Stepwise changes in flavonoids in spores/pollen contributed to terrestrial adaptation of plants.

Protecting haploid pollen and spores against UV-B light and high temperature, 2 major stresses inherent to the terrestrial environment, is critical for plant reproduction and dispersal. Here, we show flavonoids play an indispensable role in this process. First, we identified the flavanone naringenin, which serves to defend against UV-B damage, in the sporopollenin wall of all vascular plants tested. Second, we found that flavonols are present in the spore/pollen protoplasm of all euphyllophyte plants tested and that these flavonols scavenge reactive oxygen species to protect against environmental stresses, particularly heat. Genetic and biochemical analyses showed that these flavonoids are sequentially synthesized in both the tapetum and microspores during pollen ontogeny in Arabidopsis (Arabidopsis thaliana). We show that stepwise increases in the complexity of flavonoids in spores/pollen during plant evolution mirror their progressive adaptation to terrestrial environments. The close relationship between flavonoid complexity and phylogeny and its strong association with pollen survival phenotypes suggest that flavonoids played a central role in the progression of plants from aquatic environments into progressively dry land habitats.

Discovery of a Pimaradiene that Decreases Viability of MDA-MB-468 Cells Through Inhibition of EGFR Signaling Pathway.

Triple-negative breast cancer (TNBC) is characterized by strong invasiveness, high relapse rates, and poor overall survival. It occurs in approximately 15-20 % of all breast cancer cases. Natural compounds are a promising option for managing breast cancer. ent-8(14),15-Pimaradiene-2ß,19-diol (JXE-23), is a pimaradiene isolated from the fern Aleuritopteris albofusca. However, the effects and molecular mechanisms of JXE-23 on cancer cells are still unknown. Thus, this study was designed to determine the potential of JXE-23 for its anticancer properties in TNBC cells. JXE-23 was evaluated for its antiproliferative activity in vitro against human breast cancer cell lines, and showed selectively cytotoxic activity against MDA-MB-468, an EGFR-overexpressing TNBC cancer cell line, with an IC50 value of 1.17±0.04 µM. Moreover, mechanistic investigations indicated that JXE-23 was significantly capable of inhibiting cell proliferation and viability in MDA-MB-468 cells. In addition, JXE-23 exerted an anticancer effect against MDA-MB-468 cells via restraining cell migration in a dose-dependent mode. Moreover, after treatment with JXE-23, the protein expressions of pEGFR, pERK, pAkt and p-p70S6K were significantly reduced in MDA-MB-468 cells. The results underscored that JXE-23 could be a potential lead compound for the treatment of EGFR-overexpressing TNBC cells.

Commentary: Machine learning for autism spectrum disorder diagnosis - challenges and opportunities - a commentary on Schulte-Rüther et al. (2022).

The commentary cites a study by Schulte-Rüther et al. (Journal of Child Psychology and Psychiatry, 2022) that proposed a machine learning model to predict a clinical best-estimate diagnosis of ASD when existing other co-occurring diagnoses. We discuss the valuable contribution of this work to developing a reliable computer-assisted diagnosis (CAD) system for ASD and point out that related research can be integrated with other multimodal machine learning methods. For future studies on developing the CAD system for ASD, we propose problems that need to be solved and potential research directions.

Onychiol B attenuates lipopolysaccharide-induced inflammation via MAPK/NF-κB pathways and acute lung injury in vivo.

Acute lung injury (ALI) is a devastating respiratory disorder characterized by rapid alveolar injury, uncontrolled inflammatory response, etc. Onychiol B is a cyathane diterpene originally isolated from fern plants. In this study, onychiol B can inhibit the production and secretion of pro-inflammatory cytokines such as NO, iNOS, IL-6 and TNF-α in LPS-stimulated RAW264.7 cells by restraining the NF-κB and the p38 MAPK pathway. In addition, it prevents the production of ROS and reduces the loss of mitochondrial membrane potential in LPS-stimulated RAW264.7 cells. Furthermore, in the acute lung injury mouse model induced by LPS injected into the trachea, onychiol B alleviates pulmonary edema, reverses inflammatory mediator TNF-α, IL-6, and IL-ß secretion in lung. In general, our data show that significant anti-ALI effects of onychiol B would render it a potential candidate for the treatment of inflammatory diseases.

Chronic pain, psychological distress, and quality of life in males with Duchenne muscular dystrophy.

AIM: To describe chronic pain in Duchenne muscular dystrophy (DMD) from children's/adolescents' perspectives, explore patient variables associated with self-reported pain, and examine the relationship between chronic pain, psychological functioning, and health-related quality of life (HRQoL). METHOD: This observational study included a paediatric cohort (aged 8-18 years; median age 9 years 4 months) with DMD under multidisciplinary care (n = 45). Clinical data of the latest visit were extracted from the electronic health record and assessment of pain, psychological distress, and HRQoL were performed during the same visit. RESULTS: Thirty-two patients reported pain during the previous 4 weeks, and 18 reported persistent or recurrent chronic pain. Average pain intensity of chronic pain was mild, with regions of the legs (n = 12), lower back (n = 6), hips (n = 6), and shoulder (n = 6) most frequently affected. Older age, higher body mass index, being non-ambulatory, wheelchair dependency, and spinal deformities were contextual variables related to the presence of chronic pain. Furthermore, chronic pain was significantly associated with psychological distress and reduced HRQoL in paediatric patients with DMD. INTERPRETATION: Chronic pain in paediatric DMD is frequent and widespread, highlighting the need for pain to be addressed in the routine care of affected young people. Chronic pain may make a significant contribution to psychological distress and impaired HRQoL in paediatric patients with DMD. WHAT THIS PAPER ADDS: Chronic pain differs in aetiology, scope, and nature compared with acute pain in paediatric Duchenne muscular dystrophy (DMD). Older age, higher body mass index, being non-ambulatory, wheelchair dependency, and spinal deformities are important patient variables. Chronic pain is significantly associated with psychological distress and reduced health-related quality of life in paediatric DMD.

Rasch analysis of the 32-item motor function measure in ambulant patients with Duchenne muscular dystrophy.

OBJECTIVE: This study analyzed the 32-item Motor Function Measure in a cohort of ambulatory patients with Duchenne muscular dystrophy using Rasch measurement methods. DESIGN: This is a psychometric study. SETTING: Rehabilitation centre of a large public children's hospital in Shenzhen, China. PARTICIPANTS: Data from 176 genetically confirmed ambulant patients with Duchenne muscular dystrophy (mean age 7.3 years, SD 2.3 years, range 3.1-13.1 years) were analyzed. RESULTS: Rasch analyses supported the Motor Function Measure domain D1 as a reliable (person reliability = 0.88, person separation index = 2.71) and valid (acceptable targeting, little misfit, minimal category disordering) measure in ambulant patients with Duchenne muscular dystrophy. Remodelling the domain D1 by collapsing item 25 from 4 to 3 response categories addressed the problematic disordered thresholds, resulting in a rebuilt domain D1 with enhanced measurement properties. However, findings for domains D2 and D3 did not fulfil most Rasch model expectations. There were disordered thresholds for most items in domains D2 and D3, with low reliability coefficients, item mistargeting and misfit, and large ceiling effects. CONCLUSION: Rasch analyses confirmed that the Motor Function Measure domain D1 was reliable and valid and provided a unidimensional measure for motor function in ambulant Duchenne muscular dystrophy patients. Accuracy of measurement had been enhanced through remodelling, and a rebuilt domain D1 with category collapsing for item 25 was proposed. The analysis revealed multiple limitations of the domains D2 and D3 that certain essential psychometrics were poorly met and, therefore, should be used with caution in this patient group.

Structurally Diverse Diterpenes from the South China Sea Soft Coral Sarcophyton trocheliophorum.

The present investigation of the South China Sea soft coral Sarcophyton trocheliophorum resulted in the discovery of six new polyoxygenated diterpenes, namely sartrocheliols A-E (1, 3, 5-8) along with four known ones, 2, 4, 9, and 10. Based on extensive spectroscopic data analysis, sartrocheliol A (1) was identified as an uncommon capnosane diterpene, while sartrocheliols B-E (3, 5-8) were established as cembrane diterpenes. They displayed diverse structural features not only at the distinctly different carbon frameworks but also at the various types of heterocycles, including the epoxide, γ-lactone, furan, and pyran rings. Moreover, their absolute configurations were determined by a combination of quantum mechanical-nuclear magnetic resonance (QM-NMR) approach, modified Mosher's method, and X-ray diffraction analysis. In the anti-tumor bioassay, compound 4 exhibited moderate cytotoxic activities against A549, H1975, MDA-MB-231, and H1299 cells with the IC50 values ranging from 26.3 to 47.9 µM.

Synthesis and Evaluation of Piperazine-Tethered Derivatives of Alepterolic Acid as Anticancer Agents.

Alepterolic acid is a natural diterpenoid isolated from Aleuritopteris argentea with potential anti-cancer activity. In this study, alepterolic acid was modified to construct a series of arylformyl piperazinyl derivatives (3a-3p). The synthesized derivatives were fully characterized with HRMS, NMR, and IR. Four compounds with inhibition rate higher than 30 % at 10 µM (3f, 3n, 3g and 3k) were further measured to obtain the IC50 values against four cancer cell lines, including hepatoma cell lines HepG2, lung cancer cell lines A549, estrogen receptor-positive cell lines MCF7, and triple-negative breast cancer (TNBC) cell lines MDA-MB-231 by MTT assay. It was found that these compounds were more effective to HepG2 and MDA-MB-231 cells, while less toxic to A549 and MCF7 cells, and compound 3n as the most toxic derivatve against MDA-MB-231 cell lines, with IC50 value of 5.55±0.56 µM. Trypan blue staining and colony formation assay showed that compound 3n inhibited the growth of MDA-MB-231 cells and prevented colony formation. Hoechst staining, flow cytometry and western blot analysis revealed that compound 3n induced caspase-dependent apoptosis in MDA-MB-231 cells. Conclusively, compound 3n was demonstrated to be a potential anti-cancer lead compound for further investigation.

Identification of an Alepterolic Acid Derivative as a Potent Anti-Breast-Cancer Agent via Inhibition of the Akt/p70S6K Signaling Pathway.

Alepterolic acid is a diterpene occurring in the fern Aleuritopteris argentea with potential biological activity that warrants further structural modification. In the present work, sixteen alepterolic acid derivatives were synthesized and evaluated for their anticancer activities. Among them, N-[m-(trifluoromethoxy)phenyl] alepterolamide displayed comparable activity (IC50 =4.20±0.21 µM) in MCF-7 cells. Moreover, mechanistic investigations indicated this compound was significantly capable of diminishing cell proliferation and viability of MCF-7 cells. After treatment with N-[m-(trifluoromethoxy)phenyl] alepterolamide, a significant increase in cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP) and Bax/Bcl2 ratio were observed in MCF-7 cells, leading to caspase-dependent apoptotic pathways. Further studies showed this compound promoted cellular apoptosis and inhibited migration in MCF-7 cells via modulation of the Akt/p70S6K signaling pathway. All these results revealed the potential of N-[m-(trifluoromethoxy)phenyl] alepterolamide as an appealing therapeutic drug candidate for breast cancer.

Antitumor Cembrane Diterpenoids from the South China Sea Soft Coral Lobophytum sp.

Two new highly functionalized cembrane diterpenoids named ximaolobophytolides A (1) and B (2) as minor components, together with seven related known compounds (3-9), have been isolated and identified from the Ximao soft coral Lobophytum sp. They were characterized by the presence of an α-methylene-γ-lactone moiety. Based on the comprehensive analyses of 1D and 2D NMR spectroscopic data, the absolute configurations of these two new compounds were elucidated by the combination of quantum mechanical (QM)-NMR and time-dependent density functional theory/electronic circular dichroism (TDDFT-ECD) calculation approaches. In the anti-tumor bioassays, compounds 3-9 showed moderate to significant inhibitory effects (IC50 values ranging from 29.66 to 0.39 µM) against the proliferations of five tumor cells HEL, A549, H1975, MDA-MB-231, and H1299. It might be worthy to point out that compounds 4, 7, and 8 exhibited better anti-tumor activities than that of the positive control Doxorubicin.

Elucidating Flavonoid and Antioxidant Activity in Edible and Medicinal Herbs Woodwardia japonica (L.f.) Sm. Based on HPLC-ESI-TOF-MS and Artificial Neural Network Model: Response to Climatic Factors.

Woodwardia japonica is a kind of great potential edible and medicinal fern. In a previous study, it was found that flavonoid and antioxidant activity of W. japonica from different sites were different. However, the cause of the differences has still been unclear, which has restricted the utilization of W. japonica. In this paper, flavonoid and antioxidant activity of W. japonica from nine different regions were determined with the method of a colorimetric assay with UV-VIS spectrophotometry and HPLC-ESI-TOF-MS, and the effects of climate factors on flavonoids and antioxidant activities were evaluated by mathematical modeling and statistical methods. The results showed: (1) total flavonoid content (TFC) of W. japonica from Wuyi Mountain (Jiangxi) was the highest, which might be related to the low temperature; (2) the differences of antioxidant activities of W. japonica might be related to precipitation; (3) five flavonols, two flavones and one isoflavone were tentatively identified in W. japonica; (4) flavonol and isoflavone might be affected by sunshine duration, and flavones were probably related to temperature. In conclusion, the effects of climate factors on flavonoids and antioxidants are significant, which would provide an important basis for further exploring the mechanism of climate affecting secondary metabolites.

Antibacterial effect of Cu2O/TiO2 photocatalytic composite on Pseudomonas marginalis pv. marginalis.

Cu2O/TiO2 visible-light photocatalytic composite was successfully synthesized by supercritical solvothermal route. Cu2O/TiO2 presented excellent bacterial inactivation activity for Pseudomonas marginalis pv. marginalis, which was related to the concentration of bacteria and the antibacterial time. The highest sterilization ratio reached up to 100% when the bacteria was treated with 80 µg/mL of Cu2O/TiO2 photocatalytic composite for 80 min, which could be further proved by the damage of integrity and shrink of the cell membrane in transmission electron microscopy (TEM) image. When the bacterial concentration was 1 × 105 CFU/mL, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined as 16 and 32 µg/mL by agar dilution, respectively. Meanwhile, the production of reactive oxygen species (ROS), glutathione reductase (GR) and glutathione (GSH) of Pseudomonas marginalis pv. marginalis treated by Cu2O/TiO2 were determined by DCFH-DA, DTNB and kinetic method, respectively, to evaluate the anti-oxidation capacity of bacteria cell. The enzyme activity of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) in bacteria treated with Cu2O/TiO2 were measured to further confirm the overproduction of ROS. Cu2O/TiO2 was demonstrated as the excellent visible-light photocatalyst for efficiently killing Pseudomonas marginalis pv. marginalis with the low dosage. Finally, the Cu2O/TiO2 composite photocatalytic material was applied to cucumber seedlings based on field experimental, and its inhibitory effect in practical application was judged by measuring the morphology, enzyme activity and resistance index of cucumber plants. It is of great significance to the practical application as a suitable and powerful antibacterial agent for Pseudomonas marginalis pv. marginalis and other bacteria.

Synthesis and biological evaluation of cytotoxic activity of novel podophyllotoxin derivatives incorporating piperazinyl-cinnamic amide moieties.

Podophyllotoxin, as a natural lignan isolated from the dried rhizomes and roots of several plant species of Podophyllum family, exhibits potent activity of interfering polymerization of tubulin and causes cancer cell apoptosis. Structure-activity relationship research revealed that modification at 4-position was tolerable for its potency. In the present study, podophyllotoxin derivatives incorporating piperazinyl-cinnamic amide moieties at 4-position were designed and synthesized. Their structures were confirmed by 1H NMR, 13C NMR, and mass spectral data. ADMET analysis proposed that these compounds had a good distribution and high clearance profile with little toxicity. The cytotoxicity of these derivatives was evaluated against four human cancer cell lines (MCF-7, A549, HeLa and PC-3) by MTT assay. Among all the compounds, compound 6e exhibited the best anti-proliferative properties with an IC50 = 0.08 ± 0.01 µM against MCF-7 cancer cell line. Further cellular mechanism studies by cell colony formation, mitochondrial membrane potential assay, nuclear morphology analysis and western blot confirmed that compound 6e could inhibit cancer cell proliferation and induce mitochondria-associated apoptosis in MCF-7 cells. Meanwhile, immunofluorescence assay revealed that compound 6e could apparently disrupt tubulin network in MCF-7 cells, and molecular docking further supported that compound 6e was able to bind into the colchicine site of tubulin. The above results might lay a foundation for further investigation for drug discovery based on podophyllotoxin.

Casticin inhibits stemness of hepatocellular carcinoma cells via disrupting the reciprocal negative regulation between DNMT1 and miR-148a-3p.

Casticin (CAS) is a polymethyl flavonoid from Fructus viticis and has multiple pharmacological activities, including anticancer. However, whether the molecular mechanism underlying CAS represses stemness characteristics in hepatocellular carcinoma (HCC) cells involves intervention in the reciprocal negative regulation between DNA methyltransferase 1 (DNMT1) and miR-148a-3p has not yet been reported. In this study, the effect of CAS on stemness characteristics of HCC cells and its mechanism were investigated. Results showed that CAS selectively reduced the viabilities of HCC cells but not L02 cells, as determined by CCK-8 assay. Importantly, the sub-cytotoxic concentrations of CAS could inhibit the stemness characteristics in HCC cells, as demonstrated by the expression of stemness biomarkers (CD44, EpCAM, Bmi1, Nanog, and Oct4), sphere forming assay, RT-qPCR, and Western blotting. In addition, CAS repressed DNMT1 activity and expression and increased miR-148a-3p. The effect of CAS on stemness characteristics was abolished by stable DNMT1 overexpression. MiR-148a-3p overexpression enhanced the reduction of CAS on stemness characteristics. DNMT1 overexpression promoted miR-148a-3p promoter hypermethylation as detected by methylation-specific PCR (MSP), which repressed its expression. Conversely, miR-148a-3p repressed DNMT1 expression by specific site binding to 3'-UTR of DNMT1 mRNA, as determined by luciferase assay. Moreover, the combination of CAS and agomir-148a-3p had robust effects on tumor suppression as compared to the sole activity of either molecule in nude mouse xenograft experiments in vivo. The findings suggested that CAS could inhibit stemness characteristics in HCC cells by interruption of the reciprocal negative regulation between DNMT1 and miR-148a-3p.

Incorporation of amino moiety to alepterolic acid improve activity against cancer cell lines: Synthesis and biological evaluation.

Aleuritopteris argentea (S. G. Gmél.) Fée is a medicinal fern consisting of an ent-labdane diterpene, i.e. alepterolic aicd, as the major metabolite. We recently isolated grams of alepterolic acid from A. argentea enabling subsequent structural modification. By incorporation of amino moiety to alepterolic acid, fifteen amide derivatives were synthesized, characterized, and further biological evaluated regarding their activity against four cancer cells and normal human liver cells. The potency of synthesized amides dramatically improved as compared to alepterolic aicd itself. The best hit (compound 11) inhibits HeLa cells with an IC50 of 7.39 ± 0.80 µM, and is nearly nontoxic to normal cells. Compound 11 exhibits an inhibitory effect on the colony forming ability of the four cancer cells, especially of HeLa cells. Moreover, it induces apoptosis of HeLa cells by decreasing mitochondrial membrane potential and altering expression of apoptosis-associated proteins. Release of cytochrome c, activation of caspases-3, caspases-9 and alteration of Bax/Bcl-2 balance was detected in the biological assays. These results imply that compound 11 can inhibit the proliferation of cervical cancer cell line HeLa and induce apoptosis through the mitochondrial pathway. These findings encourage further rational structural modification of 15- carboxyl group of alepterolic acid.

Straightforward synthesis, characterization, and cytotoxicity evaluation of hybrids of natural alkaloid evodiamine/rutaecarpine and thieno[2,3-d]pyrimidinones.

Dozens of hybrids of natural alkaloid evodiamine/rutaecarpine and thieno[2,3-d]pyrimidinones were synthesized in a straightforward method by condensation of substituted 2H-thieno[2,3-d][1, 3]oxazine-2,4(1H)-diones or N-methyl-2H-thieno[2,3-d][1, 3]oxazine-2,4(1H)-dione with 3,4-dihydro-ß-carbolines. In vitro cytotoxic assay discovered that compounds 9a, 10e, 11a, 11d, 11f, and 12a could induce antiproliferation against four different types of human cancer cells while compounds 10f and 12e were inactive. Notably, compound 11a displayed potent cell cytotoxicity for human non-small cell lung cancer cells A549, PC-9, human prostate cancer cells PC-3, and human breast cancer cell line MCF-7. Furthermore, compound 11a exhibited strong colony formation inhibition to A549 cells. These results unfold potential anticancer therapeutic applications of hybrids of thieno[2,3-d]pyrimidinones and quinazolinones.

Chrysin Inhibits Proinflammatory Factor-Induced EMT Phenotype and Cancer Stem Cell-Like Features in HeLa Cells by Blocking the NF-κB/Twist Axis.

BACKGROUND/AIMS: TNF-α and TGF-ß associated epithelial-mesenchymal transition (EMT) occurs via NF-κB-dependent transcriptional upregulation of Twist1.Chrysin (ChR) is a major active ingredient ofpropolis, which inhibits various cancer cells and possesses anti-inflammatory activities. This study aimed to assess whether and how ChR inhibits proinflammatory cytokine-induced EMT phenotype and cancer stem-like cell (CSLC) features in the HeLa cell line. METHODS: HeLa cells were co-administered TNF-α (10.0 ng/mL) and TGF-ß (5.0 ng/mL) for 24h following TGF-ß (5.0 ng/mL) alone for 6 d in the presence or absence of ChR (5.0, 10.0 and 20.0µM). Then, the levels of EMT-related factors, multi-potential transcription factors, and stem cell markers were analyzed by immunoblot. Wound healing and tumor sphere formation assays were performed to assess the migration and self-renewal capabilities of cells, respectively. Overexpression and/or knockdown of NF-κBp65 and/or Twsit1 were used to explore the molecular mechanisms. RESULTS: The results showed that ChR inhibited EMT and CSLC properties in HeLa cells administered TNF-α after prolonged TGF-ß treatment, in a concentration-dependent fashion. NF-κBp65 knockdown and ChR(10.0µM) cooperatively enhanced the inhibition of NF-κBp65 and Twist1 expression, EMT, and CSLC properties. Conversely, overexpression of NF-κBp65 combined with ChR(10.0µM) antagonized such activities. Meanwhile, Twist1silencing or overexpression combined with ChR treatment did not affect NF-κBp65 levels, but also reduced or enhanced EMT and CSLC properties. Importantly, overexpressing Twist1 combined with ChR reversed the effects of NF-κBp65 knockdown and ChR. CONCLUSION: ChR inhibits proinflammatory cytokine-induced EMT and CSLC features in HeLa cells by blocking the NF-κB/Twist axis.

8-bromo-7-methoxychrysin suppress stemness of SMMC-7721 cells induced by co-culture of liver cancer stem-like cells with hepatic stellate cells.

BACKGROUND: Our previous works have demonstrated that 8-bromo-7-methoxychrysin suppressed stemness of human hepatocellular carcinoma (HCC) cell line SMMC-7721 induced by condition medium from hepatic stellate cell line LX-2 that was activated by liver cancer stem-like cells (LCSCs). However, whether and whereby BrMC inhibits the stemness induced by co-culture of LCSCs and LX-2 cells remains to be investigated. METHODS: The second-generation spheres by sphere culture were identified and used as SMMC-7721-and MHCC97H-derived LCSLCs. SMMC-7721-and MHCC97-derived LCSCs/LX-2 cells transwell co-culture system was treated with BrMC and its lead compound chrysin. The concentrations of IL-6, IL-8, HGF and PDGF in condition medium from co-culture were measured by enzyme-linked immunosorbent assay (ELISA). The stemness of SMMC-7721 cells was evaluated by sphere formation assay and western blot analysis for expression levels of cancer stem cell markers (CD133 and CD44).The expression levels of cancer-associated fibroblast markers (FAP-α and α-SMA) were employed to evaluate pathologic activation of LX-2 cells. Addition of IL-6 and/or HGF or deletion of IL-6 and/or HGF was conducted to investigate the mechanisms for BrMC and chrysin treatment in SMMC-7721-derived LCSLCs co-cultured with LX-2cells. RESULTS: The co-culture of LCSLCs with LX-2 cells increased sphere formation capability as well as expression of CD133 and CD44 in SMMC-7721 cells, meanwhile, upregulated expression of FAP-α in LX-2 cells. ELISA indicated that the concentrations of IL-6 and HGF were significantly elevated in Co-CM than that of condition media from co-cultured SMMC-7721 cells/LX-2 cells. Treatment of BrMC and chrysin with co-cultures of SMMC-7721- and MHCC97H-derived LCSLCs and LX-2 cells effectively inhibited the above responses. Moreover, addition of IL-6 and/or HGF induced stemness of SMMC-7721 cells and activation of LX-2 cells, conversely, deletion of IL-6 and/or HGF suppressed those. Furthermore, the inhibitory effects of BrMC and chrysin on stemness of SMMC-7721 cells and activation of LX-2 cells were attenuated by addition of IL-6 or HGF, and enhanced by deletion of IL-6 or HGF. CONCLUSIONS: Our results suggest IL-6 and HGF may be the key communication molecules for the interaction between LCSLCs and HSCs, and BrMC and chrysin could block these effects and be the novel therapeutic candidates for HCC management.

Disruption of crosstalk between LX-2 and liver cancer stem-like cells from MHCC97H cells by DFOG via inhibiting FOXM1.

Hepatic stellate cell (HSC) line LX-2 is activated by liver cancer stem-like cells (LCSLCs) and produces various cytokines that make up most of the hepatocellular carcinoma (HCC) microenvironment. The new genistein derivative, 7-difluoromethoxyl-5,4'-di-n-octylgenistein (DFOG), shows anticancer effects in multiple malignancies by controlling forkhead box M1 (FOXM1). In this study, we aimed to assess whether DFOG disrupts the crosstalk between human HSC LX-2 cells and LCSLCs. Distinct generations of MHCC97H-derived spheres were obtained with the second generation considered as LCSLCs which displayed enhanced self-renewal ability and elevated expression levels of CD133, CD44, and EpCAM proteins, as well as tumorigenicity, as revealed by colony formation assay in vitro and tumorigenicity assay in vivo. LX-2 and MHCC97H cells were co-cultured with/without DFOG (1, 5, and 10 µM, respectively) using the transwell system. FOXM1 overexpression and/or knockdown were employed for mechanistic investigations. Our results suggested that Co-CM promoted LX-2 cell transformation into liver cancer-associated HSCs. Meanwhile, FOXM1 was up-regulated and the level of hepatocyte growth factor (HGF) was increased in LX-2 cells and in the supernatant after Co-CM stimulation. Sphere and colony formation abilities in MHCC97H cells, and protein levels of CD133, CD44, and EpCAM, were also markedly elevated. DFOG dose-dependently inhibited the above effects, similar to FOXM1 knockdown in LX-2 cells. FOXM1 overexpression reversed the inhibitory effects of DFOG or FOXM1 knockdown or both on LX-2 cell activation and LCSLC feature induction in MHCC97H cells by LCSLC/LX-2 co-culture. This study demonstrated that DFOG disrupts the crosstalk between HSCs and LCSLCs to suppress LCSLC features via down-regulating FOXM1 expression and reducing HGF secretion in HSCs.