Alternative Approach for Specific Tyrosinase Inhibitor Screening: Uncompetitive Inhibition of Tyrosinase by Moringa oleifera

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.

Species-specific mechanisms of tumor suppression are fundamental drivers of vertebrate speciation: critical implications for the ‘war on cancer’

We recently reported our detection of an anthropoid primate-specific, ‘kill switch’ tumor suppression system that reached its greatest expression in humans, but that is fully functional only during the first twenty-five years of life, corresponding to the primitive human lifespan that has characterized the majority of our species' existence. This tumor suppression system is based upon the kill switch being triggered in cells in which p53 has been inactivated; such kill switch consisting of a rapid, catastrophic increase in ROS caused by the induction of irreversible uncompetitive inhibition of glucose-6- phosphate dehydrogenase (G6PD), which requires high concentrations of both inhibitor (DHEA) and G6P substrate. While high concentrations of intracellular DHEA are readily available in primates from the importation and subsequent de-sulfation of circulating DHEAS into p53-affected cells, both an anthropoid primate-specific sequence motif (GAAT) in the glucose-6-phosphatase (G6PC) promoter, and primate-specific inactivation of de novo synthesis of vitamin C by deletion of gulonolactone oxidase (GLO) were required to enable accumulation of G6P to levels sufficient to enable irreversible uncompetitive inhibition of G6PD. Malignant transformation acts as a counterforce opposing vertebrate speciation, particularly increases in body size and lifespan that enable optimized exploitation of particular niches. Unique mechanisms of tumor suppression that evolved to enable niche exploitation distinguish vertebrate species, and prevent one vertebrate species from serving as a valid model system for another. This here-to-fore unrecognized element of speciation undermines decades of cancer research data, using murine species, which presumed universal mechanisms of tumor suppression, independent of species. Despite this setback, the potential for pharmacological reconstitution of the kill switch tumor suppression system that distinguishes our species suggests that ‘normalization’ of human cancer risk, from its current 40% to the 4% of virtually all other large, long-lived species, represents a realistic near-term goal.

Taxifolin Resensitizes Multidrug Resistance Cancer Cells via Uncompetitive Inhibition of P-Glycoprotein Function

P-glycoprotein (P-gp) effluxes lots of chemotherapeutic agents and leads to multidrug resistance (MDR) in cancer treatments. The development of P-gp inhibitors from natural products provide a potential strategy for the beneficial clinical outcomes. This study aimed to evaluate the effects of the natural flavonoid taxifolin, luteolin, (−)-gallocatechin, and (−)-catechin on human P-gp activity. The kinetic interactions and underlying mechanisms of taxifolin-mediated transporter inhibition were further investigated. The transporter inhibition ability was evaluated in human P-gp stable expression cells (ABCB1/Flp-InTM-293) by calcein-AM uptake assays. The kinetics study for P-gp inhibition was evaluated by doxorubicin and rhodamine123 efflux assays. The MDR reversal ability of taxifolin were performed by SRB assays to detect the cell viability in sensitive cancer cell line (HeLaS3), and resistant cancer cell line (KB-vin). Cell cycle analysis and ABCB1 real-time RT-PCR were used for mechanical exploration. The results demonstrated that taxifolin decreased ABCB1 expression in a concentration-dependent manner. The function of P-gp was inhibited by taxifolin through uncompetitive inhibition of rhodamine 123 and doxorubicin efflux. The combination of taxifolin significantly resensitized MDR cancer cells to chemotherapeutic agents. These results suggested that taxifolin may be considered as a potential P-gp modulator for synergistic treatment of MDR cancers.

Detection of a novel, primate-specific ‘kill switch’ tumor suppression mechanism that may fundamentally control cancer risk in humans: an unexpected twist in the basic biology of TP53

The activation of TP53 is well known to exert tumor suppressive effects. We have detected aprimate-specificadrenal androgen-mediated tumor suppression system in which circulating DHEAS is converted to DHEA specifically in cells in which TP53 has beeninactivated. DHEA is anuncompetitiveinhibitor of glucose-6-phosphate dehydrogenase (G6PD), an enzyme indispensable for maintaining reactive oxygen species within limits survivable by the cell. Uncompetitive inhibition is otherwise unknown in natural systems because it becomesirreversiblein the presence of high concentrations of substrate and inhibitor. In addition to primate-specific circulating DHEAS, a unique, primate-specific sequence motif that disables an activating regulatory site in the glucose-6-phosphatase (G6PC) promoter was also required to enable function of this previously unrecognized tumor suppression system. In human somatic cells, loss of TP53 thus triggers activation of DHEAS transport proteins and steroid sulfatase, which converts circulating DHEAS into intracellular DHEA, and hexokinase which increases glucose-6-phosphate substrate concentration. The triggering of these enzymes in the TP53-affected cell combines with the primate-specific G6PC promoter sequence motif that enables G6P substrate accumulation, driving uncompetitive inhibition of G6PD to irreversibility and ROS-mediated cell death. By this catastrophic ‘kill switch’ mechanism, TP53 mutations are effectively prevented from initiating tumorigenesis in the somatic cells of humans, the primate with the highest peak levels of circulating DHEAS. TP53 mutations in human tumors therefore represent fossils of kill switch failure resulting from an age-related decline in circulating DHEAS, a potentially reversible artifact of hominid evolution.

Species specificity of an adrenal androgen-mediated kill-switch triggered by p53 inactivation

Glucose-6-phosphate dehydrogenase (G6PD) is an oncoprotein that is regulated by the p53 tumor suppressor. Mutant p53 loses the ability to inhibit G6PD, and loss of G6PD control clearly plays a role in oncogenesis. The steroid hormone precursor dehydroepiandrosterone (DHEA) is an endogenous uncompetitive inhibitor of G6PD. In humans, and a few other species, the sulfated circulatory form of DHEA (DHEAS) is present at extremely high concentrations – much higher than can be accounted for by DHEA’s function as a precursor to steroid hormones. Uncompetitive inhibition is extremely rare in natural systems because it is irreversible in the presence of high concentrations of substrate and inhibitor. What has gone unappreciated is that such uncompetitive inhibition can quickly lead to cell death when the target is an obligatory housekeeping gene such as G6PD. Cells with inactivated p53 not only lose control over G6PD, but also over hexokinase (HK), the enzyme that converts glucose into glucose-6-phosphate (G6P), the substrate of G6PD. Furthermore, loss of p53 function de-represses NFκB activity, resulting in the upregulation of steroid sulfatase (SS) which converts circulating DHEAS into active DHEA. We propose that inactivation of p53 rapidly elevates G6P and DHEA concentrations in affected cells, driving uncompetitive inhibition of G6PD to lethal irreversibility. In animals with circulating DHEAS, this kill-switch mechanism may prevent most cases of p53 inactivation from becoming tumorigenic. Tumors would thus represent instances in which this mechanism had not been triggered, but which might still be triggered by application of DHEA sufficient to uncompetitively inhibit tumor G6PD. To test this hypothesis, we performed a pilot study in which dogs with cardiac hemangiosarcoma were treated with high dose (HD) DHEA supplemented with isoprene precursors to maintain geranylation of Rac GTPase. Tumor regression and longevity observed in these dogs supported the concept that some tumors retain extraordinary sensitivity to uncompetitive inhibition by DHEA.