Effect of N-acylethanolamines with different acyl-chains on DPPC multilamellar liposomes

Relationships between lower extremity strength and stride characteristics were studied in 24 patients with post-polio syndrome. Maximum isometric torques were measured in the ankle plantar flexors, hip and knee extensors, and hip abductors. Gait velocity, stride length, and cadence were recorded during free and fast walking. Step-wise regression analysis was performed to determine which muscle groups best predicted ambulatory function. Plantar flexion torque was the best predictor of velocity (r = .55 free walking and r = .76 fast) and cadence (r = .46 free and r = .58 fast). The combination of plantar flexion and hip abduction torques was the best predictor of fast stride length (r = .78). These findings emphasize the important role of the plantar flexor muscles in gait. Knee extension torque was the poorest predictor for each of the gait parameters. Several patients demonstrated gait deviations that minimized the penalty of quadriceps weakness. Without a contracture or an orthosis, however, no adequate substitution exists for weak plantar flexion.     

Effect of lanthanide ions on the phase behavior of dipalmitoylphosphatidylcholine multilamellar liposomes

The effect of lanthanide ions (Ln3+) and their coordination compounds of diethylenetriamine pentaacetic acid (DTPA) on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multi-lamellar liposomes has been studied by differential scanning calorimetry (DSC), Raman spectroscopy, and freeze-fracture electron microscopic techniques. The displacement of Ca2+ binding on DPPC liposomes by lanthanide ions was also studied. The results show that the binding degree of four kinds of chloride salts with DPPC liposomes is: YbCl3 > GdCl3 > LaCl3 > CaCl2. Lanthanide ions increase the phase transition temperature of DPPC liposomes and decrease the membrane fluidity. Freeze-fracture electron microscopic results show that La3+ enhances the order of DPPC membrane. The effect of coordination compounds of lanthanides with DTPA on the phase behavior of DPPC liposomes is smaller than that of their chlorides. La3+, Gd3+, and Yb3+, can displace Ca2+ binding on DPPC liposomes, but there coordination compounds of DTPA can hardly displace Ca2+. Raman spectroscopic results show that a very slight effect in lateral packing order of DPPC liposomes was observed at various concentrations of lanthanides.     

Determinants of trimetrexate lethality in human colon cancer cells

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.     

Effect of O-glycosylated mucin on invasion and metastasis of HM7 human colon cancer cells

Mucinous colorectal cancers have a poorer prognosis than colorectal cancers which produce a low amount of mucin, but the exact mechanism is not well understood. The present study was undertaken to elucidate the possible mechanisms of invasion and metastasis of colon cancer cells producing high levels of mucin using mucin glycosylation inhibitor, benzyl-alpha-N-acetylgalactosamine. The binding activity of treated HM7 cells to endothelial leukocyte adhesion molecule (ELAM-1) was significantly decreased and fixed cell binding of MoAb SNH-3 and 19-9 (specific for sialyl Le(x) and sialyl Le(a), respectively) was also significantly decreased. Metalloproteinase activity in conditioned medium and invasion of matrigel-coated porous filters by treated HM7 cells were decreased. However, there was no difference between control and treated HM7 cells in terms of matrix protein binding. These results suggest that O-glycosylated mucin is important in the invasive and metastatic properties of HM7 human colon cancer cells.     

Atypical microtubule organization in undifferentiated human colon cancer cells

Immunocytochemical identification of GTH I and GTH II cells in the pituitary of the bluefin tuna (Thunnus thynnus) was performed using antisera specific for the common alpha-subunit and the two distinct beta-subunits of tuna (Thunnus obesus) GTH I and GTH II. Cells of the dorsal part of the proximal pars distalis (PPD), in close association with somatotrophs, displayed immunoreactivity of GTHIbeta. GTH IIbeta immunoreactivity was present in cells of the central part of the PPD and the external border of the pars intermedia. Anti-GTHalpha immunostained both GTH Ibeta- and GTH IIbeta-immunoreactive cells and also thyrotrophs. Both GTH Ibeta- and GTH IIbeta-immunoreactive cells were observed in immature bluefin tuna, although there were greater numbers of GTH IIbeta immunoreactive cells. These results suggest that GTH I and GTH II are synthesized in separate cells in the pituitary of the bluefin tuna. The localization and appearance of the two distinct gonadotropic cells of the tuna are compared with the salmonid arrangement.     

Antiproliferative potency of structurally distinct dietary flavonoids on human colon cancer cells

Dietary flavonoids are known to be antiproliferative and may play an important role in cancer chemoprevention, especially cancers of the gastrointestinal tract, because of a direct contact with food. This study was designed to compare the antiproliferative potency of several structurally distinct dietary flavonoids in colon cancer cells, Caco-2 and HT-29, and in rat non-transformed intestinal crypt cells, IEC-6. Flavonoids varied significantly in their antiproliferative potency depending on the structural features but the observations were consistent among the three cell lines studied. Of the two most potent flavonoids, quercetin and genistein, the effect was found to be dose-dependent and chromatin condensation, an indication of apoptosis, was noticed. Quercetin was found to distribute throughout the cell with higher amounts in the perinuclear and nucleoli areas. The lack of specific cell membrane enrichment by quercetin was consistent with its lack of effect on the transepithelial resistance. While several flavonoids including quercetin were found to be unstable, the chemical instability did not correlate with the antiproliferative potency, although it may contribute to the antiproliferative effect.     

Intracellular hyperthermia for cancer using magnetite cationic liposomes: an in vivo study

OBJECTIVE: Only two previous studies have assessed the effects of long-term GH replacement therapy on bone mineral density (BMD) in patients with adult onset GH deficiency. To date no study has looked at the long-term impact on BMD after a short course (6-12 months) of GH replacement. In two groups of patients with adult onset GH deficiency we have studied BMD either (a) after 3 years of continuous GH replacement or (b) 2 years after completion of a short course of GH. DESIGN: An open GH therapeutic study in which patients were recruited from a previous double-blind placebo-controlled study. The BMD status of all patients was unknown to the physician and patient at the time of recruitment. PATIENTS: Group A (n = 7, three females) all received GH replacement continuously for 3 years. Group B (n = 8, five females) included six patients who received GH replacement for 6 months and two who received GH replacement for 12 months with BMD being measured at 6-monthly intervals. METHODS: Single photon absorptiometry (SPA) and later single X-ray absorptiometry (SXA) were used to measure forearm cortical BMD. Dual-energy X-ray absorptiometry (DXA) was used to measure lumbar spine, trochanteric, femoral neck and Ward's area BMD. RESULTS: In group A lumbar spine and trochanter BMD had increased significantly from baseline by 3.7% (DXA: median change = 0.045 g/cm2; P = 0.028) and 4.0% (DXA: median change = 0.031 g/cm2; P = 0.046), respectively. There were non-significant decreases in femoral neck (1.9%) (DXA: median change = -0.02 g/cm2; P = 0.39), Ward's area (6.5%) (DXA: median change = -0.06 g/cm2; P = 0.09) and forearm (2.6%) (SPA/SXA: median change = -0.013 g/cm2; P = 0.18). In group B, compared with baseline, only trochanter BMD changed significantly, increasing by 5.9% (DXA: median change = 0.0485 g/cm2; P = 0.049). Lumbar spine (DXA: median change = -0.001 g/cm2) Ward's area (DXA: median change = 0.0135 g/cm2), femoral neck (DXA: median change = -0.005 g/cm2) and forearm cortical (SPA/SXA; median change = -0.01 g/cm2) BMD did not change significantly (P = 0.67, P = 0.57, P = 0.86 and P = 0.31, respectively). Median percentage changes compared with baseline were -0.1%, 1.8%, -0.5% and -2.1%, respectively. From the time of completion of GH therapy however, BMD increased significantly at lumbar spine, (median change = 0.023 g/cm2), Ward's area (median change = 0.03 g/cm2) and trochanter (median change = 0.056 g/cm2) (P = 0.036, P = 0.049 and P = 0.012, respectively) but not at the femoral neck (median change = 0.017 g/cm2; P = 0.31) or forearm (median change = 0 g/cm2; P = 0.75). CONCLUSION: Long-term GH replacement therapy for three years appears to have beneficial effects on bone in patients with adult onset GH deficiency particularly at the lumbar spine and trochanter; the effects on femoral neck and forearm cortical BMD, however, are less impressive. A short course (6-12 months) of GH replacement therapy results in an increase in trochanter BMD several years later, and after an initial decline in BMD whilst on GH replacement, lumbar spine and Ward's area BMD return towards their baseline values. These results emphasize that not all types of bone and skeletal sites respond to GH therapy identically. Furthermore a short course of GH replacement over 6-12 months may result in significant changes in BMD several years later.     

Use of amphotropic retroviral vectors for gene transfer in human colon carcinoma cells

Previous studies in rodent models have demonstrated the feasibility of gene transfer to the stem cells of the intestinal epithelium using ecotropic retroviral vectors delivered luminally. This report represents a next step toward targeting the human intestine as a site for somatic gene therapy. The first experiment assessed the viability of amphotropic retroviral vectors in the luminal environment. It was found that after 4 hr at 37 degrees C in luminal effluent, the loss of titer was no greater than when incubated in control media. Likewise, neither the vector nor the target cells were adversely affected by N-acetylcysteine, which is likely to be used as a preparatory agent for mucus removal. To determine whether human intestinal cells are transducible by these vectors, three colon carcinoma cell lines were studied: HT-29, T84, and Caco-2. All were transduced; however, the expression of the reporter gene was highest in the HT-29 cells. Subsequent studies using these cells showed that with regular stocks of vector, gene transfer peaked at a stock dilution of 1/10 and declined at full strength. This problem could be partially overcome by centrifugal concentration of the retroviral stocks. With this approach, gene transfer increased with increasing particles up to 10x regular stock titers but was inefficient at 100x. Overall, these findings provide encouraging evidence that amphotropic retroviral vectors may eventually be used for in vivo gene transfer into human intestinal epithelium. However, they also point to the need for improved methods of concentrating retroviral vectors.     

Effects of 5'-aminothymidine and leucovorin on radiosensitization by iododeoxyuridine in human colon cancer cells

We examined the effects of two biochemical modulators, 5'-aminothymidine (5'-AdThd) and leucovorin (LV), on the in vitro incorporation of iododeoxyuridine (IdUrd) into DNA and its subsequent radiosensitization in two human colon cancer cell lines, HT 29 and HCT 116. 5'-AdThd is a modulator of thymidine kinase activity while LV is an essential cofactor for thymidylate synthase activity. In HT 29 cells, the combination of 5'-AdThd (10 &mgr;m) and IdUrd (1-10 &mgr;m) resulted in a significant increase in IdUrd triphosphate pools and in IdUrd-DNA incorporation. Coadministration of LV (10 &mgr;m) with IdUrd (1-10 &mgr;m) resulted in a significant decrease in thymidine triphosphate pools and a comparable increase in IdUrd-DNA incorporation as the combination of 5'-AdThd + IdUrd. The increase in radiosensitization by clonogenic survival with either combination was a direct linear function with the percentage of IdUrd-DNA incorporation. For HCT 116 cells, however, the results were different. While 5'-AdThd + IdUrd resulted in an increase in IdUrd triphosphate and percentage of IdUrd-DNA incorporation, significant cytotoxicity was noted. The radiosensitivity of HCT 116 cells treated with 5'-AdThd + IdUrd was not a linear function above 25% IdUrd-DNA incorporation. Also, no increase in IdUrd-DNA incorporation or radiosensitization was observed with LV + IdUrd although LV enhanced the decrease in thymidine triphosphate pools by IdUrd treatment. These results indicate heterogeneity in the response of different colon cancer cells to these modulators which may be related to the regulation of deoxynucleotide metabolic enzymes.     

Wild-type p53 protein potentiates cytotoxicity of therapeutic agents in human colon cancer cells

Wild-type p53 is induced by DNA damage. In different cell types, this induction is suggested either to facilitate DNA repair by inducing a cell cycle pause or to potentiate cell death via apoptosis. Wild-type p53 in different cell types has similarly been associated with either enhancement of or increased resistance to the cytotoxicity of many cancer therapeutic agents. We have constructed a colorectal cancer cell line bearing, in addition to endogenous mutant p53 alleles, an exogenous wild-type p53 allele that is under the regulatable control of the lac repressor. Induction of wild-type p53 by isopropyl-beta-thiogalactopyranoside in these cells induces a reversible growth arrest but does not induce cell death. However, we find that the induction of wild-type p53 powerfully potentiates the cytotoxicity of both irradiation and 5-fluorouracil, two agents that are used clinically in the treatment of colorectal cancer. We also find that induction of wild-type p53 potentiates the cytotoxicity of topotecan, a member of the camptothecin family of drugs that also has clinical activity against colon cancer. These findings suggest that the common loss of wild-type p53 in many colorectal cancers may play a role in the clinical resistance of these tumors to anticancer agents. Although some cancer cells may not be directly killed by p53 gene therapy, our findings suggest that genetic alteration of some cancers to induce wild-type p53 may increase their sensitivity to cytotoxic gene therapy.     

Alterations of the PPP2R1B gene in human lung and colon cancer

The PPP2R1B gene, which encodes the beta isoform of the A subunit of the serine/threonine protein phosphatase 2A (PP2A), was identified as a putative human tumor suppressor gene. Sequencing of the PPP2R1B gene, located on human chromosome 11q22-24, revealed somatic alterations in 15% (5 out of 33) of primary lung tumors, 6% (4 out of 70) of lung tumor-derived cell lines, and 15% (2 out of 13) of primary colon tumors. One deletion mutation generated a truncated PP2A-Abeta protein that was unable to bind to the catalytic subunit of the PP2A holoenzyme. The PP2R1B gene product may suppress tumor development through its role in cell cycle regulation and cellular growth control.     

特异性下调GRP78基因表达对卵巢癌细胞耐药性的影响

目的:探讨RNA干扰技术沉默葡萄糖调节蛋白78(GRP78)基因对人卵巢癌细胞耐药性的影响,阐明GRP78基因沉默逆转肿瘤细胞耐药性的生物学机制.方法:构建pSilencerTM3.0-H1-GRP78 siRNA重组质粒,脂质体介导转染至SKOV3/DDP细胞;RT-PCR和Western blotting法检测GRP78基因的蛋白的表达;Western blotting法检测caspase-4和caspase-3蛋白表达;流式细胞术检测细胞凋亡率.结果:转染GRP78 siRNA重组质粒的SKOV3/DDP细胞GRP78基因和蛋白表达较转染空质粒组明显降低(P<0.05);加用顺铂后,转染GRP78 siRNA重组质粒细胞组较未转染GRP78 siRNA重组质粒细胞组caspase-4和caspase-3表达明显增加(P<0.05),细胞凋亡率也明显增加(P<0.05).结论:抑制GRP78基因表达能通过上调caspase-4和caspase-3表达及增加顺铂诱导的SKOV3/DDP细胞凋亡率,降低SKOV3/DDP细胞对顺铂的耐药性.     

Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells

BACKGROUND & AIMS: The epidermal growth factor receptor (EGFR) is under investigation as a therapeutic target for cancers. Colon cancer cell lines are variably dependent on autocrine stimulation of EGFR. We therefore examined the effects of a selective EGFR tyrosine kinase inhibitor, PD 153035, on proliferation and survival of five colon cancer cell lines whose autonomous proliferation is either EGFR ligand dependent or EGFR ligand independent. METHODS: Effects of inhibitors were screened by MTS growth assays, [3H]thymidine incorporation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, fluorescence microscopy, immunoblotting, and in vitro protease assays. RESULTS: PD 153035 caused dose-dependent cytostasis (200 nmol/L to 1 micromol/L) and apoptosis (>10 micromol/L) in ligand-dependent cell lines and caused variable apoptosis (>10 micromol/L) but no cytostasis in ligand-independent cell lines. Apoptosis induced by 10 micromol/L PD 153035 was not associated with induction of p53 protein expression but was accompanied by activation of caspases that cleave poly(ADP-ribose) polymerase, lamin B1, and Bcl-2. Inhibition of caspase 3-like protease activity by DEVD-fluoromethylketone significantly delayed the onset of PD 153035-induced apoptosis. CONCLUSIONS: The EGFR tyrosine kinase inhibitor PD 153035 induces cytostasis and caspase-dependent apoptosis in EGFR ligand-dependent colon cancer cell lines. These observations encourage further investigation of EGFR tyrosine kinase inhibitors for treatment of colorectal neoplasms.     

Saccharide-assisted delivery of cytotoxic liposomes to human malignant cells

The overexpression of lectins by malignant cells was applied for in vitro targeting of liposomes equipped with a saccharide vector and loaded in the lipid phase with a lipid derivative of anticancer agent sarcolysine. The lectin specificity of human leukemia HL-60 and human lung adenocarcinoma ACL cells was revealed by tests with fluorescein-labeled sugar probes. With the help of fluorescent lipid dye it was shown that active saccharide ligands increased the level of the vectored liposome binding to malignant cells by 50-80% as compared to liposomes without vector or with inactive one. The degree of liposome/cell membrane fusion was monitored fluorometrically and was shown to be complete and independent of the vectors. The targeted drug-loaded liposomes had the cytotoxic activity 2-4 times higher as compared to the vector-free ones.     

Oncogenic ras induces gastrin gene expression in colon cancer

BACKGROUND: Exposure to environmental tobacco smoke is associated with detrimental effects on pulmonary function in children. The authors investigated the relation between airway complications in children receiving general anesthesia and the passive inhalation of tobacco smoke. METHODS: Six hundred two children scheduled to receive general anesthesia were enrolled in this prospective study. The anesthesiologist and the recovery room nurse, unaware of the smoke exposure history, recorded the occurrence of airway complications. A history of passive smoking was assessed by measuring the urinary concentration of the major nicotine metabolite cotinine and by questionnaire. RESULTS: Airway complications occurred in 42% of the patients with urinary concentrations of cotinine > or =40 ng/ml, in 33% of the patients with concentrations of cotinine between 10.0 and 39.9 ng/ml, and in 24% of the patients with concentrations of cotinine < 10 ng/ml (P = 0.01 for the trend among the three groups). The gender of the child (P = 0.001) and the educational level of the child's mother (P = 0.0008) significantly modified the effect of the concentration of cotinine on the incidence of adverse respiratory events. CONCLUSIONS: There is a strong association between passive inhalation of tobacco smoke and airway complications in children receiving general anesthesia. The relationship is greatest for girls and for those whose mothers have a lower level of education. Passive smoking should be regarded as a risk factor in children undergoing general anesthesia.     

Suppression of invasive properties of colon cancer cells by a metastasis suppressor KAI1 gene

KAI1 is a potential metastatic suppressor gene for prostate cancer. We found by Northern blot analysis that six of ten (60%) gastric and colon cancer cell lines exhibited undetectable or very low expression level of KAI1 mRNA. The effects of KAI1 on the adhesion, motility and invasiveness of colon cancer cells was therefore investigated by using two kinds of stable transfectants, i.e., antisense transfectants of BM314 cells whose KAI1 mRNA expression was suppressed by transfer of antisense KAI1 cDNA and sense transfectants of DLD-1 cells with the enhanced KAI1 mRNA by sense cDNA transfer. The following results were obtained: (1) KAI1 gene expression had no significant effect on in vitro cell growth rate of colon cancer BM314 and DLD-1 cells; (2) Cell aggregation assay showed that KAI1 enhanced the Ca++-independent aggregatability of those colon cancer cells; (3) It was revealed by cell motility and invasion assays that KAI1 suppressed both the motility and in vitro invasiveness of those cells and (4) Furthermore, both the binding to fibronectin and the migration on fibronectin-coated plates of those cells were inhibited by KAI1 expression. These suggest that reduced KAI1 gene expression may contribute to the invasiveness and metastatic ability of colon cancer cells.     

Activation of c-Src by receptor tyrosine kinases in human colon cancer cells with high metastatic potential

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two different sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Specifically, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highly-metastatic cells. EGFR was found to associate with c-Src in colon cancer cells and specific inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identified in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.     

Apoptosis-prone phenotype of human colon carcinoma cells with a high level amplification of the c-myc gene

Although apoptosis can be induced by the enforced expression of exogenously introduced c-myc genes, it is not clear whether overexpression resulting from the amplification of the resident c-myc gene in tumor cells is sufficient to induce apoptosis. We have investigated the relationship between c-myc gene amplification and the propensity of tumor cells to undergo apoptosis, using the SW613-12A1 and SW613-B3 cell lines, which are representatives, respectively, of tumorigenic and non-tumorigenic clones isolated from the SW613-S human colon carcinoma cell line. Tumorigenic clones are characterized by a high level of amplification and expression of the c-myc gene, whereas cells of non-tumorigenic clones have a small number of copies and a lower level of expression of this gene. Analysis of c-myc mRNA level in cells cultured under low serum conditions indicated that the expression of the gene is tightly regulated by serum growth factors in non-tumorigenic B3 cells, whereas it is poorly regulated in tumorigenic 12A1 cells, the level of mRNAs remaining relatively high in serum-starved 12A1 cells. Under these conditions, 12A1 cells showed clear evidence of apoptosis, whereas B3 cells were completely refractory to the induction of apoptosis. Moreover, the study of cell lines derived from non-tumorigenic apoptosis-resistant clones following the introduction by transfection of exogenous c-myc gene copies showed that they have acquired an apoptosisprone phenotype. Altogether, our results strongly suggest that deregulated c-myc expression due to high-level amplification confers an apoptosis-prone phenotype to tumor cells. The possible consequences of these observations for cancer therapy are discussed.