Discovery of the tryptanthrin-derived indoloquinazoline as an anti-breast cancer agent via ERK/JNK activation.

Tryptanthrin, an alkaloid applied in traditional Chinese medicine, exhibits a variety of pharmacological activities. This study aimed to investigate the anti-tumor activity of the tryptanthrin derivative (8-cyanoindolo[2,1-b]quinazoline-6,12-dione [CIQ]) in breast cancer cells. In both MDA-MB-231 and MCF-7 breast cancer cells, CIQ inhibited cell viability and promoted caspase-dependent apoptosis. At the concentration- and time-dependent ways, CIQ increased the levels of p-ERK, p-JNK, and p-p38 in breast cancer cells. We found that exposure to the JNK inhibitor or the ERK inhibitor partially reversed CIQ's viability. We also observed that CIQ increased reactive oxygen species (ROS) generation, and upregulated the phosphorylation and expression of H2AX. However, the pretreatment of the antioxidants did not protect the cells against CIQ's effects on cell viability and apoptosis, which suggested that ROS does not play a major role in the mechanism of action of CIQ. In addition, CIQ inhibited the invasion of MDA-MB-231 cells and decreased the expression of the prometastatic factors (MMP-2 and Snail). These findings demonstrated that the possibility of this compound to show promise in playing an important role against breast cancer.

Extract of Ficus septica modulates apoptosis and migration in human oral squamous cell carcinoma cells.

According to the alarming statistical analysis of global cancer, there are over 19 million new diagnoses and more than 10 million deaths each year. One such cancer is the oral squamous cell carcinoma (OSCC), which requires new therapeutic strategies. Ficus septica extract has been used in traditional medicine to treat infectious diseases. In this study, we examined the anti-proliferative effects of an extract of F. septica bark (FSB) in OSCC cells. Our results showed that FSB caused a concentration-dependent reduction in the viability of SCC2095 OSCC cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and was less sensitive to fibroblasts. In addition, FSB induced apoptosis by activating caspases, accompanied by the modulation of Akt/mTOR/NF-κB and mitogen-activated protein kinase signaling. Moreover, FSB increased reactive oxygen species generation in a concentration-dependent manner in SCC2095 cells. Furthermore, FSB inhibited cell migration and modulated the levels of the cell adhesion molecules including E-cadherin, N-cadherin, and Snail in SCC2095 cells. Pinoresinol, a lignan isolated from FSB, showed antitumor effects in SCC2095 cells, implying that this compound might play an important role in FSB-induced OSCC cell death. Taken together, FSB is a potential anti-tumor agent against OSCC cells.

Fructose milieu undermines the therapeutic effect of Tribulus terrestris extract on neuroblastoma cell line via maintaining mitochondrial function.

Fructose overconsumption promotes tumor progression. Neuroblastoma is a common extracranial tumor with about 50% 5-year survival rate in high-risk children. The anti-tumor effect of Tribulus terrestris might bring new hope to neuroblastoma therapy. However, whether fructose disturbs the therapeutic effect of T. terrestris is currently unknown. In this study, the mouse neuroblastoma cell line, Neuro 2a (N2a) cells, was used to investigate the therapeutic effects of T. terrestris extract at various dosages (0.01, 1, 100 ng/ml) in regular EMEM medium or extra added fructose (20 mM) for 24 h. 100 ng/ml T. terrestris treatment significantly reduced the cell viability, whereas the cell viabilities were enhanced at the dosages of 0.01 or 1 ng/ml T. terrestris in the fructose milieu instead. The inhibition effect of T. terrestris on N2a migration was blunted in the fructose milieu. Moreover, T. terrestris effectively suppressed mitochondrial functions, including oxygen consumption rates, the activities of electron transport enzymes, the expressions of mitochondrial respiratory enzymes, and mitochondrial membrane potential. These suppressions were reversed in the fructose group. In addition, the T. terrestris-suppressed mitofusin and the T. terrestris-enhance mitochondrial fission 1 protein were maintained at basal levels in the fructose milieu. Together, these results demonstrated that T. terrestris extract effectively suppressed the survival and migration of neuroblastoma via inhibiting mitochondrial oxidative phosphorylation and disturbing mitochondrial dynamics. Whereas, the fructose milieu blunted the therapeutic effect of T. terrestris, particularly, when the dosage is reduced.

Tribulus terrestris fruit extract inhibits autophagic flux to diminish cell proliferation and metastatic characteristics of oral cancer cells.

Elevated autophagy is highly associated with cancer development and progression. Fruit extracts of several plants inhibit activity of autophagy-related protease ATG4B and autophagy activity in colorectal cancer cells. However, the effects of these plant extracts in oral cancer cells remain unclear. In this study, we found that the extracted Tribulus terrestris fruit (TT-(fr)) and Xanthium strumarium fruit had inhibitory effects on autophagy inhibition in both SAS and TW2.6 oral cancer cells. Moreover, the fruit extracts had differential effects on cell proliferation of oral cancer cells. In addition, the fruit extracts hampered cell migration and invasion of oral cancer cells, particularly in TT-(fr) extracts. Our results indicated that TT-(fr) extracts consistently inhibited autophagic flux, cell growth and metastatic characteristics of oral cancer cells, suggesting TT-(fr) might contain function ingredient to suppress oral cancer cells.

TCD, a triterpenoid isolated from wild bitter gourd, reduces synaptosomal release of glutamate and protects against kainic acid-induced neuronal death.

3ß,7ß,25-Trihydroxycucurbita-5,23(E)-dien-19-al (TCD) is a triterpenoid isolated from wild bitter gourd that is a common tropical vegetable with neuroprotective effects. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the aims of this study were to examine the effect of TCD on glutamate release in vitro and to examine the effect of TCD in vivo. In rat cerebrocortical synaptosomes, TCD reduced 4-aminopyridine (4-AP)-stimulated glutamate release and Ca2+ concentration elevation, but had no effect on plasma membrane potential. TCD-mediated inhibition of 4-AP-induced glutamate release was dependent on the presence of extracellular calcium; persisted in the presence of the glutamate transporter inhibitor dl-TBOA, P/Q-type Ca2+ channel blocker ω-agatoxin IVA, and intracellular Ca2+-releasing inhibitors dantrolene and CGP37157; and was blocked by the vesicular transporter inhibitor bafilomycin A1 and the N-type Ca2+ channel blocker ω-conotoxin GVIA. Molecular docking studies have demonstrated that TCD binds to N-type Ca2+ channels. TCD-mediated inhibition of 4-AP-induced glutamate release was abolished by the Ca2+-dependent protein kinase C (PKC) inhibitor Go6976, but was unaffected by the Ca2+-independent PKC inhibitor rottlerin. Furthermore, TCD considerably reduced the phosphorylation of PKC, PKCα, and myristoylated alanine-rich C kinase substrate, a major presynaptic substrate for PKC. In a rat model of kainic acid (KA)-induced excitotoxicity, TCD pretreatment substantially attenuated KA-induced neuronal death in the CA3 hippocampal region. These results suggest that TCD inhibits synaptosomal glutamate release by suppressing N-type Ca2+ channels and PKC activity and exerts protective effects against KA-induced excitotoxicity in vivo.

Antiviral activity of Sambucus FormosanaNakai ethanol extract and related phenolic acid constituents against human coronavirus NL63.

Human coronavirus NL63 (HCoV-NL63), one of the main circulating HCoVs worldwide, causes respiratory tract illnesses like runny nose, cough, bronchiolitis and pneumonia. Recently, a severe respiratory illness outbreak of HCoV-NL63 has been reported in a long-term care facility. Sambucus FormosanaNakai, a species of elderberry, is a traditional medicinal herb with anti-inflammatory and antiviral potential. The study investigated the antiviral activity of Sambucus FormosanaNakai stem ethanol extract and some phenolic acid constituents against HCoV-NL63. The extract was less cytotoxic and concentration-dependently increased anti-HCoV-NL63 activities, including cytopathicity, sub-G1 fraction, virus yield (IC50 = 1.17 µg/ml), plaque formation (IC50 = 4.67 µg/ml) and virus attachment (IC50 = 15.75 µg/ml). Among the phenolic acid constituents in Sambucus FormosanaNakai extract, caffeic acid, chlorogenic acid and gallic acid sustained the anti-HCoV-NL63 activity that was ranked in the following order of virus yield reduction: caffeic acid (IC50 = 3.54 µM) > chlorogenic acid (IC50 = 43.45 µM) > coumaric acid (IC50 = 71.48 µM). Caffeic acid significantly inhibited the replication of HCoV-NL63 in a cell-type independent manner, and specifically blocked virus attachment (IC50 = 8.1 µM). Therefore, the results revealed that Sambucus Formosana Nakai stem ethanol extract displayed the strong anti-HCoV-NL63 potential; caffeic acid could be the vital component with anti-HCoV-NL63 activity. The finding could be helpful for developing antivirals against HCoV-NL63.

Xanthium strumarium Fruit Extract Inhibits ATG4B and Diminishes the Proliferation and Metastatic Characteristics of Colorectal Cancer Cells.

Autophagy is an evolutionarily conserved pathway to degrade damaged proteins and organelles for subsequent recycling in cells during times of nutrient deprivation. This process plays an important role in tumor development and progression, allowing cancer cells to survive in nutrient-poor environments. The plant kingdom provides a powerful source for new drug development to treat cancer. Several plant extracts induce autophagy in cancer cells. However, little is known about the role of plant extracts in autophagy inhibition, particularly autophagy-related (ATG) proteins. In this study, we employed S-tagged gamma-aminobutyric acid receptor associated protein like 2 (GABARAPL2) as a reporter to screen 48 plant extracts for their effects on the activity of autophagy protease ATG4B. Xanthium strumarium and Tribulus terrestris fruit extracts were validated as potential ATG4B inhibitors by another reporter substrate MAP1LC3B-PLA2. The inhibitory effects of the extracts on cellular ATG4B and autophagic flux were further confirmed. Moreover, the plant extracts significantly reduced colorectal cancer cell viability and sensitized cancer cells to starvation conditions. The fruit extract of X. strumarium consistently diminished cancer cell migration and invasion. Taken together, the results showed that the fruit of X. strumarium may have an active ingredient to inhibit ATG4B and suppress the proliferation and metastatic characteristics of colorectal cancer cells.

Cyclocommunol induces apoptosis in human oral squamous cell carcinoma partially through a Mcl-1-dependent mechanism.

BACKGROUND: Crude extract of breadfruit has been reported to have antitumor activity against various cancer cell lines with unknown mechanism. PURPOSE: This study aims to investigate the proapoptotic effect of cyclocommunol (CYC), a prenylflavonoid from breadfruit, in two oral squamous cell carcinoma (OSCC) cell lines, SCC2095 and Ca922. METHODS: The antiproliferative effects of CYC were assessed by MTT assays and PI/annexin V analysis. SCC2095 cells were transiently transfected with Mcl-1 plasmid in overexpression experiment. Other methods used to investigate the mechanism of CYC included Western blotting, acridine orange staining and confocal microscopic visualization. RESULTS: Our results showed that CYC suppressed the viability of SCC2095 and Ca922 with IC50 values at 48 h of 4.2 and 5.0 µM, respectively. This decrease in viability occurred in a caspase-dependent apoptotic manner. In addition, CYC down-regulated the phosphorylation/expression of Akt/mTOR and Mcl-1, accompanied by reactive oxygen species generation, and autophagy induction. Notably, overexpression of Mcl-1 using Mcl-1-tag-myc partially rescued CYC-mediated caspase-3 activation, PARP cleavage, and cytotoxicity. In summary, our study demonstrated the proapoptotic activity of CYC on OSCC, partially through down-regulation of Mcl-1. CONCLUSION: CYC from breadfruit has translational value as a proapoptotic agent for OSCC.

Identification of a Triterpenoid as a Novel PPARγ Activator Derived from Formosan Plants.

Peroxisome proliferator-activated receptor γ (PPARγ), one of the transcription factors that regulate lipid metabolism and energy use in tumor cells, is a viable target for cancer therapy. In our search for potential PPARγ activator, extracts from five Formosan plants were tested. Among them, Momordica charantia L. showed the highest ability to activate PPARγ, which led us to identify its potential constituents. Among the seven compounds isolated from M. charantia, a triterpenoid, 5ß,19-epoxy-19-methoxycucurbita-6,23-dien-3ß,25-diol (compound 1), was identified as a PPARγ activator with an IC50 of 10 µM in breast cancer MCF-7 cells. Flow cytometric analysis indicated that compound 1 induced G1 cell cycle arrest which might be attributable to the modulation of phosphorylation and expression of numerous key signaling effectors, including cyclin D1, CDK6, and p53. Notably, compound 1 downregulated the expression of histone deacetylase 1, leading to increased histone H3 acetylation. Taken together, these findings suggest that compound 1 may have therapeutic applications in cancer treatment through PPARγ activation. Copyright © 2017 John Wiley & Sons, Ltd.

Ficus septica plant extracts for treating Dengue virus in vitro.

Dengue virus types 1-4 (DENV-1-4) are positive-strand RNA viruses with an envelope that belongs to the Flaviviridae. DENV infection threatens human health worldwide. However, other than supportive treatments, no specific therapy is available for the infection. In order to discover novel medicine against DENV, we tested 59 crude extracts, without cytotoxicity, from 23 plants in vitro; immunofluorescence assay revealed that the methanol extracts of fruit, heartwood, leaves and stem from Ficus septica Burm. f. had a promising anti-DENV-1 and DENV-2 effect. However, infection with the non-envelope picornavirus, Aichi virus, was not inhibited by treatment with F. septica extracts. F. septica may be a candidate antiviral drug against an enveloped virus such as DENV.

A Flavone Constituent from Myoporum bontioides Induces M-Phase Cell Cycle Arrest of MCF-7 Breast Cancer Cells.

Myoporum bontioides is a traditional medicinal plant in Asia with various biological activities, including anti-inflammatory and anti-bacterial characteristics. To identify the bioactive constituents from M. bontioides, a newly-identified flavone, 3,4'-dimethoxy-3',5,7-trihydroxyflavone (compound 1), along with eight known compounds, were investigated in human MCF-7 breast cancer, SCC4 oral cancer, and THP-1 monocytic leukemia cells. Among these compounds, compound 1 exhibited the strongest antiproliferative activity with half-maximal inhibitory concentration (IC50) values ranging from 3.3 µM (MCF-7) to 8.6 µM (SCC4). Flow cytometric analysis indicated that compound 1 induced G2/M cell cycle arrest in MCF-7 cells. Mechanistic evidence suggests that the G2/M arrest could be attributable to compound 1's modulatory effects on the phosphorylation and expression of numerous key signaling effectors, including cell division cycle 2 (CDC2), CDC25C, and p53. Notably, compound 1 downregulated the expression of histone deacetylase 2 (HDAC2) and HDAC4, leading to increased histone H3 acetylation and p21 upregulation. Together, these findings suggest the translational potential of compound 1 as a breast cancer treatment.

A triterpenoid from wild bitter gourd inhibits breast cancer cells.

The antitumor activity of 3ß,7ß,25-trihydroxycucurbita-5,23(E)-dien-19-al (TCD), a triterpenoid isolated from wild bitter gourd, in breast cancer cells was investigated. TCD suppressed the proliferation of MCF-7 and MDA-MB-231 breast cancer cells with IC50 values at 72 h of 19 and 23 µM, respectively, via a PPARγ-independent manner. TCD induced cell apoptosis accompanied with pleiotrophic biological modulations including down-regulation of Akt-NF-κB signaling, up-regulation of p38 mitogen-activated protein kinase and p53, increased reactive oxygen species generation, inhibition of histone deacetylases protein expression, and cytoprotective autophagy. Together, these findings provided the translational value of TCD and wild bitter gourd as an antitumor agent for patients with breast cancer.

Anti-tumor activities of triterpenes from Syzygium kusukusense.

In this study, we report the isolation from the stem of Syzygium kusukusense of five triterpenes, 2α-hydroxybetulinic acid (1), betulinic acid (2), platanic acid (3), ursolic acid (4), and hyptatic acid A (5). All were identified for the first time from this indigenous plant of Taiwan. Assessment of the cytotoxic activities of these compounds against a panel of human tumor cell lines, including MCF-7 breast, PC-3 prostate, and SCC2095 oral squamous cell cancers, revealed the high potency of compounds 1 (IC50, 5.7 - 7.6 µM) and, especially, 4 (IC50, 1.7 - 3.7 µM) in suppressing cell viability, which warrants further mechanistic investigations.

Identification of kazinol Q, a natural product from Formosan plants, as an inhibitor of DNA methyltransferase.

DNA methylation plays a pivotal role in the epigenetic regulation of the transcription of a number of cancer-related genes, thereby representing an important target for cancer prevention and treatment. In our search for DNA methyltransferase (DNMT) inhibitors from Formosan plants, by screening against a library consisting of 12 structurally distinct natural products, we identified kazinol Q {4-[6-(1,1-dimethyl-allyl)-7-hydroxy-chroman-2-yl]-3,6-bis-(3-methyl-but-2-enyl)-benzene-1,2-diol} as an inhibitor of recombinant DNMT1 with IC50 of 7 µM. The effect of kazinol Q on DNMT inhibition was validated by its ability to reactivate the expression of a DNA methylation-silenced gene, E-cadherin, in MDA-MB-231 breast cancer cells. Moreover, kazinol Q suppressed the proliferation of MCF-7 breast and LNCaP prostate cancer cells, in part, through apoptosis induction. The role of DNMT1 inhibition in mediating kazinol Q's antiproliferative effect was supported by the protective effect of ectopic expression of DNMT1 on kazinol Q-induced cell death. Molecular modeling analysis suggests that kazinol Q inhibited DNMT activity by competing with cytosine binding, a mechanism similar to that described for (-)-epigallocatechin-3-gallate (EGCG). Relative to EGCG, kazinol Q exhibits several desirable features for drug development, including chemical stability and increased hydrophobicity, and might have therapeutic relevance to cancer treatment.

Cucurbitane Triterpenoid from Momordica charantia Induces Apoptosis and Autophagy in Breast Cancer Cells, in Part, through Peroxisome Proliferator-Activated Receptor γ Activation.

Although the antitumor activity of the crude extract of wild bitter gourd (Momordica charantia L.) has been reported, its bioactive constituents and the underlying mechanism remain undefined. Here, we report that 3 ß ,7 ß -dihydroxy-25-methoxycucurbita-5,23-diene-19-al (DMC), a cucurbitane-type triterpene isolated from wild bitter gourd, induced apoptotic death in breast cancer cells through peroxisome proliferator-activated receptor (PPAR) γ activation. Luciferase reporter assays indicated the ability of DMC to activate PPAR γ , and pharmacological inhibition of PPAR γ protected cells from DMC's antiproliferative effect. Western blot analysis indicated that DMC suppressed the expression of many PPAR γ -targeted signaling effectors, including cyclin D1, CDK6, Bcl-2, XIAP, cyclooxygenase-2, NF- κ B, and estrogen receptor α , and induced endoplasmic reticulum stress, as manifested by the induction of GADD153 and GRP78 expression. Moreover, DMC inhibited mTOR-p70S6K signaling through Akt downregulation and AMPK activation. The ability of DMC to activate AMPK in liver kinase (LK) B1-deficient MDA-MB-231 cells suggests that this activation was independent of LKB1-regulated cellular metabolic status. However, DMC induced a cytoprotective autophagy presumably through mTOR inhibition, which could be overcome by the cotreatment with the autophagy inhibitor chloroquine. Together, the ability of DMC to modulate multiple PPAR γ -targeted signaling pathways provides a mechanistic basis to account for the antitumor activity of wild bitter gourd.

Cytotoxic constituents from Celastrus paniculatus induce apoptosis and autophagy in breast cancer cells.

Celastrus paniculatus is a traditional medicinal plant with diverse pharmacological activities. To identify its bioactive constituents, three new ß-dihydroagarofuranoid sesquiterpenes were isolated from the whole plant, of which the major constituent is (1α,2α,8ß,9ß)-1,8-bis(acetyloxy)-2,9-bis(benzoyloxy)-14-hydroxy-ß-dihydroagarofuran. It was assessed for its antiproliferative activity, and it suppressed the viability of MCF-7 breast cancer cells with an IC50 of 17±1µM. This growth inhibition was, in part, attributable to apoptosis. Moreover, this drug treatment led to LC3B-II accumulation, indicative of autophagy. Western blot analysis established its ability to target a broad range of signaling effectors related to survival and cell cycle progression, including Akt, NF-κB, p53, and MAP kinases. In addition, flow cytometry analysis indicates increased reactive oxygen species production in response to this compound. Taken together, these findings suggest a pleiotropic mode of mechanism that underlies the antiproliferative activity of this compound in MCF-7 breast cancer cells.

Cytotoxic and antioxidant constituents from Garcinia subelliptica.

Two new triterpenoids, garcinielliptones Q (1) and S (3), and a new phloroglucinol, garcinielliptone R (2), were isolated from the seed of Garcinia subelliptica. Their structures were established by analysis of their spectroscopic data. Phloroglucinol, garcinielliptone FC (4) from this plant exhibited a significant increase of antiproliferative effect, while 4 combined with cisplatin significantly caused decrease of cell inhibition induced by cisplatin in NTUB1. Exposure of NTUB1 cells to 4 cotreated with cisplatin for significantly decreased the amount of reactive oxygen species (ROS) than that of the total amount generated by 4 and cisplatin. These results suggested that 4 could protect the cisplatin toxicity through reduction of ROS in NTUB1. Phloroglucinols, garcinielliptones, A (5) and F (7), and garsubelline A (6), from this plant, revealed ABTS radical cation scavenging activity and 5 displayed an inhibitory effect on xanthine oxidase. These finding showed that 5-7 may be used as antioxidants.

Anti-inflammatory effects of Calophyllum inophyllum L. in RAW264.7 cells.

Calophyllum inophyllum L. has been used as folk medicine in the treatment of ocular burn and it has demonstrated potential to be an anti-inflammatory agent. The aim of this study was to explore the anti-inflammatory activities of an acetone extract of C. inophyllum L. leaves (CIL). The CIL extract was tested on lipopolysaccharide (LPS)-induced RAW 264.7 cells to evaluate the effect of CIL extract on the expression of nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Results showed that the CIL extract markedly suppressed the LPS-induced production of nitric oxide, as well as the expression of iNOS, cyclooxygenase (COX)-2 and nuclear factor-kappaB (NF-κB) in a dose-dependent manner. LPS-induced microRNA (miR)-146a expression was inhibited by CIL extract, while miR-155 and miR-424 expression was not affected as demonstrated using quantitative RT-PCR analysis. Taken together, these observations show that CIL extract has anti-inflammatory effect, which extends the potential application for prevention of inflammatory diseases, and its mechanism may be partially associated with blocking COX-2 and iNOS of RAW 264.7 cells.

Inhibition of hedgehog signaling induces monocytic differentiation of HL-60 cells.

There is little evidence to demonstrate the importance of the Sonic hedgehog homolog (Shh) pathway to differentiation therapy in the treatment of hematological neoplasms. Here we characterize the changes in acute myelogenous leukemia (HL-60) cells after blocking the Shh pathway by an antagonist of Smoothened, cyclopamine. Cyclopamine induces apoptosis of HL-60 cells in a dose- and time-dependent manner with increased G0/G1 cycle fraction. Treatment with cyclopamine increases the expression of monocytic cell markers CD11b and CD14, but the expression of CD13, CD33 and CD38 is unchanged. The monocytic differentiation of HL-60 cells induced by cyclopamine is also evidenced by an increase in Egr-1 expression. Importantly, cyclopamine down-regulates the phosphorylation of Akt and ERK, but activates AMP-activated protein kinase (AMPK) signaling. Further investigations should determine the clinical application of modulating the Shh pathway in the treatment of hematological malignancies.

Gypenosides inhibits migration and invasion of human oral cancer SAS cells through the inhibition of matrix metalloproteinase-2 -9 and urokinase-plasminogen by ERK1/2 and NF-kappa B signaling pathways.

Gypenosides (Gyp), found in Gynostemma pentaphyllum Makino, has been used as a folk medicine in the Chinese population for centuries and is known to have diverse pharmacologic effects, including anti-proliferative and anti-cancer actions. However, the effects of Gyp on prevention from invasion and migration of oral cancer cells are still unsatisfactory. The purpose of this study was to investigate effects of Gyp treatment on migration and invasion of SAS human oral cancer cells. SAS cells were cultured in the presence of 90 and 180 µg/mL Gyp for 24 and 48 hours. Gyp induced cytotoxic effects and inhibited SAS cells migration and invasion in dose- and time-dependent response. Wound-healing assay and boyden chamber assay were carried out to investigate Gyp-inhibited migration and invasion of SAS cells. Gyp decreased the abundance of several proteins, including nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/ 2), matrix metalloproteinase-9, -2 (MMP-9, -2), sevenless homolog (SOS), Ras, urokinase-type plasminogen activator (uPA), focal adhesion kinase (FAK) and RAC-alpha serine/threonine-protein kinase (Akt), in a time-dependent manner. In addition, Gyp decreased mRNA levels of MMP-2, MMP-7, MMP-9 but did not affect FAK and Rho A mRNA levels in SAS cells. These results provide evidences for the role of Gyp as a potent anti-metastatic agent, which can markedly inhibit the metastatic and invasive capacity of oral cancer cells. The inhibition of NF-κB and MMP-2, -7 and -9 signaling may be one of the mechanisms that is present in Gyp-inhibited cancer cell invasion and migration.