Synergistic effect of indomethacin and NGX6 on proliferation and invasion by human colorectal cancer cells through modulation of the Wnt/β-catenin signaling pathway

This study was designed to investigate whether indomethacin and NGX6 synergistically inhibit the growth and invasiveness of human colon cancer cells (HT-29 and SW620) and to elucidate the molecular mechanism of their action. Cell proliferation was assessed by MTT assay. Cell apoptosis was assessed by acridine orange/ethidium bromide staining (AO–EB) and annexin-V-FITC/PI assay. Invasive behaviors of colorectal cancer cells were examined by cell adhesion, migration, and invasion assays. Gap junctional intercellular communication (GJIC) was assessed by the scrape-loading/dye transfer technique. The subcellular localization and expression of β-catenin protein was examined by immunofluorescence staining and western blot analysis, respectively. Indomethacin and NGX6 had a synergistic effect on inhibiting proliferation and invasiveness of colon cancer HT-29 and SW620 cells, restoring GJIC of HT-29 and SW620, and suppressing translocation of β-catenin from the nucleus and cytoplasm to the plasma membrane. However, they did not have synergistic effects on enhancing apoptosis and suppressing extracellular matrix adhesion of HT-29 and SW620 cells. Indomethacin and NGX6 inhibit the proliferation and invasiveness of HT-29 and SW620 colon cancer cells by attenuating the WNT/ß-catenin signaling pathway.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

Anti-proliferative effect of chalcone derivatives through inactivation of NF-κB in human cancer cells

To investigate the anti-proliferative effect of NF-κB inhibitor, a series of analogs of (E)-1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (5a) were prepared and evaluated for their NF-κB inhibition and anti-proliferative activity against various human cancer cell lines. Compounds (E)-1-(2-(3,3-dimethylbutoxy)-6-hydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (5e) and (E)-4-(3-(2-(3,3-dimethylbutoxy)-6-hydroxyphenyl)-3-oxoprop-1-enyl)benzenesulfonamide (5p) showed good NF-κB inhibition as well as potent anti-proliferative activity. SAR studies showed that all the compounds with potent or moderate NF-κB inhibition displayed good anti-proliferative activity. All the analogs (5b–r) maintained a good correlation between their NF-κB inhibition and anti-proliferative activity though the extent is not directly proportional to each other.     

Methyl-β-cyclodextrin restores impaired autophagy flux in Niemann-Pick C1-deficient cells through activation of AMPK

The drug 2-hydroxypropyl-β-cyclodextrin (HPβCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-β-cyclodextrin (MβCD), a potent analog of HPβCD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through MβCD binding to its β-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK β1 or β2 subunit) expression and an AMPK inhibitor abolished MβCD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of MβCD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.     

Study on anti-proliferative effect of benzoxathiole derivatives through inactivation of NF-κB in human cancer cells

To investigate the anti-proliferative effect of a newly discovered NF-kB inhibitor, 6,6-dimethyl-2-(phenylimino)-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one (1a), a series of its analogs (1b-n) were prepared and evaluated for their NF-κB inhibition and anti-proliferative activity against various human cancer cell lines. Slight variation of hydrophobicity by replacement of dimethyl group of 1a at 6-position with bulky isopropyl group and introduction of para-fluoro substitution on 2-phenyl group showed good NF-κB inhibitory activity and anti-proliferative activity. However, excessive increase in hydrophobicity with 2,4,6-trichloro substituents on phenyl group resulted in the loss of both the activities. From the SAR results, 2-phenylimino-6,7-dihydrobenzo[d][1,3]oxathiol-4(5H)-one was identified as the lead scaffold for investigating new anticancer agent through inactivation of NF-κB.     

Thymosin β4 induces invasion and migration of human colorectal cancer cells through the ILK/AKT/β-catenin signaling pathway

Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and that Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.     

Downregulation of cPLA2γ expression inhibits EGF-induced chemotaxis of human breast cancer cells through Akt pathway

Phospholipids play an important role in mediating cell migration. In the present study, we investigated the role of cPLA₂γ in chemotaxis of human breast cancer cells. Inhibition of cPLA₂γ expression by small interference RNA severely inhibits EGF-induced chemotaxis in a dose-dependent manner in MDA-MB-231, MCF-7, T47D and ZR-75-30 cells. Furthermore, silencing cPLA₂γ expression also impaired directional migration, adhesion and invasion in MDA-MB-231 cells. In addition, we investigated the molecular mechanism by which cPLA₂γ regulated migration. Knockdown of cPLA₂γ suppressed the phosphorylation of Akt at both Thr308 and Ser473. Phosphorylation of PKCζ, downstream of Akt, was also dampened. Knockdown of cPLA₂γ also impaired the phosphorylation of integrin β1 and cofilin, key regulators of cell adhesion and actin polymerization, respectively. Taken together, our results suggest that cPLA₂γ plays an important role in cancer cell chemotaxis.     

Shear stress modulation of IL-1β-induced E-selectin expression in human endothelial cells

Background

Endothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated.

Methodology

We investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber.

Results and Conclusion

Naïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8–12 hr of continuous shear-cytokine activation contrary to the commonly observed 4–6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation.     

Induction of antiproliferative effect by diosgenin through activation of p53,release of apoptosis-inducing factor （AIF） and modulation of caspase-3 activity in different human cancer cells

Previously, we demonstrated that a plant steroid, diosgenin, altered cell cycle distribution and induced apoptosis in the human osteosarcoma 1547 cell line. The objective of this study was to investigate if the antiproliferative effect of diosgenin was similar for different human cancer cell lines such as laryngocarcinoma HEp-2 and melanoma M4Beu cells. Moreover, this work essentially focused on the mitochondrial pathway. We found that diosgenin had an important and similar antiproliferative effect on different types of cancer cells. In addition, our new results show that diosgenin-induced apoptosis is caspase-3 dependent with a fall of mitochondrial membrane potential, nuclear localization of AIF and poly (ADP-ribose) polymerase cleavage. Diosgenin treatment also induces p53 activation and cell cycle arrest in the different cell lines studied.     

(−)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells

(?)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1α, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3 h, EGCG caused a decrease in the expression of HIF-1α protein and VEGF, HIF-1α, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6 h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1α and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.     

CCL5 neutralization restricts cancer growth and potentiates the targeting of PDGFRβ in colorectal carcinoma

Increased CCL5 levels are markers of an unfavourable outcome in patients with melanoma, breast, cervical, prostate, gastric or pancreatic cancer. Here, we have assessed the role played by CCL5/CCR5 interactions in the development of colon cancer. To do so, we have examined a number of human colorectal carcinoma clinical specimens and found CCL5 and its receptors over-expressed within primary as well as liver and pulmonary metastases of patients compared to healthy tissues. In vitro, CCL5 increased the growth and migratory responses of colon cancer cells from both human and mouse origins. In addition, systemic treatment of mice with CCL5-directed antibodies reduced the extent of development of subcutaneous colon tumors, of liver metastases and of peritoneal carcinosis. Consistently, we found increased numbers of CD45-immunoreactive cells within the stroma of the remaining lesions as well as at the interface with the healthy tissue. In contrast, selective targeting of CCR5 through administration of TAK-779, a CCR5 antagonist, only partially compromised colon cancer progression. Furthermore, CCL5 neutralization rendered the tumors more sensitive to a PDGFRβ-directed strategy in mice, this combination regimen offering the greatest protection against liver metastases and suppressing macroscopic peritoneal carcinosis. Collectively, our data demonstrate the involvement of CCL5 in the pathogenesis of colorectal carcinoma and point to its potential value as a therapeutic target.     

APRIL depletion induces cell cycle arrest and apoptosis through blocking TGF-β1/ERK signaling pathway in human colorectal cancer cells

It is well documented that a proliferation-inducing ligand (APRIL), a newly found member of tumor necrosis factor superfamily, overexpressed in the majority of malignancies, plays a potential role in the occurrence and development of these tumors. Herein, we demonstrated that APRIL depletion by using RNA interference in human colorectal cancer (CRC) COLO 205 and SW480 cells resulted in cell proliferation inhibition and evoked cell cycle arrest in G0/G1 phase and apoptosis, coupled with decrease in CDK2, Cyclin D1, Bcl-2 expression and an increase of p21 and Bax expression. In addition, the decreased expression of transforming growth factor-β1 (TGF-β1) and p-ERK was also showed in siRNA-APRIL transfected COLO 205 and SW480 cells, whereas the protein expression levels of Smad2/3, p-Smad2/3, and ERK were not significantly changed. Taken together, our results indicate that APRIL depletion induces cell cycle arrest and apoptosis partly through blocking noncanonical TGF-β1/ERK, rather than canonical TGF-β1/Smad2/3, signaling pathway in CRC cells. Moreover, our study highlights APRIL as a potential molecular target for the therapy of CRC.