Combination of erlotinib and EGCG induces apoptosis of head and neck cancers through posttranscriptional regulation of Bim and Bcl-2.

Combinatorial approaches using two or more compounds are gaining increasing attention for cancer therapy. We have previously reported that the combination of the EGFR-TKI erlotinib and epigallocatechin-3-gallate (EGCG) exhibited synergistic chemopreventive effects in head and neck cancers by inducing the expression of Bim, p21, p27, and by inhibiting the phosphorylation of ERK and AKT and expression of Bcl-2. In the current study, we further investigated the mechanism of regulation of Bim, Bcl-2, p21 and p27, and their role in apoptosis. shRNA-mediated silencing of Bim significantly inhibited apoptosis induced by the combination of erlotinib and EGCG (p = 0.005). On the other hand, overexpression of Bcl-2 markedly protected cells from apoptosis (p = 0.003), whereas overexpression of constitutively active AKT only minimally protected cells from apoptosis induced by the combination of the two compounds. Analysis of mRNA expression by RT-PCR revealed that erlotinib, EGCG and their combination had no significant effects on the mRNA expression of Bim, p21, p27 or Bcl-2 suggesting the post-transcriptional regulation of these molecules. Furthermore, we found that erlotinib or the combination of EGCG and erlotinib inhibited the phosphorylation of Bim and stabilized Bim after inhibition of protein translation by cycloheximide. Taken together, our results strongly suggest that the combination of erlotinib and EGCG induces apoptosis of SCCHN cells by regulating Bim and Bcl-2 at the posttranscriptional level.

N-(phosphonacetyl)-L-aspartate induces TAp73-dependent apoptosis by modulating multiple Bcl-2 proteins: potential for cancer therapy.

p53 is essential for the cellular responses to DNA damage that help to maintain genomic stability. However, the great majority of human cancers undergo disruption of the p53-network. Identification and characterization of molecular components important in both p53-dependent and -independent apoptosis might be useful in developing novel therapies for cancers. In the complete absence of p53, cells treated with N-(phosphonacetyl)-L-aspartate (PALA) continue to synthesize DNA slowly and eventually progress through S-phase, suffering severe DNA damage that in turn triggers apoptosis, whereas cells with functional p53 undergo growth arrest. In this study, we investigated apoptotic signaling in response to PALA and the role of p53 expression in this pathway. We found that treatment of cells lacking p53 with PALA induced TAp73, Noxa and Bim and inactivation of these proteins with dominant-negative plasmids or small interfering RNAs significantly inhibited apoptosis, suggesting that PALA-induced apoptosis was mediated via TAp73-dependent expression of Noxa and Bim. However, PALA treatment inhibited the expression of ΔNp73 only in cells lacking p53 but not in cells expressing p53. In addition, PALA treatment inhibited Bcl-2, and overexpression of Bcl-2 significantly inhibited PALA-induced apoptosis. Moreover, expression of p53 in these cells protected them from PALA-induced apoptosis by activating p21, sustaining the expression of ΔNp73 and inhibiting the induction of Noxa and Bim. Taken together, our study identifies novel but opposing roles for the p53 and TAp73 in the induction of Noxa and Bim and regulation of apoptosis. Our data will help to develop strategies to eliminate cancer cells lacking p53 while protecting normal cells with wild-type p53.

Cancer prevention with natural compounds.

Botanical and nutritional compounds have been used for the treatment of cancer throughout history. These compounds also may be useful in the prevention of cancer. Population studies suggest that a reduced risk of cancer is associated with high consumption of vegetables and fruits. Thus, the cancer chemopreventive potential of naturally occurring phytochemicals is of great interest. There are numerous reports of cancer chemopreventive activity of dietary botanicals, including cruciferous vegetables such as cabbage and broccoli, Allium vegetables such as garlic and onion, green tea, Citrus fruits, soybeans, tomatoes, berries, and ginger, as well as medicinal plants. Several lead compounds, such as genistein (from soybeans), lycopene (from tomatoes), brassinin (from cruciferous vegetables), sulforaphane (from asparagus), indole-3-carbinol (from broccoli), and resveratrol (from grapes and peanuts) are in preclinical or clinical trials for cancer chemoprevention. Phytochemicals have great potential in cancer prevention because of their safety, low cost, and oral bioavailability. In this review, we discuss potential natural cancer preventive compounds and their mechanisms of action.

p53-Dependent p21-mediated growth arrest pre-empts and protects HCT116 cells from PUMA-mediated apoptosis induced by EGCG.

The tumor suppressor protein p53 plays a key role in regulation of negative cellular growth in response to EGCG. To further explore the role of p53 signaling and elucidate the molecular mechanism, we employed colon cancer HCT116 cell line and its derivatives in which a specific transcriptional target of p53 is knocked down by homologous recombination. Cells expressing p53 and p21 accumulate in G1 upon treatment with EGCG. In contrast, same cells lacking p21 traverse through the cell cycle and eventually undergo apoptosis as revealed by TUNEL staining. Treatment with EGCG leads to induction of p53, p21 and PUMA in p21 wild-type, and p53 and PUMA in p21(-/-) cells. Ablation of p53 by RNAi protects p21(-/-) cells, thus indicating a p53-dependent apoptosis by EGCG. Furthermore, analysis of cells lacking PUMA or Bax with or without p21 but with p53 reveals that all the cells expressing p53 and p21 survived after EGCG treatment. More interestingly, cells lacking both PUMA and p21 survived ECGC treatment whereas those lacking p21 and Bax did not. Taken together, our results present a novel concept wherein p21-dependent growth arrest pre-empts and protects cells from otherwise, in its absence, apoptosis which is mediated by activation of pro-apoptotic protein PUMA. Furthermore, we find that p53-dependent activation of PUMA in response to EGCG directly leads to apoptosis with out requiring Bax as is the case in response to agents that induce DNA damage. p21, thus can be used as a molecular switch for therapeutic intervention of colon cancer.

A role for SHPS-1/SIRPalpha in Concanavalin A-dependent production of MMP-9.

SHPS-1/SIRPalpha1 is a transmembrane glycoprotein that belongs to the immunoglobulin (Ig) super family. In the present study, we show that SHPS-1 strongly associates with Concanavalin A (Con A), a plant lectin obtained from jack beans. Further studies with SHPS-1 mutants reveal that the extracellular domain of SHPS-1 containing the Ig sequence is responsible for its association with Con A. Con A treatment induces cross-linking and multimerization of the SHPS-1 protein in the plasma membrane, accompanied by its tyrosine phosphorylation and recruitment of SHP-2. In contrast, Ricinus communis agglutinin (RCA), another lectin obtained from castor bean, does not bind or activate tyrosine phosphorylation of SHPS-1. Moreover, Con A activates Akt in a SHP-2-dependent manner. Treatment of mouse embryonic fibroblasts (MEFs) with Con A induces secretion of matrix metalloproteinase (MMP)-9, a phenomenon that is inhibited in cells expressing YF mutant of SHPS-1, a dominant negative form of Akt or in cells pre-treated with an Akt inhibitor, LY294002 or extracellular-signal regulated kinase (Erk) inhibitor, U0126. In addition, expression of the YF mutant of SHPS-1 inhibits Con A-dependent activation of Akt and Erk kinases. Taken together, our results suggest that SHPS-1 is a receptor for Con A that mediates Con A-dependent MMP-9 secretion through SHP-2-promoted activation of both Akt and Erk pathways.

DNA replication licensing factor minichromosome maintenance deficient 5 rescues p53-mediated growth arrest.

Inactivation of p53 signaling by mutation of p53 itself or abrogation of its normal function by other transfactors, such as MDM2, is a key event in the development of most human cancers. To identify novel regulators of p53, we have used a phenotype-based selection in which a total cDNA library in a retroviral vector has been introduced into TR9-7ER cells, which arrest when p53 is expressed from a tetracycline-regulated promoter. We have isolated several clones derived from cells that are not growth-arrested when p53 is overexpressed. In one clone, the levels of p53, p21, and MDM2 are comparable with those in TR9-7ER cells and, therefore, the abrogation of growth arrest by an exogenous cDNA is likely to be distal to p21. Using reverse transcription-PCR, we were able to isolate a cDNA of approximately 2.2 kb, which was found to have 99% identity to the nucleotides between about 80 and 2,288 of the open reading frame of a gene encoding DNA replication licensing factor. It encodes complete peptide of 734 residues of this protein also called minichromosome maintenance deficient 5 (MCM5) or cell division cycle 46 (Saccharomyces cerevisiae). Northern and Western blot analyses revealed that the expression of MCM5 and its transcriptional regulator, E2F1, is negatively regulated by p53. When MCM5 cDNA was reintroduced into fresh TR9-7ER cells, numerous colonies that grow in the absence of tetracycline were formed. This novel observation establishes a role for MCM5 in negating the growth arrest function of p53.

SHP-2-Erk signaling regulates concanavalin A-dependent production of TIMP-2.

To search for the signaling critical for the production of tissue inhibitor of metalloproteinase-2 (TIMP-2), we investigated the role of SHP-2 in TIMP-2 production with Concanavalin A (Con A)-treated cells. In wild-type fibroblasts, Con A-treatment dramatically activated TIMP-2 production. In contrast, production of TIMP-2 in response to Con A-treatment was severely impaired in cells expressing mutant SHP-2 whose 65 amino acids in the SH2-N domain were deleted. Con A-treatment activated dual signaling pathways, Erk and p38, in a SHP-2-dependent manner. Pretreatment of wild-type cells with U0126, a potent inhibitor of MEK1, significantly inhibited the production of TIMP-2, whereas SB203580, a specific inhibitor for p38, could not. Finally, expression of exogenous wild-type SHP-2 in SHP-2 mutant cells clearly rescued Erk activation and TIMP-2 production in response to Con A-treatment. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive modulator for the production of TIMP-2 via MEK1-Erk signaling in fibroblasts.

SH2 domain containing protein tyrosine phosphatase 2 regulates concanavalin A-dependent secretion and activation of matrix metalloproteinase 2 via the extracellular signal-regulated kinase and p38 pathways.

We investigated the role of SH2 domain containing protein tyrosine phosphatase (SHP) 2 in Concanavalin A (Con A) -dependent signaling that leads to the augmented secretion and activation of matrix metalloproteinase (MMP) 2. In cells expressing mutant SHP-2 in which 65 amino acids in the SH2-N domain were deleted, we found that production, secretion, and proteolytic activation of MMP-2 in response to Con A treatment was severely impaired. Under Con A stimulation, complex formation of SHP-2 with SOS-1 and Grb-2 together with the activation of Ras signaling was clearly observed in wild-type cells, but not in SHP-2 mutant cells. In wild-type cells, Con A-treatment activated dual signaling pathways, extracellular signal-regulated kinase (Erk) and p38, in a Ras-dependent manner, whereas Con A-dependent activation of these signaling pathways was absent in SHP-2 mutant cells. In addition, pretreatment of wild-type cells with U0126, a potent inhibitor for mitogen-activated protein/ERK kinase 1, or with SB203580, a specific inhibitor for p38, significantly inhibited the Con A-dependent secretion and activation of MMP-2. However, overexpression of active mitogen-activated protein/ERK kinase 1 in SHP-2 mutant cells could not induce clear activation of MMP-2 secretion, although these cells responded well to the Con A treatment in a p38-dependent manner. Finally, reintroduction of wild-type SHP-2 into SHP-2 mutant cells rescued Erk and p38 activation, and also MMP-2 secretion, whereas dominant-negative SHP-2 could block the Con A-dependent activation of Erk and p38. Taken together, our results strongly suggest that SHP-2 plays a critical role as a positive mediator for Con A-dependent activation of MMP-2 secretion via Ras-Erk and Ras-p38 signalings.

Secretion of matrix metalloproteinase-9 by the proinflammatory cytokine, IL-1beta: a role for the dual signalling pathways, Akt and Erk.

BACKGROUND: Matrix metalloproteinases including MMP-9 mediate matrix destruction during chronic inflammatory diseases such as arthritis and atherosclerosis. MMP-9 up-regulation by inflammatory cytokines involve interactions between several transcription factors including activator protein-1 and NFkappaB. The upstream regulatory pathways are less well understood. RESULTS: To search for the mechanism of tissue destruction in the process of inflammatory disorders, we investigated the signalling pathway critical for the activation of MMP-9 expression and secretion by IL-1beta. Treatment of Balb 3T3 cells with IL-1beta activated MMP-9 transcription and subsequent secretion in a time- and dose-dependent manner. Concomitantly, IL-1beta treatment of cells activated phosphorylation of Akt, Erk and p38. Treatment of cells with either LY294002, a PI3K inhibitor, or expression of a dominant negative form of Akt drastically suppressed the IL-1beta-dependent secretion of MMP-9. Pretreatment of cells with a MEK1 inhibitor, U0126, also strongly inhibited IL-1beta-dependent secretion of MMP-9. In contrast, pre-treatment with a specific p38 kinase inhibitor, SB203580, had no effect on IL-1beta-dependent secretion of MMP-9. In addition, cells expressing constitutively active form of Akt or MEK1 showed no clear activation of MMP-9 secretion, whereas these cells responded well to IL-1beta treatment. However, co-transfection of cells with both active Akt and MEK1 was sufficient to induce MMP-9 secretion without stimulation with IL-1beta. CONCLUSION: Taken together, our results suggest that IL-1beta stimulation of cells activates MMP-9 secretion by the activation of the dual signalling pathways, the PI3K-Akt and MEK1-Erk and constitutive activation of these pathways were sufficient to induce MMP-9 secretion.

SHPS-1, a multifunctional transmembrane glycoprotein.

Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein (SIRP) family. The amino-terminal immunoglobulin-like domain of SHPS-1 is necessary for interaction with CD47, a ligand for SHPS-1, which plays an important role in cell-cell interaction. The intracellular region of SHPS-1, on the other hand, may act as a scaffold protein, binding to various adapter proteins. Interestingly, increasing evidence has shown that SHPS-1 is involved in various biological phenomena, including suppression of anchorage-independent cell growth, negative regulation of immune cells, self-recognition of red blood cells, mediation of macrophage multinucleation, skeletal muscle differentiation, entrainment of circadian clock, neuronal survival and synaptogenesis. Recent progress has been made in attributing these novel exciting functions. Here we discuss how this interesting molecule works and consider its true role in biology.