Cell and Tissue Journal

Abstract Introduction: Colorectal cancer (CRC) is the second most commonly diagnosed malignant tumor in the world, with about 1,926,425 new cases estimated for 2022. The most therapeutic approaches for CRC include surgery, chemotherapy, targeted medicine, and radiation therapy. Each of these treatments exerts side effects such as fatigue, constipation, loss of appetite, and low blood cell counts. Natural products have provided valuable anticancer effects for years. An increasing number of studies have elucidated that plant-based natural compounds can act as an alternative chemotherapy option against CRC. It has been demonstrated that herbal bioactive compounds such as flavonoids, alkaloids, peptides, terpenoids, and steroids have the potential to effectively treat CRC. Sesquiterpenoid lactones, a subclass of terpenoid compounds, have been shown to induce anti-tumor effects. Aguerin B, a sesquiterpene lactone, exhibits cytotoxic effects against some types of tumors.
Aims: The current study aims to investigate the effect of Aguerin B on HT29 colorectal cancer, and assess the molecular mechanism of Aguerin B in colorectal cancer by evaluating the type of cell death it induces and its effect on the expression of the tumor suppressor gene p53 in HT29 colorectal cancer cells.
Materia and Methods: Colorectal cancer cells were purchased from the Iranian Biological Resource Center. HT29 cells were cultured in DMEM medium enriched with 10% fetal bovine serum and 1% antibiotics in 96-well plates. After 24 hours, the cells were treated with Aguerin B (variable concentrations from 1 to 8 μg/ml) for 24 and 48 hours. Cell viability was assessed using the MTT assay. Acridine orange-propidium iodide staining, caspase-3 and caspase-9 activity assays, using DCF-DA kits, were used to determine the type of cell death induced by Aguerin B. Additionally, qRT-PCR was used to evaluate p53 expression at the transcriptional level.
Results: The findings demonstrated that Aguerin B exerts cytotoxic effects on colorectal cancer cells in a dose- and time-dependent manner, with an IC50 (Inhibitory Concentration) value of 4.6 μg/ml. Acridine orange-propidium iodide staining confirmed apoptosis induction at the IC50 concentration of Aguerin B. The cultivated activity of caspase-3 and -9, along with elevated ROS levels, indicated apoptosis induction via the mitochondrial pathway. Furthermore, the upregulation of p53 suggests the tumor-suppressive effect of Aguerin B in colorectal cancer cells.
Discussion: Among the cell death mechanisms, the induction of apoptosis is a more profound mechanism of cell death mediated by anticancer bioactive compounds extracted from natural sources. Recent investigations have been focused on cancer therapeutic techniques that increase the apoptosis rate in tumor cells by disrupting mitochondrial biogenesis, leading to mitochondrial dysfunction. Caspases, as proteolytic enzymes, play a significant role as effector molecules in the apoptosis-induced cell death pathway. HT29 cells are sensitive to the chemotherapeutic drugs 5-fluorouracil and oxaliplatin, which are standard treatment options for colorectal cancer, and therefore were selected as chemotherapy-responsive colorectal cancer cells in this study. The results of this study exhibited that Aguerin B can induce cytotoxicity and anticancer effects in HT29 colorectal cancer cells in a dose- and time-dependent manner. This compound is also able to induce its properties through the recruitment of the intrinsic pathway, the caspase-dependent pathway in HT29 cells. Nevertheless, the role of p53 mutations in the pathogenesis of CRC has been recognized. The obtained data showed that Aguerin B can exert its anti-cancer potential. Previously, it was found that some natural products, including sesquiterpenoids, can also exert their suppressive effects against cancers by targeting the p53-MDM2 pathway.
Conclusion: Given its potent cytotoxicity and apoptosis-inducing effects, Aguerin B, which can suppress HT29 cells, appears to be an effective compound for obstructing human colorectal cancer cells, warranting further investigation in the pre-clinical phase and clinical studies.

Abstract Aim: One of main challenges in worldwide is increasing rate of depression and sexual dysfunction associated with antidepressant drug consumption. Regarding the role of Sertoli cells in spermatogenesis, this study investigated the effect of duloxetine on viability, apoptosis and expression of Bax and Cx43 (Connexin 43) expression in Sertoli cells.
Material and Methods: TM4 Sertoli cells were cultured in DMEM/F12 medium containing 2.5% FBS, 5% horse serum and 1% penicillin-streptomycin. Cells were treated with different doses of duloxetine (3.75, 7.5, 15, 30, 60 µg/ml) for 24 to 72 hours. MTT assay was performed to evaluate cell viability. The rate of apoptosis was measured by flow cytometry and RT-qPCR was performed to evaluate of Bax (proapoptotic gene) and Cx43 (essential for spermatogenesis) genes.
Results: Duloxetine reduced cell survival in a dose and time-dependent manner. On the basis of

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