Anticancer cardamonin analogs suppress the activation of NF-kappaB pathway in lung cancer cells

NF-kappaB pathway has been proven to be critical to survival of lung cancer cells, and many natural products from plants were shown to inhibit the activation of this pathway. In this study, we investigated the effects of two cardamonin analogs, 4,4′-dihydroxylchalcone (DHC) and 4,4′-dihydroxy-2′-methoxychalcone (DHMC), on survival of lung cancer cells and the involved mechanisms. MTT assay revealed that the two compounds potently decreased the survival of immortalized and primary lung cancer cells. DHC and DHMC were able to induce apoptosis in A549 and NCI-H460 cells. Immunoblotting, immunofluorescent staining, and luciferase reporter further demonstrated that the two compounds suppressed the activation of NF-kappaB pathway in lung cancer cells. PMA-mediated NF-kappaB reactivation abrogated the effect of DHC and DHMC on lung cancer cells. DHC and DHMC were also shown to suppress the growth of A549 xenograft in mice. Collectively, we verified two cardamonin analogs as novel compounds suppressing NF-kappaB signaling for lung cancer therapy.     

Butyrate induces cell apoptosis through activation of JNK MAP kinase pathway in human colon cancer RKO cells

Butyrate has been shown to display anti-cancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism involved in butyrate-induced apoptosis is still not fully understood. Here, we investigated the cytotoxicity mechanism of butyrate in human colon cancer RKO cells. The results showed that butyrate induced a strong growth inhibitory effect against RKO cells. Butyrate also effectively induced apoptosis in RKO cells, which was characterized by DNA fragmentation, nuclear staining of DAPI, and the activation of caspase-9 and caspase-3. The expression of anti-apoptotic protein Bcl-2 decreased, whereas the apoptotic protein Bax increased in a dose-dependent manner during butyrate-induced apoptosis. Moreover, treatment of RKO cells with butyrate induced a sustained activation of the phosphorylation of c-jun N-terminal kinase (JNK) in a dose- and time-dependent manner, and the pharmacological inhibition of JNK MAPK by SP600125 significantly abolished the butyrate-induced apoptosis in RKO cells. These results suggest that butyrate acts on RKO cells via the JNK but not the p38 pathway. Butyrate triggered the caspase apoptotic pathway, indicated by an enhanced Bax-to-Bcl-2 expression ratio and caspase cascade reaction, which was blocked by SP600125. Taken together, our data indicate that butyrate induces apoptosis through JNK MAPK activation in colon cancer RKO cells.     

A new regulatory mechanism of NF-kappaB activation by I-kappaBbeta in cancer cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-κB) by depleting inhibitor of NF-κBα (I-κBα) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-κB activity with canonical pathway. The major I-κB proteins such as I-κBα and I-κBβ resemble each other in both primary sequence and tertiary structure. However, I-κBβ does not degrade fully, while I-κBα degrades immediately in response to most stimuli. We found that I-κBβ does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-κBβ is independent from TGase 2-mediated polymerization. Furthermore, increased I-κBβ expression reversed NF-κB activation in cancer cells, compensating for the loss of I-κBα via TGase 2 polymerization.     

CLAP, a novel caspase recruitment domain-containing protein in the tumor necrosis factor receptor pathway, regulates NF-kappaB activation and apoptosis.

Molecules that regulate NF-kappaB activation play critical roles in apoptosis and inflammation. We describe the cloning of the cellular homolog of the equine herpesvirus-2 protein E10 and show that both proteins regulate apoptosis and NF-kappaB activation. These proteins were found to contain N-terminal caspase-recruitment domains (CARDs) and novel C-terminal domains (CTDs) and were therefore named CLAPs (CARD-like apoptotic proteins). The cellular and viral CLAPs induce apoptosis downstream of caspase-8 by activating the Apaf-1-caspase-9 pathway and activate NF-kappaB by acting upstream of the NF-kappaB-inducing kinase, NIK, and the IkB kinase, IKKalpha. Deletion of either the CARD or the CTD domain inhibits both activities. The CARD domain was found to be important for homo- and heterodimerization of CLAPs. Substitution of the CARD domain with an inducible FKBP12 oligomerization domain produced a molecule that can induce NF-kappaB activation, suggesting that the CARD domain functions as an oligomerization domain, whereas the CTD domain functions as the effector domain in the NF-kappaB activation pathway. Expression of the CARD domain of human CLAP abrogates tumor necrosis factor-alpha-induced NF-kappaB activation, suggesting that cellular CLAP plays an essential role in this pathway of NF-kappaB activation.     

Inhibition of cytosolic phospholipase A2 mRNA expression: a novel mechanism for acetylsalicylic acid-mediated growth inhibition and apoptosis in colon cancer cells

Acetylsalicylic acid (ASA) has been confirmed to inhibit proliferation and to induce apoptosis in human colorectal cancer cells in vitro. However, the mechanism by which ASA exhibits antiproliferative and proapoptotic effects in cyclooxygenase 2 (COX-2)-negative cells remains to be further elucidated. In the present study, SW480, a COX-2-negative colon cancer cell line, was treated with various concentrations of ASA (0, 2.5, 5, and 10 mM). The antiproliferative and proapoptotic effects of ASA were confirmed by MTT assay, flow cytometry of propidium iodide (PI)-stained cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. After treatment with ASA, intracellular cyclic AMP (cAMP) levels were increased and the production of prostaglandin E2 (PGE2) was decreased. RT-PCR analysis revealed that treatment of ASA induced a concentration-dependent downregulation of cytosolic phospholipase A2 (cPLA2) mRNA expression in SW480 cells and also in two other colorectal cancer cell lines, Colo320 and HT-29 cells. Intracellular calcium levels were unaffected by ASA treatment. Our results indicate that the ASA-induced downregulation of cytosolic phospholipase A2 mRNA expression might be a novel mechanism for ASA-mediated growth inhibition and apoptosis in colon cancer cells.     

Targeting the extrinsic apoptosis signaling pathway for cancer therapy

The extrinsic apoptosis pathway is triggered by the binding of death ligands of the tumor necrosis factor (TNF) family to their appropriate death receptors (DRs) on the cell surface. One TNF family member, TNF-related apoptosis-inducing ligand (TRAIL or Apo2L), seems to preferentially cause apoptosis of transformed cells and can be systemically administered in the absence of severe toxicity. Therefore, there has been enthusiasm for the use of TRAIL or agonist antibodies to the TRAIL DR4 and DR5 in cancer therapy. Nonetheless, many cancer cells are very resistant to TRAIL apoptosis in vitro. Therefore, there is much interest in identifying compounds that can be combined with TRAIL to amplify its apoptotic effects. In this review, I will provide a brief overview of apoptosis signaling by TRAIL and discuss apoptosis-sensitizing agents, focusing mainly on the proteasome inhibitor bortezomib (VELCADE) and some novel sensitizers that we have recently identified. Alternative ways to administer TRAIL or DR agonist antibodies as therapeutic agents will also be described. Finally, I will discuss some of the gaps in our understanding of TRAIL apoptosis signaling and suggest some research directions that may provide additional information for optimizing the targeting of the extrinsic apoptosis pathway for future cancer therapy.     

Sulindac inhibits activation of the NF-kappaB pathway.

Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IkappaB kinase beta (IKKbeta) that is required to activate the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-kappaB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKbeta kinase activity. Concentrations of sulindac that inhibit IKKbeta activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-kappaB pathway.     

Baicalin induced colon cancer cells apoptosis through miR-217/DKK1-mediated inhibition of Wnt signaling pathway

Molecular Biology Reports - To analyze the anti-tumor mechanism of Baicalin in human colon cancer. The MTT assay and colony formation assay demonstrated that Baicalin treatment inhibits the...     

Induction of apoptosis in colon cancer cells by a novel topoisomerase I inhibitor TopIn

In virus-infected cells, viral RNA with non-self structural pattern is recognized by DExD/Hbox RNA helicase, RIG-I. Once RIG-I senses viral RNA, it triggers a signaling cascade, resulting in the activation of genes including type I interferon, which activates antiviral responses. Overexpression of N-terminal caspase activation and recruitment domain (CARD) is sufficient to activate signaling; however basal activity of full-length RIG-I is undetectable. The repressor domain (RD), initially identified as a.a. 735–925, is responsible for diminished basal activity; therefore, it is suggested that RIG-I is under auto-repression in uninfected cells and the repression is reversed upon its encounter with viral RNA. In this report, we further delimited RD to a.a. 747–801, which corresponds to a linker connecting the helicase and the C-terminal domain (CTD). Alanine substitutions of the conserved residues in the linker conferred constitutive activity to full-length RIG-I. We found that the constitutive active mutants do not exhibit ATPase activity, suggesting that ATPase is required for de-repression but not signaling itself. Furthermore, trypsin digestion of recombinant RIG-I revealed that the wild-type, but not linker mutant conforms to the trypsin-resistant structure, containing CARD and helicase domain. The result strongly suggests that the linker is responsible for maintaining RIG-I in a “closed” structure to minimize unwanted production of interferon in uninfected cells. These findings shed light on the structural regulation of RIG-I function.     

Polyethylene glycol induces apoptosis in HT-29 cells: potential mechanism for chemoprevention of colon cancer

Recent experimental evidence suggests that polyethylene glycol (PEG) is a highly effective chemopreventive agent against colon cancer; however, the mechanism(s) remain largely unexplored. To further elucidate this issue, we evaluated the effect of PEG on two human colon cancer cell lines. PEG treatment resulted in a dose- and time-dependent reduction in cell number without alteration in markers of cell proliferation. However, there was a dramatic and specific, concentration-dependent induction of apoptosis, with 50 mM PEG rendering approximately half the cells apoptotic. This corresponded with a 17-fold induction in the expression of the pro-apoptotic protein, prostate apoptosis response-4. Our data suggest that induction of apoptosis may be responsible, at least in part, for the ability of PEG to prevent experimental colon cancer.     

Apple procyanidins activate apoptotic signaling pathway in human colon adenocarcinoma cells by a lipid-raft independent mechanism

Flavonoids are polyphenolic compounds able to favour cholesterol-lipid-raft formation and control cell signaling pathways by targeting receptors at the cell surface. Procyanidins (Pcy) are oligomeric and polymeric flavonoids formed by catechins and epicatechins monomers trigger apoptosis by activating TRAIL-death receptors in human colon adenocarcinoma SW480 cells. Here, we investigated whether the apoptotic process triggered by apple procyanidins involving the up-regulation of TRAIL-death receptors DR4/DR5 at the cell surface was dependent on cell membrane lipid-raft formation. We report that Pcy-induced apoptosis was enhanced in presence of nystatin, a cholesterol-sequestering compound inhibiting lipid-raft formation, without changing DR4/DR5 receptor expression. Treatment of SW480 cells with TRAIL caused a 3.5-fold increased level of caveolin together with a 2- to 2.5-fold increased amount of DR4/DR5 proteins in lipid rafts. Pcy-treatment did not induce any alteration in the expression of DR4/DR5 proteins as well as of caveolin present in lipid-raft fractions. Pcy induced an activation of TRAIL-death receptor-mediated apoptosis by a mechanism independent of lipid-raft formation. These results highlight the potential of Pcy as a direct activator of TRAIL-death receptors in cell membrane even in the absence of lipid rafts.     

A novel signaling mechanism for soluble CD95 ligand. Synergy with anti-CD95 monoclonal antibodies for apoptosis and NF-kappaB nuclear translocation

Soluble CD95 (Fas) ligand (sFasL) is known to be deficient in transducing signals upon engagement with membrane Fas. Here we report that sFasL tranduces, in synergy with non-cytotoxic anti-Fas monoclonal antibody (mAb), signals for apoptosis and nuclear translocation of the NF-kappaB (p65/p50) heterodimer. Activation of the specific signaling pathways correlates with target Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein expression. Synergy with anti-Fas mAb was demonstrated with a trimeric unit of sFasL bearing a single binding site for Fas trimer. In contrast, membrane-bound FasL as expressed on cell-derived vesicles was fully competent in transducing Fas-mediated signals for apoptosis and NF-kappaB nuclear translocation. We propose a model in which the trimeric sFasL signaling requires target expression of a high focal density of Fas, which is induced by the signaling-incompetent anti-Fas mAb. Membrane-bound FasL induces powerful Fas-mediated signals because it possesses both Fas-focusing and signal-transducing functions.     

The interaction of butyrate with TNF-alpha during differentiation and apoptosis of colon epithelial cells: role of NF-kappaB activation

We demonstrated that TNF-alpha suppressed differentiation and potentiated cell death induced by butyrate (NaBt) in both adenocarcinoma HT-29 and fetal FHC human colon cells in vitro. Since TNF-alpha is a typical activator of NF-kappaB pathway, we studied the role of NF-kappaB activation in cell differentiation and death during the TNF-alpha and NaBt co-treatment. TNF-alpha induced rapid NF-kappaB activation in both HT-29 and FHC cell lines and this effect was differently modulated by NaBt in these two cell lines. In HT-29 cells, NaBt potentiated NF-kappaB activity induced by TNF-alpha after 4h treatment. However, this initial potentiation of NF-kappaB activity was not observed in FHC cells. During additional time of TNF-alpha and NaBt co-treatment, NaBt decreased the TNF-alpha-mediated NF-kappaB activity in both cell types. We also detected a different response of HT-29 and FHC cells after the pre-treatment with the NF-kappaB inhibitor parthenolide. Our results indicated that NaBt-mediated differentiation and apoptosis of colon epithelial cells can be modulated by TNF-alpha. Furthermore, we found significant differences in the mechanism of the NaBt and TNF-alpha co-treatment effects between cells of non-cancer and cancer origin, suggesting that the NF-kappaB pathway may be more effectively involved in these processes in cancer cells.