Hye-Yeon Jang’s research while affiliated with Korea Research Institute of Chemical Technology and other places

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A) Structures of the marketed drugs, including Nirmatrelvir, Simnotrelvir, Leritrelvir, and Ensitrelvir, and the preclinical compounds GC‐14 and AA‐625. EC50 values and CC50 values correspond to antiviral effects and cytotoxicity, respectively. B) Illustration of the general design concept of focused compound libraries.

Structures of azides (N1 to N69) employed as starting materials.

Effects of the racemate C5N17 and its two enantiomers (C5N17A, C5N17B) on Mpro inhibition and antiviral efficacy determined in a cellular assay. A) Mpro inhibitory potency of C5N17, C5N17A, C5N17B, and Nirmatrelvir. B) Anti‐SARS‐CoV‐2 activity of C5N17, C5N17A, C5N17B, and Nirmatrelvir in Calu‐3 cells. C) Toxicity of C5N17, C5N17A, C5N17B, and Nirmatrelvir on Calu‐3 cells. D) Western blot analysis for detecting viral S and N proteins in Calu‐3 cells in the presence of C5N17B or Nirmatrelvir on day 2 post‐infection. E) Quantitative RT‐PCR for measuring SARS‐CoV‐2 viral RNA amounts accumulated in Calu‐3 cell culture supernatants in the presence of increasing concentrations of C5N17B or Nirmatrelvir on day 2 post‐infection. n.t., not tested. In all experiments, Nirmatrelvir was used as a positive control. In panels (A–C) and (E), all values are represented as means ± SD from three independent experiments.

X‐ray co‐crystal structures of the (R)‐configurated C5N17B (PDB ID: 9G0I) and its (S)‐configurated enantiomer C5N17A (PDB ID: 9G0H) in complex with Mpro. A) View of C5N17B (green) in the binding pocket. Hydrogen bonds are shown as magenta‐colored dashed lines; π–π stacking is indicated as green dashed lines. B) View of the interactions of C5N17B (green) with His41. C) View of C5N17A (red) in the binding pocket. D) Binding pose comparison of C5N17B (green) and C5N17A (red).

Comparison of the Mpro binding modes of C5N17B and selected other inhibitors with piperazine scaffolds. A) Binding pose comparison of C5N17B (green), GC‐14[¹³] (yellow, PDB ID: 8ACL), and JZD‐07[³⁰] (cyan, PDB ID: 8GTV). B) Superposition of the binding modes of C5N17B (green), nirmatrelvir (orange, PDB ID: 7VH8), and ensitrelvir (blue, PDB ID: 7VU6).

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Miniaturized Modular Click Chemistry‐enabled Rapid Discovery of Unique SARS‐CoV‐2 M Inhibitors With Robust Potency and Drug‐like Profile

September 2024

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118 Reads

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4 Citations

Advanced Science

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The COVID‐19 pandemic has required an expeditious advancement of innovative antiviral drugs. In this study, focused compound libraries are synthesized in 96‐ well plates utilizing modular click chemistry to rapidly discover potent inhibitors targeting the main protease (Mpro) of SARS‐CoV‐2. Subsequent direct biological screening identifies novel 1,2,3‐triazole derivatives as robust Mpro inhibitors with high anti‐SARS‐CoV‐2 activity. Notably, C5N17B demonstrates sub‐micromolar Mpro inhibitory potency (IC50 = 0.12 µM) and excellent antiviral activity in Calu‐3 cells determined in an immunofluorescence‐based antiviral assay (EC50 = 0.078 µM, no cytotoxicity: CC50 > 100 µM). C5N17B shows superior potency to nirmatrelvir (EC50 = 1.95 µM) and similar efficacy to ensitrelvir (EC50 = 0.11 µM). Importantly, this compound displays high antiviral activities against several SARS‐CoV‐2 variants (Gamma, Delta, and Omicron, EC50 = 0.13 – 0.26 µM) and HCoV‐OC43, indicating its broad‐spectrum antiviral activity. It is worthy that C5N17B retains antiviral activity against nirmatrelvir‐resistant strains with T21I/E166V and L50F/E166V mutations in Mpro (EC50 = 0.26 and 0.15 µM, respectively). Furthermore, C5N17B displays favorable pharmacokinetic properties. Crystallography studies reveal a unique, non‐covalent multi‐site binding mode. In conclusion, these findings substantiate the potential of C5N17B as an up‐and‐coming drug candidate targeting SARS‐CoV‐2 Mpro for clinical therapy.

Effects of fisetin on HDF and HEK viability. Cells were treated with fisetin at various concentrations, and cytotoxicity was evaluated after 24 h. (A,B) After pretreatment with fisetin, cells were irradiated with UVA and incubated for 24 h. Cell viability was analyzed using the MTT assay. Fisetin inhibited UVA-induced MMP-1 and MMP-3 expression in HDFs and HEKs. HDFs and HEKs were treated with fisetin, irradiated with UVA, and then incubated for 24 h. (C,E) MMP-1 and MMP-3 protein expression was analyzed using Western blotting; β-actin was used for loading control. (D,F) MMP-1 and MMP-3 mRNA levels were analyzed using real-time PCR; GAPDH was used for internal control. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA.

Fisetin inhibited the expression of pro-inflammatory cytokines in UVA-irradiated HDFs and HEKs. (A) HDFs were treated with fisetin (10 μM) and exposed to UVA (8 J/cm²). (B) HEKs were treated with fisetin (5 μM) and exposed to UVA (5 J/cm²). After 4 h, pro-inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) mRNA levels were analyzed using real-time PCR. GAPDH was used as an internal control. (C–F) Inhibitory effects of fisetin on UVA-induced reactive oxygen species (ROS) production in HDFs (C,D) and HEKs (E,F). HDFs and HEKs were treated with fisetin, irradiated with UVA, and then incubated for 1 h. Cells were then washed with PBS and incubated with DCF-DA for 30 min at room temperature. Intracellular ROS production was measured using DCF-DA fluorescence using a FACS flow cytometer. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA.

Fisetin suppressed UVA-induced MAPK activation in HDFs and HEKs. (A,B) Fisetin inhibits UVA-induced AP-1 activation in HDFs. (C,D) HDFs and HEKs were treated with fisetin and exposed to UVA. After 30 min, total ERK, p38, JNK and phospho-ERK, phospho-p38, and phospho-JNK levels were analyzed using Western blotting. β-actin was used for loading control. After 2 h, nuclear extracts were prepared. Western blotting was performed to determine the nuclear levels of the AP-1 (p-c-Jun, p-c-Fos) subunits. PCNA was used as an internal control for the nuclear fraction. (E,F) To assess phosphorylation levels, the density of each band was quantified using ImageJ software (ver. 1.52 for MS Windows), and the relative density ratio of each protein was calculated accordingly. To assess phosphorylation levels, the density of each band was quantified using the ImageJ software (ver. 1.52 for MS Windows), and the relative density ratio of each protein was calculated accordingly. (G) HDFs were treated with fisetin and irradiated with UVA, and the promoter activity of AP-1 was measured using the dual-luciferase reporter assay. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA. ND: not detected.

Fisetin inhibited the expression of NOX in UVA-irradiated HDFs and HEKs. (A) HDFs were treated with fisetin (10 μM) and exposed to UVA (8 J/cm²). (B) HEKs were treated with fisetin (5 μM) and exposed to UVA (5 J/cm²). NOX1–NOX5 mRNA levels were analyzed using real-time PCR. GAPDH was used as an internal control. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA. Regulation of MMP-1 and MMP-3 expression by NOX. NOX1–NOX5 were knocked down in HDFs and HEKs using NOX siRNA. Then, the cells were irradiated with UVA and incubated for 24 h. (C–F) * p < 0.01 vs. negative siRNA UVA-treated group. The data are expressed as the means ± SE (n = 3).

Schematic diagram of protective mechanisms of the fisetin on UVA-induced HDF and HEK cell damages via MMP-1 and MMP-3 expression through the NOX/ROS/MAPK pathway.

Fisetin Inhibits UVA-Induced Expression of MMP-1 and MMP-3 through the NOX/ROS/MAPK Pathway in Human Dermal Fibroblasts and Human Epidermal Keratinocytes

December 2023

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22 Reads

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6 Citations

International Journal of Molecular Sciences (IJMS)

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Fisetin is a flavonoid found in plants and has been reported to be effective in various human diseases. However, the effective mechanisms of ultraviolet-A (UVA)-mediated skin damage are not yet clear. In this study, we investigated the protective mechanisms of fisetin regarding UVA-induced human dermal fibroblasts (HDFs) and human epidermal keratinocytes (HEKs) damages. Fisetin showed a cytoprotective effect against UVA irradiation and suppressed matrix metalloproteinases (MMPs), MMP-1, and MMP-3 expression. In addition, fisetin was rescued, which decreased mRNA levels of pro-inflammatory cytokines, reactive oxygen species production, and the downregulation of MAPK/AP-1 related protein and NADPH oxidase (NOX) mRNA levels. Furthermore, UVA-induced MMP-1 and MMP-3 were effectively inhibited by siRNAs to NOX 1 to 5 in HDFs and HEKs. These results indicate that fisetin suppresses UVA-induced damage through the NOX/ROS/MAPK pathway in HDFs and HEKs.

Effects of PBE on BMM viability. (A) BMMs (1 × 10⁴ cells/well) were seeded in 96-well plates, treated with PBE (0, 10, 25, 50, and 100 µg/mL), and incubated at 37 °C with 5% CO2 for 24 h. (B) BMMs were treated with PBE (50 µg/mL) and incubated for 0, 1, 2, 3, or 4 days. After incubation, cell viability was measured using the MTT assay. Bars labeled with different superscripts indicate significant differences (* p < 0.05 vs. control or vs. 0 days). The results are expressed as the mean ± standard deviation of at least three independent experiments (n = 3).

Effects of PBE on RANKL-induced osteoclast differentiation and bone resorption. BMMs were cultured for 4 days in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) with PBE (0, 25, and 50 μg/mL). (A) TRAP staining (upper panel) and (B) bone resorption (lower panel) were performed to visualize osteoclast differentiation. Scale bar, 250 μm. (C) TRAP-positive MNCs (TRAP⁺ MNCs; nuclei > 3) were counted as mature osteoclasts. (D) TRAP activity in BMMs was analyzed on day 4 according to the manufacturer’s protocol. (E) Bone resorption areas were quantified using ImageJ software, and significant differences between the PBE and control groups are indicated (** p < 0.005; *** p < 0.001). Data are presented as the mean ± standard deviation and are representative of at least three experiments.

Effect of PBE on the expression of osteoclastogenesis-related genes. BMMs were cultured with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the absence or presence of PBE (50 μg/mL) for 0, 1, 2, 3, or 4 days. The expression of genes (Acp5 (TRAP), Oscar, CTSK (cathepsin K), Tm7sf4 (dendritic cell-specific transmembrane protein, DC-STAMP), Atp6v0d2, and NFATc1) involved in osteoclast differentiation was evaluated by qRT-PCR. Experiments were performed in triplicate, and the error bars represent the standard deviation. Significant differences between the PBE and control groups are indicated (* p < 0.05; ** p < 0.005; *** p < 0.001).

Effect of PBE on MAPK activation in BMMs. BMMs were cultured with RANKL (100 ng/mL) and M-CSF (30 ng/mL), with or without PBE (50 μg/mL), for 5, 15, 30, and 60 min. (A) Protein expression levels were analyzed by Western blotting using anti-ERK, p-ERK, p38, p-p38, JNK, and p-JNK antibodies. (B) Band intensities were quantified using ImageJ software. Bars labeled with different superscripts indicate significant differences (# p < 0.05 vs. RANKL-non treated group; ** p < 0.005 vs. control). Data are expressed as the mean ± SD of at least three independent experiments (n = 3).

Effect of PBE on NF-κB activation in BMMs. BMMs were cultured with RANKL (100 ng/mL) and M-CSF (30 ng/mL), with or without PBE (50 μg/mL), for 5, 15, 30, and 60 min. (A) Protein expression levels were analyzed by Western blotting using anti-NF-κB p65, p-NF-κB p65, IκBα, and p-IκBα antibodies. (B) Image J software was used to quantify the protein bands. Bars labeled with different superscripts indicate significant differences (# p < 0.05 vs. RANKL non-treated group; ** p < 0.005 vs. control; *** p < 0.001 vs. control). Data are presented as the mean ± SD of at least three independent experiments (n = 3).

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i>Protaetia brevitarsis Extract Attenuates RANKL-Induced Osteoclastogenesis by Inhibiting the JNK/NF-κB/PLCγ2 Signaling Pathway

July 2023

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31 Reads

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2 Citations

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Protaetia brevitarsis (PB)-derived bioactive substances have been used as food and medicine in many Asian countries because of their antioxidant, antidiabetic, anti-cancer, and hepatoprotective properties. However, the effect of PB extracts (PBE) on osteoclast differentiation is unclear. In this study, we investigated the effect of PBE on RANKL-induced osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs). To investigate the cytotoxicity of PBE, the viability of BMMs was confirmed via MTT assay. Tartrate-resistant acid phosphatase (TRAP) staining and pit assays were performed to confirm the inhibitory effect of PBE on osteoclast differentiation and bone resorption. The expression levels of osteoclast differentiation-related genes and proteins were evaluated using quantitative real-time PCR and Western blotting. PBE attenuated osteoclastogenesis in BMMs in TRAP and pit assays without cytotoxicity. The expression levels of osteoclast marker genes and proteins induced by RANKL were decreased after PBE treatment. PBE suppressed osteoclastogenesis by inhibiting the RANKL-induced activated JNK/NF-κB/PLCγ2 signaling pathway and the expression of NFATc1 and c-Fos. Collectively, these results suggest that PBE could be a potential therapeutic strategy or functional product for osteoclast-related bone disease.

Figure 2. Effects of CDDO on TPA-mediated MAPK and AP-1 activation through a PPARγ-independent pathway. (A) The transfection efficiency of si-PPARγ was confirmed in the nuclear fraction of cells. PCNA was used as the internal control. (B) Cells were transfected with PPARγ siRNA and then treated with TPA and CDDO for 24 h. MMP-9 protein expression was analyzed using western blotting. (C) Cells were treated with CDDO and/or TPA for 30 min. The phosphorylation of ERK, p38, and JNK was determined by western blotting using β-actin as a loading control. (D) Cells were treated with CDDO and/or TPA. After a 2 h incubation, prepare the extracted cytoplasmic and nuclear. The nuclei of p-c-Jun, p-c-Fos, total c-Jun, and c-Fos were quantitated by western blotting. PCNA was the internal control for the nucleus. (E) AP-1-luc reporters were co-transfected with a Renilla luciferase reporter. Cells were treated with CDDO, TPA was added for 2 h, and the AP-1 promoter activities were analyzed using a luciferase reporter assay. The ratio of western blots was quantified with ImageJ. Values are the mean ± standard error of the mean of three independent experiments. # P<0.05 vs. untreated cells, * P<0.05 vs. TPA alone. PPARγ, peroxisome proliferator-activated receptor γ; siRNA, small interfering RNA; CDDO, 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate; p-, phosphorylated; AP-1, activator protein-1.

Figure 3. NF-κB activation of CDDO in MCF-7 cells. (A) Cells were treated with CDDO and/or TPA. After incubation for 2 h, prepare the extracted cytoplasmic and nuclear. The levels of p50 and p65 in the nucleus, of p-IκBα and p-IKKα/β in the cytoplasm, and of total IκBα, IKKα, and IKKβ were analyzed by western blotting. PCNA was the internal control for the nucleus, and β-actin was the loading control for the cytoplasm. (B) NF-κB-luc reporters were co-transfected with a Renilla luciferase reporter. Cells were treated with CDDO, TPA was added for 2 h, and the NF-κB promoter activities were analyzed using a luciferase reporter assay. The ratio of western blots was quantified with ImageJ. Values are the mean ± standard error of the mean of three independent experiments.

Figure 4. CDDO suppression of migration and invasion of MCF-7 cells. (A) Chamber migration and invasion assays were implemented without or with Matrigel. Cells were treated with CDDO and/or TPA. After 24 h, cells were stained, and microscopic photography was conducted. Bar, 200 µm. (B) Data were quantified by counting the migrated cells in four randomly selected regions during the migration assay. (C) Data were quantified by counting the invading cells in four randomly selected regions during the invasion assay. Values are the mean ± standard error of the mean of three independent experiments. # P<0.05 vs. untreated cells, * P<0.05 vs. TPA alone. CDDO, 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate.

CDDO, a PPAR‑γ ligand, inhibits TPA‑induced cell migration and invasion through a PPAR‑γ‑independent mechanism

August 2022

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40 Reads

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1 Citation

Oncology Letters

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Peroxisome proliferator-activated receptor-γ (PPAR-γ) acts as a key factor in breast cancer metastasis. Notably, PPAR-γ can inhibit metalloproteinase (MMP), which is involved in cancer metastasis. Our previous study revealed that PPAR-γ was related to breast cancer metastasis. The present study aimed to investigate whether the PPAR-γ ligand 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) mediated suppression of cell invasion and reduced the expression of MMP-9 in breast cancer cells. The results indicated that CDDO reduced MMP-9 expression, cell migration and invasion of breast cancer cells by inhibiting TPA-induced phosphorylation of mitogen-activated protein kinases, and downregulating the activities of activator protein-1 and nuclear factor κB. Notably, knock-out of PPAR-γ by small interfering RNA in MCF-7 cells revealed that TPA-induced MMP-9 expression occurred through a PPAR-γ-independent pathway. These data indicated that the downregulatory effect of CDDO on MMP-9 expression was affected by a mechanism independent of PPAR-γ. In conclusion, the findings of the present study suggested that CDDO may act as a key agent in the regulation of breast cancer metastasis, suggesting CDDO as a new targeted therapy for breast cancer.

MMP-9 and SIRT6 expression in human breast cancer tissue and normal tissue. (a) Western blot analysis of MMP-9 and SIRT6 expression in normal (N) and breast cancer (T) tissues. GAPDH was used as a loading control. (b) Relative abundance of MMP-9 and SIRT6 levels in (a). (***p < 0.005). N normal breast tissue, T breast cancer tissue.

Upregulation of MMP-9 expression by TPA or TNF-α treatment. MCF-7 and MDA-MB-231 cells were treated with TPA or TNF-α for 24 h. (a, b) The amount of MMP-9 secreted into the medium was analyzed by gelatin zymography (zymo-MMP-9). MMP-9 levels were analyzed by western blotting using β-actin as an internal control. (c, d) MMP-9 mRNA levels were examined by RT-qPCR, using GAPDH as an internal control. The results are presented as means ± standard error of three independent experiments. *p < 0.05, vs. untreated controls. RT-qPCR reverse transcription quantitative polymerase chain reaction.

Effect of SIRT6 on MMP-9 expression. MCF-7 and MDA-MB-231 cells were transfected with control siRNA or SIRT6 siRNA and treated with TPA or TNF-α for 24 h. (a, b) MMP-9 secretion into the medium was analyzed by gelatin zymography (zymo-MMP-9). MMP-9 and SIRT6 levels were examined by western blotting using β-actin as an internal control. (c, d) MMP-9 and SIRT6 mRNA levels were examined by RT-qPCR, using GAPDH as an internal control. The results are presented as means ± standard error of three independent experiments. *p < 0.05, vs. TPA- or TNF-α-treated control siRNA; **p < 0.01 vs. TPA- or TNF-α-treated control siRNA.

Effect of SIRT6 overexpression on MMP-9 expression. MCF-7 and MDA-MB-231 cells were infected with adeno-SIRT6 virus or adeno-LacZ virus and then treated with TPA or TNF-α for 24 h. (a, b) MMP-9 secretion in the medium was examined by gelatin zymography (zymo-MMP-9). MMP-9 and SIRT6 protein levels were examined by western blotting using β-actin as an internal control. (c, d) MMP-9 and SIRT6 mRNA levels were analyzed by RT-qPCR, using GAPDH as an internal control. The results are presented as means ± standard error of three independent experiments. *p < 0.05 vs. TPA- or TNF-α-treated adeno-LacZ virus.

Effect of SIRT6 on MAPK. (a, b) MCF-7 and MDA-MB-231 cells transfected with control siRNA or SIRT6 siRNA were treated with TPA or TNF-α for 30 min. Phosphorylated ERK, JNK, and p38 levels and total ERK, JNK, and p38 levels were examined by western blotting. β-actin was used as an internal control.

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Inhibition of cell invasion and migration by targeting matrix metalloproteinase-9 expression via sirtuin 6 silencing in human breast cancer cells

July 2022

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93 Reads

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24 Citations

Scientific Reports

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Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on protein kinase C activator- and cytokine-mediated cancer cell invasion and migration in MCF-7 and MDA-MB-231 cells and the association between SIRT6 and matrix metalloproteinase-9 (MMP-9) expression. To assess MMP-9 and SIRT6 expression in patients, protein levels in BC tissues were analyzed. MCF-7 and MDA-MB-231 cell viability was analyzed using MTT assays. SIRT6 was silenced in both cell lines and protein secretion, expression, and mRNA levels were analyzed. Transcription factor DNA activity was investigated using luciferase assays. Matrigel invasion assays were used to assess the effects of SIRT6 in both cell lines. SIRT6 and MMP-9 expression in cancer tissues was significantly higher than in paired normal breast tissues. 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) increased MMP-9 expression and cell invasion and migration, but SIRT6 knockdown abolished these effects. SIRT6 overexpression additively increased TPA- and TNF-α-induced MMP-9 expression. SIRT6 knockdown suppressed the mitogen-activated protein kinase (MAPK) signaling pathway and thus TPA- and TNF-α-induced MMP-9 expression. SIRT6 silencing suppressed TPA- and TNF-α-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) expressions in both cell lines, and treatment with MAPK, NF-κB, and AP-1 inhibitors reduced MMP-9 expression. The anti-invasive effects of SIRT6 in BC cells might be mediated by suppression of MAPK phosphorylation and reduction in NF-κB and AP-1 DNA activities, leading to MMP-9 downregulation, suggesting that SIRT6 modulation has the potential to target BC metastasis.

Fig. 3. AURKA regulates TPA-induced invasion and migration in SW480 colon cancer cells. (A) Cells were transfected with AURKAsiRNA and were transferred into a 24-well chamber and treated with 100 nM TPA. After 24 h, the cells were fixed and stained with a toluidine blue solution. For the scratch migration assay, confluent cell monolayers were scratched at 0 h. (B) Cells were seeded in a 24-well chamber and treated with 100 nM TPA. After 24 h, the cells were fixed and stained with a toluidine blue solution.

Fig. 4. AURKA inhibits TPA-induced NF-κB and AP-1 activation in SW480 colon cancer cells. (A) Cells were transfected with AURKAsiRNA for 24 h and then treated with TPA for 3 h. The expressions of p65 and p-c-Jun were analyzed using western blotting. PCNA was used as the nuclear loading control. (B) SW480 cells were pretreated with AURK inhibitors (reversine and VX-680) for 1 h and then stimulated with TPA for 3 h. Cell nuclear extracts were obtained and subjected to western blotting to determine the nuclear levels of NF-κB (p65) and AP-1 (p-c-Jun) subunits. PCNA was used as the nuclear loading control. (C) Cells were treated with AURK inhibitors (reversine and VX-680) for 1 h and then stimulated with TPA for 3 h. The DNA binding activity of NF-κB was determined through an electrophoretic mobility gel shift assay (EMSA).

Aurora kinase A induces migration and invasion by inducing epithelial-to-mesenchymal transition in colon cancer cells

December 2021

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34 Reads

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12 Citations

BMB reports

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Aurora kinase is a family of serine/threonine kinases intimately associated with mitotic progression and the development of human cancers. Studies have shown that aurora kinases are important for the protein kinase C (PKC)-induced invasion of colon cancer cells. Recent studies have shown that aurora kinase A promotes distant metastasis by inducing epithelial-to-mesenchymal transition (EMT) in colon cancer cells. However, the role of aurora kinase A in colon cancer metastasis remains unclear. In this study, we investigated the effects of aurora kinase A on PKC-induced cell invasion, migration, and EMT in human SW480 colon cancer cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) changed the expression levels of EMT markers, increasing α-SMA, vimentin, and MMP-9 expression and decreasing E-cadherin expression, with changes in cell morphology. TPA treatment induced EMT in a PKC-dependent manner. Moreover, the inhibition of aurora kinase A by siRNAs and inhibitors (reversine and VX-680) suppressed TPA-induced cell invasion, migration, and EMT in SW480 human colon cells. Inhibition of aurora kinase A blocked TPA-induced vimentin and MMP-9 expression, and decreased E-cadherin expression. Furthermore, the knockdown of aurora kinase A decreased the transcriptional activity of NF-κB and AP-1 in PKC-stimulated SW480 cells. These findings indicate that aurora kinase A induces migration and invasion by inducing EMT in SW480 colon cancer cells. To the best of our knowledge, this is the first study that showed aurora kinase A is a key molecule in PKC-induced metastasis in colon cancer cells.

Figure 4. Triptolide inhibits the invasion of Matrigel by MCF-7 cells. (A) Matrigel invasion assays were carried out on cells treated with TPA alone or with triptolide. After 24 h, cells on the bottom of the filter were fixed and stained with crystal violet, and microscopic photography was performed (magnification, x40) (B) Results were quantified by counting the migrated cells in five randomly selected areas. Data are presented as the mean ± SEM of three independent experiments. * P<0.05 and ** P<0.01 vs. TPA alone. TPA, 12-O-tetradecanoylphorbol-13-acetate.

Triptolide inhibits matrix metalloproteinase-9 expression and invasion of breast cancer cells through the inhibition of NF-κB and AP-1 signaling pathways

May 2021

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32 Reads

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14 Citations

Oncology Letters

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Park kwang hyun

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Triptolide is a diterpenoid epoxide that is endogenously produced by the thunder god vine, Tripterygium wilfordii Hook F. Triptolide has demonstrated a variety of biological activities, including anticancer activities, in previous studies. Invasion and metastasis are the leading causes of mortality for patients with breast cancer, and the increased expression of matrix metalloproteinase-9 (MMP-9) has been shown to be associated with breast cancer invasion. Therefore, the aim of the present study was to investigate the effect of triptolide on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced cell invasion and MMP-9 expression in breast cancer cells. The expression of signal molecules was examined by western blotting, zymography and quantitative polymerase chain reaction; an electrophoretic mobility gel shift assay was also used, and cell invasiveness was measured by an in vitro Matrigel invasion assay. The MCF-7 human breast cancer cell line was treated with triptolide at the highest concentrations at which no marked cytotoxicity was evident. The results demonstrated that triptolide decreased the expression of MMP-9 through inhibition of the TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK) and the downregulation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity. In addition, a Transwell assay revealed that triptolide reduced the ability of MCF-7 cells to invade Matrigel. These data demonstrate that the anti-invasive effect of triptolide is associated with the inhibition of ERK signaling and NF-κB and AP-1 activation, and suggest that triptolide may be a promising drug for breast cancer.

Fig. 1. Effect of CME on BMM viability. BMMs were treated with CME in a dose-and time-dependent manner. (a) Cells were treated with various concentrations of CME (0, 50, 100, 150, and 300 μg/mL) and incubated for 1 days. (b) Cells were treated with 150 μg/mL CME and incubated for 0, 1, 2, 3, or 4 days. Data are presented as the mean ± standard deviation and are representative of at least three experiments.

Aqueous extract of Chrysanthemum morifolium Ramat. inhibits RANKL-induced osteoclast differentiation by suppressing the c-fos/NFATc1 pathway

February 2021

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56 Reads

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13 Citations

Archives of Oral Biology

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Objective The flower of chrysanthemum, used worldwide as a medicinal and edible product, has shown various bioactivities, such as anti-inflammatory, antioxidant, anti-tumorigenic, and hepatoprotective activities, as well as cardiovascular protection. However, the effect of Chrysanthemum morifolium Ramat. on the regulation of osteoclast differentiation has not yet been reported. In this study, we aimed to investigate the inhibitory effect of Chrysanthemum morifolium Ramat. water extract (CME) on RANKL-induced osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). Study design Bone marrow-derived macrophages (BMMs) isolated from the C57BL/6 J mice. The viability of BMMs was detected with MTT assays. Inhibitory effects of CME on osteoclast differentiation and bone resorption was measured by TRAP staining and Pit assay. Osteoclast differentiation-associated gene expression were assessed by Real-time quantitative polymerase chain reaction. Intracellular signaling molecules was assessed by western blot. Results CME significantly inhibited osteoclast differentiation in BMMs without cytotoxicity, besides inhibiting MAPK/c-fos and PLCγ2/CREB activation. The inhibitory effects of CME on differentiation-related signaling molecules resulted in significant repression of NFATc1 expression, which is a key transcription factor in osteoclast differentiation, fusion, and activation. Conclusion Our results confirmed the inhibition of RANKL-induced PLCγ2/CREB/c-fos/NFATc1 activation by CME during osteoclast differentiation. The findings collectively suggested CME as a traditional therapeutic agent for osteoporosis, RA, and periodontitis.

Roles of JNK/Nrf2 Pathway on Hemin-Induced Heme Oxygenase-1 Activation in MCF-7 Human Breast Cancer Cells

May 2020

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14 Citations

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Heme oxygenase-1 (HO-1) is highly induced in various human disease states, including cancer, indicating that HO-1 is an emerging target of cancer therapy. In this study, we investigated that the mechanisms of hemin-induced HO-1 expression and its signaling pathways in human breast cancer cell. We used MCF-7 cells, a human breast cancer cell line. Hemin increased HO-1 expression in MCF-7 cells in a dose- and time-dependent manner. Hemin enhanced HO-1 expression through the activation of c-Jun N-terminal kinases (JNK) signaling pathway. Hemin also induced activation of Nrf2, a major transcription factor of HO-1 expression. These responses in MCF-7 cells were completely blocked by pretreatment with brazilin, a HO-1 regulator. These results indicated that brazilin inhibits hemin-induced HO-1 expressions through inactivation of JNK/Nrf2 in MCF-7 cells. Thus, our findings suggest that HO-1 is an important anticancer-target of brazilin in human breast cancer.

Fig. 3. 15d-PGJ2 inhibits TPA-induced MMP-9 activation and cell invasion by means of the Nrf2/HO-1 signaling pathway in MCF-7 cells. (A, B) We treated MCF-7 cells with 15d-PGJ2 for 24 h. HO-1 protein expression was analyzed by Western blot. HO-1 mRNA levels were analyzed by RT-PCR using GAPDH mRNA as an internal control. (C) Cells were transfected with HO-1 siRNA, and then treated with 15d-PGJ2 and TPA for 24 h. HO-1 siRNA attenuated the inhibitory effects of 15d-PGJ2. MMP-9 secretion was analyzed by gelatin zymography (Zymo). We analyzed MMP-9 and HO-1 protein expression by Western blot. (D) Cells were transfected with HO-1 siRNA for 24 h. We carried out matrigel invasion assays on cells treated with TPA and 15d-PGJ2 (5 M) for 24 h. The data presented as the mean number of migrated cells. Data are presented as means ± SE of three independent experiments. **P ＜ 0.01 vs. control. *P ＜ 0.05 vs. control.

Fig. 4. HO-1 regulation affected PPAR expression in MCF-7 cells. (A, B) We treated cells with 15d-PGJ2 for 0.5, 1, 2, 3, 4 h, followed by cytosol, and then prepared nuclear extracts. Protein expression was analyzed by Western blot. (C) We treated cells with 15d-PGJ2 in the presence of ZnPP for 4 h and then prepared nuclear extracts. (D, E) Cells were transfected with Nrf2 siRNA and HO-1 siRNA for 24 h, then treated with 15d-PGJ2 for 4 h and then d nuclear extracts and whole cell lysates were prepared. PCNA was used as loading controls for nuclear and -actin were used as internal control for the whole cell. (F) Cells were transfected with HO-1 siRNA for 24 h, then treated for 3, 6, 12, and 24 h with 15d-PGJ2. (G) Cells were transfected with Nrf2 siRNA and HO-1 siRNA for 24 h. We analyzed PPAR mRNA levels by real-time PCR using GAPDH mRNA as an internal control. Data are presented as means ± SE of three independent experiments. **P ＜ 0.01 vs. control. *P ＜ 0.05 vs. control.

15d-PGJ2 inhibits NF-κB and AP-1-mediated MMP-9 expression and invasion of breast cancer cell by means of a heme oxygenase-1-dependent mechanism

January 2020

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24 Citations

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Activation of peroxisome proliferator-activated receptor γ (PPARγ) serves as a key factor in the proliferation and invasion of breast cancer cells and is a potential therapeutic target for breast cancer. However, the mechanisms underlying this effect remain largely unknown. Heme oxygenase-1 (HO-1) is induced and over-expressed in various cancers and is associated with features of tumor aggressiveness. Recent studies have shown that HO-1 is a major downstream target of PPARγ. In this study, we investigated the effects of induction of HO-1 by PPARγ on TPA-induced MMP-9 expression and cell invasion using MCF-7 breast cancer cells. TPA treatment increased NF-κB /AP-1 DNA binding as well as MMP-9 expression. These effects were significantly blocked by 15d-PGJ2, a natural PPARγ ligand. 15d-PGJ2 induced HO-1 expression in a dose-dependent manner. Interestingly, HO-1 siRNA significantly attenuated the inhibition of TPA-induced MMP-9 protein expression and cell invasion by 15d-PGJ2. These results suggest that 15d-PGJ2 inhibits TPA-induced MMP-9 expression and invasion of MCF-7 cells by means of a heme oxygenase-1-dependent mechanism. Therefore, PPARγ/HO-1 signaling-pathway inhibition may be beneficial for prevention and treatment of breast cancer.

... The protein-protein interaction (PPI) network analysis suggested that the MAPK cascade may play a central regulatory role among the crosstalk genes (Fig. 2L). For example, the MAPK cascade could regulate MMP3 expression [46] and activate POSTN [47]. Our bioinformatic analysis confirmed the vital role of the MAPK signaling pathway in the pathogenesis of both FS and OP, as well as its significant involvement in the crosstalk genes. ...
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... Among the many edible insects, Tenebrio molitor larvae has been widely studied and found to prevent hepatic steatosis [3], obesity [4], and inflammation [5]. Similarly, Protaetia brevitarsis (PB) has shown promising effects on high fat diet-induced obesity [6], oxidative stress-induced hepatotoxicity [7], and attenuation of osteoclastogenesis in mouse bone marrow-derived macrophages [8]. Therefore, PB also contains various functional compounds with potential advantages against several diseases, including obesity, liver toxicity, and osteoporosis. ...
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... Luciferase assay was performed as described in a previous study [39]. Cells were seeded into 24-well plates and then transfected with an AP-1 reporter plasmid (provided by Professor Chul-Ho Kim, SungKyunKwan University, Suwon, Republic of Korea) using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, WA, USA). ...
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... Tao et al. reported that Triptolide decreased the IL-1b-induced NF-kB DNA binding capacity and cytosolic amount of p-IkBa in subepithelial myofibroblasts (62). Furthermore, Triptolide inhibited matrix metalloproteinase-9 expression and invasion by inhibiting NF-kB and AP-1 expression in breast cancer cells (63). Similarly, Weiwei Yuan et al. reported that pretreatment with Triptolide increased apoptosis in TNF-a-induced gastric cancer cells by disrupting the H19/miR-204-5p/NF-kB/FLIP axis (64). ...
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... Brazilin can also be found in heartwood trees, including brazilwood (Caesalpinia echinata) and bralette (Caesalpinia Violaceae) (Dapson and Bain, 2015). In several studies, we found that brazilin has the ability to treat several types of cancer, such as breast cancer Hermawan and Putri, 2020;Jang et al., 2020;Chatterjee et al., 2022;Haryanti et al., 2022;Yang et al., 2023), colorectal cancer (Handayani et al., 2017), multiple myeloma (Kim et al., 2012), osteosarcoma , lung cancer (Suyatmi et al., 2022), cervical cancer (Kitdamrongtham et al., 2013;Jeon et al., 2014), bladder carcinoma (Zhang et al., 2015;Zhang et al., 2018), and others (Lee et al., 2013;Mitani et al., 2013;Zhang et al., 2014;Bello-Martínez et al., 2017;He et al., 2017;Correia Soeiro et al., 2022;Yan et al., 2022). ...
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... It has been suggested that NF-kB binding to DNA may be inhibited by PPAR (thiazolidinedione) agonists (177). It was also highlighted that 15d-PGJ2 could inhibit the expression of metalloproteinase-9 via NF-kB and AP-1 and modulate breast cancer invasion through the PPARg/HO-1 signaling pathway (178). Therefore, regarding the inhibition of NLRP1 and NLRP3 via 15d-PGJ2-mediated PPARg activation (103), the modulation of PPARg-NF-kB signaling should also be considered as an indirect regulation of NLRP1 and NLRP3 activity by 15d-PGJ2. ...
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15d-PGJ2 inhibits NF-κB and AP-1-mediated MMP-9 expression and invasion of breast cancer cell by means of a heme oxygenase-1-dependent mechanism

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... Tese cytokines can promote other signaling cascade and directly lead to lung injury. TNF-α and IL-6 are major cytokines, which stimulate other cytokine and chemokine secretion and contribute to severity of ALI [39,40]. Furthermore, IL-1β is an important early mediator in ALI and infammatory conditions [41]. ...
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