The expression of COX-2 and EP2 in HCC cells and meloxicam reduces cell viability <i>in vitro</i>.

<p>(A) The expression of COX-2 in HCC cell lines SMMC-7402, Bel-7402, HepG2, SMMC-7721 and Huh-7 was detected by Western Blotting. GAPDH served as an internal control. (B) Bel-7402, HepG2 and SMMC-7721 cells that express higher levels of COX-2 were incubated with increasing concentrations of meloxicam, and the rates of viability inhibition were measured. (C) The expression of EP2 in the above five HCC cell lines was detected by Western Blotting. GAPDH served as an internal control.</p

Meloxicam Executes Its Antitumor Effects against Hepatocellular Carcinoma in COX-2- Dependent and -Independent Pathways

<div><p>Background</p><p>Cyclooxygenase (COX)-2 is overexpressed in many types of cancers including hepatocellular carcinoma (HCC). Meloxicam, a selective COX-2 inhibitor, has shown potential therapeutic effects against HCC, but the mechanisms accounting for its anti-cancer activities remain unclear.</p><p>Methods and Findings</p><p>Meloxicam inhibited the ability of human HCC cells expressing higher levels of COX-2 to migrate, invade, adhere and form colonies through upregulating the expression of E-cadherin and downregulating the expression of matrix metalloproteinase (MMP) -2. Meloxicam induced cell apoptosis by upregulating pro-apoptotic proteins including Bax and Fas-L, and downregulating anti-apoptotic proteins including survivin and myeloid cell leukemia-1 (Mcl-1), through inhibiting phosphorylation of AKT. Addition of prostaglandin E2 (PGE2), the major product of COX-2, could abrogate the effects of meloxicam on the expression of survivin and myeloid cell leukemia-1 (Mcl-1), but not Bax and Fas-L, indicating that meloxicam induces cell apoptosis via both COX-2-dependent and -independent pathways. Meloxicam also induced cell autophagy by upregulating Beclin 1 and light chain 3-II. Specific inhibition of autophagy by 3-methyladenine and chloroquine had little effect on cell apoptosis but could enhance the pro-apoptotic effects of meloxicam by further upregulating the expression of Bax.</p><p>Conclusions</p><p>Meloxicam executes its antitumor effects by targeting the COX-2/MMP-2/E-cadherin, AKT, apoptotic and autophagic pathways in COX-2-dependent and -independent pathways, and inhibition of cell autophagy could help to overcome the resistance to meloxicam-induced apoptosis in HCC.</p></div

Meloxicam inhibits cell migration, invasion, adhesion and colony formation.

<p>(A) Representative photographs were taken from HepG2 cells incubated for 48 h with meloxicam (80 μM) or vehicle (control) and subjected to cell migration, invasion, adhesion and colony formation assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092864#s2" target="_blank">Materials and Method</a>s. (B) The above assays were quantified. Data represent three independent experiments. “**” indicates a highly significant (<i>P</i>&lt;0.001) difference from controls.</p

Meloxicam induces cell apoptosis via COX-2-dependent and -independent mechanisms.

<p>(A, B) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence or absence of PGE2 (3 μM) or rh-MMP-2 (25 ng/mL). Untreated cells served as controls. (A) Representative dot plots were taken from cytometrically analyzed cells. (B) The apoptosis rate was calculated. (C) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (D) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence or absence of PGE2 (3 μM) or MK-2206 (5 μM). (E) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (F) HepG2 cells were incubated with meloxicam (80 μM) in the presence or absence of PGE2 (3 μM) for 72 h, and harvested. The above harvested cells were subjected to Western blot analysis. The band density in each assay was measured and normalized to that of GAPDH, respectively. Data represent three independent experiments. “*” indicates a significant (<i>P</i>&lt;0.05) difference, and “**”, a highly significant (<i>P</i>&lt;0.001) difference.</p

Meloxicam upregulates E-cadherin and downregulates MMP-2 in a COX-2-dependent way.

<p>HepG2 cells were cultured for 72(80 μM), PGE2 (3 μM) or rh-MMP-2 (25 ng/mL), or the combination. Cell lysates were analyzed by Western blot analysis to detect expression of E-cadherin (A, B) and MMP-1/MMP-2 (C) proteins. The band density in each assay was measured and normalized to that of GAPDH, respectively. (D, E) The concentrations of soluble E-cadherin (sE-cad) in supernatants from the above cell culture were measured by ELISA. (F–H) The above cells were lysed and subjected to quantitative real-time RT-PCR for measuring the levels of E-Cadherin (F) and MMP-2 (G) mRNAs, and to a standard RT-PCR assay, in which PCR products of E-Cadherin (I) and MMP-2 (H) were electrophoresed. GAPDH served as an internal control. Data represent three independent experiments. “*” indicates a significant (<i>P</i>&lt;0.05) difference, and “**”, a highly significant (<i>P</i>&lt;0.001) difference.</p

Meloxicam inhibits phosphorylation of AKT in a COX-2-depdendent way.

<p>(A) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (B, C) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence and absence of PGE2 (3 μM) (B) or rh-MMP-2 (25 ng/mL) (C). The above harvested cells were subjected to Western blot analysis. The band density in each assay was measured and normalized to that of GAPDH, respectively. Data represent three independent experiments. “**”, indicates a highly significant (<i>P</i>&lt;0.001) difference.</p

Additional file 1: of Neuropilin-1 is associated with clinicopathology of gastric cancer and contributes to cell proliferation and migration as multifunctional co-receptors

Supplementary materials. (DOCX 23 kb

Proposed mechanisms by which meloxicam executes its antitumor effects in COX-2-depdendent and -independent ways.

<p>Meloxicam inhibits the production of PGE2 by inhibiting COX-2 activity. PGE2 binds to EP2 to upregulate the expression of survivin and Mcl-1 via activation of AKT. Meloxicam induces cell apoptosis by upregulating Bax and Fas-L in a COX-2-independent way. 3-MA inhibits the engulfing of Bax by autophagosome, thus blocks the inhibitory effect of autophagy on apoptosis. PGE2 binds to EP2 to upregulate MMP-2, which in turn promotes the cleavage of E-cadherin. Soluble E-cadherin binds to HER/IGF-1R to activate AKT, while full-length of E-cadherin inhibits activation of AKT. “→” indicates positive regulation or activation; “⊥”, negative regulation or blockade; COX-2, cyclooxygenase-2; E-cad, E-cadherin; EP2, prostaglandin E2 receptor; HER, human epidermal growth factor receptor; IGF-1R, insulin-like growth factor-1 receptor; Mcl-1, myeloid cell leukemia-1; MMP-2, matrix metalloproteinase-2; PGE2, prostaglandin E2; 3-MA, 3-methyladenine.</p

Meloxicam induces autophagy and inhibition of autophagy promotes the apoptosis of HepG2 cells.

<p>(A) Representative images were from HepG2 cells that were incubated for 72 h with meloxicam (Mel) (80 μM) in the presence or absence of 3-MA (2 mM), and then stained by acridine orange. Untreated cells served as control (CTL). (B) The above cells from (A) were further subjected to flow cytometry to measure the degree of autophagic lysosomes as expressed by fold change of acridine orange fluorescence intensity (FL3) in treated cells versus control cells. (C) HepG2 cells were incubated with meloxicam (80 μM), and harvested at the indicated time points. (D–F) HepG2 cells were incubated for 72 h with meloxicam (80 μM) in the presence or absence of 3-MA (2 mM). (E) Representative dot plots were taken from cytometrically analyzed cells. (F) The apoptosis rate in (E) was calculated. (G) The above harvested cells were subjected to Western blot analysis. The band density in each assay was measured and normalized to that of GAPDH, respectively. Data represent three independent experiments. “*” indicates a significant (<i>P</i>&lt;0.05) difference, and “**”, a highly significant (<i>P</i>&lt;0.001) difference.</p

Additional file 2 of LncRNA SNHG1 contributes to sorafenib resistance by activating the Akt pathway and is positively regulated by miR-21 in hepatocellular carcinoma cells

Supplementary tables and figures. (PDF 149 kb