Multi-analyte proteomic analysis identifies blood-based neuroinflammation, cerebrovascular and synaptic biomarkers in preclinical Alzheimer's disease.

BACKGROUND: Blood-based biomarkers are gaining grounds for the detection of Alzheimer's disease (AD) and related disorders (ADRDs). However, two key obstacles remain: the lack of methods for multi-analyte assessments and the need for biomarkers for related pathophysiological processes like neuroinflammation, vascular, and synaptic dysfunction. A novel proteomic method for pre-selected analytes, based on proximity extension technology, was recently introduced. Referred to as the NULISAseq CNS disease panel, the assay simultaneously measures ~ 120 analytes related to neurodegenerative diseases, including those linked to both core (i.e., tau and amyloid-beta (Aß)) and non-core AD processes. This study aimed to evaluate the technical and clinical performance of this novel targeted proteomic panel. METHODS: The NULISAseq CNS disease panel was applied to 176 plasma samples from 113 individuals in the MYHAT-NI cohort of predominantly cognitively normal participants from an economically underserved region in southwestern Pennsylvania, USA. Classical AD biomarkers, including p-tau181, p-tau217, p-tau231, GFAP, NEFL, Aß40, and Aß42, were independently measured using Single Molecule Array (Simoa) and correlations and diagnostic performances compared. Aß pathology, tau pathology, and neurodegeneration (AT(N) statuses) were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and an MRI-based AD-signature composite cortical thickness index, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA and neuroimaging-determined AT(N) biomarkers. RESULTS: NULISA concurrently measured 116 plasma biomarkers with good technical performance (97.2 ± 13.9% targets gave signals above assay limits of detection), and significant correlation with Simoa assays for the classical biomarkers. Cross-sectionally, p-tau217 was the top hit to identify Aß pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878-0.983). Fourteen markers were significantly decreased in Aß-PET + participants, including TIMP3, BDNF, MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aß PET-dependent yearly increases in Aß-PET + participants. Novel plasma biomarkers with tau PET-dependent longitudinal changes included proteins associated with neuroinflammation, synaptic function, and cerebrovascular integrity, such as CHIT1, CHI3L1, NPTX1, PGF, PDGFRB, and VEGFA; all previously linked to AD but only reliable when measured in cerebrospinal fluid. The autophagosome cargo protein SQSTM1 exhibited significant association with neurodegeneration after adjusting age, sex, and APOE ε4 genotype. CONCLUSIONS: Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes, consistent with the recently revised biological and diagnostic framework. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD.

A streamlined, resource-efficient immunoprecipitation-mass spectrometry method for quantifying plasma amyloid-ß biomarkers in Alzheimer's disease.

High-performance, resource-efficient methods for plasma amyloid-ß (Aß) quantification in Alzheimer's disease are lacking; existing mass spectrometry-based assays are resource- and time-intensive. We developed a streamlined mass spectrometry method with a single immunoprecipitation step, an optimized buffer system, and ≤75% less antibody requirement. Analytical and clinical performances were compared with an in-house reproduced version of a well-known two-step assay. The streamlined assay showed high dilution linearity (r2>0.99) and precision (< 10% coefficient of variation), low quantification limits (Aß1-40: 12.5 pg/ml; Aß1-42: 3.125 pg/ml), and high signal correlation (r2~0.7) with the two-step immunoprecipitation assay. The novel single-step assay showed more efficient recovery of Aß peptides via fewer immunoprecipitation steps, with significantly higher signal-to-noise ratios, even at plasma sample volumes down to 50 pl. Both assays had equivalent performances in distinguishing non-elevated vs. elevated brain Aß-PET individuals. The new method enables simplified yet robust evaluation of plasma Aß biomarkers in Alzheimer's disease.

Multi-analyte proteomic analysis identifies blood-based neuroinflammation, cerebrovascular and synaptic biomarkers in preclinical Alzheimer's disease.

Background: Blood-based biomarkers are gaining grounds for Alzheimer's disease (AD) detection. However, two key obstacles need to be addressed: the lack of methods for multi-analyte assessments and the need for markers of neuroinflammation, vascular, and synaptic dysfunction. Here, we evaluated a novel multi-analyte biomarker platform, NULISAseq CNS disease panel, a multiplex NUcleic acid-linked Immuno-Sandwich Assay (NULISA) targeting ~120 analytes, including classical AD biomarkers and key proteins defining various disease hallmarks. Methods: The NULISAseq panel was applied to 176 plasma samples from the MYHAT-NI cohort of cognitively normal participants from an economically underserved region in Western Pennsylvania. Classical AD biomarkers, including p-tau181 p-tau217, p-tau231, GFAP, NEFL, Aß40, and Aß42, were also measured using Single Molecule Array (Simoa). Amyloid pathology, tau pathology, and neurodegeneration were evaluated with [11C] PiB PET, [18F]AV-1451 PET, and MRI, respectively. Linear mixed models were used to examine cross-sectional and Wilcoxon rank sum tests for longitudinal associations between NULISA biomarkers and AD pathologies. Spearman correlations were used to compare NULISA and Simoa. Results: NULISA concurrently measured 116 plasma biomarkers with good technical performance, and good correlation with Simoa measures. Cross-sectionally, p-tau217 was the top hit to identify Aß pathology, with age, sex, and APOE genotype-adjusted AUC of 0.930 (95%CI: 0.878-0.983). Fourteen markers were significantly decreased in Aß-PET+ participants, including TIMP3, which regulates brain Aß production, the neurotrophic factor BDNF, the energy metabolism marker MDH1, and several cytokines. Longitudinally, FGF2, IL4, and IL9 exhibited Aß PET-dependent yearly increases in Aß-PET+ participants. Markers with tau PET-dependent longitudinal changes included the microglial activation marker CHIT1, the reactive astrogliosis marker CHI3L1, the synaptic protein NPTX1, and the cerebrovascular markers PGF, PDGFRB, and VEFGA; all previously linked to AD but only reliably measured in cerebrospinal fluid. SQSTM1, the autophagosome cargo protein, exhibited a significant association with neurodegeneration status after adjusting age, sex, and APOE ε4 genotype. Conclusions: Together, our results demonstrate the feasibility and potential of immunoassay-based multiplexing to provide a comprehensive view of AD-associated proteomic changes. Further validation of the identified inflammation, synaptic, and vascular markers will be important for establishing disease state markers in asymptomatic AD.

Alzheimer blood biomarkers: practical guidelines for study design, sample collection, processing, biobanking, measurement and result reporting.

Alzheimer's disease (AD), the most common form of dementia, remains challenging to understand and treat despite decades of research and clinical investigation. This might be partly due to a lack of widely available and cost-effective modalities for diagnosis and prognosis. Recently, the blood-based AD biomarker field has seen significant progress driven by technological advances, mainly improved analytical sensitivity and precision of the assays and measurement platforms. Several blood-based biomarkers have shown high potential for accurately detecting AD pathophysiology. As a result, there has been considerable interest in applying these biomarkers for diagnosis and prognosis, as surrogate metrics to investigate the impact of various covariates on AD pathophysiology and to accelerate AD therapeutic trials and monitor treatment effects. However, the lack of standardization of how blood samples and collected, processed, stored analyzed and reported can affect the reproducibility of these biomarker measurements, potentially hindering progress toward their widespread use in clinical and research settings. To help address these issues, we provide fundamental guidelines developed according to recent research findings on the impact of sample handling on blood biomarker measurements. These guidelines cover important considerations including study design, blood collection, blood processing, biobanking, biomarker measurement, and result reporting. Furthermore, the proposed guidelines include best practices for appropriate blood handling procedures for genetic and ribonucleic acid analyses. While we focus on the key blood-based AD biomarkers for the AT(N) criteria (e.g., amyloid-beta [Aß]40, Aß42, Aß42/40 ratio, total-tau, phosphorylated-tau, neurofilament light chain, brain-derived tau and glial fibrillary acidic protein), we anticipate that these guidelines will generally be applicable to other types of blood biomarkers. We also anticipate that these guidelines will assist investigators in planning and executing biomarker research, enabling harmonization of sample handling to improve comparability across studies.

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). In this study, we conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improves the stability of Aß42 and Aß40 across all approaches. However, the Aß42/Aß40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aß42 and Aß40, and the Aß42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.

Acinar to ß-like cell conversion through inhibition of focal adhesion kinase.

Insufficient functional ß-cell mass causes diabetes; however, an effective cell replacement therapy for curing diabetes is currently not available. Reprogramming of acinar cells toward functional insulin-producing cells would offer an abundant and autologous source of insulin-producing cells. Our lineage tracing studies along with transcriptomic characterization demonstrate that treatment of adult mice with a small molecule that specifically inhibits kinase activity of focal adhesion kinase results in trans-differentiation of a subset of peri-islet acinar cells into insulin producing ß-like cells. The acinar-derived insulin-producing cells infiltrate the pre-existing endocrine islets, partially restore ß-cell mass, and significantly improve glucose homeostasis in diabetic mice. These findings provide evidence that inhibition of the kinase activity of focal adhesion kinase can convert acinar cells into insulin-producing cells and could offer a promising strategy for treating diabetes.

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

INTRODUCTION: The reliability of plasma Alzheimer's disease (AD) biomarkers can be compromised by protease-induced degradation. This limits the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). This study conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. METHODS: We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 hours. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0h or 24h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA or P100 tubes, followed by storage at RT for 0h or 24h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. RESULTS: Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improved the stability of Aß42 and Aß40 across all approaches. Additionally, the Aß42/Aß40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. CONCLUSION: Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aß42 and Aß40, and the Aß ratio for IP-MS assay. This has crucial implications for preanalytical procedures, particularly in resource-limited settings.

Novel ultrasensitive immunoassay for the selective quantification of tau oligomers and related soluble aggregates.

INTRODUCTION: Tau aggregation into paired helical filaments and neurofibrillary tangles is characteristic of Alzheimer's disease (AD) and related disorders. However, biochemical assays for the quantification of soluble, earlier-stage tau aggregates are lacking. We describe an immunoassay that is selective for tau oligomers and related soluble aggregates over monomers. METHODS: A homogeneous (single-antibody) immunoassay was developed using a novel anti-tau monoclonal antibody and validated with recombinant and brain tissue-derived tau. RESULTS: The assay signals were concentration dependent for recombinant tau aggregates in solution but not monomers, and recognized peptides within, but not outside, the aggregation-prone microtubule binding region. The signals in inferior and middle frontal cortical tissue homogenates increased with neuropathologically determined Braak staging, and were higher in insoluble than soluble homogenized brain fractions. Autopsy-verified AD gave stronger signals than other neurodegenerative diseases. DISCUSSION: The quantitative oligomer/soluble aggregate-specific assay can identify soluble tau aggregates, including oligomers, from monomers in human and in vitro biospecimens. HIGHLIGHTS: The aggregation of tau to form fibrils and neurofibrillary tangles is a key feature of Alzheimer's disease. However, biochemical assays for the quantification of oligomers/soluble aggregated forms of tau are lacking. We developed a new assay that preferentially binds to soluble tau aggregates, including oligomers and fibrils, versus monomers. The assay signal increased corresponding to the total protein content, Braak staging, and insolubility of the sequentially homogenized brain tissue fractions in an autopsy-verified cohort. The assay recognized tau peptides containing the microtubule binding region but not those covering the N- or C-terminal regions only.

Chemical pancreatectomy in non-human primates ablates the acini and ducts and enhances beta-cell function.

Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that "chemical pancreatectomy," a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays. Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.

Chemical pancreatectomy in non-human primates ablates the acini and ducts and enhances beta-cell function.

Chronic pancreatitis is a debilitating disease affecting millions worldwide. These patients suffer from bouts of severe pain that are minimally relieved by pain medications and may necessitate major surgeries with high morbidity and mortality. Previously, we demonstrated that "chemical pancreatectomy," a pancreatic intraductal infusion of dilute acetic acid solution, ablated the exocrine pancreas while preserving the endocrine pancreas. Notably, chemical pancreatectomy resolved chronic inflammation, alleviated allodynia in the cerulein pancreatitis model, and improved glucose homeostasis. Herein, we extensively tested the feasibility of a chemical pancreatectomy in NHPs and validated our previously published pilot study. We did serial computed tomography (CT) scans of the abdomen and pelvis, analyzed dorsal root ganglia, measured serum enzymes, and performed histological and ultrastructural assessments and pancreatic endocrine function assays.  Based on serial CT scans, chemical pancreatectomy led to the loss of pancreatic volume. Immunohistochemistry and transmission electron microscopy demonstrated exocrine pancreatic ablation with endocrine islet preservation. Importantly, chemical pancreatectomy did not increase pro-nociceptive markers in harvested dorsal root ganglia. Also, chemical pancreatectomy improved insulin secretion to supranormal levels in vivo and in vitro. Thus, this study may provide a foundation for translating this procedure to patients with chronic pancreatitis or other conditions requiring a pancreatectomy.

Ionizable lipid nanoparticles deliver mRNA to pancreatic ß cells via macrophage-mediated gene transfer.

Systemic messenger RNA (mRNA) delivery to organs outside the liver, spleen, and lungs remains challenging. To overcome this issue, we hypothesized that altering nanoparticle chemistry and administration routes may enable mRNA-induced protein expression outside of the reticuloendothelial system. Here, we describe a strategy for delivering mRNA potently and specifically to the pancreas using lipid nanoparticles. Our results show that delivering lipid nanoparticles containing cationic helper lipids by intraperitoneal administration produces robust and specific protein expression in the pancreas. Most resultant protein expression occurred within insulin-producing ß cells. Last, we found that pancreatic mRNA delivery was dependent on horizontal gene transfer by peritoneal macrophage exosome secretion, an underappreciated mechanism that influences the delivery of mRNA lipid nanoparticles. We anticipate that this strategy will enable gene therapies for intractable pancreatic diseases such as diabetes and cancer.

ß-cell Smad2 null mice have improved ß-cell function and are protected from diet-induced hyperglycemia.

Understanding signaling pathways that regulate pancreatic ß-cell function to produce, store, and release insulin, as well as pathways that control ß-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-ß) signaling is involved in a broad range of ß-cell functions. The canonical TGF-ß signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-ß/smad2 signaling in regulating mature ß-cell proliferation and function using ß-cell-specific smad2 null mutant mice. ß-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased ß-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.

Mechanisms of Impaired Lung Development and Ciliation in Mannosidase-1-Alpha-2 (Man1a2) Mutants.

BACKGROUND: Ciliary defects cause heterogenous phenotypes related to mutation burden which lead to impaired development. A previously reported homozygous deletion in the Man1a2 gene causes lethal respiratory failure in newborn pups and decreased lung ciliation compared with wild type (WT) pups. The effects of heterozygous mutation, and the potential for rescue are not known. PURPOSE: We hypothesized that survival and lung ciliation, (a) would decrease progressively in Man1a2 +/- heterozygous and Man1a2 -/- null newborn pups compared with WT, and (b) could be enhanced by gestational treatment with N-Acetyl-cysteine (NAC), an antioxidant. METHODS: Man1a2+/- adult mice were fed NAC or placebo from a week before breeding through gestation. Survival of newborn pups was monitored for 24 h. Lungs, liver and tails were harvested for morphology, genotyping, and transcriptional profiling. RESULTS: Survival (p = 0.0001, Kaplan-Meier) and percent lung ciliation (p = 0.0001, ANOVA) measured by frequency of Arl13b+ respiratory epithelial cells decreased progressively, as hypothesized. Compared with placebo, gestational NAC treatment enhanced (a) lung ciliation in pups with each genotype, (b) survival in heterozygous pups (p = 0.017) but not in WT or null pups. Whole transcriptome of lung but not liver demonstrated patterns of up- and down-regulated genes that were identical in living heterozygous and WT pups, and completely opposite to those in dead heterozygous and null pups. Systems biology analysis enabled reconstruction of protein interaction networks that yielded functionally relevant modules and their interactions. In these networks, the mutant Man1a2 enzyme contributes to abnormal synthesis of proteins essential for lung development. The associated unfolded protein, hypoxic and oxidative stress responses can be mitigated with NAC. Comparisons with the developing human fetal lung transcriptome show that NAC likely restores normal vascular and epithelial tube morphogenesis in Man1a2 mutant mice. CONCLUSION: Survival and lung ciliation in the Man1a2 mutant mouse, and its improvement with N-Acetyl cysteine is genotype-dependent. NAC-mediated rescue depends on the central role for oxidative and hypoxic stress in regulating ciliary function and organogenesis during development.

Chemical pancreatectomy treats chronic pancreatitis while preserving endocrine function in preclinical models.

Chronic pancreatitis affects over 250,000 people in the US and millions worldwide. It is associated with chronic debilitating pain, pancreatic exocrine failure, and high risk of pancreatic cancer and usually progresses to diabetes. Treatment options are limited and ineffective. We developed a new potential therapy, wherein a pancreatic ductal infusion of 1%-2% acetic acid in mice and nonhuman primates resulted in a nonregenerative, near-complete ablation of the exocrine pancreas, with complete preservation of the islets. Pancreatic ductal infusion of acetic acid in a mouse model of chronic pancreatitis led to resolution of chronic inflammation and pancreatitis-associated pain. Furthermore, acetic acid-treated animals showed improved glucose tolerance and insulin secretion. The loss of exocrine tissue in this procedure would not typically require further management in patients with chronic pancreatitis because they usually have pancreatic exocrine failure requiring dietary enzyme supplements. Thus, this procedure, which should be readily translatable to humans through an endoscopic retrograde cholangiopancreatography (ERCP), may offer a potential innovative nonsurgical therapy for chronic pancreatitis that relieves pain and prevents the progression of pancreatic diabetes.

SMAD7 enhances adult ß-cell proliferation without significantly affecting ß-cell function in mice.

The interplay between the transforming growth factor ß (TGF-ß) signaling proteins, SMAD family member 2 (SMAD2) and 3 (SMAD3), and the TGF-ß-inhibiting SMAD, SMAD7, seems to play a vital role in proper pancreatic endocrine development and also in normal ß-cell function in adult pancreatic islets. Here, we generated conditional SMAD7 knockout mice by crossing insulin1Cre mice with SMAD7fx/fx mice. We also created a ß cell-specific SMAD7-overexpressing mouse line by crossing insulin1Dre mice with HPRT-SMAD7/RosaGFP mice. We analyzed ß-cell function in adult islets when SMAD7 was either absent or overexpressed in ß cells. Loss of SMAD7 in ß cells inhibited proliferation, and SMAD7 overexpression enhanced cell proliferation. However, alterations in basic glucose homeostasis were not detectable following either SMAD7 deletion or overexpression in ß cells. Our results show that both the absence and overexpression of SMAD7 affect TGF-ß signaling and modulates ß-cell proliferation but does not appear to alter ß-cell function. Reversible SMAD7 overexpression may represent an attractive therapeutic option to enhance ß-cell proliferation without negative effects on ß-cell function.

Withaferin A-mediated apoptosis in breast cancer cells is associated with alterations in mitochondrial dynamics.

Withaferin A (WA), a steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits cancer development in transgenic and chemically-induced rodent models of breast cancer but the underlying mechanism is not fully grasped. We have shown previously that WA treatment causes apoptotic cell death in human breast cancer cells that is preceded by inhibition of complex III of the mitochondrial electron transport chain. This study extends these observations to now demonstrate alterations in mitochondrial dynamics in WA-induced apoptosis. Assembly of complex III was decreased in MCF-7 and SUM159 cells but not in MDA-MB-231 as determined by native blue gel electrophoresis. Because WA is a Michael acceptor (electrophile), we explored the possibility of whether it covalently modifies cysteine residue(s) in ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1). Covalent modification of cysteine in UQCRFS1 was not observed after WA treatment. Instead, WA treatment inhibited chemically-induced mitochondrial fusion and decreased the mitochondrial volume, and this effect was accompanied by a decrease in the expression of proteins involved in fusion process, including mitofusin1, mitofusin2, and full-length optic atrophy protein 1 (OPA1). A loss of volume in fragmented mitochondria also occurred in WA-exposed cells when compared to vehicle-treated control. WA treatment also caused a decrease in protein level of mitochondrial fission-regulating protein dynamin-related protein 1 (DRP1). Functional studies revealed that DRP1 deficiency and OPA1 knockdown attenuated apoptotic potential of WA. Taken together, these results indicate that WA not only alters Complex III assembly but also inhibits mitochondrial dynamics in breast cancer cells.

Zerumbone modulates CD1d expression and lipid antigen presentation pathway in breast cancer cells.

Natural Killer T (NKT) cells based cancer immunotherapy is an evolving area of cancer therapy, but tumors escape from this treatment modality by altering CD1d expression and its antigen presentation pathway. Here, we have studied the relation of CD1d expression in various breast cancer cell lines to their viability and progression. We observed a novel phenomenon that CD1d expression level increases with the progressive stage of the cancer. A small molecule, zerumbone (ZER) caused down-regulation of CD1d that was accompanied by breast cancer cell growth in vitro. The growth inhibitory effect of ZER against breast cancer cells was augmented by treatment with anti-CD1d mAb. This effect was mediated by G1-phase cell cycle arrest and apoptosis induction coupled with an increase in mitochondrial membrane depolarization. CD1d expression and cell proliferation were inhibited by both CD1d siRNA and ZER. The α-galactosylceramide, a ligand for CD1d, showed increased CD1d expression as well as cell proliferation which was opposite to the effects of ZER. This study shows that, CD1d overexpression is associated with the progressive stages of breast cancer and ZER could be an adjuvant to potentiate cancer immunotherapy.

Disease Subtype-Independent Biomarkers of Breast Cancer Chemoprevention by the Ayurvedic Medicine Phytochemical Withaferin A.

Background: A nontoxic chemopreventive intervention efficacious against different subtypes of breast cancer is still a clinically unmet need. The present study was undertaken to determine the efficacy of an Ayurvedic medicine phytochemical (Withaferin A, [WA]) for chemoprevention of breast cancer and to elucidate its mode of action. Methods: Chemopreventive efficacy of WA (4 and 8 mg/kg body weight) was determined using a rat model of breast cancer induced by N-methyl-N-nitrosourea (MNU; n = 14 for control group, n = 15 for 4 mg/kg group, and n = 18 for 8 mg/kg group). The mechanisms underlying breast cancer chemoprevention by WA were elucidated by immunoblotting, biochemical assays, immunohistochemistry, and cytokine profiling using plasma and tumors from the MNU-rat (n = 8-12 for control group, n = 7-11 for 4 mg/kg group, and n = 8-12 for 8 mg/kg group) and/or mouse mammary tumor virus-neu (MMTV-neu) models (n = 4-11 for control group and n = 4-21 for 4 mg/kg group). Inhibitory effect of WA on exit from mitosis and leptin-induced oncogenic signaling was determined using MCF-7 and/or MDA-MB-231 cells. All statistical tests were two-sided. Results: Incidence, multiplicity, and burden of breast cancer in rats were decreased by WA administration. For example, the tumor weight in the 8 mg/kg group was lower by about 68% compared with controls (8 mg/kg vs control, mean = 2.76 vs 8.59, difference = -5.83, 95% confidence interval of difference = -9.89 to -1.76, P = .004). Mitotic arrest and apoptosis induction were some common determinants of breast cancer chemoprevention by WA in the MNU-rat and MMTV-neu models. Cytokine profiling showed suppression of plasma leptin levels by WA in rats. WA inhibited leptin-induced oncogenic signaling in cultured breast cancer cells. Conclusions: WA is a promising chemopreventative phytochemical with the ability to inhibit at least two different subtypes of breast cancer.

Mitochondrial dysfunction in cancer chemoprevention by phytochemicals from dietary and medicinal plants.

Cancer chemoprevention, a scientific term coined by Dr. Sporn in the late seventies, implies use of natural or synthetic chemicals to block, delay or reverse carcinogenesis. Phytochemicals derived from edible and medicinal plants have been studied rather extensively for cancer chemoprevention using preclinical models in the past few decades. Nevertheless, some of these agents (e.g., isothiocyanates from cruciferous vegetables like broccoli and watercress) have already entered into clinical investigations. Examples of widely studied and highly promising phytochemicals from edible and medicinal plants include cruciferous vegetable constituents (phenethyl isothiocyanate, benzyl isothiocyanate, and sulforaphane), withaferin A (WA) derived from a medicinal plant (Withania somnifera) used heavily in Asia, and an oriental medicine plant component honokiol (HNK). An interesting feature of these structurally-diverse phytochemicals is that they target mitochondria to provoke cancer cell-selective death program. Mechanisms underlying cell death induction by commonly studied phytochemicals have been discussed rather extensively and thus are not covered in this review article. Instead, the primary focus of this perspective is to discuss experimental evidence pointing to mitochondrial dysfunction in cancer chemoprevention by promising phytochemicals.