Daisy De Leon

Expression of E-FABP in PC12 cells increases neurite extension during differentiation: involvement of n-3 and n-6 fatty acids.

Epidermal fatty acid‐binding protein (E‐FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E‐FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E‐FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n‐3 docosahexaenoic acid (DHA), C20:5n‐3 eicosapentaenoic acid (EPA), and C20:4n‐6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E‐FABP expression that is blocked by mitogen‐activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor‐differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12‐AS) which exhibit low E‐FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12‐S). Replenishing NGFDPC12‐AS cells with biotinylated recombinant E‐FABP (biotin‐E‐FABP) protein restores normal neurite outgrowth. Cellular localization of biotin‐E‐FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E‐FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12‐S than in NGFDPC12‐AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E‐FABP.

Differential effect of proIGF-II and IGF-II on resveratrol induced cell death by regulating survivin cellular localization and mitochondrial depolarization in breast cancer cells

Insulin-like growth factor II (IGF-II) plays a pivotal role in fetal and cancer development by signaling through the IGF-I and insulin receptors and activating the estrogen signaling cascade. We previously showed that precursor IGF-II (proIGF-II, the predominant form expressed in cancer) and not mature IGF-II (mIGF-II) blocks resveratrol (RSV) (a phytoalexin/anticancer agent)-induced cell death in MCF-7 cells. We hypothesize that proIGF-II regulates antiapoptotic proteins and/or the mitochondria to inhibit RSV actions and promote cell survival. This study examines the effect of mIGF-II and proIGF-II on survivin expression and mitochondrial polarization in response to RSV. RSV inhibits survivin expression and stimulates mitochondrial depolarization, caspase 7 activation and cell death. These effects were completely blocked by the addition of proIGF-II. RSV treatment had no effect on transfected MCF-7 cells constitutively expressing proIGF-II, while IGF-II siRNA transfection decreased survivin levels. Our results provide new insights for the potential use of proIGF-II as target for new anticancer therapies.

Differential Insulin-like Growth Factor II (IGF-II) Expression: A Potential Role for Breast Cancer Survival Disparity

OBJECTIVE Increased risk of cancer and other adult diseases have been associated with perinatal exposure to adverse conditions such as stress and famine. Recently, Insulin-like growth factor II (IGF-II) was identified as the first gene associated with altered expression caused by fetal exposure to poor nutrition. IGF-II regulates fetal development and breast cancer cell survival, in part, by regulating anti-apoptotic proteins through activation of the IGF-I and insulin receptors. African-American (AA) women have a lower overall breast cancer (BC) incidence, however, they present with advanced disease at diagnosis, poorer prognosis and lower survival than Caucasian (CA) women. The reasons for the BC survival disparity are not well understood. We hypothesize that IGF-II plays a role in the survival disparity observed among AA breast cancer patients by stimulating rapid tumor growth, inhibiting apoptosis, and promoting metastasis. DESIGN This study examines IGF-II expression and regulation of the anti-apoptotic proteins Bcl-2, Bcl-X(L), and survivin in Hs578t (ER-), CRL 2335 (ER-), and CRL 2329 (ER+) breast cancer cells and compares with the expression of these proteins in paired breast tissue samples from AA and CA women by qRT-PCR and Western blotting. RESULTS IGF-II expression was significantly higher in AA cell lines and tissue samples when compared to Caucasians. IGF-II siRNA treatment decreased anti-apoptotic protein levels in all cell lines (regardless of ER status). These effects were blocked by the addition of recombinant IGF-II. Of significance, IGF-II expression and regulation of Bcl-X(L) and survivin in cell lines correlated with their expression in paired breast tissues. CONCLUSIONS IGF-II and the anti-apoptotic proteins differential expression among AA and CA patients may contribute to the breast cancer survival disparities observed between these ethnic groups.

Insulin-like growth factors I and II receptors in the breast cancer survival disparity among African-American women

OBJECTIVE African-American (AA) women with breast cancer are more likely to have advanced disease at diagnosis, higher risk of recurrence and poorer prognosis than Caucasian (CA) women. We have recently shown higher insulin-like growth factor II (IGF-II) expression in paired breast tissue samples from AA women as compared to CA women. IGF-II is a potent mitogen that induces cell proliferation and survival signals through activation of the IGF-I and Insulin receptors (IGF-IR, IR) while IGF-II circulating levels are regulated by cellular uptake through the IGF2 receptor. We hypothesize that differential expression of the IGF1R and IGF2R among AA and CA women potentiates IGF-II mitogenic effects, thus contributing to the health disparity observed between these ethnic groups. DESIGN We examined IGF-IR and IGF2R mRNA, protein expression and IGF1R phosphorylation in paired breast tissue samples from AA and CA women by Real Time-PCR, Western blot analysis, immunohistochemistry and ELISA techniques. RESULTS Our results showed significantly increased expression of IGF1R in AA normal tissues as compared to CA normal tissues. IGF1R expression was similar between AA normal and malignant tissues, while IGF1R, IRS-1 and Shc phosphorylation was significantly higher in AA tumor samples. Significantly higher levels of IGF2R were found in CA tumor samples as compared to AA tumor samples. CONCLUSIONS We conclude that IGF1R and IGF2R differential expression may contribute to the increased risk of malignant transformation in young AA women and to the more aggressive breast cancer phenotype observed among AA breast cancer patients and represent, along with IGF-II, potential therapeutic targets in breast cancer.

Lipotoxicity-mediated cell dysfunction and death involve lysosomal membrane permeabilization and cathepsin L activity.

Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity.

Differential expression and signaling activation of insulin receptor isoforms A and B: A link between breast cancer and diabetes

We showed that when insulin-like growth factor II (IGF-II) is highly expressed in breast tissues and cell lines, the IGF-I receptor signaling pathway is highly activated. Since IGF-II activates the insulin receptor (INSR), we propose that the INSR signaling is also activated in this system. We examined the expression of both INSR isoforms, insulin receptor A (INSR-A) and insulin receptor B (INSR-B), and the downstream signaling pathways in breast cancer (BC) cells and in paired (normal/tumor) breast tissues from 100 patients. Analysis was performed by real-time PCR, Western blot, immunohistochemistry, and phospho-ELISA techniques. Tumor tissues and cell lines from African-American patients expressed higher levels of INSR-A, but lower levels of INSR-B. Accordingly, insulin receptor substrate 1 and focal adhesion kinase activation were significantly increased in these women. We conclude that higher INSR-A and lower INSR-B contribute to higher proliferation and lower metabolic response. Thus, differential expression of INSR isoforms represents a potential biological link between BC and diabetes.

Underrepresented Minority High School and College Students Report STEM-Pipeline Sustaining Gains After Participating in the Loma Linda University Summer Health Disparities Research Program

An urgent need exists for graduate and professional schools to establish evidence-based STEM (science, technology, engineering, and math) pipeline programs to increase the diversity of the biomedical workforce. An untapped yet promising pool of willing participants are capable high school students that have a strong STEM interest but may lack the skills and the guided mentoring needed to succeed in competitive STEM fields. This study evaluates and compares the impact of the Loma Linda University (LLU) Summer Health Disparities Research Program on high school (HS) and undergraduate (UG) student participants. The primary focus of our summer research experience (SRE) is to enhance the research self-efficacy of the participants by actively involving them in a research project and by providing the students with personalized mentoring and targeted career development activities, including education on health disparities. The results of our study show that our SRE influenced terminal degree intent and increased participant willingness to incorporate research into future careers for both the HS and the UG groups. The quantitative data shows that both the HS and the UG participants reported large, statistically significant gains in self-assessed research skills and research self-efficacy. Both participant groups identified the hands-on research and the mentor experience as the most valuable aspects of our SRE and reported increased science skills, increased confidence in science ability and increased motivation and affirmation to pursue a science career. The follow-up data indicates that 67% of the HS participants and 90% of the UG participants graduated from college with a STEM degree; for those who enrolled in graduate education, 61% and 43% enrolled in LLU, respectively. We conclude that structured SREs can be highly effective STEM strengthening interventions for both UG and HS students and may be a way to measurably increase institutional and biomedical workforce diversity.

Insulin-like growth factor II mediates resveratrol stimulatory effect on cathepsin D in breast cancer cells

Cathepsin D (CD) is an enzyme that promotes breast cancer. CD is stored intracellularly; however, we demonstrated that IGF-II promotes CD secretion in estrogen receptor positive (ER+) breast cancer cells. We also showed that resveratrol (RSV) stimulates IGF-II in ER(+) breast cancer cells. Thus, we designed this study to determine whether RSV regulates CD in MCF-7, T47D (ER+) breast cancer cells as well as in Hs578t (cancer) and MCF-10A (normal) ER − cell lines. RSV (10− 6 M) increased CD and IGF-II secretion in ER+ but not ER − cells. RSV treatment (10− 4 M) inhibited CD in ER+ but not in ER − cells. Transfection of ER − cells with proIGF-II increased CD secretion. RSV (10− 6 M) modulates CD secretion through IGF-II while RSV (10− 4 M) inhibits CD in ER+ but not ER − cells. Regulation of CD by RSV represents a novel mechanism by which RSV may protect against breast cancer.

Precursor IGF-II (proIGF-II) and mature IGF-II (mIGF-II) induce Bcl-2 and Bcl-XL expression through different signaling pathways in breast cancer cells

IGF-II plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor. We have shown that inhibition of IGF-II by resveratrol (RSV) induced apoptosis and that proIGF-II (highly expressed in cancer) was more potent than mIGF-II in inhibiting this effect. Thus, we hypothesized that IGF-II differentially regulates the signaling cascade of the IGF-I receptor to stimulate the anti-apoptotic proteins Bcl-2 and Bcl-XL to prevent apoptosis. RSV treatment to breast cancer cells inhibited Bcl-2 and Bcl-XL expression and induced mitochondrial membrane depolarization. ProIGF-II was more potent than mIGF-II in: (1) activating the PI3/Akt pathway, (2) regulating Bcl-2 and Bcl-XL expression, and (3) inducing phosphorylation/nuclear translocation of Cyclic AMP-responsive element binding protein. Furthermore, IGF-II differentially regulated the intracellular translocation of Bcl-2 and Bcl-XL, a critical process in breast cancer progression to hormone-independence. Our study provides a novel mechanism of how proIGF-II promotes progression and chemoresistance in breast cancer development.

Function of SERCA mediated calcium uptake and expression of SERCA3 in cerebral cortex from young and old rats.

Previous work on peripheral sympathetic neurons indicated that a decline in sarco/endoplasmic reticulum calcium ATPase (SERCA) function occurs with advancing age. Therefore, an age-related decline in mechanisms controlling intracellular calcium homeostasis could contribute to altered neuronal function and/or degeneration. In this study we sought to extend the findings on peripheral neurons and to detect possible age-related declines in SERCA function and expression of SERCA3 in central neurons from cerebral cortex from young (6-month) and old (20-month) rats. Functional studies compared ATP-dependent 45Ca(2+)-uptake into microsomes and plasma membrane vesicles (PMVs). We and found no significant difference in 45Ca(2+)-uptake between microsomes or PMVs between young and old animals. On the other hand expression of SERCA3 mRNA in rat cerebral cortex showed a significant decline with advancing age. However, comparison of SERCA3 protein content did not reveal a corresponding decline; implying that SERCA mRNA turnover rates may be greater in the younger group. Although the present work with rat cerebral cortex does not indicate an age-related decline in SERCA function, previous work from our laboratory on sympathetic nerves and by others on the hippocampus indicate such a decline. In light of our previous and current studies, aging may affect calcium homeostatic mechanisms in central and peripheral autonomic neurons differently.