Citrus peel flavonoids as potential cancer prevention agents

Citrus fruit and in particular flavonoid compounds from citrus peel have been identified as agents with utility in the treatment of cancer. This review provides a background and overview regarding the compounds found within citrus peel with putative anticancer potential as well as the associated in vitro and in vivo studies. Historical studies have identified a number of cellular processes that can be modulated by citrus peel flavonoids including cell proliferation, cell cycle regulation, apoptosis, metastasis, and angiogenesis. More recently, molecular studies have started to elucidate the underlying cell signaling pathways that are responsible for the flavonoids' mechanism of action. These growing data support further research into the chemopreventative potential of citrus peel extracts, and purified flavonoids in particular. This critical review highlights new research in the field and synthesizes the pathways modulated by flavonoids and other polyphenolic compounds into a generalized schema

Phospholipase

Phospholipase A2 (PLA2) enzymes are a family of proteins and to date at least 20 members have been identified in mammals. The family can be classified into four classes on the basis of their nucleotide and amino acid sequence homology. First, there are at present ten secreted phospholipase A2 enzymes (sPLA2-IB, -IIA, -IIC, -IID, -IIE, -IIF, -III, -V, -X, and -XII), which are of low molecular weight (13–18 kDa) with a catalytic histidine in their active site and a requirement for calcium for enzyme activity. Second, there are three characterized human cytosolic PLA2 enzymes (cPLA2-α, -β, and -γ, also known as Group IVA, IVB, and IVC PLA2) that use a catalytic serine in their active site. cPLA2-α and -β contain a C2 calcium binding domain and enzyme activity is calcium-dependent while cPLA2-γ lacks this domain and is thus a calcium-independent PLA2. Recently, a comprehensive homology search against the murine genome and EST databases using conserved sequences of cPLA2 as the query, led to the identification of cPLA2-δ, cPLA2-ε, and cPLA2-ξ (also known as Group IVD, IVE, and IVF PLA2), all of which are calcium-dependent enzymes. Third, three calcium-independent cytosolic PLA2 enzymes (iPLA2-α, -β, and -γ also known as Group VIA-1, VIA-2, and VIB) with an active-site serine, and fourth, four platelet-activating factor acetylhydrolase (PAF-AH) enzymes (Group VIIA, VIIB, VIIIA, and VIIIB) also involve a catalytic serine. Many of the different forms of PLA2 are differentially expressed in a tissue-, species-, and/or genotype-specific manner

Targeted amplification of mutant strands for efficient site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (e.g. cDNA, genomic DNA, and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligonucleotides (oligos) are incorporated together with the desired mutagenic oligos into the newly synthesized mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening

Proliferating cell unclear antigen-associated factor (PAF15) : a novel oncogene

Proliferating cell nuclear antigen (PCNA)-Associated Factor (PAF15) is a small protein containing a PCNA interacting motif and sequences for association with ubiquitin enzymes. In interaction with PCNA, PAF15 plays a key role in recruiting DNA replicative polymerase by double monoubiquitination at Lys15 and Lys24. Under DNA damage conditions, PAF15 regulates the switch from DNA replicative polymerase to translesion synthesis polymerase in order to bypass the replication-blocking lesions. Overexpression of PAF15 promotes the repair of ultraviolet-induced DNA damage and prevents cell death, whereas attenuation of PAF15 decreases DNA replication and cell survival. Ectopic expression of PAF15 in mouse fibroblasts increases colony formation and tumourigenicity. PAF15 is aberrantly increased in various human malignancies with poor prognosis. Collectively, PAF15 may contribute to carcinogenesis and represents one of the potential therapeutic targets in the treatment of cancer

The arachidonic acid pathway and its role in prostate cancer development and progression

Purpose: The arachidonic acid pathway incorporates phospholipase, cyclooxygenase, lipoxygenase and epoxygenase enzymes. This pathway has been shown to have a major role in the development and progression of a number of cancers, including prostate cancer. We discuss the current status of research of this pathway in the area of prostate cancer, ranging from preclinical in vitro studies to human clinical trials. Materials and Methods: We performed an online search of the current and past peer reviewed literature on prostate cancer and arachidonic acid, phospholipase, cyclooxygenase, lipoxygenase, epoxygenase, platelet activating factor, prostaglandin and eicosanoid. We retrieved and evaluated all full-length articles published in English from the 1980s to January 2007. Results: Epidemiological evidence suggested that nonsteroidal anti-inflammatory drugs may decrease the risk of prostate cancer. This effect, presumably through the inhibition of cyclooxygenase-2, has been validated in preclinical studies. Cyclooxygenase-2 inhibition has also decreased the rate of prostate specific antigen increase in men with biochemical recurrence after treatment for prostate cancer. Although lipoxygenase and secretory phospholipase A2 inhibition was also effective for decreasing prostate cancer growth in preclinical studies, to our knowledge these strategies have not yet been used in clinical trials. Cytosolic phospholipase A2, platelet activating factor and epoxygenase need further investigation to determine a role in prostate cancer. Conclusions: Evolving data suggest a significant role for some areas of the arachidonic acid pathway in prostate cancer. Inhibiting 1 or a number of these enzymes in combination may hold promise for future prostate cancer treatment

Pitfalls in the use of arachidonic acid oxidation products to assign lipoxygenase activity in cancer cells

Arachidonic acid (AA) reaction with cyclooxygenase (COX) and lipoxygenases (LOX) yield eicosanoids that can mediate prostate cancer proliferation and enhance both tumour vascularization and metastasis. Increasingly measurement of eicosanoids with liquid chromatography is employed to implicate LOX activity in different biological systems and in particular link LOX activity to the progression of cancer in experimental models. This study demonstrates that simply identifying patterns of eicosanoid regio-isomerism is insufficient to designate LOX activity in prostate cancer cells and the analysis must include complete stereochemical assignment of the various isomers in order to validate the assignment of LOX activity

Effect of citrus peel extracts on the cellular quiescence of prostate cancer cells

The re-entry of quiescent cancer cells to the cell cycle plays a key role in cancer recurrence, which can have a high risk after primary treatment. Citrus peel extracts (CPEs) contain compounds that can impair tumour growth, however the mechanism of action and effects on cell cycle regulation remain unclear. In this study, the capacity of ethyl acetate: hexane extract (CPE/hexane) and water extract (CPE/water) to modulate cell cycle re-entry of quiescent (PC-3 and LNCaP) prostate cancer cells was tested in an in vitro culture system. Cell cycle analysis showed that the quiescent PC-3 and LNCaP cancer cells in the presence of CPE/water were impaired in their ability to enter S phase where only 2-3% reduction of G0/G1 cells was noted compared to 12-18% reduction for control cells. In contrast, the CPE/hexane did not show any cell cycle inhibition activity in both cell lines. A low DNA synthesis rate and weak apoptosis were observed in quiescent cancer cells treated with CPEs. Hesperidin and narirutin, the predominant flavonoids found in citrus, were not responsible for the observed biological activity, implicating alternative bioactive compounds. Notably, citric acid was identified as one of the compounds present in CPE that acts as a cell cycle re-entry inhibitor. Citric acid exhibited a higher cell toxicity effect on PC-3 prostate cancer cells than non-cancerous RWPE-1 prostate cells, suggesting specific benefits for cancer treatment. In conclusion, CPE containing citric acid together with various bioactive compounds could be used as a chemopreventive agent for post-therapy cancer patients

A novel splice variant of the β-tropomyosin (TPM2) gene in prostate cancer

Decreased expression of high molecular weight isoforms of tropomyosin (Tm) is associated with oncogenic transformation and is evident in cancers, with isoform Tm1 seemingly an important tumor suppressor. Tm1 expression in prostate cancer has not previously been described. In this study, while demonstrating suppressed levels of Tm1 in the prostate cancer cell lines LNCaP, PC3, and DU-145 compared to normal prostate epithelial cell primary isolates (PrEC), a novel splice variant of the TPM2 gene was identified. Quantitative RT-PCR determined significantly greater levels of the transcript variant in all three prostate cancer cell lines than in normal prostate epithelial cells. Characterization of this novel variant demonstrated it to include exon 6b, previously thought unique to the muscle-specific β-Tm isoform, with an exon arrangement of 1-2-3-4-5-6a-6b-7-8-10. Inclusion of exon 6b introduces a premature stop codon directly following the 6a-6b exon boundary. Western blot analysis demonstrated the presence of a truncated protein in prostate cancer cell lines that was absent in normal prostate epithelial cells. It is hypothesized that this truncated protein will result in suppression of Tm1 polymer formation required for actin filament association. The lack of Tm polymer-actin association will result in loss of the stable actin microfilament organization and stress fiber formation, a state associated with cell transformation

Effect of arachidonic acid and its producing enzyme phospholipase A2α on key oncogenic pathways in prostate cancer

The dietary arachidonic acid (AA) is stored at the sn-2 position in the glycerolphospholipid of cell membrane. AA is released via the cleavage by phospholipase A2 in response to stimuli such as cytokines, growth factors and hormones. The freed AA is metabolised typically by cyclooxygenase and lipoxygenase enzymes to form eicosanoids. Of all the phospholipase A2 that catalyse the hydrolysis of fatty acid at sn-2 bond of glycerophospholipids, cytosolic phospholipase A2α (cPLA2α) is preferentially targeting AA-containing phospholipids. Hence, cPLA2α action represents the key step for AA release and eicosanoid synthesis. Stimulation of cells with agents that mobilise intracellular calcium and/or promote the phosphorylation of cPLA2α leads to increased AA release and eicosanoid production

Pharmacological targeting of the integrated protein kinase B, phosphatase and tensin homolog deleted on chromosome 10, and transforming growth Factor-β pathways in prostate cancer

Prostate cancer is a highly heterogenous disease in which a patient-tailored care program is much desired. Central to this goal is the development of novel targeted pharmacological interventions. To develop these treatment strategies, an understanding of the integration of cellular pathways involved in both tumorigen-esis and tumor suppression is crucial. Of further interest are the events elicited by drug treatments that exploit the underlying molecular pathology in cancer. This review briefly describes the evidence that suggests integration of three established pathways: the tumorigenic phosphoinositide 3-kinase/protein kinase B (AKT) pathway, the tumor suppressive phosphatase and tensin homolog deleted on chromosome 10 pathway, and the tumor suppressive transforming growth factor-β pathway. More importantly, we discuss novel pharmaceutical agents that target key points of integration in these three pathways. These new therapeutic strategies include the use of agents that target iron to inhibit proliferation via multiple mechanisms and suppression of AKT by cytosolic phos-pholipase A2-α inhibitors