RECK Antibody

What is RECK and why are RECK antibodies important in cancer research?

RECK (Reversion-Inducing-Cysteine-Rich Protein with Kazal Motifs) is a membrane-anchored glycoprotein that functions as a key regulator of extracellular matrix remodeling. It is particularly important as a negative regulator of matrix metalloproteinases (MMPs), which are enzymes involved in degrading the extracellular matrix. RECK antibodies are critical tools in cancer research because:

• RECK is frequently downregulated in various cancer types, and reduced expression often correlates with poorer prognoses

• RECK serves as a potent inhibitor of tumor invasion, metastasis, and angiogenesis

• Forced expression of RECK in tumor xenografts results in suppressed tumor angiogenesis, invasion, and metastasis in mouse models

• RECK mutations are rare in cancer genomes, suggesting its expression is transcriptionally or epigenetically regulated, making it a potential therapeutic target

What are the major types of RECK antibodies available for research?

Several types of RECK antibodies are available for research applications, including:

• Rabbit polyclonal antibodies targeting various amino acid regions (e.g., AA 23-212, AA 110-210, AA 430-456, AA 801-900)

• Mouse polyclonal antibodies against full-length RECK protein

• Goat anti-human RECK antigen affinity-purified polyclonal antibodies

These antibodies come in different formats:

• Unconjugated antibodies for general applications

• Conjugated versions (HRP, FITC, Biotin) for specialized applications like ELISA

What is the molecular structure and function of RECK protein?

RECK is a GPI-anchored glycoprotein with multiple functional domains:

• Contains multiple epidermal growth factor-like (EGF-like) repeats and serine-protease inhibitor (SPI) motifs

• Anchored to the cell membrane via a C-terminal glycosylphosphatidylinositol (GPI) modification

• Functions as a natural inhibitor of MMPs, particularly MMP-9, MMP-2, and MMP-14 (MT1-MMP)

• Besides MMP inhibition, RECK:

  • Functions together with ADGRA2 to enable brain endothelial cells to selectively respond to Wnt7 signals

  • Acts as a Wnt7-specific coactivator of canonical Wnt signaling

  • Plays roles in cell migration, proliferation, and apoptosis

How do different RECK isoforms influence cell migration and cancer progression?

Research has revealed that RECK has multiple isoforms with opposing effects on cell behavior:

• The long RECK isoform (canonical, 110 kDa) inhibits cell migration and has tumor-suppressive properties

• The short RECK isoform (25 kDa) promotes cell migration

• These isoforms are regulated differently during cellular states:

  • Long RECK expression increases during quiescence

  • Short RECK expression increases during proliferation

  • TGF-β treatment can alter the balance between isoforms

The opposing functions appear to be mediated through protein-protein interactions:

• Short and long RECK isoforms can interact with each other

• This interaction occurs in the ER, Golgi, and cell surface

• Both isoforms can also form homodimers

• The short RECK isoform may inhibit the anti-migratory function of long RECK through these interactions

What mechanisms regulate RECK expression in normal and cancer cells?

RECK expression is regulated through multiple mechanisms:

• Transcriptional regulation:

  • SP1 sites in the RECK promoter are involved in its expression

  • KLF2 may mediate RECK induction

  • Histone deacetylase (HDAC) inhibitors can upregulate RECK expression

• Epigenetic regulation:

  • RECK downregulation in cancer often occurs without mutations in the gene itself

  • Hypoxia suppresses RECK expression, while HDAC inhibition can restore it

• MicroRNA regulation:

  • miR-21 negatively regulates RECK expression

  • Compounds like nimbolide can upregulate RECK by targeting miR-21 and HIF-1α

How does RECK contribute to the regulation of tumor microenvironment and angiogenesis?

RECK's role in the tumor microenvironment is multifaceted:

What are the optimal protocols for detecting RECK protein in different experimental systems?

Detection methods vary based on the experimental system:

Western Blot Analysis:

• Use reducing conditions with Immunoblot Buffer Group 1

• Expected molecular weight: approximately 130 kDa for the long RECK isoform

• Recommended antibody dilutions: 1:500 - 1:2000

• Positive control samples: 293T, MCF-7, H460, Mouse lung, Mouse kidney

Immunohistochemistry:

• Recommended dilution: 1:200-1:500

• Applications include paraffin-embedded (IHC-p) and frozen (IHC-fro) sections

Immunofluorescence:

• Recommended dilution: 1:50-1:200

• Applicable for both cell cultures (IF-cc) and tissue sections (IF-p)

ELISA:

• Unconjugated or HRP/Biotin-conjugated antibodies can be used

• Protein G-purified antibodies (>95% purity) yield optimal results

How can researchers differentiate between RECK isoforms in experimental settings?

Distinguishing between RECK isoforms requires specific approaches:

RNA Detection:

• Design isoform-specific primers for RT-PCR:

  • Primers targeting the unique 3' UTR of the short RECK isoform

  • Primers for regions present in long but not short RECK

  • Universal primers recognizing both isoforms for comparison

Protein Detection:

• Use antibodies targeting specific regions:

  • Long RECK-specific antibodies can target regions absent in short RECK

  • Short RECK-specific antibodies should recognize the unique 13-amino-acid C-terminal exon

• Expected molecular weights:

  • Long RECK: ~110 kDa

  • Short RECK: ~25 kDa

Co-immunoprecipitation Studies:

• For interaction studies, use differentially tagged constructs:

  • Flag-tagged short RECK and S-tagged long RECK have been successfully used

  • PI-PLC treatment can cleave the GPI anchor of long RECK to study surface interactions

What experimental models are most appropriate for studying RECK's role in metastasis inhibition?

Several validated experimental models are useful for studying RECK's anti-metastatic properties:

In Vitro Models:

• Matrigel invasion assays: Evaluate the ability of cells with different RECK expression levels to invade through basement membrane matrix

• Flat reversion assays: Assess morphological changes in v-K-RAS-transformed cells upon RECK modulation

• Suspension culture systems: Study the effects of RECK on anchorage-independent growth and aggregation

• Zymography: Monitor MMP activity in response to RECK expression or modulating compounds

In Vivo Models:

• Mouse xenograft models:

  • Spontaneous lung metastasis model with RECK-expressing tumor cells

  • HPV-transformed cell lines with modulated RECK expression show different tumor establishment rates and animal survival

• DMBA-induced hamster buccal pouch carcinogenesis model: A paradigm for oral oncogenesis that shows sequential RECKlessness stimulating angiogenesis and Notch signaling

What are common issues with RECK detection in Western blots and how can they be resolved?

Challenge: Multiple or unexpected bands

• Cause: Detection of different RECK isoforms or degradation products

• Solution: Use reducing conditions, fresh samples, and protease inhibitors during extraction

• Verification: Compare with positive control samples (293T, MCF-7, H460 lysates)

Challenge: Weak signal detection

• Cause: Low RECK expression in many cancer cell lines

• Solution: Use sensitive detection methods and longer exposure times

• Optimization: Consider using PVDF membrane and HRP-conjugated secondary antibodies as successful combinations

Challenge: Non-specific binding

• Cause: Antibody cross-reactivity or improper blocking

• Solution: Increase blocking time/concentration and optimize antibody dilution

• Alternative: Try different anti-RECK antibodies targeting various epitopes (AA 23-212, AA 801-900, etc.)

How can researchers address variability in RECK expression across different cell types?

Challenge: Cell type-dependent expression patterns

• Approach: Establish baseline RECK expression for your specific cell type using qPCR before protein analysis

• Consideration: RECK is downregulated in many transformed cells but expressed in normal tissues

Challenge: Hypoxic conditions affecting RECK expression

• Cause: Hypoxia suppresses RECK expression via HIF-1α

• Solution: Control oxygen conditions during experiments and consider using HDAC inhibitors to restore RECK expression in hypoxic conditions

Challenge: Growth conditions affecting isoform ratios

• Observation: Proliferating vs. contact-inhibited cells show different RECK isoform patterns

• Approach: Standardize growth conditions and cell density for comparable results

How should researchers interpret changes in RECK expression in relation to cancer progression?

When analyzing RECK expression data:

• Consider the balance between long and short isoforms, not just total RECK levels

• Correlate RECK expression with:

  • MMP activity (particularly MMP-2, MMP-9, and MT1-MMP)

  • Invasive/metastatic potential

  • Angiogenic markers (microvascular density, VEGF expression)

  • Patient survival data

• Interpret RECK restoration as potentially beneficial:

  • In xenograft models, RECK expression delayed tumor establishment and growth

  • 30% of animals injected with SW765 RECK+ cells exhibited tumor regression

  • RECK expression increased animal survival

What are the most promising approaches for targeting RECK in cancer therapeutics?

Current research highlights several strategies:

RECK Induction Approaches:

• Small molecule HDAC inhibitors:

  • DSK638 has shown promise in inducing RECK expression and inhibiting metastasis

  • Other benzamide-type HDAC inhibitors (CI-994/Tacedinaline, MS275/Entinostat, QSS) also induce RECK expression

• Natural compounds:

  • Nimbolide (from Azadirachta indica) upregulates RECK by targeting miR-21 and HIF-1α

  • This results in reduced MMP activity and blockade of VEGF and Notch signaling

Combination Approaches:

• Target both RECK and MXI1:

  • DSK638 upregulates both RECK and MXI1 (an endogenous MYC-antagonist)

  • Inhibition of metastasis by DSK638 is dependent on both proteins

Screening Platforms:

• RECK-promoter reporter assays have been successful in identifying compounds that activate RECK expression

• Quantitative reversion assays and suspension culture systems can evaluate the anti-oncogenic/anti-metastatic activities of test compounds

How can RECK antibodies contribute to understanding the relationship between RECK and the Wnt signaling pathway?

RECK antibodies are valuable tools for investigating RECK's role in Wnt signaling:

Detection of RECK-Wnt Interactions:

• Co-immunoprecipitation with RECK antibodies can identify interactions with:

  • Wnt7 proteins (RECK interacts specifically with the disordered linker region of Wnt7)

  • ADGRA2, which is required to deliver RECK-bound Wnt7 to frizzled receptors

  • Components of the RECK-ADGRA2-Fzd-LRP5-LRP6 complex

Functional Studies:

• RECK antibodies can be used to:

  • Block RECK function in central nervous system (CNS) angiogenesis experiments

  • Study RECK's role as a Wnt7-specific coactivator of canonical Wnt signaling

  • Investigate blood-brain barrier regulation

Combined Approaches:

• Using RECK antibodies with Wnt pathway inhibitors or activators can help dissect:

  • How RECK confers ligand selectivity for Wnt7

  • The dual role of RECK in protease inhibition and Wnt signaling

  • Potential crosstalk between these pathways in cancer progression

Research Data Table: RECK Antibody Comparative Applications