Understanding the molecular mechanisms of cancer prevention by dietary phytochemicals:From experimental models to clinical trials

Chemoprevention is one of the cancer prevention approaches wherein natural/synthetic agent(s) are prescribed with the aim to delay or disrupt multiple pathways and processes involved at multiple steps, i.e., initiation, promotion, and progression of cancer. Amongst environmental chemopreventive compounds, diet/beverage-derived components are under evaluation, because of their long history of exposure to humans, high tolerability, low toxicity, and reported biological activities. This compilation briefly covers and compares the available evidence on chemopreventive efficacy and probable mechanism of chemoprevention by selected dietary phytochemicals(capsaicin, curcumin, diallyl sulphide, genistein, green/black tea polyphenols, indoles, lycopene, phenethyl isocyanate, resveratrol, retinoids and tocopherols) in experimental systems and clinical trials. All the dietary phytochemicals covered in this review have demonstrated chemopreventive efficacy against spontaneous or carcinogen-induced experimental tumors and/or associated biomarkers and processes in rodents at several organ sites. The observed anti-initiating, anti-promoting and anti-progression activity of dietary phytochemicals in carcinogen-induced experimental models involve phytochemical-mediated redox changes, modulation of enzymes and signaling kinases resulting to effects on multiple genes and cell signaling pathways. Results from clinical trials using these compounds have not shown them to be chemopreventive. This may be due to our:(1) inability to reproduce the exposure conditions, i.e., levels, complexity, other host and lifestyle factors; and(2) lack of understanding about the mechanisms of action and agent-mediated toxicity in several organs and physiological processes in the host. Current research efforts in addressing the issues of exposure conditions, bioavailability, toxicity and the mode of action of dietary phytochemicals may help address the reason for observed mismatch that may ultimately lead to identification of new chemopreventive agents for protection against broad spectrum of exposures.     

A survey of antiprion compounds reveals the prevalence of non-PrP molecular targets

Prion diseases are fatal neurodegenerative diseases caused by the accumulation of the misfolded isoform (PrP(Sc)) of the prion protein (PrP(C)). Cell-based screens have identified several compounds that induce a reduction in PrP(Sc) levels in infected cultured cells. However, the molecular targets of most antiprion compounds remain unknown. We undertook a large-scale, unbiased, cell-based screen for antiprion compounds and then investigated whether a representative subset of the active molecules had measurable affinity for PrP, increased the susceptibility of PrP(Sc) to proteolysis, or altered the cellular localization or expression level of PrP(C). None of the antiprion compounds showed in vitro affinity for PrP or had the ability to disaggregate PrP(Sc) in infected brain homogenates. These observations suggest that most antiprion compounds identified in cell-based screens deploy their activity via non-PrP targets in the cell. Our findings indicate that in comparison to PrP conformers themselves, proteins that play auxiliary roles in prion propagation may be more effective targets for future drug discovery efforts.     

A clinical and molecular genetic analysis of the fragile X syndrome

Results of phenotypical, patho-psychological and molecular-genetic analysis of the 53 probands with clinical features of the fragile X syndrome and 10 female carriers are presented. The clinical heterogeneity, diagnostic criteria, methods of genetic risk estimation, perspectives of prevention of this disease are discussed.     

A multivariate test of interaction for use in clinical trials

An important issue in clinical trials is whether the effect of treatment is essentially homogeneous as a function of baseline covariates. Covariates that have the potential for an interaction with treatment may be suspected on the basis of treatment mechanism or may be known risk factors, as it is often thought that the sickest patients may benefit most from treatment. If disease severity is more accurately determined by a collection of baseline covariates rather than a single risk factor, methods that examine each covariate in turn for interaction may be inadequate. We propose a procedure whereby treatment interaction is examined along a single severity index that is a linear combination of baseline covariates. Formally, we derive a likelihood ratio test based on the null beta0 = beta1 versus the alternative abeta0 = beta1, where X'beta(k) (k = 0, 1) corresponds to the severity index in arm k and X is a vector of baseline covariates. While our explicit test requires a Gaussian response, it can be readily implemented whenever the estimates of beta0,beta1 are approximately multivariate normal. For example, it is appropriate for large clinical trials where beta(k) is based on a logisitic or Cox regression of response on X.     

Identification of two loci in tomato reveals distinct mechanisms for salt tolerance

Salt stress is one of the most serious environmental factors limiting the productivity of crop plants. To understand the molecular basis for salt responses, we used mutagenesis to identify plant genes required for salt tolerance in tomato. As a result, three tomato salt-hypersensitive (tss) mutants were isolated. These mutants defined two loci and were caused by single recessive nuclear mutations. The tss1 mutant is specifically hypersensitive to growth inhibition by Na(+) or Li(+) and is not hypersensitive to general osmotic stress. The tss2 mutant is hypersensitive to growth inhibition by Na(+) or Li(+) but, in contrast to tss1, is also hypersensitive to general osmotic stress. The TSS1 locus is necessary for K(+) nutrition because tss1 mutants are unable to grow on a culture medium containing low concentrations of K(+). Increased Ca(2)+ in the culture medium suppresses the growth defect of tss1 on low K(+). Measurements of membrane potential in apical root cells were made with an intracellular microelectrode to assess the permeability of the membrane to K(+) and Na(+). K(+)-dependent membrane potential measurements indicate impaired K(+) uptake in tss1 but not tss2, whereas no differences in Na(+) uptake were found. The TSS2 locus may be a negative regulator of abscisic acid signaling, because tss2 is hypersensitive to growth inhibition by abscisic acid. Our results demonstrate that the TSS1 locus is essential for K(+) nutrition and NaCl tolerance in tomato. Significantly, the isolation of the tss2 mutant demonstrates that abscisic acid signaling is also important for salt and osmotic tolerance in glycophytic plants.     

Anticancer properties of tocotrienols: A review of cellular mechanisms and molecular targets

Vitamin E is composed of two groups of compounds: α-, β-, γ-, and δ-tocopherols (TPs), and the corresponding unsaturated tocotrienols (TTs). TTs are found in natural sources such as red palm oil, annatto seeds, and rice bran. In the last decades, TTs (specifically, γ-TT and δ-TT) have gained interest due to their health benefits in chronic diseases, based on their antioxidant, neuroprotective, cholesterol-lowering, anti-inflammatory activities. Several in vitro and in vivo studies pointed out that TTs also exert a significant antitumor activity in a wide range of cancer cells. Specifically, TTs were shown to exert antiproliferative/proapoptotic effects and to reduce the metastatic or angiogenic properties of different cancer cells; moreover, these compounds were reported to specifically target the subpopulation of cancer stem cells, known to be deeply involved in the development of resistance to standard therapies. Interestingly, recent studies pointed out that TTs exert a synergistic antitumor effect on cancer cells when given in combination with either standard antitumor agents (i.e., chemotherapeutics, statins, “targeted” therapies) or natural compounds with anticancer activity (i.e., sesamin, epigallocatechin gallate (EGCG), resveratrol, ferulic acid). Based on these observations, different TT synthetic derivatives and formulations were recently developed and demonstrated to improve TT water solubility and to reduce TT metabolism in cancer cells, thus increasing their biological activity. These promising results, together with the safety of TT administration in healthy subjects, suggest that these compounds might represent a new chemopreventive or anticancer treatment (i.e., in combination with standard therapies) strategy. Clinical trials aimed at confirming this antitumor activity of TTs are needed.     

The molecular mechanisms of cellular tolerance to delta-opioid agonists. A minireview

Chronic treatment with deltaopioid agonists, similar to other agonist drugs, causes tolerance. Tolerance is a complex adaptation process that consists of multiple, cellular and neural-system adaptations. Cellular tolerance to delta-opioid agonists involves feedback-regulation of the function, concentration, and localization of the delta-opioid receptors (receptor desensitization) as well as of intracellular effectors (functional desensitization). We are using a recombinant Chinese hamster ovary cell line expressing the human delta-opioid receptors (hDOR/CHO) to investigate the molecular mechanisms of cellular tolerance. We found that the structurally distinct delta-opioid agonists mediate receptor down-regulation by different mechanisms. Thus, truncation of the last 35 C-terminal amino acids of the hDOR completely abolished DPDPE, but not SNC 80-mediated receptor down-regulation. In addition, down-regulation of the wild type-, and the truncated hDORs exhibited different inhibitor sensitivity-profile. Chronic delta-opioid agonist treatment also causes functional desensitization of forskolin-stimulated cAMP formation and cAMP overshoot in the hDOR/CHO cells. We have demonstrated that chronic SNC 80 treatment also causes concurrent phosphorylation of the adenylyl cyclase (AC) VI isoenzyme hDOR/CHO cells. Both AC superactivation and AC VI phosphorylation were SNC 80 dose-dependent, naltrindole-sensitive, and exhibited similar time course-, and protein kinase inhibitor-sensitivity profile. We hypothesize that phosphorylation of AC VI plays an important role in delta-opioid agonist-mediated AC superactivation in hDOR/CHO cells.     

A homogeneity test in overviews with group sequentially monitored clinical trials

It is known that using statistical stopping rules in clinical trials can create an artificial heterogeneity of treatment effects in overviews of related trials (Hughes, Freedman, and Pocock, 1992, Biometrics 48, 41-53). If the true treatment effect being tested is small, as is often the case, the homogeneity test by DerSimonian and Laird (1986, Controlled Clinical Trials 7, 177-188) violates the size of the test very severely. This paper provides a new homogeneity test, which preserves the size of the test more accurately. The operating characteristics of the new test are examined through simulations.     

Ubiquitin-proteasome system reveals clinical subtypes with distinct molecular characteristics and prognoses in gastric cancer

Modulators in ubiquitin-proteasome system (UPS) has been implicated in regulating cancer-related genes, immune responses, and oncogenesis. However, the global UPS expression pattern and its role in gastric cancer (GC) pathology remain elusive. Herein, we integrated the modulators in UPS and dissected their associations with tumor microenvironment (TME), therapeutic response and prognosis in GC. Ten eligible GC cohorts (n = 2161) were collected in this comprehensive analysis. Unsupervised clustering based on expression profile of ubiquitination regulators was performed to identify distinct expression pattern. Then, pathway activation, and TME characteristics and prognosis were explored for patients in each pattern. Finally, a UPS scoring system in GC, termed UPSGC, is developed for individualized quantification of UPS expression pattern. Two prognosis-distinctive UPS expression patterns were identified and validated. Multiple interdependent characteristics were found in each pattern. Patients in the pattern with poor prognosis were found with activation of EMT, TNFα/NF-κB and IL6/JAK/STAT3 signaling, and more infiltration of immunosuppressive M2 macrophages and Th2 cells in TME. And another pattern was characterized by upregulation of angiogenesis, Notch and Wnt-β/catenin signaling, as well as enrichment of microvessels in TME. Based on the UPSGC system, two pattern-related clinical subtypes were identified. Finally, the UPSGC subtypes were validated as robust biomarker to predict patient's therapeutic responses and survival outcomes. In conclusion, this study proposes two previously unexplored UPS expression patterns in GC, in which patients have distinct survival outcomes and molecular characteristics. The findings provide new evidences to support the clinical relevance of ubiquitination with personalized therapy.     

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear. Inhibition of either of these pathways has been proposed as a strategy to increase cytotoxicity of platinating agents in cancer treatment. Here, we defined the importance of PCNA-ubiquitination and REV1 for DNA in mammalian ICL repair. In mice, loss of PCNA-ubiquitination, but not REV1, resulted in germ cell defects and hypersensitivity to cisplatin. Loss of PCNA-ubiquitination, but not REV1 sensitized mammalian cancer cell lines to cisplatin. We identify polymerase Kappa as essential in tolerating DNA damage-induced lesions, in particular cisplatin lesions. Polk-deficient tumors were controlled by cisplatin treatment and it significantly delayed tumor outgrowth and increased overall survival of tumor bearing mice. Our results indicate that PCNA-ubiquitination and REV1 play distinct roles in DNA damage tolerance. Moreover, our results highlight POLK as a critical TLS polymerase in tolerating multiple genotoxic lesions, including cisplatin lesions. The relative frequent loss of Polk in cancers indicates an exploitable vulnerability for precision cancer medicine.     

Anti‐cancer targets of formononetin and molecular mechanisms in osteosarcoma: Findings of bioinformatic and experimental assays

In current study, a bioinformatic‐based network pharmacology was used to identify the osteosarcoma (OGS)‐pathological targets and formononetin (FN)‐treated targets before the main core predictive biotargets were screened. In addition, all core targets were selected through a number of bioinformatic databases, followed by identification of predominant biological processes and signalling pathways of FN anti‐OGS. Further, top three core targets of FN anti‐OGS were determined as oestrogen receptor 1 (ESR1), tumour protein p53 (TP53), receptor tyrosine‐protein kinase erbB‐2 (ERBB2) respectively. In clinical biochemical data, the plasma samples of OGS showed the increased trends of alkaline phosphatase, triglyceride, blood glucose, lactate dehydrogenase, high‐sensitive C‐reactive protein and some immune cell counts when referenced to medical criteria. In clinicopathological examination, histological OGS sections resulted in increased positive cell counts of neoplastic ESR1, TP53, ERBB2. To further validate these corn proteins in experimental study in vivo, FN‐treated tumour‐bearing nude mice showed intracellular reductions of ESR1, TP53, ERBB2 positive expressions, accompanied with visibly reduced tumour weights. Collectively, our bioinformatic and experimental findings disclosed main core targets, biological processes and signalling pathways of FN anti‐OGS. Interestingly, the top core targets were representatively validated following FN treatment in vivo. Therefore, we reasoned that these predictive targets might be the potential biomarkers for screening and treating osteosarcoma.     

High-throughput molecular profiling of a P-cadherin overexpressing breast cancer model reveals new targets for the anti-cancer bacterial protein azurin

Azurin is a bacterial protein from Pseudomonas aeruginosa which exerts an inhibitory activity in cancer cells. In P-cadherin-overexpressing models, a bad prognosis marker in breast cancer increasing invasion and other malignant features, azurin decreases the invasion of cancer cells.We performed a microarray analysis to compare the expression profile of azurin treated cells with different P-cadherin expression levels. Azurin up-regulated apoptosis mediated by p53 protein, endocytosis and vesicle-mediated transport. In the contrary, in invasive MCF-7/AZ.Pcad cells, azurin decreased the expression of genes associated with cell surface receptors and signal transduction, as well as biological adhesion. Further, azurin decreased adhesion of cells to proteins from the extracellular matrix (ECM) and altered protein expression of integrins α6, β4 and β1 and interfered with the ability of these cells to form mammospheres. Altogether, our results further enlighten the anti-cancer effects mediated by azurin in P-cadherin overexpression breast cancer models.     

CDC25A pathway toward tumorigenesis: Molecular targets of CDC25A in cell-cycle regulation

The cell division cycle 25 (CDC25) phosphatases regulate key transitions between cell-cycle phases during normal cell division, and in the case of DNA damage, they are key targets of the checkpoint machinery that ensure genetic stability. Little is known about the mechanisms underlying dysregulation and downstream targets of CDC25. To understand these mechanisms, we silenced the CDC25A gene in breast cancer cell line MDA-MB-231 and studied downstream targets of CDC25A gene. MDA-MB-231 breast cancer cells were transfected and silenced by CDC25A small interfering RNA. Total messenger RNA (mRNA) was extracted and analyzed by quantitative real-time polymerase chain reaction. CDC25A phosphatase level was visualized by Western blot analysis and was analyzed by 2D electrophoresis and LC-ESI-MS/MS. After CDC25A silencing, cell proliferation reduced, and the expression of 12 proteins changed. These proteins are involved in cell-cycle regulation, programmed cell death, cell differentiation, regulation of gene expression, mRNA editing, protein folding, and cell signaling pathways. Five of these proteins, including ribosomal protein lateral stalk subunit P0, growth factor receptor bound protein 2, pyruvate kinase muscle 2, eukaryotic translation elongation factor 2, and calpain small subunit 1 increase the activity of cyclin D1. Our results suggest that CDC25A controls the cell proliferation and tumorigenesis by a change in expression of proteins involved in cyclin D1 regulation and G1/S transition.     

In vitro selection of RNase P RNA reveals optimized catalytic activity in a highly conserved structural domain.

In vitro selection techniques are useful means of dissecting the functions of both natural and artificial ribozymes. Using a self-cleaving conjugate containing the Escherichia coli ribonuclease P RNA and its substrate, pre-tRNA (Frank DN, Harris ME, Pace NR, 1994, Biochemistry 33:10800-10808), we have devised a method to select for catalytically active variants of the RNase P ribozyme. A selection experiment was performed to probe the structural and sequence constraints that operate on a highly conserved region of RNase P: the J3/4-P4-J2/4 region, which lies within the core of RNase P and is thought to bind catalytically essential magnesium ions (Harris ME et al., 1994, EMBO J 13:3953-3963; Hardt WD et al., 1995, EMBO J 14:2935-2944; Harris ME, Pace NR, 1995, RNA 1:210-218). We sought to determine which, if any, of the nearly invariant nucleotides within J3/4-P4-J2/4 are required for ribozyme-mediated catalysis. Twenty-two residues in the J3/4-P4-J2/4 component of RNase P RNA were randomized and, surprisingly, after only 10 generations, each of the randomized positions returned to the wild-type sequence. This indicates that every position in J3/4-P4-J2/4 contributes to optimal catalytic activity. These results contrast sharply with selections involving other large ribozymes, which evolve improved catalytic function readily in vitro (Chapman KB, Szostak JW, 1994, Curr Opin Struct Biol 4:618-622; Joyce GF, 1994, Curr Opin Struct Biol 4:331-336; Kumar PKR, Ellington AE, 1995, FASEB J 9:1183-1195). The phylogenetic conservation of J3/4-P4-J2/4, coupled with the results reported here, suggests that the contribution of this structure to RNA-mediated catalysis was optimized very early in evolution, before the last common ancestor of all life.