Association of nursing overtime, nurse staffing and unit occupancy with medical incidents and outcomes of very preterm infants.

OBJECTIVE: To examine the association of nursing overtime, nursing provision and unit occupancy rate with medical incident rates in the neonatal intensive care unit (NICU) and the risk of mortality or major morbidity among very preterm infants. STUDY DESIGN: Single center retrospective cohort study of infants born within 23 to 29 weeks of gestational age or birth weight <1000 g admitted at a 56 bed, level III NICU. Nursing overtime ratios (nursing overtime hours/total nursing hours), nursing provision ratios (nursing hours/recommended nursing hours based on patient dependency categories) and unit occupancy rates were pooled for all shifts during NICU hospitalization of each infant. Log-binomial models assessed their association with the composite outcome (mortality or major morbidity). RESULTS: Of the 257 infants that met the inclusion criteria, 131 (51%) developed the composite outcome. In the adjusted multivariable analyses, high (>3.4%) relative to low nursing overtime ratios (⩽3.4%) were not associated with the composite outcome (relative risk (RR): 0.93; 95% confidence interval (CI): 0.86 to 1.02). High nursing provision ratios (>1) were associated with a lower risk of the composite outcome relative to low ones (⩽1) (RR: 0.81; 95% CI: 0.74 to 0.90). NICU occupancy rates were not associated with the composite outcome (RR: 0.98; 95% CI: 0.89 to 1.07, high (>100%) vs low (⩽100%)). Days with high nursing provision ratios (>1) were also associated with lower risk of having medical incidents (RR: 0.91; 95% CI: 0.82 to 0.99). CONCLUSION: High nursing provision ratio during NICU hospitalization is associated with a lower risk of a composite adverse outcome in very preterm infants.

Educación sobre la prevención del dolor de espalda impartida a niños en las escuelas desde la Asistencia Primaria / Education in the prevention of backache imparted to school children by primary care workers

Objetivo: Estudio sobre consejos de higiene, con respecto a la postura y la prevención del dolor de espalda, impartidos a niños en el ámbito escolar desde la Asistencia Primaria. Metodología: Durante dos años, se efectuaron clases dirigidas a escolares del último curso de primer grado en las tres escuelas públicas pertenecientes al Área Básica de Salud. En estos escolares se realizó un estudio descriptivo de tipo observacional, seleccionando una muestra de 37 alumnos escogida aleatoriamente, según el método aleatorizado simple, que se llevó a cabo distribuyendo una encuesta previamente validada, que constaba de 7 ítems con preguntas sobre higiene postural, dolor de espalda y práctica de ejercicio físico. A 21 de estos alumnos se les distribuyó la encuesta antes de efectuar la clase de educación sanitaria, y a otros 16 se les distribuyó después de haber realizado la clase; posteriormente, se les pusieron tres ejemplos prácticos para ver sí los consejos impartidos se habían asimilado o no: recoger un objeto del suelo, coger un objeto puesto en lo alto de una estantería y sentarse en la mesa para escribir. Resultados: Estas clases se dirigieron a un total de 108 escolares que asistían al último curso de primer grado, en las tres escuelas públicas del Área Básica de Salud, durante los dos últimos años y también a los padres en el propio Centro de Salud, con muy buena aceptación por parte de todos, manifestando quedar satisfechos el 98 por ciento de los entrevistados. Por otra parte, al menos uno de cada dos niños manifestó haber tenido, al menos una vez en su vida, dolor de espalda. La distribución por sexos era del 51 por ciento de niños y del 49 por ciento de niñas. Respecto a los niños a los que se les distribuyó la encuesta antes de la clase, sólo el 1 por ciento consideraba que el dolor de espalda era debido a mala higiene de la postura, el 60 por ciento de ellos consideró que era mejor dormir en cama blanda, y sólo el 19 por ciento manifestó no efectuar habitualmente ejercicio físico. Todos los niños que contestaron la encuesta después de recibir la clase de educación sanitaria consideraban que el dolor de espalda era debido a una mala postura (excepto uno que lo atribuía también a enfermedad), el 63 por ciento estimo que era mejor dormir en cama dura y el 18 por ciento estimó que no hacían ejercicio regularmente. Después de la educación sanitaria, el 99 por ciento de ellos sabía que lo mejor para coger un objeto del suelo era hacerlo agachado en cuclillas, el 81 por ciento sabía que no podía estirar demasiado la espalda para coger un objeto que estuviera alto y que lo mejor era subirse a una banqueta, y el 99 por ciento había aprendido a sentarse correctamente en la mesa, con la espalda recostada en el respaldo de la silla. Conclusiones: La valoración de la educación sanitaria ha sido muy buena tanto por parte de los escolares, como de los padres y los profesores. Parece que, al menos a corto plazo, se asimilaron bien los cambios de conocimiento y actitud para prevenir el dolor de espalda. Se ha considerado, por tanto, que el dar consejos sobre higiene postural y práctica de ejercicio físico puede resultar una medida muy eficaz en la prevención del dolor de espalda (AU)

Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT(388)-granulocyte-macrophage colony-stimulating factor (GM-CSF), a truncated diphtheria toxin fused to human GM-CSF.

DT(388)-GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia. HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT(388)-GM-CSF on metabolism of ceramide, a modulator of apoptosis. After 48 hours with DT(388)-GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence. Similar results were obtained in HL-60 cells. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT(388)-GM-CSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50%. Exposure to C(6)-ceramide inhibited protein synthesis (EC(50) approximately 5 microM) and decreased viability (EC(50) approximately 6 microM). Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis. Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT(388)-GM-CSF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.

High-dose tamoxifen added to concurrent biochemotherapy with decrescendo interleukin-2 in patients with metastatic melanoma.

BACKGROUND: In vitro cell culture data and preclinical models suggest that tamoxifen modulates tumor cell sensitivity to a wide range of therapeutic agents. In the current study, the authors examined whether high-dose tamoxifen (HDT) improved the overall and complete response in patients with metastatic melanoma who were treated with concurrent biochemotherapy. METHODS: Forty-nine patients were treated with a biochemotherapy regimen of dacarbazine, vinblastine, cisplatin, decrescendo interleukin-2, interferon-alpha-2b, and tamoxifen. The study had a 2-step design, beginning with a tamoxifen dose escalation from 40 mg to 320 mg (17 subjects) to evaluate safety and tolerability, followed by Phase II accrual of 32 patients to HDT (320 mg) to assess clinical efficacy. Efficacy was compared with a similar modified biochemotherapy regimen with low-dose tamoxifen (LDT). Pharmacokinetic studies were performed to determine in vivo tamoxifen levels. RESULTS: Tamoxifen dose escalation was completed without any reported dose-limiting toxicity. The overall response rate in the HDT group was 50% (95% confidence interval, 33.2%-66.8%), with a complete response rate of 6% and a median survival of 9.5 months. The overall response rate was not improved and the complete response and survival appeared inferior compared with that of patients recently treated with concurrent biochemotherapy and LDT. Serum tamoxifen levels were found to correlate with the dose administered, with a mean of 0.9 microM at the 40-mg dose to 4.6 microM at the 320-mg dose. Ultrafiltered protein-free sera demonstrated low (< 0.01 microM) concentrations of tamoxifen. CONCLUSIONS: The addition of HDT to a regimen of concurrent biochemotherapy did not appear to improve response rates or overall survival, despite reaching the targeted plasma concentration. Unknown drug interactions or high protein binding of tamoxifen may account for the lack of clinical effectiveness.

N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138-1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [(3)H]palmitic acid, caused sequential formation of [(3)H]sphinganine (220% over control) and [(3)H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B(1), an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB(1) were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 microM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 microM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.

Taxol-induced ceramide generation and apoptosis in human breast cancer cells.

PURPOSE: Taxol has emerged as a valuable antimitotic chemotherapeutic agent, particularly in advanced breast and ovarian cancers. Although much is known about cytotoxic mechanisms, the effectiveness of Taxol cannot be solely explained by microtubular interaction. This study was undertaken to determine whether ceramide generation plays a role in Taxol-induced apoptosis. METHODS: Hormone-independent MDA-MB-468 and hormone-dependent MCF-7 breast cancer cell lines were employed, and ceramide metabolism was characterized using [3H]palmitic acid as lipid precursor. RESULTS: Exposure of cells to Taxol resulted in enhanced formation of [3H]ceramide. Ceramide increased nearly 2-fold in MDA-MB-468 cells exposed to 50 nM Taxol, and more than 2.5-fold in MCF-7 cells exposed to 1.0 microM Taxol. These concentrations mirrored the EC50 (amount of drug eliciting 50% cell kill) for Taxol in the two cell lines. Use of cell-permeable C6-ceramide as a medium supplement revealed that MDA-MB-468 cells were 20-fold more sensitive to ceramide than MCF-7 cells (P < 0.001). Ceramide was generated as early as 6 h after exposure to Taxol in MDA-MB-468 cells, whereas the earliest signs of apoptosis were detected 12 h after treatment, and by 24 h the apoptotic index was six times that of untreated cells. Both fumonisin B1, a ceramide synthase inhibitor, and L-cycloserine, a serine palmitoyltransferase inhibitor, blocked Taxol-induced ceramide generation, whilst sphingomyelin levels remained unchanged, indicating a de novo pathway of ceramide formation. L-Cycloserine reduced Taxol-induced apoptosis by 30% in MDA-MB-468 cells and totally blocked Taxol-induced apoptosis in MCF-7 cells. CONCLUSIONS: These results suggest that Taxol-induced apoptosis is, in part, attributable to ceramide and sphingoid bases. This is of relevance to drug mechanism studies, as ceramide is a known messenger of apoptosis. Clinical use of Taxol with ceramide-enhancing agents may maximize cytotoxic potential.

Targeting ceramide metabolism--a strategy for overcoming drug resistance.

Inherent or acquired drug resistance, which frequently characterizes cancer cells, is caused by multiple mechanisms, including dysfunctional metabolism of the lipid second messenger ceramide. Ceramide, the basic structural unit of the sphingolipids, plays a role in activating cell death signals initiated by cytokines, chemotherapeutic agents, and ionizing radiation. Recent discoveries about the metabolism of ceramide suggest that this agent may have an important influence on the effectiveness of various cancer therapeutics. In particular, the cytotoxic effect of chemotherapy is decreased when generation of ceramide is impaired but is increased when the degradation of ceramide is blocked. Herein, we review the mechanisms of resistance to chemotherapeutic agents in terms of ceramide metabolism.

Ceramide glycosylation potentiates cellular multidrug resistance.

Ceramide glycosylation, through glucosylceramide synthase (GCS), allows cellular escape from ceramide-induced programmed cell death. This glycosylation event confers cancer cell resistance to cytotoxic anticancer agents [Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140-1146]. We previously found that glucosylceramide, the glycosylated form of ceramide, accumulates in adriamycin-resistant breast carcinoma cells, in vinblastine-resistant epithelioid carcinoma cells, and in tumor specimens from patients showing poor response to chemotherapy. Here we show that multidrug resistance can be increased over baseline and then totally reversed in human breast cancer cells by GCS gene targeting. In adriamycin-resistant MCF-7-AdrR cells, transfection of GCS upgraded multidrug resistance, whereas transfection of GCS antisense markedly restored cellular sensitivity to anthracyclines, Vinca alkaloids, taxanes, and other anticancer drugs. Sensitivity to the various drugs by GCS antisense transfection increased 7- to 240-fold and was consistent with the resumption of ceramide-caspase-apoptotic signaling. GCS targeting had little influence on cellular sensitivity to either 5-FU or cisplatin, nor did it modify P-glycoprotein expression or rhodamine-123 efflux. GCS antisense transfection did enhance rhodamine-123 uptake compared with parent MCF-7-AdrR cells. This study reveals that GCS is a novel mechanism of multidrug resistance and positions GCS antisense as an innovative force to overcome multidrug resistance in cancer chemotherapy.

Synergistic cytotoxicity in solid tumor cell lines between N-(4-hydroxyphenyl)retinamide and modulators of ceramide metabolism.

BACKGROUND: We previously reported that N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) treatment caused large increases of ceramide levels in neuroblastoma cell lines and induced cell death by a combination of apoptosis and necrosis through p53 (also known as TP53)-independent and caspase-independent pathways. Our goal was to determine if several molecules that inhibit enzymes involved in ceramide metabolism-L-threo-dihydrosphingosine (safingol), d, l-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), and tamoxifen-enhanced 4-HPR-mediated cytotoxicity and/or affected ceramide levels. METHODS: Cellular lipids were quantified by radiolabeling and thin-layer chromatography. Cytotoxicity and cytotoxic synergy (expressed as combination index, where combination index <1 indicates synergy and >1 indicates antagonism) were measured in cultured cancer cell lines with the use of a fluorescence-based assay of cell viability employing digital imaging microscopy. Statistical tests were two-sided. RESULTS: 4-HPR increased ceramide levels by de novo synthesis. Safingol (1-4 microM) was incorporated into a stereochemical variant of ceramide and synergized with a 3:1 molar ratio of 4-HPR (3-12 microM), to produce a 100-fold to 10 000-fold (2 to 4 logs) increase in cytotoxicity relative to 4-HPR alone in neuroblastoma (combination index <0.1), lung (combination index <0.1-0.2), melanoma (combination index <0.1-0.2), prostate (combination index <0.1-1.0), colon (combination index 0.1-0.3), breast (combination index = 0.1-0.5), and pancreas (combination index = 0.2) cell lines, including p53 mutant and alkylator-resistant cell lines. The 4-HPR and safingol combination was cytotoxic in low-oxygen conditions and was minimally toxic to normal fibroblasts and bone marrow myeloid progenitor cells. Addition of agents that retard ceramide glucosylation and/or acylation, such as PPMP or tamoxifen, to 4-HPR or to the combination of 4-HPR and safingol further increased cytotoxicity to tumor cells. CONCLUSIONS: Combinations of 4-HPR and modulators of ceramide metabolism may form the basis for a novel chemotherapy that is functional under hypoxic conditions (e.g., such as those within tumors) and is p53 independent and caspase independent.

SDZ PSC 833 the drug resistance modulator activates cellular ceramide formation by a pathway independent of P-glycoprotein.

SDZ PSC 833 (PSC 833) is a new multidrug resistance modulator. Recent studies have shown that the principal mechanism of action of PSC 833 is to bind P-glycoprotein (P-gp) and prevent cellular efflux of chemotherapeutic drugs. We previously reported that PSC 833 increases cellular ceramide levels. The present study was conducted to determine whether the impact of PSC 833 on ceramide generation is dependent on P-gp. Work was carried out using the drug-sensitive P-gp-deficient human breast adenocarcinoma cell line, MCF-7, and drug resistant MCF-7/MDR1 clone 10.3 cells (MCF-7/MDR1), which show a stable MDR1 P-gp phenotype. Overexpression of P-gp in MCF-7/MDR1 cells did not increase the levels of glucosylceramide, a characteristic which has been associated with multidrug resistant cells. Treatment of MCF-7 and MCF-7/MDR1 cells with PSC 833 caused similar ceramide elevation, in a dose-responsive manner. At 5.0 microM, PSC 833 increased ceramide levels 4- to 5-fold. The increase in ceramide levels correlated with a decrease in survival in both cell lines. The EC50 (concentration of drug that kills 50% of cells) for PSC 833 in MCF-7 and MCF-7/MDR1 cells was 7.2 +/- 0.6 and 11.0 +/- 1.0 microM, respectively. C6-Ceramide exposure diminished survival of MCF-7 cells; whereas, MCF-7/MDR1 cells were resistant to this short chain ceramide analog. Preincubation of cells with cyclosporine A, which has high affinity for P-gp, did not diminish the levels of ceramide generated upon exposure to PSC 833. These results demonstrate that PSC 833-induced cellular ceramide formation occurs independently of P-gp. As such, these data indicate that reversal of drug resistance by classical P-gp blockers may be modulated by factors unrelated to drug efflux parameters.

Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance.

Previous work from our laboratory demonstrated that increased competence to glycosylate ceramide conferred adriamycin resistance in MCF-7 breast cancer cells (Liu, Y. Y., Han, T. Y., Giuliano, A. E. , and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140-1146). This was achieved by cellular transfection with glucosylceramide synthase (GCS), the enzyme that converts ceramide to glucosylceramide. With this, we hypothesized that a decrease in cellular ceramide glycosylation would result in heightened drug sensitivity and reverse adriamycin resistance. To down-regulate ceramide glycosylation potential, we transfected adriamycin-resistant breast cancer cells (MCF-7-AdrR) with GCS antisense (asGCS), using a pcDNA 3.1/his A vector and developed a new cell line, MCF-7-AdrR/asGCS. Reverse transcription-polymerase chain reaction assay and Western blot analysis revealed marked decreases in both GCS mRNA and protein in MCF-7-AdrR/asGCS cells compared with the MCF-7-AdrR parental cells. MCF-7-AdrR/asGCS cells exhibited 30% less GCS activity by in vitro enzyme assay (19.7 +/- 1.1 versus 27.4 +/- 2.3 pmol GC/h/microg protein, p < 0.001) and were 28-fold more sensitive to adriamycin (EC(50), 0.44 +/- 0.01 versus 12.4 +/- 0.7 microM, p < 0. 0001). GCS antisense transfected cells were also 2.4-fold more sensitive to C(6)-ceramide compared with parental cells (EC(50) = 4. 0 +/- 0.03 versus 9.6 +/- 0.5 microM, p < 0.0005). Under adriamycin stress, GCS antisense transfected cells compared with parental cells displayed time- and dose-dependent increases in endogenous ceramide and dramatically higher levels of apoptotic effector, caspase-3. Western blotting showed that adriamycin sensitivity, introduced by asGCS gene transfection, was independent of P-glycoprotein and Bcl-2 expression. In summary, this work shows that transfection of GCS antisense tempers the expression of native GCS and restores cell sensitivity to adriamycin. Therefore, limiting the potential to glycosylate ceramide, which is an apoptotic signal in chemotherapy and radiotherapy, provides a promising approach to combat drug resistance.

Glycosylation of ceramide potentiates cellular resistance to tumor necrosis factor-alpha-induced apoptosis.

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-alpha has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC(50) for TNF-alpha, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-alpha had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-alpha resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-alpha.

Multidrug resistance modulators and doxorubicin synergize to elevate ceramide levels and elicit apoptosis in drug-resistant cancer cells.

BACKGROUND: To provide insight for the development of more effective clinical agents, the authors attempted to elucidate the mechanisms of action of multidrug resistance (MDR) modulators. Previously, the authors found that MDR modulators blocked the conversion of ceramide to glucosylceramide in MDR cells, thereby enhancing cytotoxicity. Because ceramide is a critical component of the apoptosis signaling cascade, the current study examined the impact of therapy using agents that elicit ceramide formation combined with agents that block ceramide glycosylation. METHODS: Doxorubicin-resistant human breast carcinoma cells (MCF-7-AdrR) were treated with either doxorubicin, tamoxifen, cyclosporine A, or the cyclosporine A analog SDZ PSC 833 (PSC 833) or with combinations thereof, and ceramide and glucosylceramide metabolisms were measured by cell radiolabeling. Cell viability was quantitated spectrophotometrically and apoptosis was evaluated analyzing DNA integrity by gel electrophoresis. RESULTS: Whereas cyclosporine A blocked the generation of glucosylceramide in MCF-7-AdrR cells, a chemical cousin, PSC 833, elicited a 3-fold increase in glucosylceramide and a 5-fold increase in ceramide levels at 24 hours. The PSC 833 response was time-dependent(as early as 30 minutes) and dose-dependent (as low as 0.1 microM). The appearance of ceramide foreran the generation of glucosylceramide. Sphingomyelin levels were not decreased in response to PSC 833; however, Fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Adding tamoxifen, which blocks ceramide glycosylation, to the PSC 833 regimen boosted ceramide levels 11-fold over controls and caused DNA fragmentation. A 3-component regimen comprised of tamoxifen, doxorubicin, and PSC 833 increased ceramide levels 26-fold and brought cell viability to zero. CONCLUSIONS: These results demonstrate that MDR modulators can be used separately, in combination, or in conjunction with chemotherapy at clinically relevant concentrations to manipulate cellular ceramide levels and restore sensitivity in the drug resistant setting. As such, this represents a new direction in the treatment of cancer.

Ceramide toxicity and metabolism differ in wild-type and multidrug-resistant cancer cells.

Previously we demonstrated that multidrug-resistant (MDR) cancer cells have elevated levels of a glycosylated form of ceramide, glucosylceramide. Here we compared ceramide metabolism and ceramide toxicity in MCF-7 and in adriamycin-resistant (MCF-7-AdrR) human breast cancer cells. MCF-7-AdrR cells were resistant to C6-ceramide (1-10 microM); however, in MCF-7 cells treated with C6-ceramide, viability dropped sharply. Ceramide, when supplemented, was not metabolized by MCF-7 cells. In contrast, ceramide was efficiently converted to glucosylceramide by MCF-7-AdrR cells. Analysis of extracellular [3H]ceramide in radiolabeled cells showed that MCF-7-AdrR cells do not have an enhanced capacity to efflux ceramide compared with MCF-7 cells. Triphenylethylene anti-estrogens, known modulators of drug resistance, were effective inhibitors of ceramide conversion to glucosylceramide, suggesting that blocking ceramide metabolism plays a role in chemosensitization. The anti-progestine, RU486, also blocked glucosylceramide synthesis in cells; however, LY117018, a raloxifene analog, was without influence. We propose that an enhanced capacity to glycosylate ceramide as evidenced in MCF-7-AdrR cells, is a molecular determinant of drug resistance, particularly as regards resistance to ceramide-enhancing agents such as anthracyclines, ionizing radiation, and tumor necrosis factor-alpha.

Modification of ceramide metabolism increases cancer cell sensitivity to cytotoxics.

In the preceding report we demonstrated that MCF-7-AdrR cells (adriamycin resistant) were insensitive to ceramide, whereas MCF-7 wild-type cells were sensitive. It was also shown that the drug resistant cells had an increased capacity to convert ceramide to glucosylceramide. Here we demonstrate that blocking the conversion of ceramide to glucosylceramide increases MCF-7-AdrR cell sensitivity to ceramide as well as to antitumor agents. Treatment of MCF-7 cells with adriamycin elicited a 5-fold increase in ceramide, and caused oligonucleosomal fragmentation, characteristic to apoptosis. Under similar treatment conditions, ceramide was not generated in MCF-7-AdrR cells. In MCF-7-AdrR cells neither C6-ceramide nor tamoxifen was cytotoxic; however, the addition of tamoxifen to the C6-ceramide treatment regimen reduced cell viability to 42% and elicited apoptosis. Treatment of MCF-7-AdrR cells with Adriamycin promoted an increase in ceramide only if tamoxifen was present, in which case ceramide increased 7-fold, and cell viability decreased to 50%. The employment of another agent, RU486 (Mifepristone), which blocks ceramide glycosylation, increased MCF-7-AdrR cell sensitivity to adriamycin in a dose-dependent manner. Our data show that agents that block ceramide glycosylation potentiate cellular sensitivity to ceramide and to chemotherapeutic drugs, and suggest that the ceramide metabolic pathway is an important target for anticancer drug development.

Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)- retinamide in neuroblastoma cell lines.

BACKGROUND: The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR or fenretinide) is toxic to myeloid leukemia and cervical carcinoma cell lines, probably in part due to its ability to increase levels of reactive oxygen species (ROS). We have studied the effects of 4-HPR on neuroblastoma cell lines. Since neuroblastomas commonly relapse in bone marrow, a hypoxic tissue compartment, and many chemotherapeutic agents are antagonized by hypoxia, our purpose was to study in these cell lines several factors influencing 4-HPR-induced cytotoxicity, including induced levels of ROS, effects of physiologic hypoxia and antioxidants, levels of ceramide, and the mechanism of cell death. METHODS: ROS generation was measured by carboxydichlorofluorescein diacetate fluoresence. Ceramide was quantified by radiolabeling and thin-layer chromatography. Immunoblotting was used to assess p53 protein levels. Apoptosis (programmed cell death) and necrosis were analyzed by nuclear morphology and internucleosomal DNA fragmentation patterns. Cytotoxicity was measured by a fluorescence-based assay employing digital imaging microscopy in the presence or absence of the pancaspase enzyme inhibitor BOC-d-fmk. Statistical tests were two-sided. RESULTS/CONCLUSIONS: In addition to increasing ROS, 4-HPR (2.5-10 microM) statistically significantly increased the level of intracellular ceramide (up to approximately 10-fold; P<.001) in a dose-dependent manner in two neuroblastoma cell lines, one of which is highly resistant to alkylating agents and to etoposide. Cell death induced by 4-HPR was reduced but not abrogated by hypoxia in the presence or absence of an antioxidant, N-acetyl-L-cysteine. Expression of p53 protein was not affected by 4-HPR. Furthermore, the pan-caspase enzyme inhibitor BOC-d-fmk prevented apoptosis, but not necrosis, and only partially decreased cytotoxicity induced by 4-HPR, indicating that 4-HPR induced both apoptosis and necrosis in neuroblastoma cells. IMPLICATIONS: 4-HPR may form the basis for a novel, p53-independent chemotherapy that operates through increased intracellular levels of ceramide and that retains cytotoxicity under reduced oxygen conditions.

SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells.

Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effective treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and [3H]palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833) activates ceramide synthesis. In a short time course experiment, ceramide was generated as early as 15 min (40% increase) after the addition of PSC 833 (5.0 microM), and by 3 h, [3H]ceramide was >3-fold that of control cells. A 24-h dose-response experiment showed that at 1.0 and 10 microM PSC 833, ceramide levels were 2.5- and 13.6-fold higher, respectively, than in untreated cells. Concomitant with the increase in cellular ceramide was a progressive decrease in cell survival, suggesting that ceramide elicited a cytotoxic response. Analysis of DNA in cells treated with PSC 833 showed oligonucleosomal DNA fragmentation, characteristic of apoptosis. The inclusion of fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Assessment of ceramide mass by TLC lipid charring confirmed that PSC 833 markedly enhanced ceramide synthesis, not only in KB-V-1 cells but also in wild-type KB-3-1 cells. The capacity of PSC 833 to reverse drug resistance was demonstrated with vinblastine. Whereas each agent at a concentration of 1.0 microM reduced cell survival by approximately 20%, when PSC 833 and vinblastine were coadministered, cell viability fell to zero. In parallel experiments measuring ceramide metabolism, it was shown that the PSC 833/vinblastine combination synergistically increased cellular ceramide levels. Vinblastine toxicity, also intensified by PSC 833 in wild-type KB-3-1 cells, was as well accompanied by enhanced ceramide formation. These data demonstrate that PSC 833 has mechanisms of action in addition to P-glycoprotein chemotherapy efflux pumping.

Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells.

Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682-1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an 11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to adriamycin and ceramide in MCF-7 breast cancer cells.

The multidrug resistance modulator SDZ PSC 833 is a potent activator of cellular ceramide formation.

In this study we demonstrate that the multidrug resistance (MDR) modulator PSC 833 is a potent agonist of ceramide metabolism. When added with [3H]serine or [3H]palmitic acid to the culture medium of MCF-7 cells, PSC 833, in a dose-responsive fashion (1-10 microM), increased the levels of [3H]ceramide as much as 16-fold over control. The actual increase in ceramide mass was verified by thin-layer chromatographic chars. Cellular sphingomyelin radioactivity did not decrease during treatment, indicating that PSC 833 does not elicit ceramide formation through a sphingomyelinase pathway. Inclusion of fumonisin B1, an inhibitor of ceramide synthase, blocked formation of ceramide by PSC 833. The results of cell proliferation assays demonstrated a clear correlation between PSC 833 elicitation of ceramide formation and increased cytotoxicity. The MDR modulator and chemical cousin of PSC 833, cyclosporin A, had little impact on cellular ceramide formation. At a concentration of 2.5 microM, cyclosporin A and PSC 833 treatment increased ceramide formation by 20% and 7.5-fold, respectively. These results reveal a new action of PSC 833 which may contribute to its potency as a drug resistance modulator.

Tamoxifen induces selective membrane association of protein kinase C epsilon in MCF-7 human breast cancer cells.

Tamoxifen, a synthetic antiestrogen, is known for its antitumoral action in vivo; however, it is well accepted that many tamoxifen effects are elicited via estrogen receptor-independent routes. Previously, we reported that tamoxifen induces PKC translocation in fibroblasts. In the present study, we investigated the influence of tamoxifen, and several triphenylethylene derivatives, on protein kinase C (PKC) in MCF-7 human breast cancer cells. As measured by Western blot analysis, tamoxifen elicited isozyme-specific membrane association of PKC-epsilon, which was time-dependent (as early as 5 min post-treatment) and dose-dependent (5.0-20 microM). Tamoxifen did not influence translocation of alpha, beta, gamma, delta or zeta PKC isoforms. Structure-activity relationship studies demonstrated chemical requirements for PKC-epsilon translocation, with tamoxifen, 3-OH-tamoxifen and clomiphene being active. Compounds without the basic amino side chain, such as triphenylethylene, or minus a phenyl group, such as N,N-dimethyl-2-[(4-phenylmethyl)phenoxy]ethanamine, were not active. In vitro cell growth assays showed a correlation between agent-induced PKC-epsilon translocation and inhibition of cell growth. Exposure of cells to clomiphene resulted in apoptosis. Since PKC-epsilon has been associated with cell differentiation and cellular growth-related processes, the antiproliferative influence of tamoxifen on MCF-7 cells may be related to the interaction with PKC-epsilon.