Patients with advanced cervical cancer had significantly higher frequency of leukocyte alterations, although they may occur apart from the preinvasive stages. Overall, neutrophilia was the best indicator of cancer invasiveness.
Neutrophil migration is a key event in the inflammatory response of any origin, and neutrophils may present antitumor activity. We investigated the number and function of circulating neutrophils obtained from patients with cervical neoplasia at different stages. Patients with preinvasive (cervical intraepithelial neoplasia, CIN3, n= 6) or microinvasive ([MICRO] stage IA1, n= 4) neoplasia were evaluated together as CIN/MICRO group (n= 10), while patients at stages II-IV were evaluated as invasive group (INV, n= 12). Healthy women served as controls (n= 15). For patients, analysis of leukogram on diagnosis showed a significant elevated neutrophil count in INV group compared with that in CIN/MICRO group. A neutrophil/lymphocyte ratio >/=5 was observed in 67% patients from INV group compared with only 10% from CIN/MICRO group. Neutrophil migration, assayed in a microchemotaxis chamber in response to the chemoattractants (10(-7) M) N-formyl-l-methionyl-l-leucyl-l-phenylalanine, leukotriene B(4), or interleukin-8, was reduced in INV group than in controls or CIN/MICRO group. Surgical treatment in randomly selected patients from CIN/MICRO group (four CIN, one MICRO) increased neutrophil migration to all chemoattractants compared with time on diagnosis. The serum levels of nitric oxide (NO) metabolites, assayed by the Griess reaction, were higher in patients (n= 19) than in controls (n= 15), without differences related to tumor stage, but were reduced in patients after surgery compared with pretreatment (n= 10). Taken together, the results suggest that neutrophils play a role in the host response in cervical cancer. Soluble circulating mediators released by tumor cells, such as NO, could interfere early in the capacity of neutrophils to migrate, thus impairing host immune response.
SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 μM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.
This study aims to examine the effects of a new 1,4-dihydropyridine derivative, VdiE-2N, on cell signaling pathways and mitochondrial events in head and neck squamous cell carcinoma (HNSCC) cells, and on a mice model of xenograft tumor growth/cell proliferation. Four HNSCC cell lines (HN13, HN12, HN6, and CAL27), HEK293 cells (human embryonic kidney 293 cells), and human oral healthy mucosa fibroblasts (OHMF) were used for in vitro assessment of cell viability (resazurin assay) and invasion capacity (modified Boyden chamber assay), and mitochondrial membrane potential (JC-1 fluorescence assay), morphology (transmission electron microscopy), and number of mitochondria (MitoTracker® imaging). SET and pDRP1 proteins were analyzed by immunofluorescence, and proteins involved in cell death/survival pathways were analyzed by Western blotting. HN12 xenograft tumors were established in the flank of Balb/c nude mice, and their characteristics and sensitivity to VdiE-2N were determined by immunohistochemistry and histology. VdiE-2N decreased cell viability in HNSCC cells (IC50 = 9.56 and 22.45µM for HN13 and HN12 cells, respectively) more strongly than it decreased cell viability in OHMF and HEK293 cells (IC50 = 32.90 and > 50µM, respectively). In HN13 cells, VdiE-2N dissipated mitochondrial membrane potential and altered the mitochondria size, shape, and number in a concentration-dependent manner, as well as it induced apoptosis and reduced their invasion capacity. Treatment of mice bearing xenograft tumors with VdiE-2N significantly diminished proliferation of cancer cells. Therefore, VdiE-2N induces HNSCC cell death in vitro through mitochondria-mediated apoptotic pathways and dampens tumor growth in vivo, thus supporting a potential anti-cancer effect.
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