14 research outputs found
Evaluation of the mirn23a Cluster through an iTRAQ-based Quantitative Proteomic Approach
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are implicated in a number of disease states. MiRNAs can exist as individual entities, or may be clustered and transcribed as a single polycistron. The mirn23a cluster consists of three miRNAs, miR-23a, miR-24-2, and miR-27a. While these miRNAs are transcribed together, they often exist at varying levels in the cell. Despite the fact that the mirn23a cluster is known to play a role in a number of diseases and developmental processes, few direct targets have been identified. In this study, we examined the effects of miR-23a, miR-24-2, miR-27a, or the mirn23a cluster overexpression on the proteome of 70Z/3 pre-B lymphoblast cells. Quantitative mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ) allowed for global profiling of cell lines after miRNA overexpression. We identified a number of targets of each miRNA that contained predicted miRNA seed sequences and are likely direct targets. In addition, we discovered a cohort of shared miRNA targets and cluster targets, demonstrating the importance of studying miRNA clusters in their entirety
ColoType: a forty gene signature for consensus molecular subtyping of colorectal cancer tumors using whole-genome assay or targeted RNA-sequencing
Colorectal cancer (CRC) tumors can be partitioned into four biologically distinct consensus molecular subtypes (CMS1-4) using gene expression. Evidence is accumulating that tumors in different subtypes are likely to respond differently to treatments. However, to date, there is no clinical diagnostic test for CMS subtyping. In this study, we used novel methodology in a multi-cohort training domain (n = 1,214) to develop the ColoType scores and classifier to predict CMS1-4 based on expression of 40 genes. In three validation cohorts (n = 1,744, in total) representing three distinct gene-expression measurement technologies, ColoType predicted gold-standard CMS subtypes with accuracies 0.90, 0.91, 0.88, respectively. To accommodate for potential intratumoral heterogeneity and tumors of mixed subtypes, ColoType was designed to report continuous scores measuring the prevalence of each of CMS1–4 in a tumor, in addition to specifying the most prevalent subtype. For analysis of clinical specimens, ColoType was also implemented with targeted RNA-sequencing (Illumina AmpliSeq). In a series of formalin-fixed, paraffin-embedded CRC samples (n = 49), ColoType by targeted RNA-sequencing agreed with subtypes predicted by two independent methods with accuracies 0.92, 0.82, respectively. With further validation, ColoType by targeted RNA-sequencing, may enable clinical application of CMS subtyping with widely-available and cost-effective technology
Calcitriol Supplementation Causes Decreases in Tumorigenic Proteins and Different Proteomic and Metabolomic Signatures in Right versus Left-Sided Colon Cancer
Vitamin D deficiency is a common problem worldwide. In particular, it is an issue in the Northern Hemisphere where UVB radiation does not penetrate the atmosphere as readily. There is a correlation between vitamin D deficiency and colorectal cancer incidence and mortality. Furthermore, there is strong evidence that cancer of the ascending (right side) colon is different from cancer of the descending (left side) colon in terms of prognosis, tumor differentiation, and polyp type, as well as at the molecular level. Right-side tumors have elevated Wnt signaling and are more likely to relapse, whereas left-side tumors have reduced expression of tumor suppressor genes. This study seeks to understand both the proteomic and metabolomic changes resulting from treatment of the active metabolite of vitamin D, calcitriol, in right-sided and left-sided colon cancer. Our results show that left-sided colon cancer treated with calcitriol has a substantially greater number of changes in both the proteome and the metabolome than right-sided colon cancer. We found that calcitriol treatment in both right-sided and left-sided colon cancer causes a downregulation of ribosomal protein L37 and protein S100A10. Both of these proteins are heavily involved in tumorigenesis, suggesting a possible mechanism for the correlation between low vitamin D levels and colon cancer
Comparison of In-Solution, FASP, and S‑Trap Based Digestion Methods for Bottom-Up Proteomic Studies
Bottom-up proteomic
strategies rely on efficient digestion of proteins
into peptides for mass spectrometry analysis. In-solution and filter-based
strategies are commonly used for proteomic analysis. In recent years,
filter-aided sample preparation (FASP) has become the dominant filter-based
method due to its ability to remove SDS prior to mass spectrometry
analysis. However, the time-consuming nature of FASP protocols have
led to the development of new filter-based strategies. Suspension
traps (S-Traps) were recently reported as an alternative to FASP and
in-solution strategies as they allow for high concentrations of SDS
in a fraction of the time of a typical FASP protocol. In this study,
we compare the yields from in-solution, FASP, and S-Trap based digestions
of proteins extracted in SDS and urea-based lysis buffers. We performed
label-free quantification to analyze the differences in the portions
of the proteome identified using each method. Overall, our results
show that each digestion method had a high degree of reproducibility
within the method type. However, S-Traps outperformed FASP and in-solution
digestions by providing the most efficient digestion with the greatest
number of unique protein identifications. This is the first work to
provide a direct quantitative comparison of two filter-based digestion
methods and a traditional in-solution approach to provide information
regarding the most efficient proteomic preparation
Comparison of In-Solution, FASP, and S‑Trap Based Digestion Methods for Bottom-Up Proteomic Studies
Bottom-up proteomic
strategies rely on efficient digestion of proteins
into peptides for mass spectrometry analysis. In-solution and filter-based
strategies are commonly used for proteomic analysis. In recent years,
filter-aided sample preparation (FASP) has become the dominant filter-based
method due to its ability to remove SDS prior to mass spectrometry
analysis. However, the time-consuming nature of FASP protocols have
led to the development of new filter-based strategies. Suspension
traps (S-Traps) were recently reported as an alternative to FASP and
in-solution strategies as they allow for high concentrations of SDS
in a fraction of the time of a typical FASP protocol. In this study,
we compare the yields from in-solution, FASP, and S-Trap based digestions
of proteins extracted in SDS and urea-based lysis buffers. We performed
label-free quantification to analyze the differences in the portions
of the proteome identified using each method. Overall, our results
show that each digestion method had a high degree of reproducibility
within the method type. However, S-Traps outperformed FASP and in-solution
digestions by providing the most efficient digestion with the greatest
number of unique protein identifications. This is the first work to
provide a direct quantitative comparison of two filter-based digestion
methods and a traditional in-solution approach to provide information
regarding the most efficient proteomic preparation
Evaluation of the mirn23a Cluster through an iTRAQ-based Quantitative Proteomic Approach
Glucose Restriction Combined with Autophagy Inhibition and Chemotherapy in HCT 116 Spheroids Decreases Cell Clonogenicity and Viability Regulated by Tumor Suppressor Genes
Drug
resistance is a prevalent phenomenon that decreases the efficacy
of cancer treatments and contributes to cancer progression and metastasis.
Weakening drug-resistant cancer cells prior to chemotherapy is a potential
strategy to combat chemoresistance. One approach to damage resistant
cancer cells is modulation of nutritional intake. The combination
of nutrient restriction with targeted compound treatment results in
pronounced molecular changes. This study provides valuable information
about augmenting existing chemotherapeutic regimes with simultaneous
glucose restriction and autophagy inhibition in colorectal cancer
cells. In this study, we explore the chemical pathways that drive
the cellular response to nutrient restriction, autophagy inhibition,
and the chemotherapy irinotecan using global quantitative proteomics
and imaging mass spectrometry. We determined that significant pathways
were altered including autophagy and metabolism via glycolysis, gluconeogenesis,
and sucrose degradation. We also found that period circadian clock
2 (PER2), a tumor suppressor protein, was significantly up-regulated
only when glucose was restricted with autophagy inhibition and chemotherapy.
The upstream regulators of these differentially regulated pathways
were determined to have implications in cancer, showing an increase
in tumor suppressor proteins and a decrease in nuclear protein 1 (NUPR1)
an important protein in chemoresistance. We also evaluated the phenotypic
response of these cells and discovered autophagy inhibition and chemotherapy
treatment increased apoptosis and decreased cell clonogenicity and
viability. When glucose restriction was combined with autophagy inhibition
and chemotherapy, all of the phenotypic results were intensified.
In sum, our results indicate that glucose metabolism is of great importance
in the ability of cancer cells to survive chemotherapy. By weakening
cancer cells with glucose restriction and autophagy inhibition prior
to chemotherapy, cancer cells become more sensitive to therapy
iTRAQ Quantitative Proteomic Profiling and MALDI–MSI of Colon Cancer Spheroids Treated with Combination Chemotherapies in a 3D Printed Fluidic Device
For
a patient with metastatic colorectal cancer there are limited
clinical options aside from chemotherapy. Unfortunately, the development
of new chemotherapeutics is a long and costly process. New methods
are needed to identify promising drug candidates earlier in the drug
development process. Most chemotherapies are administered to patients
in combinations. Here, an in vitro platform is used to assess the
penetration and metabolism of combination chemotherapies in three-dimensional
colon cancer cell cultures, or spheroids. Colon carcinoma HCT 116
cells were cultured and grown into three-dimensional cell culture
spheroids. These spheroids were then dosed with a common combination
chemotherapy, FOLFIRI (folinic acid, 5-fluorouracil, and irinotecan)
in a 3D printed fluidic device. This fluidic device allows for the
dynamic treatment of spheroids across a semipermeable membrane. Following
dosing, the spheroids were harvested for quantitative proteomic profiling
to examine the effects of the combination chemotherapy on the colon
cancer cells. Spheroids were also imaged to assess the spatial distribution
of administered chemotherapeutics and metabolites with MALDI–imaging
mass spectrometry. Following treatment, we observed penetration of
folinic acid to the core of spheroids and metabolism of the drug in
the outer proliferating region of the spheroid. Proteomic changes
identified included an enrichment of several cancer-associated pathways.
This innovative dosing device, along with the proteomic evaluation
with iTRAQ-MS/MS, provides a robust platform that could have a transformative
impact on the preclinical evaluation of drug candidates. This system
is a high-throughput and cost-effective approach to examine novel
drugs and drug combinations prior to animal testing
Over 2300 Phosphorylated Peptide Identifications with Single-Shot Capillary Zone Electrophoresis-Tandem Mass Spectrometry in a 100 min Separation
Ultraperformance liquid chromatography
(UPLC)-electrospray ionization
(ESI)-tandem mass spectrometry (MS/MS) is typically employed for phosphoproteome
analysis. Alternatively, capillary zone electrophoresis (CZE)-ESI-MS/MS
has great potential for phosphoproteome analysis due to the significantly
different migration times of phosphorylated and unphosphorylated forms
of peptides. In this work, we systematically compared UPLC-MS/MS and
CZE-MS/MS for phosphorylated peptide identifications (IDs) using an
enriched phosphoproteome from the MCF-10A cell line. When the sample
loading amount of UPLC was 10 times higher than that of CZE (2 ÎĽg
vs 200 ng), UPLC generated more phosphorylated peptide IDs than CZE
(3313 vs 1783). However, when the same sample loading amounts were
used for CZE and UPLC (2–200 ng), CZE-MS/MS consistently and
significantly outperformed UPLC-MS/MS in terms of phosphorylated peptide
and total peptide IDs. This superior performance is most likely due
to the higher peptide intensity generated by CZE-MS/MS. More importantly,
compared with UPLC data from a 2 ÎĽg sample, CZE-MS/MS can identify
over 500 unique phosphorylated peptides from a 200 ng sample, suggesting
that CZE and UPLC are complementary for phosphorylated peptide IDs.
With further improved loading capacity via a dynamic pH junction method,
2313 phosphorylated peptides were identified with single-shot CZE-MS/MS
in a 100 min analysis. This number of phosphorylated peptide IDs is
over 1 order of magnitude higher than the number of phosphorylated
peptide IDs previously reported by single-shot CZE-MS/MS
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Promoting healthy cardiovascular aging: emerging topics
The development of age-related cardiovascular (CV) dysfunction increases the risk of CV disease as well as other chronic age-associated disorders, including chronic kidney disease, and Alzheimer’s disease and related dementias. Major manifestations of age-associated CV dysfunction that increase disease risk are vascular dysfunction, primarily vascular endothelial dysfunction and arterial stiffening, and elevated systolic blood pressure. Declines in nitric oxide bioavailability secondary to increased oxidative stress and inflammation are established mechanisms of CV dysfunction with aging. Moreover, fundamental mechanisms of aging, termed the “hallmarks of aging” extend to the CV system and, as such, may be considered “hallmarks of CV aging”. These mechanisms represent viable therapeutic targets for treating CV dysfunction with aging. Healthy lifestyle behaviors, such as regular aerobic exercise and certain dietary patterns, are considered “first-line” strategies to prevent and/or treat age-associated CV dysfunction. Despite the well-established benefits of these strategies, many older adults do not meet the recommended guidelines for exercise or consume a healthy diet. Therefore, it is important to establish alternative and/or complementary evidence-based approaches to prevent or reverse age-related CV dysfunction. Targeting fundamental mechanisms of CV aging with interventions such as time-efficient exercise training, food-derived molecules, termed nutraceuticals, or select synthetic pharmacological agents represents a promising approach. In the present review, we will highlight emerging topics in the field of healthy CV aging with a specific focus on how exercise, nutrition/dietary patterns, nutraceuticals and select synthetic pharmacological compounds may promote healthy CV aging, in part, by targeting the hallmarks of CV aging