Gene-Directed Generation of Unprecedented Bioactive Compounds

Bioactive compounds with previously undescribed frameworks are highly desired for the discovery and development of new drugs and agrochemicals, but very few attempts have been reported to generate such molecules in biological contexts. Here, we present a gene-directed generation of architecturally unprecedented polyketide–indole hybrids (PIHs), which was conceptualized and materialized by employing polyketide synthases expressed in a heterologous vector, with simultaneous exposure to exogenous chemicals. To make an exemplification to this generally applicable approach, the ChrA and ChrB genes of Daldinia eschscholzii IFB-TL01 were integrated into the Aspergillus oryzae (AO) cell, and the resultant ChrA/ChrB-AO transformant was cultured in the indole-3-carbinol (I3C)-supplemented medium, leading to the production of seven skeletally undescribed PIHs named aochrabines A–G. Among them, aochrabines A–C exhibited a broad spectrum in inhibiting the growth of Gram-positive bacteria, whereas aochrabines B, C, and G showed moderate antitumor activities. Unexpectedly, the construction of such aochrabine molecules was achieved by the regioselective Michael addition of 3-methyleneindolium (3MI, generated from I3C in the AO culture) to different polyketide precursors with the yields (much) higher than those in the D. eschscholzii culture where comparable. Chemically, the benzyl-methine carbons in the precursor molecules were found to be made more vulnerable to the 3MI attack by the hydrogen-bonding between the ortho-hydroxyl and meta-carbonyl groups. Collectively, this is the first report of the ortho- and meta-substituent co-driven regioselective Michael addition of electrophilic methylene compounds to heterologous PKS production platform to in situ multiply the chemodiversity of microbial cultures, thus showing great potential in producing valuable compounds with new chemical space

β3 promotes TGF-β1/H₂O₂/HOCl-induced expression of α3 and SNAI2.

<p>(<b>A</b>) HepG2 cells were stimulated with H₂O₂/HOCl, TGF-β1, and T/H/H (TGF-β1/H₂O­₂/HOCl). <i>ITGA3</i> expression was detected by real-time RT-PCR at the indicated time points, or by flow cytometry and Western blot after 8-d culture. (<b>B</b>) HepG2 cells were untreated or treated for 8 days with T/H/H in absence or presence of SB203580 (10 µM), PD98059 (10 µM), SP600125 (10 µM), wortmannin (WT, 40 nM), QNZ (40 nM), and SIS3 (2 µM). <i>ITGA3</i> expression was detected by real-time RT-PCR. (<b>C and D</b>) HepG2 cells were stimulated with H₂O₂/HOCl, TGF-β1, and T/H/H. Phosphorylated ERK was detected by Western blot at the indicated time points (C). <i>SNAI2</i> expression was detected by real-time RT-PCR at the indicated time points, or by Western blot after 8-d culture (D). (<b>E</b>) Control HepG2 cells and the HepG2 cells expressing control shRNA or β3 shRNA were untreated or treated for 8 days with T/H/H. The relative activation of ERK (p-ERK/ERK) was calculated after densitometric analysis of Western blots. The expression of <i>ITGA3</i> and <i>SNAI2</i> was detected by real-time RT-PCR. <i>P</i> values, *<i>P</i><0.05, **<i>P</i><0.01.</p

β3 Integrin Promotes TGF-β1/H₂O₂/HOCl-Mediated Induction of Metastatic Phenotype of Hepatocellular Carcinoma Cells by Enhancing TGF-β1 Signaling

<div><p>In addition to being an important mediator of migration and invasion of tumor cells, β3 integrin can also enhance TGF-β1 signaling. However, it is not known whether β3 might influence the induction of metastatic phenotype of tumor cells, especially non-metastatic tumor cells which express low level of β3. Here we report that H₂O₂ and HOCl, the reactive oxygen species produced by neutrophils, could cooperate with TGF-β1 to induce metastatic phenotype of non-metastatic hepatocellular carcinoma (HCC) cells. TGF-β1/H₂O₂/HOCl, but not TGF-β1 or H₂O₂/HOCl, induced β3 expression by triggering the enhanced activation of p38 MAPK. Intriguingly, β3 in turn promoted TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of HCC cells by enhancing TGF-β1 signaling. β3 promoted TGF-β1/H₂O₂/HOCl-induced expression of itself via positive feed-back effect on p38 MAPK activation, and also promoted TGF-β1/H₂O₂/HOCl-induced expression of α3 and SNAI2 by enhancing the activation of ERK pathway, thus resulting in higher invasive capacity of HCC cells. By enhancing MAPK activation, β3 enabled TGF-β1 to augment the promoting effect of H₂O₂/HOCl on anoikis-resistance of HCC cells. TGF-β1/H₂O₂/HOCl-induced metastatic phenotype was sufficient for HCC cells to extravasate from circulation and form metastatic foci in an experimental metastasis model in nude mice. Inhibiting the function of β3 could suppress or abrogate the promoting effects of TGF-β1/H₂O₂/HOCl on invasive capacity, anoikis-resistance, and extravasation of HCC cells. These results suggest that β3 could function as a modulator to promote TGF-β1/H₂O₂/HOCl-mediated induction of metastatic phenotype of non-metastatic tumor cells, and that targeting β3 might be a potential approach in preventing the induction of metastatic phenotype of non-metastatic tumor cells.</p></div

β3 augments the promoting effect of TGF-β1/H₂O₂/HOCl on invasive capacity.

<p>(<b>A</b>) HepG2 cells were untreated or treated with T/H/H (TGF-β1/H₂O₂/HOCl) for 10 days, and then used for Matrigel invasion assay in absence or presence of control antibody, anti-α3 antibody, anti-αvβ3 antibody. (<b>B</b>) Control HepG2 cells and the HepG2 cells expressing control shRNA or β3 shRNA were untreated or treated for 10 days with T/H/H, and then used for Matrigel invasion assay in absence or presence of anti-αvβ3 antibody. (<b>C</b>) HepG2 cells were untreated or treated for 10 days with T/H/H in absence or presence of SU6656 (10 µM). The cells were then used for Matrigel invasion assay. <i>P</i> values, *<i>P</i><0.05, **<i>P</i><0.01.</p

β3 enables TGF-β1 to promote anoikis-resistance of HCC cells.

<p>(<b>A</b>) HepG2 cells were untreated or treated for 10 days with TGF-β1, H₂O­₂/HOCl, or T/H/H (TGF-β1/H₂O₂/HOCl). The cells were then used for the assay of anoikis as described in Methods. (<b>B</b>) Control HepG2 cells and the HepG2 cells expressing control shRNA or β3 shRNA were untreated or treated for 10 days with H₂O­₂/HOCl or T/H/H. The cells were then used for the assay of anoikis. (<b>C</b>) HepG2 cells were treated with T/H/H for 10 days in absence or presence of SB203580 (10 µM), PD98059 (10 µM), and SIS3 (2 µM), and then used for the assay of anoikis. Untreated and H₂O­₂/HOCl-treated cells were used as control. <i>P</i> values, *<i>P</i><0.05, **<i>P</i><0.01.</p

TGF-β1/H₂O₂/HOCl facilitates invasive capability of HCC cells.

<p>(<b>A</b>) Tumor cells were cultured in presence of TGF-β1 or T/H/H (TGF-β1, 5 ng/ml, H₂O₂, 100 µM, HOCl, 50 µM) for the indicated time, and then used for Matrigel invasion assay. (<b>B</b>) After 10-d culture in absence or presence of TGF-β1, H₂O₂ and HOCl, tumor cells were used for Matrigel invasion assay. (<b>C</b>) Tumor cells were treated for 10 days with H₂O₂/HOCl, TGF-β1, or T/H/H, and then incubated in presence of matrigel for 5 h. The cells with highly polymerized actin were visualized by staining with rhodamine-phalloidin (left). Their percentage in total cells was calculated (right). (<b>D</b>) Tumor cells were treated as described in C, and then cultured in presence of matrigel for 48 h. The MMP-2 and MMP-9 in supernatants were detected by zymography assay. The fold difference of active MMP-2 and MMP-9 was calculated after densitometric analysis of the gel. <i>P</i> values, *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

H₂O₂/HOCl cooperates with TGF-β1 to promote β3 expression and p38 MAPK activation.

<p>(<b>A</b>) HepG2 cells were stimulated with H₂O₂ (100 µM)/HOCl (50 µM), TGF-β1 (5 ng/ml), and T/H/H (TGF-β1/H₂O₂/HOCl). <i>ITGB3</i> expression was detected by real-time RT-PCR at the indicated time points, or by flow cytometry and Western blot after 8-d culture. (<b>B</b>) HepG2 cells were untreated or treated for 8 days with T/H/H in absence or presence of SB203580 (10 µM), PD98059 (10 µM), SP600125 (10 µM), wortmannin (WT, 40 nM), QNZ (40 nM), and SIS3 (2 µM). <i>ITGB3</i> expression was detected by real-time RT-PCR. (<b>C</b>) HepG2 cells were stimulated with H₂O₂/HOCl, TGF-β1, and T/H/H. The phosphorylation of p38 MAPK was detected by Western blot at the indicated time points. (<b>D and E</b>) Control HepG2 cells and the HepG2 cells expressing control shRNA or β3 shRNA were untreated or treated for 8 days with T/H/H. β3 expression was detected by flow cytometry (D). The relative activation of p38 MAPK (p-p38 MAPK/p38 MAPK) was calculated after densitometric analysis of Western blots (E). (<b>F</b>) HepG2 cells were untreated or treated for 8 days with T/H/H in absence or presence of SU6656 (10 µM). The relative activation of p38 MAPK was calculated after densitometric analysis of Western blots. (<b>G</b>) HepG2 cells were untreated or treated with TGF-β1 in presence of H₂O₂/HOCl within the indicated time-frame. <i>ITGB3</i> expression was detected by real-time RT-PCR. The relative activation of p38 MAPK was calculated after densitometric analysis of Western blots. <i>P</i> values, *<i>P</i><0.05, **<i>P</i><0.01.</p