Shikonin is a commonly used traditional Chinese medicine in clinic. It was first recorded in the Shennong herbal classic. It tastes bitter and cold. It has functions of cooling blood, activating blood circulation, detoxifying and so on. It can be used for clearing heat on the body surface, detoxifying by oral administration, activating blood circulation and removing blood stasis in local wounds^[1]Many studies have found that shikonin has significant antioxidant, anti-inflammatory, antibacterial, antiviral and anti-tumor pharmacological effects^[2]The active ingredients of Arnebia euchroma are mainly naphthoquinones extracted from its dried roots（naphthoquinone）Pigment, common and studied ones include Arnebia euchromaLithospermum erythrorhizon Sieb. etZucc. （D-shikonin，shikonin，1, fig1）; Arnebia Euchroma Arnebia euchroma JohnstThe main component of acarnin (L-shikonin,alkannin，2, fig1）, which is the enantiomer of Shikonin^[3]And Arnebia euchroma from Inner MongoliaArebia guttata BungeIsovaleryl shikonin extracted from（isovaleryl shikonin）^[4], other shikonin natural derivatives such asAcetylshikonin, deoxyshikonin, isobutyryl shikoninβ-Acetoxyisovaleryl shikonin, beta, beta dimethylacrylyl shikonin, etc^[5]。

In recent years, the pharmacological activities of shikonin, especially the anti-tumor effect, have beenTo extensive research^[6-7]。In order to reduce its toxicity to normal cells and improve the efficiency of tumor inhibition, researchers have modified the structure of shikonin, and the sites in its molecular structure that can be modified mainly include the mother nucleus naphthyl ring, containing6The side chains of carbon atoms and the hydroxyl groups on the side chains, thus obtainingA variety of different synthetic derivatives^[8-9], e.g2-dithiocarbamate- 3-Coumarin produced by modification of methyl substituted naphthoquinone compounds and side chain hydroxyl groups-Carboxyl shikonate derivativesPMMB229-240、1,4-Dioxime-5,8-Dimethoxynaphthalene, aryl dihydrothiazolyl shikonin ester derivatives, and their toxicity to cancer cells and normal cells were studied. The results showed that shikonin synthetic derivatives had better application prospects. In this paper, the effects of shikonin and its derivatives on4The anti-tumor effects and mechanisms of action of breast cancer, cervical cancer, ovarian cancer and endometrial cancer are reviewed.

1  Study on the anti gynecological tumor effect of Shikonin

1.1  Anti breast cancer

Chenetc.^[10]Studies on shikonin, acetylshikonin, andβ,β-Effect of Dimethylacrylate shikonin on human triple negative breast cancerMDA-MB-231Cell and mouse breast cancer4T1The cytotoxic effect of shikonin on4T1Cell growth was significantly inhibited. 72 hright4T1Half inhibitory concentration of cells（IC₅₀）For（0.83±0.03）μmol/L, rightMDA-MB-231CellularIC₅₀For（1.13±0.06）μmol/L, while it is less toxic to normal mammary epithelial cells,IC₅₀For（3.76±0.09）μmol/LShikonin was continuously administered to mice with lung metastasis of breast cancer（10 mg/kg）14 dAfter treatment, the total number of lung metastatic nodules in the treatment group was significantly less than that in the control group, the volume of nodules was smaller, and the area of lung metastatic foci was also significantly reduced. The results showed that shikonin could inhibit lung metastasis of breast cancer cells.

Yangetc.^[11]Discovery of human breast cancerSK-BR-3Cells were treated with shikonin at different concentrations48 hThe proliferation rate decreased and the apoptosis rate increased in a concentration dependent manner after Shikonin1 μmol/L（24±4）%, and the apoptosis rate was11.6%The difference was significant compared with the control group. In addition, Shikonin0.1、1、10 μmol/LTreatment of human breast cancerMCF-7cell24 hafter^[12]With the increase of shikonin concentration, the cell proliferation inhibition rate and apoptosis rate increased significantly. These results indicated that shikonin could inhibit the growth of breast cancer cells in a dose-dependent manner.

1.2  Anti cervical cancer

Shikonin derivatives were usedβ-Hydroxyisovaleryl Shikonin（β-hydroxyisovalerylshikonin，β-HIVS）1、5、10 μmol/LTreatment of human cervical cancerHeLaCells after different times^[13], the results showed that,β-HIVSIt inhibited cell proliferation and promoted apoptosis in a time - and dose-dependent manner, and the cell cycle distribution also changed significantly,G₀/G₁The number of phase cells decreased,SThe number of phase cells increased, whileG₂/MThere was no significant change in the number of phase cells, demonstrating thatβ-HIVSIt can arrest the cell cycle of cervical cancer cells atS.Linetc.^[14]Pyruvate dehydrogenase（PDH）Cofactor ofα-Lipoic acid was introduced into shikonin to synthesize a double effect compound with anti mitosis and glycolysis18speciesα-Shikonin lipoate derivatives, in which compounds1C(Fig2）RightHeLaCells have the strongest cytotoxicity,IC₅₀The value is（3.14±0.58）μmol/LOn the one hand, it acts as a tubulin inhibitorG₂/MPhase inducedHeLaCell cycle arrest.HeLaThe tumor bearing mice were treated with different doses（0.5～2.0 mg/kg）1CAfter treatment, the tumor volume was smaller than that of the control group, of which2 mg/kgThe average tumor volume of mice in the treatment group was smaller than that in the control group5Times, indicating that1C. On the other hand, through phosphoinositide dependent protein kinase（PDK）Inhibition of activity promotedPDHActivity, forcingHeLaCells undergo more aerobic metabolism and induce apoptosis.

Shikonin acts on human cervical cancer in vitroSiHaPost cellular^[15], cell proliferation index decreased,G₀/G₁Phase cells increased,SPhase cells decreased in a concentration dependent manner, which proved that shikonin could arrestSiHaCellularG₀/G₁Phase progression and inhibit cell proliferation. Shikonin treatment of human cervical cancerCaskicell12、24、36 hafter^[16]It was found that the inhibition rate of cell proliferation gradually increased, the number of invasion decreased, and phosphatase and tensin homologues（PTEN）The expression increased in a time - and dose-dependent manner. Shikonin can inhibit the proliferation and invasion of cervical cancer cells.

1.3  Anti ovarian cancer

Human ovarian cancer cellsSKOV3andA2780Shikonin（5、10 μmol/L）After treatment^[17]Compared with the control group, the apoptosis rate had no obvious change, but the levels of necroptosis related proteins in cells were significantly up-regulated, and the proportion of necrotic cells was significantly increased. IOSE-80Cells have little effect.

Shikonin can significantly reverse cisplatin resistant human ovarian cancer cellsSKOV3/DDPIn addition, the drug resistance in the cisplatin concentration was12.5 μmol/LShikonin combined with chemotherapy can significantly inhibit the cancer cell cycleG₁/STransformation, compared with cisplatin alone group, the early apoptosis rate of shikonin and cisplatin combined group increased^[18]。P-Glycoprotein（P-gp）It is an energy dependent transmembrane transporter and often acts as a tumor multidrug resistance（MDR）Markers of. Shikonin1 μmol/L, paclitaxel1 μmol/LTreatment of drug-resistant human ovarian cancer cells alone or in combinationA2780/PTX 24 hIt can significantly induce cancer apoptosis, and the apoptosis rate is6.3%、10.6%and30.9%, further study found that shikonin did not rely onP-gpThe manner of protein reversalA2780/PTXCell resistance to paclitaxel^[19]This suggests that shikonin combined with other anticancer drugs may provide a new therapeutic strategy for the treatment of ovarian cancer and other cancers.

1.4  Anti endometrial cancer

Endometrial cancer has70%～80%For estrogen dependent type, progesterone treatment is the main endocrine treatment measure, but more than30%Of early-stage patients were insensitive to progesterone^[20-21]Human endometrial cancer positive for estrogen receptorIshikawaCells have the effect of promoting proliferation, but in the presence of10 nmol/LWhen the concentration of shikonin was gradually increased in the culture medium of estrogen, the cell proliferation inhibition rate was significantly increased in a concentration and time-dependent manner^[22]In addition, different concentrations of shikonin were used to treat4Human endometrial cancer cellsIshikawa、HEC-1A、KLEandRL95-2 24 h, shikonin was found to reduce the viability of cancer cells in a dose-dependent manner, which was beneficial to4Seed cellIC₅₀Respectively3.63、4.81、8.22、8.96 μmol/L。IshikawaandHEC-1ACells are more sensitive to shikonin, which5 μmol/Lhandle24 hAfter, the apoptosis rates were significantly increased^[23]Shikonin can inhibit human endometrial cancerRL95-2Cell proliferation and promote its apoptosis^[24], in a dose - and time-dependent manner, where1.2 μg/mLThe apoptosis promoting effect was the best, and the apoptosis rate was（29.57±3.24）%The above results suggest that shikonin has different degrees of Pro apoptotic effects on different endometrial cancer cells in a concentration - and time-dependent manner, among which shikonin has a significant effect onIshikawaThe inhibitory growth and pro apoptotic activities of cells were stronger.

2  Mechanism of shikonin on gynecological tumor

2.1  Inhibition of tumor cell proliferation

Malignant proliferation is the most prominent feature of cancer cells. A large number of experiments have shown that shikonin can inhibit tumor cell proliferation through multiple target molecules and signaling pathways. There are research findings^[25]Shikonin can be used inmRNAAnd protein levels to induce bispecific phosphatases（DUSP1）AndDUSP2, which reduces its downstream signaling moleculesc-jun NTelokinase（JNK）Andp38Of activity, which blocks theG₁Phase, which in turn inhibitsMCF-7、MDA-MB-231andSK-BR-3Cell proliferation. Multiple studies^[15,18,20]It was found that shikonin at different concentrations acted onSiHa、SKOV3/DDP、IshikawaAfter cells,G₀/G₁The proportion of stage cells was significantly increased,G₂/MThe proportion of stage cells was significantly reduced,SThe mechanism of action is similar to that ofG（GPER）, cyclinsD1（Cyclin D1）, cell cycle dependent protein kinase2（CDK2）Related to the down-regulation of the expression level of isoproteins.

Cell division cycle proteins25（Cdc25）By regulating its substratesCDKThe phosphorylation status of α - Catenin plays a crucial role in cell cycle progression. Shikonin can inhibitCdc25Phosphatase activity, concentration dependent inhibitionCDK1Dephosphorylation, thereby inhibitingMCF-7andHeLaCell proliferation, shikonin was proved by in vivo experiments1、5、10 mg/kgIt has a good anti-cancer cell proliferation effect on xenograft tumor model mice, and blocks cells inG₂/Mstage^[26]。

FromMCF-7The exosomes of cells can be absorbed by tumor cells themselves and rapidly obtained from the microenvironmentmiR-128Exosomes downregulate Pro apoptotic genesBax, protect cancer cells from programmed death, andmiR-128Transmission promotion ofMCF-7Cell proliferation.qRT-PCRShikonin was detected by HPLC1 μmol/LProcessedMCF-7cellmiR-128The decreased exosome release suggested that shikonin could inhibit tumor proliferation by changing the tumor microenvironment^[27]。

2.2  Induction of tumor apoptosis

Apoptosis is a natural way to remove senescent cells from the body. However, the deregulation of apoptotic signals and the activation of anti apoptotic system in cancer cells allow cancer cells to evade the apoptotic program and proliferate uncontrolled, leading to tumor survival. Many anticancer therapies work by inducing apoptosis and its associated cell death network, shikonin is no exception.

Shikonin on4T1andMDA-MB-231Apoptosis of cells was induced. through2 μmol/LAfter shikonin treatment,4T1In cellsp38、JNKAnd phosphorylationJNKThe protein level was not significantly different from that of the control group,andPhosphorylationp38The protein expression level was increased, suggesting that shikonin is through activatingp38Signaling pathway instead ofJNKsignaling pathway，increaseCaspase-3/7Protein expression levels, induction4T1apoptosis^[28]。Shikonin9 μmol/LCan make human ovarian cancer resistant to cisplatin cellsA2780-CRDecreased survival of50%, which increased in a time-dependent mannerJNK, mitogen activated protein kinase（MAPK）And extracellular signal regulated kinase（ERK）The phosphorylation level of shikonin, indicating that shikonin can activateMAPKSignaling pathway inductionA2780-CRapoptosis。 Meanwhile, shikonin was found to reduce the intracellularBcl-2Level, improveBax、Caspase-9andCaspase-3Levels, reducing mitochondrial membrane potential（Δψ_m）, elevated cytochrome released from mitochondria to the cytosolCLevels, suggesting that shikonin induces cancer apoptosis through a mitochondria dependent pathway^[29]。

Solid tumors have very unique physiological characteristics, such as vascular abnormalities and intratumoral hypoxia. To prevent cell death caused by oxygen depletion, cancer cells often overexpress hypoxia inducible factors（HIF-1α）. Synthetic coumarin-Carboxyl shikonate derivativesPMMB232Able to promote dose dependentlyHeLaapoptosis， The proportion of apoptotic cells was38.0%, which was significantly higher than the proportion of apoptotic cells in shikonin treated group（20.8%）。PMMB2328 μmol/LhandleHeLaPost cellularHIF-1αThe expression was significantly decreased, which was similar to the results of shikonin treatment group at the same concentration. Molecular docking calculation found thatPMMB232Can be associated withHIF-1αStable combination and promotionHIF-1αTo induce tumor apoptosis due to the degradation of^[30]。

Phosphatidylinositol-3Kinases（PI3K）/protein kinaseB（Akt）/Mammalian rapamycin protein（mTOR）/PTENThe pathway is activated in a variety of cancers and plays a key role in inhibiting apoptosis and promoting proliferation. It is a relatively independent and complex pathway, which is similar toHIF-1αAnd other pathways are also closely related. Shikonin inhibits proto oncogenesmiR-106bIn endometrial cancer cellsHEC-1A、KLEandRL95-2To upregulate tumor suppressor genesPTEN, upregulatedPTENAntagonisticPI3KSignal transduction, inhibitionAkt/mTORaccessthusPromote apoptosis, whilemiR-106Overexpression of shikonin significantly attenuated apoptosis induced by Shikonin^[23]。

2.3  Inhibit cancer cell invasion and metastasis

Controlling cancer cell invasion and tumor metastasis is of great significance for improving the prognosis of cancer patients. Shikonin can up regulate glycogen synthase kinase3β（GSK-3β）Horizontal and enhancedβ-Catenin（β-catenin）β-cateninThe expression and nuclear accumulation of α - Catenin, inhibiting its interaction withTCytokines（TCF）, thereby inhibiting epithelial mesenchymal transition（EMT）Transcription of related genes, downregulation of interstitial markersN-Cadherin expression, decreased vimentin andSnailProtein levels, elevated epithelial markersE-Cadherin levels, indicating that shikonin inhibitsWnt/β-cateninSignaling pathway reversalMDA-MB-231and4T1CellularEMT, thereby inhibiting breast cancer cell metastasis and invasion^[10]Shikonin9 μmol/LhandleA2780-CRcell48 hafter^[29]Detection findingsE-Increased cadherin levels,N-The level of cadherin decreased, indicating that shikonin can also reverse ovarian cancer cellsEMTThus weakening its migration ability.

Shikonin derivativesPMMB232ProcessedHeLacell^[30]inHIF-1αThe expression of α - Catenin was decreased compared with the control group3Times, whileE-The expression of cadherin was significantly upregulated, the cell-cell adhesion was strengthened, and the migration and metastasis of tumor cells were slowed down, indicating that shikonin through downregulationHIF-1α.

Other studies found that shikonin effectively inhibited signaling and transcriptional activator proteins in a dose-dependent manner3（STAT3）（FAK）The activity andSrcThe expression ofMDA-MB-231、MDA-MB-468、MCF-7and4T1Invasive growth and metastasis of cells^[31]。

2.4  Induced necrosis、Autophagy and cellular immunity

Unlike apoptosis, necrosis is an independentCaspaseOf non programmed cell death, which is mainly regulated by receptor interacting serine protein kinases1（RIP1）AndRIP3The regulation of ROS is also related to intracellular ROS（ROS）,ROSElevation causes severeDNAInjury, gene mutation and active inflammation of tumor cells can promote the proliferation, invasion, metastasis and angiogenesis of tumor cells^[32]。

Shikonin can promoteMDA-MB-468Intracellular tumor necrosis factor-α（TNF-α）αreceptor1（TNFR1）Binding, stimulating the formation of death complexes on the cytoplasmic membraneI(includesTRADD、TRAF2、RIP1KandCIAP1）. coverNecrostatin-1Blocked necrotic complexIIbThe formation ofRIP1KThe kinase activity of shikonin makes the cytoplasmicROSIncrease in contentRIP1K、RIP3KAnd mixed series of protein kinase like domains（mixed lineage kinase domain-like，MLKL）Of phosphorylation, leading to this3Necrotic complex mediated by seed protein interactionIIbFormation, which in turn increases mitochondrialROSGeneration of, reduceΔψ_m, leading to tumor cell necrosis^[33]. useDCFH-DAfluorescenceProbe detection revealed that shikonin treatedSKOV3andA2780IntracellularROSThe level of shikonin was significantly increased, indicating that shikonin promoted the proliferation of ovarian cancer cells byROSExcessive accumulation and activation of necroptosis process^[17]。

（ICD）Generally by releasing damage associated molecular patterns（DAMPs）Mediated, macroautophagy activity enhanced byOften accompanied by necrosis^[34]. treatment of mouse breast cancer with Shikonin4T1-luc2Cells, primingRIPK1andRIPK3Dependent necrosis^[35-36]，Concomitant with enhanced autophagy causes membrane surfaceDAMP（ectoDAMP）Upregulated, thereby activating co cultured dendritic cells（DCs）, to inhibit the metastasis of primary cancer cells, and were treated with shikonin and chloroquine in combination4T1-luc2Cells can enhance immunogen activity more than shikonin alone and based onDCsCancer vaccine effects.

2.5  Inhibition of glycolysis

Tumors usually use glycolysis to provide energy for their own metabolism and growth, i.eWarburgEffect, pyruvate kinaseM2（PKM2）Not only is it the rate limiting enzyme that controls the last step of glycolysis^[37], which is also a protein kinase regulating gene expression in cancer cells^[38]. by epidermal growth factor receptor（EGFR）ActivatedPKM2Translocation from the cytoplasm to the nucleus, activatingβ-catenin, induced to encode cyclinsD1Genes of（CCND1）And oncogenesc-MycExpression, upregulation of glucose transporters1（GLUT1）And lactate dehydrogenaseA（LDHA）Levels, increased glucose consumption and lactate production, which in turn promoted tumorigenesis^[39]。

Shikonin can inhibitMCF-7Pyruvate kinase, hexokinase, phosphofructokinase in cells-1, fructose bisphosphate aldolase, glyceraldehyde-3-The activity of phosphate dehydrogenase, thereby reducing the sugar content of cancer cellsFermentation rate^[40]。1 μmol/LShikonin treatment24 hafterMCF-7andMDA-MB-231In cellsPKM2Decreased activity,HIF-1αAnd vascular endothelial growth factor（VEGF）The reduced expression and cell migration ability suggested that shikonin could targetPKM2/ HIF-1αAxis to inhibit the migration of breast cancer cells^[41]In addition, shikonin could also inhibit theSKOV3andCP70IntracellularPKM2Activity of^[42], significantly reduce the glucose consumption of cancer cells and reduce the extracellular acidification rate, thus causing metabolic disorders and. Researchers designed a series of novel naphthoquinone derivatives^[43], in which piperidinyl substituted compounds3KWith stronger inhibition than ShikoninPKM2The ability of activity, itsIC₅₀For（2.95±0.53）μmol/L, while shikonin had no effect onPKM2OfIC₅₀For（8.82±2.62）μmol/L；3KAlso has stronger inhibitionHeLaThe ability of cells to proliferate,IC₅₀For（0.29±0.11）μmol/L, shikonin vsHeLaCellularIC₅₀For（2.45±0.81）μmol/L。

3  epilogue

A large number of studies at home and abroad have found that shikonin can pass through multiple signaling pathways and multiple molecular targets (e.gWnt/β-catenin、PI3K/Akt/ mTOR/PTEN、p38-MAPK、Bcl-2/Bax、ROS/HIF-1α、STAT3、RIPK1/RIPK3andPKM2Etc.) inhibit the proliferation, invasion and metastasis of a variety of gynecological tumors, induce cancer apoptosis, necrosis and autophagy, thus exerting anti-tumor effects.

Although a number of in vitro and in vivo animal experiments have confirmed the anti-tumor effect and mechanism of shikonin, shikonin or its derivatives have not been independently applied in clinic as anti-tumor drugs, which are usually accompanied by chemotherapy drugs to interact to enhance the anti-cancer effect^[18-19]The main limiting factors are that the effective concentration of shikonin is relatively high, and there may be some side effects, lacking the specific selectivity of antitumor effect. In recent years, researchers have been committed to changing its structure to obtain shikonin and naphthoquinone derivatives with stronger potency and less side effects, and have made some progress, such as1C、3K、PMMB232Synthetic derivatives such asHeLaCells have a stronger inhibitory effect^[14,30,43]In addition, shikonin is insoluble in water, soluble in ethanol, organic solvents and vegetable oils, and the difference in the mode of administration will also affect its efficacy. In recent years, the field of medicinal chemistry and nanotechnology has developed rapidly, creating new derivatives and nanoformulations based on shikonin, which has improved its pharmacological function. The researchers prepared shikonin microemulsion using the classic emulsification process（SKN-MEs）, and then passEDC/NHSPrepared by condensation technologyCD133Antibody modified shikonin microemulsion^[44], can significantly inhibitMDA-MB-231Xenograft experiments showed that the in vivo tumor inhibition rate of stem cells was78.5%, which was significantly higher than that of shikonin group（31.3%）。

At present, the anti-tumor research of shikonin mostly stays at the molecular level. With the emergence of new technologies such as whole genome sequencing, genome editing and quantitative proteomic analysis, we can have a more thorough and accurate understanding of the molecular targets and signal pathways of shikonin's anticancer function. This information is helpful to develop shikonin or its derivatives as effective anticancer drugs. More animal models and clinical trials are also needed to verify the effectiveness and safety of shikonin for clinical treatment.

References (omitted)  

Come   Source: Chen   Jing, Hou   Yao, wuchunlian. Research progress of shikonin and its derivatives on gynecological tumor [j]. Chinese herbal medicine, 2020, 51 (14): 3814-3820