CELLFOOD? induces apoptosis in human mesothelioma and colorectal cancer cells by modulating p53, c-myc and pAkt signaling pathways

Background

CELLFOOD™ (CF) is a nutraceutical non-addictive, non-invasive, and completely non-toxic unique proprietary colloidal-ionic formula. Little is known about its effect on cancer cells in solid tumors. The aim of this study was to evaluate the effect that CF has on different cancer cell lines and the mechanism by which the nutraceutical works.

Methods

The effect of CF on HFF (normal fibroblasts), Met5A (mesothelium), MSTO-211H, NCI-2452, Ist-Mes1, MPP89, Ist-Mes2 (mesothelioma), M14 (melanoma), H1650, H1975 (lung cancer), SKRB3 (breast cancer), and HCT-116 (colorectal cancer) cell growth was tested by cell proliferation and clonogenic assay. Among all of them, MSTO-211 and HCT-116 were analyzed for cell cycle by flow cytometry and western blot.

Results

All human cancer lines were suppressed on cell growth upon 1:200 CF treatment for 24 and 48 hours. Death was not observed in HFF and Met5A cell lines. Cell cycle analysis showed an increased sub-G1 with reduction of G1 in MSTO-211 and a cell cycle arrest of in G1 in HCT116. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for both cell lines. Increased expression levels of p53, p21, and p27, downregulation of c-myc and Bcl-2, and inhibition of Akt activation were also found in CF-treated MSTO-211 and HCT-116 cells.

Conclusions

These findings ascertained an interaction between p53, c-myc, p21, p27, Bcl-2, PI3K/Akt pathway, and CF-induced apoptosis in MSTO-211H and HCT-116 cells, suggesting that CF acts as an important regulator of cell growth in human cancer cell lines. CF could be a useful nutraceutical intervention for prevention in colon cancer and mesothelioma.     

IL-32α suppresses colorectal cancer development via TNFR1-mediated death signaling

Inflammation is associated with cancer-prone microenvironment, leading to cancer. IL-32 is expressed in chronic inflammation-linked human cancers. To investigate IL-32α in inflammation-linked colorectal carcinogenesis, we generated a strain of mice, expressing IL-32 (IL-32α-Tg). In IL-32α-Tg mice, azoxymethane (AOM)-induced colon cancer incidence was decreased, whereas expression of TNFR1 and TNFR1-medicated apoptosis was increased. Also, IL-32α increased ROS production to induce prolonged JNK activation. In colon cancer patients, IL-32α and TNFR1 were increased. These findings indicate that IL-32α suppressed colon cancer development by promoting the death signaling of TNFR1.     

α-TEA induces apoptosis of human breast cancer cells via activation of TRAIL/DR5 death receptor pathway

Vitamin E derivative RRR‐α‐tocopherol ether‐linked acetic acid analog (α‐TEA) induces apoptosis in MCF‐7 and HCC‐1954 human breast cancer cells in a dose‐ and time‐dependent manner. α‐TEA induces increased levels of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and death receptor‐5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE‐like inhibitory protein (c‐FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c‐FLIP (L), caspase‐8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase‐9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α‐TEA to enhance DR5 protein levels, downregulate c‐FLIP(L) protein levels and induce apoptosis. Combination of α‐TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c‐FLIP (L) protein levels. siRNA knockdown of c‐FLIP produces a low level of spontaneous apoptosis and enhances α‐TEA‐ and TRAIL‐induced apoptosis. Taken together, these studies show that α‐TEA induces TRAIL/DR5 mitochondria‐dependent apoptosis in human breast cancer cells, and that TRAIL/DR5‐dependent increases in DR5 and decreases in c‐FLIP expression are triggered by TRAIL or α‐TEA treatments. © 2010 Wiley‐Liss, Inc.     

c-Src dependency of NSAID-induced effects on NF-κB-mediated apoptosis in colorectal cancer cells

Long-term aspirin or related non-steroidal anti-inflammatory drugs (NSAIDs) ingestion can protect against colorectal cancer (CRC). NSAIDs have a pro-apoptotic activity and we have shown that stimulation of the nuclear factor-kappaB (NF-κB) pathway is a key component of this pro-apoptotic effect. However, the upstream pathways have yet to be fully elucidated. Here, we demonstrate that aspirin activates the c-Src tyrosine kinase pathway in CRC cells. We show that c-Src activation occurs in a time- and dose-dependent manner, preceding aspirin-mediated degradation of IκBα, nuclear/nucleolar translocation of NF-κB/RelA and induction of apoptosis. Furthermore, inhibition of c-Src activity, by chemical inhibition or expression of a kinase dead form of the protein abrogates aspirin-mediated degradation of IκBα, nuclear translocation of RelA and apoptosis, suggesting a causal link. Expression of constitutively active c-Src mimics aspirin-induced stimulation of the NF-κB pathway. The NSAIDs sulindac, sulindac sulphone and indomethacin all similarly activate a c-Src-dependent NF-κB and apoptotic response. These data provide compelling evidence that c-Src is an upstream mediator of aspirin/NSAID effects on NF-κB signalling and apoptosis in CRC cells and have relevance to the development of future chemotherapeutic/chemopreventative agents.     

Hsp27 overexpression inhibits doxorubicin–induced apoptosis in human breast cancer cells

Previously we demonstrated that heat shock protein 27 (hsp27) overexpression confers resistance to the chemotherapeutic agent doxorubicin in MDA–MB–231 breast cancer cells. Since induction of apoptosis is one underlying mechanism of chemotherapeutic drug action, we investigated the effect of hsp27 overexpression on doxorubicin–induced apoptosis, finding that hsp27 protects MDA–MB–231 cells from apoptosis. We also examined expression of the doxorubicin target, topoisomerase II (topo II), in control and hsp27–overexpressing stable transfectants, as topo II expression is important for both drug sensitivity and the initiation of apoptosis by doxorubicin. The relative levels of both topo II and were higher in the controls than the hsp27–overexpressing clones, suggesting that the apoptotic protective effect of hsp27 overexpression in MDA–MB–231 cells is associated with altered topo II expression.abstract     

The complex interplay between autophagy and NF-κB signaling pathways in cancer cells

Tight regulation of both the NF-κB pathway and the autophagy process is necessary for maintenance of cellular homeostasis. Deregulation of both pathways is frequently observed in cancer cells and is associated with tumorigenesis and tumor cell resistance to cancer therapies. Autophagy is involved in several cellular functions regulated by NF-κB including cell survival, differentiation, senescence, inflammation, and immunity. On a molecular level, autophagy and NF-κB share common upstream signals and regulators and can control each other through positive or negative feedback loops, thus ensuring homeostatic responses. Here, we summarize and discuss the most recent discoveries that shed new light on the complex interplay between autophagy and NF-κB signaling pathways; this certainly has functional relevance in tumorigenesis and tumor responses to therapy.     

5-Aminosalicylic acid inhibits TGF-β1 signalling in colorectal cancer cells

The transforming growth factor-β (TGF-β) pathway is an important pathway in the initiation and progression of colorectal cancer. We aimed to determine the effects of 5-aminosalicylic acid (5-ASA) on TGF-β signalling in colorectal cancer cells in vitro. 5-ASA inhibited TGF-β1 signalling in HCT116 cells and colonic fibroblasts, as judged by a TGF-β-specific reporter gene assay, plasminogen activator inhibitor-1 mRNA and protein levels, fibroblast trans-differentiation, Smad3 phosphorylation and nuclear translocation. We conclude that 5-ASA inhibits TGF-β1 signalling in colorectal cancer cells, and might be a potent adjuvant therapeutic drug, interfering with aberrant TGF-β signalling in colorectal cancer.     

Xanthine oxidoreductase – Clinical significance in colorectal cancer and in vitro expression of the protein in human colon cancer cells

Xanthine oxidoreductase (XOR) is a key enzyme in degradation of DNA and RNA, and has previously been shown to be decreased in aggressive breast and gastric cancer. In this study, XOR expression was assessed in tissue microarray specimens of 478 patients with colorectal cancer and related to clinical parameters. In addition, we performed in vitro studies of XOR activity, protein and mRNA in colon cancer cells (Caco-2). Results from the tissue expression analyses show that XOR was decreased in 62% and undetectable in 22% of the tumours as compared to normal tissue. Loss of XOR was associated with poor grade of differentiation (p = 0.006) and advanced Dukes stage (p = 0.03). In multivariate survival analysis, XOR was a prognostic factor (p = 0.008), independent of Dukes stage, histological grade, age and tumour location. The in vitro analyses show that XOR is not measurable in undifferentiated Caco-2 cells, but appears and increases with differentiation. We conclude that XOR expression is associated with histological grade of differentiation and extent of disease in colorectal cancer, and it provides significant prognostic information independently of established factors.     

STAT3 regulates 5-Fu resistance in human colorectal cancer cells by promoting Mcl-1–dependent cytoprotective autophagy

Chemoresistance to 5-fluorouracil (5-Fu)-based chemotherapy is one of the primary reasons for the failure of colorectal cancer (CRC) management. STAT3 can mediate tumor drug resistance through a variety of diverse mechanisms. Nonetheless, the underlying mechanisms of STAT3-induced 5-Fu resistance in CRC are still poorly understood. Here, we aimed to investigate the potential mechanism(s) of STAT3-induced 5-Fu resistance in CRC. Quantitative RT-PCR and Western blot were used to test the expression of STAT3 and Mcl-1 in chemosensitive and chemoresistant CRC tissues and cell lines. After overexpression or knockdown of STAT3 or Mcl-1, and/or treatment with or without 5-Fu or chloroquine (CQ), we tested cell viability, inhibitory concentration 50% (IC₅₀) value of 5-FU, cell apoptosis, proliferation, migration, and autophagy. STAT3 and Mcl-1 were significantly upregulated in the chemoresistant CRC tissues and cell lines, and STAT3 positively regulated Mcl-1. Functional studies demonstrated that STAT3 promoted 5-Fu resistance in CRC. Mechanistically, STAT3 triggered autophagy via Mcl-1 to induce cancer chemoresistance. Our results show that STAT3 regulates 5-Fu resistance in CRC by promoting Mcl-1–dependent cytoprotective autophagy. Our results provide a novel role of STAT3 and may offer a new approach for managing CRC 5-Fu resistance.     

Bid integrates intrinsic and extrinsic signaling in apoptosis induced by α-tocopheryl succinate in human gastric carcinoma cells

The underlying mechanisms of α-tocopheryl succinate (α-TOS)-mediated apoptosis are not understood in detail, although the redox-silent vitamin E analog is a potent apoptogen and anti-cancer agent. Our previous studies showed the important role of Fas signaling in apoptosis induced by the mitocan. The objective of the present study was to investigate whether apoptosis triggered by α-TOS in gastric carcinomas cells involves both mitochondria- and death receptor-dependent pathways. α-TOS induced apoptosis and mitochondrial permeability transition in a concentration- and time-dependent manner. As a consequence, cytochrome c and the apoptosis-inducing factor were released and caspases were activated. Bax was translocated from the cytosol to mitochondria and Bid was cleaved into its truncated form, tBid. Knocking down Bid by RNAi and Fas antisense oligodeoxynucleotides resulted in a decreased release and cleavage. The results imply that Bid may serve as a critical integrating factor of the death receptor and mitochondrial pathway in α-TOS-mediated apoptosis.     

Breast cancer cells can switch between estrogen receptor α and ErbB signaling and combined treatment against both signaling pathways postpones development of resistance

The majority of breast cancers are estrogen responsive, but upon progression of disease other growth promoting pathways are activated, e.g., the ErbB receptor system. The present study focuses on resistance to the pure estrogen antagonist fulvestrant and strategies to treat resistant cells or even circumvent development of resistance. Limited effects were observed when targeting EGFR and ErbB2 with the monoclonal antibodies cetuximab, trastuzumab, and pertuzumab, whereas the pan-ErbB inhibitor CI-1033 selectively inhibited growth of fulvestrant resistant cell lines. CI-1033 inhibited Erk but not Akt signaling, which as well as Erk is important for antiestrogen resistant cell growth. Accordingly, combination therapy with CI-1033 and the Akt inhibitor SH-6 or the Protein Kinase C inhibitor RO-32-0432 was applied and found superior to single agent treatment. Further, the resistant cell lines were more sensitive to CI-1033 treatment when grown in the presence of fulvestrant, as withdrawal of fulvestrant restored signaling through the estrogen receptor α (ERα), partly overcoming the growth inhibitory effects of CI-1033. Thus, the resistant cells could switch between ERα and ErbB signaling for growth promotion. Although parental MCF-7 cell growth primarily depends on ERα signaling, a heregulin-1β induced switch to ErbB signaling rescued MCF-7 cells from the growth inhibition exerted by fulvestrant-mediated blockade of ERα signaling. This interplay between ERα and ErbB signaling could be abrogated by combined therapy targeting both receptor systems. Thus, the present study indicates that upon development of antiestrogen resistance, antiestrogen treatment should be continued in combination with signal transduction inhibitors. Further, upfront combination of endocrine therapy with pan-ErbB inhibition may postpone or even prevent development of treatment resistance.     

Neurotensin signaling induces intracellular alkalinization and interleukin‐8 expression in human pancreatic cancer cells

Pancreatic adenocarcinomas express neurotensin receptors in up to 90% of cases, however, their role in tumor biology and as a drug target is not clear. In the present study, a stable neurotensin (NT) analog induced intracellular calcium release and intracellular alkalinization in BxPC‐3 and PANC‐1 pancreatic cancer cells that was abolished by inhibitors of NT receptor (NTR) and sodium–proton exchanger 1 (NHE1), amiloride and SR 142948, respectively. Activation of NHE1 involved increased phosphorylation of dimethylfumarate‐sensitive mitogen‐ and stress‐activated kinase 1/2 (MSK1/2). NTR signaling appears to promote a metastatic phenotype in pancreatic cancer cells by induction of localized extracellular acidification in normoxic cells, preceeding acidosis induced by hypoxia and switch to glycolysis in addition to increased expression of interleukin‐8 (IL‐8).     

β-adrenoceptor signaling and its control of cell replication in MDA-MB-231 human breast cancer cells

MDA-MB-231 human breast cancer cells express high -adrenoceptor levels, predominantly the ₂ subtype. Receptor stimulation by isoproterenol evoked immediate reductions in DNA synthesis which were blocked completely by propranolol and were of the same magnitude as effects elicited by high concentrations of 8-Br-cAMP. Isoproterenol-induced inhibition of DNA synthesis was maintained throughout several days of exposure, resulting in a decrement in total cell number, and the effects were augmented by cotreatment with dexamethason; an even greater effect was seen when cAMP breakdown was inhibited by theophylline, with or without addition of isoproterenol. Despite the persistent effect of isoproterenol, receptor downregulation was evident with as little as 1 h of treatment, and over 90% of the receptors were lost within 24 h. Receptor downregulation was paralleled by homologous desensitization of the adenylyl cyclase response to -adrenoceptor stimulation. Dexamethasone augmented the effects of isoproterenol on DNA synthesis but did not prevent receptor downregulation or desensitization. These results indicate that -adrenoceptors are effectively linked, through cAMP, to the termination of cell replication in MDA-MB-231 human breast cancer cells, and that activation of only a small number of receptors is sufficient for a maximal effect. Novel pharmacologic strategies that focus on cell surface receptors operating through adenylyl cyclase may offer opportunities to combat cancers that are unresponsive to hormonal agents, or that have developed multidrug resistance.     

MicroRNA-520/373 family functions as a tumor suppressor in estrogen receptor negative breast cancer by targeting NF-κB and TGF-β signaling pathways

MicroRNAs (miRNAs) as modulators of gene expression have been described to display both tumor-promoting and tumor-suppressive functions. Although their role has been studied in different tumor types, little is known about how they regulate nuclear factor κB (NF-κB) signaling in breast cancer. Here, we performed an unbiased whole genome miRNA (miRome) screen to identify novel modulators of NF-κB pathway in breast cancer. The screen identified 13 miRNA families whose members induced consistent effects on NF-κB activity. Among those, the miR-520/373 family inhibited NF-κB signaling through direct targeting of RELA and thus strongly reduced expression and secretion of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. With a combination of in vitro and in vivo approaches, we propose a metastasis-suppressive role of miR-520/373 family. miR-520c and miR-373 abrogated both in vitro cell invasion and in vivo intravasation of highly invasive MDA-MB-231 cells. However, knockdown of RELA did not affect their metastatic ability. mRNA profiling of MDA-MB-231 cells on overexpression of miR-520/373 members revealed a strong downregulation of transforming growth factor-β (TGF-β) signaling. Mechanistically, the metastasis-suppressive role of miR-520/373 can be attributed to direct suppression of TGFBR2, as the silencing of TGFBR2 phenocopied the effects of miR-520/373 overexpression on suppression of Smad-dependent expression of the metastasis-promoting genes parathyroid hormone-related protein, plasminogen activator inhibitor-1 and angiopoietin-like 4 as well as tumor cell invasion, in vitro and in vivo. A negative correlation between miR-520c and TGFBR2 expression was observed in estrogen receptor negative (ER(-)) breast cancer patients but not in the ER positive (ER(+)) subtype. Remarkably, decreased expression of miR-520c correlated with lymph node metastasis specifically in ER(-) tumors. Taken together, our findings reveal that miR-520/373 family has a tumor-suppressive role in ER(-) breast cancer by acting as a link between the NF-κB and TGF-β pathways and may thus contribute to the interplay of tumor progression, metastasis and inflammation.     

Anti-apoptotic effect of claudin-1 on TNF-α-induced apoptosis in human breast cancer MCF-7 cells

Accumulating evidence reveals that aberrant expression of claudins manifests in various tumors; however, their biological functions are poorly understood. Here, we report on the elevated expression of claudin-1 in human breast cancer MCF-7 cells under tumor necrosis factor (TNF)-?? treatment. Interestingly, the increased expression of claudin-1 contributes to an anti-apoptotic role in TNF-??-induced apoptosis. In line with this, upon TNF-?? stimulus, downregulation of claudin-1 by siRNA knockdown results in a significant increase in cleavage of caspase-8 and poly (ADP-ribose) polymerase, a decrease of cyclinD1 expression, and DNA fragmentation. Consistently, TdT-mediated dUTP nick end labeling assay also shows that loss of claudin-1 increases the susceptibility of MCF-7 cells to TNF-??-induced apoptosis. However, there is no obvious effect on the expression of Bax and p53 after the treatment aforementioned. In addition, TNF-?? increases the amount of claudin-1 and the cytoplasmic accumulation of ??-catenin, while claudin-1 siRNA increases the amount of ??-catenin in the cell membrane as well as the amount of E-cadherin in the cytoplasm. In conclusion, our data reveal a novel role of claudin-1 in regulating apoptosis in MCF-7 cells.     

WISP-2 in human gastric cancer and its potential metastatic suppressor role in gastric cancer cells mediated by JNK and PLC-γ pathways

Background:

It has recently been shown that WISP proteins (Wnt-inducted secreted proteins), a group of intra- and extra-cellular regulatory proteins, have been implicated in the initiation and progression of a variety of tumour types including colorectal and breast cancer. However, the role of WISP proteins in gastric cancer (GC) cells and their clinical implications have not yet been elucidated.

Methods:

The expression of WISP molecules in a cohort of GC patients was analysed using real-time quantitative PCR and immunohistochemistry. The expression of a panel of recognised epithelial–mesenchymal transition (EMT) markers was quantified using Q-PCR in paired tumour and normal tissues. WISP-2 knockdown (kd) sublines using ribozyme transgenes were created in the GC cell lines AGS and HGC27. Subsequently, several biological functions, including cell growth, adhesion, migration and invasion, were studied. Potential pathways for the interaction of EMT, extracellular matrix and MMP were evaluated.

Results:

Overexpression of WISP-2 was detected in GC and significantly correlated with early tumour node-metastasis staging, differentiation status and positively correlated with overall survival and disease-free survival of the patients. WISP-2 expression was inversely correlated with that of Twist and Slug in paired samples. Kd of WISP-2 expression promoted the proliferation, migration and invasion of GC cells. WISP-2 suppressed GC cell metastasis through reversing EMT and suppressing the expression and activity of MMP9 and MMP2 via JNK and ERK. Cell motility analysis indicated that WISP-2 kd contributed to GC cells'' motility and can be attenuated by PLC-γ and JNK small inhibitors.

Conclusions:

Increased expression of WISP-2 in GC is positively correlated with favourable clinical features and the survival of patients with GC and is a negative regulator of growth, migration and invasion in GC cells. These findings suggest that WISP-2 is a potential tumour suppressor in GC.