C2orf40 Antibody

What is C2orf40 and what is its significance in cancer biology?

C2orf40 is a tumor suppressor gene that has been identified in various cancers, most notably in nasopharyngeal carcinoma (NPC). Research has demonstrated that C2orf40 expression is significantly downregulated in NPC tissues compared to normal nasopharyngeal epithelial tissues, and this downregulation is inversely associated with poor prognosis . Mechanistically, C2orf40 functions by inhibiting cancer cell migration and invasion while enhancing sensitivity to radiotherapy and chemotherapy treatments .

The biological significance of C2orf40 extends beyond NPC, as it plays a crucial role in cellular functions and signaling cascades, making it an important target for research in areas including cellular metabolism, gene regulation, and protein interactions .

What experimental applications can C2orf40 antibodies be used for?

C2orf40 antibodies, such as the PACO05519 polyclonal antibody, have been validated for multiple experimental applications, including:

• ELISA (recommended dilution 1:20000)

• Immunohistochemistry (IHC) (recommended dilution 1:100-1:300)

• Immunofluorescence (IF) (recommended dilution 1:200-1:1000)

• Western blotting

These applications allow researchers to detect and analyze C2orf40 protein expression in various experimental contexts. For IHC applications specifically, researchers typically perform antigen retrieval using citrate solution, followed by overnight incubation with the primary antibody at 4°C, and subsequent visualization using appropriate secondary antibodies and detection systems such as EnVision peroxidase reagent and 3,3-diaminobenzidine (DAB) staining .

How does C2orf40 expression correlate with patient outcomes in cancer?

Studies on nasopharyngeal carcinoma have revealed a strong correlation between C2orf40 expression and patient prognosis. Kaplan-Meier analysis of NPC patients (n=96) demonstrated that higher C2orf40 expression predicts better prognosis with a hazard ratio (HR) of 0.1595 (p < 0.01) . This correlation was established through immunohistochemical analysis, which categorized patients into high C2orf40 expression (n=48) and low C2orf40 expression (n=48) groups .

This significant association between expression levels and clinical outcomes suggests that C2orf40 could serve as a potential prognostic biomarker in NPC and possibly other cancers where this gene functions as a tumor suppressor.

What are the optimal protocols for C2orf40 overexpression in cancer cell lines?

For C2orf40 overexpression in cancer cell lines, researchers have successfully employed the following methodology:

• Vector Construction:

  • Use pcDNA-3.1(+) vectors for transient overexpression

  • Clone C2orf40 using specific primers:

    • Forward: 5′-CTAGCTAGCCCACCGATGGCTGCCTCCCCCGCGCGGCC-3′

    • Reverse: 5′-TTAGTAGTCATCGTAGTTGACGCTGATATCCCG-3′

  • Use restriction enzymes AgeI and EcoRI for cloning into the vector

• Transient Transfection Protocol:

  • Seed 3×10^5 cells in 6-well plates and incubate at 37°C for 24h

  • Transfect 2000 ng of vector containing C2orf40 or empty vector using Lipofectamine 3000 reagent

  • Repeat experiments at least three times for validation

• Stable Overexpression Protocol:

  • Subclone the full-length human C2orf40 gene into lentiviral vector pLV

  • Co-transfect pLV-C2orf40, psPAX2, and pMD2.G into HEK-293T cells

  • Collect virus-containing supernatants and infect target NPC cells for 48h

  • Select stable clones with 0.5 μg/ml puromycin

Verification of overexpression should be performed using both qRT-PCR for mRNA expression and Western blotting for protein expression to confirm successful transfection before proceeding with functional assays.

How can researchers accurately evaluate C2orf40 methylation status in clinical samples?

To evaluate C2orf40 methylation status in clinical samples, researchers can follow this methodological approach:

• Sample Collection and DNA Extraction:

  • Obtain paired tumor and normal tissue samples (e.g., 24 NPC tissues and 24 normal nasopharyngeal epithelial tissues as described in the literature)

  • Extract genomic DNA using standard procedures ensuring high purity

• Methylation Analysis:

  • Employ pyrosequencing for quantitative methylation level detection

  • Focus analysis on the promoter region of C2orf40

  • Compare methylation levels between tumor and normal tissues using appropriate statistical methods

• Functional Validation:

  • Treat cancer cell lines with DNA methylation inhibitors such as decitabine (DAC)

  • Measure C2orf40 mRNA levels post-treatment using qRT-PCR

  • A significant elevation in C2orf40 expression following DAC treatment confirms methylation-dependent regulation

Research has demonstrated that the DNA methylation level at the promoter region of C2orf40 in NPC tissues is notably higher than in normal nasopharyngeal epithelial tissues, suggesting that hypermethylation is a key mechanism for C2orf40 downregulation in NPC .

What are the optimal immunohistochemistry protocols for detecting C2orf40 in paraffin-embedded tissues?

For optimal detection of C2orf40 in paraffin-embedded tissues, researchers should follow this detailed IHC protocol:

• Sample Preparation:

  • Deparaffinize tissue sections

  • Rehydrate using standard procedures

  • Perform antigen retrieval using citrate solution

• Antibody Incubation:

  • Apply primary C2orf40 antibody at a dilution of 1:100 to 1:300 (optimal dilution 1:400 has been reported)

  • Incubate overnight at 4°C

  • Apply appropriate secondary antibody and incubate for 30 minutes in a humid chamber

  • Wash thoroughly with Tris-buffered saline (TBS)

• Detection and Visualization:

  • Incubate slides with EnVision peroxidase reagent for 30 minutes

  • Stain with 3,3-diaminobenzidine (DAB) for 5 minutes

  • Counterstain with Mayer's hematoxylin solution

  • Mount slides with appropriate mounting medium

• Quantification:

  • Assess percentage of positively stained cells in multiple images (at least three)

  • Use ImageJ software for quantitative analysis

  • Categorize samples as high or low expression based on established cutoffs

This protocol has been successfully employed to determine C2orf40 expression levels in NPC patients, with results showing significant correlation with prognosis.

How does C2orf40 influence cell cycle regulation in cancer cells?

C2orf40 has been demonstrated to influence cell cycle regulation in cancer cells through the following mechanisms:

• Cell Cycle Arrest Induction:

  • Overexpression of C2orf40 induces cell cycle arrest specifically at the G2/M phase in cancer cell lines including HONE-1 and SUNE-1

• Regulation of Cell Cycle-Related Proteins:

  • C2orf40 expression is negatively correlated with cell cycle regulators CCNE1 and CDK1 across multiple GEO datasets (GSE12452, GSE53819, GSE12452)

  • C2orf40 overexpression leads to downregulation of:

    • CDK1 (Cyclin-dependent kinase 1)

    • CCNE1 (Cyclin E1)

    • CCNB1 (Cyclin B1)

  • Additionally, C2orf40 inhibits phosphorylation of:

    • CDK1

    • Rb (Retinoblastoma protein)

• Bioinformatic Evidence:

  • Gene co-expression analysis identified 544 genes negatively correlated with C2orf40 (|R|> 0.3, P < 0.05)

  • GO and KEGG pathway enrichment analyses revealed these genes were significantly associated with cell cycle regulation, platinum resistance, DNA repair, and PI3K signaling pathway

This cell cycle regulatory function of C2orf40 provides a mechanistic explanation for its role in enhancing chemosensitivity and radiosensitivity in cancer cells.

What signaling pathways does C2orf40 interact with to exert its tumor-suppressive effects?

C2orf40 exerts its tumor-suppressive effects through interaction with multiple signaling pathways:

• PI3K/AKT/mTOR Signaling Pathway:

  • C2orf40 overexpression inhibits the activation of the PI3K/AKT/mTOR pathway

  • Specifically, it reduces phosphorylation levels of:

    • PI3K

    • AKT

    • mTOR

  • This inhibitory effect persists even when cells are exposed to radiation (2 Gy)

• Homologous Recombination Repair (HRR) Pathway:

  • C2orf40 expression negatively correlates with genes involved in HRR:

    • BRCA1 (P < 0.01)

    • BRCA2 (P < 0.05)

    • CDC25A (P < 0.01)

    • RAD51 (P < 0.05)

  • Overexpression of C2orf40 downregulates expression of these HRR-related proteins

• Apoptotic Pathway:

  • C2orf40 overexpression increases expression of apoptosis-associated proteins:

    • C-caspase-3

    • C-PARP

    • Bax

  • This effect is enhanced when cells are treated with cisplatin

The interaction with these pathways collectively contributes to C2orf40's ability to inhibit cancer cell migration and enhance sensitivity to chemotherapy and radiotherapy.

How does C2orf40 influence chemosensitivity and radiosensitivity in cancer cells?

C2orf40 enhances both chemosensitivity and radiosensitivity in cancer cells through multiple interconnected mechanisms:

• Chemosensitivity Enhancement:

  • Cisplatin Sensitivity:

    • C2orf40-overexpressing cells show increased sensitivity to cisplatin treatment across various concentrations (0-8 μM)

    • CCK-8 assays and colony formation assays confirm enhanced sensitivity

  • Apoptosis Induction:

    • TUNEL assays reveal increased cisplatin-induced apoptosis in C2orf40-overexpressing cells

    • Western blot analysis shows upregulation of apoptosis-associated proteins (C-caspase-3, C-PARP, Bax) in C2orf40-overexpressing cells, especially after cisplatin treatment

• Radiosensitivity Enhancement:

  • DNA Damage Response:

    • C2orf40 overexpression inhibits the repair of radiation-induced DNA damage

    • Comet assay reveals increased DNA damage in irradiated C2orf40-overexpressing cells

  • Homologous Recombination Inhibition:

    • C2orf40 downregulates HRR-related proteins (BRCA1, BRCA2, RAD51, CDC25A)

    • Impaired HRR leads to inadequate repair of radiation-induced double-strand breaks

• Cell Cycle Regulation:

  • G2/M phase arrest induced by C2orf40 increases cell vulnerability to both chemotherapy and radiotherapy

  • Downregulation of cell cycle proteins (CDK1, CCNE1, CCNB1) contributes to this effect

These mechanisms collectively explain how C2orf40 functions as a potential molecular target for improving cancer treatment efficacy.

What are common issues encountered when detecting C2orf40 in clinical samples and how can they be addressed?

Researchers may encounter several challenges when detecting C2orf40 in clinical samples:

• Low Expression Levels:

  • Issue: C2orf40 is frequently downregulated in cancer tissues, making detection challenging

  • Solution:

    • Use amplification steps in IHC protocols (e.g., EnVision peroxidase system)

    • Optimize antibody concentrations (1:100-1:400 dilution range)

    • Consider extended primary antibody incubation (overnight at 4°C)

• Variable Methylation Status:

  • Issue: Hypermethylation of C2orf40 promoter varies between samples

  • Solution:

    • Perform pyrosequencing to quantify methylation levels

    • Consider treating cell lines with demethylating agents like decitabine as positive controls

    • Correlate methylation status with expression levels in each sample

• Tissue Heterogeneity:

  • Issue: Variable expression across different areas of the same tumor

  • Solution:

    • Analyze multiple regions (at least three images per sample)

    • Use ImageJ software for quantitative assessment

    • Report percentage of positively stained cells rather than binary results

• Antigen Retrieval Effectiveness:

  • Issue: Inadequate antigen retrieval leading to false negatives

  • Solution:

    • Optimize citrate buffer concentration and pH

    • Carefully control retrieval time and temperature

    • Consider alternative retrieval methods for difficult samples

How can researchers validate the specificity of C2orf40 antibodies in their experimental systems?

To ensure antibody specificity for C2orf40 detection, researchers should implement the following validation procedures:

• Positive and Negative Controls:

  • Positive Controls:

    • Use cell lines with confirmed C2orf40 expression

    • Include C2orf40-overexpressing transfected cells alongside empty vector controls

    • Consider normal tissue samples known to express C2orf40

  • Negative Controls:

    • Omit primary antibody in parallel samples

    • Use cell lines with confirmed C2orf40 downregulation

    • Consider samples with C2orf40 promoter hypermethylation

• Multiple Detection Methods:

  • Confirm findings using different detection techniques:

    • Western blotting to confirm antibody specificity by molecular weight

    • qRT-PCR to correlate protein detection with mRNA levels

    • IF to confirm cellular localization patterns

• Knockdown/Overexpression Validation:

  • Generate cells with:

    • C2orf40 overexpression using pcDNA-3.1(+) vectors or lentiviral systems

    • C2orf40 knockdown using siRNA or CRISPR/Cas9

  • Confirm signal changes correlate with genetic manipulation

• Peptide Competition Assays:

  • Pre-incubate antibody with purified C2orf40 peptide

  • Observe signal reduction in Western blot or IHC

  • Confirm specificity through signal blocking

What controls should be included when studying C2orf40's effect on chemosensitivity and radiosensitivity?

When investigating C2orf40's impact on treatment sensitivity, the following controls are essential:

• Expression Controls:

  • Vector Controls:

    • Empty vector-transfected cells (pcDNA-3.1(+) or pLV)

    • Verify absence of C2orf40 overexpression by Western blot and qRT-PCR

  • Expression Verification:

    • Confirm C2orf40 expression levels before all functional assays

    • Document both protein and mRNA levels

• Treatment Controls:

  • Dose-Response Curves:

    • Include multiple concentrations of chemotherapy agents (e.g., cisplatin 0-8 μM)

    • Test various radiation doses (e.g., 0, 2, 4, 6, and 8 Gy)

  • Timing Controls:

    • Document treatment duration

    • Perform time-course experiments to capture optimal response windows

• Pathway Validation Controls:

  • Pathway Inhibitors:

    • Include PI3K/AKT/mTOR pathway inhibitors to confirm pathway involvement

    • Compare effects with C2orf40 overexpression

  • Protein Expression:

    • Monitor expression of pathway-related proteins:

      • For HRR: BRCA1, BRCA2, RAD51, CDC25A

      • For cell cycle: CDK1, CCNE1, CCNB1

      • For apoptosis: C-caspase-3, C-PARP, Bax

• Technical Controls:

  • Viability Assays:

    • Include multiple assay types (CCK-8, colony formation, TUNEL)

    • Perform each in at least triplicate

  • Statistical Validation:

    • Apply appropriate statistical tests

    • Report p-values and confidence intervals

How should researchers interpret contradictory results between in vitro and in vivo C2orf40 studies?

When encountering contradictions between in vitro and in vivo C2orf40 studies, researchers should consider:

What statistical approaches are most appropriate for analyzing C2orf40 expression in relation to patient outcomes?

For robust statistical analysis of C2orf40 expression and patient outcomes:

• Survival Analysis:

  • Kaplan-Meier Method:

    • Stratify patients into high and low C2orf40 expression groups

    • Calculate hazard ratios (e.g., HR = 0.1595 as reported)

    • Report log-rank p-values for significance testing

  • Cox Proportional Hazards Model:

    • Incorporate multiple variables including C2orf40 expression

    • Report adjusted hazard ratios

    • Include confidence intervals

• Expression Categorization:

  • Continuous vs. Categorical:

    • Consider analyzing C2orf40 expression both as a continuous variable and categorical (high/low)

    • Determine optimal cutoff points using:

      • Median split

      • ROC curve analysis

      • Minimum p-value approach

  • Sample Balancing:

    • Ensure balanced groups (e.g., n=48 in both high and low expression groups)

• Correlation Analysis:

  • Gene Expression Correlations:

    • Use Pearson or Spearman correlation to identify genes inversely correlated with C2orf40

    • Apply strict significance criteria (|R|> 0.3, P < 0.05)

  • Multivariate Approaches:

    • Principal component analysis

    • Hierarchical clustering

    • Consider adjusting for confounding variables

• Pathway Enrichment Analysis:

  • GO and KEGG Analysis:

    • Apply to genes correlated with C2orf40 expression

    • Focus on relevant pathways (cell cycle, DNA repair, PI3K signaling)

  • Gene Set Enrichment Analysis:

    • Use to identify affected biological processes

    • Report enrichment scores and p-values

How can researchers integrate C2orf40 methylation and expression data to develop predictive biomarkers?

To develop predictive biomarkers integrating C2orf40 methylation and expression:

What are promising approaches for targeting C2orf40 expression or function in cancer therapy?

Several promising therapeutic approaches targeting C2orf40 warrant further investigation:

• Epigenetic Modulation:

  • Demethylating Agents:

    • Decitabine (DAC) has been shown to significantly elevate C2orf40 mRNA levels in NPC cell lines

    • Clinical trials combining demethylating agents with conventional therapies could enhance treatment efficacy

  • Histone Deacetylase Inhibitors:

    • May complement demethylating agents

    • Could provide synergistic restoration of C2orf40 expression

• Combination Therapies:

  • Chemosensitization Strategy:

    • C2orf40 restoration paired with cisplatin or other platinum agents

    • Lower doses may achieve greater efficacy with reduced toxicity

  • Radiosensitization Approach:

    • C2orf40 induction prior to radiotherapy

    • May allow for lower radiation doses while maintaining efficacy

• Pathway-Based Interventions:

  • PI3K/AKT/mTOR Pathway Inhibitors:

    • Since C2orf40 inhibits this pathway, combining C2orf40 restoration with pathway inhibitors could enhance effectiveness

    • May overcome resistance mechanisms

  • HRR Pathway Targeting:

    • PARP inhibitors could synergize with C2orf40's inhibition of homologous recombination

    • Synthetic lethality approach for tumors with C2orf40 downregulation

• Nanotechnology-Based Delivery:

  • Targeted Delivery Systems:

    • Nanoparticles carrying C2orf40 expression vectors

    • Tumor-specific targeting to minimize off-target effects

  • Controlled Release Strategies:

    • Sustained expression in tumor microenvironment

    • Combination with conventional therapies

What cell-type specific effects of C2orf40 require further investigation?

Several cell-type specific effects of C2orf40 warrant deeper investigation:

• Cancer Type Specificity:

  • Beyond Nasopharyngeal Carcinoma:

    • While C2orf40's role in NPC is established, its function in other cancer types requires systematic evaluation

    • Comparative studies across multiple cancer types could reveal tissue-specific mechanisms

  • Histological Subtypes:

    • Evaluation across different histological variants within cancer types

    • Identification of subtypes most responsive to C2orf40-based interventions

• Tumor Microenvironment Interactions:

  • Immune Cell Interactions:

    • How C2orf40 expression influences tumor immunogenicity

    • Potential impact on immune checkpoint inhibitor efficacy

  • Stromal Interactions:

    • Effects on cancer-associated fibroblasts

    • Influence on extracellular matrix composition and stiffness

• Stem Cell Populations:

  • Cancer Stem Cells:

    • C2orf40's impact on cancer stem cell properties

    • Potential role in tumor initiation and recurrence

  • Differentiation Effects:

    • Whether C2orf40 influences cellular differentiation programs

    • Therapeutic implications for differentiation therapy approaches

• Cell Lineage Effects:

  • Epithelial vs. Mesenchymal Phenotypes:

    • C2orf40's role in epithelial-mesenchymal transition

    • Lineage-specific response patterns

  • Developmental Context:

    • Expression patterns during normal development

    • Relevance to embryonal tumors

How might high-throughput screening approaches identify novel modulators of C2orf40 function?

Advanced high-throughput screening approaches to identify novel C2orf40 modulators include:

• Epigenetic Modifier Screens:

  • Compound Libraries:

    • Screen epigenetic modifier libraries beyond conventional demethylating agents

    • Include histone modifiers, chromatin remodelers, and RNA modifiers

  • Readout Systems:

    • Develop reporter systems with C2orf40 promoter driving fluorescent or luminescent markers

    • Quantify expression changes in response to compound treatment

• Functional Genomic Screens:

  • CRISPR-Based Approaches:

    • Genome-wide CRISPR screens to identify genes that modulate C2orf40 expression

    • Focused screens targeting epigenetic regulators or signaling pathways

  • RNA Interference:

    • siRNA or shRNA libraries targeting transcription factors

    • Identification of upstream regulators of C2orf40 expression

• Pathway-Focused Screens:

  • PI3K/AKT/mTOR Pathway:

    • Screen for compounds that synergize with C2orf40's inhibitory effects on this pathway

    • Identify nodes where intervention maximizes therapeutic benefit

  • DNA Repair Modifiers:

    • Screen for compounds that enhance C2orf40's effects on homologous recombination

    • Identify synthetic lethal interactions in C2orf40-deficient cells

• Computational Approaches:

  • In Silico Screening:

    • Virtual screening for compounds that bind to C2orf40 or its interacting partners

    • Structural modeling to identify potential binding pockets

  • Network Analysis:

    • Systems biology approaches to map the C2orf40 interactome

    • Identification of hub proteins that may serve as alternative therapeutic targets