Pro-Oxidant Auranofin and Glutathione-Depleting Combination Unveils Synergistic Lethality in Glioblastoma Cells with Aberrant Epidermal Growth Factor Receptor Expression.

Glioblastoma (GBM) is the most prevalent and advanced malignant primary brain tumor in adults. GBM frequently harbors epidermal growth factor receptor (EGFR) wild-type (EGFRwt) gene amplification and/or EGFRvIII activating mutation. EGFR-driven GBM relies on the thioredoxin (Trx) and/or glutathione (GSH) antioxidant systems to withstand the excessive production of reactive oxygen species (ROS). The impact of EGFRwt or EGFRvIII overexpression on the response to a Trx/GSH co-targeting strategy is unknown. In this study, we investigated Trx/GSH co-targeting in the context of EGFR overexpression in GBM. Auranofin is a thioredoxin reductase (TrxR) inhibitor, FDA-approved for rheumatoid arthritis. L-buthionine-sulfoximine (L-BSO) inhibits GSH synthesis by targeting the glutamate-cysteine ligase catalytic (GCLC) enzyme subunit. We analyzed the mechanisms of cytotoxicity of auranofin and the interaction between auranofin and L-BSO in U87MG, U87/EGFRwt, and U87/EGFRvIII GBM isogenic GBM cell lines. ROS-dependent effects were assessed using the antioxidant N-acetylsteine. We show that auranofin decreased TrxR1 activity and increased ROS. Auranofin decreased cell vitality and colony formation and increased protein polyubiquitination through ROS-dependent mechanisms, suggesting the role of ROS in auranofin-induced cytotoxicity in the three cell lines. ROS-dependent PARP-1 cleavage was associated with EGFRvIII downregulation in U87/EGFRvIII cells. Remarkably, the auranofin and L-BSO combination induced the significant depletion of intracellular GSH and synergistic cytotoxicity regardless of EGFR overexpression. Nevertheless, molecular mechanisms associated with cytotoxicity were modulated to a different extent among the three cell lines. U87/EGFRvIII exhibited the most prominent ROS increase, P-AKT(Ser-473), and AKT decrease along with drastic EGFRvIII downregulation. U87/EGFRwt and U87/EGFRvIII displayed lower basal intracellular GSH levels and synergistic ROS-dependent DNA damage compared to U87MG cells. Our study provides evidence for ROS-dependent synergistic cytotoxicity of auranofin and L-BSO combination in GBM in vitro. Unraveling the sensitivity of EGFR-overexpressing cells to auranofin alone, and synergistic auranofin and L-BSO combination, supports the rationale to repurpose this promising pro-oxidant treatment strategy in GBM.

Molecular Analysis of the Superior Efficacy of a Dual Epidermal Growth Factor Receptor (EGFR)-DNA-Targeting Combi-Molecule in Comparison with Its Putative Prodrugs 6-Mono-Alkylamino- and 6,6-Dialkylaminoquinazoline in a Human Osteosarcoma Xenograft Model.

Background: ZR2002 is a dual EGFR-DNA-targeting combi-molecule that carries a chloroethyl group at the six-position of the quinazoline ring designed to alkylate DNA. Despite its good pharmacokinetics, ZR2002 is metabolized in vivo into dechlorinated metabolites, losing the DNA-alkylating function required to damage DNA. To increase the DNA damage activity in tumor cells in vivo, we compared ZR2002 with two of its 6-N,N-disubstituted analogs: "JS61", with a nitrogen mustard function at the six-position of the quinazoline ring, and "JS84", with an N-methyl group. Methods: Tumor xenografts were performed with the human Saos-2 osteosarcoma cell line expressing EGFR. Mice were treated with ZR2002, JS84 or JS61, and the tumor burden was measured with a caliper and CT/PET imaging. Drug metabolism was analyzed with LC-MS. EGFR and ɣ-H2AX phosphorylation were quantified via Western blot analysis and immunohistochemistry. Results: In vivo analysis showed that significant tumor growth inhibition was only achieved when ZR2002 was administered in its naked form. The metabolic dealkylation of JS61 and JS84 did not release sufficient concentrations of ZR2002 for the intratumoral inhibition of P-EGFR or enhanced levels of P-H2AX. Conclusions: The results in toto suggest that intratumoral concentrations of intact ZR2002 are correlated with the highest inhibition of P-EGFR and induction of DNA damage in vivo. ZR2002 may well represent a good drug candidate for the treatment of EGFR-expressing osteosarcoma.

Molecular Profiling of Peanut under Raw, Roasting, and Autoclaving Conditions Using High-Resolution Magic Angle Spinning and Solution 1H NMR Spectroscopy.

Higher rates of peanut allergy have been observed in countries that commonly roast peanuts prior to consumption. Despite the importance of understanding the role of thermal processing in allergy and on peanut composition, studies toward generating signatures that identify molecular contents following processing are scant. Here, we identified spectral signatures to track changes and differences in the molecular composition of peanuts under raw, roasted, and high-pressure and high-temperature autoclaved conditions. We analyzed both the solid flesh of the seed and solutions derived from soaking peanuts using High-Resolution Magic Angle Spinning (HR-MAS) and solution 1H Nuclear Magnetic Resonance (NMR) spectroscopy, respectively. The NMR spectra of intact peanuts revealed triglycerides as the dominant species, assigned on the basis of multiplets at 4.1 and 4.3 ppm, and corresponding defatted flours revealed the presence of sugars. Sucrose assigned based on a doublet at 5.4 ppm (anomeric proton), and triglycerides were the most abundant small molecules observed, with little variation between conditions. Soaked peanut solutions were devoid of lipids, and their resulting spectra matched the profiles of defatted peanuts. Spectral signatures resulting from autoclaving differed strikingly between those from raw and roasted peanuts, with considerable line-broadening in regions corresponding to proteins and amino-acid side chains, from 0.5 to 2.0 ppm and 6.5 to 8.5 ppm. Taken together, by using complementary NMR methods to obtain a fingerprint of the molecular components in peanuts, we demonstrated that autoclaving led to a distinct composition, likely resulting from the hydrolytic cleavage of proteins, the most important molecule of the allergic reaction.

Quantitative Analysis of the Potency of Equimolar Two-Drug Combinations and Combi-Molecules Involving Kinase Inhibitors In Vitro: The Concept of Balanced Targeting.

The median-effect principle proposed by Chou and Talalay is the most effective approach to parameterize interactions between several agents in combination. However, this method cannot be used to evaluate the effectiveness of equimolar drug combinations, which are comparative references for dual-targeting molecular design. Here, using data acquired through the development of "combi-molecules" blocking two kinases (e.g., EGFR-c-Src and EGFR-c-Met), we established potency indices for equimolar and dual-targeted inhibitors. If the fold difference (κ) between the IC50 of the two individual kinase inhibitors was >6, the IC50 of their equimolar combination resembled that of the more potent inhibitor. Hence, the "combi-targeting" of the two kinases was considered "imbalanced" and the combination ineffective. However, if κ ≤ 6, the IC50 of the combination fell below that of each individual drug and the combi-targeting was considered "balanced" and the combination effective. We also showed that combi-molecules should be compared with equimolar combinations only under balanced conditions and propose a new parameter Ω for validating their effectiveness. A multi-targeted drug is effective if Ω < 1, where Ω is defined as the IC50 of the drug divided by that of the corresponding equimolar combination. Our study provides a methodology to determine the in vitro potency of equimolar two-drug combinations as well as combi-/hybrid molecules inhibiting two different kinase targets.

Subcellular distribution and mechanism of action of AL906, a novel and potent EGFR inhibitor rationally designed to be green fluorescent.

To enhance the potency of EGFR inhibitors, we developed a novel strategy that seeks to conjugate EGFR to a bioactive moiety leading to a molecule termed "combi-molecule". In order to mimic the penetration of this type of molecules, based upon previously reported structure activity relationship studies, we designed a new molecule containing a quinazoline moiety tethered to a p-nitrobenzoxadiazole (NBD) moiety [molecular weight (MW) 700]. Despite its size, AL906 growth inhibitory activity was superior to that of the clinical drug gefitinib. Furthermore, AL906 retained significant EGFR inhibitory activity and good cellular penetration with abundant distribution in the perinuclear region of the cells. In an isogenic NIH3T3 transfected cell panel, it selectively inhibited the growth  of the NIH3T3-EGFR and HER2 transfectants. Confocal microscopy analysis revealed that it was capable of penetrating multilayer aggregates although to a lesser extent than FD105, a small inhibitor of EGFR inhibitor of the same class (MW 300). Its ability to inhibit EGFR auto-phosphorylation in monolayer culture was stronger than in the aggregates. The results suggest that our strategy did not negatively affect EGFR inhibitory potency, EGFR selectivity and growth inhibition. However, its molecular size may account for its decreased aggregate penetration when compared with a smaller EGFR inhibitor of the quinazoline class.

Design and Synthesis of a Trifunctional Molecular System "Programmed" to Block Epidermal Growth Factor Receptor Tyrosine Kinase, Induce High Levels of DNA Damage, and Inhibit the DNA Repair Enzyme (Poly(ADP-ribose) Polymerase) in Prostate Cancer Cells.

Resistance to chemotherapy in advanced cancers can be mediated by different factors such as epidermal growth factor receptor (EGFR) overexpression and DNA repair enzymes. Therefore, current standards of care usually involve combinations of multiple treatments. Here, to reduce the adverse effects of multiple drug combinations and improve outcome, we proposed a single drug approach to block multiple overlapping effects that characterize chemoresistance. Thus, we designed a new linker that allows assembly of multiple functions (e.g., inhibition of EGFR phosphorylation, induction of DNA lesions, and blockade of their repair) into a single molecule. This led to the successful synthesis of a novel and potent combi-molecule JS230. Here, we demonstrated that in resistant prostate cancer cells overexpressing EGFR, it was capable of (a) inhibiting EGFR in a dose-dependent manner, (b) damaging DNA, and (c) sustaining the damage by inhibiting the DNA repair protein poly(ADP-ribose) polymerase (PARP). The triple mechanism of action of JS230 cumulated into growth inhibitory potency superior to that of classical two- or three-drug combinations.

Comparative analysis of the dual EGFR-DNA targeting and growth inhibitory properties of 6-mono-alkylamino- and 6,6-dialkylaminoquinazoline-based type II combi-molecules.

Over the past decade, we described a novel tumour targeted approach that sought to design "combi-molecules" to hit two distinct targets in tumour cells. Here, to generate small combi-molecules with strong DNA damaging potential while retaining EGFR inhibitory potency, we developed the first synthetic strategy to access the 6-N, N-disubstituted quinazoline scaffold and designed JS61 to possess a nitrogen mustard function directly attached to the 6-position of the quinazoline ring. We compared its biological activity with that of structures containing either a hemi mustard or a non-alkylating substituent. Surprisingly, the results showed that JS61, while capable of inducing strong DNA damage, exhibited moderate EGFR inhibitory potency. In contrast, "combi-molecules" with no bulky substituent at the N-6 position (e.g. ZR2002 and JS84) showed stronger EGFR and growth inhibitory potency than JS61 in a panel of lung cancer cells. To rationalize these results, X-ray crystallography and molecular modeling studies were undertaken, and the data obtained indicated that bulkiness of the 6-N,N-disubstituted moieties hinder its binding to the ATP site and affects binding reversibility.

Radiomics in Glioblastoma: Current Status and Challenges Facing Clinical Implementation.

Radiomics analysis has had remarkable progress along with advances in medical imaging, most notability in central nervous system malignancies. Radiomics refers to the extraction of a large number of quantitative features that describe the intensity, texture and geometrical characteristics attributed to the tumor radiographic data. These features have been used to build predictive models for diagnosis, prognosis, and therapeutic response. Such models are being combined with clinical, biological, genetics and proteomic features to enhance reproducibility. Broadly, the four steps necessary for radiomic analysis are: (1) image acquisition, (2) segmentation or labeling, (3) feature extraction, and (4) statistical analysis. Major methodological challenges remain prior to clinical implementation. Essential steps include: adoption of an optimized standard imaging process, establishing a common criterion for performing segmentation, fully automated extraction of radiomic features without redundancy, and robust statistical modeling validated in the prospective setting. This review walks through these steps in detail, as it pertains to high grade gliomas. The impact on precision medicine will be discussed, as well as the challenges facing clinical implementation of radiomic in the current management of glioblastoma.

Molecular analysis of the dual targeting of the epidermal growth factor receptor and the O6-methylguanine-DNA methyltransferase with a double arm hybrid molecule.

Disordered expression of the epidermal growth factor receptor (EGFR) has been associated with induction of DNA repair genes (e.g. XRCC1, ERCC1) and resistance to radiation and genotoxic drugs. However, our previous work showed that EGFR inhibition did not affect O6-methylguanine-DNA methyltransferase (MGMT)-mediated resistance. In order to block uncoupled events associated with EGFR and MGMT, we designed MR30, a single molecule termed "combi-molecule" that contains a quinazoline arm targeted to EGFR and an O6-benzylguanine (O6-BG) moiety to block MGMT. Molecular analysis of the mechanism of action of its two arms showed that: (a) it could block EGFR phosphorylation, (b) down-regulate the RAF-MAPK and the PI3K-AKT pathways, and (c) covalently modify MGMT through S-benzylation, as confirmed by MALDI analysis of a direct binding assay with isolated MGMT, (d) it induced a dose-dependent down-regulation of MGMT in lung and melanoma cells. The pleiotropic mechanism of action of MR30 culminated into strong growth inhibition (IC50: 0.018-6.02 µM), with superior activity when compared with an equimolar combination of gefitinib (a clinical EGFR inhibitor) and O6-BG (a known MGMT inhibitor). Pulse exposure experiments were required to attenuate the contribution of EGFR inhibition to the strong potency of MR30, thereby allowing to achieve the dose level required to sensitize cells to temozolomide (TMZ). Indeed, MR30 significantly sensitized EGFR-MGMT co-expressing cells to TMZ (p<0.05-0.0001). The results in toto suggest that MR30 is the first prototype of agents that may be used against tumours addicted to EGFR and to sensitize resistant tumours co-expressing EGFR and MGMT to TMZ.

Design, synthesis and biological activity of novel molecules designed to target PARP and DNA.

In order to enhance the cytotoxic potential of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a PARP targeting scaffold. A cell-based selectivity assay involving a BRCA2-deficient Chinese hamster cell line and its corresponding BRCA2 wild type transfectant, was used to predict the PARP targeting potential of the latter agents. The results showed that adding a DNA damaging function to the PARP inhibitors decreased but did not abrogate the selective targeting of the BRCA2-deficient cells. The DNA damaging moiety augmented the potency in BRCA2 deficient cells by 2-20 fold. The most selective dual PARP-DNA targeting agent 14b was found to possess dual DNA and PARP targeting properties.

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model.

PURPOSE: ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. METHODS AND MATERIALS: The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Western blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. RESULTS: Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. CONCLUSIONS: Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.

Target modulation by a kinase inhibitor engineered to induce a tandem blockade of the epidermal growth factor receptor (EGFR) and c-Src: the concept of type III combi-targeting.

Cancer cells are characterized by a complex network of interrelated and compensatory signaling driven by multiple kinases that reduce their sensitivity to targeted therapy. Therefore, strategies directed at inhibiting two or more kinases are required to robustly block the growth of refractory tumour cells. Here we report on a novel strategy to promote sustained inhibition of two oncogenic kinases (Kin-1 and Kin-2) by designing a molecule K1-K2, termed "combi-molecule", to induce a tandem blockade of Kin-1 and Kin-2, as an intact structure and to be further hydrolyzed to two inhibitors K1 and K2 directed at Kin-1 and Kin-2, respectively. We chose to target EGFR (Kin-1) and c-Src (Kin-2), two tyrosine kinases known to synergize to promote tumour growth and progression. Variation of K1-K2 linkers led to AL776, our first optimized EGFR-c-Src targeting prototype. Here we showed that: (a) AL776 blocked EGFR and c-Src as an intact structure using an in vitro kinase assay (IC50 EGFR = 0.12 µM and IC50 c-Src = 3 nM), (b) it could release K1 (AL621, a nanomolar EGFR inhibitor) and K2 (dasatinib, a clinically approved Abl/c-Src inhibitor) by hydrolytic cleavage both in vitro and in vivo, (c) it could robustly inhibit phosphorylation of EGFR and c-Src (0.25-1 µM) in cells, (d) it induced 2-4 fold stronger growth inhibition than gefitinib or dasatinib and apoptosis at concentrations as low as 1 µM, and, (e) blocked motility and invasion at sub-micromolar doses in the highly invasive 4T1 and MDA-MB-231 cells. Despite its size (MW = 1032), AL776 blocked phosphorylation of EGFR and c-Src in 4T1 tumours in vivo. We now term this new targeting model consisting of designing a kinase inhibitor K1-K2 to target Kin-1 and Kin-2, and to further release two inhibitors K1 and K2 of the latter kinases, "type III combi-targeting".

Positional isomerization of a non-cleavable combi-molecule dramatically altered tumor cell response profile.

To potentiate the quinazoline-based inhibitor of the epidermal growth factor receptor (EGFR), a chloroethyl alkylating moiety was appended to its 6-position. This led to molecules with extremely strong EGFR inhibitory potency and anomalously strong DNA-damaging potential. To assess the role of the chloroethyl group on potency, we designed a molecule in which it is shifted to the 7-position where it would be less reactive and away from the cys773 of the EGFR ATP site. The results showed that (i) ZR2009 was 10-fold less potent than its positional isomer ZR2003 in EGFR tyrosine kinase inhibition, (ii) it consistently exhibited significantly weaker antiproliferative potency than ZR2003, (iii) in reversibility assays, while ZR2003 induced sustained inhibition of EGFR phosphorylation, ZR2009 inhibitory activity was partially reversed, and (iv) likewise, ZR2009 significantly lost its activity in short exposure growth inhibitory assays and induced lower levels of DNA damage than ZR2003. Molecular modeling suggested that while the chloroethylamino group in ZR2003 was at 3.5 Å away from Cys773, that of ZR2009 was at 6.3 Å. The results in toto suggest that, while the chloroethyl is a strong alkylating group, its appendage to the 6-position is optimal for DNA damage, sustained EGFR, and growth inhibition.

ZRX1, the first EGFR inhibitor-capecitabine based combi-molecule, requires carboxylesterase-mediated hydrolysis for optimal activity.

Capecitabine, an orally available prodrug of 5-FU, requires activation by carboxylesterase (CES) enzymes present in the liver to generate 5'-deoxy-5-flurocytidine ribose (5'-DFCR). The deamination of the latter by cytidine deaminase gives 5'-deoxy-5-fluorouridine ribose (5'-DFUR). Finally, the conversion of 5'-DFUR to the cytotoxic drug 5-FU, occurs primarily in the tumour and is catalyzed by thymidine phosphorylase (TP). Accordingly, it was surmised that events associated with an increase of TP levels should enhance the potency of capecitabine and its metabolites. EGFR inhibition was found to be one such event. The observed synergy between gefitinib and 5'-DFUR has inspired the design of single molecules capable of acting as prodrugs of both an EGFR inhibitor and 5-FU. Here, we report on the synthesis and characterization of one such molecule, ZRX1, that consists of an acetylated 5'-DFCR moiety linked to a quinazoline inhibitor of EGFR through an alkyl dicarbamate spacer that requires CES activation to generate the two active metabolites. Our results showed that ZRX1 was ineffective as an intact molecule. However, when CES was present, ZRX1 induced an increase in EGFR inhibition, TP expression, DNA damage and apoptosis. ZRX1 was, at least, 3-fold more potent than capecitabine and 5'-DFUR and recapitulated the effects of the combination treatments. LC-MS analysis showed that in the presence of CES, ZRX1 is metabolized into a mixture of bioactive quinazoline derivatives and 5'-DFCR derived metabolites. Our results in toto, suggest that capecitabine-based EGFR targeting combi-molecules of the same type than ZRX1, have the potential to induce stronger growth inhibitory potency than capecitabine, 5'-DFUR or single EGFR inhibitors and equivalent potency when compared with combinations of EGFR inhibitors + 5'-DFUR.

Biological effects of AL622, a molecule rationally designed to release an EGFR and a c-Src kinase inhibitor.

In breast cancer cells expressing c-Src and EGFR, a control of one of the two oncogenes over proliferation and invasion is observed, whereas in others, the synergistic interaction between them is required for tumor progression. With the purpose of developing molecules with the highest probability for blocking the adverse effects of these two oncogenes, we designed AL622, which contains a quinazoline head targeted to EGFR and a linker that bridges it to the PP2-like structure for targeting c-Src. In case the entire molecule would not be capable of blocking c-Src, we designed AL622 to hydrolyze to an intact c-Src-targeting PP2 molecule. After confirming its binary c-Src-EGFR targeting potency of AL622, we analyzed its potency in isogenic NIH3T3 cells transfected with EGFR and HER2 and human breast cancer cells known to be dominated by c-Src function. The results showed that in EGFR/HER-2-driven cells, it was more potent than PP2 and its activity was in the same range as the latter in more c-Src-driven cells. Its ability to block motility and invasion was comparable with that of PP2 and corresponding combinations, indicating that AL622 could be a better antitumor agent in cells where c-Src and/or EGFR play a role.

The Concept of Divergent Targeting through the Activation and Inhibition of Receptors as a Novel Chemotherapeutic Strategy: Signaling Responses to Strong DNA-Reactive Combinatorial Mimicries.

Recently, we reported the combination of multitargeted ErbB1 inhibitor-DNA damage combi-molecules with OCT in order to downregulate ErbB1 and activate SSTRs. Absence of translation to cell kill was believed to be partially due to insufficient ErbB1 blockage and DNA damage. In this study, we evaluated cell response to molecules that damage DNA more aggressively and induce stronger attenuation of ErbB1 phosphorylation. We used three cell lines expressing low levels (U87MG) or transfected to overexpress wildtype (U87/EGFR) or a variant (U87/EGFRvIII) of ErbB1. The results showed that Iressa ± HN2 and the combi-molecules, ZRBA4 and ZR2003, significantly blocked ErbB1 phosphorylation in U87MG cells. Addition of OCT significantly altered cell cycle distribution. Analysis of the DNA damage response pathway revealed strong upregulation of p53 by HN2 and the combi-molecules. Apoptosis was only induced by a 48 h exposure to HN2. All other treatments resulted in cell necrosis. This is in agreement with Akt-Bad pathway activation and survivin upregulation. Despite strong DNA damaging properties and downregulation of ErbB1 phosphorylation by these molecules, the strongest effect of SSTR activation was on cell cycle distribution. Therefore, any enhanced antiproliferative effects of combining ErbB1 inhibition with SSTR activation must be addressed in the context of cell cycle arrest.

Enhancement of the cytotoxic potential of the mixed EGFR and DNA-targeting 'combi-molecule' ZRBA1 against human solid tumour cells by a bis-quinazoline-based drug design approach.

ZRBA1 is a quinazoline-based molecule termed 'combi-molecule' designed to block the epidermal growth factor receptor (EGFR) and further degrade to FD105, an EGFR inhibitor plus a DNA-alkylating agent. To augment the potency of ZRBA1, we designed JDE52, a bistriazene that, following degradation, was 'programmed' to yield higher concentrations of the free inhibitor FD105 and a more cytotoxic bifunctional DNA-damaging species. The results indicated that JDE52 was capable of inducing significant blockade of EGFR phosphorylation, DNA strand breaks and interstrand cross-links in human cells. The fluorescent property of FD105, the secondary inhibitor that both JDE52 and ZRBA1 are capable of releasing, has permitted the analysis of its levels in tumour cells by ultraviolet flow cytometry. It was found that JDE52 was indeed capable of significantly releasing higher levels of fluorescence (P<0.05) in human tumour cells when compared with ZRBA1. Apoptosis was triggered by JDE52 at a faster rate than ZRBA1 and led to higher levels of cell killing. The results in toto suggest that the superior potency of JDE52, when compared with ZRBA1, may be imputed to mechanisms associated with the generation of higher intracellular concentrations of FD105 and to the induction of DNA cross-links. These combined mechanisms (blockade of EGFR-tyrosine kinase and induction of cross-links) contributed to an accelerated rate of apoptosis by JDE52. This study conclusively demonstrated that designing molecules as prodrugs of high levels of quinazoline inhibitors of EGFR and bifunctional DNA cross-linking species is a valid strategy to enhance the potency of mixed EGFR-DNA-targeting combi-molecules.

"Combi-targeting" mitozolomide: conferring novel signaling inhibitory properties to an abandoned DNA alkylating agent in the treatment of advanced prostate cancer.

PURPOSE: At the preclinical stage, mitozolomide (MTZ) showed exciting preclinical activity but failed later in clinical trial due to toxic side effects. We surmised that by targeting MTZ to epidermal growth factor receptor (EGFR), we may not only alter its toxicity profile, but also enhance its potency in EGFR-overexpressing tumors. To test this hypothesis, we designed JDF12, studied its mechanism of action in human prostate cancer (PCa) cells and determined its potency in vivo. EXPERIMENTAL DESIGN: To analyze its mixed EGFR-DNA targeting potential, we performed an enzyme linked immunosorbent assay (ELISA) and western blotting analysis of EGFR phosphorylation in cells stimulated with EGF. DNA damage was analyzed using the comet assay, and apoptosis quantitated by annexin V binding assay. Growth inhibition in vitro was determined by the sulforhodamine B (SRB) assay and in vivo efficacy analyzed in male CD-1 nude mice. RESULTS: The results showed that: Under physiological conditions, JDF12 was hydrolyzed to JDF04R and both agents were capable of inhibiting isolated EGFR tyrosine kinase (TK) and EGFR phosphorylation in EGF-stimulated cells. JDF12 significantly damaged DNA, induced apoptosis in DU145 cells and was up to 2-10-fold more potent than equieffective combinations of MTZ and JDF04R or Iressa in a panel that also included LNCaP and its EGFR and ErbB2 transfectants. In vivo, it induced significant antitumor activity in a DU145 xenograft model. CONCLUSIONS: The results suggest that the superior cytotoxicity of JDF12 when compared with MTZ and JDF04R may be imputed to its potent EGFR-DNA targeting properties and confirm the ability of this novel strategy to confer EGFR targeting properties to a classical alkylator.

Inhibition of EGFR phosphorylation in a panel of human breast cancer cells correlates with synergistic interactions between gefitinib and 5'-DFUR, the bioactive metabolite of Xeloda.

Capecitabine (Xeloda) is a prodrug of 5-FU used in the clinical management of advanced breast cancer. It is metabolized first in the liver by carboxylesterases to generate 5'-deoxy-5-flurocytidine ribose (5'-DFCR), which is subsequently converted to 5'-deoxy-5-fluorouridine ribose (5'-DFUR) by cytidine deaminase in tumour and normal tissues. The conversion of 5'-DFUR to the cytotoxic 5-FU, occurs primarily in the tumour and is catalyzed by thymidine phosphorylase (TP). Prior work in head and neck cancer showed that cell treatment with an inhibitor of the epidermal growth receptor (EGFR) gefitinib led to an increase in TP expression and sensitized them to 5'-DFUR. This work seeks to investigate the factors influencing the potency of gefitinib + 5'-DFUR combination. Here, we studied these factors in a panel of six human breast cancer cell lines, with varied levels of sensitivity to gefitinib. Our results first confirmed that 5'-DFUR potency linearly correlates with TP basal levels in the panel of cell lines. In contrast, the strength of the synergistic effect of the gefitinib + 5'-DFUR combination, as measured by their combination indices (CI) correlates with pEGFR percent inhibition and with the modulation of TP expression by gefitinib (as quantitated by TP fold change) rather than TP basal levels. The results, in toto, suggest that the extent of modulation of TP by gefitinib may be used as a predictor of tumour sensitivity to gefitinib + capecitabine/5'-DFUR combinations.

Synthesis, characterization and anticancer studies of mixed ligand dithiocarbamate palladium(II) complexes.

Six mixed ligand dithiocarbamate Pd(II) complexes (1-6) of general formula [(DT)Pd(PR(3))Cl], where DT = dimethyldithiocarbamate (1, 5), diethyldithiocarbamate (2, 3), dicyclohexyldithiocarbamate (4), bis(2-methoxyethyl)dithiocarbamate (6); PR(3) = benzyldiphenylphosphine (1), diphenyl-2-methoxyphenylphosphine (2), diphenyl-p-tolylphosphine (3), diphenyl-m-tolylphosphine (4), tricyclohexylphosphine (5), diphenyl-2-pyridylphosphine (6) have been synthesized and characterised using Elemental analysis, FT-IR, Raman and multinuclear magnetic resonance (NMR) spectroscopy. Compounds 1 and 2 were also characterized by single crystal X-ray diffraction technique (XRD). The XRD study reveals that the Pd(II) moiety has a pseudo square-planar geometry, in which two positions are occupied by the dithiocarbamate ligand in a bidentate fashion, while at the remaining two positions organophosphine and chloride are present. The anticancer activity of the synthesized metallodrugs was checked against DU145 human prostate carcinoma (HTB-81) cells, the IC(50) values indicate that the compounds are highly active against these cells. These Pd(II) complexes also show moderate antibacterial activity against gram positive and gram negative bacteria.