Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation.

There is increasing evidence to suggest that reduced folate status may be a causative factor in carcinogenesis, particularly colorectal carcinogenesis. Folate is essential for the synthesis of S-adenosylmethionine, the methyl donor required for all methylation reactions in the cell, including the methylation of DNA. Global DNA hypomethylation appears to be an early, and consistent, molecular event in carcinogenesis. We have examined the effects of folate depletion on human-derived cultured colon carcinoma cells using 2 novel modifications to the Comet (single cell gel electrophoresis) assay to detect global DNA hypomethylation and gene region–specific DNA hypomethylation. Colon cells cultured in folate-free medium for 14 d showed a significant increase in global DNA hypomethylation compared with cells grown in medium containing 3µmol/L folic acid. This was also true at a gene level, with folate-deprived cells showing significantly more DNA hypomethylation in the region of the p53 gene. In both cases, the effects of folate depletion were completely reversed by the reintroduction of folic acid to the cells. These results confirm that decreased folate levels are capable of inducing DNA hypomethylation in colon cells and particularly in the region of the p53 gene, suggesting that a more optimal folate status in vivo may normalize any DNA hypomethylation, offering potential protective effects against carcinogenesis. This study also introduces 2 novel functional biomarkers of DNA hypomethylation and demonstrates their suitability to detect folate depletion–induced molecular changes.

Alteration of GSH level, gene expression and cell transformation in NIH3T3 cells by chronic exposure to low dose of arsenic

It is well established that arsenic toxicity is postulated to be primarily due to the binding of As(III) to sulfhydryl-containing enzymes. However, the mechanism of carcinogenesis induced by arsenic is still unclear. The interaction of arsenic with GSH and related enzymes seems a very important issue regarding mechanism of arsenical induced toxicity or carcinogenesis. The purpose of this work is to investigate the effect of chronic exposure to low dose of As(III) on GSH level, gene expression and cell transformation in NIH3T3 cells. The results showed that long-term, low dose arsenic treatment makes 3T3 cell more resistant to acute arsenic treatment. There were morphology changes after long-term arsenic treatment. First, partially immortalized 3T3 cell became immortalized. In addition, the cells were doubling more quickly than the control cells and attained higher density than the control cells at confluence. Second, cells treated with 0.1 µ.M As(III) exhibited anchorage-independent growth. Arsenic could enhance GSH level at 0.5 -10 µM dose of arsenic in 24 h treatment and decrease it at 25 µM and above. In long-term treatment with low dose of arsenic, GSH levels were decreased. As(I1I) can increase both glutathione S-transferase (GST) and glutathione reductase (GR) activities at low dose (0.5-10 M), but decreased GST and GR activities at 25 M and higher dose of arsenic, while in long-term As(III) treatment, GST and GR activities are increased. Both long-term and short-term treatments with As(III) can induce GR gene expression. GPx mRNA levels were decreased both in acute and chronic arsenic treated cells. Chronic treatment with As(III) also decreased the p53 mRNA level. Taken together, our results suggest that As(III) can alter GST, GR enzyme activities as well as GSH level and related gene expression both in long-term and short-term treatment but in a different manner in different doses. Alteration of cellular GSH level by As(III) might play all important role in gene expression and arsenic induced cell transformation.

Early induction of chalpains in rotenone-mediated neuronal apoptosis

Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson's disease (PD), where neurons undergo apoptosis by caspase-dependent and/or caspase-independent pathways. Inhibition of calpains has recently been shown to attenuate neuronal apoptosis. This study aims to establish for the first time, the time-point of calpain activation with respect to the caspase activation and the possibility of cell cycle re-entry in rotenone-mediated cell death. Immunoblot results revealed calpain activation occurred at 5, 10 h prior to caspase-3 activation (at 15 h), suggesting calpain activation was an earlier cellular event compared to caspase activation in the rotenone-mediated apoptosis. In addition, an upregulation of phospho-p53 was observed at 21 h. However, no expression or upregulation of cell cycle regulatory proteins including cdc25a, cyclin-D1 and cyclin-D3 were observed, strongly suggesting that cell cycle re-entry did not occur. These findings provide new insights into the differential patterns of calpain and caspase activation that result from rotenone poisoning and which may be relevant to the therapeutic management of PD.

Microbial-based therapy of cancer: a new twist to age old practice

The use of bacteria in the regression of tumors has long been known. Various approaches for using bacteria in cancer therapy include the use of bacteria as sensitizing agents for chemotherapy, as delivery agents for cancer drugs and as agents for gene therapy. The tumor regression stimulated by infecting microorganisms has been attributed to activation of the immune system of the host. However, recent studies indicate that when tumor-harboring mice with defective immune systems are infected with certain microorganisms, the regression of the tumor is still observed, suggesting that there are other host factors contributing to the microbial associated regression of tumors. Since the use of live or attenuated bacteria for tumor regression has associated toxic effects, studies are in progress to identify a pure microbial metabolite or any component of the microbial cell that might have anti-cancer activity. It has now been demonstrated that a redox protein from Pseudomonas aeruginosa, a cupredoxin, can enter into human cancer cells and trigger the apoptotic cell death. In vivo, this cupredoxin can lead to the regression of tumor growth in immunodeficient mice harboring xenografted melanomas and breast cancer tumors without inducing significant toxic effects, suggesting that it has potential anti-cancer activity. This bacterial protein interacts with p53 and modulates mammalian cellular activity. Hence, it could potentially be used as an anti-cancer agent for solid tumors and has translational value in tumor-targeted or in combinational-biochemotherapy strategies for cancer treatments. Here, we focus on diverse approaches to cancer biotherapy, including bacteriolytic and bacterially-derived anti-cancer agents with an emphasis on their mechanism of action and therapeutic potential.

Tissue specific roles for the Ribosome Biogenesis Factor Wdr43 in zebrafish development

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells.

Plumbagin (PLB) has shown anti-cancer activity but the mechanism is unclear. This study has found that PLB has a potent pro-apoptotic and pro-autophagic effect on A549 and H23 cells. PLB arrests cells in G2/M phase, and increases the intracellular level of reactive oxygen species in both cell lines. PLB dose-dependently induces autophagy through inhibition of PI3K/Akt/mTOR pathway as indicated by reduced phosphorylation of Akt and mTOR. Inhibition or induction of autophagy enhances PLB-induced apoptosis. There is crosstalk between PLB-induced apoptosis and autophagy. These findings indicate that PLB initiates both apoptosis and autophagy in NSCLC cells through coordinated pathways.

MicroRNAs: biogenesis, roles for carcinogenesis and as potential biomarkers for cancer diagnosis and prognosis

MicroRNAs (miRNAs) are short non-coding RNAs of 20-24 nucleotides that play important roles in carcinogenesis. Accordingly, miRNAs control numerous cancer-relevant biological events such as cell proliferation, cell cycle control, metabolism and apoptosis. In this review, we summarize the current knowledge and concepts concerning the biogenesis of miRNAs, miRNA roles in cancer and their potential as biomarkers for cancer diagnosis and prognosis including the regulation of key cancer-related pathways, such as cell cycle control and miRNA dysregulation. Moreover, microRNA molecules are already receiving the attention of world researchers as therapeutic targets and agents. Therefore, in-depth knowledge of microRNAs has the potential not only to identify their roles in cancer, but also to exploit them as potential biomarkers for cancer diagnosis and identify therapeutic targets for new drug discovery.

Epigallocatechin-3-gallate induces the apoptosis of hepatocellular carcinoma LM6 cells but not non-cancerous liver cells

Epigallocatechin-3-gallate (EGCG) is a constituent of green tea and has been associated with anticancer activity. In the present study, the inhibitory effect of EGCG on human hepatocellular cancer cells was examined by cell viability assay, in vitro apoptosis assay and cell cycle analysis. In addition, gene expression was measured to elucidate the molecular mechanisms of action of EGCG by mitochondrial membrane potential (MMP) determination and western blot analysis. We demonstrated that EGCG induced apoptosis, decreased mitochondrial membrane potential and promoted G0/G1 phase cell cycle arrest of HCCLM6 cells but not that of non-cancerous liver cells (HL-7702). The EGCG-induced apoptosis of HCCLM6 cells was associated with a significant decrease in Bcl-2 and NF-κB expression. In addition, the expression of Bax, p53, caspase-9 and caspase-3 increased, and cytochrome c was released. These results suggest that EGCG inhibits the progression of cancer through cytocidal activity and that it is a potential therapeutic compound for hepatocellular carcinoma (HCC).

Bardoxolone methyl induces apoptosis and autophagy and inhibits epithelial-to-mesenchymal transition and stemness in esophageal squamous cancer cells

Natural and synthetic triterpenoids have been shown to kill cancer cells via multiple mechanisms. The therapeutic effect and underlying mechanism of the synthetic triterpenoid bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me) on esophageal cancer are unclear. Herein, we aimed to investigate the anticancer effects and underlying mechanisms of CDDO-Me in human esophageal squamous cell carcinoma (ESCC) cells. Our study showed that CDDO-Me suppressed the proliferation and arrested cells in G2/M phase, and induced apoptosis in human ESCC Ec109 and KYSE70 cells. The G2/M arrest was accompanied with upregulated p21Waf1/Cip1 and p53 expression. CDDO-Me significantly decreased B-cell lymphoma-extra large (Bcl-xl), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9, and cleaved poly ADP ribose polymerase (PARP) levels but increased the expression level of Bcl-2-associated X (Bax). Furthermore, CDDO-Me induced autophagy in both Ec109 and KYSE70 cells via suppression of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. There were interactions between the autophagic and apoptotic pathways in Ec109 and KYSE70 cells subject to CDDO-Me treatment. CDDO-Me also scavenged reactive oxygen species through activation of the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) pathway in Ec109 and KYSE70 cells. CDDO-Me inhibited cell invasion, epithelial-mesenchymal transition, and stemness in Ec109 and KYSE70 cells. CDDO-Me significantly downregulated E-cadherin but upregulated Snail, Slug, and zinc finger E-box-binding homeobox 1 (TCF-8/ZEB1) in Ec109 and KYSE70 cells. CDDO-Me significantly decreased the expression of octamer-4, sex determining region Y-box 2 (Sox-2), Nanog, and B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), all markers of cancer cell stemness, in Ec109 and KYSE70 cells. Taken together, these results indicate that CDDO-Me is a promising anticancer agent against ESCC. Further studies are warranted to explore the molecular targets, efficacy and safety of CDDO-Me in the treatment of ESCC.

The pan-inhibitor of Aurora kinases danusertib induces apoptosis and autophagy and suppresses epithelial-to-mesenchymal transition in human breast cancer cells

Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and β-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy.

Regionally specific changes in levels of cortical S100β in bipolar 1 disorder but not schizophrenia

Objective: To determine if levels of the glial-derived proteins S100β and glial acidic fibrillary protein (GFAP) and the pro- and antiapoptotic proteins p53 and Bcl-2 were altered in the cortex of subjects with schizophrenia or bipolar 1 disorder.
Method: Levels of S100β, GFAP, p53 and Bcl-2 were measured in cortex (Brodmann's Areas (BAs) 9, 10, 46 and 40) of control subjects and subjects with schizophrenia, bipolar 1 disorder and in the cortex of rats treated with haloperidol or lithium using protein-specific antibodies and western blot analysis.
Results: Levels of S100β were decreased in BA 9 and increased in BA 40 from subjects with bipolar 1 disorder. Levels of this protein were not altered in other CNS regions, in schizophrenia or in the cortex of rats treated with haloperidol or lithium. No changes in levels of the other three proteins were detected across diagnoses.
Conclusions: Regionally selective changes in cortical S100β may be associated with the pathology of bipolar 1 disorder and may reflect derangements in neuronal death or survival

Iron-free and iron-saturated bovine lactoferrin inhibit survivin expression and differentially modulate apoptosis in breast cancer

BACKGROUND: Iron binding, naturally occurring protein bovine lactoferrin (bLf) has attracted attention as a safe anti-cancer agent capable of inducing apoptosis. Naturally, bLf exists partially saturated (15-20%) with Fe(3+) however, it has been demonstrated that manipulating the saturation state can enhance bLf's anti-cancer activities. METHODS: Apo-bLf (Fe(3+) free) and Fe-bLf (>90% Fe(3+) Saturated) were therefore, tested in MDA-MB-231 and MCF-7 human breast cancer cells in terms of cytotoxicity, proliferation, migration and invasion. Annexin-V Fluos staining was also employed in addition to apoptotic protein arrays and Western blotting to determine the specific mechanism of bLf-induced apoptosis with a key focus on p53 and inhibitor of apoptosis proteins (IAP), specifically survivin. RESULTS: Apo-bLf induced significantly greater cytotoxicity and reduction in cell proliferation in both cancer cells showing a time and dose dependent effect. Importantly, no cytotoxicity was detected in normal MCF-10-2A cells. Both forms of bLf significantly reduced cell invasion in cancer cells. Key apoptotic molecules including p53, Bcl-2 family proteins, IAP members and their inhibitors were significantly modulated by both forms of bLf, though differentially in each cell line. Most interestingly, both Apo-bLf and Fe-bLf completely inhibited the expression of survivin protein (key IAP), after 48 h at 30 and 40 nM in cancer cells. CONCLUSIONS: The capacity of these forms of bLf to target survivin expression and modulation of apoptosis demonstrates an exciting potential for bLf as an anti-cancer therapeutic in the existing void of survivin inhibitors, with a lack of successful inhibitors in the clinical management of cancer.

LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging

This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin, survivin splice variant (survivin 2B) and other anti-apoptotic proteins Bad, Bcl-2 and XIAP. Apart from the promising anti-cancer efficacy and the exceptional tumor targeting ability observed by multimodal imaging using near-infrared (NIR) imaging, magnetic resonance imaging (MRI) and computerized tomographic (CT) techniques, these NCs also maintained the immunomodulatory benefits of bLf as they were able to increase the RBC, hemoglobin, iron calcium and zinc levels in mice.

Molecular regulation of survivin targeted nano-therapy in advanced prostate cancer

 The increasing complexities of prostate cancer disease progression necessitates more stable and less toxic therapeutic strategies. The current study demonstrated for the first time, the survivin targeted anti-cancer therapeutic activity of the bio-molecular drugs such as SurR9-C84A and bovine lactoferrin in inducing prostate cancer specific apoptosis. Moreover, improved therapeutic efficacy was conferred to these bio-molecules either by their encapsulation in stem cell targeted bio-compatible nanoparticles, or by the synthesis of protein-cytotoxic drug conjugates. This study also highlighted the role played by miRNAs in the regulation of iron metabolism and apoptosis, mediated by the selective activation of p53 and PTEN pathways.