Ummi Maryam Zulfin

Neuroblastomas evade apoptosis due to oncogene mutations and antiapoptotic proteins, necessitating novel therapeutics that work in concert with other forms of cell death. Pyroptosis has potential as a strategic cell death mechanism in neuroblastoma. This study aimed to identify compounds that modulate pyroptosis, specifically those that target gasdermin D (GSDMD) oligomerization. The study employed computational analysis and in vitro screening. The COCONUT database provides the natural compound dataset. Drug-likeness analysis and pharmacophore fitting were applied to screen potential GSDMD oligomerization modulators. Hit compounds underwent molecular docking with MOE and molecular dynamic (MD) simulations with NAMD 2.14 to analyse structural changes. The computational screening results were corroborated by in vitro assays, including the WST-8 assay, Western blot, and immunofluorescence, which target pyroptosis-specific caspase-1. Pharmacophore fitting, molecular docking, and molecular dynamics simulations identified the top five compounds, namely, quercetin, naringenin, hesperetin, curcumin, and galangin, as potent modulators of GSDMD oligomerization. Among these compounds, curcumin, quercetin, and galangin exerted potent cytotoxic effects on GSDMD-expressing neuroblastoma SK-N-AS cells, with IC₅₀ values of 21, 37, and 49 µM, respectively. Curcumin and quercetin also promoted apoptosis via increased caspase-3 cleavage and reduced procaspase-7 and -8 levels, as shown by immunoblotting. Curcumin and galangin upregulated caspase-1 expression, as demonstrated by the detection of a fluorescent-labelled inhibitor of caspase-1 by immunostaining, suggesting that these two compounds could induce pyroptosis. Hesperetin and naringenin showed low cytotoxicity, had no effect on caspase activation, and did not exhibit signs of pyroptosis in SK-NA-S cells. Our study successfully identified curcumin as a strong regulator of both apoptosis and pyroptosis, quercetin as a strong modulator of apoptosis, and galangin as a strong modulator of pyroptosis. Further research on these compounds is crucial for the development of novel therapeutic strategies for neuroblastoma treatment.

Purpose: The HLF and HuH-6 cell lines represent hepatocellular carcinoma (HCC) with different characteristics in chromosome content that may give different drug responses. Here, PGV-1 was intended to challenge the growth-suppressing effect on HLF and HuH-6 and trace the molecular target mechanism of action compared to sorafenib. Methods: We applied MTT cytotoxic assay, colony forming assay, flow cytometry analysis, immunofluorescence assay and western blot assay. Results: PGV-1 exhibited cytotoxic effects on HLF and HuH-6 with IC-50 values of 1 µM and 2 µM, respectively, whereas sorafenib showed less cytotoxicity with IC-50 values of 5 µM and 8 µM respectively. PGV-1 suppressed the cell growth permanently but not for sorafenib. Sorafenib did not change the cell cycle profiles on both cells, but PGV-1 arrested the cells at G2/M with the characteristic of senescent cells and mitotic disarrangement. PGV-1 and sorafenib showed the same effect in downregulating p-EGFR, indicating that both compounds have the same target on EGFR activation or as Tyrosine kinase inhibitors. PGV-1 suppressed the MYCN expression in HuH-6 and HLF cells but stabilized cMYC-T58 indicating that even though the MYCN was downregulated, the cells maintained the active form of cMYC. In this regard, PGV-1 also stabilized the expression of PLK-1 and AurA. Conclusion: PGV-1 elicits stronger cytotoxic properties compared to sorafenib. The lower the MYCN expression, the higher the cytotoxic effect of PGV-1. PGV-1 abrogates cell cycle progression of both cells in mitosis through EGFR inhibition and stabilizes PLK-1 and AurA in correlation with the suppression of MYCN expression.

Many chemotherapeutic agents cause various side effects, including nephrotoxicity. Doxorubicin, for example, increases the level of reactive oxygen species (ROS), leading to cell senescence in the kidneys. Cardamom essential oil (Amomum compactum Soland. ex Maton) contains compounds that exhibit antioxidant activity, such as 1.8-cineole, α-pinene, α-terpineol, and linalool. This study focused on exploring the potency of cardamom essential oil (CEO) as an anti-senescence induced by doxorubicin using Vero cells. CEO was obtained by steam distillation. The cytotoxic assay was carried out using trypan blue exclusion assay. We performed the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and the senescence-associated beta-galactosidase (SA-β-gal) staining to measure the effect of CEO on intracellular ROS level and cell senescence, respectively. Analysis of the compounds with gas chromatography-mass spectrophotometry (GC-MS), revealed seven compounds with significant abundance, namely 1.8-cineole (50.82%), ß-pinene (12.43%), α-terpineol (8.50%), fenchone (4.10%), α-pinene (4.00%), sabinene (3.00%), and linalool (1.98%). The cytotoxicity assay of CEO on Vero cells showed an IC50 value of 178 μg/mL. Thus, the CEO is considered to be not cytotoxic and safe for normal kidney cells. Concentrations of 50 and 100 μg/mL reduced the senescence induced by doxorubicin. Therefore, the CEO has the potency as a nephroprotective agent in doxorubicin-induced senescence.

Pentagamavunone-1 (PGV-1) is a curcumin analog with a prominent anti-cancer potency in vitro and in vivo for several cancer types, including colon cancer. Combining PGV-1 with natural compounds such as diosmin, galangin, and piperine can enhance its effectiveness due to their promising chemoprevention properties. We aimed to evaluate the effectiveness of combining PGV-1 with diosmin, galangin, or piperine for colon cancer by using in vitro and bioinformatic approaches to predict their target proteins. WiDr cells were used as a model for colon adenocarcinoma (COAD). The cell viability under a single or combination treatment of PGV-1 and diosmin, galangin, or piperine was evaluated using direct counting by the trypan blue exclusion test. SwissTargetProtein, UALCAN, and OncoLnc were utilized to predict target proteins of the compounds in COAD, the expression level of target proteins in COAD, and the survival rate of patients with overexpressed target proteins, respectively. The IC50 values for PGV-1, diosmin, galangin, and piperine were 2.8´10-2 µg/mL, 81 µg/mL, 7 µg/mL, and 172 µg/mL, respectively. All the tested natural compounds showed synergistic effects when combined with PGV-1 at low concentrations. Eleven proteins that were overexpressed in COAD were identified as potential targets. Overlapped predicted targets of PGV-1 and galangin or piperine were CDK1, MET, and TOP2A. The high expression of another set of predicted target proteins, SCD, CA9, and SQLE, led to lower survival rates in COAD patients. We concluded that combinations of PGV-1 with natural compounds can synergistically enhane its anti-cancer activity for colon cancer.

BACKGROUND: Cardamom (Amomum cardamom) essential oil (CEO) contains monoterpenes with antioxidant activity and is reported to exhibit anticancer activity against some cancer cell lines. Triple-negative breast cancer (TNBC) has the lowest prognosis among breast cancer types due to its aggressive characteristics. This study was conducted to explore the potency of CEO in inhibiting 4T1 cell proliferation and migration and compared its activity to sappan (Caesalpinia sappan) wood extract (CSE). METHODS: We used the 4T1 cell line as the TNBC cell model and tested the cytotoxicity of CEO by using a trypan blue exclusion assay. We studied the senescence induction ability of CEO using SA-β-Gal assay, the migratory inhibition activity using scratch wound healing assay, and inhibition of matrix metalloproteinase 9 (MMP-9) expression using gelatin zymography. RESULTS: CEO showed cytotoxicity toward 4T1 cells with the IC50 values of 60 µg/mL. CEO at ½ IC50 and IC50 concentration significantly increased cell senescence, but CSE did not. CEO at IC50 also reduced cell ability to migrate and also considerably reduced MMP-9 activity. Moreover, these activities related to the inhibition of the cell metastasis process were weaker compared than that of CSE. CONCLUSION: CEO showed potency as a chemopreventive agent on the TNBC 4T1 cell line model with moderate cytotoxicity. CEO induced 4T1 cell senescence, inhibited cell migration and suppressed MMP-9 expression. KEYWORDS: Amomum cardamom, Caesalpinia sappan, 4T1, senescence, cell migration, triple-negative breast cancer