Synthesis and biological evaluation of novel isoxazoloquinone derivatives as potent STAT3-targeting antipsoriasis agents.

Psoriasis is a troublesome scaling skin disease with no high-effective medication available by far. Signal transducer and activator of transcription 3 (STAT3) has recently been revealed as a crucial player in the pathogenesis and progression of psoriasis and emerged as an intriguing antipsoriatic drug target. Naturally occurring lapachol and its quinone analogs had been discovered as effective STAT3 inhibitors, however, their antipsoriatic effects are not well investigated. Previously, we have reported a series of isothiazoloquinone lapachol derivatives. Here, the antipsoriastic potentials of these isothiazoloquinones were investigated and, in addition, 35 novel isoxazoloquinone derivatives were prepared and studied for their anti-psoriasis properties. Among them, the most potent antipsoriatic compound B20 determined by in vitro test on HaCaT cells could directly bind to STAT3, reduce STAT3 level and inhibit STAT3 nuclear translocation. In vivo studies showed that topical application of B20 could effectively alleviate IMQ-induced psoriasis in mice with no obvious side effects. In addition, B20 inhibited the production of interleukin 17 (IL-17A), a STAT3-downstream cytokine essential for the progression of psoriasis, both in vitro and in vivo. Thus, isoxazoloquinone B20 is a potent STAT3-targeting antipsoriatic agent worth of further investigation.

ASS1 inhibits triple-negative breast cancer by regulating PHGDH stability and de novo serine synthesis.

Argininosuccinate synthase (ASS1), a critical enzyme in the urea cycle, acts as a tumor suppressor in many cancers. To date, the anticancer mechanism of ASS1 has not been fully elucidated. Here, we found that phosphoglycerate dehydrogenase (PHGDH), a key rate-limiting enzyme in serine synthesis, is a pivotal protein that interacts with ASS1. Our results showed that ASS1 directly binds to PHGDH and promotes its ubiquitination-mediated degradation to inhibit serine synthesis, consequently suppressing tumorigenesis. Importantly, the tumor suppressive effects of ASS1 were strongly abrogated by PHGDH knockout. In addition, ASS1 knockout and knockdown partially rescued cell proliferation when serine and glycine were depleted, while the inhibitory effect of ASS1 overexpression on cell proliferation was restored by the addition of serine and glycine. These findings unveil a novel role of ASS1 and suggest that the ASS1/PHGDH serine synthesis pathway is a promising target for cancer therapy.

Spinosyn A and Its Derivative Inhibit Colorectal Cancer Cell Growth via the EGFR Pathway.

Spinosyn A (SPA), derived from a soil microorganism, Saccharopolyspora spinosa, and its derivative, LM2I, has potential inhibitory effects on a variety of cancer cells. However, the effects of SPA and LM2I in inhibiting the growth of human colorectal cancer cells and the molecular mechanisms underlying these effects are not fully understood. Cell viability was tested by using a 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) assay and a colony formation assay. On the basis of the IC50 values of SPA and LM2I in seven colorectal cancer (CRC) cell lines, sensitive (HT29 and SW480) and insensitive (SW620 and RKO) cell lines were screened. The GSE2509 and GSE10843 data sets were used to identify 69 differentially expressed genes (DEGs) between sensitive and insensitive cell lines. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interactions (PPI) were performed to elucidate the molecular mechanisms of the DEGs. The hub gene of the DEGs was detected by Western blot analysis and verified using the CRISPR/Cas9 system. Our data indicate that SPA and its derivative LM2I have significant antiproliferative activity in seven colorectal cancer cell lines and colorectal xenograft tumors. On the basis of bioinformatics analysis, it was demonstrated that epidermal growth factor receptor (EGFR) was the hub gene of the DEGs and was associated with the inhibitory effects of SPA and LM2I in CRC cell lines. The study also revealed that SPA and LM2I inhibited the EGFR pathway in vitro and in vivo.

An Isoxazoloquinone Derivative Inhibits Tumor Growth by Targeting STAT3 and Triggering Its Ubiquitin-Dependent Degradation.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with shorter five-year survival than other breast cancer subtypes, and lacks targeted and hormonal treatment strategies. The signal transducer and activator of transcription 3 (STAT3) signaling is up-regulated in various tumors, including TNBC, and plays a vital role in regulating the expression of multiple proliferation- and apoptosis-related genes. RESULTS: By combining the unique structures of the natural compounds STA-21 and Aulosirazole with antitumor activities, we synthesized a class of novel isoxazoloquinone derivatives and showed that one of these compounds, ZSW, binds to the SH2 domain of STAT3, leading to decreased STAT3 expression and activation in TNBC cells. Furthermore, ZSW promotes STAT3 ubiquitination, inhibits the proliferation of TNBC cells in vitro, and attenuates tumor growth with manageable toxicities in vivo. ZSW also decreases the mammosphere formation of breast cancer stem cells (BCSCs) by inhibiting STAT3. CONCLUSIONS: We conclude that the novel isoxazoloquinone ZSW may be developed as a cancer therapeutic because it targets STAT3, thereby inhibiting the stemness of cancer cells.

Molecular Cloning and Identification of NADPH Cytochrome P450 Reductase from Panax ginseng.

Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology.

Naturally-occurring spinosyn A and its derivatives function as argininosuccinate synthase activator and tumor inhibitor.

Argininosuccinate synthase (ASS1) is a ubiquitous enzyme in mammals that catalyzes the formation of argininosuccinate from citrulline and aspartate. ASS1 genetic deficiency in patients leads to an autosomal recessive urea cycle disorder citrullinemia, while its somatic silence or down-regulation is very common in various human cancers. Here, we show that ASS1 functions as a tumor suppressor in breast cancer, and the pesticide spinosyn A (SPA) and its derivative LM-2I suppress breast tumor cell proliferation and growth by binding to and activating ASS1. The C13-C14 double bond in SPA and LM-2I while the Cys97 (C97) site in ASS1 are critical for the interaction between ASS1 and SPA or LM-2I. SPA and LM-2I treatment results in significant enhancement of ASS1 enzymatic activity in breast cancer cells, particularly in those cancer cells with low ASS1 expression, leading to reduced pyrimidine synthesis and consequently the inhibition of cancer cell proliferation. Thus, our results establish spinosyn A and its derivative LM-2I as potent ASS1 enzymatic activator and tumor inhibitor, which provides a therapeutic avenue for tumors with low ASS1 expression and for those non-tumor diseases caused by down-regulation of ASS1.

ZLM-7 inhibits the occurrence and angiogenesis of breast cancer through miR-212-3p/Sp1/VEGFA signal axis.

BACKGROUND: Breast cancer (BC) is a common malignant tumor with poor prognosis. Angiogenesis is related to the growth and progression of solid tumors and associated with prognosis. ZLM-7, SP1, VEGFA and miR-212-3p were associated with BC angiogenesis and proliferation, however the detailed mechanism was not clear. This study aimed to reveal the regulatory mechanism of angiogenesis of BC. METHODS: BC cell lines were treated with 10 nM ZLM-7 for 8 h. We detected protein expression level by western blot and RNA expression level by qRT-PCR. Overexpression or inhibition of miR-212-3p is performed using miR-212-3p mimics or miR-212-3p inhibitor, Sp1 overexpression using pcDNA3.1 vector. Angiogenesis was analyzed by co-culturing BC cell lines and HUVEC cells. To evaluate regulatory relationship between miR-212-3p and Sp1, dual luciferase assay was performed. Besides, the direct interaction between Sp1 and VEGFA was analyzed by ChIP. Migration and invasion were analyzed by transwell assay and proliferation was detected by clone formation assay. In mice xenograft model developed using BC cells, we also detected angiogenesis marker CD31 through immunohistochemistry. RESULTS: ZLM-7 up-regulated miR-212-3p and inhibited invasion, migration, proliferation and angiogenesis of BC, while miR-212-3p inhibitor antagonized such effects. Binding sequence was revealed between miR-212-3p and Sp1, and expression of Sp1 was inhibited by miR-212-3p on both protein and mRNA level. Sp1 could interact with VEGFA and promoted its expression. Overexpression of miR-212-3p inhibited migration, invasion, proliferation and angiogenesis of BC cell lines, while Sp1 overexpression showed the opposite effect and could antagonize these effects of miR-212-3p overexpression. ZLM-7 decreased VEGFA expression, which was rescued by co-transfection with miR-212-3p inhibitor. Similar, ZLM-7 could inhibit tumor growth and angiogenesis through the miR-212-3p/Sp1/VEGFA axis in vivo. CONCLUSIONS: ZLM-7 could directly up-regulate miR-212-3p in BC. MiR-212-3p could inhibit VEGFA expression through Sp1, thereby inhibiting angiogenesis and progression of BC.

Synthesis and biological evaluation of novel isothiazoloquinoline quinone analogues.

Natural quinones and their analogues have attracted growing attention because of their novel anticancer activities. A series of novel isothiazoloquinoline quinone analogues were synthesized and evaluated for antitumor activities against four different kind of cancer cells. Among them, isothiazoloquinolinoquinones inhibited cancer cells proliferation effectively with IC50 values in the nanomolar range, and isothiazoloquinolinoquinone 13a induced the cell apoptosis. Further exploration of possible mechanism of action indicates that 13a not only activates ROS production through NQO1-directed redox cycling but also inhibits the phosphorylation of STAT3. These findings indicate that 13a has potential use for the development of new skeleton drug candidate as an efficient substrate of NQO1 and STAT3 inhibitor.