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  • Oltipraz has been shown to

    2022-06-23

    Oltipraz has luteolin sale been shown to inhibit the growth of HCC luteolin sale and is being evaluated in clinical trials as a potential anti-cancer drug for HCC (Mann et al., 2009; Piton et al., 2005; Yates and Kensler, 2007). The present observation that pre-treatment of tumorigenic liver cells with oltipraz causes a large induction of HO-1 and Prx-1 suggests that one mechanism by which oltipraz inhibits the growth of liver cancer may involve the reduction of ROS. This would be expected to reduce the downstream actions of ROS in promoting the progression of HCC.
    Conclusion
    Conflicts of interest
    Free heme (protein-unbound heme) is known to be a prooxidant molecule that can catalyze the production of reactive oxygen species. Determining the exact concentrations of free heme in living cells and organisms is important for understanding the physiological role of free heme, although the usual concentration of free heme in normal cells is too low to be determined by the conventional methods. Sassa mentioned that the concentration of free heme in cells should not be greater than 10 M . We recently developed a novel heme sensor based on rat heme oxygenase-1 (HO-1) that allowed for detection of heme at a concentration level lower than 10 nM in a microplate-based assay. Thus, the sensor could become a powerful diagnostic tool in diseases such as colon cancer and Alzheimer's disease where free heme needs to be determined , , . HO-1 catalyzes the O-dependent degradation of heme into biliverdin IXα, carbon monoxide, and free ferrous iron in the presence of mediators that donate electrons such as NADPH–cytochrome P450 reductase and ascorbate. However, the heme degradation property of the heme sensor is unfavorable for its application to biological samples that contain reducing reagents. To overcome this problem, we attempted to reduce the heme degrading capability of the heme sensor in the current study. The Ortiz de Montellano group reported that Asp140 on the distal helix of human HO-1 participates in the hydrogen bonding network that stabilizes the Fe(III)–OOH reactive species in the active site , . In fact, the mutation of Asp140 with histidine (D140H) disturbed the O binding, thereby diminishing the enzymatic activity of HO-1 , . Nevertheless, the heme binding property was maintained in the mutated enzyme. These authors also reported that alanine mutant (D140A) showed similar property to that of D140H . On the basis of these previous studies, we examined whether the mutation of Asp140 was beneficial for improvement of our heme sensor. Thus, Lys18 of rat HO-1 that lacks a C-terminal membrane-binding region was replaced with Cys; the original truncated HO-1 and the obtained mutant are referred to as tHO-1 and K18C, respectively. The Cys residue of K18C was used for modification with thiol-reactive fluorescent dyes . The K18C was further mutated at Asp140 by His or Ala; these double mutants are referred to as K18C/D140H and K18C/D140A, respectively. The desired proteins were purified from cells transfected with the respective expression plasmids according to a procedure reported previously . Detailed information on construction of the expression plasmids and purification of proteins is described in the online supplementary material. Single turnover reactions of the tHO-1 and its mutants in the complexes with heme were performed using the system that used ascorbate as an electron donor , . The system was composed of 2 μM heme complex, 100 mM phosphate buffer (pH 7.4), 10 μM bovine serum albumin, catalase (400 U), and 50 mM ascorbate. shows the ultraviolet–visible (UV–Vis) spectra of the complexes in the wavelength region from 350 to 450 nm before and after the addition of ascorbate at 25 °C. The intensities of the Soret bands at 405 nm for tHO-1 and K18C decreased within 5 min of the addition of ascorbate, indicating that heme degradation occurred. By contrast, significant decreases in the Soret bands of the double mutants, K18C/D140A and K18C/D140H, were not observed. In addition, the Soret peak for K18C/D140H was red-shifted from 405 to 413 nm compared with other proteins, suggesting that the distal water ligand on the ferric iron of heme was replaced with His140, so that His140 becomes the sixth ligand of the ferric heme iron atom . K18C/D140A and K18C/D140H substantially exhibited no heme degradation properties in the single turnover experiment, so we decided to employ K18C/D140H in the subsequent experiments. K18C/D140A was not employed as a heme sensor because Lightning and coworkers reported that D140A possesses high peroxidation activity .