Human Fibroblast WI-38 Cells Research Articles

Elimination of radiation-induced senescent cancer cells and stromal cells invitro by near-infrared photoimmunotherapy.

Therapy-induced senescent cancer and stromal cells secrete cytokines and growth factors to promote tumor progression. Therefore, senescent cells may be novel targets for tumor treatment. Near-infrared photoimmunotherapy (NIR-PIT) is a highly tumor-selective therapy that employs conjugates of a molecular-targeting antibody and photoabsorber. Thus, NIR-PIT has the potential to be applied as anovel senolytic therapy. This study aims to investigate the efficacy of NIR-PIT treatment on senescent cancer and stromal cells. Two cancer cell lines (human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells) and two normal cell lines (mouse fibroblast transfected with human epidermal growth factor receptor 2 [HER2] cells and human fibroblast WI38 cells) were used. The cytotoxicity of NIR-PIT was evaluated using anti-epidermal growth factor receptor (EGFR) antibody panitumumab and anti-HER2 antibody transtuzumab. Cellular senescence was induced in A549 and MIA PaCa-2 cells by 10 Gy γ-irradiation. The up-regulation of cellular senescence markers and characteristic morphological changes in senescent cells, including enlargement, flattening, and multinucleation, were observed in cancer cells after 5 days of γ-irradiation. Then, NIR-PIT targeting EGFR was performed on these senescent cancer cells. The NIR-PIT induced morphological changes, including bleb formation, swelling, and the inflow of extracellular fluid, and induced a significant decrease in cellular viability. These results suggested that NIR-PIT may induce cytotoxicity using the same mechanism in senescent cancer cells. In addition, similar morphological changes were also induced in radiation-induced senescent 3T3-HER2 fibroblasts by NIR-PIT targeting human epidermal growth factor receptor 2. NIR-PIT eliminates both senescent cancer and stromal cells invitro suggesting it may be a novel strategy for tumor treatment.

In Vitro of Melaleuca viridiflora Sol. ex Gaertn Plant Investigation of Antimicrobial, Anticancer and Cytotoxic Activities

In this study; it was aimed to investigate the anticancer and cytotoxic activities on DU-145 prostate cancer and MCF-7 breast cancer cell lines and WI-38 human fibroblast cell line of essential oil (Nioli) obtained from Melaleuca viridiflora Sol. ex Gaertn. plant and its antimicrobial effects on various bacteria and yeast cells. Content analyzes of Nioli essential oil were made by Gas Chromatography-Mass Spectrometry (GK-KS). The antimicrobial effects of the essential oil were determined using Disk Diffusion and Minimum Inhibition Concentration (MIC) tests. Its cytotoxic effects were determined by the XTT (2,3-bis-(2-methoxy-4-) nitro-5)-sulfophenyl)-2H-tetrazolium-5-carboxanilide) test; were investigated at different concentrations in breast cancer (MCF-7), prostate cancer (DU-145) and healthy human fibroblast (WI-38) cell lines. In the disc diffusion method of essential oil; It was determined that it formed an inhibition zone against Klebsiella pneumoniae bacteria at 200, 100, 50, 25 and 12.5 µg/mL concentrations, and against Candida albicans yeast at 200 and 100 µg/mL concentrations. Nioli essential oil showed the highest antibacterial effect against Bacillus cereus. While it was moderately effective against Pseudomonas aeruginosa and Candida albicans, it reached MIC values effective against other bacteria. It was observed that the anticancer activity of Nioli essential oil was more effective in DU-145 prostate cancer cells compared to MCF-7 breast cancer cells, and it had no toxicity in WI-38 healthy human fibroblast cells.

Anti-Cancer Effect of Moroccan Cobra Naja haje Venom and Its Fractions against Hepatocellular Carcinoma in 3D Cell Culture.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of Moroccan cobra Naja haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with a low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of Moroccan cobra Naja haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cells proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.

Effect of Vitamin C Administration 24 Hours After 5-Fluorouracil Versus its Concomitant Administration on Cellular Proliferation, Cell Death and Lipid Peroxidation in WI-38 Human Fibroblast Cell Line

Abstract:5-Fluorouracil (5-FU) is a routine anticancer agent for several cancer types, its short half-life requires continuous administration in high doses. This results in toxic side-effects to the normal tissues in the body. Vitamin C is an antioxidant which believed to impart cyto-protection to normal tissues in cancer patients treated with chemotherapeutic drugs.Aim of the study: to assess the potential benefit of vitamin C administration 24 hours after 5-FU treatment compared to its immediate supplementation on normal fibroblast cell line.Materials and methods: fibroblast cell line (WI-38) was divided into 5 groups. Group I not treated (control group), group II treated with 5-FU only and group III received vitamin C only. Groups IV and V received 5-FU with vitamin C simultaneously and after 24 hours of 5-FU administration, respectively. The viability of cells in all groups was determined using MTT assay. MDA Quantification Assay was used to determine lipid peroxidation. Flow cytometry was used for evaluation of the cell cycle and cell death. All recorded data was statistically analyzed.Results: Cells in group IV seems like group II fewer and rounded while in group V some of them adherent spindle and the others rounded non-adherent. The least viability was noted in group II. MDA levels were significantly lower in groups III compared to all the other groups. Cell cycle analysis revealed the presence of statistically significant differences between groups in different phases of the cycle. Flowcytometry analysis showed the highest apoptosis in groups II and IV. Conclusion: Co-treatment of normal fibroblasts with vitamin C along with 5-FU reduces cell death and lipid peroxidation while enhancing viability and proliferation in treated fibroblasts, indicating healing and repair. Vitamin C administration 24 hours after 5-FU is more effective in reducing cell death and lipid peroxidation compared to its concomitant administration with 5-FU.

IN-VITRO SCREENING OF SOME PLANT EXTRACTS FOR THEIR POTENTIAL ANTICANCER ACTIVITY

Background: Natural products have been shown to be reliable sources of anticancer medicines although there is still a consistent demand for new therapeutic natural products for cancer treatment with minimal side-effects.Materials and Methods: In this study, six plant extracts (Grevillea robusta; Euphorbia millii; Euphorbia royleana; Aloe grandidentata; Bauhinia corniculate; and Cassia fistula) were screened for the presence of phytochemical metabolites as saponins, tannins, cardiac glycosides, alkaloids, flavonoids, anthraquinones and sterols, using qualitative tests. Antiproliferative screening assay was performed on a panel of three cancer cell-lines (HepG-2, HCT-116 and MCF-7) using MTT assay, and cytotoxicity was determined using WI-38 human fibroblast cell- line.Results: Some plant extracts reduced cellular growth for the selected cancerous cell-lines. For example, E. royleana and A. grandidentata extracts reduced HepG-2 cellular growth with IC50 of 0.42 and 0.53μg/mL, respectively. Moreover, A. grandidentata and C. fistula reduced cellular growth of MCF-7 with IC50 of 0.37 and 0.67 μg/mL, respectively. Conclusion: E. royleana, A. grandidentata and C. fistula showed significant anti-proliferative activity against HepG-2 and MCF-7 cell-lines with non-cytotoxic nature. This suggests their potential role as anticancer agents against these types of cancer. The presence of flavonoids, sterols and anthraquinones may suggest their enhanced anti-proliferative activities. Therefore, this study has shed light on the possible use of these extracts as potential sources of natural products-based therapy for cancer.

Amoebicidal activity of benzothiazole on Acanthamoeba castellanii cysts and trophozoites and its cytotoxic potentials

Acanthamoeba spp. are free-living amoebae isolated from many ecological areas such as swimming pools, dams and lakes, and soil. Granulomatous amebic encephalitis and amebic keratitis, caused by Acanthamoeba spp., usually occurs in chronically ill, debilitated individuals, in immunosuppressed patients and treatment is quite difficult. This study aimed to determine the effect of benzothiazole on trophozoite and cyst forms of Acanthamoeba castellanii (A.castellanii). Axenic cultures of A. castellanii trophozoites and cysts were prepared to test the amoebicidal activity of benzothiazole. The concentrations of benzothiazole in 24-well plates were prepared as 0.08%, 0.04%, 0.02%, 0.01%, 0.005%, and A. castellanii cysts and trophozoites were added to these cultures. Parasites were counted at 0, ½, 1, 3, 6, 12, 24 and 48 h in the cell counter after staining with trypan blue. Cytotoxicity of benzothiazole on the WI-38 human fibroblast cell line was also tested. Between 0.08% and 0.01% concentrations of benzothiazole showed a strong amoebicidal activity at 24 and 48 h. A significant decrease in 0.005% concentration in the number of live trophozoites and cysts was detected between 6 and 48 h. As a result of the cytotoxicity studies, benzothiazole did not show any cytotoxic effect on the WI-38 human fibroblast cell line even at 1% concentration. Benzothiazole could be concluded as a new therapeutic agent against Acanthamoeba. On the other hand, in vivo studies are needed to confirm the efficacy of the biological effect.

MicroRNA-194 Regulates Lipopolysaccharide-Induced Cell Viability by Inactivation of Nuclear Factor-κ B Pathway

The present study explored the functional role of microRNA (miR)-194 in lipopolysaccharide (LPS) induced lung cell injury, along with the underlying mechanisms and to reveal the potential role in infantile pneumonia. Human fibroblasts WI38 cells were transfected with miR-194 mimic or miR-194 inhibitor, and the transfection efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). Thereafter, the cells were treated with or without LPS, and then cell viability, cell apoptosis and mRNA and protein expressions of key proteins of nuclear factor kappa B (NF-κB) pathway including inhibitor of NF-κB (IκB) α, p-65, and B-cell CLL/lymphoma (Bcl) 3 were analyzed. Results showed that overexpression and suppression of miR-194 was effective. Administration of LPS significantly decreased the cell viability and statistically promoted the percentages of apoptotic cells and increased the mRNA and protein expressions of p-65 and Bcl-3 but downregulated IκBα compared to the control group (P < 0.05 or P < 0.01). LPS in combination with miR-194 suppression further enhanced the effects of LPS on cell viability and cell apoptosis compared to the LPS group (P < 0.05). In contrast, LPS in combination with miR-194 overexpression observably reversed the effects of LPS on cell viability, cell apoptosis and mRNA and protein expressions of the key proteins (P < 0.05 or P < 0.01). In conclusion, miR-194 increases the LPS-induced the inhibition of cell viability and increasing of the cell apoptosis by inhibition of NF-κB pathway in WI38 cells. MiR-194 might be a potential targeted therapy for treatment of infantile pneumonia.

The bystander cell-killing effect mediated by nitric oxide in normal human fibroblasts varies with irradiation dose but not with radiation quality

Purpose: To investigate the dependence of the bystander cell-killing effect on radiation dose and quality, and to elucidate related molecular mechanisms.Materials and methods: Normal human fibroblast WI-38 cells were irradiated with 0.125 - 2 Gy of γ-rays or carbon ions and were co-cultured with non-irradiated cells. Survival rates of bystander cells were investigated using the colony formation assays, and nitrite concentrations in the medium were measured using the modified Saltzman method.Results: Survival rates of bystander cells decreased with doses of γ-rays and carbon ions of ≤ 0.5 Gy. Treatment of the specific nitric oxide (NO) radical scavenger prevented reductions in survival rates of bystander cells. Moreover, nitrite concentrations increased with doses of less than 0.25 Gy (γ-rays) and 1 Gy (carbon ions). The dose responses of increased nitrite concentrations as well as survival reduction were similar between γ-rays and carbon ions. In addition, negative relationships were observed between survival rates and nitrite concentrations.Conclusion: The bystander cell-killing effect mediated by NO radicals in normal human fibroblasts depends on irradiation doses of up to 0.5 Gy, but not on radiation quality. NO radical production appears to be an important determinant of γ-ray- and carbon-ion-induced bystander effects.

Microbeam Studies of Soft X-ray Induced Bystander Cell Killing Using Microbeam X-ray Cell Irradiation System at CRIEPI

The radiation induced bystander response is defined as a response in cells which have not been directly targeted by radiation, but which are in the neighborhood of cells which have been directly exposed. In many cases, the bystander response is saturated with increasing dose and is observed when only one cell in a population is targeted by high-LET particle radiations or ultrasoft X-rays (278 eV). However, in our studies using synchrotron X-ray microbeams (5.35 keV), the bystander cell killing effect in normal human fibroblast WI-38 cells had a parabolic relationship to the irradiating dose and was detected if 5 or more cell nuclei were irradiated. To evaluate the feature of the X-ray-induced bystander cell killing effect at a wider dose range and the existence of photon energy dependence, the effects were assessed by irradiating cell nuclei in confluent WI-38 cells with AlK X-ray microbeams (1.49 keV). The surviving fraction decreased when only a single cell nucleus was irradiated, suggesting the minimal number of targeted cells to induce the effect may depend on the energy of photons used. In this study, we found that the bystander cell killing effect showed a biphasic relationship to the irradiating dose. The decrease in bystander cell survival at the doses higher than 0.23 Gy was partially suppressed between 2.3 and 7.0 Gy, followed by level-off around 90% above 14 Gy, suggesting that the X-ray-induced bystander response is dose dependent. In addition, NO is one of chief initiators/mediators of the effect at least 0.47 Gy.

Antiplasmodial Alkaloids from the Stem Bark ofStrychnos malacoclados

From the stem bark of Strychnos malacoclados, one new bisindole alkaloid, 3-hydroxylongicaudatine Y (1), was isolated along with the known alkaloids vomicine (2), bisnordihydrotoxiferine (3), divarine (4), longicaudatine (5), longicaudatine Y (6), and longicaudatine F (7). All the compounds were tested for their antimalarial activity against the chloroquine-sensitive 3D7 and -resistant W2 strains of Plasmodium falciparum. Longicaudatine was the most active compound with IC₅₀ values of 0.682 and 0.573 µM, respectively. The activity of compounds 1, 3, 4, 6, and 7 against the two strains ranged from 1.191 to 6.220 µM and 0.573 to 21.848 µM, respectively. Vomicine (2), the only monomer isolated, was inactive. The alkaloids of the longicaudatine-type ( 1, 5-7) were more active than those of the caracurine-type (3- 4). The presence of the ether bridge in the molecule seems to increase the antiplasmodial activity. Compounds 1, 5, and 7 were tested against the WI-38 human fibroblast cell line. Longicaudatine was the most cytotoxic compound with an IC₅₀ of 2.721 µM. Longicaudatine F was 40-46 times more active against the two strains of P. falciparum than against the human fibroblasts and was thus considered as the more selective alkaloid. The structures of the compounds were determined based on the analysis of their spectral data.

38 Epoxyeicosatrienoic acid protect mitochondrial functions and dynamics during in vitro cellular stress

RationaleEpoxyeicosatrienoic acid (EETs), cytochrome P450 derived metabolites of arachidonic acid, are vital in maintaining cardiac homeostasis. Previously, we demonstrated that EETs mediated cardiac protection involved preserving mitochondrial integrity and attenuating...

EDD Inhibits ATM-mediated Phosphorylation of p53

The EDD (E3 identified by differential display) gene, first identified as a progestin-induced gene in T-47D breast cancer cells, encodes an E3 ubiquitin ligase with a HECT domain. It was reported that EDD is involved in the G(2)/M progression through ubiquitination of phospho-katanin p60. Previous study has also shown that EDD can act as a transcription cofactor independently of its E3 ligase activity. In this study, we uncover a new role for EDD during cell cycle progression in an E3 ligase-independent manner. We demonstrate that EDD can physically interact with p53 and that this interaction blocks the phosphorylation of p53 by ataxia telangiectasia mutated (ATM). Silencing of EDD induces phosphorylation of p53 at Ser(15) and activates p53 target genes in fibroblasts and some transformed cells without activation of DNA damage response. The G(1)/S arrest induced by EDD depletion depends on p53. On the other hand, overexpression of EDD inhibits p53-Ser(15) phosphorylation and suppresses the induction of p53 target genes during DNA damage, and this effect does not require its E3 ligase activity. Thus, through binding to p53, EDD actively inhibits p53 phosphorylation by ATM and plays a role in ensuring smooth G(1)/S progression.

Sonodynamic and photodynamic mechanisms of action of the novel hypocrellin sonosensitizer, SL017: mitochondrial cell death is attenuated by 11, 12-epoxyeicosatrienoic acid

Development of sonosensitizers for sonodynamic therapy (SDT) which selectively target abnormal cells can limit undesired side effects in chemotherapeutic applications. Hypocrellin-B (HB) derivatives are low molecular weight compounds which belong to the perylenequinone family of photosensitizing and sonosensitizing compounds. In this study, we investigate the cytotoxic mechanisms of a novel HB-derived photo- and sonosensitizer, SL017. Human fibroblast WI-38 cells were treated with SL017 (0μM, 0.1μM or 10μM) and subjected to photodynamic therapy (PDT) or SDT. Studies demonstrate that maximal uptake of SL017 occurs within 30min, with a mitochondrial subcellular localization. Activation of SL017 by either visible light or ultrasound resulted in significant increases in reactive oxygen species (ROS) production as measured by CM-H2-DCFDA (5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester). Co-administration of the antioxidant, ascorbic acid, attenuated ROS production. Low concentrations of SL017 (100nM) induced a rapid (<90s) loss of mitochondrial membrane potential (ΔΨm). Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid (AA) involved in maintaining homeostasis and protection against cell injury, were able to attenuate loss of ΔΨm, however ascorbic acid was not. SL017 treatment resulted in increased mitochondrial fragmentation which followed loss of ΔΨm. Our studies demonstrate that SL017 targets mitochondria, triggering collapse of mitochondrial membrane potential, generates ROS and subsequently results in mitochondrial fragmentation.

Genome-wide Response of Normal WI-38 Human Fibroblast Cells to 1,763 MHz Radiofrequency Radiation

Increased exposure of human to RF fields has raised concerns for its potential adverse effects on our health. To address the biological effects of RF radiation, we used genome wide gene expression as the indicator. We exposed normal WI-38 human fibroblast cells to 1763 MHz mobile phone RF radiation at a specific absorption rate (SAR) of 60 W/kg with an operating cooling system for 24 h. There were no alterations in cell numbers or morphology after RF exposure. Through microarray analysis, we identified no differentially expressed genes (DEGs) at the 0.05 significance level after controlling for multiple testing errors with the Benjaminiochberg false discovery rate (BH FDR) method. Meanwhile, 82 genes were differentially expressed between RF-exposed cells and controls when the significance level was set at 0.01 without correction for multiple comparisons. We found that 24 genes (0.08% of the total genes examined) were changed by more than 1.5-fold on RF exposure. However, significant enrichment of any gene set or pathway was not observed from the functional annotation analysis. From these results, we did not find any evidence that non-thermal RF radiation at a 60-W/kg SAR significantly affects cell proliferation or gene expression in WI-38 cells.

Bystander Cell Killing in Normal Human Fibroblasts is Induced by Synchrotron X-Ray Microbeams

Abstract The radiation-induced bystander response is defined as a response in cells that have not been directly targeted by radiation but that are in the neighborhood of cells that have been directly exposed. In the work described here, it is shown that bystander cell killing of normal human fibroblast WI-38 cells was induced by synchrotron microbeam X radiation. Cell nuclei in confluent WI-38 cells were irradiated with the microbeam. All of the cells on the dish were harvested and plated 24 h after irradiation. It was found that the bystander cell killing effect showed a parabolic relationship to the radiation dose when five cells were irradiated. At doses above 1.9 Gy, the surviving fraction increased to approximately 1.0. This suggests that induction of bystander cell killing may require some type of activity in the targeted cells, because the dose resulting in 37% cell survival was about 2.0 Gy. Bystander cell killing was suppressed by a pretreatment with aminoguanidine [an inhibitor of inducible nitric oxide (NO) synthase] or carboxy-PTIO (a scavenger of NO). These results suggest that NO is the chief initiator/mediator of bystander cell killing induced by X-ray microbeams.

Selective tumor imaging by a novel tumor specific aralin-infrared-to-visible phosphor conjugate

Aralin is a novel cytotoxic protein from Aralia elata and selectively induces apoptosis in transformed cells as compared to normal cells (1). Aralin is a lectin specific for sugar chain such as galactose and possesses RNA N-glycosidase activity. In this study, antitumor potency of aralin was analyzed using the poly(ethyleneglycol) (PEG)/streptavidin co-immobilized infrared-to-visible upconversion phosphors, Y2O3 nanoparticles (2). Cy3-conjugated aralin could clearly detect the surface of SV40-transformed VA13 and human cervical carcinoma HeLa cells, but to a lesser extent on the normal human fibroblast WI-38 cells. Conjugation of aralin with PEGylated Y2O3 nanophosphor was carried out via biotin-avidin binding. The Y2O3-conjugated aralin also clearly visualize by a fluorescence microscope measurements equipped with near-infrared excitation source scanning in HeLa cells. It is also important to note that no remarkable damage to the cells was observed during these observations. Thus, these data imply that the Y2O3-conjugated aralin would potentially be useful material for tumor detection in vivo.

Ribosomal Protein S9 Is a Novel B23/NPM-binding Protein Required for Normal Cell Proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.

C-phycocyanin transcriptionally regulates uPA mRNA through cAMP mediated PKA pathway in human fibroblast WI-38 cells

We have previously demonstrated the efficacy of c-phycocyanin in up-regulation of urokinase-type plasminogen activator (uPA) in bovine endothelial cell line. However, the mechanism of action and pathway elucidation in uPA regulation is unclear. In experiments reported here, we have investigated the mechanism of action of c-phycocyanin (c-pc) induced uPA gene modulation in human fibroblast (WI-38) cell line. ELISA test confirmed that c-pc increased the uPA antigen whereas PAI-1 antigen level was unaffected. Treatment of cells with c-pc significantly ( P < 0.05) enhanced the uPA mRNA level in a dose (50 μg/ml) and time dependent (up to 4 h) manner. This effect of c-pc was abolished by treatment with dichloro-1-β- d-ribofuranosyl benzamidazole (DRB) (10 μg/ml). Co-treatment of c-pc with 200 μg/ml cycloheximide (CHX), translation inhibitor, resulted in over accumulation of uPA mRNA. These results suggest that uPA induction by c-pc is transcriptionally regulated and does not require de novo protein synthesis. We also provide evidence that c-pc stimulates uPA gene through cAMP dependent pathway as adenylyl cyclase (AC) inhibitor, dideoxyadenosine (DDA) significantly inhibited the uPA mRNA expression and co-treatment with adenylyl cyclase analogue, dBcAMP recovered the effect of c-pc on gene activity. Furthermore, the present investigation provides evidence on the regulatory pathway involved in the c-pc stimulus. C-pc induced uPA expression was completely inhibited by PKA inhibitor (KT 5200), indicating the regulation is dependent on PKA pathway. Elimination of PKC pathway components by prolonged incubation with excess amount of phorbol 12-myristate 13-acetate (PMA) failed to abolish the c-pc effect on uPA expression indicating the regulation is independent of PKC pathway. Taken together, our data indicate that uPA gene regulation by c-pc is transcriptionally controlled through cAMP mediated PKA pathway.

Purification and ATPase Activity of Human ABCA1

ATP-binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein metabolism. Apolipoprotein A-I binds to ABCA1 and cellular cholesterol and phospholipids, mainly phosphatidylcholine, are loaded onto apoA-I to form pre-beta high density lipoprotein (HDL). It is proposed that ABCA1 translocates phospholipids and cholesterol directly or indirectly to form pre-beta HDL. To explore the mechanism of ABCA1-mediated pre-beta HDL formation, we expressed human ABCA1 in insect Sf9 cells and purified it. Trypsin limited-digestion of purified ABCA1 in the detergent-soluble form suggested that it retained conformation similar to ABCA1 expressed in the membranes of human fibroblast WI-38 cells. Purified ABCA1 showed robust ATPase activity when reconstituted in liposomes made of synthetic phosphatidylcholine. ABCA1 showed lower ATPase activity when reconstituted in liposomes containing phosphatidylserine, phosphatidylethanolamine, or phosphatidylglycerol and also showed weak specificity in acyl chain species. ATPase activity was reduced by the addition of cholesterol and decreased by 25% in the presence of 20% cholesterol. Beta-sitosterol and campesterol showed similar inhibitory effects but stigmasterol did not, suggesting structure-specific interaction between ABCA1 and sterols. Glibenclamide suppressed ABCA1 ATPase, suggesting that it inhibits apoA-I-dependent cellular cholesterol efflux by suppressing ABCA1 ATPase activity. These results suggest that the ATPase activity of ABCA1 is stimulated preferentially by phospholipids with choline head groups, phosphatidylcholine and sphingomyelin. This study with purified human ABCA1 provides the first biochemical basis of the mechanism for HDL formation mediated by ABCA1.

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts

Differential regulation has been suggested for cellular cholesterol and phospholipid release mediated by apolipoprotein A-I (apoA-I)/ABCA1. We investigated various factors involved in cholesterol mobilization related to this pathway. ApoA-I induced a rapid decrease of the cellular cholesterol compartment that is in equilibrium with the ACAT-accessible pool in cells that generate cholesterol-rich HDL. Pharmacological and genetic inactivation of ACAT enhanced the apoA-I-mediated cholesterol release through upregulation of ABCA1 and through cholesterol enrichment in the HDL generated. Pharmacological activation of protein kinase C (PKC) also decreased the ACAT-accessible cholesterol pool, not only in the cells that produce cholesterol-rich HDL by apoA-I (i.e., human fibroblast WI-38 cells) but also in the cells that generate cholesterol-poor HDL (mouse fibroblast L929 cells). In L929 cells, the PKC activation caused an increase in apoA-I-mediated cholesterol release without detectable change in phospholipid release and in ABCA1 expression. These results indicate that apoA-I mobilizes intracellular cholesterol for the ABCA1-mediated release from the compartment that is under the control of ACAT. The cholesterol mobilization process is presumably related to PKC activation by apoA-I.