Cl, NaH, DMF. c) LiAlH4, Et2O. d) PDC, CH2Cl2. e) C3H5Br, Zn, THF. f) TBSCl, imidazole, DMF. g) OsO4. K3[Fe(CN)6], K2CO3, t-BuOH, H2O. h) NaIO4, EtOH, H2O. i) Ph3PCHI(CH3)2, n-BuLi, Et2O, N2. j) i-PrOH, THF, HCl. k) AgO, HNO3 (conc.), 1,4-dioxane. 333

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Scheme 3. Total synthesis of Shikonin (1) and Alkannin (2) [154]. Reagents and conditions: (a) LiAlH4, THF, rt, yield 90%.; (b) Py-SO3, Et3N, DMSO, 40 °C, 90%, (c) TMSOI, NaH, DMSO, 50 °C, yield 70%; (d) N2, 2,2-dimethylvinylmagnesium bromide, THF, rt, yield 50%; (e) AgO, 6M HNO3, THF, 0 °C→rt, yield 30%.

Scheme 4. Asymmetric synthesis of Shikonin (1) and Alkannin (2) [157,158]. Reagents and conditions: a) EtMgBr, CH2Cl2, 91% d.e., 70%; b) Ti[OCH(CH3)2]4, toluene, 90% d.e., 65%.

Nevertheless, the production of ROS and bioreductive alkylation of Shikonin (1) and Alkannin (2) were usually nonselective and gave rise to their toxicity to normal cells while killing cancer cells. To reduce the toxicity to normal cells, a series of modification has been made on the phenol hydroxyl and carbonyl groups to reduce ROS production and alkylation (Fig. 3). Li et al [22] prepared a series of 5,8-O-dimethyl acyl and 5,8-O-dimethyl Shikonin and found that most of these compounds displayed selective cytotoxicity towards MCF-7 cancer cells while sparing normal cells. Further, in vivo results reveal that these dimethylated Shikonin derivatives showed more potent and selective anti-cancer activity than Shikonin when combined with 5-Fu [22]. To minimize the cytotoxicity of Shikonin arising through the generation of ROS and alkylation of the naphthazarin ring, Li et al [23] synthesized dimethylated and diacetyl derivatives of Shikonin and Alkannin, and found that these derivatives showed significantly higher selectivity than Shikonin in vitro. These results suggest that reducing the capability of ROS generation and alkylation could enhance the anti-cancer activity of

the compound to exhibit cytotoxic activities against drug resistant K562, MCF-7 and HL60 cancer cell lines. Lin et al synthesized acetyl-βD-thio-glycoside Shikonin derivatives and showed that they inhibited tubulin polymerization as potent as colchicine [35]. Jiang et al reported that N-acetyl glucosaminosides of Shikonin bound more strongly to human telomeric G-quadruplex DNA than the parent compound [160]. In addition, incorporation of privileged fragments such as cinnamic acyl and phenoxyacetic acid derivatives showed comparable cytotoxic activity with Shikonin [31,34]. Quinone derivatives seem to be toxic to cells through oxidative stress [161] and/or bioreductive alkylation [162]. The naphthazarin ring of Shikonin is capable of producing ROS and alkylation. Thus, aside from the hydroxyl group of the side chain, the phenol hydroxyl group of the naphthazarin ring, including the carbonyl group, also attracted researchers’ attention in Shikonin derivatization (Fig. 3). Couladouros et al [163] reported that naphthoquinones bearing at least one phenolic hydroxyl group are potent inhibitors of topoisomerase I.

Fig. 3. The structure-activity relationship (SAR) and modification strategies of Shikonin (1). 334

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(PHBA) and geranylpyrophosphate (GPP), respectively (Fig. 4) [166,167]. Several genes that encode enzymes involved in Shikonin biosynthesis have been identified, such as acetoacetyl-CoA thiolase (ACTH), hydroxymethylglutaryl-CoA synthase gene (HMGCS), hydroxymethylglutaryl-CoA reductase gene (HMGCR), phenylalanine ammonia lyase (PAL), cinnamate 4-monooxygenase (C4H), 4-coumaroylCoA ligase (4CL), and p-hydroxybenzoate geranyltransferase gene (PGT). With the development of new technologies, increasing number of genes are continuously identified in the biogenic production of Shikonin. For instance, a recent RNA-seq study revealed two additional genes (G10H and 12OPR) that are involved in Shikonin biosynthesis [168]. We expect that more enzymes will be identified for the biogenic synthesis steps whose enzymes remain unknown at this moment (Fig. 4). Formation of m-geranyl-β-hydroxybenzoic acid (GBA) is key for Shikonin synthesis. This step requires β-hydroxybenzoate geranyltransferase (PGT), which shows high specificity towards GPP and PHBA [169]. PGT and other enzymes involved in Shikonin synthesis in Lithospermum erythrorhizon seem to locate in non-soluble membrane vessels [169], which is consistent with the observation that Shikonin is likely produced in the endoplasmic reticulum [170]. This pathway best works in the presence of magnesium at pH 7.1-9.3 [169]. Ultrasound treatment of Lithospermum erythrorhizon cells was reported to stimulate Shikonin production probably through increased expression of PHB geranyltransferase [171,172]. A number of culture methods such as, seed germination, callus induction and hairy roots, have been developed to increase the yield of Shikonin in plants [173,174]. LeMRP, an ATP-binding cassette transporter from Lithospermum erythrorhizon, may regulate the membrane transport of Shikonin and overexpression of this gene increased Shikonin production through increasing its biosynthesis enzymes [175]. Shikonin has been commercially produced by aseptic system from plant

Shikonin while reducing its side effect in vivo. Oxime is one of the traditional nitric oxide (NO) donors that display anti-neoplastic function. Oxidation of the carbonyl group in Shikonin not only reduces the levels of ROS and alkylation, but also retains its anti-tumor activity. Huang et al synthesized a series of sulfur-containing Shikonin oxime derivatives and found that some of the analogues displayed comparable or even stronger anticancer effect than the parent compound against HCT-15, MGC-803 and Bel7402 cancer cell lines [32]. Among them, the dioxime derivative with 3,4,5-trimethoxybenzoylthio substitutions arrested the cell cycle at G1 phase and induced apoptotic cell death through the down-regulation of Bcl-2 and the up-regulation of Bax, Caspase 3 and 9 [32]. In addition, it is well known that permethylation of naphthazarin derivatives could form 6and 2-isomers. Li et al prepared several 6-isomers of 5,8-O-dimethyl acylshikonin derivatives and found that the majority of the compounds were more active than or at least comparative to Shikonin. Further, these compounds retained their selectivity towards cancer cells while showing little toxicity to normal cells [24]. In vivo study showed that 6isomers of 5,8-O-dimethyl acylshikonin derivatives were more potent than their corresponding 2-isomers. Based on these results, Duan et al [164] simplified the synthesis with arylsulfonaide side chains on Shikonin and showed that most of the analogues exhibited stronger inhibitory effect on HeLa and HL60 cancer cells than the lead compound. Together, these findings suggest that Shikonin derivatives display elevated anti-cancer activities, holding the promise of being developed as lead anti-cancer compounds for further clinical evaluation.

4. Biogenic synthesis The biogenic synthesis of Shikonin, first reported by Inouye et al in 1979 [165], starts from phenylalanine and involves both the shikimate and the mevalonate pathways to produce β-hydroxybenzioc acid

Fig. 4. Biogenic synthesis of Shikonin (1) and Alkannin (2). PHBA, β-hydroxybenzioc acid; GPP, geranylpyrophosphate; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-monooxygenase; 4CL, 4-coumaroyl-CoA ligase, PGT, p-hydroxybenzoate geranyltransferase gene; ACTH, acetoacetyl-CoA thiolase, GBA, m-geranyl-βhydroxybenzoic acid; GHQ, geranyl hydroquinone. 335

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secondary metabolites. Identification of the identity and understanding the function of these biogenic enzymes may provide specific approaches to biogenically synthesize Shikonin in the near future, which offers an alternative strategy from chemical synthesis to produce Shikonin for clinical application.

[6]

[7] [8]

5. Conclusions and discussion

[9]

The wide variety of activities at one hand demonstrates the effectiveness of Shikonin; on the other hand, they may indicate a difficulty in pinpointing the true biological event that accounts for the effect of this compound class. This situation is further complicated by contradictory research findings. For instance, Shikonin was reported to both inhibit [89] and increase [87,176] the JNK and p38 pathway. Similarly, Shikonin and its derivatives were reported to be able to inhibit or activate the ERK signaling. Shikonin increased the drug efflux pump in hepatocytes [176], while reducing it in cancer cells [139,140]. Although it is thought that Shikonin produces ROS, there are reports showing antioxidant properties of Shikonin [1]. Another issue in the development of Shikonin is that the efficacy of Shikonin and its derivatives are relatively low, which often requires μM level of concentrations to be functional. Thus, further efforts are needed to derivatize Shikonin for more potent compounds. In addition, Shikonin may increase the expression of certain cytochrome P450 family metabolic enzymes [176], indicating a potential drug-drug interaction with other drugs that rely on this enzyme for metabolism. Nonetheless, Shikonin could be developed as a potent anti-cancer agent given the large body of evidence presented here and elsewhere. Further, with the advent of new technologies such as the whole genome sequencing, genome editing and quantitative proteomic analysis, we can envisage the thorough understanding of the precise biological targets and the molecular pathways of Shikonin not only in the anti-cancer function, but also in other biological activities. In combination with these newly developed synthetic approaches and the SAR insight, we anticipate that these lines of information may lead to the development of Shikonin or its derivatives as potent anti-cancer agents in the clinic in near future.

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[15]

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Consent for publication

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Not applicable Conflict of interest

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We declare no conflict of interest Acknowledgements

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We apologize to colleagues whose work was not cited due to space limitations. Work in J Tang’s lab was supported by National Science Foundation of China (No 81728022, 81673320). X.Yao. is supported by the National 111 Project of China. Y Zhang is supported by National Institute of Health (R01 163214) and the American Cancer Society (ACS RSG-15-042 DMC).

[25]

[26]

[27]

References

[28]

[1] I. Andujar, J.L. Rios, R.M. Giner, M.C. Recio, Pharmacological properties of shikonin - a review of literature since 2002, Planta Med 79 (2013) 1685–1697. [2] X. Chen, L. Yang, J.J. Oppenheim, O.M.Z. Howard, Cellular pharmacology studies of shikonin derivatives, Phytother Res 16 (2002) 199–209. [3] V.P. Papageorgiou, A.N. Assimopoulou, E.A. Couladouros, D. Hepworth, K.C. Nicolaou, The chemistry and biology of alkannin, shikonin, and related naphthazarin natural products, Angew Chem Int Ed 38 (1999) 270–301. [4] Y. Masuda, A. Nishida, K. Hori, T. Hirabayashi, S. Kajimoto, S. Nakajo, T. Kondo, M. Asaka, K. Nakaya, Beta-hydroxyisovalerylshikonin induces apoptosis in human leukemia cells by inhibiting the activity of a polo-like kinase 1 (PLK1), Oncogene 22 (2003) 1012–1023. [5] W. Han, L. Li, S. Qiu, Q. Lu, Q. Pan, Y. Gu, J. Luo, X. Hu, Shikonin circumvents

[29]

[30]

[31]

336

cancer drug resistance by induction of a necroptotic death, Mol Cancer Ther 6 (2007) 1641–1649. X.P. Guo, X.Y. Zhang, S.D. Zhang, [Clinical trial on the effects of shikonin mixture on later stage lung cancer], Zhong xi yi jie he za zhi, Chin J Modern Dev Tradit Med 11 (1991) 598–599. C. Fan, Y. Xie, Y. Dong, Y. Su, Z. Upton, Investigating the potential of Shikonin as a novel hypertrophic scar treatment, J Biomed Sci 22 (2015) 70. Y. Xie, C. Fan, Y. Dong, E. Lynam, D.I. Leavesley, K. Li, Y. Su, Y. Yang, Z. Upton, Functional and mechanistic investigation of Shikonin in scarring, Chem-biol Interact 228 (2015) 18–27. X. Deng, Q. Chen, L. Qiang, M. Chi, N. Xie, Y. Wu, M. Yao, D. Zhao, J. Ma, N. Zhang, Y. Xie, Development of a porcine full-thickness burn hypertrophic scar model and investigation of the effects of shikonin on hypertrophic scar remediation, Front Pharmacol 9 (2018) 590. T. Liu, L. Xu, C. Wang, K. Chen, Y. Xia, J. Li, S. Li, L. Wu, J. Feng, S. Xu, W. Wang, X. Lu, X. Fan, W. Mo, Y. Zhou, Y. Zhao, C. Guo, Shikonin alleviates hepatic fibrosis and autophagy via inhibition of TGF-beta1/Smads pathway, J Gastroen Hepatol (2018), https://doi.org/10.1111/jgh.14299. Y. Nie, Y. Yang, J. Zhang, G. Cai, Y. Chang, G. Chai, C. Guo, Shikonin suppresses pulmonary fibroblasts proliferation and activation by regulating Akt and p38 MAPK signaling pathways, Biomed Pharmacother 95 (2017) 1119–1128. H. Zhang, D. Wang, M. Li, L. Plecita-Hlavata, A. D'Alessandro, J. Tauber, S. Riddle, S. Kumar, A. Flockton, B.A. McKeon, M.G. Frid, J.A. Reisz, P. Caruso, K.C. El Kasmi, P. Jezek, N.W. Morrell, C.J. Hu, K.R. Stenmark, Metabolic and proliferative state of vascular adventitial fibroblasts in pulmonary hypertension is regulated through a MicroRNA-124/PTBP1 (polypyrimidine tract binding Protein 1)/pyruvate kinase muscle axis, Circulation 136 (2017) 2468–2485. S. Wang, Y. Zhu, R. Qiu, Shikonin protects H9C2 cardiomyocytes against hypoxia/ reoxygenation injury through activation of PI3K/Akt signaling pathway, Biomed Pharmacother 104 (2018) 712–717. Z. Wang, T. Liu, L. Gan, T. Wang, X. Yuan, B. Zhang, H. Chen, Q. Zheng, Shikonin protects mouse brain against cerebral ischemia/reperfusion injury through its antioxidant activity, Eur J Pharmacol 643 (2010) 211–217. Y. Tong, L. Bai, R. Gong, J. Chuan, X. Duan, Y. Zhu, Shikonin protects PC12 cells against beta-amyloid peptide-induced cell injury through antioxidant and antiapoptotic activities, Sci Rep 8 (2018) 26. Y. Bi, Y. Zhu, M. Zhang, K. Zhang, X. Hua, Z. Fang, J. Zhou, W. Dai, Y. Cui, J. Li, T. You, Effect of Shikonin on spinal cord injury in rats via regulation of HMGB1/ TLR4/NF-kB signaling pathway, Cell Physiol Biochem 43 (2017) 481–491. X. Zhang, J. Cui, Q. Meng, S. Li, W. Zhou, S. Xiao, Advance in anti-tumor mechanisms of Shikonin, alkannin and their derivatives, Mini-Rev Med Chem 18 (2018) 164–172. I. Andujar, M.C. Recio, R.M. Giner, J.L. Rios, Traditional chinese medicine remedy to jury: the pharmacological basis for the use of shikonin as an anticancer therapy, Curr Med Chem 20 (2013) 2892–2898. S. Tanaka, M. Tajima, M. Tsukada, M. Tabata, A comparative study on anti-inflammatory activities of the enantiomers, shikonin and alkannin, J Nat Prod 49 (1986) 466–469. Y. Su, J. Xie, Y. Wang, X. Hu, X. Lin, Synthesis and antitumor activity of new shikonin glycosides, Eur J Med Chem 45 (2010) 2713–2718. H.Y. Qiu, J.Y. Fu, M.K. Yang, H.W. Han, P.F. Wang, Y.H. Zhang, H.Y. Lin, C.Y. Tang, J.L. Qi, R.W. Yang, X.M. Wang, H.L. Zhu, Y.H. Yang, Identification of new shikonin derivatives as STAT3 inhibitors, Biochem Pharmacol 146 (2017) 74–86. W. Zhou, Y. Peng, S.S. Li, Semi-synthesis and anti-tumor activity of 5,8-O-dimethyl acylshikonin derivatives, Eur J Med Chem 45 (2010) 6005–6011. R.B. Wang, W. Zhou, Q.Q. Meng, X. Zhang, J. Ding, Y. Xu, H.L. Song, K. Yang, J.H. Cui, S.S. Li, Design, synthesis, and biological evaluation of shikonin and alkannin derivatives as potential anticancer agents via a prodrug approach, ChemMedChem 9 (2014) 2798–2808. W. Zhou, X. Zhang, L. Xiao, J. Ding, Q.H. Liu, S.S. Li, Semi-synthesis and antitumor activity of 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives, Eur J Med Chem 46 (2011) 3420–3427. G. Huang, H. Zhao, Q. Meng, W. Zhou, Q. Zhang, J. Dong, J. Cui, S. Li, Cerium (IV) ammonium nitrate (CAN)-mediated regioselective synthesis and anticancer activity of 6-substituted 5,8-dimethoxy-1,4-naphthoquinone, Chin Chem Lett 28 (2017) 1553–1558. X. Zhang, J.H. Cui, W. Zhou, S.S. Li, Design, synthesis and anticancer activity of shikonin and alkannin derivatives with different substituents on the naphthazarin scaffold, Chem Res Chin Univ 31 (2015) 394–400. G. Huang, Q.Q. Meng, W. Zhou, Q.J. Zhang, J.Y. Dong, S.S. Li, Design and synthesis of biotinylated dimethylation of alkannin oxime derivatives, Chin Chem Lett 28 (2017) 453–457. H.W. Han, C.S. Zheng, S.J. Chu, W.X. Sun, L.J. Han, R.W. Yang, J.L. Qi, G.H. Lu, X.M. Wang, Y.H. Yang, The evaluation of potent antitumor activities of shikonin coumarin-carboxylic acid, PMMB232 through HIF-1 alpha-mediated apoptosis, Biomed Pharmacother 97 (2018) 656–666. L.M. Zhao, T.P. Xie, Y.Q. He, D.F. Xu, S.S. Li, Synthesis and antitumor activity of 6and 2-(1-substituted-thio-4-methylpent-3-enyl)-5,8-dimethoxynaphthalene-1,4diones, Eur J Med Chem 44 (2009) 1410–1414. X. Ning, H. Qi, R. Li, Y. Li, Y. Jin, M.A. McNutt, J. Liu, Y. Yin, Discovery of novel naphthoquinone derivatives as inhibitors of the tumor cell specific M2 isoform of pyruvate kinase, Eur J Med Chem 138 (2017) 343–352. H.Y. Lin, W. Chen, J. Shi, W.Y. Kong, J.L. Qi, X.M. Wang, Y.H. Yang, Design, synthesis and biological evaluation of cinnamic acyl shikonin derivatives, Chem Biol Drug Des 81 (2013) 275–283.

Fitoterapia 134 (2019) 329–339

F. Wang, et al.

[32] G. Huang, H.R. Zhao, Q.Q. Meng, Q.J. Zhang, J.Y. Dong, B.Q. Zhu, S.S. Li, Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as potential antineoplastic agents, Eur J Med Chem 143 (2018) 166–181. [33] G. Huang, H.R. Zhao, Q.Q. Meng, W. Zhou, Q.J. Zhang, J.Y. Dong, J.H. Cui, S.S. Li, Cerium (IV) ammonium nitrate (CAN)-mediated regioselective synthesis and anticancer activity of 6-substituted 5,8-dimethOxy-1,4-naphthoquinone, Chinese Chem Lett 28 (2017) 1553–1558. [34] J. Guo, X.F. Chen, J. Liu, H.Y. Lin, H.W. Han, H.C. Liu, S.C. Huang, B.K. Shahla, A. Kulek, J.L. Qi, X.M. Wang, L.J. Ling, Y.H. Yang, Novel shikonin derivatives targeting tubulin as anticancer agents, Chem Biol Drug Des 84 (2014) 603–615. [35] H.Y. Lin, H.W. Han, L.F. Bai, H.Y. Qiu, D.Z. Yin, J.L. Qi, X.M. Wang, H.W. Gu, Y.H. Yang, Design, synthesis and biological evaluation of shikonin thio-glycoside derivatives: new anti-tubulin agents, Rsc Adv 4 (2014) 49796–49805. [36] Q. Zhao, N. Kretschmer, R. Bauer, T. Efferth, Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinib, Int J Cancer 137 (2015) 1446–1456. [37] H. Zheng, Q. Huang, S. Huang, X. Yang, T. Zhu, W. Wang, H. Wang, S. He, L. Ji, Y. Wang, X. Qi, Z. Liu, L. Lu, Senescence inducer Shikonin ROS-dependently suppressed lung cancer progression, Front Pharmacol 9 (2018) 519. [38] J.C. Tang, Y.G. Ren, J. Zhao, F. Long, J.Y. Chen, Z. Jiang, Shikonin enhances sensitization of gefitinib against wild-type EGFR non-small cell lung cancer via inhibition PKM2/stat3/cyclinD1 signal pathway, Life Sci 204 (2018) 71–77. [39] H.J. Kim, K.E. Hwang, D.S. Park, S.H. Oh, H.Y. Jun, K.H. Yoon, E.T. Jeong, H.R. Kim, Y.S. Kim, Shikonin-induced necroptosis is enhanced by the inhibition of autophagy in non-small cell lung cancer cells, J Transl Med 15 (2017) 123. [40] H.Y. Lin, H.W. Han, W.X. Sun, Y.S. Yang, C.Y. Tang, G.H. Lu, J.L. Qi, X.M. Wang, Y.H. Yang, Design and characterization of alpha-lipoic acyl shikonin ester twin drugs as tubulin and PDK1 dual inhibitors, Eur J Med Chem 144 (2018) 137–150. [41] J.C. Tang, J. Zhao, F. Long, J.Y. Chen, B. Mu, Z. Jiang, Y. Ren, J. Yang, Efficacy of Shikonin against esophageal cancer cells and its possible mechanisms in vitro and in vivo, J Cancer 9 (2018) 32–40. [42] C. Chen, W. Xiao, L. Huang, G. Yu, J. Ni, L. Yang, R. Wan, G. Hu, Shikonin induces apoptosis and necroptosis in pancreatic cancer via regulating the expression of RIP1/RIP3 and synergizes the activity of gemcitabine, Am J Transl Res 9 (2017) 5507–5517. [43] W. Liang, J. Cui, K. Zhang, H. Xi, A. Cai, J. Li, Y. Gao, C. Hu, Y. Liu, Y. Lu, N. Wang, X. Wu, B. Wei, L. Chen, Shikonin induces ROS-based mitochondriamediated apoptosis in colon cancer, Oncotarget 8 (2017) 109094–109106. [44] M.Y. Li, C. Mi, K.S. Wang, Z. Wang, H.X. Zuo, L.X. Piao, G.H. Xu, X. Li, J. Ma, X. Jin, Shikonin suppresses proliferation and induces cell cycle arrest through the inhibition of hypoxia-inducible factor-1alpha signaling, Chem-Biol Interact 274 (2017) 58–67. [45] F. Ni, X. Huang, Z. Chen, W. Qian, X. Tong, Shikonin exerts antitumor activity in Burkitt's lymphoma by inhibiting C-MYC and PI3K/AKT/mTOR pathway and acts synergistically with doxorubicin, Sci Rep 8 (2018) 3317. [46] B. Lu, Z. Wang, Y. Ding, X. Wang, S. Lu, C. Wang, C. He, M. Piao, G. Chi, Y. Luo, P. Ge, RIP1 and RIP3 contribute to shikonin-induced glycolysis suppression in glioma cells via increase of intracellular hydrogen peroxide, Cancer Lett 425 (2018) 31–42. [47] S. Li, T. Zhang, W. Xu, J. Ding, F. Yin, J. Xu, W. Sun, H. Wang, M. Sun, Z. Cai, Y. Hua, Sarcoma-targeting peptide-decorated polypeptide nanogel intracellularly delivers shikonin for upregulated osteosarcoma necroptosis and diminished pulmonary metastasis, Theranostics 8 (2018) 1361–1375. [48] K. Kitayama, M. Yashiro, T. Morisaki, Y. Miki, T. Okuno, H. Kinoshita, T. Fukuoka, H. Kasashima, G. Masuda, T. Hasegawa, K. Sakurai, N. Kubo, K. Hirakawa, M. Ohira, Pyruvate kinase isozyme M2 and glutaminase might be promising molecular targets for the treatment of gastric cancer, Cancer Sci 108 (2017) 2462–2469. [49] Z. Zhang, Z. Zhang, Q. Li, H. Jiao, D. Chong, X. Sun, P. Zhang, Q. Huo, H. Liu, Shikonin induces necroptosis by reactive oxygen species activation in nasopharyngeal carcinoma cell line CNE-2Z, J Bioenerg Biomembr 49 (2017) 265–272. [50] X. Wang, F. Zhang, X.R. Wu, Inhibition of pyruvate kinase M2 markedly reduces chemoresistance of advanced bladder cancer to cisplatin, Sci Rep 7 (2017) 45983. [51] S. Shi, H. Cao, Shikonin promotes autophagy in BXPC-3 human pancreatic cancer cells through the PI3K/Akt signaling pathway, Oncol Lett 8 (2014) 1087–1089. [52] M. Gu, X. Li, X. Tang, Effect of autophagy on the shikonin induced apoptosis of human medullary thyroid carcinoma TT cells, Int J Clin Exp Med 9 (2016) 17428–17434. [53] D. Hong, S.Y. Jang, E.H. Jang, B. Jung, I.H. Cho, M.J. Park, S.Y. Jeong, J.H. Kim, Shikonin as an inhibitor of the LPS-induced epithelial-to-mesenchymal transition in human breast cancer cells, Int J Mol Med 36 (2015) 1601–1606. [54] Y.S. Hsieh, C.H. Liao, W.S. Chen, J.T. Pai, M.S. Weng, Shikonin inhibited migration and invasion of human lung cancer cells via suppression of c-met-mediated epithelial-to-mesenchymal transition, J Cell Biochem 118 (2017) 4639–4651. [55] B. Wiench, T. Eichhorn, M. Paulsen, T. Efferth, Shikonin directly targets mitochondria and causes mitochondrial dysfunction in cancer cells, Evid-based Compl Alt Med: eCAM 2012 (2012) 726025. [56] H. Wang, Z. Liu, X. Li, R. Zhao, Y. Pu, H. Wu, W. Guan, Shikonin causes apoptosis by disrupting intracellular calcium homeostasis and mitochondrial function in human hepatoma cells, Exp Ther Med 15 (2018) 1484–1492. [57] R.K. Gara, V.K. Srivastava, S. Duggal, J.K. Bagga, M. Bhatt, S. Sanyal, D.P. Mishra, Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway, J Biomed Sci 22 (2015) 26. [58] Z.L. Shan, L. Zhong, C.L. Xiao, L.G. Gan, T. Xu, H. Song, R. Yang, L. Li, B.Z. Liu, Shikonin suppresses proliferation and induces apoptosis in human leukemia NB4

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66] [67] [68]

[69] [70]

[71]

[72] [73] [74] [75]

[76]

[77]

[78]

[79] [80]

[81]

[82]

[83] [84]

[85]

[86]

[87]

337

cells through modulation of MAPKs and cMyc, Mol Med Rep 16 (2017) 3055–3060. B. Gupta, S. Chakraborty, S. Saha, S.G. Chandel, A.K. Baranwal, M. Banerjee, M. Chatterjee, A. Chaudhury, Antinociceptive properties of shikonin: in vitro and in vivo studies, Can J Physiol Pharm 94 (2016) 788–796. X. Han, K.A. Kang, M.J. Piao, A.X. Zhen, Y.J. Hyun, H.M. Kim, Y.S. Ryu, J.W. Hyun, Shikonin exerts cytotoxic effects in human colon cancers by inducing apoptotic cell death via the endoplasmic reticulum and mitochondria-mediated pathways, Biomol Ther (2018), https://doi.org/10.4062/biomolther.2018.047. Z. Fu, B. Deng, Y. Liao, L. Shan, F. Yin, Z. Wang, H. Zeng, D. Zuo, Y. Hua, Z. Cai, The anti-tumor effect of shikonin on osteosarcoma by inducing RIP1 and RIP3 dependent necroptosis, BMC cancer 13 (2013) 580. S. Park, H. Shin, Y. Cho, Shikonin induces programmed necrosis-like cell death through the formation of receptor interacting protein 1 and 3 complex, Food Chem Toxicol 55 (2013) 36–41. B. Lu, X. Gong, Z.Q. Wang, Y. Ding, C. Wang, T.F. Luo, M.H. Piao, F.K. Meng, G.F. Chi, Y.N. Luo, P.F. Ge, Shikonin induces glioma cell necroptosis in vitro by ROS overproduction and promoting RIP1/RIP3 necrosome formation, Acta Pharmacol Sin 38 (2017) 1543–1553. B. Gupta, S. Chakraborty, A. Chaudhury, Identification of novel targets for Shikonin as a potent drug for inflammation and cancer, Pharmacologia 7 (2016) 350–360. J. Chen, J. Xie, Z. Jiang, B. Wang, Y. Wang, X. Hu, Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2, Oncogene 30 (2011) 4297–4306. B. Altenberg, K.O. Greulich, Genes of glycolysis are ubiquitously overexpressed in 24 cancer classes, Genomics 84 (2004) 1014–1020. O. Warburg, F. Wind, E. Negelein, The Metabolism of Tumors in the Body, J Gen Physiol 8 (1927) 519–530. H.R. Christofk, M.G. Vander Heiden, M.H. Harris, A. Ramanathan, R.E. Gerszten, R. Wei, M.D. Fleming, S.L. Schreiber, L.C. Cantley, The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth, Nature 452 (2008) 230–233. W. Yang, Z. Lu, Nuclear PKM2 regulates the Warburg effect, Cell cycle 12 (2013) 3154–3158. X. Zhao, Y. Zhu, J. Hu, L. Jiang, L. Li, S. Jia, K. Zen, Shikonin inhibits tumor growth in mice by suppressing pyruvate kinase M2-mediated aerobic glycolysis, Sci Rep 8 (2018) 14517. H. Zhang, D.Z. Qian, Y.S. Tan, K. Lee, P. Gao, Y.R. Ren, S. Rey, H. Hammers, D. Chang, R. Pili, C.V. Dang, J.O. Liu, G.L. Semenza, Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth, Proc Nat Acad Sci U S A 105 (2008) 19579–19586. W. Peti, R. Page, Molecular basis of MAP kinase regulation, Protein Sci 22 (2013) 1698–1710. Y. Zhang, C. Dong, Regulatory mechanisms of mitogen-activated kinase signaling, Cell Mol Life Sci: CMLS 64 (2007) 2771–2789. Y.D. Shaul, R. Seger, The MEK/ERK cascade: from signaling specificity to diverse functions, Biochim Biophys Acta 1773 (2007) 1213–1226. Q. Zhao, A.N. Assimopoulou, S.M. Klauck, H. Damianakos, I. Chinou, N. Kretschmer, J.L. Rios, V.P. Papageorgiou, R. Bauer, T. Efferth, Inhibition of cMYC with involvement of ERK/JNK/MAPK and AKT pathways as a novel mechanism for shikonin and its derivatives in killing leukemia cells, Oncotarget 6 (2015) 38934–38951. H. Jing, W. Sun, J. Fan, Y. Zhang, J. Yang, J. Jia, J. Li, J. Guo, S. Luo, Y. Zheng, Shikonin induces apoptosis of HaCaT cells via the mitochondrial, Erk and Akt pathways, Mol Med Rep 13 (2016) 3009–3016. T.Y. Wang, Q.L. Zhou, M. Li, Y.X. Shang, Shikonin alleviates allergic airway remodeling by inhibiting the ERK-NF-kappaB signaling pathway, Int Immunopharmacol 48 (2017) 169–179. S.Y. Gwon, J.Y. Ahn, C.H. Jung, B.K. Moon, T.Y. Ha, Shikonin suppresses ERK 1/2 phosphorylation during the early stages of adipocyte differentiation in 3T3-L1 cells, BMC Complem Altern Med 13 (2013) 207. S. Zhuang, R.G. Schnellmann, A death-promoting role for extracellular signalregulated kinase, J Pharmacol Exp Ther 319 (2006) 991–997. X.J. Shen, H.B. Wang, X.Q. Ma, J.H. Chen, Beta,beta-Dimethylacrylshikonin induces mitochondria dependent apoptosis through ERK pathway in human gastric cancer SGC-7901 cells, PloS One 7 (2012) e41773. Z. Wu, L.J. Wu, S. Tashiro, S. Onodera, T. Ikejima, Phosphorylated extracellular signal-regulated kinase up-regulated p53 expression in shikonin-induced HeLa cell apoptosis, Chinese Med J 118 (2005) 671–677. I.C. Chang, Y.J. Huang, T.I. Chiang, C.W. Yeh, L.S. Hsu, Shikonin induces apoptosis through reactive oxygen species/extracellular signal-regulated kinase pathway in osteosarcoma cells, Biol Pharm Bull 33 (2010) 816–824. R.J. Davis, Signal transduction by the JNK group of MAP kinases, Cell 103 (2000) 239–252. C. Davies, C. Tournier, Exploring the function of the JNK (c-Jun N-terminal kinase) signalling pathway in physiological and pathological processes to design novel therapeutic strategies, Biochem Soc Trans 40 (2012) 85–89. J.J. Ventura, A. Hubner, C. Zhang, R.A. Flavell, K.M. Shokat, R.J. Davis, Chemical genetic analysis of the time course of signal transduction by JNK, Mol Cell 21 (2006) 701–710. X. Mao, C.R. Yu, W.H. Li, W.X. Li, Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia (CML) cells, Cell Res 18 (2008) 879–888. G. Zhou, Z. Yang, X. Wang, R. Tao, Y. Zhou, TRAIL enhances shikonin induced apoptosis through ROS/JNK signaling in cholangiocarcinoma cells, Cell Physiol

Fitoterapia 134 (2019) 329–339

F. Wang, et al.

[118] D. Duan, B. Zhang, J. Yao, Y. Liu, J. Fang, Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL-60 cells, Free Radical Biol Med 70 (2014) 182–193. [119] W.R. Huang, Y. Zhang, X. Tang, Shikonin inhibits the proliferation of human lens epithelial cells by inducing apoptosis through ROS and caspase-dependent pathway, Molecules 19 (2014) 7785–7797. [120] L. Liu, Y. Wu, K. Cao, Y.Y. Xu, X.H. Gao, H.D. Chen, L. Geng, Shikonin inhibits IFNgamma-induced K17 over-expression of HaCaT cells by interfering with STAT3 signaling, Int J Clin Exp Pathol 8 (2015) 9202–9207. [121] S.L. Furtek, D.S. Backos, C.J. Matheson, P. Reigan, Strategies and approaches of targeting STAT3 for cancer treatment, ACS Chem Biol 11 (2016) 308–318. [122] G.L. Semenza, Regulation of oxygen homeostasis by hypoxia-inducible factor 1, Physiology 24 (2009) 97–106. [123] L.L. Zhang, L. Zhan, Y.D. Jin, Z.L. Min, C. Wei, Q. Wang, Y.J. Chen, Q.M. Wu, X.M. Hu, Q. Yuan, SIRT2 mediated antitumor effects of shikonin on metastatic colorectal cancer, Eur J Pharm 797 (2017) 1–8. [124] S.Y. Jang, D. Hong, S.Y. Jeong, J.H. Kim, Shikonin causes apoptosis by up-regulating p73 and down-regulating ICBP90 in human cancer cells, Biochem Biophys Res Commun 465 (2015) 71–76. [125] P.C. Hsu, Y.T. Huang, M.L. Tsai, Y.J. Wang, J.K. Lin, M.H. Pan, Induction of apoptosis by shikonin through coordinative modulation of the Bcl-2 family, p27, and p53, release of cytochrome c, and sequential activation of caspases in human colorectal carcinoma cells, J Agric Food Chem 52 (2004) 6330–6337. [126] Z. Wu, L. Wu, L. Li, S. Tashiro, S. Onodera, T. Ikejima, p53-mediated cell cycle arrest and apoptosis induced by shikonin via a caspase-9-dependent mechanism in human malignant melanoma A375-S2 cells, J Pharm Sci 94 (2004) 166–176. [127] F. Yang, Y. Chen, W. Duan, C. Zhang, H. Zhu, J. Ding, SH-7, a new synthesized shikonin derivative, exerting its potent antitumor activities as a topoisomerase inhibitor, Int J Cancer 119 (2006) 1184–1193. [128] Z.F. Plyta, T. Li, V.P. Papageorgiou, A.S. Mellidis, A.N. Assimopoulou, E.N. Pitsinos, E.A. Couladouros, Inhibition of topoisomerase I by naphthoquinone derivatives, Bioorg Med Chem Lett 8 (1998) 3385–3390. [129] B.Z. Ahn, K.U. Baik, G.R. Kweon, K. Lim, B.D. Hwang, Acylshikonin analogues: synthesis and inhibition of DNA topoisomerase-I, J Med Chem 38 (1995) 1044–1047. [130] J. Chen, X. Hu, J. Cui, Shikonin, vitamin K3 and vitamin K5 inhibit multiple glycolytic enzymes in MCF-7 cells, Oncol Lett 15 (2018) 7423–7432. [131] R. Min, Z. Zun, Y. Min, D. Wenhu, Y. Wenjun, Z. Chenping, Shikonin inhibits tumor invasion via down-regulation of NF-kappaB-mediated MMP-9 expression in human ACC-M cells, Oral Dis 17 (2011) 362–369. [132] Y. Chen, L. Zheng, J. Liu, Z. Zhou, X. Cao, X. Lv, F. Chen, Shikonin inhibits prostate cancer cells metastasis by reducing matrix metalloproteinase-2/-9 expression via AKT/mTOR and ROS/ERK1/2 pathways, Int Immunopharmacol 21 (2014) 447–455. [133] P.L. Wei, C.C. Tu, C.H. Chen, Y.S. Ho, C.T. Wu, H.Y. Su, W.Y. Chen, J.J. Liu, Y.J. Chang, Shikonin suppresses the migratory ability of hepatocellular carcinoma cells, J Agric Food Chem 61 (2013) 8191–8197. [134] C. Hasenoehrl, G. Schwach, N. Ghaffari-Tabrizi-Wizsy, R. Fuchs, N. Kretschmer, R. Bauer, R. Pfragner, Anti-tumor effects of shikonin derivatives on human medullary thyroid carcinoma cells, Endocr Connect 6 (2017) 53–62. [135] D. Matias, J. Balca-Silva, L.G. Dubois, B. Pontes, V.P. Ferrer, L. Rosario, A. do Carmo, J. Echevarria-Lima, A.B. Sarmento-Ribeiro, M.C. Lopes, V. Moura-Neto, Dual treatment with shikonin and temozolomide reduces glioblastoma tumor growth, migration and glial-to-mesenchymal transition, Cell Oncol 40 (2017) 247–261. [136] B. Deng, Y. Feng, B. Deng, TIPE2 mediates the suppressive effects of Shikonin on MMP13 in osteosarcoma cells, Cell Physiol Biochem 37 (2015) 2434–2443. [137] G. He, G. He, R. Zhou, Z. Pi, T. Zhu, L. Jiang, Y. Xie, Enhancement of cisplatininduced colon cancer cells apoptosis by shikonin, a natural inducer of ROS in vitro and in vivo, Biochem Biophys Res Commun 469 (2016) 1075–1082. [138] K. Shilnikova, M.J. Piao, K.A. Kang, Y.S. Ryu, J.E. Park, Y.J. Hyun, A.X. Zhen, Y.J. Jeong, U. Jung, I.G. Kim, J.W. Hyun, Shikonin induces mitochondria-mediated apoptosis and attenuates epithelial-mesenchymal transition in cisplatin-resistant human ovarian cancer cells, Oncol Lett 15 (2018) 5417–5424. [139] S.A. Mirzaei, S. Reiisi, P. Ghiasi Tabari, A. Shekari, F. Aliakbari, E. Azadfallah, F. Elahian, Broad blocking of MDR efflux pumps by acetylshikonin and acetoxyisovalerylshikonin to generate hypersensitive phenotype of malignant carcinoma cells, Sci Rep 8 (2018) 3446. [140] X. Liu, G. Sun, Shikonin enhances Adriamycin antitumor effects by inhibiting efflux pumps in A549 cells, Oncol Lett 14 (2017) 4270–4276. [141] Q. Yang, S. Li, Z. Fu, B. Lin, Z. Zhou, Z. Wang, Y. Hua, Z. Cai, Shikonin promotes adriamycin induced apoptosis by upregulating caspase3 and caspase8 in osteosarcoma, Mol Med Rep 16 (2017) 1347–1352. [142] W. Li, J. Liu, K. Jackson, R. Shi, Y. Zhao, Sensitizing the therapeutic efficacy of taxol with shikonin in human breast cancer cells, PloS one 9 (2014) e94079. [143] B. Li, Z. Yuan, J. Jiang, Y. Rao, Anti-tumor activity of Shikonin against afatinib resistant non-small cell lung cancer via negative regulation of PI3K/Akt signaling pathway, Bioscience Rep 38 (2018), https://doi.org/10.1042/BSR20181693. [144] Y. Li, H. Lu, Y. Gu, Z. Ning, T. Cao, C. Chen, C. Hu, M. Tao, Enhancement of NK cells proliferation and function by Shikonin, Immunopharm Immunot 39 (2017) 124–130. [145] X. Wen, J. Li, D. Cai, L. Yue, Q. Wang, L. Zhou, L. Fan, J. Sun, Y. Wu, Anticancer efficacy of targeted Shikonin liposomes modified with RGD in breast cancer cells, Molecules 23 (2018), https://doi.org/10.3390/molecules23020268. [146] H. Li, Y. Tong, L. Bai, L. Ye, L. Zhong, X. Duan, Y. Zhu, Lactoferrin functionalized PEG-PLGA nanoparticles of shikonin for brain targeting therapy of glioma, Int J

Biochem 42 (2017) 1073–1086. [88] H. Ko, S.J. Kim, S.H. Shim, H. Chang, C.H. Ha, Shikonin induces apoptotic cell death via regulation of p53 and Nrf2 in AGS human stomach carcinoma cells, Biomol Ther 24 (2016) 501–509. [89] K.H. Lin, M.Y. Huang, W.C. Cheng, S.C. Wang, S.H. Fang, H.P. Tu, C.C. Su, Y.L. Hung, P.L. Liu, C.S. Chen, Y.T. Wang, C.Y. Li, RNA-seq transcriptome analysis of breast cancer cell lines under shikonin treatment, Sci Rep 8 (2018) 2672. [90] T. Zhai, Z. Hei, Q. Ma, H. Liang, Y. Xu, Y. Zhang, L. Jin, C. Han, J. Wang, Shikonin induces apoptosis and G0/G1 phase arrest of gallbladder cancer cells via the JNK signaling pathway, Oncol Rep 38 (2017) 3473–3480. [91] Q. Yang, M. Ji, H. Guan, B. Shi, P. Hou, Shikonin inhibits thyroid cancer cell growth and invasiveness through targeting major signaling pathways, J Clin Endocr Metab 98 (2013) E1909–E1917. [92] T.F. Franke, D.R. Kaplan, L.C. Cantley, A. Toker, Direct regulation of the Akt protooncogene product by phosphatidylinositol-3,4-bisphosphate, Science 275 (1997) 665–668. [93] A. Klippel, W.M. Kavanaugh, D. Pot, L.T. Williams, A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain, Mol Cell Biol 17 (1997) 338–344. [94] N.N. Ahmed, H.L. Grimes, A. Bellacosa, T.O. Chan, P.N. Tsichlis, Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase, Proc Nat Acad Sci U S A 94 (1997) 3627–3632. [95] A. Bellacosa, T.O. Chan, N.N. Ahmed, K. Datta, S. Malstrom, D. Stokoe, F. McCormick, J. Feng, P. Tsichlis, Akt activation by growth factors is a multiplestep process: the role of the PH domain, Oncogene 17 (1998) 313–325. [96] D.R. Alessi, M. Deak, A. Casamayor, F.B. Caudwell, N. Morrice, D.G. Norman, P. Gaffney, C.B. Reese, C.N. MacDougall, D. Harbison, A. Ashworth, M. Bownes, 3Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase, Curr Biol: CB 7 (1997) 776–789. [97] D.R. Alessi, S.R. James, C.P. Downes, A.B. Holmes, P.R. Gaffney, C.B. Reese, P. Cohen, Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha, Curr Biol: CB 7 (1997) 261–269. [98] D.D. Sarbassov, D.A. Guertin, S.M. Ali, D.M. Sabatini, Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex, Science 307 (2005) 1098–1101. [99] R.C. Hresko, M. Mueckler, mTOR.RICTOR is the Ser473 kinase for Akt/protein kinase B in 3T3-L1 adipocytes, J Biol Chem 280 (2005) 40406–40416. [100] A. Patke, I. Mecklenbrauker, H. Erdjument-Bromage, P. Tempst, A. Tarakhovsky, BAFF controls B cell metabolic fitness through a PKC beta- and Akt-dependent mechanism, J Exp Med 203 (2006) 2551–2562. [101] J. Joshi, P.J. Fernandez-Marcos, A. Galvez, R. Amanchy, J.F. Linares, A. Duran, P. Pathrose, M. Leitges, M. Canamero, M. Collado, C. Salas, M. Serrano, J. Moscat, M.T. Diaz-Meco, Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis, EMBO J 27 (2008) 2181–2193. [102] L. Bozulic, B.A. Hemmings, PIKKing on PKB: regulation of PKB activity by phosphorylation, Curr Opin Cell Biol 21 (2009) 256–261. [103] Y. Liao, M.C. Hung, Physiological regulation of Akt activity and stability, Am J Transl Res 2 (2010) 19–42. [104] B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream, Cell 129 (2007) 1261–1274. [105] D.A. Fruman, H. Chiu, B.D. Hopkins, S. Bagrodia, L.C. Cantley, R.T. Abraham, The PI3K pathway in human disease, Cell 170 (2017) 605–635. [106] F.Y. Zhang, Y. Hu, Z.Y. Que, P. Wang, Y.H. Liu, Z.H. Wang, Y.X. Xue, Shikonin inhibits the migration and invasion of human glioblastoma cells by targeting phosphorylated beta-catenin and phosphorylated PI3K/Akt: A potential mechanism for the anti-glioma efficacy of a traditional chinese herbal medicine, Int J Mol Sci 16 (2015) 23823–23848. [107] Y. Chen, T. Wang, J. Du, Y. Li, X. Wang, Y. Zhou, X. Yu, W. Fan, Q. Zhu, X. Tong, Y. Wang, The critical role of PTEN/PI3K/AKT signaling pathway in shikonin-induced apoptosis and proliferation inhibition of chronic myeloid leukemia, Cell Physiol Biochem 47 (2018) 981–993. [108] Y. Zhang, B. Sun, Z. Huang, D.W. Zhao, Q. Zeng, Shikonin inhibites migration and invasion of thyroid cancer cells by downregulating DNMT1, Med Sci Monit 24 (2018) 661–670. [109] K. Nigorikawa, K. Yoshikawa, T. Sasaki, E. Iida, M. Tsukamoto, H. Murakami, T. Maehama, K. Hazeki, O. Hazeki, A naphthoquinone derivative, shikonin, has insulin-like actions by inhibiting both phosphatase and tensin homolog deleted on chromosome 10 and tyrosine phosphatases, Mol Pharmacol 70 (2006) 1143–1149. [110] J. Zhu, J. Blenis, J. Yuan, Activation of PI3K/Akt and MAPK pathways regulates Myc-mediated transcription by phosphorylating and promoting the degradation of Mad1, Proc Nat Acad Sci U S A 105 (2008) 6584–6589. [111] M.W. Gramling, C.M. Eischen, Suppression of Ras/Mapk pathway signaling inhibits Myc-induced lymphomagenesis, Cell Death Differ 19 (2012) 1220–1227. [112] F. Morrish, N. Neretti, J.M. Sedivy, D.M. Hockenbery, The oncogene c-Myc coordinates regulation of metabolic networks to enable rapid cell cycle entry, Cell cycle 7 (2008) 1054–1066. [113] A. Wolfer, S. Ramaswamy, MYC and metastasis, Cancer Res 71 (2011) 2034–2037. [114] N. Meyer, L.Z. Penn, Reflecting on 25 years with MYC, Nat Rev Cancer 8 (2008) 976–990. [115] D.M. Miller, S.D. Thomas, A. Islam, D. Muench, K. Sedoris, c-Myc and cancer metabolism, Clin Cancer Res 18 (2012) 5546–5553. [116] G. Bretones, M.D. Delgado, J. Leon, Myc and cell cycle control, Biochim Biophys Acta 1849 (2015) 506–516. [117] T.R. Henry, K.B. Wallace, Differential mechanisms of induction of the mitochondrial permeability transition by quinones of varying chemical reactivities, Toxicol Appl Pharm 134 (1995) 195–203.

338

Fitoterapia 134 (2019) 329–339

F. Wang, et al.

Biol Macromol 107 (2018) 204–211. [147] Y. Su, N. Huang, D. Chen, L. Zhang, X. Dong, Y. Sun, X. Zhu, F. Zhang, J. Gao, Y. Wang, K. Fan, P. Lo, W. Li, C. Ling, Successful in vivo hyperthermal therapy toward breast cancer by Chinese medicine shikonin-loaded thermosensitive micelle, Int J Nanomed 12 (2017) 4019–4035. [148] A. Terada, Y. Tanoue, A. Hatada, H. Sakamoto, Total synthesis of Shikalkin [(+/-)-Shikonin], J Chem Soc Chem Comm (1983) 987–988. [149] R. Wang, R. Yin, W. Zhou, D. Xu, S. Li, Shikonin and its derivatives: a patent review, Exper Opin Ther Pat 22 (2012) 977–997. [150] E.A. Couladouros, A.T. Strongilos, V.P. Papageorgiou, Z.F. Plyta, A new efficient route for multigram asymmetric synthesis of alkannin and shikonin, Chem Eur J 8 (2002) 1795–1803. [151] K.C. Nicolaou, D. Hepworth, Concise and efficient total syntheses of alkannin and Shikonin, Angew Chem Int Ed 37 (1998) 839–841. [152] D.F. Xu, X.J. Guo, Z.H. Xu, S.S. Li, A novel total synthesis of (+/-)shikonin, Chinese Chem Lett 17 (2006) 871–873. [153] H. Yang, Q. Li, J.X. Bao, W. Lu, H.A. Aisa, J.C. Cai, New synthesis of (+/-)shikalkin, Chinese Chem Lett 13 (2002) 515–518. [154] Q. Lu, H.L. Tang, Y.Q. Shao, J.C. Cai, A new facile synthesis of shikalkin, Chinese Chem Lett 19 (2008) 172–174. [155] R.B. Wang, H. Guo, J.H. Cui, S.S. Li, A novel and efficient total synthesis of shikonin, Tetrahedron Lett 53 (2012) 3977–3980. [156] R.B. Wang, S.S. Zhou, H.D.G.L. Jiang, X.G. Zheng, W. Zhou, S.S. Li, An efficient multigram synthesis of alkannin and Shikonin, Eur J Org Chem (2012) 1373–1379. [157] J.G. Zhang, Q. Lu, W.H. Duan, J.C. Cai, New asymmetric synthesis of alkannin and shikonin, Chinese Chem Lett 16 (2005) 465–467. [158] X.H. Wu, L. Xu, J.C. Cai, Asymmetric synthesis and antitumor activity of cycloalkanin, Bioorg Med Chem Lett 9 (1999) 2635–2638. [159] Z. Rao, X. Liu, W. Zhou, J. Yi, S.S. Li, Synthesis and antitumor activity of β-hydroxyisovalerylshikonin analogues, Eur J Med Chem 46 (2011) 3934–3941. [160] H. He, L.P. Bai, Z.H. Jiang, Synthesis and human telomeric G-quadruplex DNAbinding activity of glucosaminosides of shikonin/alkannin, Bioorg Med Chem Lett 22 (2012) 1582–1586. [161] M.G. Miller, A. Rodgers, G.M. Cohen, Mechanisms of toxicity of naphthoquinones to isolated hepatocytes, Biochem Pharmacol 35 (1986) 1177–1184. [162] H.W. Moore, Bioactivation as a model for drug design bioreductive alkylation, Science 197 (1977) 527–532. [163] Z.F. Plyta, T.H. Li, V.P. Papageorgiou, A.S. Mellidis, A.N. Assimopoulou, E.N. Pitsinos, E.A. Couladouros, Inhibition of topoisomerase I by naphthoquinone derivatives, Bioorg Med Chem Lett 8 (1998) 3385–3390. [164] W. Wang, M. Dai, C. Zhu, J. Zhang, L. Lin, J. Ding, W. Duan, Synthesis and

[165]

[166]

[167]

[168]

[169]

[170]

[171]

[172] [173]

[174]

[175]

[176]

339

biological activity of novel shikonin analogues, Bioorg Med Chem Lett 19 (2009) 735–737. H. Inouye, S. Ueda, K. Inoue, H. Matsumura, Quinones and related compounds in higher-plants .8. Biosynthesis of Shikonin in Callus-Cultures of LithospermumErythrorhizon, Phytochemistry 18 (1979) 1301–1308. A. Rai, T. Nakaya, Y. Shimizu, M. Rai, M. Nakamura, H. Suzuki, K. Saito, M. Yamazaki, De Novo transcriptome assembly and characterization of lithospermum officinale to discover putative genes involved in specialized metabolites biosynthesis, Planta Med 84 (2018) 920–934. S. Wang, R. Wang, T. Liu, Z. Zhan, L. Kang, Y. Wang, C. Lv, D. Werck-Reichhart, L. Guo, L. Huang, Production of 3-geranyl-4-hydroxybenzoate acid in yeast, an important intermediate of shikonin biosynthesis pathway, FEMS Yeast Res 17 (2017), https://doi.org/10.1093/femsyr/fox064. F.Y. Wu, C.Y. Tang, Y.M. Guo, Z.W. Bian, J.Y. Fu, G.H. Lu, J.L. Qi, Y.J. Pang, Y.H. Yang, Transcriptome analysis explores genes related to shikonin biosynthesis in Lithospermeae plants and provides insights into Boraginales' evolutionary history, Sci Rep 7 (2017) 4477. L. Heide, M. Tabata, Geranylpyrophosphate - para-hydroxybenzoate geranyltransferase activity in extracts of lithospermum-erythrorhizon cell-cultures, Phytochemistry 26 (1987) 1651–1655. M. Tsukada, M. Tabata, Intracellular-localization and secretion of naphthoquinone pigments in cell-cultures of lithospermum-erythrorhizon, Planta Med 50 (1984) 338–341. L. Lin, J. Wu, Enhancement of shikonin production in single- and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound, Biotechnol Bioeng 78 (2002) 81–88. M.M. Hasan, T. Bashir, H. Bae, Use of ultrasonication technology for the increased production of plant secondary metabolites, Molecules 22 (2017) 1046. F. Bagheri, R. Tahvilian, N. Karimi, M. Chalabi, M. Azami, Shikonin production by callus culture of onosma bulbotrichom as active pharmaceutical ingredient, Iran J Pharm Res: IJPR 17 (2018) 495–504. M. Jeziorek, K. Syklowska-Baranek, A. Pietrosiuk, Hairy root cultures for the production of anti-cancer naphthoquinone compounds, Curr Med Chem 25 (2018) 4718–4739 https https://doi.org/10.2174/0929867324666170821161844. Y. Zhu, S.J. Chu, Y.L. Luo, J.Y. Fu, C.Y. Tang, G.H. Lu, Y.J. Pang, X.M. Wang, R.W. Yang, J.L. Qi, Y.H. Yang, Involvement of LeMRP, an ATP-binding cassette transporter, in shikonin transport and biosynthesis in Lithospermum erythrorhizon, Plant Biol 20 (2018) 365–373. C.S. Huang, H.W. Chen, T.Y. Lin, A.H. Lin, C.K. Lii, Shikonin upregulates the expression of drug-metabolizing enzymes and drug transporters in primary rat hepatocytes, J Ethnopharmacol 216 (2018) 18–25.