In vitro and in vivo reversal of resistance to 5-fluorouracil in colorectal cancer cells with a novel stealth double-liposomal formulation

Drug resistance is a major cause of treatment failure in cancer chemotherapy, including that with the extensively prescribed antimetabolite, 5-fluorouracil (5-FU). In this study, we tried to reverse 5-FU resistance by using a double-punch strategy: combining 5-FU with a biochemical modulator to improve its tumoural activation and encapsulating both these agents in one same stealth liposome. Experiments carried out in the highly resistant, canonical SW620 human colorectal model showed a up to 80% sensitisation to 5-FU when these cells were treated with our liposomal formulation. Results with this formulation demonstrated 30% higher tumoural drug uptake, better activation with increased active metabolites including critical-5-fluoro-2-deoxyuridine-5-monophosphate, superior inhibition (98%) of tumour thymidylate synthase, and subsequently, higher induction of both early and late apoptosis. Drug monitoring showed that higher and sustained exposure was achieved in rats treated with liposomal formulation. When examined in a xenograft animal model, our dual-agent liposomal formulation caused a 74% reduction in tumour size with a mean doubling in survival time, whereas standard 5-FU failed to exhibit significant antiproliferative activity as well as to increase the lifespan of tumour-bearing mice. Taken collectively, our data suggest that resistance to 5-FU can be overcome through a better control of its intratumoural activation and the use of an encapsulated formulation

Kinetic Analysis of Substrate Utilization by Native and TNAP-, NPP1-, or PHOSPHO1-Deficient Matrix Vesicles

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast-derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PPi by isolated wild-type (WT) as well as TNAP-, NPP1- and PHOSPHO1-deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1-deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PPi in the MV compartment. NPP1 does not have a major role in PPi generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research

Association between Variations in Cell Cycle Genes and Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating and progressive lung disease. Its aetiology is thought to involve damage to the epithelium and abnormal repair. Alveolar epithelial cells near areas of remodelling show an increased expression of proapoptotic molecules. Therefore, we investigated the role of genes involved in cell cycle control in IPF. Genotypes for five single nucleotide polymorphisms (SNPs) in the tumour protein 53 (TP53) gene and four SNPs in cyclin-dependent kinase inhibitor 1A (CDKN1A), the gene encoding p21, were determined in 77 IPF patients and 353 controls. In peripheral blood mononuclear cells (PBMC) from 16 healthy controls mRNA expression of TP53 and CDKN1A was determined

Silibinin induces mitochondrial NOX4-mediated endoplasmic reticulum stress response and its subsequent apoptosis

Background: Silibinin, a biologically active compound of milk thistle, has chemopreventive effects on cancer cell lines. Recently it was reported that silibinin inhibited tumor growth through activation of the apoptotic signaling pathway. Although various evidences showed multiple signaling pathways of silibinin in apoptosis, there were no reports to address the clear mechanism of ROS-mediated pathway in prostate cancer PC-3 cells. Several studies suggested that reactive oxygen species (ROS) play an important role in various signaling cascades, but the primary source of ROS was currently unclear. Methods: The effect of silibinin was investigated on cell growth of prostate cell lines by MTT assay. We examined whether silibinin induced apoptosis through production of ROS using flow cytometry. Expression of apoptosis-, endoplasmic reticulum (ER)-related protein and gene were determined by western blotting and RT-PCR, respectively. Results: Results showed that silibinin triggered mitochondrial ROS production through NOX4 expression and finally led to induce apoptosis. In addition, mitochondrial ROS caused ER stress through disruption of Ca2+ homeostasis. Co-treatment of ROS inhibitor reduced the silibinin-induced apoptosis through the inhibition of NOX4 expression, resulting in reduction of both Ca2+ level and ER stress response. Conclusions: Taken together, silibinin induced mitochondrial ROS-dependent apoptosis through NOX4, which is associated with disruption of Ca2+ homeostasis and ER stress response. Therefore, the regulation of NOX4, mitochondrial ROS producer, could be a potential target for the treatment of prostate cancer.ope

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families: functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2

IGF1 activates cell cycle arrest following irradiation by reducing binding of ΔNp63 to the p21 promoter

Radiotherapy for head and neck tumors often results in persistent loss of function in salivary glands. Patients suffering from impaired salivary function frequently terminate treatment prematurely because of reduced quality of life caused by malnutrition and other debilitating side-effects. It has been previously shown in mice expressing a constitutively active form of Akt (myr-Akt1), or in mice pretreated with IGF1, apoptosis is suppressed, which correlates with maintained salivary gland function measured by stimulated salivary flow. Induction of cell cycle arrest may be important for this protection by allowing cells time for DNA repair. We have observed increased accumulation of cells in G2/M at acute time-points after irradiation in parotid glands of mice receiving pretreatment with IGF1. As p21, a transcriptional target of the p53 family, is necessary for maintaining G2/M arrest, we analyzed the roles of p53 and p63 in modulating IGF1-stimulated p21 expression. Pretreatment with IGF1 reduces binding of ΔNp63 to the p21 promoter after irradiation, which coincides with increased p53 binding and sustained p21 transcription. Our data indicate a role for ΔNp63 in modulating p53-dependent gene expression and influencing whether a cell death or cell cycle arrest program is initiated

Aberrant CDKN1A transcriptional response associates with abnormal sensitivity to radiation treatment

Normal tissue reactions to radiation therapy vary in severity among patients and cannot be accurately predicted, limiting treatment doses. The existence of heritable radiosensitivity syndromes suggests that normal tissue reaction severity is determined, at least in part, by genetic factors and these may be revealed by differences in gene expression. To test this hypothesis, peripheral blood lymphocyte cultures from 22 breast cancer patients with either minimal (11) or very severe acute skin reactions (11) have been used to analyse gene expression. Basal and post-irradiation expression of four radiation-responsive genes (CDKN1A, GADD45A, CCNB1, and BBC3) was determined by quantitative real-time PCR in T-cell cultures established from the two patient groups before radiotherapy. Relative expression levels of BBC3, CCNB1, and GADD45A 2 h following 2 Gy X-rays did not discriminate between groups. However, post-irradiation expression response was significantly reduced for CDKN1A (P<0.002) in severe reactors compared to normal. Prediction of reaction severity of ∼91% of individuals sampled was achieved using this end point. Analysis of TP53 Arg72Pro and CDKN1A Ser31Arg single nucleotide polymorphisms did not show any significant association with reaction sensitivity. Although these results require confirmation and extension, this study demonstrates the possibility of predicting the severity of acute skin radiation toxicity in simple tests

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer

Rigorous quality control steps, termed checkpoints, tightly regulate progression through the cell cycle. DNA-damaging chemotherapy and radiation activate functional cellular checkpoints. These checkpoints can facilitate DNA repair and promote cell death in unrepaired cells. There are at least three DNA damage checkpoints – at G1/S, S, and G2/M – as well as a mitotic spindle checkpoint. Most cancer cells harbour mutations in tumour suppressors and/or oncogenes, which impair certain cell checkpoints. Inhibiting the remaining cell checkpoints – particularly after exposure of cancer cells to chemotherapy and/or radiation – allows cell death, a strategy now being employed in cancer therapeutics. With our increasing knowledge of cell cycle regulation, many compounds have been developed to inhibit specific checkpoint components, particularly at the G2/M transition. One such target is checkpoint kinase-1 (Chk1). We review here the molecular framework of the cell cycle, the rationale for targeting Chk1, the preclinical concepts related to the development of Chk1 inhibitors, and the efficacy and safety results from Chk1 inhibitors now in phase I/II trials

Altered expression of the suppressors PML and p53 in glioblastoma cells with the antisense-EGF-receptor

Gene amplification and enhanced expression of the epidermal growth factor receptor (EGFR) represent the major molecular genetic alteration in glioblastomas and it may play an essential role in cell growth and in the carcinogenic process. On the other hand, the nuclear suppressor proteins PML and p53 are also known to play critical roles in cancer development and in suppressing cell growth. Here we report that, in glioblastoma cells with defective EGFR function, the expressions of both promyelocytic leukaemia (PML) and p53 were altered. Cells that were transfected with the antisense-cDNA of EGFR were found to have more cells in G1 and fewer cells in S phase. In addition, the transfected cells were found to be non-responsive to EGF-induced cell growth. Interestingly, the expression of the suppressors p53 and PML were found to be significantly increased by immunohistochemical assay in the antisense-EGFR cells. Moreover, the PML expression in many of the cells was converted from the nuclear dot pattern into fine-granulated staining pattern. In contrast, the expressions of other cell cycle regulated genes and proto-oncogene, including the cyclin-dependent kinase 4 (cdk4), retinoblastoma, p16INK4a and p21H-ras, were not altered. These data indicate that there are specific inductions of PML and p53 proteins which may account for the increase in G1 and growth arrest in antisense-EGFR treated cells. It also indicates that the EGF, p53 and PML transduction pathways were linked and they may constitute an integral part of an altered growth regulatory programme. The interactions and cross-talks of these critical molecules may be very important in regulating cell growth, differentiation and cellular response to treatment in glioblastomas. © 1999 Cancer Research Campaig