Subchronic exposure to arsenic increases the incidence of human cancers such as skin, lung, colon, and rectal cancer. The mechanism for arsenic-induced tumorigenesis is still not clear. It is generally believed that DNA damage and genomic instability, generated by arsenic-promoted oxidative stress, account largely for this process. The major sources of reactive oxygen species (ROS) are arsenic-damaged mitochondria. Autophagy is a catabolic process functioning in turnover of long-lived proteins and dysfunctional organelles such as mitochondria. Defects of autophagy under stress conditions promote genomic instability and increase the risk of tumorigenesis. In the present study using a human bronchial epithelial cell line, BEAS-2B cells, we investigated the role of autophagy in arsenic-induced cell transformation, an important step in arsenic tumorigenesis. Our results show that subchronic arsenic exposure induces BEAS-2B cell transformation accompanied with increased ROS generation and autophagy activation. However, the patterns for ROS and autophagy alteration are different. Arsenic exposure generated a prolonged and steady increase of ROS levels, whereas the activation of autophagy, after an initial boost by arsenic administration, decreases in response to subchronic arsenic exposure, although the activity is still higher than a nontreated control. Further stimulation of autophagy increases mitochondria turnover and decreases ROS generation and arsenic-induced cell transformation. Contrarily, inhibition of autophagy activity decreases mitochondria turnover and enhances arsenic-induced ROS generation and cell transformation. In addition, the mammalian target of rapamycin signaling pathway is involved in arsenic-mediated autophagy activation. Our results suggest that autophagy is a cell self-protective mechanism against arsenic-induced cell transformation.
Ultraviolet B (UVB) exposure causes damage to skin and represents the primary etiological agent for skin cancer formation. UVB induces DNA damage and apoptosis in epidermal cells. In this study, we demonstrated that UVB activated autophagy in JB6 epidermal cells, which was evident by the formation of LC3 puncta, the induction of LC3 lipidation, the increase in beclin 1 expression, and the decrease in the levels of p62. Autophagy appeared to be a protective response to UVB-induced damage because inhibition of autophagy exacerbated UVB-induced cell death, and stimulation of autophagy offered protection. Furthermore, we demonstrated that glycogen synthase kinase 3β (GSK3β) was involved in UVB-induced autophagy. UVB inhibited GSK3β activation by simultaneously enhancing phosphorylation at Ser9 and suppressing Tyr216 phosphorylation. GSK3β negatively regulated autophagy; overexpression of wild-type or S9A (constitutive-active) GSK3β mutant inhibited UVB-mediated autophagy, while overexpression of a dominant-negative K85R mutant enhanced UVB-mediated autophagy. Inhibition of GSK3β also offered protection against UVB-mediated damage. UVB activated AMP-activated protein kinase (AMPK), an important regulator of autophagy through the inhibition of GSK3β. Taken together, our results suggest that UVB-stimulated autophagy is a protective response for epidermal cells and is mediated by the GSK3β/AMPK pathway.
Both epidemiological and experimental studies indicate that ethanol exposure enhances tumor progression. Ethanol exposure promotes cancer cell invasion and is implicated in tumor metastasis. Metastasis consists of multiple processes involving intravasation and extravasation of cancer cells across the blood vessel walls. The integrity of the vascular endothelial barrier that lines the inner surface of blood vessels plays a critical role in cancer cell intravasation/extravasation. We examined the effects of ethanol on the endothelial integrity in vitro. Ethanol at physiologically relevant concentrations did not alter cell viability but disrupted the endothelial monolayer integrity, which was evident by a decrease in the electric resistance and the appearance of intercellular gaps in the endothelial monolayer. The effect of ethanol was reversible once ethanol was removed. The disruption of the endothelial monolayer integrity was associated with an increased invasion of cancer cells through the endothelial monolayer. Ethanol induced the formation of stress fibers; stabilization of actin filaments by jasplakinolide prevented ethanol-induced disruption of endothelial integrity and cancer cell invasion. VE-cadherin is a critical component of the adherens junctions, which regulates vascular endothelial integrity. Ethanol induced the endocytosis of VE-cadherin and the effect was blocked by jasplakinolide. Our results indicate that ethanol may facilitate cancer metastasis by disrupting the vascular endothelial barrier.
Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for beta-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.
Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.
SummaryThe experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/ VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space.
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