AKR1C1 Human, His

What is AKR1C1 and what are its primary biochemical functions?

AKR1C1 is a member of the aldo-keto reductase superfamily, which contains over 40 enzymes that catalyze the conversion of aldehydes and ketones to their corresponding alcohols by utilizing NADH and/or NADPH as cofactors. It plays several critical roles in human metabolism:

• Primarily responsible for progesterone metabolism, catalyzing the conversion of progesterone to its inactive form, 20-alpha-hydroxy progesterone (20α-OHP)

• Monitors intrahepatic bile acid concentration in the liver and intestine, though with relatively low bile-binding ability

• Contributes to myelin formation in the nervous system

• Participates in steroid hormone metabolism and detoxification pathways

Research methods for studying these functions typically involve enzyme activity assays with specific substrates, measuring reaction rates using spectrophotometric techniques that track cofactor oxidation/reduction.

What nomenclature and synonyms are used for AKR1C1 in scientific literature?

AKR1C1 is known by multiple names in scientific literature, which can complicate literature searches and comparative analyses. The most common synonyms include:

• DDH1, DDH (Dihydrodiol dehydrogenase 1/2)

• HAKRC (Chlordecone reductase homolog)

• 20-alpha-HSD (20-alpha-hydroxysteroid dehydrogenase)

• DD1/DD2, HBAB, C9, H-37, MBAB, MGC8954

• 2-ALPHA-HSD

• Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase

• Indanol dehydrogenase

• High-affinity hepatic bile acid-binding protein

When designing literature searches, researchers should include these alternative designations to ensure comprehensive coverage of relevant studies.

What are the structural and biochemical properties of recombinant AKR1C1?

Recombinant AKR1C1 Human protein:

• Comprises a single, non-glycosylated polypeptide chain containing 343 amino acids (1-323 a.a. of the native protein plus a 20 a.a. His-tag)

• Has a molecular mass of approximately 38.9 kDa

• When produced in E. coli expression systems, the protein is typically fused to a 20-amino acid His-Tag at the N-terminus for purification purposes

• Optimal storage conditions include 20mM Tris-HCl pH-8, 1mM DTT, and 20% glycerol at 4°C for short-term use (2-4 weeks) or at -20°C for longer periods

• For extended stability, addition of carrier protein (0.1% HSA or BSA) is recommended to prevent degradation during freeze-thaw cycles

These properties are critical considerations when designing experiments utilizing recombinant AKR1C1.

How can AKR1C1 substrate specificity be characterized experimentally?

AKR1C1 demonstrates selective substrate preferences that can be experimentally determined through:

• Comparative enzyme kinetics with multiple potential substrates, measuring Km and kcat values

• Assessment of stereospecificity in reduction reactions, particularly with steroid substrates

• Analysis of progesterone reduction to 20α-dihydroprogesterone (20α-DHP) as a primary activity marker

• Evaluation of dydrogesterone conversion to 20α-dihydrodydrogesterone as an alternative substrate reaction

In a systematic substrate specificity study, AKR1C1 showed high activity toward progesterone but negligible activity with pregnenolone, 17α-hydroxyprogesterone, 11-deoxycortisol, cortisol, 11-deoxycorticosterone, corticosterone, and aldosterone, demonstrating its selectivity for certain 20-keto steroids .

What mechanisms underlie AKR1C1's role in cancer metastasis?

AKR1C1 has been identified as a key promoter of metastasis in several cancer types, particularly non-small cell lung cancer (NSCLC). The mechanisms include:

• Direct interaction with STAT3, facilitating its phosphorylation and subsequent binding to promoter regions of metastasis-related target genes

• Reinforcement of STAT3 binding to target gene promoters, leading to transcriptional activation of genes promoting tumor metastasis

• Facilitation of the interaction between STAT3 and its upstream kinase JAK2

• Enhancement of cancer cell invasion and migration capabilities

Notably, these pro-metastatic effects occur in a catalytic-independent manner, suggesting that AKR1C1's role in cancer progression extends beyond its enzymatic functions . Researchers investigating these mechanisms typically employ knockdown/overexpression studies combined with invasion assays, phosphorylation analysis, and protein-protein interaction studies.

How does AKR1C1 interact with the STAT3 pathway in metastatic progression?

The interaction between AKR1C1 and STAT3 represents a critical mechanism in metastatic progression:

• AKR1C1 directly binds to STAT3, as demonstrated through co-immunoprecipitation experiments

• This interaction promotes STAT3 phosphorylation, a key activation step in the pathway

• Activated STAT3 then binds more effectively to promoter regions of target genes involved in metastasis

• AKR1C1 may also facilitate interaction between STAT3 and its upstream activator JAK2, creating a positive feedback loop

• A significant correlation between AKR1C1 and STAT3 pathway activation has been observed in metastatic foci of NSCLC patients

Research methods to study this pathway include phospho-specific western blotting, chromatin immunoprecipitation, and protein interaction studies using both recombinant proteins and cancer cell models.

What is AKR1C1's role in bladder cancer progression?

In bladder cancer, AKR1C1 shows a distinct pattern of expression and function:

• Immunohistochemistry studies have demonstrated high AKR1C1 expression in metastatic lesions of human bladder cancer patients

• AKR1C1 is upregulated in metastatic sublines derived from orthotopic xenograft models

• Its expression correlates with elevated levels of EMT-associated markers (Snail, Slug, CD44)

• Knockdown of AKR1C1 significantly decreases invasion capacity in bladder cancer cells

• Mechanistically, AKR1C1 appears to regulate Rac1, Src, and Akt signaling in these cells

• The inflammatory cytokine interleukin-1β induces AKR1C1 expression in bladder cancer cell lines

These findings suggest AKR1C1 as a potential therapeutic target in invasive bladder cancer, particularly through modulation of inflammatory signaling pathways.

How does AKR1C1 contribute to chemotherapy resistance?

AKR1C1 plays significant roles in chemotherapy resistance through several mechanisms:

• In bladder cancer models, AKR1C1 increases cisplatin resistance in metastatic cell lines

• Non-steroidal anti-inflammatory drugs (NSAIDs), particularly flufenamic acid, can antagonize AKR1C1 and decrease cisplatin resistance

• AKR1C1 has been implicated in pathophysiological roles in the development of cisplatin resistance in human colon cancers

• In leukemia models, induction of AKR1C1 (and AKR1C3) abolishes the efficacy of daunorubicin chemotherapy

Methodological approaches to study these effects include combination treatment studies with AKR1C1 inhibitors and chemotherapeutic agents, cell viability assays, and molecular analysis of resistance mechanisms in patient-derived samples.

What expression systems are optimal for recombinant AKR1C1 production?

Several expression systems have been successfully used for recombinant AKR1C1 production:

• Escherichia coli expression systems are commonly used for producing His-tagged AKR1C1 (38.9 kDa) for biochemical and structural studies

• Schizosaccharomyces pombe (fission yeast) provides a eukaryotic system that allows functional expression of human AKR1C1 with proper folding and activity

• In S. pombe, recombinant AKR1C1 efficiently catalyzes the reduction of progesterone to 20α-dihydroprogesterone (20α-DHP) and dydrogesterone to 20α-dihydrodydrogesterone without detectable byproducts or back reactions

For large-scale production, optimized fed-batch fermentation with S. pombe can achieve 20α-DHP production rates of approximately 300 μM/day over a 72-hour biotransformation period, significantly higher than the 90 μM/day observed in shake flask cultures .

How can AKR1C1 enzymatic activity be reliably measured?

Reliable measurement of AKR1C1 activity requires careful consideration of assay conditions:

• Standard spectrophotometric assays track the oxidation of NADPH at 340 nm when AKR1C1 reduces its substrates

• Specific activity toward progesterone can be measured by monitoring conversion to 20α-DHP using HPLC or LC-MS/MS

• Optimal reaction conditions typically include pH 7.0-7.5, 25-37°C, and cofactor concentrations of 0.1-0.5 mM NADPH

• For whole-cell biotransformation assays using recombinant systems, substrate conversion rates can be quantified (e.g., 90±26 μM/day for 20α-DHP and 244±93 μM/day for 20α-DHD in S. pombe shake flask cultures)

• Inhibition studies often employ salicylic acid-based compounds as reference inhibitors, with activity measured as IC50 or Ki values

Control experiments should include enzyme-free and substrate-free reactions to account for non-enzymatic conversions and background activity.

What approaches are effective for studying AKR1C1's role in cancer models?

Effective approaches for studying AKR1C1 in cancer include:

• Gene expression manipulation: siRNA knockdown and overexpression systems to evaluate phenotypic changes

• Orthotopic xenograft models: Particularly useful for establishing metastatic sublines, as demonstrated in bladder cancer studies where metastatic cells were isolated from liver, lung, and bone lesions

• Microarray analysis: Used to identify AKR1C1 upregulation in metastatic lesions, with verification in human cancer specimens

• Functional invasion assays: Transwell and Matrigel invasion assays to quantify invasive potential following AKR1C1 modulation

• Protein interaction studies: Co-immunoprecipitation and proximity ligation assays to investigate interactions with signaling proteins like STAT3

• Pharmacological inhibition: Using selective inhibitors like flufenamic acid to probe AKR1C1's contribution to drug resistance and invasion

These approaches enable comprehensive characterization of AKR1C1's roles in cancer progression across multiple model systems.

How can researchers develop and evaluate AKR1C1 inhibitors?

Development and evaluation of AKR1C1 inhibitors follows a systematic process:

• Structure-based design: Utilizing crystal structures of AKR1C1 to identify potential binding pockets, particularly those that can confer selectivity over other AKR family members

• Salicylic acid-based scaffolds: These have proven effective as AKR1C1 inhibitors and can serve as starting points for structure optimization

• Non-steroidal anti-inflammatory drugs (NSAIDs): Compounds like flufenamic acid antagonize AKR1C1 and can decrease cisplatin resistance and invasion potential in cancer cell models

• In vitro enzyme inhibition assays: Determining IC50 and Ki values using purified recombinant enzyme

• Cellular activity: Evaluating effects on AKR1C1-dependent processes in cellular models, particularly cancer cells with high AKR1C1 expression

• Selectivity profiling: Assessing activity against other AKR family members to ensure specific targeting

Effective inhibitors should demonstrate both potent enzyme inhibition and meaningful modulation of AKR1C1-dependent cellular phenotypes.

How does AKR1C1 contribute to inflammatory processes?

AKR1C1's role in inflammation involves bidirectional regulation:

• Inflammatory cytokines like interleukin-1β induce AKR1C1 expression in multiple cell types, including bladder cancer cells

• This induction appears to be part of the cellular response to inflammatory conditions

• AKR1C1 may in turn modulate inflammatory signaling by affecting prostaglandin metabolism

• The relationship between AKR1C1 and inflammation creates potential for intervention with anti-inflammatory drugs

• NSAIDs like flufenamic acid can antagonize AKR1C1, suggesting a mechanistic link between anti-inflammatory effects and AKR1C1 inhibition

Research methods include cytokine stimulation studies, analysis of inflammatory gene expression following AKR1C1 modulation, and evaluation of prostaglandin metabolism in AKR1C1-expressing cells.

What is the potential of AKR1C1 as a biomarker or therapeutic target?

AKR1C1 shows significant potential as both a biomarker and therapeutic target:

• Diagnostic/prognostic biomarker: AKR1C1 expression correlates with metastatic potential and poor prognosis in NSCLC and bladder cancer patients

• Therapeutic target: Given its roles in both metastasis and drug resistance, inhibition of AKR1C1 could simultaneously address two major challenges in cancer treatment

• Combinatorial therapy: AKR1C1 inhibitors like flufenamic acid can sensitize cancer cells to conventional chemotherapeutics such as cisplatin

• Stratification marker: AKR1C1 expression levels could help identify patients who might benefit from specific therapeutic approaches

• Multi-cancer relevance: Beyond NSCLC and bladder cancer, AKR1C1 has been implicated in leukemia and colon cancer, suggesting broader applicability

The dual role of AKR1C1 in both metastasis and drug resistance makes it particularly valuable as a therapeutic target, potentially addressing two major challenges in cancer treatment simultaneously.

How do AKR1C1 functions differ between normal physiology and pathological states?

AKR1C1 functions show notable differences between normal and pathological states:

• Normal tissues: Primarily functions in steroid hormone metabolism, particularly progesterone inactivation, and participates in bile acid homeostasis and myelin formation

• Cancer: Acquires non-enzymatic functions in promoting metastasis through STAT3 pathway activation and enhances chemoresistance

• Tissue specificity: Normal expression is highest in liver, intestine, and reproductive tissues, while pathological upregulation occurs in multiple cancer types

• Regulatory mechanisms: Normal expression appears tightly regulated by metabolic needs, while pathological expression can be driven by inflammatory signals and oncogenic pathways

• Functional consequences: In normal physiology, functions largely within metabolic pathways; in cancer, impacts cell migration, invasion, and survival mechanisms

Understanding these context-dependent functions is essential for targeted therapeutic development that minimizes impact on normal physiological processes.

What genetic variants of AKR1C1 exist and how do they affect enzyme function?

Several naturally occurring variants of AKR1C1 have been identified with potential functional consequences:

• Genetic polymorphisms in AKR1C1 can alter enzyme activity, substrate specificity, or protein stability

• Some variants show reduced in vitro metabolism of chemotherapeutic agents like daunorubicin and doxorubicin

• These functional differences may contribute to inter-individual variability in drug metabolism and response

• Variants may also affect normal physiological processes involving steroid hormone metabolism

• Research methods include recombinant expression of variant proteins, comparative enzyme kinetics, and cellular phenotype studies

Characterization of these variants provides insight into structure-function relationships and may have implications for personalized medicine approaches.

How does AKR1C1 interact with other members of the AKR family in metabolic networks?

AKR1C1 functions within a network of related enzymes with overlapping yet distinct activities:

• The AKR1C subfamily includes AKR1C1, AKR1C2, AKR1C3, and AKR1C4, all involved in steroid metabolism but with different substrate preferences

• While AKR1C1 primarily catalyzes the conversion of progesterone to 20α-DHP, other family members have distinct substrate preferences (e.g., AKR1C3 plays a key role in androgen metabolism)

• These enzymes may function redundantly in some contexts but show tissue-specific expression patterns

• Co-expression of multiple AKR enzymes can create complex metabolic networks with implications for both normal physiology and disease states

• Comparative studies of substrate specificity show that among seven steroids with a 20-keto group, only progesterone and dydrogesterone are effectively reduced by AKR1C1