ABCC2 Antibody

What is ABCC2 and why is it significant in cancer research?

ABCC2 is an ATP-binding cassette multidrug resistance transporter that mediates the efflux of various compounds from cells. In cancer research, ABCC2 has gained prominence because it is frequently overexpressed in multiple human cancers and contributes significantly to chemotherapy resistance. Studies have demonstrated that ABCC2 upregulation is associated with resistance to platinum-based drugs like cisplatin in non-small cell lung cancer (NSCLC) . The transporter has important clinical value in evaluating chemotherapy effectiveness, as its expression levels can predict treatment outcomes. For example, in urothelial carcinoma of the urinary bladder, cisplatin treatment can induce expression of ABCC2, resulting in decreased drug efficacy . Similar relationships between ABCC2 expression and drug resistance have been observed in ovarian cancer, head and neck squamous cell carcinoma, and other malignancies .

How does ABCC2 contribute to multidrug resistance mechanisms?

ABCC2 contributes to multidrug resistance through several mechanisms:

• Active drug efflux: ABCC2 utilizes ATP to pump various anticancer drugs out of cancer cells, reducing intracellular drug concentrations below therapeutic thresholds.

• Altered drug disposition: ABCC2 expression in liver and kidney influences the pharmacokinetics of many compounds, potentially affecting their bioavailability and efficacy .

• Polymorphic variations: Genetic polymorphisms like ABCC2 (G1249A) can enhance transport capabilities for certain drugs. Research demonstrates that cells expressing the 1249A variant show higher efflux ratios for paclitaxel and doxorubicin compared to those with the 1249G wild-type, resulting in greater drug resistance .

The role of ABCC2 in multidrug resistance makes it a valuable target for antibody-based detection and quantification in experimental studies examining resistance mechanisms and potential therapeutic interventions.

What are the common applications of ABCC2 antibodies in research?

ABCC2 antibodies have several important applications in cancer and pharmacological research:

• Expression analysis: Western blot analysis to quantify ABCC2 protein levels in cell lines, patient samples, and tissue homogenates (typically using 1:1000 dilution of antibodies like ab205718) .

• Tissue localization: Immunohistochemistry to visualize the distribution and expression patterns of ABCC2 in tissue sections, particularly in comparing normal versus tumor tissues .

• Mechanism studies: Investigation of resistance mechanisms through correlation of ABCC2 expression with drug sensitivity and treatment outcomes.

• Genetic variant studies: Examination of how polymorphic variants affect ABCC2 function and localization .

• Drug development: Screening potential ABCC2 inhibitors or modulators that might reverse multidrug resistance.

How can ABCC2 antibodies be utilized to investigate cisplatin resistance in non-small cell lung cancer?

Investigating cisplatin resistance in NSCLC using ABCC2 antibodies involves several sophisticated experimental approaches:

• Comparative expression analysis: Using validated ABCC2 antibodies in western blot analysis to compare expression levels between cisplatin-sensitive (A549) and cisplatin-resistant (A549/DDP) cell lines. Research has shown that ABCC2 is significantly upregulated (7.86-fold increase at mRNA level) in resistant cells .

• Functional validation through knockdown studies: After ABCC2 knockdown, antibodies can confirm protein reduction and correlate this with changes in drug sensitivity. Studies demonstrate that ABCC2 knockdown significantly reduces the IC50 value of cisplatin in resistant cells .

• Mechanistic investigations: Antibodies can be used to examine how ABCC2 expression correlates with apoptotic markers (PARP, caspase-3) and cell cycle distribution following drug treatment .

• In vivo verification: ABCC2 antibodies in immunohistochemistry can evaluate ABCC2 expression in xenograft tumor tissues and correlate this with tumor growth and response to treatment. Ki-67 staining can be performed in parallel to assess proliferation .

This experimental framework provides comprehensive insights into how ABCC2 contributes to cisplatin resistance mechanisms and potential strategies for overcoming resistance.

What methodological approaches should be used when studying ABCC2 polymorphisms and their impact on drug transport?

When investigating ABCC2 polymorphisms and their effects on drug transport, researchers should implement a multifaceted methodological approach:

• Recombinant expression systems: Generate cell lines (such as LLC-PK1) expressing different ABCC2 variants (e.g., G1249A) to isolate the effects of specific polymorphisms .

• Comparative transport assays: Measure drug sensitivity (IC50), intracellular accumulation, and transmembrane transport of relevant substrates across wild-type and variant ABCC2-expressing cells. For example, studies with the G1249A polymorphism revealed significantly different transport characteristics for paclitaxel and doxorubicin, but not for docetaxel .

• Antibody validation: Confirm equivalent ABCC2 expression levels between wild-type and variant cell lines using antibodies to ensure differences in transport are due to functional changes rather than expression differences.

• Structure-function analysis: Correlate polymorphism location with known structural domains of ABCC2 to understand the molecular basis of altered transport.

This methodological framework enables precise characterization of how specific genetic variations impact ABCC2 transport function for different substrates.

How can researchers differentiate the effects of ABCC2 from other ABC transporters in multidrug resistance studies?

Differentiating ABCC2's specific contribution from other ABC transporters requires careful experimental design:

• Selective inhibition: Use relatively selective inhibitors for different ABC transporters and measure their impact on drug accumulation or efflux. For example, MK571 has been used as an ABCC2 inhibitor in studies examining cisplatin sensitivity in head and neck squamous cell carcinoma .

• Genetic manipulation: Employ siRNA or CRISPR techniques targeting specific transporters individually and in combination. The search results indicate that knockdown of ABCC2 using shRNA constructs (sh1-ABCC2 and sh2-ABCC2) can isolate its effects on drug resistance .

• Substrate specificity profiling: Utilize the different substrate preferences of ABC transporters. The vesicular transport assay with probes like β-estradiol 17-(β-D-glucuronide) (EG) and 2′,7′-dichlorofluorescin (CDCF) can help distinguish ABCC2 activity .

• Antibody specificity: Use highly specific antibodies that do not cross-react with other ABC family members for unambiguous identification of ABCC2.

• Correlated expression analysis: Analyze the expression patterns of multiple transporters simultaneously and correlate them with resistance phenotypes to deconvolute individual contributions.

What are the optimal conditions for using ABCC2 antibodies in western blot analysis?

For optimal western blot analysis of ABCC2, researchers should consider the following parameters:

• Sample preparation: Lyse cells on ice for 30 minutes in RIPA buffer containing 1% phenylmethylsulfonyl fluoride (1 mM). Centrifuge at 12,000 rpm at 4°C for 15 minutes and collect the supernatant .

• Protein quantification: Determine protein concentrations using a bicinchoninic acid assay and mix the supernatant with 5× SDS at a 4:1 ratio, followed by incubation at 100°C for 10 minutes .

• Electrophoresis and transfer: Separate proteins (20 μg) on 12% SDS-polyacrylamide gels and transfer to polyvinylidene fluoride (PVDF) membranes .

• Blocking: Block PVDF membranes with 5% skim milk (dissolved in TBST) for 2 hours at room temperature .

• Primary antibody: Use ABCC2 antibody (e.g., ab205718) at 1:1000 dilution. Incubate overnight at 4°C for optimal signal-to-noise ratio .

• Controls: Include both negative controls (cells with low ABCC2 expression) and positive controls (cells with known high ABCC2 expression) to validate results.

• Normalization: Use appropriate housekeeping genes (β-actin, GAPDH) for normalization of expression levels.

These optimized conditions ensure reliable and reproducible detection of ABCC2 protein in experimental samples.

What are the key considerations for ABCC2 antibody use in immunohistochemistry?

When performing immunohistochemistry (IHC) with ABCC2 antibodies, researchers should follow these critical steps:

• Tissue preparation: Fix tissues with paraffin, followed by dewaxing and hydration using xylene and ethanol .

• Antigen retrieval: Perform antigen retrieval at 95°C for 20 minutes to expose epitopes that may be masked during fixation .

• Endogenous peroxidase blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide to reduce background signal .

• Primary antibody incubation: Apply optimized dilution of ABCC2 antibody and incubate according to validated protocol (typically overnight at 4°C).

• Secondary antibody and detection: Use appropriate secondary antibody system and develop signal according to manufacturer's recommendations.

• Counterstaining: Use hematoxylin for counterstaining to visualize tissue architecture .

• Controls: Include positive control tissues (liver, kidney) with known ABCC2 expression and negative controls (antibody omission or isotype controls).

• Quantification: Score ABCC2 expression in multiple randomly chosen fields (at least 8) under consistent magnification (×200 is commonly used) to ensure representative results .

• Parallel markers: Consider staining serial sections for related markers (e.g., Ki-67 for proliferation) to correlate ABCC2 expression with other biological parameters .

How should vesicular transport assays be designed to evaluate ABCC2 function and modulation?

Vesicular transport assays are valuable tools for studying ABCC2 function and modulation, requiring careful experimental design:

• Membrane vesicle preparation: Express human ABCC2 in Spodoptera frugiperda (Sf9) insect cells using systems like Bac-to-Bac® expression system. Prepare inside-out membrane vesicles containing ABCC2 following established protocols .

• Probe selection: Use appropriate fluorescent or radiolabeled probes. β-estradiol 17-(β-D-glucuronide) (EG, radiolabeled) and 2′,7′-dichlorofluorescin (CDCF, fluorescent) are commonly used and provide complementary information, with CDCF typically yielding more robust measurements .

• Reaction conditions: Optimize ATP concentration, incubation time, temperature, and DMSO concentration (should not exceed 1.5% to avoid interference with transport) .

• Controls: Include both ATP-dependent and ATP-independent transport measurements to determine specific ABCC2-mediated transport.

• Modulator screening: For inhibitor studies, test compounds at multiple concentrations (typically 3-7 concentrations in triplicate) to generate reliable dose-response curves .

• Data analysis: Calculate modulation effect as the ratio between ATP-dependent probe transport with and without the tested compound. IC50 values can be determined from dose-response curves .

The vesicular transport assay provides valuable information about compound interactions with ABCC2 without necessarily indicating whether compounds are transported substrates themselves.

How can ABCC2 antibodies contribute to developing strategies to overcome chemotherapy resistance?

ABCC2 antibodies play crucial roles in developing strategies to overcome chemotherapy resistance:

• Target validation: Confirm ABCC2 overexpression in resistant tumors and cell lines to establish its role in specific resistance mechanisms .

• Knockdown studies: Validate the effects of ABCC2 reduction on reversing drug resistance. Research has demonstrated that ABCC2 knockdown enhances the cytotoxicity of cisplatin to subcutaneous tumors, providing proof-of-concept for ABCC2-targeting approaches .

• Inhibitor development: Screen and evaluate potential ABCC2 inhibitors, using antibodies to confirm that effects are specifically related to ABCC2 inhibition rather than off-target effects.

• Biomarker development: Establish ABCC2 expression levels as potential predictive biomarkers for treatment response. Studies indicate that ABCC2 expression is associated with cisplatin resistance and could become a valuable clinical marker .

• Combination therapy assessment: Evaluate the efficacy of combining ABCC2 inhibitors with standard chemotherapeutics, using antibodies to monitor changes in ABCC2 expression or localization.

• Cell cycle effects: Investigate how ABCC2 modulation affects cell cycle distribution. Research has shown that ABCC2 knockdown promotes G1 phase arrest in cisplatin-resistant cells, suggesting complex interactions with cell cycle regulation machinery .

What insights can structure-activity relationship studies provide about ABCC2 modulation and inhibition?

Structure-activity relationship (SAR) studies offer valuable insights into ABCC2 modulation:

• Modulator identification: Screening compound libraries (such as the 432 compounds examined in the vesicular transport assay) has identified numerous ABCC2 modulators with varying potencies .

• Chemical features: SAR studies reveal that approximately 22% of tested compounds exhibit modulatory effects on ABCC2 at 80 μM concentration, with 84 potential inhibitors and 12 stimulators identified through primary screening .

• Probe selectivity: Most inhibitors affect transport of both EG and CDCF probes, but some compounds show probe selectivity, indicating possible binding to different sites within the ABCC2 protein .

• Binding site analysis: Analysis of the three-dimensional structure of ABCC2 suggests modulators may exert their action by binding directly to the central cavity, competing with transported substrates, or binding to either of the two nucleotide binding domains .

• Prediction models: Classification models built from screening data can highlight descriptors important for distinguishing inhibitors from inactive compounds, guiding rational design of more potent and selective inhibitors .

The high percentage of compounds showing ABCC2 modulation (62% with milder boundaries) suggests greater than previously thought implication of ABCC2 in pharmacokinetics and drug-drug interactions .

How can researchers investigate the relationship between ABCC2 polymorphisms and clinical drug response?

Investigating ABCC2 polymorphism effects on clinical drug response requires a multidisciplinary approach:

• In vitro transport models: Use recombinant cell lines expressing different ABCC2 variants (such as the G1249A polymorphism) to characterize transport differences for clinically relevant drugs .

• Drug-specific effects: Recognize that polymorphism effects may be substrate-dependent. For example, the G1249A variant significantly affects transport of paclitaxel and doxorubicin but not docetaxel .

• Functional parameters: Assess multiple parameters including drug sensitivity (IC50), intracellular accumulation, and transmembrane transport to comprehensively characterize how polymorphisms affect ABCC2 function .

• Antibody-based verification: Use ABCC2 antibodies to confirm equivalent expression levels between variant forms, ensuring functional differences are not due to expression variations.

• Clinical correlation: Correlate in vitro findings with clinical outcomes in patients with different ABCC2 genotypes treated with relevant drugs.

• Personalized medicine implications: Develop predictive models based on ABCC2 genotype that could guide treatment selection and dosing for individual patients.

These approaches can provide valuable insights into how genetic variations in ABCC2 contribute to interindividual differences in drug response and toxicity, potentially enabling more personalized and effective cancer treatment strategies.