Application of a Specific and Sensitive NQO1 Turn-On Near-Infrared Fluorescence Probe for Live Cancer Cell and Xenografted Tumor Imaging in Nude Mice.

Sensitive activity stains for enzymes selectively expressed in human cancers offer valuable tools for imaging with wide applications in experimental, diagnostic, and therapeutic settings. The scant expression of the antioxidant enzyme NQO1 in normal tissues and its great abundance in malignant counterparts due to the increased redox stress and hypoxia is one such example. Previously, we described a potent nontoxic probe that remains nonfluorescent but releases an intense fluorogenic compound after intracellular cleavage by NQO1 catalysis. This infrared probe with a 644 nm emission has excellent tissue penetrating ability and low background absorption. Described here are methods (fluorescence microscopy, flow cytometry, and in vivo animal imaging) to rapidly image NQO1 activity in hypoxic and non-hypoxic cancer cells and tumors developed in live mouse xenograft models. The specificity of the dye for NQO1 in all three procedures was verified, and the methods should be useful for both in vitro and in vivo studies.

Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression.

BACKGROUND: The molecular regulation of increased MGMT expression in human brain tumors, the associated regulatory elements, and linkages of these to its epigenetic silencing are not understood. Because the heightened expression or non-expression of MGMT plays a pivotal role in glioma therapeutics, we applied bioinformatics and experimental tools to identify the regulatory elements in the MGMT and neighboring EBF3 gene loci. RESULTS: Extensive genome database analyses showed that the MGMT genomic space was rich in and harbored many undescribed RNA regulatory sequences and recognition motifs. We extended the MGMT's exon-1 promoter to 2019 bp to include five overlapping alternate promoters. Consensus sequences in the revised promoter for (a) the transcriptional factors CTCF, NRF1/NRF2, GAF, (b) the genetic switch MYC/MAX/MAD, and (c) two well-defined p53 response elements in MGMT intron-1, were identified. A putative protein-coding or non-coding RNA sequence was located in the extended 3' UTR of the MGMT transcript. Eleven non-coding RNA loci coding for miRNAs, antisense RNA, and lncRNAs were identified in the MGMT-EBF3 region and six of these showed validated potential for curtailing the expression of both MGMT and EBF3 genes. ChIP analysis verified the binding site in MGMT promoter for CTCF which regulates the genomic methylation and chromatin looping. CTCF depletion by a pool of specific siRNA and shRNAs led to a significant attenuation of MGMT expression in human GBM cell lines. Computational analysis of the ChIP sequence data in ENCODE showed the presence of NRF1 in the MGMT promoter and this occurred only in MGMT-proficient cell lines. Further, an enforced NRF2 expression markedly augmented the MGMT mRNA and protein levels in glioma cells. CONCLUSIONS: We provide the first evidence for several new regulatory components in the MGMT gene locus which predict complex transcriptional and posttranscriptional controls with potential for new therapeutic avenues.

Double-component diazeniumdiolate derivatives as anti-cancer agents.

In this study, we synthesized a series of double-component O2-aryl diazeniumdiolate (DDNO) derivatives, of which each molecule can release up to four nitric oxide molecules. These compounds showed cytotoxic activities to cancer cells, such as human leukemia, breast cancer and lung cancer. Among them, compound 1 (DDNO-1) showed the highest specific activity to human leukemia cells. It induced cell apopotosis and arrest cell cycle of G2/M phase. The JNK and p38 protein kinases were activated by compound 1 to induce cancer cell apoptosis. Compound 1 also increased pro-apoptotic Bax level, which is a same function compared to a reported NO donor, JS-K. More interestingly, it decreased the level of an anti-apoptotic member Bcl-2, which is an opposite effect compared to JS-K. Compound 1 could be developed as a new anti-cancer agent since it increases the Bax/Bcl-2 ratio to overcome the drug resistance.

Characterization of a highly specific NQO1-activated near-infrared fluorescent probe and its application for in vivo tumor imaging.

The Near-infrared Fluorescence (NIRF) molecular imaging of cancer is known to be superior in sensitivity, deeper penetration, and low phototoxicity compared to other imaging modalities. In view of an increased need for efficient and targeted imaging agents, we synthesized a NAD(P)H quinone oxidoreductase 1 (NQO1)-activatable NIR fluorescent probe (NIR-ASM) by conjugating dicyanoisophorone (ASM) fluorophore with the NQO1 substrate quinone propionic acid (QPA). The probe remained non-fluorescent until activation by NQO1, whose expression is largely limited to malignant tissues. With a large Stokes shift (186 nm) and a prominent near-infrared emission (646 nm) in response to NQO1, NIR-ASM was capable of monitoring NQO1 activity in vitro and in vivo with high specificity and selectivity. We successfully employed the NIR-ASM to differentiate cancer cells from normal cells based on NQO1 activity using fluorescence microscopy and flow cytometry. Chemical and genetic approaches involving the use of ES936, a specific inhibitor of NQO1 and siRNA and gene transfection procedures unambiguously demonstrated NQO1 to be the sole target activating the NIR-ASM in cell cultures. NIR-ASM was successfully used to detect and image the endogenous NQO1 in three live tumor-bearing mouse models (A549 lung cancer, Lewis lung carcinoma, and MDMAMB 231 xenografts) with a high signal-to-low noise ratiometric NIR fluorescence response. When the NQO1-proficient A549 tumors and NQO1-deficient MDA-MB-231 tumors were developed in the same animal, only the A549 malignancies activated the NIR-ASM probe with a strong signal. Because of its high sensitivity, rapid activation, tumor selectivity, and nontoxic properties, the NIR-ASM appears to be a promising agent with clinical applications.

Cancer-Specific Biomarker hNQO1-Activatable Fluorescent Probe for Imaging Cancer Cells In Vitro and In Vivo.

Human NAD(P)H quinone oxidoreductase-1 (hNQO1) is an important cancer-related biomarker, which shows significant overexpression in malignant cells. Developing an effective method for detecting NQO1 activity with high sensitivity and selectivity in tumors holds a great potential for cancer diagnosis, treatment, and management. In the present study, we report a new dicyanoisophorone (DCP) based fluorescent probe (NQ-DCP) capable of monitoring hNQO1 activity in vitro and in vivo in both ratiometric and turn-on model. NQ-DCP was prepared by conjugating dicyanoisophorone fluoroprobe with hNQO1 activatable quinone propionic acid (QPA), which remain non-fluorescent until activation by tumor-specific hNQO1. NQ-DCP featured a large Stokes shift (145 nm), excellent biocompatibility, cell permeability, and selectivity towards hNQO1 allowed to differentiate cancer cells from healthy cells. We have successfully employed NQ-DCP to monitor non-invasive endogenous hNQO1 activity in brain tumor cells in vitro and in xenografted tumors developed in nude mice.

Selective killing of human breast cancer cells by the styryl lactone (R)-goniothalamin is mediated by glutathione conjugation, induction of oxidative stress and marked reactivation of the R175H mutant p53 protein.

The molecular basis of anticancer and apoptotic effects of R-goniothalamin (GON), a plant secondary metabolite was studied. We show that induction of oxidative stress and reactivation of mutant p53 underlie the strong cytotoxic effects of GON against the breast cancer cells. While GON was not toxic to the MCF10a breast epithelial cells, the SKBR3 breast cancer cells harboring an R175H mutant p53 were highly sensitive (IC50 = 7.3 µM). Flow cytometry and other pertinent assays showed that GON-induced abundant reactive oxygen species (ROS), glutathione depletion, protein glutathionylation and activation of apoptotic markers. GON was found to conjugate with glutathione both in vitro and in cells and the product was characterized by mass spectrometry. We hypothesized that the redox imbalance induced by GON may affect the structure of the R175H mutant p53 protein, and account for greater cytotoxicity. Using the SKBR3 breast cancer and p53-null H1299 lung cancer cells stably expressing the R175H p53 mutant protein, we demonstrated that GON triggers the appearance of a wild-type-like p53 protein by using conformation-specific antibodies, immunoprecipitation, DNA-binding assays and target gene expression. p53 restoration was associated with a G2/M arrest, senescence, reduced cell migration, invasion and increased cell death. GON elicited a highly synergistic cytotoxicity with cisplatin in SKBR3 cells. In SKBR3 xenografts developed in nude mice, there was a marked tumor growth delay by GON alone and GON + cisplatin combination. Our studies highlight the impact of tumor redox-stress generated by GON in activating the mutant p53 protein for greater antitumor efficacy.

Conception, synthesis, and characterization of a rofecoxib-combretastatin hybrid drug with potent cyclooxygenase-2 (COX-2) inhibiting and microtubule disrupting activities in colon cancer cell culture and xenograft models.

Tumor heterogeneity and drug resistance pose severe limitations to chemotherapy of colorectal cancers (CRCs) necessitating innovative approaches to trigger multiple cytocidal events for increased efficacy. Here, we developed a hybrid drug called KSS19 by combining the COX-2 selective NSAID rofecoxib with the cis-stilbene found in combretastatin A4 (CA4), a problematic, but potent antimicrotubule and anti-angiogenesis agent. The structural design of KSS19 completely prevented the isomerization of CA4 its biologically inactive trans-form. Molecular modeling showed that KSS19 bound avidly to the COX-2 active site and colchicine -binding site of tubulin, with similar docking scores of rofecoxib and CA4 respectively. KSS-19 showed potent anti-proliferative activity against a panel of colon cancer cell lines; HT29 cells, which are resistant to CA4 were 100 times more sensitive to KSS19. The hybrid drug potently inhibited the tubulin polymerization in vitro and in cells inducing a G2/M arrest and aberrant mitotic spindles. Both the basal and LPS-activated levels of COX-2 in colon cancer cells were highly suppressed by the KSS-19. The cancer cell migration/invasion was inhibited and accompanied by increased E-cadherin levels and activated NF-kB/Snail pathways in KSS19-treated cells. The drug also curtailed the formation of endothelial tubes in three-dimensional cultures of the HUVE cells at 250 nM, indicating strong anti-angiogenic properties. In subcutaneous HT29 colon cancer xenografts, KSS19, as a single agent (25 mg/kg/day) significantly inhibited the tumor growth and downregulated the intratumoral COX-2, Ki-67, the angiogenesis marker CD31, however, the cleaved caspase-3 was elevated. Collectively, KSS19 represents a rational hybrid drug with clinical relevance to CRC.

Beyond Brooding on Oncometabolic Havoc in IDH-Mutant Gliomas and AML: Current and Future Therapeutic Strategies.

Isocitrate dehydrogenases 1 and 2 (IDH1,2), the key Krebs cycle enzymes that generate NADPH reducing equivalents, undergo heterozygous mutations in >70% of low- to mid-grade gliomas and ~20% of acute myeloid leukemias (AMLs) and gain an unusual new activity of reducing the α-ketoglutarate (α-KG) to D-2 hydroxyglutarate (D-2HG) in a NADPH-consuming reaction. The oncometabolite D-2HG, which accumulates >35 mM, is widely accepted to drive a progressive oncogenesis besides exacerbating the already increased oxidative stress in these cancers. More importantly, D-2HG competes with α-KG and inhibits a large number of α-KG-dependent dioxygenases such as TET (Ten-eleven translocation), JmjC domain-containing KDMs (histone lysine demethylases), and the ALKBH DNA repair proteins that ultimately lead to hypermethylation of the CpG islands in the genome. The resulting CpG Island Methylator Phenotype (CIMP) accounts for major gene expression changes including the silencing of the MGMT (O6-methylguanine DNA methyltransferase) repair protein in gliomas. Glioma patients with IDH1 mutations also show better therapeutic responses and longer survival, the reasons for which are yet unclear. There has been a great surge in drug discovery for curtailing the mutant IDH activities, and arresting tumor proliferation; however, given the unique and chronic metabolic effects of D-2HG, the promise of these compounds for glioma treatment is uncertain. This comprehensive review discusses the biology, current drug design and opportunities for improved therapies through exploitable synthetic lethality pathways, and an intriguing oncometabolite-inspired strategy for primary glioblastoma.

Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo.

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC50) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.