Baicalein mediates inhibition of migration and invasiveness of skin carcinoma through Ezrin in A431 cells

<p>Abstract</p> <p>Background</p> <p>Ezrin is highly expressed in skin cancer and promotes tumor metastasis. Ezrin serves as a promising target for anti-metastasis therapy. The aim of this study is to determine if the flavonoid bacailein inhibits the metastasis of skin cancer cells through Ezrin.</p> <p>Methods</p> <p>Cells from a cutaneous squamous carcinoma cell line, A431, were treated with baicalein at 0-60 μM to establish the non-cytotoxic concentration (NCC) range for baicalein. Following treatment with baicalein within this range, total Ezrin protein (both phosphorylated and unphosphorylated forms) and phosphorylated-Ezrin (phos-Ezrin) were detected by western blotting, and Ezrin RNA was detected in A431 cells using reverse transcription-polymerase chain reaction (RT-PCR). Thereafter, the motility and invasiveness of A431 cells following baicalein treatment were determined using wound-healing and Boyden chamber invasion assays. Short-interfering RNA (si-RNA) specifically targeting Ezrin was transfected into A431 cells, and a si-RNA Ezrin-A431 cell line was established by G418 selection. This stable cell line was transiently transfected with Ezrin and mutant Ezrin plasmids, and its motilityand invasiveness was subsequently determined to clarify whether bacailein inhibits these processes through Ezrin.</p> <p>Results</p> <p>We determined the range of NCCs for baicalein to be 2.5-40 μM in A431 cells. Baicalein displayed a dose- and time-dependent inhibition of expressions of total Ezrin and phos-Ezrin within this range NCCs. In addition, it exerted this inhibitory effect through the reduction of Ezrin RNA transcript. Baicalein also inhibited the motility and invasiveness of A431 skin carcinoma cells within the range of NCCs, in a dose- and time-dependent manner. A431 cell motility and invasiveness were inhibited by 73% and 80% respectively when cells were treated with 20 μM baicalein. However, the motility and invasiveness of A431 cells containing the Ezrin mutant were not effectively inhibited by baicalein.</p> <p>Conclusions</p> <p>Baicalein reduces the migration and invasiveness of A431 cells through the inhibition of Ezrin expression, which leads to the suppression of tumor metastasis.</p

N,N'-dinitrosopiperazine--mediated heat-shock protein 70-2 expression is involved in metastasis of nasopharyngeal carcinoma.

N,N'-Dinitrosopiperazine (DNP) is invovled in nasopharyngeal carcinoma (NPC) development and metastasis, and it shows organ specificity to the nasopharyngeal epithelium. Herein, we demonstrate that DNP induces heat-shock protein (HSP) 70-2 expression in NPC cells (6-10B) at a non-cytotoxic concentration. DNP induced HSP70-2 expression in a dose- and time- dependent manner, but showed no effect on other HSP70 family members. Furthermore, DNP also increased HSP70-2 RNA transcription through directly binding to the hypoxia-responsive elements (HRE) and heat shock elements (HSE) located in the HSP70-2 promoter. DNP-mediated HSP70-2 expression might act through enhancing the transcription of HSP70-2 RNA. Importantly, DNP induced motility and invasion of 6-10B cells dose- and time-dependently, and DNP-mediated NPC metastasis was confirmed in nude mice, which showed high HSP70-2 expression in the metastatic tumor tissue. However, the motility and invasion of NPC cells that were stably transfected using short interfering RNA against HSP70-2 could not effectively induce DNP. These results indicate that DNP induces HSP70-2 expression through increasing HSP70-2 transcription, increases the motility and invasion of cells, and promotes NPC tumor metastasis. Therefore, DNP mediated HSP70-2 expression may be an important factor of NPC-high metastasis

Dinitrosopiperazine-Mediated Phosphorylated-Proteins Are Involved in Nasopharyngeal Carcinoma Metastasis

N,N\u27-dinitrosopiperazine (DNP) with organ specificity for nasopharyngeal epithelium, is involved in nasopharyngeal carcinoma (NPC) metastasis, though its mechanism is unclear. To reveal the pathogenesis of DNP-induced metastasis, immunoprecipitation was used to identify DNP-mediated phosphoproteins. DNP-mediated NPC cell line (6-10B) motility and invasion was confirmed. Twenty-six phosphoproteins were increased at least 1.5-fold following DNP exposure. Changes in the expression levels of selected phosphoproteins were verified by Western-blotting analysis. DNP treatment altered the phosphorylation of ezrin (threonine 567), vimentin (serine 55), stathmin (serine 25) and STAT3 (serine 727). Furthermore, it was shown that DNP-dependent metastasis is mediated in part through ezrin at threonine 567, as DNP-mediated metastasis was decreased when threonine 567 of ezrin was mutated. Strikingly, NPC metastatic tumors exhibited a higher expression of phosphorylated-ezrin at threonine 567 than the primary tumors. These findings provide novel insight into DNP-induced NPC metastasis and may contribute to a better understanding of the metastatic mechanisms of NPC tumors

DNP-mediated NPC cell metastasis and HSP70-2 expression <i>in vivo</i>.

<p>A, 20 BABL/c nude mice were injected with 6-10B cells in Matrigel through the tail vein (1×10⁴ cells/mouse), and randomly divided into 2 groups (DNP-treated and control groups) with 10 mice per group. The DNP-treated group was abdominally injected using DNP at a dose of 40 mg/kg twice a week for 60 days. The control group was injected using 0.1% DMSO-PBS. After 60 days, metastatic tumors from the mediastinal lymph nodes were weighed (*, p<0.05). B, HSP70-2 expression was detected in the metastatic tumor samples using immunohistochemistry. Paraffin sections were stained using hematoxylin and eosin as well as with antibodies against HSP70-2. The upper panel represents staining with hematoxylin and eosin; the lower panel represents immunohistochemistry data. a and c represent untreated 6-10B cells as a negative control; b and d represent DNP-treated 6-10B cells; Arrows = positive cell. Original magnification, ×400. Scale bar = 5 µm. C, Schematic illustration of DNP-induced HSP70-2. DNP-mediated HSP70-2 expression through binding to HSP70-2 promoter, promotes motility and invasion, leading to metastasis of NPC cells.</p

Dose- and time- dependent DNP-mediated HSP70 expression.

<p>A, HSP70-2 expression was detected in NPC cell lines 5-8F, 6-10B, CNE1, CNE2, HONE1, HNE1, and HNE2 by Western blotting. B, DNP-mediated HSP70-2 expression was detected using immunofluorescence. a, 6-10B cells treated with 0.1% DMSO-PBS served as a negative control; b, 6-10B cells treated with DNP; c, 5-8F served as a positive control; d, 6-10B cells stained with DAPI. Scale bar = 50 µm. C, DNP-mediated HSP70-2 expression was detected in 6-10B and CNE2 cells. D, Time- and dose-dependency of DNP-induced HSP70-2 expression. a, HSP70-2 expression in 6-10B cells treated with the indicated concentration; b, HSP70-2 expression in 6-10B cells with 4 µM DNP for the indicated time. Three independent experiments were performed, the abundance ratio to β-actin was counted, and data are represented as means ± SD from three experiments. *, p<0.05. E, Expression of HSP70-2, HSP70t, HSC70, GRP75, GRP78, and HSP70-4 was detected in 6-10B cells after DNP treatment.</p

Non-cytotoxic concentration of <i>N</i>,<i>N</i>′-dinitrosopiperazine (DNP) in 6-10B cells.

<p>A, Structure of DNP, an <i>N</i>-nitroso compound. B, 6-10B cells were treated with the indicated concentration DNP for 48 h before being subjected to the MTT cell viability assay. C, After 6-10B cells were treated with the indicated DNP concentration for 48 h, cell media were subjected to the LDH assay. The optical density (OD) indicates the relative OD at 563 nm. Lactate dehydrogenase (LDH) activity is expressed per 1 L of media. Data are presented as means ± SD from 3 independent experiments. Statistical analysis was performed using the Student’s <i>t</i> test (*, <i>p</i><0.05).</p

DNP-mediated HSP70 RNA transcription through HSP70-2 promoter interaction.

<p>A, DNP-mediated Hsp70 transcription was detected in 6-10B and CNE1 cells by RT-PCR. B, Schematic of the HSP70-2 promoter and its mutations. HSP70-2 promoter, the full length of promoter; HSP70-2M1, HER1 promoter mutation; HSP70-2M2, HER1, and HER2 mutation; HSP70-2M3, HER1, 2 and HSE mutation. HSP70-2 promoter and its mutations were respectively incubated with ³H-DNP, and the reactive complexes were separated by SDS-PAGE for autoradiography. C, The affinity was determined by displacement of ³H-DNP from the HSP70-2 promoter. IC50 of DNP interaction with HSP70-2 promoter was 273.3 nM, and Ki was 107 nM. D, Relative firefly luciferase activities were normalized by the Renilla luciferase activity (mean ± SD). The relative luciferase activity indicates the transcriptional activity of the HSP70-2 promoter and its mutation with or without DNP treatment.</p