BACKGROUND: The IGF-1 signaling pathway has been deeply involved in the aging mechanism. The insulin-like growth factor binding protein 3 (IGFBP-3) is a protein that binds to IGF-1 that regulates growth, survival, and aging.
OBJECTIVE: The purpose of this study was to investigate the impact of the IGFBP3 gene knockout (KO) on the expressions of aging-related proteins and genes using the CRISPR/Cas9 system.
METHODS: The IGFBP3 gene knockout (KO) was performed by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses were used to verify the induction of mutation.
RESULTS: First, Sanger DNA sequencing was used to analyze the IGFBP3 gene knockout in murine cells (B16F1). The isolation of three colonies with the mutated DNA sequences in the IGFBP3 gene was validated. In addition, the expression levels of the IGFBP3 gene and protein in the edited B16F1 cells were lower than in those of normal B16F1 cells in western blot analysis as well as RT-PCR and qPCR. Moreover, IGFBP3 gene KO cells enhanced the level of SA-ß-gal staining and short telomere length compared to normal B16F1 cells. In particular, it was found that the expression levels of senescence-related proteins such as PI3K, AKT1, PDK1, and p53 were higher in IGFBP3 gene KO cells than in normal cells in both the absence and presence of IGF-1.
CONCLUSIONS: Therefore, the above findings could provide a clue that IGFBP3 could play a key role in the aging mechanism.

BACKGROUND: Cancer is one of the most dangerous and widespread diseases in the world today and it has risen to the position of the leading cause of death around the globe in the last few decades. Due to the inherent resistance of many types of cancer to conventional radiotherapy and chemotherapy, it is vital to develop innovative anticancer medications. Recently, a strategy based on nanotechnology has been used to improve the effectiveness of both old and new cancer drugs.
OBJECTIVE: The present study aimed to design and synthesize a series of phenyl-isoxazole-Carboxamide derivatives, evaluate their anticancer properties, and improve the permeability of potent compounds into cancer cells by using a nano-emulgel strategy.
METHODS: The coupling reaction of aniline derivatives and isoxazole-Carboxylic acid was used to synthesize a series of isoxazole-Carboxamide derivatives. IR, HRMS, 1H-NMR, and 13C-NMR spectroscopy techniques, characterized all the synthesized compounds. The in-vitro cytotoxic evaluation was performed by using the MTS assay against seven cancer cell lines, including hepatocellular carcinoma (Hep3B and HepG2), cervical adenocarcinoma (HeLa), breast carcinoma (MCF-7), melanoma (B16F1), colorectal adenocarcinoma (Caco-2), and colon adenocarcinoma (Colo205), as well as human hepatic stellate (LX-2) in addition to the normal cell line (Hek293T). A nano-emulgel was developed for the most potent compound, using a self-emulsifying technique.
RESULTS: All synthesized compounds were found to have potent to moderate activities against B16F1, Colo205, and HepG2 cancer cell lines. The results revealed that the 2a compound has broad spectrum activity against B16F1, Colo205, HepG2, and HeLa cancer cell lines with an IC50 range of 7.55-40.85 µM. Moreover, compound 2e was the most active compound against B16F1 with an IC50 of 0.079 µM compared with Dox (IC50 = 0.056 µM). Nanoemulgel was used to increase the potency of the 2e molecule against this cancer cell line, and the IC50 was reduced to 0.039 µM. The antifibrotic activities were investigated against the LX-2 cell line, and it was found that our synthesized molecules showed better antifibrotic activities at 1 µM than 5-FU, and the cell viability values were 67 and 95%, respectively.
CONCLUSIONS: This study suggests that a 2e nano-formalized compound is a potential and promising anti-melanoma agent.

Melanoma is the most aggressive type of cutaneous malignancies. In addition to its role as a regulator of extracellular matrix (ECM) integrity, lumican, a small leucine-rich proteoglycan, also exhibits anti-tumor properties in melanoma. This work focuses on the use of infrared spectral imaging (IRSI) and histopathology (IRSH) to study the effect of lumican-derived peptide (L9Mc) on B16F1 melanoma primary tumor growth. Female C57BL/6 mice were injected with B16F1 cells treated with L9Mc (n = 10) or its scrambled peptide (n = 8), and without peptide (control, n = 9). The melanoma primary tumors were subjected to histological and IR imaging analysis. In addition, immunohistochemical staining was performed using anti-Ki-67 and anti-cleaved caspase-3 antibodies. The IR images were analyzed by common K-means clustering to obtain high-contrast IRSH that allowed identifying different ECM tissue regions from the epidermis to the tumor area, which correlated well with H&E staining. Furthermore, IRSH showed good correlation with immunostaining data obtained with anti-Ki-67 and anti-cleaved caspase-3 antibodies, whereby the L9Mc peptide inhibited cell proliferation and increased strongly apoptosis of B16F1 cells in this mouse model of melanoma primary tumors.

Murine melanoma cells B16F1 were exposed to the flame retardant and wood preservative chemical 2,4,6-tribromophenol (TBP) during 24 and 48 h, at the concentrations found in human diet. TBP-exposed cells had increased MTT and Alamar blue® metabolism and ABCB5 mRNA levels (qPCR), but the cells had decreased proliferation (crystal violet assay), migration (scratch assay), and drug-effux transporters activity (rhodamine B efflux assay). Exposure to TBP did not affect the cell viability (neutral red and annexin V-PI assays), colony formation (colony number, clonogenic assay), and the levels of reactive oxygen species (DCF probe) or P53 mRNA (qPCR). The tested TBP concentrations had low toxicity to melanoma cells B16F1. However, dual effect on metastatic profile and chemoresistance suggests that the increase of ABCB5 positively modulates the cell chemoresistance, but decreases cell migration and proliferation. These findings may be explored in cancer therapy.