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3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), as the rate-limiting enzyme in the mevalonate pathway, is essential for the biosynthesis of shikonin in Lithospermum erythrorhizon. However, in the absence of sufficient data, the principles of a genome-wide in-depth evolutionary exploration of HMGR family members in plants, as well as key members related to shikonin biosynthesis, remain unidentified. In this study, 124 HMGRs were identified and characterized from 36 representative plants, including L. erythrorhizon. Vascular plants were found to have more HMGR family genes than nonvascular plants. The phylogenetic tree revealed that during lineage and species diversification, the HMGRs evolved independently and intronless LerHMGRs emerged from multi-intron HMGR in land plants. Among them, Pinus tabuliformis and L. erythrorhizon had the most HMGR gene duplications, with 11 LerHMGRs most likely expanded through WGD/segmental and tandem duplications. In seedling roots and M9 cultured cells/hairy roots, where shikonin biosynthesis occurs, LerHMGR1 and LerHMGR2 were expressed significantly more than other genes. The enzymatic activities of LerHMGR1 and LerHMGR2 further supported their roles in catalyzing the conversion of HMG-CoA to mevalonate. Our findings provide insight into the molecular evolutionary properties and function of the HMGR family in plants and a basis for the genetic improvement of efficiently produced secondary metabolites in L. erythrorhizon. [ABSTRACT FROM AUTHOR]

(A) The structural maps of pBI121-LeBAHD1/LeSAT1-eGFP overexpression vector and pYLCRISPR/Cas9-LeBAHD1/LeSAT1 knockout vectors. However, not all members of the BAHD acyltransferase gene family could be screened out from the I L. erythrorhizon i genome by only using AAT/SAT amino acid sequences as a query. Identification and Characterization of LeBAHD Genes in L. erythrorhizon Genome In order to have a comprehensive understanding of the characteristics and functions of BAHD family genes in I L. erythrorhizon i , 104 members were initially screened out from the I L. erythrorhizon i genome based on sequences homology and the conserved BAHD superfamily feature domain (Pfam: PF02458). [Extracted from the article]

Triple-negative breast cancer (TNBC) is a malignant tumor without specific therapeutic targets and a poor prognosis. Chemotherapy is currently the first-line therapeutic option for TNBC. However, due to the heterogeneity of TNBC, not all of TNBC patients are responsive to chemotherapeutic agents. Therefore, the demand for new targeted agents is critical. β -tubulin isotype III (Tubb3) is a prognostic factor associated with cancer progression, including breast cancer, and targeting Tubb3 may lead to improve TNBC disease control. Shikonin, the active compound in the roots of Lithospermun erythrorhizon suppresses the growth of various types of tumors, and its efficacy can be improved by altering its chemical structure. In this work, the anti-TNBC effect of a shikonin derivative (PMMB276) was investigated, and its mechanism was also investigated. This study combines flow cytometry, immunofluorescence staining, immunoblotting, immunoprecipitation, siRNA silencing, and the iTRAQ proteomics assay to analyze the inhibition potential of PMMB276 on TNBC. In vivo study was performed, Balb/c female murine models with or without the small molecule treatments. Herein, we screened 300 in-house synthesized analogs of shikonin against TNBC and identified a novel small molecule, PMMB276; it suppressed cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase, suggesting that it could have a tumor suppressive role in TNBC. Tubb3 was identified as the target of PMMB276 using proteomic and biological activity analyses. Meanwhile, PMMB276 regulated microtubule dynamics in vitro by inducing microtubule depolymerization and it could act as a tubulin stabilizer by a different process than that of paclitaxel. Moreover, suppressing or inhibiting Tubb3 with PMMB276 reduced the growth of breast cancer in an experimental mouse model, indicating that Tubb3 plays a significant role in TNBC progression. The findings support the therapeutic potential of PMMB276, a Tubb3 inhibitor, as a treatment for TNBC. Our findings might serve as a foundation for the utilization of shikonin and its derivatives in the development of anti-TNBC. A small novel molecule, i.e., α-lipoic acyl shikonin, modified form of a natural product (shikonin), acts as a novel inhibitor of Tubb3. [Display omitted] [ABSTRACT FROM AUTHOR]

Shikonin and its derivatives are the main components of traditional Chinese medicine, Zicao. The pharmacological potential of shikonin and its derivatives have been extensively studied. Yet, less is known about the microbial assemblages associated with shikonin producing Borage plants. We studied microbial profiles of two Borage species, Echium plantagineum (EP) and Lithospermum erythrorhizon (LE), to identify the dynamics of microbial colonization pattern within three rhizo-compatments and two distinct soil types. Results of α and β-diversity via PacBio sequencing revealed significantly higher microbial richness and diversity in the natural soil along with a decreasing microbial gradient across rhizosphere to endosphere. Our results displayed genotype and soil type–dependent fine-tuning of microbial profiles. The host plant was found to exert effects on the physical and chemical properties of soil, resulting in reproducibly different micro-biota. Analysis of differentially abundant microbial OTUs displayed Planctomycetes and Bacteroidetes to be specifically enriched in EP and LE rhizosphere while endosphere was mostly prevailed by Cyanobacteria. Network analysis to unfold co-existing microbial species displayed different types of positive and negative interactions within different communities. The data provided here will help to identify microbes associated with different rhizo-compartments of potential host plants. In the future, this might be helpful for manipulating the keystone microbes for ecosystem functioning. [ABSTRACT FROM AUTHOR]

Colorectal cancer is a common malignancy and the second leading cause of cancer-related death. Emerging evidence suggests that the Wnt/β-catenin pathway has a significant role in cancer development. Shikonin is one of the main bioactive components of the plant, Lithospermum erythrorhizon, which possesses various biological properties. In this study, we identified a shikonin derivative, E5, as a novel suppressor of colorectal carcinoma that acted by inhibiting cell proliferation and migration, and promoting apoptosis. Mechanistically, the drug activated caspase-dependent pathways, negatively regulating the Wnt/β-catenin pathway, and inhibited epithelial–mesenchymal transition. The findings suggest the potential of β-catenin as a molecular target in colon carcinoma and propose E5 as a promising therapeutic strategy for targeting Wnt/β-catenin [Display omitted] ● A novel thioether shikonin derivative (E5), modified form of shikonin, acts as a novel inhibitor of β-catenin. ● E5 suppressed cancer cell proliferation and migration and promoted cell apoptosis via caspase-dependent pathways. ● E5 can be a drug candidate and has the potential to inhibit Wnt/β-catenin signaling pathway in the colon carcinoma. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel series of dual-target benzenesulfonyl shikonin derivative has been designed and synthesized. • Z10 could activate activity of PKM2 and inhibit activity against PDK1 by direct intereaction. • Z10 showed significant anticancer activity in colorectal cancer in comparison with shikonin, PKM2 or PDK1 target agent. • Z10 exhibited lower toxicity than shikonin at 4 μM in vitro and at a dosage of 4.2 mg/kg in vivo. • Z10 could significantly induce apoptosis in colorectal cells and in xenograft mice models. Warburg effect provides energy and material essential for tumor proliferation, the reverse of Warburg effect provides insights into the development of a novel anti-cancer strategy. Pyruvate kinase 2 (PKM2) and pyruvate dehydrogenase kinase 1 (PDK1) are two key enzymes in tumor glucose metabolism pathway that not only contribute to the Warburg effect through accelerating aerobic glycolysis, but also serve as druggable target for colorectal cancer (CRC). Considering that targeting PKM2 or PDK1 alone does not seem to be sufficient to remodel abnormal glucose metabolism and achieve significant antitumor activity, a series of novel benzenesulfonyl shikonin derivatives were designed to regulate PKM2 and PDK1 simultaneously. By means of molecular docking and antiproliferative screen, we found that compound Z10 could act as the combination of PKM2 activator and PDK1 inhibitor, thereby significantly inhibited glycolysis that reshaping tumor metabolism. Moreover, Z10 could inhibit proliferation, migration and induce apoptosis in CRC cell HCT-8. Finally, the in vivo anti-tumor activity of Z10 was evaluated in a colorectal cancer cell xenograft model in nude mice and the results demonstrated that Z10 induced tumor cell apoptosis and inhibited tumor cell proliferation with lower toxicity than shikonin. Our findings indicated that it is feasible to alter tumor energy metabolism through multi-target synergies, and the dual-target benzenesulfonyl shikonin derivative Z10 could be a potential anti-CRC agent. [ABSTRACT FROM AUTHOR]

Pyruvate kinase 2 (PKM2) and pyruvate dehydrogenase kinase 1 (PDK1) are two key enzymes in tumor glucose metabolism pathway that not only promote tumor growth and proliferation through accelerating aerobic glycolysis, but also contribute to drug resistance of non-small cell lung cancer (NSCLC). Considering that targeting PKM2 or PDK1 alone seems insufficient to remodel abnormal glucose metabolism to achieve significant antitumor activity, we proposed a "two-step approach" that regulates PKM2 and PDK1 synchronously. Firstly, we found that the combination of ML265 (PKM2 activator) and AZD7545 (PDK1 inhibitor) could synergistically inhibit proliferation and induce apoptosis in H1299 cells. Base on this, we designed a series of novel shikonin (SK) thioether derivatives as PKM2/PDK1 dual-target agents, among which the most potent compound E5 featuring a 2-methyl substitution on the benzene ring exerted significantly increased inhibitory activity toward EGFR mutant NSCLC cell H1975 (IC 50 = 1.51 μmol/L), which was 3 and 17-fold more active than the lead compound SK (IC 50 = 4.56 μmol/L) and the positive control gefitinib (IC 50 = 25.56 μmol/L), respectively. Additionally, E5 also showed good anti-tumor activity in xenografted mouse models, with significantly lower toxicity side effects than SK. Moreover, E5 also inhibited the entry of PKM2 into nucleus to regulate the transcriptional activation of oncogenes, thus restoring the sensitivity of H1975 cell to gefitinib. Collectively, these data demonstrate that E5 , a dual inhibitor of PKM2/PDK1, may be a promising adjunct to gefitinib in the treatment of EGFR-TKIs resistant NSCLC, deserving further investigation. [Display omitted] • A synergistic effect of PKM2 activation and PDK1 inhibition was first demonstrated. • A series of PKM2/PDK1 dual-target shikonin thioether derivatives were developed. • The most active compound E5 significantly inhibit tumor growth in vitro and in vivo. • The most active compound E5 enhance the sensitivity of H1975 cells to gefitinib. [ABSTRACT FROM AUTHOR]

PDK1 is one of the key enzymes in the glucose metabolism pathway, which is abnormally high expressed in breast cancer tissues and can promote tumor proliferation and metastasis. PDK1 and the PDHC/PDK axis are important targets for regulating glucose metabolism and anti-tumor activity. In this study, we evaluated the anti-tumor activities of a series of semi-synthesized shikonin (SK) derivatives against human breast cancer cells. The anti-proliferation activity of SK derivatives against human breast cancer cell lines was tested by CCK-8 and EdU assay. Flow cytometry was utilized to evaluate cell apoptosis, reactive oxygen species and cell cycle distribution. Cell migration ability was determined by wound healing and trans-well assay. PDK1 targeting effect was confirmed by western bolting, molecular docking, bio-layer interferometry and PDK1 enzyme activity assay. Nude-mouse transplanted tumor model was used to evaluate their anti-tumor effect in vivo. Findings revealed that SK derivatives had good anti-proliferation ability against MDA-MB-231 cell. They induced cell apoptosis by regulating the mitochondrial apoptosis and death receptor pathway. They also inhibited cell migration by suppressing EMT progression. Molecular docking, PDK1 affinity and enzyme activity demonstrated their PDK1 targeting. In vivo antitumor experiment showed that E2 could significantly inhibit tumor growth with lower side-effect on mice than SK. In conclusion, the novel SK derivatives E2 and E5 inhibited tumor glycolysis by targeting PDK1 and ultimately induced apoptosis. Our data demonstrated that E2 would be a good lead compound for the treatment of human TNBC as a novel PDK1 inhibitor. [ABSTRACT FROM AUTHOR]

[Display omitted] • SK and acetyl-SK relieve colonic injuries inAOM / DSS -induced colitis-associated colorectal cancer (CAC) model of C57BL/6J mice. • SK and acetyl-SK remodel the gut microbial communities of CAC mice models. • SK and acetyl-SK rescued the increased Ileibacterium valens and g_norank_f_norank_o_Clostridia_UCG-014 in CAC mice. • SK downregulated the expression of ACTN2, FGG and PKM2 in CAC mice. • SK upregulated the expression of Aldh1a1 and Aldh1b1 which involved in the Wnt/ β -catenin pathway in CAC mice. Colorectal cancer (CRC) and inflammatory bowel disease (IBD) are the most common diseases of human digestive system. Nowadays, the influence of the inflammatory microenvironment on tumorigenesis has become a new direction, and the exploration of relative molecular mechanism will facilitate the discovery and identification of novel potential anti-cancer molecules. Natural shikonin (SK) and acetyl-shikonin (acetyl-SK) was administered to azoxymethane (AOM)/dextran sodium sulphate (DSS)-induced colitis-associated colorectal cancer (CAC) mice model by gavage to investigate their therapeutic effects. Moreover, fresh feces and colon tissues were collected for determining the function of SK and acetyl-SK on the gut microbes and protein expression, respectively. Both SK and acetyl-SK decreased AOM/DSS-induced CAC, and regulated the intestinal flora structure in CAC mouse model. They, especially SK, improved species richness, evenness and diversity of intestinal flora, recovered the upregulated ratio of Firmicutes to Bacteroidota (F/B ratio) which symbolizes gut microbiota dysbiosis. SK and its derivative increased the beneficial bacteria g__norank_f__Muribaculaceae, Lactobacillus, Lachnospiraceae_NK4A136_Group, and reduced those harmful ones including Ileibacterium and Coriobacteriaceae UCG–002. Notably, AOM/DSS caused significant increase in the abundance of Ileibaterium valens and g__norank_f__norank_o__Clostridia_UCG-014 , which were not previously reported in studies of colonic inflammation or cancer, and the disorder was reversed by 20 mg/kg of SK. In our current study, the action of SK and acetyl-SK is dose-dependent, and 20 mg/kg SK exhibited the most effective functions, even better than the positive drug mesalazine. Moreover, differential proteomics and ELISA results showed that SK could recover the increase of pro-inflammatory cytokines (including IL-1β, IL-6 and TNF-α), the upregulation of pyruvate kinase isozyme type M2 (PKM2) and some other proteins (mainly concentrated in transcriptional mis-regulation in cancer and IL-17 signaling pathways), and the downregulation of Aldh1b1-Acc3-Maoa and Μgt2b34-Aldh1a1-Aldh1a7 involved in Wnt/ β -catenin signaling pathway. Our study identified SK and acetyl-SK, especially SK, as potential preventive agents for CAC through regulating both gut microbes and pathways involved in inflammation and cancer such as Wnt/ β -catenin signaling pathway. [ABSTRACT FROM AUTHOR]

PMMB-302 has an effect on the expression of apoptosis-related proteins such as Bcl2, Bcl-XL, caspase-9, caspase3 and FADD. Moreover, PMMB-302 inhibited expression of telomerase core proteins, dyskerin and NHP2, and telomerase reverse transcriptase RNA. These results suggested that PMMB-302 has tumor suppressive roles in lung cancer cells. [Display omitted] • A small novel molecule modified form of a natural product (shikonin), acts as a novel inhibitor of telomerase. • The small molecule suppresses cancer cell proliferation in vitro via inducing apoptosis and arresting cell cycle at G2/M phase. • This small molecule induces apoptosis in A549 cells through the mitochondrial and death receptor pathways. In this study, a series of novel shikonin N -benzyl matrinic acid ester derivatives (PMMB-299–PMMB-310) were synthesized and tested for their ability to inhibit the proliferation of cancer cells. Compared with shikonin and matrine, some of the ester derivatives were found to exhibit better anti-proliferative activity against seven different cancer cell lines, with less cytotoxicity toward non-cancerous cells. The strongest anti-proliferative activity was exhibited by PMMB-302, which had an IC 50 value of 2.71 μM against A549 cells. The compound caused cell cycle arrest in the G2/M phase and induced apoptosis. Effects on the expression of apoptosis-related molecules such as Bcl2, Bcl-XL, caspase-3, caspase-9 and FADD suggested that PMMB-302 has tumor suppressive roles in lung cancer cells. In addition, PMMB-302 inhibited expression of telomerase core proteins, dyskerin and NHP2, and telomerase reverse transcriptase RNA. Moreover, molecular docking of PMMB-302 was subsequently conducted to determine the probable binding mode with telomerase. Taken together, the results indicate that PMMB-302 acts as a tumor suppressor in lung cancer cells by negatively regulating telomerase expression. [ABSTRACT FROM AUTHOR]

[Display omitted] • A series of novel anilide (dicarboxylic acid) shikonin esters were designed and synthesized as potential antitumor agents. • Representative compound M9 displayed potent anti-proliferative activity in vitro. • M9 could significantly induce apoptosis and arrest cell cycle in G2/M phase on MDA-MB-231 cells. • M9 inhibited the migration of MDA-MB-231 cells through Wnt/β-catenin signaling pathway in vitro. • M9 down-regulates PI3K/Akt/mTOR pathway by targeting Akt, thereby inhibiting cell proliferation and migration. Triple-negative breast cancer (TNBC) has an unfavorable prognosis attribute to its low differentiation, rapid proliferation and high distant metastasis rate. PI3K/Akt/mTOR as an intracellular signaling pathway plays a key role in the cell proliferation, migration, invasion, metabolism and regeneration. In this work, we designed and synthesized a series of anilide (dicarboxylic acid) shikonin esters targeting PI3K/Akt/mTOR signaling pathway, and assessed their antitumor effects. Through three rounds of screening by computer-aided drug design method (CADD), we preliminarily obtained sixteen novel anilide (dicarboxylic acid) shikonin esters and identified them as excellent compounds. CCK-8 assay results demonstrated that compound M9 exhibited better antiproliferative activities against MDA-MB-231, A549 and HeLa cell lines than shikonin (SK), especially for MDA-MB-231 (M9: IC 50 = 4.52 ± 0.28 μM; SK: IC 50 = 7.62 ± 0.26 μM). Moreover, the antiproliferative activity of M9 was better than that of paclitaxel. Further pharmacological studies showed that M9 could induce apoptosis of MDA-MB-231 cells and arrest the cell cycle in G2/M phase. M9 also inhibited the migration of MDA-MB-231 cells by inhibiting Wnt/β-catenin signaling pathway. In addition, western blot results showed that M9 could inhibit cell proliferation and migration by down-regulating PI3K/Akt/mTOR signaling pathway. Finally, a three-dimensional quantitative structure–activity relationship (3D-QSAR) model was also constructed to provide a basis for further development of shikonin derivatives as potential antitumor drugs through structure–activity relationship analysis. To sum up, M9 could be a potential candidate for TNBC therapy. [ABSTRACT FROM AUTHOR]