Preclinical study on hypoxic radiosensitizing effects of glycididazole in comparison with those of doranidazole in vitro and in vivo.

To overcome the radioresistance of hypoxic cells in solid tumor, numerous types of radiosensitizers specifically against them have been developed. Glycididazole has a chemical structure in which two metronidazole forms are combined, and is widely used as a hypoxic radiosensitizer in China. However, a detailed investigation of its radiosensitizing properties has not been performed. The present study reported a comparative assessment of glycididazole and doranidazole, another hypoxic radiosensitizer. All experiments were performed using the murine squamous cell carcinoma cell line SCCVII. Prior to X-irradiation, the cells were treated with the test drugs at concentrations of 10 mM and 200 mg/kg in vitro and in vivo, respectively. Uptake and their intratumor chemical forms were analyzed by high performance liquid chromatography (HPLC). Both drugs enhanced the reproductive cell death induced by X-irradiation under hypoxia. However, the growth delay assay of the transplanted tumor revealed the combination of X-irradiation and glycididazole showed a similar antitumor effect to that of X-irradiation alone, whereas doranidazole significantly sensitized the cells to X-irradiation. HPLC analysis revealed that incorporated glycididazole was decomposed to metronidazole and was therefore present at a lower concentration compared with that of doranidazole. The decomposition of glycididazole to metronidazole reduced its radiosensitizing efficiency in vivo. Elucidation of the kinetics of drugs containing metabolizable chemical forms is necessary for the optimization of clinical treatment.

Potentiality of syringetin for preferential radiosensitization to cancer cells.

PURPOSE: To examine the enhancing effects of syringetin on the radiosensitivity of normal and cancer cells, and the related mechanism. MATERIALS AND METHODS: We used normal human lung and mouse fibroblasts as well as human lung and mouse cancer cells derived from the above normal fibroblasts. Cell radiosensitivity was measured using a colony formation assay. Apoptosis was analyzed with DAPI staining and Western blots. DNA lesions were analyzed with γH2AX immunofluorescent staining. RESULTS: The colony formation assay showed that syringetin enhanced radiosensitivity more effectively in cancer cells (H1299 and C3H/MCA clone 15) compared with normal cells (HFL-III and C3H/10T1/2). The radiosensitizing effect of syringetin was observed in mutated p53 and wild-type p53-transfected H1299 cells regardless of p53 status. Apoptosis was more frequently observed in X-ray-irradiated H1299 cells combined with syringetin compared with X-ray-only-treated cells. Enhanced apoptosis by syringetin was not observed in HFL-III cells. Western blot analysis showed that X-ray-induced Caspase-3 activation was enhanced by syringetin in H1299 cells. The number of X-ray-induced DNA double-strand breaks (DSB) measured by quantitative analysis of γH2AX foci was the same for H1299 cells treated with X-rays with or without syringetin. CONCLUSIONS: This study supports the hypothesis that syringetin enhances radiosensitivity more effectively in cancer cells than in normal cells through enhancement of the Caspase-3-mediated apoptosis pathway. Syringetin could be useful in the development of novel efficacious radiosensitizers.

Comparison of Characteristics of Two Topical Therapeutic Agents for Onychomycosis.

Two topical therapeutic agents were approved in Japan from 2015 to 2016, adding new options for onychomycosis therapy in the clinical field. In order to confirm the differences of formulation properties and nail pharmacokinetics between 5% luliconazole solution and 10% efinaconazole solution, drug concentration and antifungal activity in the nail were measured after topical treatment using human nail plates. In the in vitro permeation study, concentration of each drug was measured in the transversely sliced nail after single treatment with the two topical therapeutic agents. The results showed that concentration of luliconazole is higher than that of efinaconazole at all nail layers, differing by 1.7-8.4 times at each measurement point. Next, we examined antifungal activities of each drug in sliced nail after 14-day topical treatment. Mean rates of formation of inhibition zones for 5% luliconazole solution and 10% efinaconazole solution were 71.0% and 12.6%, respectively, and were statistically different. These results show that the two topical therapeutic agents have different properties, and suggest that 5% luliconazole solution has good nail permeation and retention characteristics. Moreover, luliconazole was found to retain enough antifungal activity in the nail plate against Trichophyton spp. after treatment with the topical agent.

[Distribution of Luliconazole in Nail Plate by In Vitro Permeation and Efficacy by Zone of Inhibition Test after Treatment of Luliconazole Nail Solution].

To clarify the character of luliconazole nail solution we have developed, we investigated luliconazole distribution and antifungal activity in nail plate. An in vitro permeation study which measured luliconazole concentration of sliced nail in the transverse direction after treatment of luliconazole nail solution was conducted to investigate for concentration dependency and the influences of nail thickness and treatment duration. When 0.2, 1, 3, 5, and 7.5% luliconazole nail solutions were used, luliconazole was detected in the all the layers of nail and there was a concentration gradient from the dorsal side to deep nail layers. The luliconazole concentration was almost same after 14-day treatment with 5% luliconazole nail solution when using nails of different thicknesses. And we confirmed that concentration of luliconazole into the nail was increased depending on the treatment duration. In zone of inhibition test after 14-day treatment, 5% luliconazole nail solution showed statistically high formation rate of zones of inhibition compared to 8% ciclopirox nail lacquer. Above all, these data suggested that 5% luliconazole nail solution has the potential to show high therapeutic effect for onychomycosis.

[Antifungal Activity of Luliconazole Nail Solution on in vitro and in vivo Onychomycosis Model].

We evaluated luliconazole nail solution, originally generated formulation, for the topical treatment of onychomycosis by two infection models. First, a suspension of Trichophyton mentagrophytes was dropped onto the ventral layer of human nail plate and these nails were set in Franz diffusion cells. After 9-day culture, luliconazole nail solutions (1, 3, and 5%) were applied to the dorsal surface of the nails once a day for 7 days. After application, fungal viability was assessed by measuring the ATP contents of the samples. The dose-dependent efficacy was confirmed, with 3% and 5% luliconazole nail solutions producing significantly lower ATP levels at 7-day treatment. When 3% and 5% luliconazole nail solutions were evaluated in a rabbit model of onychomycosis, both concentrations completely inhibited the recovery of fungi on culture after 4-week treatment. We therefore think these results indicate that 5% luliconazole nail solution is sufficiently potent for treatment of onychomycosis.

Plastic induction of CD133AC133-positive cells in the microenvironment of glioblastoma spheroids.

Recent studies showed that the stemness of cancer stem cells is maintained under a hypoxic microenvironment. However, the relationship of the hypoxic microenvironment in a three-dimensional cell mass and the induction of cancer stem cell-like phenotype is not well known. We examined the relationship between CD133 expression and the hypoxic microenvironment using glioblastoma spheroids formed with the T98G cell line. CD133(AC133)- and HIF-1α-positive cells were observed in the marginal region of the central hypoxic area positive for HIF-1α 10 days after plating T98G cells. CD133(AC133)-positive cells were positive for nestin. Quantitative PCR analysis showed that the CD133 expression level is not different in spheroids during the tested period after spheroid formation, indicating that post-translational regulation of the CD133 protein mediates positivity to CD133(AC133). When spheroids were trypsinized and the dissociated cells were cultured under the adherent monolayer conditions, the CD133(AC133)-positive cells gradually disappeared. These results show that CD133(AC133)-positive cells, which may incline toward undifferentiated cells because of nestin positivity, are plastically induced under the different culture conditions, spheroid and monolayer. In this plasticity, HIF-1α is involved in the induction and maintenance of CD133(AC133)-positive cells. Spheroids as an in vitro tumor model are useful to study the dynamic changes in the tumor cell phenotype in the different cell microenvironments.

Radiosensitization of human lung cancer cells by the novel purine-scaffold Hsp90 inhibitor, PU-H71.

The molecular chaperone heat shock protein 90 (Hsp90) is involved in the maturation and stabilization of a wide range of oncogenic client proteins for oncogenesis and malignant cell proliferation, which renders this protein a promising target in the development of cancer therapeutics. PU-H71 is a purine-scaffold Hsp90 inhibitor with less toxicity in normal cells than in cancer cells. In this study, we examined the in vitro radiosensitizing activity and molecular mechanisms of action of PU-H71 in human lung cancer cell lines. PU-H71 enhanced the sensitivity of the SQ-5 and A549 cancer cells to radiation. When the cancer cells were pre-treated with PU-H71, the repair of DNA double-strand breaks (DSBs) was markedly inhibited after irradiation compared with the cells that were not pre-treated with PU-H71, as evaluated by counting the foci of phosphorylated histone H2AX (γ-H2AX). We further demonstrated that post-irradiation, PU-H71 inhibited Rad51 foci formation, a critical protein for the homologous recombination pathway of DNA DSB repair. These data indicate that targeting Hsp90 with PU-H71 may be novel therapeutic strategy for radioresistant carcinomas.

Functional expression of choline transporter-like protein 1 (CTL1) in small cell lung carcinoma cells: a target molecule for lung cancer therapy.

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine and the neurotransmitter acetylcholine (ACh). Elevated levels of choline and up-regulated choline kinase activity have been detected in cancer cells. Thus, the intracellular accumulation of choline through choline transporters is the rate-limiting step in phospholipid metabolism and a prerequisite for cancer cell proliferation. However, the uptake system for choline and the functional expression of choline transporters in lung cancer cells are poorly understood. We examined the molecular and functional characterization of choline uptake in the small cell lung carcinoma cell line NCI-H69. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in choline uptake under the Na(+)-free conditions was inhibited by dimethylamiloride (DMA), a Na(+)/H(+) exchanger (NHE) inhibitor. Various organic cations and the choline analog hemicholinium-3 (HC-3) inhibited the choline uptake and cell viability. A correlation analysis of the potencies of organic cations for the inhibition of choline uptake and cell viability showed a strong correlation (R=0.8077). RT-PCR revealed that choline transporter-like protein 1 (CTL1) mRNA and NHE1 are mainly expressed. HC-3 and CTL1 siRNA inhibited choline uptake and cell viability, and increased caspase-3/7 activity. The conversion of choline to ACh was confirmed, and this conversion was enhanced under Na(+)-free conditions, which in turn was sensitive to HC-3. These results indicate that choline uptake through CTL1 is used for ACh synthesis. Both an acetylcholinesterase inhibitor (eserine) and a butyrylcholinesterase inhibitor (ethopropazine) increased cell proliferation, and these effects were inhibited by 4-DAMP, a mAChR3 antagonist. We conclude that NCI-H69 cells express the choline transporter CTL1 which uses a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. This system primarily supplies choline for the synthesis of ACh and secretes ACh to act as an autocrine/paracrine growth factor, and the functional inhibition of CTL1 could promote apoptotic cell death. Identification of this new CTL1-mediated choline transport system provides a potential new target for therapeutic intervention.

Comparative study of radioresistance between feline cells and human cells.

Radioresistance of cats has been seen in animal radiotherapy. Feline radioresistance and its underlying mechanism(s) were investigated in fibroblast cells and lymphocytes. We hypothesized that radioresistance was attributable to an increase in the cells ability to repair DNA damage. To investigate this hypothesis, fibroblast cells were exposed to various doses of X rays and then colony formation assays were performed. Survival curves showed that potential lethal damage repair (PLDR) for feline cells were greater than that for human cells. γ-H2AX foci assays were performed to evaluate DNA double-strand breaks (DSBs) formation and repair kinetics. After PLDR, feline cells displayed a decreased residual amount of γ-H2AX foci. Formation of chromosome aberrations (dicentrics) after PLDR as an indicator of radiation-induced DNA damage and repair; human, feline and canine lymphocytes were evaluated. Human and canine lymphocytes showed two to three times the number of dicentrics compared to feline lymphocytes. Finally, micronuclei assays were performed to further confirm the radioresistant nature of feline lymphocytes. In concordance with the results of the chromosome aberration assay, the number of micronuclei in feline lymphocytes was less than observed in human and canine lymphocytes. Taken together, these results show that DNA and chromosome damage induced by X irradiation is more effectively repaired in feline cells, resulting in less residual damage. Our results suggest that both feline fibroblasts and lymphocytes are more radioresistant compared to human cells of similar tissues, and this resistance can be contributed, at least in part, to greater ability for PLDR.

Comparison of the bromodeoxyuridine-mediated sensitization effects between low-LET and high-LET ionizing radiation on DNA double-strand breaks.

The incorporation of halogenated pyrmidines such as bromo- and iodo-deoxyuridines (BrdU, IdU) into DNA as thymidine analogs enhances cellular radiosensitivity when high-linear energy transfer (LET) radiation is not used. Although it is known that high-LET ionizing radiation confers fewer biological effects resulting from halogenated pyrimidine incorporation, the exact mechanisms of reduced radiosensitivity with high-LET radiation are not clear. We investigated the radiosensitization effects of halogenated pyrimidines with high-LET radiation using accelerated carbon and iron ions. Cells synchronized into the G1 phase after unifilar (1 cell cycle) and bifilar (2 cell cycles) substitution with 10 µM BrdU were exposed to various degrees of LET with heavy ions and X-rays. We then carried out a colony formation assay to measure cell survival. The γ-H2AX focus formation assay provided a measure of DNA double-strand break (DSB) formation and repair kinetics. Chromosomal aberration formations for the first post-irradiation metaphase were also scored. For both low-LET X-rays and carbon ions (13 keV/µm), BrdU incorporation led to impaired DNA repair kinetics, a larger initial number of DNA DSBs more frequent chromosomal aberrations at the first post-irradiated metaphase, and increased radiosensitivity for cell lethality. The enhancement ratio was higher after bifilar substitution. In contrast, no such synergistic enhancements were observed after high-LET irradiation with carbon and iron ions (70 and 200 keV/µm, respectively), even after bifilar substitution. Our results suggest that BrdU substitution did not modify the number and quality of DNA DSBs produced by high-LET radiation. The incorporation of halogenated pyrimidines may produce more complex/clustered DNA damage along with radicals formed by low-LET ionizing radiation. In contrast, the severity of damage produced by high-LET radiation may undermine the effects of BrdU and account for the observed minimal radiosensitization effects.

The prospective application of a hypoxic radiosensitizer, doranidazole to rat intracranial glioblastoma with blood brain barrier disruption.

BACKGROUND: Glioblastoma is one of the intractable cancers and is highly resistant to ionizing radiation. This radioresistance is partly due to the presence of a hypoxic region which is widely found in advanced malignant gliomas. In the present study, we evaluated the effectiveness of the hypoxic cell sensitizer doranidazole (PR-350) using the C6 rat glioblastoma model, focusing on the status of blood brain barrier (BBB). METHODS: Reproductive cell death in the rat C6 glioma cell line was determined by means of clonogenic assay. An intracranial C6 glioma model was established for the in vivo experiments. To investigate the status of the BBB in C6 glioma bearing brain, we performed the Evans blue extravasation test. Autoradiography with [(14)C]-doranidazole was performed to examine the distribution of doranidazole in the glioma tumor. T2-weighted MRI was employed to examine the effects of X-irradiation and/or doranidazole on tumor growth. RESULTS: Doranidazole significantly enhanced radiation-induced reproductive cell death in vitro under hypoxia, but not under normoxia. The BBB in C6-bearing brain was completely disrupted and [(14)C]-doranidazole specifically penetrated the tumor regions. Combined treatment with X-irradiation and doranidazole significantly inhibited the growth of C6 gliomas. CONCLUSIONS: Our results revealed that BBB disruption in glioma enables BBB-impermeable radiosensitizers to penetrate and distribute in the target region. This study is the first to propose that in malignant glioma the administration of hydrophilic hypoxic radiosensitizers could be a potent strategy for improving the clinical outcome of radiotherapy without side effects.

Animal model of dermatophytosis.

Dermatophytosis is superficial fungal infection caused by dermatophytes that invade the keratinized tissue of humans and animals. Lesions from dermatophytosis exhibit an inflammatory reaction induced to eliminate the invading fungi by using the host's normal immune function. Many scientists have attempted to establish an experimental animal model to elucidate the pathogenesis of human dermatophytosis and evaluate drug efficacy. However, current animal models have several issues. In the present paper, we surveyed reports about the methodology of the dermatophytosis animal model for tinea corporis, tinea pedis, and tinea unguium and discussed future prospects.

Benzyl isothiocyanate sensitizes human pancreatic cancer cells to radiation by inducing apoptosis.

Isothiocyanates are a class of naturally occurring chemopreventive agents known to suppress proliferation of cancer cells in culture. The present study was undertaken in order to examine the effects of benzyl isothiocyanate (BITC), one of the common dietary isothiocyanates, on the radiosensitivity of human pancreatic cancer cells and to gain insights into the underlying molecular mechanism of BITC-induced radiosensitization. Two human pancreatic cancer cell lines, PANC-1 and MIAPaCa-2, were treated with BITC and irradiated with X-rays. Radiation sensitivity, apoptosis, and protein levels were determined by a clonogenic assay, fluorescence microscopic analysis with DAPI staining and Western blotting, respectively. MIAPaCa-2 cells were relatively more sensitive to BITC treatment compared with PANC-1 cells. Radiosensitization was observed in both PANC-1 and MIAPaCa-2 cells incubated with BITC at 5 to 10 µM and 2.5 to 5 µM for 24 h, respectively. The combination treatments with BITC and X-rays also revealed an increased percentage of apoptotic cells. In addition, treatment with BITC and X-rays resulted in a decrease in the protein levels of the X-linked inhibitor of apoptosis (XIAP), inhibitor of apoptosis (IAP) family protein, and in a marked increase in the apoptosis protease activating factor-1 (Apaf-1), essential for activation of caspase-9 in stress-induced apoptosis. BITC may be a useful radiosensitizer for radiotherapy of pancreatic cancers.

Establishment of a novel model of onychomycosis in rabbits for evaluation of antifungal agents.

We developed a novel model of onychomycosis in which we observed fungi in the deep layer of the nail, and we used the model to evaluate the efficacy of two topical antifungal drugs. To establish an experimental, in vivo model of onychomycosis, we applied Trichophyton mentagrophytes TIMM2789 to the nails of the hind limbs of rabbits that underwent steroid treatment. The nails were taken from the rabbits' feet at 0, 2, and 6 weeks after a 2-week infection. The localization of the fungi was evaluated histopathologically. Some fungi were seen to penetrate to the nail bed, and the infection rate in the sample at 0, 2, and 6 weeks after infection was 57, 87, and 93%, respectively. In addition, fungi proliferated and moved proximally into the nail plate in a manner that depended on the duration of infection. Second, using this model we evaluated antifungal efficacy both by the culture recovery method and histopathological examination. Two topical antifungal drugs, 8% ciclopirox nail lacquer and 5% amorolfine nail lacquer, were applied to the nail for 4 weeks in each group. On histopathological examination, two antifungal treatment groups showed no significant difference against the nontreated control group. However, there were a significantly low fungus-positive rate and intensity of the recovery of fungi on culture between antifungal treatment and nontreated control groups. We therefore suggest that we have established an in vivo model of onychomycosis that is useful for the evaluation of the efficacy of antifungal agents.

Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells).

INTRODUCTION: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of (14)C(U)-L-tyrosine ((14)C-Tyr), (125)I-4-iodo-L-meta-tyrosine (4-(125)I-mTyr), (125)I-6-iodo-L-meta-tyrosine (6-(125)I-mTyr), (125)I-3-iodo-α-methyl-L-tyrosine ((125)I-IMT) and (125)I-3-iodo-L-tyrosine (3-(125)I-Tyr) using Chinese hamster ovary cells (CHO-K1). METHODS: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na(+) at 37°C or 4°C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na(+)-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na(+)-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN(3) and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and L- and D-isomers of natural amino acids. RESULTS: (14)C-Tyr exhibited affinity for systems L, A and ASC. 4-(125)I-mTyr and 3-(125)I-Tyr exhibited high specificity for system L, whereas 6-(125)I-mTyr and (125)I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-(125)I-mTyr was markedly reduced by incubation at 4 °C, and was not significantly inhibited by NaN(3), DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids indicated that system L mediates the transport of 4-(125)I-mTyr. CONCLUSIONS: 4-(125)I-mTyr exhibited the greatest system L specificity (93.46 ± 0.13%) of all of the tested amino acids.

Ascorbic acid gives different protective effects in human cells exposed to X-rays and heavy ions.

We studied the effects and mechanisms of ascorbic acid as a radiation protector. Cell survival, repair of DNA double strand breaks (DSBs), and sister chromatid exchanges (SCEs) were examined in normal human fibroblasts irradiated with X-rays and heavy ions. Post-irradiation treatment with 5mM ascorbic acid for 24 h in plateau phase (non-cycling) cells enhanced cell survival and DNA double strand break repair, and reduced SCEs after X-rays irradiation. On the other hand, only reduced SCEs were observed after heavy ion exposure such as to carbon ions. Judging from our data, it is possible that the radioprotective action of ascorbic acid would be effective in non-complex type DNA damage such as induced by X-rays. These findings provide new insight into the mechanism of DNA damage and repair produced by heavy ion irradiation.

Stimulation of 125I-3-iodo-alpha-methyl-L-tyrosine uptake in Chinese hamster ovary (CHO-K1) cells by tyrosine esters.

INTRODUCTION: Transport of the amino acid analog (123)I-3-iodo-alpha-methyl-L-tyrosine, which is used in clinical SPECT imaging, occurs mainly via L-type amino acid transporter type 1 (LAT1; an amino acid exchanger). As LAT1 is highly expressed in actively proliferating tumors, we made a preliminary investigation of the effects of amino acid esters on enhancement of (125)I-3-iodo-alpha-methyl-L-tyrosine (IMT) uptake via LAT1 in Chinese hamster ovary (CHO-K1) cells. METHODS: Because the sequence of the CHO-K1 LAT1 gene is not available, we confirmed LAT1 expression through IMT (18.5 kBq) uptake mechanisms using specific inhibitors. L-Gly, L-Ser, L-Leu, L-Phe, L-Met, L-Tyr, D-Tyr, L-Val and L-Lys ethyl/methyl esters were tested in combination with IMT. Time-course studies over a 3-h period were conducted, and the concentration dependence of L-Tyr ethyl and methyl esters (0.001 to 10 mM) in combination with IMT was also examined. For a proof of de-esterification of L- and D-Tyr ethyl and methyl esters in the cells (by enzymatic attack or other cause), the concentration of L- and D-Tyr was analyzed by high-performance liquid chromatography of the esters in phosphate buffer (pH 7.4) and cell homogenates at 37 degrees C or under ice-cold conditions. RESULTS: Inhibition tests suggested that LAT1 is involved in IMT uptake by CHO-K1 cells. Co-administration of 1 mM of l-Tyr ethyl or methyl ester with IMT produced the greatest enhancement. The de-esterification reaction was stereo selective and temperature dependent in the homogenate. De-esterification kinetics were very fast in the homogenate and very slow in the phosphate buffer. CONCLUSIONS: The L-Tyr ethyl or methyl esters were the most effective enhancers of IMT uptake into CHO-K1 cells and acted by trans-stimulation of the amino acid exchange function of LAT1. This result suggests that de-esterification in the cells may be caused by enzymatic attack. We will use IMT and L-Tyr ethyl or methyl esters to examine LAT1 function in tumor cells or tissues in vivo.

Uptake of 3-[125I]iodo-alpha-methyl-L-tyrosine into colon cancer DLD-1 cells: characterization and inhibitory effect of natural amino acids and amino acid-like drugs.

INTRODUCTION: We examined 3-[(123)I]iodo-alpha-methyl-L-tyrosine ([(123)I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure. METHODS: Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [(125)I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [(125)I]IMT uptake were examined. RESULTS: Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B(0)AT was not detected. [(125)I]IMT uptake in DLD-1 cells involved Na(+)-independent system L primarily and Na(+)-dependent system(s). Uptake of [(125)I]IMT in Na(+)-free buffer followed Michaelis-Menten kinetics, with a K(m) of 78 microM and V(max) of 333 pmol/10(6) cells per minute. Neutral D- and L-amino acids with branched or aromatic large side chains inhibited [(125)I]IMT uptake. Tyrosine analogues, tryptophan analogues, L-phenylalanine and p-halogeno-L-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-L-phenylalanine (L-DOPA), DL-threo-beta-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [(125)I]IMT uptake, but L-tyrosine methyl ester and R(+)/S(-)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [(125)I]IMT uptake except L-serine and D/L-cysteine. CONCLUSIONS: [(125)I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1.

p53 independent radio-sensitization of human lymphoblastoid cell lines by Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin.

Inhibition of heat shock protein 90 (Hsp90) is an attractive modality for cancer therapy. Recent studies presented that an Hsp90 inhibitor, 17AAG (17-allylamino-17-demethoxygeldanamycin), enhanced tumor radio-sensitivity, while this was not observed in normal cells. One of the studies reported that the effect of this drug was only observed in tumor cells carrying the wild-type p53 gene, thus demonstrating p53-dependent tumor radio-sensitization by 17AAG. We have now tested the effects of 17AAG on two human lymphoblastoid cell lines from the same donor, TK6 cells with the wild-type p53 gene and WTK1 cells with the mutated p53 gene. The effects of 17AAG were tested at concentrations of 10 and 100 nM on various parameters, including growth inhibition of the cells, enhancement of radio-sensitivity by colony formation assay, apoptosis and chromosomal radio-sensitivity and abrogation of radiation induced G2/M checkpoint. When 100 nM 17AAG was applied, all of these parameters were enhanced in a similar fashion in both cell lines, indicating that the drug effect is p53-independent. Our results suggest that 17AAG is likely to be an effective sensitizer for radiotherapy, even on tumors with mutated p53.

Scriptaid, a novel histone deacetylase inhibitor, enhances the response of human tumor cells to radiation.

A group of histone deacetylase (HDAC) inhibitors has been shown to suppress the growth of a variety of human tumor lines in vitro and in vivo and they are among the most promising candidates for anti-cancer therapeutic agents. We investigated the ability of scriptaid, a novel HDAC inhibitor and trichostatin A (TSA) to enhance cell killing by radiation in radioresistant SQ-20B cells derived from human head and neck squamous carcinoma. SQ-20B cells were treated with scriptaid or TSA in combination with radiation. Cell survival was determined by a colony formation assay and protein levels were examined by Western blotting. DNA double strand breaks were measured by a gamma-H2AX focus assay. Radiosensitization was observed for SQ-20B cells incubated with scriptaid at 5 microM or TSA at 0.1 microM for 24 h. Radiosensitization by scriptaid was accompanied by a prolonged retention of gamma-H2AX foci, suggesting that the enhancement of radiation cell killing by scriptaid involved inhibition of DNA double strand break repair. In addition, treatment with scriptaid suppressed expression of Ku80, but not Ku70. Scriptaid may be a useful radiosensitizer in the treatment of radioresistant human carcinomas.