<p class="Abstract">The aim of the present investigation was to examine and demonstrate in detail the anti-cancer and apoptotic effects of daucosterol in human colon cancer cell line HCT-116. The effects of this compound on cell migration, cell invasion, cell cycle analysis and caspase signalling pathway were also studied. Cell viability was evaluated by MTT assay using different doses of the drug. In vitro wound healing assay was used to study cell migration. Flow cytometry was involved to examine cell apoptosis as well as cell cycle phase distribution. Daucosterol induced significant, dose-dependent as well as time-dependent cytotoxic effects with IC<sub>50</sub> values of 26.6 and 47.3 µM at 24 and 48 hours time intervals respectively. The percent of cells that migrated decreased from 99% in case of untreated control to 84.2, 45.2, 39.5 and 14.4% in groups treated with 0, 5, 50, 75 and 100 µM of daucosterol respectively. Percentage of apoptotic cells increased from 2.5% in untreated control cells to 23.6, 46.9 and 74.2% in cells treated with 5, 50 and 100 µM dose of daucosterol respectively. Daucosterol at different doses induced cell cycle arrest at sub-G1 phase of the cell cycle.</p><p><strong>Video clip</strong></p><p><a href="https://youtube.com/v/CUkLDjyNUFw">Cell Migration Assay</a>: 1 min</p>
Background: Colorectal cancer is a common digestive tract malignancy. This study aimed to expound the functional role of fatty-acid-binding protein 4 (FABP4) and the potential underlying mechanisms in the development of colorectal cancer. Methods: Several techniques were utilized to investigate the role of FABP4 in colorectal cancer. FABP4 mRNA expression was quantified using Real time-quantitative PCR (RT-qPCR). Cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), sphere formation assays and flow cytometry evaluated cell growth, stemness, and apoptosis in SW480 and HT29 cells. Glycolysis was assessed via extracellular acidification rate (ECAR) , lactate production, glucose uptake, adenosine triphosphate (ATP)/adenosine 5'-diphosphate (ADP) ratio, and Glut1 and Elevated lactate dehydrogenase A (LDHA) protein expression. Reactive oxygen species (ROS) levels were analyzed by flow cytometry. Western blot measured the protein expression of FABP4, Proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, Glut1, LDHA, stemness makers (Sox2, Oct4, and ALDHA1), and extracellular regulated protein kinase (ERK)/mammalian target of rapamycin (mTOR) pathway proteins. In vivo experiments, BALB/c nude mice (n = 12) were inoculated with 200 µL HT29 cells (5 × 10 6 cells) transfected with sh-FABP4 or short hairpin (sh)-negative control (NC), forming two groups with 6 mice each. The in vivo mice tumor model allowed for evaluating FABP4's impact on tumor growth.Results: FABP4 was significantly upregulated in colorectal cancer tissues and cells (p < 0.05). FABP4 knockdown markedly inhibited cell proliferation, stemness, and glycolysis, while promoting apoptosis in these cells (p < 0.05). Additionally, FABP4 depletion led to a significant increase in ROS level (p < 0.05). However, N-acetyl-L-cysteine (NAC) (p < 0.05), a ROS scavenger, mitigates these effects. Furthermore, the effects of FABP4 depletion on cell growth, stemness, glycolysis, and apoptosis in colorectal cancer cells were also retarded by NAC (p < 0.05). Notably, FABP4 knockdown also suppressed the ERK/mTOR pathway, suggesting its regulation via ROS (p < 0.05). In vivo study results showed, FABP4 depletion significantly curbed tumor growth in colorectal cancer (p < 0.05). Conclusions: These results suggest that FABP4 depletion inhibits colorectal cancer progression by modulating cell growth, stemness, glycolysis and apoptosis. This regulation occurs through the ROS/ERK/mTOR pathway.
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