P-glycoprotein (P-gp), a 170 kDa plasma membrane protein, is one of the most relevant ABC transporters involved in the development of multidrug resistance (MDR). Understanding its mechanism of transport as well as its interactions with various substrates are basic requirements for the development of adequate therapeutic approaches to overcome this kind of resistance against a broad spectrum of structurally unrelated cytostatic drugs. P-gp modulators (activators) that exert various effects on the intracellular accumulation of distinct P-gp substrates are useful tools for investigating the interactions between multiple drug binding sites of this transport protein. In this study, a series of 27 different imidazobenzothiazoles and imidazobenzimidazoles structurally related to the known P-gp activators QB102 and QB11 was designed, and their modulating properties were investigated. Most of them were able to stimulate P-gp-mediated efflux of daunorubicin and rhodamine 123 in a concentration-dependent manner, but some compounds also displayed weak inhibitory effects. Additionally, P-gp-mediated efflux of vinblastine and colchicine was inhibited by several compounds. Therefore, we concluded that the novel compounds bind to the H site of P-gp and activate the efflux of specific substrates of the R site in a positive cooperative manner, whereas binding of H-type substrates is inhibited competitively. This hypothesis is confirmed by the observation that the modulators do not influence hydrolysis of ATP or its affinity toward P-gp.

Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS-PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity.

Integrins have become a target for novel therapeutic strategies against malignant gliomas. Cilengitide, a synthetic Arg-Gly-Asp (RGD)-motif peptide, interferes with ligand binding to avb3 and avb5 integrins and is currently investigated in clinical trials. Integrins may also be involved in the activation of transforming growth factor (TGF)-b, a mediator of invasiveness and immune escape of glioma cells. Using flow cytometry, we demonstrate that the target integrins of cilengitide are expressed not only in glioblastoma blood vessels, but also by tumor cells. After exposure of glioma cells to cilengitide, we noticed reduced phosphorylation of Smad2 in most glioma cell lines, including stem-like glioma cells. Phophorylation of Smad2, but not cilengitide-induced detachment, is rescued by addition of recombinant TGF-b. Administration of cilengitide to glioma cells results in reduced TGF-b-mediated reporter gene activity. Furthermore, exposure to cilengitide leads to decreased TGF-b 1 and TGF-b 2 mRNA and protein expression. These effects are mimicked by blocking av, b3 or b5 antibodies or by silencing of integrins av, b3, b5 or b8 using RNA interference. Treatment of mice bearing experimental LN-308 glioma xenografts with cilengitide results in reduced pSmad2 levels. Taken together, cilengitide may exert anti-invasive and immune stimulatory activity in human glioblastoma patients by its anti-TGF-b properties.

Mutations in the p53 gene occur in a variety of human cancers with remarkably high frequencies (as high as 80%), depending on the type and stage of the tumors (www-p53.iarc.fr). The majority of mutations are missense that are localized to six “hotspot” residues, which play a role either in the structural integrity (structural mutants, e.g., R175H) or in the DNA binding domain (contact mutants, e.g., R273H). Previously, we showed that phenethyl isothiocyanate (PEITC), a naturally occurring dietary constituent present abundantly in watercress, selectively depletes p53 mutant protein, but not the WT p53, and induces apoptosis in tumor cells expressing p53 mutants. In this study we demonstrated that PEITC can reactivate p53R175 mutant protein and inhibit tumor growth in a mutant p53R175H -dependent manner. PEITC displayed a 175 allele preference of inhibition of cell proliferation as compared to other hotspot mutants as detected by WST-1 assay and induced apoptosis in a p53R175 mutant -dependent manner. Immunofluorescence and co-immunoprecipitation assay on PEITC treated p53R175 cells using conformation-specific antibodies (PAB1620 and PAB240) showed restoration of the “WT-like” conformation to p53 mutant. Reactivation of the WT like functions to the p53R175 mutant was established by its ability to bind DNA, transactivate p53 regulated downstream target genes (p21, PUMA, MDM2, NOXA, BAX and BCL2), and overexpression of p21 protein. PEITC treatment induced DNA damage response as detected by pATM-S1981 and pCHK2-Thr68, G2/M and S-phase cell-cycle arrest and oxidative stress as measured by decrease in the GSH levels in the cells. In contrast, co-treatment of cells with PEITC and reducing agents alleviated the inhibitory effects of PEITC on cell proliferation. Elevated oxidative stress due to the combined effect of PEITC and elevated ROS levels in p53 mutant cells might be responsible for the activation of restored “WT-like” p53R175 mutant protein and induction of apoptosis. PEITC induced proteasomal degradation and autophagy of the p53R175 mutant. Animals fed with PEITC diet showed a statistically significant reduction in xenograft tumor volumes, proliferation marker Ki67, and mutant p53 stained cells. Elevated mRNA levels of p53 regulated genes from animals fed PEITC diet provide in vivo evidence for the p53R175H mutant reactivation and inhibition of xenograft growth in a mutant p53-dependent manner. These are important findings because they demonstrated that a dietary compound can inhibit the growth of tumor cells in vivo by restoring the tumor suppressor functions to mutant p53, and thus provide a target for the development of novel chemopreventive strategies through dietary interventions. Citation Format: Monika Aggarwal, Elizabeth Sinclair, Anna Jacobs, Ying Fu, Marcin Dyba, Xiantao Wang, Idalia M. Cruz, Deborah Berry, Kallakury Bhaskar, Susette C. Mueller, Maria Laura Avantaggiati, Fung-Lung Chung. Reactivation of p53R175 mutant by dietary phenethyl isothiocyanate (PEITC) impairs tumor growth in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2015-1910