Effect Of Shikonin Research Articles

Shikonin Inhibits Oral Cancer Progression Through Suppression of Metastasis and Angiogenesis and Induction of Autophagy and Apoptosis

Background: Shikonin, a compound extracted from Lithospermum erythrorhizon, has demonstrated therapeutic effects on cancer; however, its effects on oral cancer remain unclear. Objectives: This study aimed to explore the therapeutic value of shikonin for the treatment of oral cancer. Methods: MTT, colony formation, and Transwell assays were employed to evaluate the inhibitory effects of shikonin on the proliferation and migration abilities of human oral cancer cell lines (SCC-4 and SAS). Apoptosis and cell viability were assessed using the TdT-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. To investigate the potential anticancer mechanisms of shikonin, RNA sequencing, quantitative PCR, and western blotting were performed to analyze changes in the expression levels of oral cancer cell-related genes and proteins (matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), VEGF-A, VEGF-C, Beclin-1, autophagy-related genes (ATG5), and light chain-3 (LC-3)). Additionally, animal xenograft experiments were conducted to examine the in vivo antitumor effects of shikonin. Results: The findings revealed that the external application of shikonin specifically targeted oral cancer cells without affecting normal cells and led to a dose-dependent inhibition of their growth. Even at non-lethal doses, shikonin effectively suppressed the production of metalloproteinases, thereby inhibiting cancer cell migration and wound healing. Furthermore, shikonin treatment reduced levels of tumor progression factors, such as vascular endothelial growth factor (VEGF)-A and VEGF-C, which are released during the early stages of cancer cell angiogenesis and lymphangiogenesis. Meanwhile, higher doses of shikonin induced cell autophagy and activated proteins such as ATG-5, LC-3B, and Beclin-1. At lethal doses, shikonin further decreased mitochondrial membrane potential, released calcium ions, and triggered apoptotic pathways. However, the administration of a calcium ion chelator (BAPTA-AM) inhibited shikonin-induced apoptosis. Conclusions: These results demonstrate that shikonin induces autophagy and activates apoptotic pathways in oral cancer cells. Shikonin treatment significantly inhibited oral cancer growth and induced apoptosis in a rat model. In conclusion, shikonin effectively inhibited oral cancer cell growth, metastasis, and the expression of tumor progression-related proteins. Given the ease of drug delivery to the affected area in oral cancer, shikonin holds substantial potential for future applications that may improve patient recovery and enhance cure rates.

Shikonin protects skin cells against oxidative stress and cellular dysfunction induced by fine particulate matter.

Shikonin, an herbal naphthoquinone, demonstrates a broad spectrum of pharmacological properties. Owing to increasingly adverse environmental conditions, human skin is vulnerable to harmful influences from dust particles. This study explored the antioxidant capabilities of shikonin and its ability to protect human keratinocytes from oxidative stress induced by fine particulate matter (PM2.5). We found that shikonin at a concentration of 3 µM was nontoxic to human keratinocytes and effectively scavenged reactive oxygen species (ROS) while increasing the production of reduced glutathione (GSH). Furthermore, shikonin enhanced GSH level by upregulating glutamate-cysteine ligase catalytic subunit and glutathione synthetase mediated by nuclear factor-erythroid 2-related factor. Shikonin reduced ROS levels induced by PM2.5, leading to recovering PM2.5-impaired cellular biomolecules and cell viability. Shikonin restored the GSH level in PM2.5-exposed keratinocytes via enhancing the expression of GSH-synthesizing enzymes. Notably, buthionine sulphoximine, an inhibitor of GSH synthesis, diminished effect of shikonin against PM2.5-induced cell damage, confirming the role of GSH in shikonin-induced cytoprotection. Collectively, these findings indicated that shikonin could provide substantial cytoprotection against the adverse effects of PM2.5 through direct ROS scavenging and modulation of cellular antioxidant system.

Effect and mechanisms of shikonin on breast cancer cells in vitro and in vivo

BackgroundBreast cancer seriously affects physical and mental health of women. Despite advances in the clinical use of different treatments, breast cancer remains a major cause of mortality. Therefore, it is imperative to identify promising treatment options. In the present study, we investigated the effects of shikonin on 4T1 breast cancer cells and its potential mechanisms of action.MethodsBALB/c-derived mouse breast cancer 4T1 is very close to human breast cancer in growth characteristics and systemic response, so 4T1 cells were selected for further experiments. Cell viability, apoptosis, intracellular reactive oxygen species (ROS), mitochondrial activity, and cellular calreticulin (CRT) exposure were assessed to evaluate the antitumor effects and mechanisms of shikonin in vitro. Orthotopic tumor growth inhibition and splenic immune cell regulation by shikonin were evaluated in 4T1 breast cancer orthotopic mice in vivo.ResultsIn vitro, shikonin could inhibit cell proliferation, cause apoptosis, disrupt mitochondrial activity, and induce ROS production and CRT exposure. In vivo, shikonin inhibited tumor growth, increased the proportion of CD8+ T cells, and reduced the proportion of regulatory cells (CD25+ Foxp3+ T cells) in the spleen.ConclusionsShikonin inhibits the growth of 4T1 breast cancer cells by disrupting mitochondrial activity, promoting oxidative stress, and regulating immune function.

Shikonin ameliorated LPS-induced acute lung injury in mice via modulating MCU-mediated mitochondrial Ca2+ and macrophage polarization

BackgroundMacrophages play a pivotal role in the development and recovery of acute lung injury (ALI), wherein their phenotypic differentiation and metabolic programming are orchestrated by mitochondria. Specifically, the mitochondrial calcium uniporter (MCU) regulates mitochondrial Ca2+ (mCa2+) uptake and may bridge the metabolic reprogramming and functional regulation of immune cells. However, the precise mechanism on macrophages remains elusive. Shikonin, a natural naphthoquinone, has demonstrated efficacy in mitigating ALI and suppressing glycolysis in macrophages, yet which mechanism remains to be fully elucidated. PurposeThis study explored whether Shikonin ameliorated ALI via modulating MCU-mediated mCa2+ and macrophage polarization. MethodsThis study firstly examined the protective effects of Shikonin on LPS-induced ALI mice, and investigated whether it is depends on macrophage by depleting macrophage using clodronate liposomes. The regulatory effect of Shikonin on macrophage polarization and mitochondrial MCU/Ca2+ signal was testified on RAW264.7 cells, and further validated by knocking-down MCU expression or by using RU360, an MCU inhibitor. Additionally, the crucial role of MCU in the therapeutic effect of Shikonin, along with its regulation on macrophage polarization was validated in mice with LPS-induced ALI under the intervention of RU360. ResultsShikonin alleviated LPS-induced mice ALI, down-regulated inflammatory cytokines and inhibited the pro-inflammatory polarization of macrophages. Intravenous injection of clodronate liposomes on mice abolished the protective effects of Shikonin on ALI. On RAW264.7 cells, LPS&IFN decreased the protein expression of MCU, while induced pro-inflammatory polarization and glycolytic metabolism. In contrast, Shikonin increased MCU expression, activated MCU-mediated mCa2+ signal, promoted the polarization of macrophages to anti-inflammatory M2 phenotype, and driven a metabolic shift from glycolysis to oxidative phosphorylation. Either knocking-down MCU expression or pharmacological inhibiting MCU by using RU360 mitigated the effects of Shikonin on Raw 264.7 cells. Furthermore, RU360 counteracted the ameliorative effect of Shikonin on ALI mice. ConclusionThe current data showed that Shikonin alleviated LPS-induced mice ALI by activating mitochondrial MCU/mCa2+ signal and regulating macrophage metabolism.

Systematic bioinformatics analysis reveals the role of shikonin in blocking colon cancer progression by identifying senescence-induced genes.

Shikonin, a naturally occurring naphthoquinone compound extracted from comfrey plants, has antitumor, anti-inflammatory, and antimicrobial properties. Cell senescence plays a key role in preventing tumor progression. It is unclear whether shikonin has an effect on cell senescence in colon cancer. In the current study, we first determine the IC50 values of shikonin on colon cancer cell lines HT29 and HCT116. Then, we verified the inhibitory effects of shikonin on the proliferation and migration abilities of colon cancer cell lines HT29 and HCT116 using cell counting kit-8, colony formation, and wound healing assays. Next, we identified a series of potential targets using high-throughput mRNA sequencing and identified 210 upregulated and 296 downregulated genes. KEGG profiling revealed eight downregulated genes associated with cell senescence: CCNB3, IL-1α, CXCL8, CDKN2A, MYC, IGFBP3, SQSTM1, and GADD45G. Among them, CXCL8 and CDKN2A were associated with poor prognosis in patients with colon cancer, suggesting that their downregulation by shikonin could improve patient survival. Furthermore, SA-β-galactosidase staining revealed that the percentage of cellular senescence in colon cancer cells was significantly increased after shikonin treatment. Molecular docking revealed that shikonin suppressed colon cancer progression by blocking CXCL8 activity. Based on these findings, we deem that shikonin might induce senescence and exert antitumor activity in colon cancer cells by downregulating CDKN2A and CXCL8. This provides a new molecular mechanism and potential therapeutic target for shikonin to inhibit colon cancer progression.

Shikonin Suppresses Cell Tumorigenesis in Gastric Cancer Associated with the Inhibition of c-Myc and Yap-1.

The study aimed to study the potential roles and mechanisms of shikonin in gastric cancer by network pharmacology and biological experiments. The key genes and targets of shikonin in gastric cancer were predicted by network pharmacology and molecular docking study. The effect of shikonin on the proliferation, migration, and invasion of gastric cancer cells was detected by the CCK8 method, and wound healing and transwell assays. The expression levels of c-Myc and Yap-1 were detected via western blotting in gastric cancer cells after shikonin intervention. The results of network pharmacology revealed the key target genes of shikonin on gastric cancer cells to be c-Myc, Yap-1, AKT1, etc. GO and KEGG analysis showed regulation of cell migration, proliferation, adhesion, and other biological processes, including the PI3K-Akt signaling pathway, HIF-1 signaling pathway, necroptosis, and other cancer pathways. Molecular docking showed shikonin to be most closely combined with protooncogenes c-Myc and Yap-1. In vitro experiments showed that the proliferation rate, migration, and invasion ability of the gastric cancer cell group decreased significantly after shikonin intervention for 24h. The expression levels of c-Myc and Yap-1 in gastric cancer cells were found to be significantly decreased after shikonin intervention. This study showed protooncogenes c-Myc and Yap-1 to be the core target genes of shikonin on gastric cancer cells. Shikonin may suppress gastric cancer cells by inhibiting the protooncogenes c-Myc and Yap-1. This suggests that shikonin may be a good candidate for the treatment of gastric cancer.

Shikonin ameliorates depressive- and anxiogenic-like behaviors in rats via the suppression of inflammation in the hippocampus

Shikonin is an active naphthoquinone with antioxidative, anti-inflammatory, and anticancer properties. In this study, we investigated the effects of shikonin on depressive- and anxiety-like behaviors in lipopolysaccharide- (LPS-) induced depression and chronic unpredictable mild stress (CUMS) rat models and explored the potential mechanism. First, a 14-day intraperitoneal administration of shikonin (10 mg/kg) significantly decreased immobility time in forced swimming test (FST) and increased open arm entries in elevated plus maze (EPM) test, without affecting line crossings in open field test (OFT), indicating that shikonin has anti-depressant- and anxiolytic-like effects. Second, chronic shikonin administration (10 mg/kg) reversed depressive- and anxiety-like behaviors in LPS-induced and CUMS depression models, as shown in the sucrose preference test (SPT), FST, EPM, and novel object recognition test (NORT). Finally, shikonin significantly reduced the levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) in hippocampus, indicating that the anti-depressant- and anxiolytic-like effects of shikonin are related to the reduction of neuroinflammation in hippocampus. These findings suggest that shikonin exerts anti-depressant- and anxiolytic-like effects via an anti-inflammatory mechanism of shikonin in the hippocampus.

Qingxiong Ointment and its Active Ingredient, Shikonin Treat Psoriasis through HIF-1 Signaling Pathway.

Psoriasis is a common chronic inflammatory skin disorder. Qingxiong ointment (QX) is a natural medicinal combination frequently employed in clinical treatment of psoriasis. However, the active ingredients of QX and its precise mechanisms of improving psoriasis remain unclear. This study elucidated the effects of QX on an Imiquimod (IMQ)-induced mouse model of psoriasis while also exploring the regulation of the active ingredient of QX, shikonin, on the HIF-1 signaling pathway in HaCaT cells. A mouse model of psoriasis was established through topical application of IMQ, and the local therapeutic effect of QX was evaluated using dorsal skin tissue with mouse psoriatic lesion and Psoriasis Area Severity Index (PASI) scores, hematoxylin-eosin (HE) staining, and immunohistochemical staining. Elisa and qPCR were employed to identify changes in the expression of inflammation-related factors in the mouse dorsal skin. Immunofluorescence was used to assess changes in the expression of T cell subsets before and after treatment with various doses of QX. HPLC was used to analyze the content of shikonin, and network pharmacology was employed to analyze the main targets of shikonin. Immunofluorescence was used to identify the effects of shikonin on the HIF-1 signaling pathway in IL6-induced psoriasis HaCaT cells. Finally, qPCR was used to identify the differential expression of the HIF-1 signaling pathway in skin tissues. QX significantly reduces PASI scores on the backs of IMQ-induced psoriasis mice. HE staining reveals alleviated epidermal thickness in the QX group. Immunohistochemical analysis shows a significant reduction in ICAM, KI67, and IL17 expression levels in the QX group. Immunofluorescence results indicate that QX can notably decrease the proportions of CD4+ T cells, γδ T cells, and CD8+ T cells while increasing the proportion of Treg cells. Network pharmacology analysis demonstrates that the main targets of shikonin are concentrated in the HIF-1 signaling pathway. Molecular docking results show favorable binding affinity between shikonin and key genes of the HIF-1 signaling pathway. Immunofluorescence results reveal that shikonin significantly reduces p-STAT3, SLC2A1, HIF1α, and NOS2 expression levels. qPCR results show significant downregulation of the HIF-1 signaling pathway at cellular and tissue levels. Our study revealed that QX can significantly reduce the dorsal inflammatory response in the IMQ-induced psoriasis mouse model. Furthermore, we discovered that its main component, shikonin, exerts its therapeutic effect by diminishing the HIF-1 signaling pathway in HaCaT cells.

Shikonin blocks CAF-induced TNBC metastasis by suppressing mitochondrial biogenesis through GSK-3β/NEDD4-1 mediated phosphorylation-dependent degradation of PGC-1α

BackgroundTriple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore.MethodsWe investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan–Meier plotter database and clinical breast cancer tissue samples.ResultsWe demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3β-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo.ConclusionsOur findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.

Effect of Shikonin on Autophagy and Apoptosis of Human Promyelocytic Leukemia Cells

To explore the effect of shikonin on autophagy and apoptosis of human promyelocytic leukemia cells and its possible mechanism. Human promyelocytic leukemia cells NB4 in the logarithmic growth phase were divided into control group (untreated NB4 cells), shikonin group (0.3 µmol/L shikonin treatment), 740Y-P group (15 µmol/L PI3K/Akt/mTOR pathway activator 740Y-P treatment), shikonin+740Y-P group (0.3 µmol/L shikonin and 15 µmol/L 740Y-P co-treatment), after 24 hours of treatment, the cells were used for subsequent experiments. CCK-8 method was used to detect cell viability, monodansylcadaverine (MDC) staining to detect the aggregation of autophagic vesicles, flow cytometry to detect cell apoptosis, and Western blot to detect the expression of Beclin1, LC3, p62, Bax, cleaved caspase-3, Bcl-2 and PI3K/Akt/mTOR pathway related proteins. Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were increased in the shikonin group, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were decreased (all P < 0.05). Compared with the control group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the 740Y-P group (all P < 0.05). Compared with the shikonin group, the purple punctate fluorescence intensity, apoptosis rate, Beclin1, LC3-Ⅱ/LC3-Ⅰ, cleaved caspase-3, and Bax protein expression in NB4 cells were decreased, while OD450 value (24, 48 h) and the expressions of Bcl-2 and p62 proteins were increased in the shikonin+740Y-P group (all P < 0.05). Compared with the control group, the expression of PI3K/Akt/mTOR pathway related proteins p-PI3K, p-Akt, and p-mTOR in NB4 cells were significantly decreased in the shikonin group, while those in the 740Y-P group were increased (all P < 0.05). Compared with the shikonin group, the expressions of p-PI3K, p-Akt, and p-mTOR proteins in NB4 cells were significantly increased in the shikonin+740Y-P group (all P < 0.05). Shikonin may promote autophagy and apoptosis of NB4 cells by inhibiting PI3K/Akt/mTOR pathway.

Shikonin attenuates cerebral ischemia/reperfusion injury via inhibiting NOD2/RIP2/NF-κB-mediated microglia polarization and neuroinflammation

ObjectivesMicroglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, which still lacks effective therapeutic agents. Shikonin possesses anti-inflammatory and neuroprotective properties. However, its underlying mechanism remains elusive. This study aimed to investigate whether Shikonin confers protection against cerebral ischemia/reperfusion (I/R) injury by modulating microglial polarization and elucidate the associated mechanisms. MethodsThis study employed an oxygen-glucose deprivation and reoxygenation (OGD/R) BV2 microglial cellular model and a middle cerebral artery occlusion/reperfusion (MCAO/R) animal model to investigate the protection and underlying mechanism of Shikonin against ischemic stroke. ResultsThe results demonstrated that Shikonin treatment significantly reduced brain infarction volume and improved neurological function in MCAO/R rats. Simultaneously, Shikonin treatment significantly reduced microglial proinflammatory phenotype and levels of proinflammatory markers (inducible-NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-6), increased microglial anti-inflammatory phenotype and levels of anti-inflammatory markers (Arginase-1 (Arg1), transforming growth factor-beta (TGF-β), and IL-10), reversed the expression of Nucleotide-binding oligomerization domain 2 (NOD2) and phosphorylation receptor interacting protein 2 (p-RIP2), and suppressed nuclear factor kappa-B (NF-κB) signaling activation in the ischemic penumbra regions. These effects of Shikonin were further corroborated in OGD/R-treated BV2 cells. Furthermore, overexpression of NOD2 markedly attenuated the neuroprotective effects of Shikonin treatment in MCAO/R rats. NOD2 overexpression also attenuated the regulatory effects of Shikonin on neuroinflammation, microglial polarization, and NF-κB signaling activation. ConclusionThis study illustrates that Shikonin mitigates inflammation mediated by microglial proinflammatory polarization by inhibiting the NOD2/RIP2/NF-κB signaling pathway, thereby exerting a protective role. The findings uncover a potential molecular mechanism for Shikonin in treating ischemic stroke.

Mechanism of Shikonin on spinal cord injury in rats based on TNFR/RIPK1 pathway

To explore the effect of shikonin on the recovery of nerve function after acute spinal cord injury(SCI) in rats. 96 male Sprague-Dawley(SD)rats were divided into 4 groups randomly:sham operation group (Group A), sham operation+shikonin group (Group B), SCI+ DMSO(Group C), SCI+shikonin group (Group D).The acute SCI model of rats was made by clamp method in groups C and D . After subdural catheterization, no drug was given in group A. rats in groups B and D were injected with 100 mg·kg-1 of shikonin through catheter 30 min after modeling, and rats in group C were given with the same amount of DMSO, once a day until the time point of collection tissue. Basso-Beattie-Bresnahan(BBB) scores were performed on 8 rats in each group at 6, 12, and 3 d after moneling, and oblique plate tests were performed on 1, 3, 7 and 14 d after modeling, and then spinal cord tissues were collected. Eight rats were intraperitoneally injected with propidine iodide(PI) 1 h before sacrificed to detection PI positive cells at 24 h in each group. Eight rats were sacrificed in each group at 24 h after modeling, the spinal cord injury was observed by HE staining.The Nissl staining was used to observe survivor number of nerve cells. Western-blot technique was used to detect the expression levels of Bcl-2 protein and apoptosis related protein RIPK1. After modeling, BBB scores were normal in group A and B, but in group C and D were significantly higher than those in group A and B. And the scores in group D were higher than those in group C in each time point (P<0.05). At 12 h after modeling, the PI red stained cells in group D were significantly reduced compared with that in group C, and the disintegration of neurons was alleviated(P<0.05). HE and Nissl staining showed nerve cells with normal morphology in group A and B at 24h after operation. The degree of SCI and the number of neuronal survival in group D were better than those in group C, the difference was statistically significant at 24h (P<0.05). The expression of Bcl-2 and RIPK1 proteins was very low in group A and B;The expression of RIPK1 was significantly increased in Group C and decreased in Group D, with a statistically significant difference (P<0.05);The expression of Bcl-2 protein in group D was significantly higher than that in group C (P<0.05). Shikonin can alleviate the pathological changes after acute SCI in rats, improve the behavioral score, and promote the recovery of spinal nerve function. The specific mechanism may be related to the inhibition of TNFR/RIPK1 signaling pathway mediated necrotic apoptosis.

Shikonin alleviates doxorubicin-induced cardiotoxicity via Mst1/Nrf2 pathway in mice

Doxorubicin (DOX) is a popular and potent anticancer drug, but its cardiotoxicity limits its clinical application. Shikonin has a wide range of biological functions, including antioxidant and anti-inflammatory effects. The aim of this study was to investigate the effects of shikonin on DOX-induced cardiac injury and to identify the underlying mechanisms. Mice receiving shikonin showed reduced cardiac injury response and enhanced cardiac function after DOX administration. Shikonin significantly attenuated DOX-induced oxidative damage, inflammation accumulation and cardiomyocyte apoptosis. Shikonin protects against DOX-induced cardiac injury by inhibiting Mammalian sterile 20-like kinase 1 (Mst1) and oxidative stress and activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. In conclusion, shikonin alleviates DOX-induced cardiotoxicity by inhibiting Mst1 and activating Nrf2. Shikonin may be used to treat DOX-induced cardiac injury.

The Anti-tumor Function of Shikonin by Targeting EGFR/AKT/mTOR Signaling in Human Osteosarcoma Cells

Background: Shikonin, a purified naphthoquinone separated from a Traditional Chinese medicinal herb Lithospermum erythrorhixon, which exhibits anticancer properties. Objectives: To clarify the molecular mechanisms of therapeutic effects of shikonin against osteosarcoma. Materials and Methods: Cell Counting Kit-8 (CCK-8) assay was employed to evaluate cell viability. Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) double-staining was conducted to evaluate the apoptotic ratio of the MG-63 cells. The effects of shikonin on the invasiveness of osteosarcoma cells were implemented by a transwell cell migration assay. In the meantime, a western blot assay was employed to detect alterations in the relevant mechanism proteins within osteosarcoma cells. Molecular docking analysis was conducted to anticipate the binding interaction between shikonin and EGFR/protein kinase B (AKT)/mTOR. Results: We observed that shikonin suppressed proliferation and induced apoptosis in the MG-63 cells in a dose-dependent manner. Pursuing these findings, the potential molecular mechanisms were detected. Shikonin intervention blocked epidermal growth factor receptor (EGFR) phosphorylation and decreased epidermal growth factor (EGF)-induced activation of downstream signaling molecules, such as AKT and mammalian target of rapamycin (mTOR) in the MG-63 cells. However, the additional recombinant human epidermal growth factor (rHuEGF) could stimulate the activation of EGFR/AKT/mTOR signaling and reverse cell apoptosis caused by shikonin. Molecular docking analysis showed that shikonin presented the highest bonding ability with EGFR, AKT, and mTOR. Conclusion: Our results show that shikonin inhibits human osteosarcoma development via inactivating EGFR/AKT/mTOR signaling. It demonstrates that shikonin may act as a potential therapeutic agent in osteosarcoma treatment.

Shikonin reactivates TSGs GADD45B and PPP3CC to block NSCLC cell proliferation and migration through JNK/P38/MAPK signaling pathways

BackgroundShikonin, a natural naphthoquinone compound extracted from the Chinese traditional herbal medicine “Lithospermum erythrorhizon”, possesses antitumor activity against various cancer types. Tumor-suppressor genes (TSGs) negatively regulate cell growth, proliferation, and differentiation, thereby inhibiting tumor formation. However, the molecular mechanism of action of shikonin on TSGs in non–small-cell lung cancer (NSCLC) remains unclear.MethodsThe inhibitory effect of shikonin on the proliferation and migration abilities of lung cancer cells were measured by Cell Counting Kit 8 (CCK8) and wound healing assays. The alteration of genes by shikonin treatment was detected by mRNA high-throughput sequencing and further confirmed by qPCR and western blotting experiments. The dominant functions of the upregulated genes were analyzed by GO and KEGG profiling.ResultsShikonin inhibited the proliferation and migration of A549 and H1299 NSCLC cells in a dose-dependent manner. mRNA high-throughput sequencing revealed a total of 1794 upregulated genes in shikonin-treated NSCLC cells. Moreover, bioinformatic analysis of GO and KEGG profiling revealed that the up-regulated genes were mostly involved in the JNK/P38/MAPK signaling pathway, among which the expression of GADD45B and PPP3CC was significantly enhanced. Finally, we confirmed that GADD45B and PPP3CC were indeed upregulated in JNK/P38/MAPK pathway.ConclusionsTaken together, these results suggested that shikonin might affect the expression of GADD45B and PPP3CC through the JNK/P38/MAPK pathway, therefore exerting an inhibitory effect on the proliferation and migration of cancer cells. To our knowledge, this is the first study reporting the role of shikonin in upregulating TSGs to activate the JNK/P38/MAPK signaling pathways in NSCLC.

The effect of shikonin on the metabolism of lapatinib in vitro, and in vivo

PurposeThe purpose of this study was to develop an UPLC-MS/MS assay for simultaneous determination of lapatinib and its metabolites (N-dealkylated lapatinib and O-dealkylated lapatinib) by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and to determine the interaction between shikonin and lapatinib in vitro, in vivo, in silico and its mechanism of action. MethodsA new UPLC-MS/MS method for the determination of the concentrations of lapatinib and its metabolites was developed. In vivo, Sprague-Dawley (SD) rats were given lapatinib with or without shikonin. In vitro, to study the interaction mechanism, rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant human CYP3A4.1 were used for determining enzyme kinetics. Lastly, we used in silico molecular docking to investigate the molecular mechanism of inhibition. ResultsThe selectivity, precision, accuracy, stability, matrix effect and recovery of UPLC-MS/MS all met the requirements of quantitative analysis of biological samples. Administration of lapatinib combined with shikonin resulted in significantly increased pharmacokinetic parameters (AUC(0-t) and Cmax) of lapatinib, indicating that shikonin increased the exposure of lapatinib in rats. Moreover, in vitro kinetic measurements indicated that shikonin was a time-independent inhibitor, which inhibited the metabolism of lapatinib through a competitive mechanism in RLMs, while noncompetitive inhibition type in both HLMs and CYP3A4.1. Molecular docking analysis further verified the non-competitive inhibition of shikonin on lapatinib in CYP3A4.1. ConclusionWe developed an UPLC-MS/MS assay for simultaneous determination of lapatinib and its metabolites. It could be successfully applied to the study of pharmacokinetic interaction of shikonin on the inhibition of lapatinib metabolism in vivo and in vitro. In the end, Further studies are needed to determine if such interactions are indeed valid in humans and if the interaction is clinically relevant.

Pharmacological Effects of Shikonin and Its Potential in Skin Repair: A Review.

Currently, skin injuries have a serious impact on people's lives and socio-economic stress. Shikonin, a naphthoquinone compound derived from the root of the traditional Chinese medicine Shikonin, has favorable biological activities such as anti-inflammatory, antibacterial, immunomodulatory, anticancer, and wound-healing-promoting pharmacological activities. It has been reported that Shikonin can be used for repairing skin diseases due to its wide range of pharmacological effects. Moreover, the antimicrobial activity of Shikonin can play a great role in food and can also reduce the number of pathogenic bacteria in food. This paper summarizes the research on the pharmacological effects of Shikonin in recent years, as well as research on the mechanism of action of Shikonin in the treatment of certain skin diseases, to provide certain theoretical references for the clinical application of Shikonin, and also to provides research ideas for the investigation of the mechanism of action of Shikonin in other skin diseases.

Shikonin reverses cancer-associated fibroblast-induced gemcitabine resistance in pancreatic cancer cells by suppressing monocarboxylate transporter 4-mediated reverse Warburg effect

BackgroundGemcitabine is a first-line chemotherapeutic agent for pancreatic cancer (PC); however, most patients who receive adjuvant gemcitabine rapidly develop resistance and recurrence. Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor stroma that contribute to gemcitabine-resistance. There is thus an urgent need to find a novel therapeutic strategy to improve the efficacy of gemcitabine in PC cells under CAF-stimulation. PurposeTo investigate if shikonin potentiates the therapeutic effects of gemcitabine in PC cells with CAF-induced drug resistance. MethodsPC cell-stimulated fibroblasts or primary CAFs derived from PC tissue were co-cultured with PC cells to evaluate the ability of shikonin to improve the chemotherapeutic effects of gemcitabine in vitro and in vivo. Glucose uptake assay, ATP content analysis, lactate measurement, real-time PCR, immunofluorescence staining, western blot, and plasmid transfection were used to investigate the underlying mechanism. ResultsCAFs were innately resistant to gemcitabine, but shikonin suppressed the PC cell-induced transactivation and proliferation of CAFs, reversed CAF-induced resistance, and restored the therapeutic efficacy of gemcitabine in the co-culture system. In addition, CAFs underwent a reverse Warburg effect when co-cultured with PC cells, represented by enhanced aerobic glycolytic metabolism, while shikonin reduced aerobic glycolysis in CAFs by reducing their glucose uptake, ATP concentration, lactate production and secretion, and glycolytic protein expression. Regarding the mechanism underlying these sensitizing effects, shikonin suppressed monocarboxylate transporter 4 (MCT4) expression and cellular membrane translocation to inhibit aerobic glycolysis in CAFs. Overexpression of MCT4 accordingly reversed the inhibitory effects of shikonin on PC cell-induced transactivation and aerobic glycolysis in CAFs, and reduced its sensitizing effects. Furthermore, shikonin promoted the effects of gemcitabine in reducing the growth of tumors derived from PC cells and CAF co-inoculation in BALB/C mice, with no significant systemic toxicity. ConclusionThese results indicate that shikonin reduced MCT4 expression and activation, resulting in inhibition of aerobic glycolysis in CAFs and overcoming CAF-induced gemcitabine resistance in PC. Shikonin is a promising chemosensitizing phytochemical agent when used in combination with gemcitabine for PC treatment. The results suggest that disrupting the metabolic coupling between cancer cells and stromal cells might provide an attractive strategy for improving gemcitabine efficacy.

Antimicrobial and antibiofilm effects of shikonin with tea tree oil nanoemulsion against Candida albicans and Staphylococcus aureus

The current work aims to develop a shikonin and tea tree oil loaded nanoemulsion system stabilized by a mixture of GRAS grade surfactants (Tween 20 and monoolein) and a cosurfactant (Transcutol P). This system was designed to address the poor aqueous solubility and photostability issues of shikonin. The authenticity of shikonin employed in this study was confirmed using nuclear magnetic resonance (NMR) spectroscopy. The optimized nanoemulsion exhibited highly favorable characteristics in terms of zeta potential (−23.8 mV), polydispersity index (0.216) and particle size (22.97 nm). These findings were corroborated by transmission electron microscopy (TEM) micrographs which confirmed the spherical and uniform nature of the nanoemulsion globules. Moreover, attenuated total reflectance (ATR) and X-ray diffraction analysis (XRD) analysis affirmed improved chemical stability and amorphization, respectively. Photodegradation studies were performed by exposing pure shikonin and the developed nanoemulsion to ultraviolet light for 1 h using a UV lamp, followed by high performance liquid chromatography (HPLC) analysis. The results confirmed that the developed nanoemulsion system imparts photoprotection to pure shikonin in the encapsulated system. Furthermore, the research investigated the effect of the nanoemulsion on biofilms formed by Candida albicans and methicillin resistant Staphylococcus aureus (MRSA). Scanning electron microscopy, florescence microscopy and phase contrast microscopy unveiled a remarkable reduction in biofilm area, accompanied by disruptions in the cell wall and abnormalities on the cell surface of the tested microorganisms. In conclusion, the nanoencapsulation of shikonin with tea tree oil as the lipid phase showcased significantly enhanced antimicrobial and antibiofilm potential compared to pure shikonin against resistant strains of Candida albicans and Staphylococcus aureus.

Shikonin Exerts an Antileukemia Effect against FLT3-ITD Mutated Acute Myeloid Leukemia Cells via Targeting FLT3 and Its Downstream Pathways

Introduction: Acute myeloid leukemia (AML) with internal tandem duplication (ITD) mutations in Fms-like tyrosine kinase 3 (FLT3) has an unfavorable prognosis. Recently, using newly emerging inhibitors of FLT3 has led to improved outcomes of patients with FLT3-ITD mutations. However, drug resistance and relapse continue to be significant challenges in the treatment of patients with FLT3-ITD mutations. This study aimed to evaluate the antileukemic effects of shikonin (SHK) and its mechanisms of action against AML cells with FLT3-ITD mutations in vitro and in vivo. Methods: The CCK-8 assay was used to analyze cell viability, and flow cytometry was used to detect cell apoptosis and differentiation. Western blotting and real-time polymerase chain reaction were used to examine the expression of certain proteins and genes. Leukemia mouse model was created to evaluate the antileukemia effect of SHK against FLT3-ITD mutated leukemia in vivo. Results: After screening a series of leukemia cell lines, those with FLT3-ITD mutations were found to be more sensitive to SHK in terms of proliferation inhibition and apoptosis induction than those without FLT3-ITD mutation. SHK suppresses the expression and phosphorylation of FLT3 receptors and their downstream molecules. Inhibition of the NF-κB/miR-155 pathway is an important mechanism through which SHK kills FLT3-AML cells. Moreover, a low concentration of SHK promotes the differentiation of AML cells with FLT3-ITD mutations. Finally, SHK could significantly inhibit the growth of MV4-11 cells in leukemia bearing mice. Conclusion: The findings of this study indicate that SHK may be a promising drug for the treatment of FLT3-ITD mutated AML.