Anti-inflammatory constituents from <i>Psychotria prainii</i> H. Lév

<p>One new and three known compounds were isolated from the ethanol extract of <i>Psychotria prainii</i> aerial parts. By means of spectroscopic methods, their structures were elucidated to be deacetylasperulosidic acid 6-ethyl ether (<b>1</b>), asperulosidic acid (<b>2</b>), asperuloside (<b>3</b>) and obtucarbamates C (<b>4</b>). The isolated compounds were evaluated for their inhibitory effect on NO production in LPS-stimulated RAW264.7 cells. Among them, compounds <b>2</b> and <b>4</b> exhibited strong effect with the IC₅₀ values of 5.75 ± 0.85 and 6.92 ± 0.43 μM, respectively. This is the first report for the chemical composition and biological activity of <i>P. prainii</i>.</p

Molecular mechanism of inhibitory effects of bovine lactoferrin on the growth of oral squamous cell carcinoma

<div><p>Background</p><p>Lactoferrin (LF), a member of the transferrin family, recently has been demonstrated to have anticancer effects on various cancers including oral squamous cell carcinoma (OSCC). However, little is known about the underlying mechanisms of its effects on OSCC. Therefore, we aimed to investigate the mechanism of the suppressive effects of bovine LF (bLF) on the growth of OSCC cells.</p><p>Methods</p><p>In the current study, HSC2, HSC3, HSC4 and normal human oral keratinocytes (RT7) cell lines were tested with bLF 1, 10, and 100 μg/ml. The effects and detail mechanisms of bLF on proliferation and apoptosis of cells were investigated using flow cytometry and western blotting.</p><p>Results</p><p>We found that bLF (1, 10, and 100 μg/ml) induced activation of p53, a tumor suppressor gene, is associated with the induction of cell cycle arrest in G1/S phase and apoptosis in OSCC. Moreover, bLF downregulated the phosphorylation of Akt and activated suppressor of cytokine signaling 3 (SOCS3), thereby attenuating multiple signaling pathways including mTOR/S6K and JAK/STAT3. Interestingly, we revealed that bLF exerted its effect selectively against HSC3 but not on RT7 via different effects on the phosphorylation status of NF-κB and Akt.</p><p>Conclusion</p><p>This is the first report showing that bLF selectively suppresses proliferation through mTOR/S6K and JAK/STAT3 pathways and induction of apoptosis in OSCC. This study provides important new findings, which might be useful in the prevention and treatment of OSCC.</p></div

Schema of inhibitory effects of bLF on growth of OSCC cells and its underlying molecular mechanisms.

<p>In OSCCs, bLF internalizes into cells through LRP1 and suppressed NF-kB p65/Akt pathways. The attenuation of p65/Akt by bLF may inhibit G1/S cell cycle through the inhibition of downstream signaling pathways including mTOR/S6K and STAT3 and induce apoptosis in OSCC. Moreover, SOCS3, which is a negative regulator of STAT3, is upregulated. The overexpression of of SOCS3 in OSCC by bLF may downregulate JAK2/STAT3 pathway leading to the induction of G1/S cell cycle arrest.</p

Effect of bovine lactoferrin on the proliferation of OSCC and normal mucosal cells.

<p>OSCC and RT7 cells were treated with different concentrations of bLF (1 μg/ml, 10 μg/ml, and 100 μg/ml) and cell number was counted on days 1, 2, 4 and 6. bLF significantly prohibited the cell proliferation of OSCC cell lines in a dose-dependent manner; (<b>A</b>) HSC2, (<b>B</b>) HSC3, and (<b>C</b>) HSC4. bLF did not affect the cell growth of RT7 cells (<b>D</b>). Data represented as mean ± S.D; * p < 0.05 and ** p < 0.01 <i>vs</i> control (0 μg/ml of bLF).</p

Bovine lactoferrin suppressed the phosphorylation of growth and survival related kinase NF-kB p65 and Akt in HSC3 cells.

<p>HSC3 cells were treated with different concentrations of bLF. (<b>A</b>) After 48 h, bLF inhibited the activation of growth and survival related kinase p65 and Akt in HSC3 cells. (<b>B</b>) Number of HSC3 cells treated with and without CAPE 10μg/ml and bLF were counted. (<b>C</b>) G1/S cell cycle arrest related proteins were observed using western blot. (<b>D</b>) Proliferation assay were conducted using HSC3 cells under treatment of LY294002 (10 μM) and bLF (100 μg/ml). Cells were counted on day 1, 2, 4, and 6. (<b>E</b>) G1/S cell cycle related molecules were examined using western blot after 48 h stimulations of LY294002 and bLF. β-actin was used as loading control. Image is representative of 3 independent experiments (n = 3).</p

Bovine lactoferrin induced apoptosis in OSCC cells.

<p>(<b>A</b>) To check the effect of bLF on cell apoptosis, HSC3 cells were incubated with bLF (1 μg/ml, 10 μg/ml, and 100 μg/ml) for 48 h. Percentage of apoptotic cells were then analyzed by flow cytometry using PE Annexin V and 7-AAD. Cell populations were analyzed in four quadrants, Q1: necrotic cells, Q2: late apoptotic cells, Q3: viable cells, and Q4: early apoptotic cells. Total apoptotic cells were measured as the sum of early and late apoptotic cells. A dose-dependent increase in apoptotic cell number was observed after bLF treatment. Data were shown as the mean ± S.D (n = 3); * p < 0.05, ** p < 0.01 vs control. (<b>B</b>) HSC3 cells were treated with or without bLF (1, 10, and 100 μg/ml) for 48 h. Expressions of apoptosis-related genes were analyzed by western blotting. bLF treatment inhibited the phosphorylation of BAD and Bcl2 in HSC3 cells whereas expression of cleaved caspases 9, 3, 6, and 7 was upregulated. β-actin was used as a loading control. Data are representative of 3 independent experiments (n = 3).</p

Bovine lactoferrin inhibited expression of mTOR and S6K pathway thereby suppressed growth in HSC3 cells.

<p>HSC3 cells were treated with (1 μg/ml, 10 μg/ml, and 100 μg/ml) or without bLF. (<b>A</b>) Protein levels of p-mTOR and p-S6K were investigated by western blot after 48 h of stimulations. Expression of phosphorylated mTOR and S6K was decreased after bLF treatment. (<b>B</b>) To check the combinatorial effect of bLF and the inhibitor of mTOR, rapamycin, HSC3 cells were treated with 10 nM of rapamycin along with 100 μg/ml of bLF. No additive change was observed on p-mTOR and p-S6K expressions as shown by western blot. (<b>C</b>) Number of HSC3 cells was counted on day 1, 2, 4, and 6 with and without rapamycin and bLF. No additional effects on growth of cells in rapamycin and its combination. (<b>D</b>) G1/S cell cycle related molecules were investigated using western blot after 48 h of stimulations. Either rapamycin (10 nM) or its combination with bLF accumulated p21 expression but suppressed cyclin D1. (<b>E</b>) The growth of HSC3 cells was investigated under treatments of PF-4708671 (10 μM) and bLF 100 μg/ml. PF-4708671 remarkably attenuated number of HSC3 cells. There were no differences in number of cells between PF-4708671 and PF-4708671 with bLF. (<b>F</b>) Protein expression of p21 and cyclin D1 were examined using PF-4708671. PF-4708671 induced G1/S cell cycle arrest in HSC3 cells whereas no additive changes in the expressions p21 and cyclin D1 between PF-4708671 and its combination with bLF. β-actin was used as a loading control. The blots represent 3 independent experiments.</p

Bovine lactoferrin induced G1/S phase arrest, activation of p53, and regulated cell cycle-related proteins in HSC3 cells.

<p>HSC3 cells were treated with various concentrations of bLF (0 μg/ml, 1 μg/ml, 10 μg/ml, and 100 μg/ml). After 48 h, cell cycle analysis was done using flow cytometry. (<b>A</b>) bLF induced significant increase in cell population in G0/G1 phase in a dose-dependent manner suggesting the inhibition of G1/S phase transition. Bar graphs are representative for the same. Data were shown as the mean ± S.D; <i>*</i> p < 0.05, <i>*</i>* p < 0.01 <i>vs</i> control (0 μg/ml of bLF). (<b>B</b>) Expression of p-p53 and total p53 was analyzed 48 h post bLF treatment using western blotting. bLF induced activation of p53 in a dose-dependent manner. (<b>C</b>) bLF downregulates expression of Cyclin D1 whereas it enhances p21 expression as determined by western blots. β-actin was used as a loading control. Images are representative of three independent experiments (n = 3).</p

Bovine lactoferrin induced expression of SOCS3 to attenuate JAK2/STAT3 activation involving in inhibition of growth OSCC cells.

<p>HSC3 cells were treated with bLF (1 μg/ml, 10 μg/ml, and 100 μg/ml) for 24 h for mRNA and 48 h for protein expressions. (<b>A</b>) Expression of SOCS3 was found to be enhanced after bLF treatment checked by RT-PCR and western blot. (<b>B</b>) Protein levels of p-JAK2 and p-STAT3 were examined by western blot. bLF inhibited the activation of JAK/STAT3 pathway in a dose-dependent manner. (<b>C</b>) Number of HSC3 cells were counted under presence or absence of S3I-201 (50 μM) and bLF (100 μg/ml). Either S3I-201 or bLF-S3I-201 remarkably reduced number of HSC3 cells. On the other hand, no significant suppressive effect of bLF on HSC3 S3I-201 treated cells was observed. (<b>D</b>) G1/S cell cycle related molecules were analyzed by western blot in S3I-201 and S3I-201-bLF stimulated HSC3 cells. There were no significant differences in expression of p21 and cyclin D1 in HSC3 cells with S3I-201 and its combination groups. GAPDH and β-actin were used as a loading control. Images are representative of 3 independent experiments (n = 3).</p

Roles of VEGF-Flt-1 signaling in malignant behaviors of oral squamous cell carcinoma

<div><p>Background</p><p>Vascular endothelial growth factor (VEGF) is a highly specific signaling protein for vascular endothelial cells that plays a critical role in tumor growth and invasion through angiogenesis, and may contribute to cell migration and activation of pre-osteoclasts, osteoclasts and some tumor cells.</p><p>Objectives</p><p>We aimed to clarify the detailed roles of VEGF-Flt-1 signaling in bone invasion of oral squamous cell carcinoma (OSCC) cells.</p><p>Results</p><p>Forty-two (42) of 54 cases with gingival SCC (77.8%) strongly expressed VEGF, and had a significantly increased number of Flt-1+ osteoclasts (p<0.01) and more aggressive bone invasion (p<0.05). PlGF, a ligand of Flt-1, induced osteoclastogenesis in single culture of bone marrow cells (BMCs), and inhibition of Flt-1-signaling by VEGF tyrosine kinase inhibitor and It’s down stream (Akt and ERK1/2) inhibitos reduced osteoclastogenesis in PlGF-stimulated BMCs (p<0.01). In molecular level, PlGF stimulation significantly upregulated RANKL expression in Flt-1-expressing HSC2 cells via phosphorylation of Akt and ERK1/2. In the co-culture of VEGF-producing HSC2 cells and BMCs, number of TRAP-positive osteoclasts markedly increased (p<0.01). The osteoclastogenesis was significantly inhibited by RANKL-neutralizing antibody (p<0.01) as well as by VEGF tyrosine kinase inhibitor (p<0.01) and it’s downstream (Akt and ERK1/2) inhibitors (p<0.01, p<0.05, respectively).</p><p>Conclusion</p><p>VEGF-Flt-1 signaling induces osteoclastogenesis in OSCC through two possible ways: 1) VEGF produced from OSCC cells can directly stimulate the Flt-1 pathway in preosteoclasts to induce migration to future bone resorbing area and differentiation into osteoclasts, and 2) VEGF-Flt-1 signaling upregulates RANKL expression in OSCC cells, which indirectly leads to osteoclast differentiation. Therefore, blocking of the VEGF-Flt-1 signaling may help inhibit bone invasion of OSCC.</p></div