Apolipoprotein A-II Plus Lipid Emulsion Enhance Cell Growth via SR-B1 and Target Pancreatic Cancer In Vitro and In Vivo

Background

Apolipoprotein A-II (ApoA-II) is down regulated in the sera of pancreatic ductal adenocarcinoma (PDAC) patients, which may be due to increase utilization of high density lipoprotein (HDL) lipid by pancreatic cancer tissue. This study examined the influence of exogenous ApoA-II on lipid uptake and cell growth in pancreatic cancer (PC) both in vitro and in vivo.

Methods

Cryo transmission electron microscopy (TEM) examined ApoA-II’s influence on morphology of SMOFLipid emulsion. The influence of ApoA-II on proliferation of cancer cell lines was determined by incubating them with lipid+/-ApoA-II and anti-SR-B1 antibody. Lipid was labeled with the fluorophore, DiD, to trace lipid uptake by cancer cells in vitro by confocal microscopy and in vivo in PDAC patient derived xenograft tumours (PDXT) by fluorescence imaging. Scavenger receptor class B type-1(SR-B1) expression in PDAC cell lines and in PDAC PDXT was measured by western blotting and immunohistochemistry, respectively.

Results

ApoA-II spontaneously converted lipid emulsion into very small unilamellar rHDL like vesicles (rHDL/A-II) and enhanced lipid uptake in PANC-1, CFPAC-1 and primary tumour cells as shown by confocal microscopy. SR-B1 expression was 13.2, 10.6, 3.1 and 2.3 fold higher in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell lines than the normal pancreatic cell line (HPDE6) and 3.7 fold greater in PDAC tissue than in normal pancreas. ApoA-II plus lipid significantly increased the uptake of labeled lipid and promoted cell growth in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells which was inhibited by anti SR-B1 antibody. Further, ApoA-II increased the uptake of lipid in xenografts by 3.4 fold.

Conclusion

Our data suggest that ApoA-II enhance targeting potential of lipid in pancreatic cancer which may have imaging and drug delivery potentialities.     

AMG-232, a New Inhibitor of MDM-2, Enhance Doxorubicin Efficiency in Pre-B Acute Lymphoblastic Leukemia Cells

Background:Doxorubicin (DOX)-induced cardiotoxicity appears to be a growing concern for extensive use in acute lymphoblastic leukemia (ALL). The new combination treatment strategies, therefore might be an effective way of decreasing its side effects as well as improving efficacy. AMG232 (KRT-232) is a potential MDM-2 inhibitor, increasing available p53 through disturbing p53-MDM-2 interaction. In this study, we examined the effects of AMG232 on DOX-induced apoptosis of NALM-6 cells.Methods:The anti-leukemic effects of Doxorubicin on NALM-6 cells, either alone or in combination with AMG232, were confirmed by MTT assay, Annexin/PI apoptosis assay, and cell cycle analysis. Expression of apoptosis and autophagy-related genes were further evaluated by Real time-PCR method. To investigate the effect of AMG232 on NALM-6 cells, the activation of p53, p21, MDM-2, cleaved Caspase-3 proteins was evaluated using western blot analysis.Results:The results showed that AMG232 inhibition of MDM-2 enhances Doxorubicin-induced apoptosis in NALM-6 cells through caspase-3 activation in a time and dose-dependent manner. Furthermore, co-treatment of AMG232 with Doxorubicin hampered the transition of NALM-6 cells from G1 phase through increasing p21 protein. In addition, this combination treatment led to enhanced expression of apoptosis and autophagy-related genes in ALL cell lines.Conclusion:The results declared that AMG232 as an MDM-2 inhibitor could be an effective approach to enhance antitumor effects of Doxorubicin on NALM-6 cells as well as an effective future treatment for ALL patients.Key Words: Acute Lymphoblastic Leukemia, AMG 232, Autophagy, Doxorubicin, p53     

Frequency of benign neutropenia among Black versus White individuals undergoing a bone marrow assessment

Healthy individuals in the United States identified as having Black race have lower neutrophil counts, on average, than individuals identified as having White race, which could result in more negative diagnostic evaluations for neutropenia. To test this hypothesis, the proportion of evaluations where the final diagnosis was clinically insignificant neutropenia for Black and White individuals who underwent an evaluation by a haematologist that included a bone marrow (BM) biopsy to investigate neutropenia was assessed. 172 individuals without prior haematological diagnoses who underwent a haematological evaluation to investigate neutropenia. Individuals diagnosed with clinically insignificant neutropenia between Black and White individuals were compared using a propensity‐score‐adjusted logistic regression. Of 172 individuals, 42 (24%) were classified as Black race, 86 (50%) were males, and the 79 (46%) were over 18 years old. A BM biopsy did not identify pathology in 95% (40 of 42) of Black individuals and 68% (89 of 130) of White Individuals. Black individuals (25 of 42 [60%]) received a final diagnosis of clinically insignificant neutropenia, compared to White individuals (12 of 130 [9%]) (adjusted odds ratio =7.9, 95% CI: 3.1 – 21.1). We conclude that black individuals were more likely to receive a diagnosis of clinically insignificant neutropenia after haematological assessment.     

Primary Spermatocyte-Specific Cre Recombinase Activity in Transgenic Mice

We have evaluated the specificity of Cre recombinase activity in transgenic mice expressing Cre under the control of the synatonemal complex protein 1 (Sycp1) gene promoter. Sycp1Cre mice were crossed with the ROSA26 reporter line R26R, to monitor the male germ cell stage-specificity of Cre activity as well as to verify that Cre was not active previously during development of other tissues. X-gal staining detected Cre-mediated recombination only in testis. Detailed histological examination indicated that weak Cre-mediated recombination occurred as early as in zygotene spermatocytes at stage XI of the cycle of the seminiferous epithelium. Robust expression of X-gal was detected in early to mid-late spermatocytes at stages V-VIII. We conclude that this transgenic line is a powerful tool for deleting genes of interest specifically during male meiosis.     

Interleukin-15 interactions with interleukin-15 receptor complexes: characterization and species specificity

Interleukin (IL-) 2 and IL-15 share the IL-2 receptor betagamma c subunits (IL-2Rbetagamma c) but have specific, unique alpha receptor subunits. We studied species specificity of human (hu), simian (si), and mouse (mu) IL-15 and found that hu and si IL-15 behaved similarly in all systems investigated. Hu and mu IL-15 bound hu or mu IL-15Ralpha with equal high affinity in the presence or absence of IL-2Rbetagamma c and exhibited similar proliferative activities on cells containing all three subunits. However, quantitative differences were noted in the specific activity of hu and mu IL-15 in both in vitro and in vivo systems utilizing IL-2Rbetagamma c in the absence of IL-15Ralpha. These data show that hu IL-15 may be used in mouse model systems, however care must be taken when comparing the efficacy and toxicity of cytokines across species.     

Activity of the Bcr GTPase-activating domain is regulated through direct protein/protein interaction with the Rho guanine nucleotide dissociation inhibitor

The cycling of Rac GTPases, alternating between an active GTP- and an inactive GDP-bound state, is controlled by guanine nucleotide exchange factors, GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). Little is known about how these controlling activities are coordinated. Studies using null mutant mice have demonstrated that Bcr and Abr are two physiologically important GAPs for Rac. Here, we report that in the presence of RhoGDIalpha, Bcr is unable to convert Rac-GTP to Rac-GDP because RhoGDI forms a direct protein complex with Bcr. Interestingly, RhoGDIalpha binds to the GAP domain in Bcr and Abr, a domain that also binds to Rac-GTP and catalyzes conversion of the bound GTP to GDP on Rac. The presence of activated Rac diminished the Bcr/RhoGDIalpha interaction. Moreover, a Bcr mutant that lacks the ability to promote hydrolysis of Rac-GTP bound to its GAP domain did not bind to RhoGDIalpha in cell lysates, indicating that binding of RhoGDIalpha and Rac-GTP to the Bcr GAP domain is mutually exclusive. Our results provide the first identification of a protein that regulates BcrGAP activity.