Human Beta-Defensin-1 Suppresses Tumor Migration and Invasion and Is an Independent Predictor for Survival of Oral Squamous Cell Carcinoma Patients

<div><p>Background</p><p>Human beta-defensin-1 (hBD-1) has recently been considered as a candidate tumor suppressor in renal and prostate cancer. The aim of this study was to investigate the role of hBD-1 in the progression of oral squamous cell carcinoma (OSCC) and its potential as diagnostic/prognostic biomarker and therapeutic target for OSCC.</p><p>Methods</p><p>HBD-1 expression in tissues at different stages of oral carcinogenesis, as well as OSCC cell lines was examined. HBD-1 was overexpressed in HSC-3, UM1, SCC-9 and SCC-25 cells and subjected to cell growth, apoptosis, migration and invasion assays. Tissue microarray constructed with tissues from 175 patients was used to examine clinicopathological significance of hBD-1 expression in OSCC.</p><p>Results</p><p>HBD-1 expression decreased from oral precancerous lesions to OSCC and was lower in OSCC with lymph node metastasis than those without metastasis. <i>In vitro</i>, the expression of hBD-1 was related to the invasive potential of OSCC cell lines. Induction of exogenous expression of hBD-1 inhibited migration and invasion of OSCC cells, probably by regulation of RhoA, RhoC and MMP-2; but had no significant effect on proliferation or apoptosis. In a cohort of patients with primary OSCC, cases with no expression of hBD-1 had more chance to be involved in lymph node metastasis. Eventually, the positive expression of hBD-1 was associated with longer survival of patients with OSCC, and multivariate analysis and ROC curve analysis confirmed hBD-1 positivity to be an independent prognostic factor of OSCC, especially OSCC at early stage.</p><p>Conclusions</p><p>Overall, these data indicated that hBD-1 suppressed tumor migration and invasion of OSCC and was likely to be a prognostic biomarker and a potential target for treatment of OSCC.</p></div

Association of hBD-1 expression in OSCC with patients' clinicopathological characteristics (n = 175).

<p>Association of hBD-1 expression in OSCC with patients' clinicopathological characteristics (n = 175).</p

Figure 3

<p><b>Antitumor activity of SAHA-DDP/PECE in vivo.</b> In the mice model of oral squamous cell carcinoma xenografts, the female nude mice received 2 × 10⁶ HSC-3 cells via subcutaneously into the right flank regions. (A) The mice were then treated with NS, PECE, SAHA, DDP, SAHA-DDP and SAHA-DDP/PECE every two weeks for a total of two doses starting on day 7 (n = 6 mice per group). Tumor volumes of mice from different groups of HSC-3 tumor model. (B) Representative tumors of OSCC mice model from NS, PECE, SAHA, DDP and SAHA-DDP control group and SAHA-DDP/PECE treated mice. Data are representative of at least two separate experiments. Bars, means ± SD (*P<0.05).(C) When tumors were palpable, the mice were randomly assigned to two independent treatment groups( n = 12 mice per group): mice treated with 100 ml combine SAHA with DDP (SAHA-DDP), or treated with 100 ml SAHA and DDP in thermosensitive hydrogel (SAHA-DDP/PECE). At the date of 1, 3, 7, 14 and 21 three mice were sacrificed separately. The ribonucleoprotein of the tumor tissues was extracted and the expression of acetyl-Histone H3 and Histone H3 was detected by western blot.</p

Immunostaining of hBD-1 protein in different tissue samples.

<p>A) Examples of typical images of hBD-1 in normal oral mucosa (negative); B) OLK (negative); C) OLK (weak); D) OLK (strong); E) OSCC (negative); F) (weak); G) OSCC (strong). H) Number of samples with negative, weak and strong staining of hBD-1 in normal oral mucosa, OLK and OSCC. I) Percentage of negative, weak and strong staining of hBD-1 in OSCC with or without metastasis.</p

Figure 4

<p><b>Systematic toxicity in the SAHA-DDP/PECE treated mice.</b> H&E staining of section of major tissues obtained from tumor beard mice which received two doses of NS, PECE, SAHA, DDP, SAHA-DDP and SAHA-DDP/PECE. Heart, liver, spleen, lung and kidney were harvested at day 28 after intravenous injection. (A) Mean body weights on days 7, 10, 13, 16, 19, 22, 25 and 28 of mice treated with two doses of NS, PECE, SAHA, DDP, SAHA-DDP and SAHA-DDP/PECE; error bars correspond to 95% confidence intervals, Values are means±SD (n  =  10 mice per group).</p

Figure 5

<p><b>SAHA-DDP/PECE inhibited cell proliferation and intratumoral angiogenesis in vivo.</b> (A) PCNA-positive cells were rich in NS and PECE groups. Whereas, the percentage of PCNA-positive cells in SAHA, DDP, SAHA-DDP and SAHA-DDP/PECE groups were significantly decreased in turn (*P<0.05). (magnification, × 200) (B) Angiogenesis within tumors was detected by CD34 staining of micro vessels. The average number of micro vessels per vascular hot spot was significantly decreased in SAHA-DDP/PECE treated tissues compared with those in the three control groups (*P<0.05).</p

OSCC cell lines with low hBD-1 expression tended to have higher invasive potential.

<p>A) hBD-1 mRNA expression level in OSCC cell lines. B) hBD-1 protein expression level in the supernatant of OSCC cell lines. Immortalized oral keratinocyte HOK16E6E7 was used as control. C–D) Assays showing the relative migration ability of OSCC cell lines. Haematoxylin staining (C) and the relative numbers of cells migrated to the lower surface of the cell culture insert (D). E–F) Assays showing the relative invasion ability of OSCC cell lines. Haematoxylin staining (E) and the relative numbers of cells migrated to the lower surface of the matrigel-coated cell culture insert (F). All assays were carried out three times in triplicates.</p

Cox's Proportional hazard model analysis of prognostic factors in patient with OSCC.

<p>Cox's Proportional hazard model analysis of prognostic factors in patient with OSCC.</p

Figure 6

<p><b>SAHA-DDP/PECE induced apoptosis in vivo.</b> (A) Induction of apoptosis was indicated by TUNEL assay. The TUNEL-positive cells display dark green nuclei and are observed under a fluorescence microgroup (×400 magnification). TUNEL-positive nuclei were significantly increased in SAHA-DDP/PECE treated tissues compared with those in the control groups (*P<0.05). (B) Detection of caspase-3, Caspase-8 and Caspase-9 by Western blots. GAPDH was used as equal loading control.</p

Analysis of samples with complex matrix by comprehensive gas chromatography

Komprehenzivní plynová chromatografie (GC×GC) je technika vhodná pro separaci komplikovaných organických směsí a nabízí mnoho výhod oproti běžné plynové chromatografii. Umožňuje kompletní analýzu vzorku současně na dvou odlišných kolonách, mezi kterými je rozhraní nazvané modulátor. Byla provedena analýza dvou éterických levandulových olejů a extraktu z levandule pomocí plynového chromatografu s pulzním tlakovým modulátorem a dvojicí detektorů: plamenově ionizačním detektorem a hmotnostním spektrometrem. Pro efektivní separaci vybraných olejů byly nalezeny optimální podmínky. Vstupní tlak na kolonu byl 250 kPa, první a druhý tlak nastavený na modulátoru byl 155 kPa a 175 kPa. Byl zvolen pomalejší teplotní program s počáteční teplotou 70 řC a nárůstem teploty 2 řC/min. Jako vhodná se ukázala modulační perioda v délce 5 s a s pulzem trvajícím 300 ms. Analyty byly identifikovány podle jejich hmotnostních spekter z MS detektoru. Nejvíce zastoupenými látkami v levandulových olejích byly terpeny. Složení levandulových olejů se liší v závislosti na druhu levandule, podmínkách pěstovaní i způsobu přípravy oleje. Klíčová slova: komprehenzivní plynová chromatografie, pulzní tlakový modulátor, levandulový ole