AKR1C3 inhibition by curcumin, demethoxycurcumin, and bisdemethoxycurcumin: an investigation Item Type Thesis or dissertation Authors Calhoon, Brecken Citation Calhoon, B. (2019). AKR1C3 inhibition by curcumin, demethoxycurcumin, and bisdemethoxycurcumin: an investigation. (Masters thesis). University of Chester, United Kingdom. Publisher University of Chester Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 25/09/2021 00:32:38 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/10034/623327 1 DECLARATION This work is original and has not been previously submitted in support of a Degree qualification or other course. Signed ……Brecken Elizabeth Calhoon………. Date…02/10/2019….. Word count: 4188 2 Research Article AKR1C3 inhibition by curcumin, demethoxycurcumin, and bisdemethoxycurcumin: an investigation Brecken Calhoon Abstract Background: Aldo-keto reductase 1C3 (AKR1C3) has been shown to be overexpressed in cancers due to its regulatory roles in cell proliferation and differentiation. Curcumin, known to have anti-tumour properties, and its analogues demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) were studied to determine their inhibitory effects on the AKR1C3 enzyme. Methods: AKR1C3 was purified and analysed to determine its protein concentration in transformed Escherichia coli (E. coli) cells. Enzyme assays equaling 1 mL contained 2.82 mg/mL AKR1C3, 50 μM of 3 mM NADPH, and varying volumes of potassium phosphate (50 mM, pH 6.5), 9,10-phenanthrenequinone (PQ), and inhibitors were measured at 340 nm. The Vmax, Km, KI, and 2 of AKR1C3 in the presence and absence of inhibitors were determined using a non- linear regression analysis on Fig.P Software. Results: PQ alone found Vmax = 0.47 IU/mg, Km = .435 μM, Ki = 6.29 μM, and 2 = 0.946. Inhibitor potency was BDMC > DMC > curcumin in the presence of 1 μM PQ. Further analysis of BDMC indicated mixed inhibition (Vmax = 0.46 IU/mg, Km = .406 μM, Ki = 1.59 μM, and 2 = 0.970). Further analysis of PQ at higher concentrations found a divergence from Michaelis-Menten kinetics, with a decrease in AKR1C3 activity after Vmax was reached. Conclusions: BDMC was the more potent inhibitor of AKR1C3 in transformed E. coli compared to DMC and curcumin. The results suggest mixed inhibition of AKR1C3 in the presence of BDMC. Additional analysis of PQ at higher concentrations saw a loss of Michaelis-Menten kinetics as the activity of AKR1C3 decreased after reaching Vmax. This requires further examination. Keywords: Aldo-keto reductase 1C3 (AKRIC3); curcumin; demethoxycurcumin (DMC); bisdemethoxycurcumin (BDMC) Introduction proliferation and differentiation. Prostaglandins (PG) PGH2 and PGD2 are Aldo-keto reductase 1C3 (AKR1C3) is a 36.8 converted to PGF2α and 9α,11β-PGF2α, kD member of the aldo/keto reductase respectively, in the presence of AKR1C3 superfamily AKR1C that catalyses alcohols (Fig. 1a) [2, 3], while weak androgens and from aldehydes and ketones in the oestrogens (4-androstone-3,17-dione and presence of NAD(P)H [1]. AKR1C3 has an oestrone) are converted into potent ones influential role in several cell signalling pathways that can influence cell 3 Figure 1. AKR1C3 influence over cellular pathways. (a) Prostaglandin synthesis in the presence and absence of AKR1C3. (b) Androgen synthesis in the presence of AKR1C3. Abbreviations: Aldo-keto reductase 1C3 (AKR1C3; Androgen receptor (AR); Oestrogen receptor (ER); Mitogen-activated protein kinase (MAPK); Nuclear factor κB (NF-κB); Peroxisome proliferator- activated γ receptor (PPARγ). (testosterone and 17β-oestradiol) due to curcumin’s efficacy as a therapeutic the co-activation of the androgen option for cancer [12-14]. However, due to receptor (AR) and oestrogen receptor (ER) its poor bioavailability and stability in vivo (Fig. 1b) [4, 5]. [12, 15-16], it has not seen much success in clinical trials. As such, curcumin analogues These conversions can be problematic in have been used to greater effect. tumorous cells, as the resulting products can influence the rate of gene transcription The naturally occurring curcumin [6]. Overactivation of gene transcription analogues demethoxycurcumin (DMC) due to AKR1C3 overexpression has been (Fig. 2b) and bisdemethoxycurcumin found in a myriad of cancers including (BDMC) (Fig. 2c), have not been examined breast, colon, colorectal, endometrial, in the AKR1C superfamily, but have tested prostate, and acute myeloid luekeaima [7, on cancer cells to a positive effect [17]. 8]. In the absence of AKR1C3 PGD2 can be BDMC has also shown to be more stable in converted to 15-deoxy-Δ12,14-PGJ2 (PGJ2), rat livers than curcumin and DMC [15]. This which can bind to the peroxisome suggests BDMC may have a greater proliferator-activated receptor (PPAR) γ [9, bioavailability as well. While curcumin has 10]. PPARγ is then able to promote cellular been studied in prostate cells on AKR1C2 differentiation and apoptosis [9]. This activity [18], neither curcumin, DMC, nor relationship between AKR1C3 and cell BDMC has been utilised to inhibit AKR1C3 signalling pathways suggests activities, despite AKR1C3 overexpression in overexpression of tumorous cells could be a variety of cancers. impeded with the inhibition of AKR1C3. This study aimed to examine the inhibitory Curcumin(Fig. 2a), a polyphenol from the effects of curcumin, DMC, and BDMC on rhizome of the turmeric plant, has been AKR1C3 expression in transformed shown to contain anti-inflammatory, Escherichia coli (E. coli) cells and determine antioxidant, and anti-tumorous activities the type of inhibition that occurred. Such [11]. Several in vitro and in vivo studies evidence could provide future studies with have been performed to determine valuable information for the development 4 aside as Solubilised Bacteria (SB). The remaining sample was split into two eppendorf tubes and spun at 30,000 g for 25 minutes at 10°C. The resulting supernatant was removed and set aside as Supernatant (Sup). Both the Sup and SB were frozen. The homogenate was further purified using a His-select Ni2+ resin column (Sigma-Aldrich, Darmstadt, Germany) per the manufacturer’s instructions. The wash buffer comprised of 50 mM sodium phosphate monobasic monohydrate at 300 mM NaCl, pH 8.0, while the elution buffer additionally contained 100 mM of Imidazole, pH 8.0. Eight elutions of 1 mL were collected and analysed for protein (P) concentration. Protein Estimation A standard curve was created using a Bradford Assay and bovine serum albumin (BSA) from Bio-Rad Laboratories, as seen in the supplementary material (Fig. 1). SB, Sup, and P samples were additionally Figure 2. Chemical structure of curcumin (a), analysed (10 μL of sample, 40 μl distilled demethoxycurcumin (b), and bisdemethoxycurcumin water, and 950 μL of Bradford Reagent). All (c) [13]. measurements were taken at 595 nm on a and optimisation of curcumin-based Jenway 7200 visible spectrophotometer therapeutics in cancer patients. (Stone, Staffordshire, UK) and performed in triplicate. Methods To determine the protein concentrations of Cell Culture and Purification AKR1C3 in SB, Sup, and P, each sample was diluted to 1 mg/ml with distilled water. The E. coli cells BL21(DE3) (New England Biolabs samples were further diluted to 0.5 mg/mL UK) containing the human AKR1C3 enzyme [100 μL sample, 100 μL sample buffer (pET21b-AKR1C3) with a His-tag were (Sigma S3401, Sigma-Aldrich, Darmstadt, grown overnight in an orbital shaker at 300 Germany), and 100 μL Laemmli Blue]. They RPM and 37°C and transferred into three 6 were then heated at 90°C for mL bottles of L-broth and ampicillin (Amp)R approximately 7 minutes and placed on (100 μg/mL). Cells were shaken and grown ice before electrophoresis was performed. overnight at 37°C before transferring into 400 mL L-broth+Amp and centrifuged for 45 Gel Electrophoresis minutes at 4500 RPM. The resulting pellet was stored at -80°C. The Bio-Rad Laboratories gel electrophoresis tank was assembled and The pellet was resuspended in accordance loaded according to the manufacturer’s with the manufacturer’s instructions upon instructions. SB, Sup, and P samples were addition of 8 mL of BugBuster, a protease loaded at 10 μg per lane. The gel was run inhibitor cocktail tablet, supplied by Sigma- at 150 V for 45 minutes before being left Aldrich (Darmstadt, Germany). Upon overnight in 30 mL of EZ blue staining removal from the room temperature water reagent (Sigma-Aldrich, Darmstadt, bath, the suspension was extracted and set Germany). 5 Activity of AKR1C3 obtained from the Protein Data Bank, file number 1RY0. Protein activities of AKR1C3 were determined by measuring the oxidation of Results NADPH (ε = 6220 M-1 cm-1) at 340 nm on a Protein Purification Jenway 7315 spectrophotometer (Stone, Staffordshire, UK) at room temperature. The The AKR1C3 enzyme was purified and NADPH solution had been made in distilled eluted into eight different eppendorf tubes. water from a 1M NADPH stock to achieve a The elution profile (Fig. 2 supplementary final concentration of 3 mM. The resulting material) shows the highest protein solution was made slightly alkaline with the concentration of AKR1C3 was collected addition of NaOH (4 μL) to stabilise said during the second and third elutions, which solution. The assay buffer was potassium were pooled together. The resulting gel phosphate (50 mM, pH 6.5). The binding (Fig. 3) showed the purified AKR1C3 to be substrate was 1 mM stock of 9,10- roughly 35.5 kD, which is near the phenanthrenequinone (PQ) in dimethyl approximate molecular mass of 37 kD for sulfoxide (DMSO). 50 μL of 3 mM NADPH AKR1C3, indicating the protein has been was used for every assay, while 20 μL of 2.82 successfully purified. The Sup and SB also mg/mL AKR1C3 was also used. Volumes of had bands around 35.5 kD, in addition to potassium phosphate, PQ, and inhibitors bands with greater and less molecular varied to ensure the total volume of the weight.