VOPP1 Human

What is VOPP1 and what are its known alternative designations in the literature?

VOPP1 (Vesicular Overexpressed in Cancer Prosurvival Protein 1) was previously known under several alternative names including EGFR-coamplified and overexpressed protein (ECOP), Glioblastoma-amplified secreted protein (GASP), and Putative NF-kappa-B-activating protein 055N. The protein is encoded by a gene that has been identified through multiple research initiatives, leading to these various designations before standardization of nomenclature . When conducting literature searches, researchers should include these alternative terms to ensure comprehensive coverage of existing research. The protein has been characterized as having pro-survival functions in cancer cells and is predominantly localized to intracellular vesicles, which explains the "vesicular" component of its current name .

In which cancer types has VOPP1 overexpression been documented?

Research has consistently demonstrated VOPP1 overexpression across multiple cancer types. Most prominently, VOPP1 has been found overexpressed in:

• Squamous cell carcinoma (SCC) - confirmed in multiple SCC cell lines including SCC-9, FaDu, and H2170

• Glioblastoma multiforme - showing amplification-mediated expression

• Potentially in lung adenocarcinoma - with expression patterns correlating with proliferation factors

To study VOPP1 expression in cancer samples, researchers typically employ immunoblotting techniques to compare protein levels between benign tissue samples and malignant specimens. For example, one study demonstrated significant VOPP1 overexpression in SCC-9, FaDu, H2170, and HeLa cell lines relative to benign squamous mucosa tissue samples . This methodological approach allows for quantification of relative expression levels across different cancer types and normal tissues.

What experimental evidence exists for VOPP1's role in cell survival?

The critical role of VOPP1 in cancer cell survival has been demonstrated through multiple experimental approaches, particularly using siRNA-mediated knockdown studies. The methodology for establishing this role typically involves:

• siRNA transfection to reduce VOPP1 expression (using at least two different siRNA constructs to confirm specificity)

• Measurement of cell viability over a time course (24h, 48h, 72h)

• Assessment of cell death using multiple complementary assays

Research findings show that VOPP1 knockdown consistently induces significant cell death at approximately 72 hours post-transfection across multiple cancer cell lines . This phenotype has been validated using three independent methodologies:

• Trypan blue exclusion assays - showing increased percentage of dead cells

• LIVE/DEAD fluorescence assays - demonstrating decreased numbers of viable cells and increased dead/damaged cells

• Protease activity-based assays (MultiTox) - quantifying the fold change in dead-to-live cell ratios

These consistent findings across multiple methodologies and cell lines provide robust evidence for VOPP1's role as a critical pro-survival factor in cancer cells .

What molecular mechanisms underlie VOPP1-mediated cancer cell survival?

VOPP1 appears to promote cancer cell survival through multiple mechanisms, with redox regulation emerging as a primary function based on current evidence. Experimental approaches to elucidate these mechanisms have included:

• Gene expression profiling following VOPP1 knockdown

• Pathway analysis of differentially expressed genes

• Functional assays to validate predicted mechanisms

Research shows that VOPP1 knockdown results in significant changes to the intracellular redox state, leading to oxidative stress and mitochondrial dysfunction. Microarray analysis of gene expression following VOPP1 knockdown revealed 280 differentially expressed genes, with enrichment in annotations related to oxidative stress and mitochondrial dysfunction .

Importantly, the introduction of antioxidants such as N-acetyl cysteine was able to abrogate the apoptosis induced by VOPP1 knockdown in a dose-responsive manner, providing strong evidence that VOPP1's pro-survival function is mediated through regulation of cellular redox state .

While some studies have suggested VOPP1 may modulate NF-κB signaling, this appears to be cell-type dependent, occurring in HeLa cells but not in SCC cell lines, indicating potential tissue-specific functions of VOPP1 .

How does VOPP1 knockdown induce apoptosis in cancer cells?

VOPP1 knockdown induces apoptosis through the intrinsic (mitochondrial) pathway, as demonstrated by a series of carefully designed experiments. The evidence for this mechanism includes:

• Temporal analysis of caspase activation following VOPP1 knockdown

• Specific assessment of caspase-9 (intrinsic pathway) versus other apoptotic pathways

• Correlation with mitochondrial dysfunction markers

Research demonstrates that VOPP1 knockdown results in:

• Activation of caspase-9 at 48 hours post-knockdown, indicating apoptosome activation through the intrinsic pathway

• Subsequent activation of effector caspases-3/7, confirming execution of the classical apoptotic program

• Correlation between the timing of caspase activation and observable cell death phenotypes (absent at 24h, present at 72h post-knockdown)

This temporal sequence provides strong evidence that VOPP1's role in cancer cell survival is mediated through protection against intrinsic apoptotic pathway activation, rather than through extrinsic death receptor pathways .

What is the relationship between VOPP1 and reactive oxygen species (ROS) in cancer cells?

VOPP1 appears to be a crucial regulator of intracellular redox state, with knockdown resulting in elevated ROS levels. This relationship has been demonstrated using multiple experimental approaches:

• Direct measurement of ROS using the fluorescent probe CM-H2DCF-DA

• Assessment of ROS-mediated cellular damage

• Rescue experiments using antioxidants

The data show that ROS levels become significantly elevated following VOPP1 knockdown, occurring at time points before the activation of effector caspases and cell death seen at later time points . This temporal relationship suggests that ROS elevation is a cause rather than a consequence of apoptosis.

The causal relationship between VOPP1-mediated ROS regulation and cell survival was confirmed through antioxidant rescue experiments. The addition of N-acetyl cysteine was able to abrogate the induction of apoptosis observed with VOPP1 knockdown in a dose-responsive manner, definitively linking VOPP1's pro-survival function to its role in regulating cellular redox state .

For researchers investigating this relationship, the recommended methodological approach includes:

• siRNA-mediated VOPP1 knockdown in relevant cancer cell models

• ROS measurement at multiple time points (24h, 48h, 72h) using fluorescent probes

• Concurrent assessment of mitochondrial function and apoptotic markers

• Antioxidant rescue experiments with dose-response analysis

How does VOPP1 influence mitochondrial function in cancer cells?

VOPP1 appears to play a critical role in maintaining mitochondrial integrity in cancer cells. Loss of VOPP1 expression leads to significant mitochondrial dysfunction, which precedes apoptotic cell death. This relationship has been characterized using:

• Mitochondrial membrane potential measurements

• Assessment of mitochondrial viability

• Temporal analysis of mitochondrial dysfunction relative to apoptosis activation

Research using MitoTracker Red CMXRos, a fluorescent probe that accumulates in mitochondria with intact membrane potential, demonstrated that VOPP1 knockdown results in loss of mitochondrial membrane potential . This dysfunction occurs prior to caspase activation and cell death, suggesting it is a causative event in the apoptotic cascade.

For researchers studying VOPP1's impact on mitochondrial function, the recommended methodological approach includes:

• siRNA-mediated VOPP1 knockdown in relevant cancer models

• Assessment of mitochondrial membrane potential using potentiometric dyes

• Quantification of mitochondrial mass and morphology

• Measurement of mitochondrial respiration and ATP production

• Time-course analysis to establish causative relationships

What are the optimal siRNA designs for effective VOPP1 knockdown studies?

Based on validated research, effective VOPP1 knockdown can be achieved using at least two different siRNA constructs to control for potential off-target effects. Successfully employed siRNA sequences include:

• 5′-GGACUCUAUCCAACCUAUU-3′ (targeting the coding sequence)

• 5′-GACAGGAGAAGUACUGACU-3′ (targeting the 3′-UTR)

The recommended transfection protocol involves:

• Using 5 pmol siRNA in 96-well format or 20 pmol in 24-well format

• Including GC content-matched siRNA controls

• Verifying knockdown efficiency via immunoblotting at 24h post-transfection

• Maintaining cultures for at least 72h to observe phenotypic effects

Using this methodology, researchers have achieved reduction of VOPP1 protein levels to approximately 16% of controls, which is sufficient to observe significant phenotypic effects .

How can researchers effectively measure the impact of VOPP1 on cell viability and death?

To comprehensively assess the impact of VOPP1 on cell viability and death, a multi-modal approach using complementary assays is recommended. Based on published research, the following methodology provides robust results:

• Trypan blue exclusion assay:

  • Conduct at multiple time points (24h, 48h, 72h post-knockdown)

  • Calculate percentage of dead cells relative to total cell count

  • Compare between VOPP1 knockdown and control conditions

• Fluorescence microscopy-based LIVE/DEAD assay:

  • Use calcein-AM to identify live cells

  • Use ethidium homodimer-1 to mark dead/damaged cells

  • Quantify dead-to-live ratios from multiple microscopic fields

  • Calculate fold changes relative to control conditions

• Protease activity-based viability assay (MultiTox):

  • Measure specific proteases associated with living and dead cells

  • Calculate fold changes in dead-to-live signal ratios

  • Perform in multi-well format for higher throughput assessment

• Caspase activation assays:

  • Measure caspase-3/7 activity to confirm apoptotic mechanism

  • Assess caspase-9 activity to confirm intrinsic pathway involvement

  • Perform at multiple time points to establish temporal relationships

What methods are appropriate for measuring ROS levels in VOPP1 studies?

For accurate assessment of ROS levels in VOPP1 functional studies, the following methodology has been validated:

• CM-H2DCF-DA fluorescent probe assay:

  • Culture cells on glass coverslips following VOPP1 siRNA knockdown

  • Label with 5 μM CM-H2DCF-DA and 1 μM Hoechst 33342 in PBS for 30 minutes

  • Rinse in distilled water and return to normal culture media

  • Image via fluorescence microscopy

  • Analyze digital images using ImageJ with standard segmentation protocols

  • Calculate percentage of ROS-positive cells using constant threshold values for comparability

• Complementary approaches to consider:

  • Flow cytometry-based ROS detection for higher throughput quantification

  • Specific ROS detection probes for mitochondrial versus cytosolic ROS

  • Protein carbonylation or lipid peroxidation assays as markers of ROS-induced damage

• Validation experiments:

  • Include positive controls (e.g., H2O2 treatment)

  • Perform antioxidant rescue experiments

  • Conduct time-course analyses to establish cause-effect relationships

This methodological approach provides reliable quantification of ROS levels following VOPP1 modulation and helps establish the causal relationship between VOPP1 function, ROS regulation, and cell survival .

What are the recommended approaches for studying VOPP1 protein expression in clinical samples?

For optimal analysis of VOPP1 protein expression in clinical samples, a multi-faceted approach combining different protein detection methods is recommended:

• Immunoblotting protocol:

  • Use affinity-purified polyclonal rabbit anti-VOPP1 antibodies

  • Include appropriate loading controls (e.g., anti-tubulin, anti-GAPDH)

  • Perform protein extraction using standardized protocols

  • Quantify relative expression levels using densitometry

  • Compare expression between malignant samples and matched normal tissues

• Tissue analysis considerations:

  • Include adequate numbers of benign (n≥4) and malignant (n≥9) tissue samples

  • Ensure matched normal-tumor pairs when possible

  • Consider tissue microarrays for higher throughput analysis

• Cell line validation:

  • Confirm VOPP1 expression status in potential model systems

  • Select cell lines with confirmed endogenous VOPP1 overexpression

  • Exclude cell lines without significant VOPP1 overexpression

This methodological approach allows for accurate assessment of VOPP1 expression patterns across different cancer types and identification of appropriate model systems for functional studies .

What are the emerging therapeutic strategies targeting VOPP1 in cancer?

Given VOPP1's critical role in cancer cell survival, several potential therapeutic approaches are emerging:

• Direct VOPP1 inhibition:

  • Small molecule inhibitors targeting VOPP1 protein function

  • Antisense oligonucleotides or siRNA-based approaches for transcript reduction

  • Protein degradation strategies (e.g., PROTACs)

• Exploiting VOPP1-mediated redox vulnerability:

  • Combination therapies with ROS-inducing agents

  • Mitochondrial-targeted compounds to enhance VOPP1 inhibition effects

  • Antioxidant inhibitors to prevent adaptive responses

• Rational combination strategies:

  • EGFR inhibitors plus VOPP1 targeting in contexts with co-amplification

  • NF-κB pathway modulators in cell types where VOPP1 regulates this pathway

  • Apoptosis sensitizers to enhance VOPP1 inhibition effects

Research suggests that amplification-mediated VOPP1 expression, such as that occurring in tumors with amplified EGFR, might impact resistance to apoptosis . This indicates that VOPP1 targeting could be particularly effective in EGFR-amplified cancers or as a strategy to overcome resistance to existing therapies.

How does VOPP1 interact with other cancer-associated pathways?

Current research suggests complex interactions between VOPP1 and other cancer-associated pathways:

• NF-κB signaling:

  • Cell-type dependent effects (active in HeLa cells but not in SCC cell lines)

  • Potential context-specific regulatory mechanisms

  • Requires investigation in diverse cancer models

• EGFR pathway:

  • Co-amplification patterns in certain cancer types

  • Potential cooperative effects on cancer cell survival

  • Implications for resistance to EGFR-targeted therapies

• Proliferation networks:

  • In lung adenocarcinoma, VOPP1 shows increased expression correlating with multiple proliferation factors

  • Associated with genes YKT6, KLHL7, and FAM220A in specific cancer contexts

  • Suggests potential role in coordinating proliferative signaling

Future research should employ network analysis approaches, co-immunoprecipitation studies, and functional genomics screens to further elucidate these complex interactions and identify potential synthetic lethal relationships that could be therapeutically exploited.

What is the structural and functional significance of VOPP1's vesicular localization?

The vesicular localization of VOPP1 presents intriguing questions about its function:

• Current structural knowledge:

  • Human recombinant VOPP1 contains 112 amino acids (positions 82-172)

  • Molecular mass of approximately 12kDa

  • N-terminal His tag in recombinant protein systems

• Functional implications of vesicular localization:

  • Challenges the model of direct VOPP1 interaction with cytoplasmic NF-κB proteins

  • Suggests potential roles in vesicular trafficking or organelle function

  • May explain cell-type specific effects observed in different cancer models

• Research approaches to address this question:

  • Detailed co-localization studies with various vesicular markers

  • Structure-function analyses with domain deletion/mutation constructs

  • Proximity labeling approaches to identify interacting partners in the vesicular context

Understanding the significance of VOPP1's vesicular localization may provide critical insights into its mechanism of action and guide more effective therapeutic targeting strategies.

What recombinant VOPP1 protein resources are available for research?

For researchers studying VOPP1 function or developing assays, the following recombinant protein resources are available: