Inhibition of cytochrome P450s enhances (+)‐usnic acid cytotoxicity in primary cultured rat hepatocytes

Shi, Qiang; Greenhaw, James; Salminen, William F.

doi:10.1002/jat.2892

Cited by 23 publications

(14 citation statements)

References 24 publications

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“…To measure cells' ATP content, 7500 A549 cells were seeded per well in a 384-well plate. After 36 h, including stimulation with TNFα and IFNγ for the indicated time, a half volume of the CellTiter-Glo reagent (Promega) was added to each well following (82). The reagent was incubated with the cells for 10 min per the manufacturer's protocol and then luminescence levels were measured over time using BioTek Synergy H1 plate reader.…”

Section: Methodsmentioning

confidence: 99%

Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression

et al. 2019

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Antigen presentation on HLA molecules is a major mechanism by which the immune system monitors self and non-self-recognition. Importantly, HLA-I presentation has gained much attention through its role in eliciting anti-tumor immunity. Several determinants controlling the peptides presented on HLA have been uncovered, mainly through the study of model substrates and large-scale immunopeptidome analyses. These determinants include the relative abundances of proteins in the cell, the stability or turnover rate of these proteins and the binding affinities of a given peptide to the HLA haplotypes found in a cell. However, the regulatory principles involved in selection and regulation of specific antigens in response to tumor pro-inflammatory signals remain largely unknown. Here, we chose to examine the effect that TNFα and IFNγ stimulation may exert on the immunopeptidome landscape of lung cancer cells. We show that the expression of many of the proteins involved in the class I antigen presentation pathway are changed by pro-inflammatory cytokines. Further, we could show that increased expression of the HLA-B allomorph drives a significant change in HLA-bound antigens, independently of the significant changes observed in the cellular proteome. Finally, we observed increased HLA-B levels in correlation with tumor infiltration across the TCGA lung cancer cohorts. Taken together, our results suggest that the immunopeptidome landscape should be examined in the context of anti-tumor immunity whereby signals in the microenvironment may be critical in shaping and modulating this important aspect of host-tumor interactions.

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Section: Methodsmentioning

confidence: 99%

Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression

et al. 2019

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“…Briefly, hepatocytes seeded in 100 Â 20 mm dishes were washed by PBS and detached by trypsinization. Cells were re-suspended in respiration buffer at 2 Â 10 6 cell/ml and 0.0075% digitonin was added for 3 min to permeablize the cell membrane (Shi et al, 2014). The stock solution of 7.5% digitonin in water was prepared by heating at 95 C for 5 min immediately before use.…”

Section: Hepatocyte Culture and Drug Treatmentmentioning

confidence: 99%

“…Compound C (10 mM) was used to inhibit AMPK activation (Saberi et al, 2014). A previously published procedure (Shi et al, 2014) was modified to collect cells for measuring oxygen consumption. Briefly, hepatocytes seeded in 100 Â 20 mm dishes were washed by PBS and detached by trypsinization.…”

Section: Hepatocyte Culture and Drug Treatmentmentioning

confidence: 99%

Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis

et al. 2015

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The tyrosine kinase inhibitor regorafenib was approved by regulatory agencies for cancer treatment, albeit with strong warnings of severe hepatotoxicity included in the product label. The basis of this toxicity is unknown; one possible mechanism, that of mitochondrial damage, was tested. In isolated rat liver mitochondria, regorafenib directly uncoupled oxidative phosphorylation (OXPHOS) and promoted calcium overload-induced swelling, which were respectively prevented by the recoupler 6-ketocholestanol (KC) and the mitochondrial permeability transition (MPT) pore blocker cyclosporine A (CsA). In primary hepatocytes, regorafenib uncoupled OXPHOS, disrupted mitochondrial inner membrane potential (MMP), and decreased cellular ATP at 1h, and triggered MPT at 3h, which was followed by necrosis but not apoptosis at 7h and 24h, all of which were abrogated by KC. The combination of the glycolysis enhancer fructose plus the mitochondrial ATPase synthase inhibitor oligomycin A abolished regorafenib induced necrosis at 7h. This effect was not seen at 24h nor with the fructose or oligomycin A separately. CsA in combination with trifluoperazine, both MPT blockers, showed similar effects. Two compensatory mechanisms, activation of AMP-activated protein kinase (AMPK) to ameliorate ATP shortage and induction of autophagy to remove dysfunctional mitochondria, were found to be mobilized. Hepatocyte necrosis was enhanced either by the AMPK inhibitor Compound C or the autophagy inhibitor chloroquine, while autophagy inducer rapamycin was strongly cytoprotective. Remarkably, all toxic effects were observed at clinically-relevant concentrations of 2.5-15μM. These data suggest that uncoupling of OXPHOS and the resulting ATP shortage and MPT induction are the key mechanisms for regorafenib induced hepatocyte injury, and AMPK activation and autophagy induction serve as pro-survival pathways against such toxicity.

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“…As shown in Fig. 5, alpha-naphthoflavone, a CYP1A-specific inhibitor in human and rat liver microsomes (19), showed the most potent inhibition against DMAKO-05 metabolism. At a concentration of as low as 10 μM, the inhibitory activity was more than 80%.…”

Section: Identification Of P450 Isoform(s) Responsible For Dmako-05 Mmentioning

confidence: 80%

Antitumor Activity of DMAKO-05, a Novel Shikonin Derivative, and Its Metabolism in Rat Liver Microsome

Wang

Zhou

et al. 2014

AAPS PharmSciTech

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Abstract. The antitumor activity of shikonin derivatives is largely dependent on the generation of superoxide radicals and the alkylation activity of their naphthoquinone moiety. However, our recent study showed that 1,4-dioxime-5,8-dimethoxynaphthalene (DMAKO-05), a novel shikonin derivative, displayed more potential antitumor activity and less toxicity compared to fluorouracil (5-FU) both in vitro and in vivo, even though the hydroxyl and carbonyl groups of its naphthoquinone structure were shielded. To understand the underlying mechanisms, we investigated the metabolism of DMAKO-05 in rat liver microsomes. The kinetic analysis indicated that DMAKO-05 underwent a biphasic metabolism in rat liver microsomes. The inhibition experiments showed that CYP1A and CYP3A were the major enzymes in the metabolism of DMAKO-05, along with partial contribution from CYP2A. In addition, the structures of eight DMAKO-05 metabolites, which were characterized by accurate mass and MS/MS fragmentograms, implied that DMAKO-05 was mainly metabolized through the oxygenation of its naphthoquinone nucleus and the hydrolysis of its side chain, instead of the oxidation of hydroxyimine to ketone. Therefore, DMAKO-05 should not be considered as a traditional naphthoquinone prodrug.

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Inhibition of cytochrome P450s enhances (+)‐usnic acid cytotoxicity in primary cultured rat hepatocytes

Cited by 23 publications

References 24 publications

Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression

Pro-inflammatory Cytokines Alter the Immunopeptidome Landscape by Modulation of HLA-B Expression

Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis

Antitumor Activity of DMAKO-05, a Novel Shikonin Derivative, and Its Metabolism in Rat Liver Microsome

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