A modified natural small molecule inhibits triple-negative breast cancer growth by interacting with Tubb3.

BACKGROUND: 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. ß-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. PURPOSE: In this work, the anti-TNBC effect of a shikonin derivative (PMMB276) was investigated, and its mechanism was also investigated. STUDY DESIGN/METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Functional characterization of two chalcone isomerase (CHI) revealing their responsibility for anthocyanins accumulation in mulberry.

Mulberry (Morus sp., Moraceae) is an important economic crop plant and mulberry fruits are rich in anthocyanidins. Chalcone isomerase (CHI) catalyzes the conversion of chalcones to flavanones providing precursors for biosynthesis of anthocyanidins. In this study, bona fide CHIs were cloned and characterized from different Morus species with differently colored fruits (Morus multicaulis, Mm and Morus alba variety LvShenZi, LSZ). Enzymatic assay of MmCHI1 and MmCHI2 showed that they can utilize naringenin chalcone as substrate. The catalytic efficiency of MmCHI2 and LSZCHI2 are approximately 200 and 120-fold greater than that of MmCHI1 respectively. Phylogenetic analysis showed the two mulberry CHIs belonged to different sub-clade of Type I CHI1 named type IA (CHI2) and type IB (CHI1). Type IB CHIs are mulberry specific. MmCHI1 and MmCHI2 had similar expression profiles and showed preferred expression in fruits. In addition, both mulberry CHI1 and CHI2 played roles in the response to excess zinc stress and sclerotiniose pathogen infection. Both MmCHI1 and MmCHI2 expression levels showed positive close relationship with anthocyanins content during fruit ripening process. The co-expression of MmCHI1 and MmCHI2 was observed during fruit ripening process and in transgenic mulberry. VIGS (virus induced gene silence) targeting on MmCHI1 and MmCHI2 showed significant down-regulation of MmCHI2 instead of MmCHI1 would result in significant (about 50%) decrease in anthocyanins content. MmCHI2 is the dominant CHI for anthocyanins accumulation in mulberry. The results presented in this work provided insight on bona fide CHIs in mulberry and reveal their roles in anthocyanins accumulation.

Establishment of the hairy root culture of Echium plantagineum L. and its shikonin production.

Echium plantagineum L. (Boraginaceae) is an invasive species in Australia and contains medicinal shikonins in its roots. In this study, the hairy root lines of E. plantagineum were established using Agrobacterium rhizogenes strain ATCC15834 and confirmed by the amplification of the rolB gene. Results showed significant difference in shikonin production between the hairy root lines in the 1/2B5 and M9 media. The biomass of the lines in the 1/2B5 medium was fivefold of that in the M9 medium. However, the components of detected shikonins were similar in these two liquid media. By contrast, different accumulation profiles appeared in the hairy root lines. HPLC analysis revealed the presence of nine possible related compounds, including shikonins, and acetylshikonin was the most abundant shikonin derivative. The content of acetylshikonin in the 1/2B5 medium (36.25 mg/L on average) was twofold of that in the M9 medium. Our results showed that the hairy root cultures of E. plantagineum can be used in enhancing the production of potential pharmaceutical compounds, such as acetylshikonin.

High throughput deep degradome sequencing reveals microRNAs and their targets in response to drought stress in mulberry (Morus alba).

MicroRNAs (miRNAs) play important regulatory roles by targeting mRNAs for cleavage or translational repression. Identification of miRNA targets is essential to better understanding the roles of miRNAs. miRNA targets have not been well characterized in mulberry (Morus alba). To anatomize miRNA guided gene regulation under drought stress, transcriptome-wide high throughput degradome sequencing was used in this study to directly detect drought stress responsive miRNA targets in mulberry. A drought library (DL) and a contrast library (CL) were constructed to capture the cleaved mRNAs for sequencing. In CL, 409 target genes of 30 conserved miRNA families and 990 target genes of 199 novel miRNAs were identified. In DL, 373 target genes of 30 conserved miRNA families and 950 target genes of 195 novel miRNAs were identified. Of the conserved miRNA families in DL, mno-miR156, mno-miR172, and mno-miR396 had the highest number of targets with 54, 52 and 41 transcripts, respectively, indicating that these three miRNA families and their target genes might play important functions in response to drought stress in mulberry. Additionally, we found that many of the target genes were transcription factors. By analyzing the miRNA-target molecular network, we found that the DL independent networks consisted of 838 miRNA-mRNA pairs (63.34%). The expression patterns of 11 target genes and 12 correspondent miRNAs were detected using qRT-PCR. Six miRNA targets were further verified by RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-5' RACE). Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these target transcripts were implicated in a broad range of biological processes and various metabolic pathways. This is the first study to comprehensively characterize target genes and their associated miRNAs in response to drought stress by degradome sequencing in mulberry. This study provides a framework for understanding the molecular mechanisms of drought resistance in mulberry.

Transgenic studies reveal the positive role of LeEIL-1 in regulating shikonin biosynthesis in Lithospermum erythrorhizon hairy roots.

BACKGROUND: The phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation. RESULTS: The mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production. CONCLUSIONS: The results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.

Transgenic analysis reveals LeACS-1 as a positive regulator of ethylene-induced shikonin biosynthesis in Lithospermum erythrorhizon hairy roots.

The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.

Molecular cloning of a dehydration-responsive protein gene (MRD22) from mulberry, and determination of abiotic stress patterns of MRD22 gene expression.

A full-length cDNA sequence coding for Dehydration-responsive protein gene of mulberry tree, which we designated was MRD22 (GenBank accession number: JQ804833) was cloned based on mulberry expressed sequence tags (ESTs). MRD22 is 1503 bp long, contains a 334 bp 5'-UTR (untranslated region) and a 563 bp 3'-UTR, encodes 201 amino acids with a predicted molecular weight of 54.28 kDa and an isoelectric point of 9.35. Phylogenetic analysis based on MRD22 sequences from different species showed that mulberry has close relationship with Populus trichocarpa, Ricinus communis, Camellia sinensis, Gossypium hirsutum, Gossypium barbadense and so on. The expression level of the MRD22 gene under conditions of drought, low temperature and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.

De novo transcriptome analysis of mulberry (Morus L.) under drought stress using RNA-seq technology.

A large-scale RNA sequencing (RNA-seq) of mulberry (Morus L.) was carried out between two samples in regular and drought stress condition. In this research, de novo assembly was performed, and totally 54736 contigs were obtained from the reads, including the scaffolded regions. 1051 genes were identified that were significantly differently expressed between the two samples. As determined by Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes pathway mapping, 10110 GO terms and 247 pathways were assigned and then analyzed. Thousands of SSR markers produced in this study will enable genetic linkage mapping construction and gene-based association studies. Seven unique genes showing different expression level in control and drought stress groups were subsequently analyzed and identified by real-time PCR. For lack of mulberry whole genome information, transcriptome and de novo analysis from the two samples will provide important and useful information for later research and help genetic breeding of mulberry.

Mulberry (Morus L.) methionine sulfoxide rreductase gene cloning, sequence analysis, and expression in plant development and stress response.

Methionine sulfoxide reductase plays a regulatory role in plant growth and development, especially in scavenging reactive oxygen species by restoration of the oxidation of methionine in protein. A full-length cDNA sequence encoding methionine sulfoxide reductase (MSR) from mulberry, which we designated MMSR, was cloned based on mulberry expressed sequence tags (ESTs). Sequence analysis showed that the MMSR is 810 bp long, encoding 194 amino acids with a predicted molecular weight of 21.6 kDa and an isoelectric point of 6.78. The expression level of the MMSR gene under conditions of drought and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.