Ketai Wang

Inhibitory and Stimulatory Bystander Effects are Differentially Induced by Iodine-125 and Iodine-123

Abstract Kishikawa, H., Wang, K., Adelstein, S. J. and Kassis, A. I. Inhibitory and Stimulatory Bystander Effects are Differentially Induced by Iodine-125 and Iodine-123. Radiat. Res. 165, 688–694 (2006). The bystander effect, originating from cells irradiated in vitro, describes responses of surrounding cells not targeted by the radiation. Previously we demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated 125I inhibits growth of co-injected LS174T cells (inhibitory bystander effect; Proc. Natl. Acad. Sci. USA 99, 13765–13770, 2002). We have repeated these studies using cells exposed to lethal doses of 123I, an Auger electron emitter whose emission spectrum is identical to that of 125I, and report herein that the decay of 123I within tumor cell DNA stimulates the proliferation of neighboring unlabeled tumor cells growing subcutaneously in nude mice (stimulatory bystander effect). Similar inhibitory bystander effects (125I) and stimulatory bystander effects (123I) are obtained in vitro. Moreover, supernatants from cultures with 125I-labeled cells are positive for tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), and those from cultures with 123I-labeled cells are positive for angiogenin. These findings call for the re-evaluation of current dosimetric approaches for the estimation of dose–response relationships in individuals after radiopharmaceutical administration or radiocontamination and demonstrate a need to adjust all “calculated” dose estimates by a dose modification factor (DMF), a radionuclide-specific constant that factors in hitherto not-so-well recognized biophysical processes.

In silico design, synthesis, and biological evaluation of radioiodinated quinazolinone derivatives for alkaline phosphatase–mediated cancer diagnosis and therapy

As part of the development of enzyme-mediated cancer imaging and therapy, a novel technology to entrap water-insoluble radioactive molecules within solid tumors, we show that a water-soluble, radioactive quinazolinone prodrug, ammonium 2-(2′-phosphoryloxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ2-P), is hydrolyzed by alkaline phosphatase to a water-insoluble, radiolabeled drug, 2-(2′-hydroxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone (125IQ2-OH). Biodistribution data suggest the existence of two isoforms of the prodrug (IQ2-P(I) and IQ2-P), and this has been confirmed by their synthesis and characterization. Structural differences of the two isoforms have been examined using in silico molecular modeling techniques and docking methods to describe the interaction/binding between the isoforms and human placental alkaline phosphatase (PLAP), a tumor cell, membrane-associated, hydrolytic enzyme whose structure is known by X-ray crystallographic determination. Docking data show that IQ2-P, but not IQ2-P(I), fits the active binding site of PLAP favorably and interacts with the catalytic amino acid Ser92, which plays an important role in the hydrolytic process. The binding free energies (ΔGbinding) of the isoforms to PLAP predict that IQ2-P will be the better substrate for PLAP. The in vitro incubation of the isoforms with PLAP leads to the rapid hydrolysis of IQ2-P only and confirms the in silico expectations. Fluorescence microscopy shows that in vitro incubation of IQ2-P with mouse and human tumor cells causes the extracellular, alkaline phosphatase–mediated hydrolysis of the molecule and precipitation of fluorescent crystals of IQ2-OH. No hydrolysis is seen in the presence of normal mouse and human cells. Furthermore, the intratumoral injection of 125IQ2-P into alkaline phosphatase–expressing solid human tumors grown s.c. in nude rats results in efficient hydrolysis of the compound and retention of ∼70% of the injected radioactivity, whereas similar injection into normal tissues (e.g., muscle) does not produce any measurable hydrolysis (∼1%) or retention of radioactivity at the injected site. These studies support the enzyme-mediated cancer imaging and therapy technology and show the potential of such quinazolinone derivatives in the in vivo radiodetection (123I/124I) and therapy (131I) of solid tumors. [Mol Cancer Ther 2006;5(12):3001–13]

Effect of distance between decaying 125I and DNA on Auger-electron induced double-strand break yield

Abstract Purpose: To determine the possible effects of 125I-to-DNA distance on the magnitude and mechanism of Auger-electron induced-double-strand break (DSB) production. Materials and methods: We have synthesized a series of 125I-labeled Hoechst (H) derivatives (125IE–H, 125IB–H, 125I-C8–H and 125I-C12–H). While all four molecules share a common DNA minor groove binding bis-benzimidazole motif, they are designed to position 125I at varying distances from the DNA helix. Each Hoechst derivative was incubated at 4°C in phosphate buffered saline (PBS) together with supercoiled (SC) 3H-pUC19 plasmid DNA (ratio 3:1) ± the •OH scavenger dimethyl sulfoxide (DMSO) (0.2 M). Aliquots were analyzed on agarose gels over time and DSB yields per decay of 125I atom were determined. Docking of the iodinated compounds on a DNA molecule was carried out to determine the distance between the iodine atom and the central axis of DNA. Results: In the absence of DMSO, the results show that the DSB yields decrease monotonically as the 125I atom is distanced – by 10.5 Å to 13.9 Å – from the DNA helix (125IEH: 0.52 ± 0.01; 125IB–H: 0.24 ± 0.03; 125I-C8–H: 0.18 ± 0.02; 125I-C12–H: 0.10 ± 0.00). In the presence of DMSO, DSB yields for 125IEH (0.49 ± 0.02) and 125IB-H (0.26 ± 0.04) remain largely unchanged indicating that DSB are entirely produced by direct effects. Strikingly, 125I-C8–H or 125I-C12–H, did not produce detectable DSB in the presence of DMSO under similar conditions suggesting when 125I atom is positioned > 12 Å from the DNA, DSB are entirely produced by indirect effects. Conclusion: These results suggest that at a critical distance between the 125I atom and the DNA helix, DSB production switches from an ‘all’ direct to an ‘all’ indirect mechanism, the latter situation being comparable to the decay of 125I free in solution. These experimental findings were correlated with theoretical expectations based on microdosimetry.

Therapeutic potential of 5-[125I]iodo-2'-deoxyuridine and methotrexate in the treatment of advanced neoplastic meningitis.

Purpose: To assess the therapeutic potential of methotrexate (MTX) and 5‐[125I]iodo‐2′‐deoxyuridine (125IdUrd) administered sequentially in rats bearing advanced (ten‐day‐old) intrathecal (i.t.) TE671 human rhabdomyosarcoma tumours. Materials and methods: Nude rats were injected with TE671 cells through an i.t. placed catheter. Ten days later, the animals were injected i.t. over a 12‐day period with (i) saline daily, (ii) MTX every other day, (iii) 125IdUrd every other day, or (iv) MTX and 125IdUrd on alternating days. Onset of paralysis was determined as a function of time, and the medians for onset (M), percentage of cells killed (% kill), and log cell kill were calculated. Results: The data show that (i) injection of MTX leads to a moderate delay in the onset of paralysis (MMTX=29 d versus Msaline=20 d), (ii) administration of 125IdUrd is more effective (MIdUrd=36 d), and (iii) sequential administration of MTX–125IdUrd further increases the therapeutic efficacy of 125IdUrd (MMTX–IdUrd=47 d). Conclusions: Intrathecal injection of MTX–125IdUrd is efficacious in the therapy of advanced intrathecal tumours.

First human treatment of resistant neoplastic meningitis by intrathecal administration of MTX Plus 125 IUdR

Purpose: Neoplastic meningitis is often the final outcome of disseminated cancer and is rapidly lethal. Its limited treatment relies on systemic or intrathecal chemotherapy with methotrexate (MTX) or thiotepa. When 5-iodo-2′-deoxyuridine labeled with 125I (125IUdR) is incorporated into the DNA of mitotic tumor cells, the Auger electrons emitted during iodine decay are highly cytotoxic. The radiotherapeutic efficacy of 125IUdR administered intrathecally has also been established in animals bearing spinal cord tumors, and MTX is known to potentiate the response. This approach has not been tested in the clinic. Methods: A 44-year-old woman, with locally advanced pancreatic cancer, was treated for three years with complete systemic remission, but then relapsed with cytologically proven neoplastic meningitis. The patient was given four successive intrathecal injections of MTX (10 mg) every 12 h and, with the fourth dose, 1850 MBq 125IUdR, followed by four additional MTX doses. The response was monitored by cytology and CA19.9 (carbohydrate antigen 19.9) levels in the cerebrospinal fluid (CSF) as well as by clinical status of the patient. Results: The follow-up of cytology and CA19.9 levels in the CSF showed dramatic improvement within 26 days followed by a biological relapse on Day +36. There was no evidence of local central nervous system toxicity. Three months later, neoplastic meningitis recurred and meningeal tumor infiltration was observed on magnetic resonance imaging. Six months after MTX–125IUdR treatment, the patient died. Conclusion: 125IUdR treatment proved to be feasible without acute neurological toxicity and seemed to have produced a biological response. This attempt provides the basis for designing prospective clinical trials.

Using Hoechst 33342 to Target Radioactivity to the Cell Nucleus

Abstract Yasui, L. S., Chen, K., Wang, K., Jones, T. P., Caldwell, J., Guse, D. and Kassis, A. I. Using Hoechst 33342 to Target Radioactivity to the Cell Nucleus. Radiat. Res. 167, 167–175 (2007). We have explored the use of Hoechst 33342 (H33342) to carry radioactivity to the cell nucleus. H33342 enters cells and targets DNA at adenine-thymine-rich regions of the minor groove. Considerable membrane blebbing and ruffling occur in CHO cells within minutes after its addition to the culture medium in micromolar quantities. Blue vesicles are apparent in the cell cytoplasm, and by 30 min the nuclei are stained dark blue. Upon its binding to DNA, a visible emission shift of the dye can be observed with fluorescence microscopy. We have radioiodinated (125I) H33342 and specifically irradiated nuclear DNA by incubating CHO cells with 125I-H33342 at 37°C and accumulating 125I decays at −90°C. At various times, the cells are thawed and assayed for survival (clonogenicity) and DSB (γ-H2AX) formation. 125I-H33342 decay leads to a monoexponential decrease in cell survival with a D0 of 122 125I decays per cell and a linear increase in DNA DSB induction (equivalent to 15 γ-H2AX foci/cell). Cell death is not modified by the radioprotective effects of H33342 because we use considerably lower concentrations than those that provide a slight protection against γ radiation. We conclude that cell killing by 125I-H33342 and the induction of γ-H2AX foci are highly correlated.

DNA double-strand breaks induced by decay of (123)I-labeled Hoechst 33342: role of DNA topology.

Purpose: To determine double-strand-break (DSB) yields produced by decay of minor-groove-bound 123I-labeled Hoechst 33342 (123IEH) in supercoiled (SC) and linear (L) forms of pUC19 DNA, to compare strand-break efficiency of 123IEH with that of 125IEH, and to examine the role of DNA topology in DSB induction by these Auger electron emitters. Materials and methods: Tritium-labeled SC and L pUC19 DNA were incubated with 123IEH (0–10.9 MBq) at 4°C. After 123I had completely decayed (10 days), samples were analyzed on agarose gel, and single-strand-break (SSB) and DSB yields were measured. Results: Each 123I decay in SC DNA produces a DSB yield of 0.18 ± 0.01. On the basis of DSB yields for 125IEH (0.52 ± 0.02 for SC and 1.62 ± 0.07 for L, reported previously) and dosimetric expectations, a DSB yield of ∼0.5 (3 × 0.18) per 123I decay is expected for L DNA. However, no DSB are observed for the L form, even after ∼2 × 1011 decays of 123I per μg DNA, whereas a similar number of 125I decays produces DSB in ∼40% of L DNA. Conclusion: 123IEH-induced DSB yield for SC but not L DNA is consistent with the dosimetric expectations for Auger electron emitters. These studies highlight the role of DNA topology in DSB production by Auger emitters and underscore the failure of current theoretical dosimetric methods per se to predict the magnitude of DSB.

Novel Prodrugs for Targeting Diagnostic and Therapeutic Radionuclides to Solid Tumors

Most cancer therapeutics (chemo, radiation, antibody-based, anti-angiogenic) are at best partially and/or temporarily effective. In general, the causes for failure can be summarized as: (i) poor diffusion and/or nonuniform distribution of drug/prodrug molecules in solid tumors; (ii) high drug concentration and retention in normal tissues (leading to side effects); (iii) requirement for plasma-membrane permeability and/or internalization of drug/prodrug molecules; (iv) low uptake of drug by tumor; (v) lack of retention of drug within tumor (most have gradient-driven reversible binding); and (vi) multidrug resistance. We are developing an innovative technology that aims to surmount these problems by actively concentrating and permanently entrapping radioimaging and radiotherapeutic prodrugs specifically within solid tumors. The approach will enable noninvasive sensing (imaging) and effective therapy of solid tumors, allowing tumor detection, diagnosis, and treatment to be closely coupled (personalized medicine).

Bystander effect in tumor cells produced by Iodine-125 labeled human lymphocytes.

Abstract Purpose: To investigate the ability of human lymphocytes labeled with DNA-incorporated 125I to exert an inhibitory (antiproliferative) bystander effect on co-cultured human colon adenocarcinoma LS174T cells in vitro. Materials and methods: Human peripheral blood lymphocytes were stimulated to synthesize DNA in the presence of phytohemagglutinin (PHA) and labeled with 5-[125I]iodo-2′-deoxyuridine. Human colon adenocarcinoma LS174T cells were co-cultured with the 125I-labeled lymphocytes in various ratios for 5 days and the proliferation of the LS174T cells was assessed. Further, the supernatant media from these co-cultures were: (i) Transferred to LS174T cells and their proliferation measured after 5 days, (ii) used to assess the clonogenic survival of LS174T cells, and (iii) screened for factors that suppress growth. Results: A significant reduction in the proliferation of LS174T cells was observed when co-cultured either with 125I-labeled lymphocytes (56 ± 3.5%) or the supernatant media (52.5 ± 1.3%) obtained from these co-cultures. Clonogenic survival of LS174T cells grown in the supernatant media corroborated the decrease in tumor cell growth. Conclusion: The observed reduction in the proliferation of LS174T cells in presence of 125I-labeled lymphocytes or media obtained from such co-cultures can be attributed to an inhibitory (antiproliferative) bystander effect, probably mediated by factor(s) released from the dying 125I-labeled lymphocytes.

Activation of diverse pathways to apoptosis by 125IdUrd and γ-photon exposure

Purpose: To delineate the mechanisms underlying induction of apoptosis in malignant cells irradiated by DNA‐incorporated iodine‐125 or γ‐photons. Materials and methods: Human tumor cells (RKO, LS174T, TE671, and MCF7) were irradiated by DNA‐incorporated 5‐[125I]iodo‐2′‐deoxyuridine (125IdUrd) or by γ‐photons. Clonogenic survival was determined by the colony‐forming assay. Caspase‐3 induction was measured with a fluorogenic substrate assay, and DNA fragmentation was determined by ligation‐mediated polymerase chain reaction. DNA arrays were used to assess the expression of the B‐cell lymphoma/leukaemia‐2 (Bcl‐2) family and related genes in RKO cells and in caspase‐3‐gene‐defective MCF7 cells. Results: After 125IdUrd or γ‐photon exposure, the highest induction of caspase‐3 was observed in the radiation‐sensitive cell lines (RKO and LS174T). DNA fragmentation was prominent in the radiosensitive cells and undetectable in TE671 (125IdUrd and γ‐photons) and MCF7 (125IdUrd only) cells. Exposure of RKO and MCF7 cells to 125I decay led to up‐regulation of several pro‐apoptotic and anti‐apoptotic Bcl‐2 family genes whereas γ‐irradiation produced minimal activation. Conclusions: Apoptosis generated by a DNA‐incorporated Auger electron emitter is induced through the mitochondrial/caspase‐3‐mediated pathway and correlates with cellular radiosensitivity. Apoptosis caused by γ‐radiation can be signaled without activation of Bcl‐2 family genes, and DNA fragmentation occurs with or without caspase‐3 activation.