In Situ In Vivo radiolabeling of polymer-coated hydroxyapatite nanoparticles to track their biodistribution in mice.

The imaging of healthy tissues and solid tumors benefits from the application of nanoparticle probes with altered pharmacokinetics, not available to low molecular weight compounds. However, the distribution and accumulation of nanoprobes in vivo typically take at least tens of hours to be efficient. For nanoprobes bearing a radioactive label, this is contradictory to the requirement of minimizing the radiation dose for patients by using as-short-as-feasible half-life radionuclides in diagnostics. Thus, we developed a two-stage diagnostic concept for monitoring long-lasting targeting effects with short-lived radioactive labels using bone-mimicking biocompatible polymer-coated and colloidally fully stabilized hydroxyapatite nanoparticles (HAP NPs) and bone-seeking radiopharmaceuticals. Within the pretargeting stage, the nonlabeled nanoparticles are allowed to circulate in the blood. Afterward, 99mTc-1-hydroxyethylidene-1.1-diphosphonate (99mTc-HEDP) is administered intravenously for in situ labeling of the nanoparticles and subsequent single-photon emission computed tomography/computed tomography (SPECT/CT) visualization. The HAP NPs, stabilized with tailored hydrophilic polymers, are not cytotoxic in vitro, as shown by several cell lines. The polymer coating prolongs the circulation of HAP NPs in the blood. The nanoparticles were successfully labeled in vivo with 99mTc-HEDP, 1 and 24 h after injection, and they were visualized by SPECT/CT over time in healthy mice.

Synthesis and Cytotoxic and Antiviral Profiling of Pyrrolo- and Furo-Fused 7-Deazapurine Ribonucleosides.

Three series of isomeric pyrrolo- and furo-fused 7-deazapurine ribonucleosides were synthesized and screened for cytostatic and antiviral activity. The synthesis was based on heterocyclizations of hetaryl-azidopyrimidines to form the tricyclic heterocyclic bases, followed by glycosylation and final derivatizations through cross-coupling reactions or nucleophilic substitutions. The pyrrolo[2',3':4,5]pyrrolo[2,3- d]pyrimidine and furo[2',3':4,5]pyrrolo[2,3- d]pyrimidine ribonucleosides were found to be potent cytostatics, whereas the isomeric pyrrolo[3',2',4,5]pyrrolo[2,3- d]pyrimidine nucleosides were inactive. The most active were the methyl, methoxy, and methylsulfanyl derivatives exerting submicromolar cytostatic effects and good selectivity toward cancer cells. We have shown that the nucleosides are activated by intracellular phosphorylation and the nucleotides get incorporated to both RNA and DNA, where they cause DNA damage. They represent a new type of promising candidates for preclinical development toward antitumor agents.

Proteomic profiling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells.

Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, pancreatic, gastric, and hepatocellular cancers. Despite its routine clinical use, little is known about the responses it induces in cancer cells. Therefore the whole-cell proteomics study was conducted to characterize the cellular response induced by oxaliplatin. Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3µM (5×IC50) for 240min (half-time to caspase activation). The proteomes of un-/treated cells were then compared by high-resolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in at least two replicates. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. BIOLOGICAL SIGNIFICANCE: We have performed a whole-cell proteomic study of cellular response to oxaliplatin treatment, which is the drug predominantly used in the treatment of colorectal cancer. Compared to its predecessors, cisplatin and carboplatin, there is only a small fraction of studies dedicated to oxaliplatin. From those studies, most of them are focused on modification of treatment regimens or study of oxaliplatin in new cancer diagnoses. Cellular response hasn't been studied deeply and to our best knowledge, this is the first whole-cell proteomics study focused exclusively to this important topic, which can help to understand molecular mechanisms of action.

Cellular effects of the microtubule-targeting agent peloruside A in hypoxia-conditioned colorectal carcinoma cells.

BACKGROUND: Hypoxia is a prominent feature of solid tumors, dramatically remodeling microtubule structures and cellular pathways and contributing to paclitaxel resistance. Peloruside A (PLA), a microtubule-targeting agent, has shown promising anti-tumor effects in preclinical studies. Although it has a similar mode of action to paclitaxel, it binds to a distinct site on ß-tubulin that differs from the classical taxane site. In this study, we examined the unexplored effects of PLA in hypoxia-conditioned colorectal HCT116 cancer cells. METHODS: Cytotoxicity of PLA was determined by cell proliferation assay. The effects of a pre-exposure to hypoxia on PLA-induced cell cycle alterations and apoptosis were examined by flow cytometry, time-lapse imaging, and western blot analysis of selected markers. The hypoxia effect on stabilization of microtubules by PLA was monitored by an intracellular tubulin polymerization assay. RESULTS: Our findings show that the cytotoxicity of PLA is not altered in hypoxia-conditioned cells compared to paclitaxel and vincristine. Furthermore, hypoxia does not alter PLA-induced microtubule stabilization nor the multinucleation of cells. PLA causes cyclin B1 and G2/M accumulation followed by apoptosis. CONCLUSIONS: The cellular and molecular effects of PLA have been determined in normoxic conditions, but there are no reports of PLA effects in hypoxic cells. Our findings reveal that hypoxia preconditioning does not alter the sensitivity of HCT116 to PLA. GENERAL SIGNIFICANCE: These data report on the cellular and molecular effects of PLA in hypoxia-conditioned cells for the first time, and will encourage further exploration of PLA as a promising anti-tumor agent.

7-(2-Thienyl)-7-Deazaadenosine (AB61), a New Potent Nucleoside Cytostatic with a Complex Mode of Action.

7-(2-Thienyl)-7-deazaadenosine (AB61) showed nanomolar cytotoxic activities against various cancer cell lines but only mild (micromolar) activities against normal fibroblasts. The selectivity of AB61 was found to be due to inefficient phosphorylation of AB61 in normal fibroblasts. The phosphorylation of AB61 in the leukemic CCRF-CEM cell line proceeds well and it was shown that AB61 is incorporated into both DNA and RNA, preferentially as a ribonucleotide. It was further confirmed that a triphosphate of AB61 is a substrate for both RNA and DNA polymerases in enzymatic assays. Gene expression analysis suggests that AB61 affects DNA damage pathways and protein translation/folding machinery. Indeed, formation of large 53BP1 foci was observed in nuclei of AB61-treated U2OS-GFP-53BP1 cells indicating DNA damage. Random incorporation of AB61 into RNA blocked its translation in an in vitro assay and reduction of reporter protein expression was also observed in mice after 4-hour treatment with AB61. AB61 also significantly reduced tumor volume in mice bearing SK-OV-3, BT-549, and HT-29 xenografts. The results indicate that AB61 is a promising compound with unique mechanism of action and deserves further development as an anticancer agent. Mol Cancer Ther; 15(5); 922-37. ©2016 AACR.

Targeting the DNA double strand break repair machinery in prostate cancer.

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.

Antidepressant-like activity of zinc: further behavioral and molecular evidence.

Zinc exhibits antidepressant-like activity in preclinical tests (the forced swim test and tail suspension test) and in olfactory bulbectomy and chronic unpredictable stress; two models of depression. Zinc also enhances the treatment of depression in humans. In the present study we evaluated the antidepressant activity of zinc in another model of depression-chronic mild stress (CMS) and the effect of zinc treatment on BDNF protein and the mRNA level. In CMS zinc hydroaspartate (10 mg/kg) exhibited a rapid (after 1 week of treatment) antidepressant-like effect. Chronic treatment with zinc induced a 17-39% increase in the BDNF mRNA and protein level in the hippocampus. These data indicate a rapidly acting antidepressant-like activity of zinc in CMS and the involvement of zinc in the regulation of BDNF.