Caspase-dependent signaling underlies glioblastoma cell death in response to the fungal metabolite, fusarochromanone

Fungal metabolites continue to show promise as a viable class of anticancer agents. In the present study, we investigated the efficacy of the fungal metabolite, fusarochromanone (FC101), for its antitumor activities in glioblastomas, which have a median survival of less than two years and a poor clinical response to surgical resection, radiation therapy and chemotherapy. Using clinically applicable doses, we demonstrated that FC101 induced glioblastoma apoptotic cell death via caspase dependent signaling, as indicated by the cleavage of poly(ADP-ribose) polymerase, glioblastoma (PARP). FC101 also induced differential reactive oxygen species (ROS) levels in glioblastoma cells, contrasting a defined role of oxidative stress in apoptotic cell death observed with other fungal metabolites. Furthermore, the antitumorigenic effects of FC101 on tumor cell migration were assessed. Cell migration assays revealed that FC101 significantly reduced the migratory capacity of glioblastomas, which are incredibly invasive tumors. Taken together, the present study establishes FC101 as a candidate anticancer agent for the cooperative treatment of glioblastomas

Biological activities of fusarochromanone: a potent anti-cancer agent

Background Fusarochromanone (FC101) is a small molecule fungal metabolite with a host of interesting biological functions, including very potent anti-angiogenic and direct anti-cancer activity. Results Herein, we report that FC101 exhibits very potent in-vitro growth inhibitory effects (IC50 ranging from 10nM-2.5 μM) against HaCat (pre-malignant skin), P9-WT (malignant skin), MCF-7 (low malignant breast), MDA-231 (malignant breast), SV-HUC (premalignant bladder), UM-UC14 (malignant bladder), and PC3 (malignant prostate) in a time-course and dose-dependent manner, with the UM-UC14 cells being the most sensitive. FC101 induces apoptosis and an increase in proportion of cells in the sub-G1 phase in both HaCat and P9-WT cell lines as evidenced by cell cycle profile analysis. In a mouse xenograft SCC tumor model, FC101 was well tolerated, non-toxic, and achieved a 30% reduction in tumor size at a dose of 8 mg/kg/day. FC101 is also a potent anti-angiogenenic agent. At nanomolar doses, FC101 inhibits the vascular endothelial growth factor-A (VEGF-A)-mediated proliferation of endothelial cells. Conclusions Our data presented here indicates that FC101 is an excellent lead candidate for a small molecule anti-cancer agent that simultaneously affects angiogenesis signaling, cancer signal transduction, and apoptosis. Further understanding of the underlying FC101’s molecular mechanism may lead to the design of novel targeted and selective therapeutics, both of which are pursued targets in cancer drug discovery

TNBC Therapeutics Based on Combination of Fusarochromanone with EGFR Inhibitors

Fusarochromanone is an experimental drug with unique and potent anti-cancer activity. Current cancer therapies often incorporate a combination of drugs to increase efficacy and decrease the development of drug resistance. In this study, we used drug combinations and cellular phenotypic screens to address important questions about FC101′s mode of action and its potential therapeutic synergies in triple negative breast cancer (TNBC). We hypothesized that FC101′s activity against TNBC is similar to the mTOR inhibitor, everolimus, because FC101 downregulates the phosphorylation of two mTOR substrates, S6K and S6. Since everolimus synergistically enhances the anti-cancer activities of two known EGFR inhibitors (erlotinib or lapatinib) in TNBC, we performed analogous studies with FC101. Phenotypic cellular assays helped assess whether FC101 acts similarly to everolimus, in both single and combination treatments with the two inhibitors. FC101 outperformed all other single treatments in both cell proliferation and viability assays. However, unlike everolimus, FC101 produced a sustained decrease in cell viability in drug washout studies. None of the other drugs were able to maintain comparable effects upon removal. Although we observed slightly additive effects when the TNBC cells were treated with FC101 and the two EGFR inhibitors, those effects were not truly synergistic in the manner displayed with everolimus

FC101 downregulates protein expression of cyclin D1, Cdc25A, CDK4/6 and upregulates expression of p21^Cip1 and p27^Kip1, leading to hypophosphorylation of Rb.

<p>COS7 cells were treated with FC101 for 24 h at indicated concentrations (A), or treated with FC101 at 1 µM for indicated time (B), followed by Western blotting with indicated antibodies. β-Tubulin was used for loading control. Representative blots are shown. Similar results were observed in at least 3 independent experiments.</p

FC101 inhibits cell proliferation and reduces cell viability.

<p>COS7 and HEK 293 cells were treated with FC101 (0–5 µM) for 6 days (for COS7) or 4 days (for HEK 293) (A, B), or 48 h (C, D), followed by cell number counting (A), morphological analysis (B), one solution assay (C), and trypan blue exclusion assay (D). For (A), (C), and (D), data represents mean ± SE (n = 6). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, difference with the control group (FC101 = 0 µM).</p

FC101 induces apoptosis. COS7 cells were treated with FC101 (0–5 µM) for 72 h.

<p>The cells were harvested and processed for apoptosis assay using the Annexin V-FITC Apoptosis Detection Kit. The cell distribution was analyzed by flow cytometry. (A) Histograms from a representative experiment show the apoptotic effect of FC101 on COS7 cells. The percentages of necrotic, late apoptotic, viable, and early apoptotic cells are displayed in Q1, Q2, Q3 and Q4, respectively. (B) Bar graphs show that FC101 induced apoptosis of COS7 cells in a concentration-dependent manner. Quantitative results (Q2+Q4) are displayed as fold change compared with control. Data represents mean ± SE (n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, difference with the control group (FC101 = 0 µM).</p

FC101 arrests cells at G₀/G₁ phase of the cell cycle.

<p>COS7 cells were treated with FC101 (0–5 µM) for 24 h. The cells were then harvested and processed for cell cycle analysis using Cellular DNA Flow Cytometric Analysis Kit and flow cytometry. (A) Histograms from a representative experiment show the effect of FC101 on cell cycle profile in COS7 cells. (B) Bar graphs show the effect of FC101 on the distribution (%) of COS7 cells in the G₀/G₁, S and G₂/M phases of the cell cycle. Data represents mean ± SE (n = 3). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, difference with the control group (FC101 = 0 µM).</p

FC101 induces caspase-dependent apoptosis.

<p>(A–D) COS7 cells, pretreated with or without Z-VAD-FMK (10 µM) for 1 h, were incubated with FC101 at indicated concentrations for 24 h, followed by caspase 3/7 activity assay (A), for 48 h, followed by trypan blue exclusion assay (B), or for 72 h, followed by Annexin V-FITC/PI staining and flow cytometry (C, D). (C) Histograms from a representative experiment show the effect of Z-VAD-FMK on FC101-induced apoptosis of COS7 cells. The percentages of necrotic, late apoptotic, viable, and early apoptotic cells are displayed in Q1, Q2, Q3 and Q4, respectively. (D) Bar graphs show that Z-VAD-FMK partially prevented FC101-induced apoptosis of COS7 cells. Quantitative results (Q2+Q4) were displayed as fold change compared with control. For (A), (B), and (D), data represents mean ± SE (n = 3). ^a<i>P</i><0.05, ^b<i>P</i><0.01, ^c<i>P</i><0.001, difference with the control group (FC101 = 0 µM). ^d<i>P</i><0.01, difference with Z-VAD-FMK group.</p

Fusarochromanone Induces G₁ Cell Cycle Arrest and Apoptosis in COS7 and HEK293 Cells

<div><p>Fusarochromanone (FC101), a mycotoxin produced by the fungus <i>Fusarium equiseti</i>, is frequently observed in the contaminated grains and feedstuffs, which is toxic to animals and humans. However, the underlying molecular mechanism remains to be defined. In this study, we found that FC101 inhibited cell proliferation and induced cell death in COS7 and HEK293 cells in a concentration-dependent manner. Flow cytometric analysis showed that FC101 induced G₁ cell cycle arrest and apoptosis in the cells. Concurrently, FC101 downregulated protein expression of cyclin D1, cyclin-dependent kinases (CDK4 and CDK6), and Cdc25A, and upregulated expression of the CDK inhibitors (p21^Cip1 and p27^Kip1), resulting in hypophosphorylation of Rb. FC101 also inhibited protein expression of Bcl-2, Bcl-xL, Mcl-1 and survivin, and induced expression of BAD, leading to activation of caspase 3 and cleavage of PARP, indicating caspase-dependent apoptosis. However, Z-VAD-FMK, a pan-caspase inhibitor, only partially prevented FC101-induced cell death, implying that FC101 may induce cell death through both caspase-dependent and -independent mechanisms. Our results support the notion that FC101 executes its toxicity at least by inhibiting cell proliferation and inducing cell death.</p></div