Simultaneous silencing of lysophosphatidylcholine acyltransferases 1-4 by nucleic acid nanoparticles (NANPs) improves radiation response of melanoma cells.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) upregulation in breast carcinoma contributes to tumor progression and predicts early tumor recurrence.

Breast cancer is the most common cancer in women worldwide. Aberrant lipid metabolism is an established hallmark of cancer cells. The recently isolated lysophosphatidylcholine acyltransferase 1 (LPCAT1), the most important enzyme in membrane biogenesis, has been currently implicated in cancer development and progression. The published literature lacks comprehensive reports on LPCAT1 expression in breast cancer and its impact on patients' outcome. We evaluated the immunohistochemical expression of LPCAT1 in 80 primary breast carcinomas, 24 metastatic lymph nodes, and 30 non-neoplastic breast tissue specimens and statistically analyzed the association between LPCAT1 expression and clinicopathological variables and patients' outcome. LPCAT1 protein was significantly upregulated in primary breast carcinoma and showed a significant ascending pattern being the lowest in normal breast tissues, relatively increased in fibrocystic disease, and the highest in primary carcinoma. LPCAT1 expression was significantly higher at tumor's advancing edge and correlated positively with tumor's grade and TNM stage. Compared to primary tumor, LPCAT1 expression was significantly lower in ductal carcinoma in situ and significantly higher in metastatic lymph nodes. LPCAT1 overexpression was significantly associated with increased proliferative activity, negative estrogen receptor (ER) and progesterone receptor (PR) status, positive human epidermal growth factor receptor 2 (HER2) status, as well as triple-negative and HER2 disease molecular subtypes. Multivariate analysis showed that advanced stage, high grade, and LPCAT1 overexpression were independent predictors of early tumor recurrence. We conclude that LPCAT1 is implicated in breast cancer pathogenesis, evolution, and progression and appears to play a potentially crucial role as a determinant of local invasiveness and metastasis. LPCAT1 is an independent predictor of early tumor recurrence of breast carcinoma and represents a novel prognostic biomarker that reflects underlying biological alterations and thus constitutes a potentially promising target for new therapeutic strategies.

High lysophosphatidylcholine acyltransferase 1 expression independently predicts high risk for biochemical recurrence in prostate cancers.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) has been suggested to play a role in cancer. To assess its role in prostate cancer, LPCAT1 expression was analyzed on a tissue microarray containing samples from 11,152 prostate cancer patients. In benign prostate glands, LPCAT1 immunostaining was absent or weak. In prostate cancer, LPCAT1 positivity was found in 73.8% of 8786 interpretable tumors including 29.2% with strong expression. Increased LPCAT1 expression was associated with advanced tumor stage (pT3b/T4) (p < 0.0001), high Gleason score (≥4 + 4) (p < 0.0001), positive nodal involvement (p = 0.0002), positive surgical margin (p = 0.0005), and early PSA recurrence (p < 0.0001). High LPCAT1 expression was strongly linked to ERG-fusion type prostate cancer. Strong LPCAT1 staining was detected in 45.3% of ERG positive but in only 16.7% of ERG negative tumors (p < 0.0001). Within ERG negative cancers, LPCAT1 staining was strongly increased within the subgroup of PTEN deleted cancers (p < 0.0001). Further subgroup analyses revealed that associations of high LPCAT1 expression with PSA recurrence and unfavorable tumor phenotype were largely driven by ERG negative cancers (p < 0.0001) while these effects were substantially mitigated in ERG positive cancers (p = 0.0073). The prognostic impact of LPCAT1 expression was independent of histological and clinical parameters. It is concluded, that LPCAT1 measurement, either alone or in combination, may be utilized for better clinical decision-making. These data also highlight the potentially important role of lipid metabolism in prostate cancer biology.

Effects of platelet-activating factor and its differential regulation by androgens and steroid hormones in prostate cancers.

BACKGROUND: Platelet-activating factor (PAF) is an arachidonic acid metabolite that plays an important role in cell proliferation, migration and neoangiogenesis, but whether it is involved in the progression of prostate cancer remains undiscovered. METHODS: Clinical prostate specimens were investigated with immunohistochemistry method and in vitro cell experiments referred to MTS cell proliferation assay, invasion and migration experiment, quantitative real-time RT-PCR assay, western blotting analysis and ELISA assay. RESULTS: Platelet-activating factor synthetase, lyso-PAF acetyl transferase (LPCAT1), increased significantly in castration-resistant prostate cancer (CRPC) specimens and CRPC PC-3 cells than that in controls. Intriguingly, PAF induced invasion and migration of PC-3 cells but not LNCaP cells. The PAF receptor antagonist inhibited proliferation of LNCaP and PC-3 cells. Dihydrotestosterone (DHT) treatment caused a decrease in LPCAT1 expression and PAF release in LNCaP cells, which could be blocked by androgen receptor antagonists. Finally, DHT increased LPCAT1 expression and PAF release in PC-3 cells in a Wnt/ß-catenin-dependent manner. CONCLUSION: For the first time, our data supported that PAF might play pivotal roles in the progression of prostate cancer, which might throw a new light on the treatment of prostate cancer and the prevention of the emergence of CRPC.

Efficient and rapid isolation and purification of mouse alveolar type II epithelial cells.

The alveolar surface is covered by an epithelium composed of 2 main cell types: type I and type II cells. Alveolar type II (ATII) cells have a distinct morphology with apical microvilli and characteristic lamellar bodies, which are the intracellular storage form of pulmonary surfactant. ATII cells play an important role in innate immunity and produce and secrete pulmonary surfactant. They proliferate to restore the epithelium after damage to the more sensitive type I cells. We developed an efficient and rapid method to isolate and purify ATII cells from mice. Alveolar epithelial cells were dissociated in the murine lung with dispase and lung tissue was gently minced with a GentleMACS Dissociator. ATII cell purification was performed using negative depletion with CD45 MicroBeads and positive selection for the epithelial-cell adhesion molecule (Ep-CAM) by magnetic labeling with Streptavidin MicroBeads in MACS LS columns. The purity of these cells as measured by flow cytometry was up to 92.1% and 91.1% for co-staining with Ep-CAM and cytokeratin and co-staining with Ep-CAM and SP-A, respectively. The resulting ATII cell population has a high purity, viability, and yield. The phenotype of isolated and cultured ATII cells was confirmed by electron micrographs, expression of surfactant proteins (SP-A, proSP-B, mature SP-B, proSP-C, SP-D), and lysophosphatidylcholine acyltransferase (LPCAT) by western blotting and immunocytofluorescence. This protocol is based on surface antigens and our data demonstrated that murine ATII cells can be rapidly isolated, efficiently purified, and effectively cultured.

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer.

The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p < 10(-8)) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [(14)C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p < 10(-5)) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy.

Platelet-activating factor production in the spinal cord of experimental allergic encephalomyelitis mice via the group IVA cytosolic phospholipase A2-lyso-PAFAT axis.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a critical role in inflammatory disorders including experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Although PAF accumulation in the spinal cord (SC) of EAE mice and cerebrospinal fluid of MS patients has been reported, little is known about the metabolic processing of PAF in these diseases. In this study, we demonstrate that the activities of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase (LysoPAFAT) are elevated in the SC of EAE mice on a C57BL/6 genetic background compared with those of naive mice and correlate with disease severity. Correspondingly, levels of groups IVA, IVB, and IVF cytosolic PLA(2)s, group V secretory PLA(2), and LysoPAFAT transcripts are up-regulated in the SC of EAE mice. PAF acetylhydrolase activity is unchanged during the disease course. In addition, we show that LysoPAFAT mRNA and protein are predominantly expressed in microglia. Considering the substrate specificity and involvement of PAF production, group IVA cytosolic PLA(2) is likely to be responsible for the increased PLA(2) activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA(2)/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.

The activities of monocyte lysophosphatidylcholine acyltransferase and coenzyme A-independent transacylase are changed by the inflammatory cytokines tumor necrosis factor alpha and interferon gamma.

Alteration of membrane phospholipid fatty acid compositions has been shown to be important for leukocyte inflammatory responses. Such modification of the molecular species of these lipid classes requires deacylation and reacylation reactions and for phosphatidylcholines, lysophosphatidylcholine acyltransferase (LPCAT) and a coenzyme A-independent transacylase (CoAIT) can each be involved. Since previous studies have shown a significant IFNgamma- and TNFalpha-induced modification of phosphatidylcholine species, we have examined whether these inflammatory cytokines alter the activity of reacylation enzymes in the human monocyte cell line MonoMac 6 (MM6). IFN-gamma caused a significant increase in the activity of the LPCAT and CoAIT enzymes in the microsomal fraction at concentrations and over a time-course consistent with an important role for these enzymes in the sensitization (priming) of monocytes. In contrast, TNFalpha was found to significantly increase the activity of the CoAIT by 50% over controls in MM6 cells after 30 min incubation with the cytokine, but decreased LPCAT activity by 65% after 24 h incubation. Such data imply that CoAIT is important for the remodelling of phospholipid composition, which is seen during the acute response of cells to TNFalpha. The results provide further information to emphasise the role of acyltransferases as part of the molecular mechanism underlying inflammation.