In vitro cytotoxic and biochemical effects of 5-aza-2'-deoxycytidine.

The in vitro effect of 5-aza-2'-deoxycytidine (5-aza-CdR) on cytotoxicity and macromolecular synthesis in A(T1)C1-3 hamster fibrosarcoma cells was investigated. The in vitro concentrations that produce 50% cell kill for 5-aza-CdR were about 1.0 and 0.01 microng/ml for a 2- and 24-hr exposure, respectively. 5-aza-CdR inhibited the growth of the fibrosarcoma cells by 40% at a concentration of 0.05 microng/ml. Deoxycytidine, but not cytidine, was a potent antagonist of the cytotoxicity produced by 5-aza-CdR. At cytotoxic concentrations 5-aza-CdR did not appear to inhibit DNA, RNA, or protein synthesis during a 1-hr incubation as measured by the incorporation of radioactive thymidine, uridine,, or leucine into acid-insoluble material. At a concentration of 10 microng/ml, 5-aza-CdR stimulated the incorporation of radioactive thymidine into DNA by more than 50%. These results indicate that 5-aza-CdR is a very potent cytotoxic agent to tumor cells in vitro at concentrations that do not inhibit macromolecular synthesis.     

Radiation, pool size and incorporation studies in mice with 5-chloro-2'-deoxycytidine

Bolus doses of 5-chlorodeoxycytidine (CldC) administered with modulators of pyrimidine metabolism, followed by X-irradiation, resulted in a 2-fold dose increase effect against RIF-1 tumors in C3H mice. Pool size studies of the fate of [14C]-CldC in BDF1 mice bearing Sarcoma-180 tumors, which demonstrated the rapid formation of 5-chlorodeoxycytidylate (CldCMP), and incorporation of CldC as such in RIF-1 tumor DNA, indicate that CldC is a substrate for deoxycytidine kinase, as our past Km studies have shown. Our data indicate that 5-chlorodeoxyuridine triphosphate (CldUTP) accumulates from both the cytidine deaminase-thymidine kinase pathway, as well as from the deoxycytidine kinase-dCMP deaminase pathway, in tumor tissue. As shown in a previous study, tetrahydrouridine (H4U), a potent inhibitor of cytidine deaminase, can effectively inhibit the enzyme in the normal tissues of BDF1 mice. When H4U was administered with the modulators N-(phosphonacetyl)-L-aspartic acid (PALA) and 5-fluorodeoxycytidine (FdC), the levels of CldC-derived RNA and DNA directed metabolites increased in tumor and decreased in normal tissues compared to when CldC was administered alone. These modulators inhibit the de novo pathway of thymidine biosynthesis, lowering thymidine triphosphate (TTP) levels, which compete with CldUTP for incorporation into DNA. 5-Benzylacyclouridine (BAU), an inhibitor of uridine phosphorylase, was also utilized. DNA incorporation studies using C3H mice bearing RIF-1 tumors showed that the extent of incorporation of 5-chlorodeoxyuridine (CldU) into DNA correlates with the levels of cytidine and dCMP deaminases; this is encouraging in view of their high activity in many human malignancies and the low activities in normal tissues, including those undergoing active replication. Up to 3.9% replacement of thymidine by CldU took place in RIF-1 tumors, whereas incorporation into bone marrow was below our limit of detection. CldC did not result in photosensitization under conditions in cell culture in which radiosensitization to X rays was obtained. Thus, the combination of CldC with modulators of its metabolism has potential as a modality of selective radiosensitization for ultimate clinical use in a wider range of tumors than those of the brain.     

Demethylating agent 5-aza-2'-deoxycytidine enhances expression of TNFRI and promotes TNF-mediated apoptosis in vitro and in vivo

Promoter hypermethylation within CpG islands plays an important role in the silencing of numerous genes involved in tumor growth including tumor suppressor genes and genes encoding proteins involved in the execution of apoptosis. Here we show that CpG islands are also found within the promoter regions of both human and mouse TNFR1 (TNFRSF1) genes. Selective inhibition of methyltransferases with 5-aza-2'-deoxycytidine increases the expression of TNFR1 in human (WM35) and murine (B16F10) melanoma cells and sensitizes them to TNF-induced apoptosis both in vitro and in vivo. Treatment of mice with the combination of 5-aza-2'-deoxycytidine and recombinant TNF leads to potentiated antitumor effects. Importantly the antitumor efficacy of the combination treatment is shown when both drugs are used in doses that do not exert any antitumor effects when used alone. Altogether our studies show that the combination treatment with 5-aza-2'-deoxycytidine and TNF might be effective in the treatment of melanoma.     

Effects of the alpha-D-anomer of 5-aza-2'-deoxycytidine on L1210 mouse leukemic cells in vitro and in vivo

The alpha-D-anomer of 5-aza-2'-deoxycytidine inhibited cell growth in vitro in L1210 mouse leukemic cells (concentration causing 50% inhibition about 1 X 10(-6) M) and was also active in vivo in increasing the life span of mice with L1210 leukemia by 100% after a single i.p. injection (800 to 1000 mg/kg). This effect could be reversed by 2'-deoxycytidine. The alpha-D-anomer produced approximately 100-fold less host toxicity than did 5-aza-2'-deoxycytidine. It was observed that alpha-D-anomer, when kept at 23 degrees for 72 hr, inhibited the uptake of radioactive 2'-deoxycytidine by L1210 cells in vitro as well as the phosphorylation of 2'-deoxycytidine by a cell-free extract from L1210 cells. These effects could not be obtained with the fresh solution of the drug. It is proposed that the action of alpha-D-anomer is due to its conversion to 5-aza-2'-deoxycytidine as shown by high-performance liquid chromatography performed on water solutions of both anomers.     

Sensitization to X ray by 5-chloro-2'-deoxycytidine co-administered with tetrahydrouridine in several mammalian cell lines and studies of 2'-chloro derivatives

5-Chloro-2'-deoxycytidine (CldC) + tetrahydrouridine (H4U) sensitizes mammalian cells (HEp-2, RIF-1, S-180) to X ray. This sensitization, as demonstrated previously with HEp-2 cells, is heightened when cells are pre-incubated with inhibitors of pyrimidine synthesis. CHO cells, which intrinsically lack both cytidine deaminase (CD) and deoxycytidylate deaminase (dCMPD), are sensitized to X ray by 5-chlorodeoxyuridine (CldU) but display no significant sensitization with CldC + H4U. The presence and level of these deaminases appears to correlate with X ray sensitization in cell culture. From experiments in cell culture, it can be inferred that one pathway of conversion, deoxycytidine kinase----dCMPD, or CD----thymidine kinase, may be sufficient for metabolizing CldC to a radiosensitizer. However, if both pathways are blocked, as in CHO cells, no X ray sensitization results. In addition to HEp-2 cells, which are extremely elevated in both CD and dCMPD activities, we have examined the sensitization of S-180 and RIF-1 cells to X ray by CldC + H4U. Both cell lines possess an enzymatic profile consistent with their sensitization to X ray by CldC + H4U. Dose enhancement ratios of 1.5 to 1.9 for cells treated with CldC + H4U and ratios of 2.0-2.7 for cells pre-treated with inhibitors of pyrimidine synthesis prior to CldC + H4U have been obtained. Based on reports of the marked X ray sensitization of bacteria by 2'-chloro-2'-deoxythymidine, we obtained 2',5-dichloro-2'-deoxycytidine and 5-bromo-2'-chloro-2-deoxyuridine and found these analogs to be X ray sensitizers of mammalian cells. The strategy that we propose with CldC + H4U and the related 2'-chloro derivatives, based on the elevation of CD and dCMPD in human tumors, offers a degree of selectivity that is not necessarily related to differences in cell kinetics; such that malignancies other than brain tumors may be amenable to this therapy.     

Overexpression of pyrimidine nucleoside phosphorylase enhances the sensitivity to 5'-deoxy-5-fluorouridine in tumour cells in vitro and in vivo

5-Fluorouracil (5-FU) and 5'-deoxy-5-fluorouridine (5'-DFUR), a prodrug of 5-FU, are representative of the chemotherapeutic agents for colorectal adenocarcinomas. Pyrimidine nucleoside phosphorylase (PyNPase) catalyses the conversion of 5'-DFUR to 5-FU, the activated form. Murine adenocarcinoma CT26 cells were transfected with human PyNPase cDNA. The engineered transfectants producing PyNPase augmented the response to 5'-DFUR in vitro and in vivo. Animals were administered by means of intraperitoneal (i.p.) injection, and not orally, in order to obtain a better efficiency of absorption. The tumours of the transfected cells nearly all disappeared, even following treatment with quite a small amount of the anticancer agent. The animals injected with the tranfected cells were protected against subsequent challenge with the parental tumour cell line. These findings demonstrate that PyNPase gene transfection increases the sensitivity to 5'-DFUR, and thereby decreases the toxicity of the agent.     

In vivo and in vitro mechanisms of radiation sensitization,drug synthesis and screening: Can we learn it all from the high dose data?

The evidence for a decreased enhancement ratio of oxygen and an electron affinic radiosensitizer (misonidazole) at low doses is presented, and the mechanism of this effect is discussed. The factors which influence the magnitude of this effect, as well as the dose levels at which the effect will be significant, are identified. This will allow further characterization of this phenomenon in the future. An approach by which present and new hypoxic radiosensitizers could be made more effective at low doses is indicated.     

In vitro and in vivo evaluation of novel antitumor prodrugs of 5-fluoro-2'-deoxyuridine activated by hypoxic irradiation

PURPOSE: We previously developed a novel antitumor prodrug that has a 2-oxopropyl substituent at the N(1) position of 5-fluorouracil (5-FU) and releases 5-FU via one-electron reduction on hypoxic irradiation. Although the compound was effective in vivo, its activity against murine tumors was not high enough to warrant clinical studies. Therefore, we developed a similar family of radiation-activated prodrugs of 5-fluoro-2'-deoxyuridine (FdUrd), which is generally more potent than 5-FU, and investigated their radiation chemical reactivity and in vitro and in vivo effects. METHODS AND MATERIALS: Compounds bearing various 2-oxoalkyl substituents at the N(3) position of FdUrd were synthesized and investigated. After aerobic or hypoxic irradiation to the prodrugs dissolved in water or culture medium, release of FdUrd was measured using high-performance liquid chromatography. To investigate in vitro cytotoxicity, SCCVII and EMT6 cells in culture were irradiated in the presence of the prodrug under aerobic or hypoxic conditions, and then kept with the compound for 24 h. Cell survival was then measured using a colony assay. To investigate in vivo effects, the drug was injected intraperitoneally at a dose of 100 or 300 mg/kg into Balb/c mice bearing EMT6 tumors 30 min before irradiation. The tumor growth delay-time was then assessed. RESULTS: In vitro, the prodrugs released FdUrd at G-values (molar numbers of molecules produced by 1 J of radiation energy) of 1.6-2.0 x 10(-7) mol/J after hypoxic irradiation. The G-values for FdUrd release with hypoxic irradiation were about 100-fold greater than those with aerobic irradiation. Among the prodrugs tested, OFU106 bearing a 2-oxocyclopentyl substituent released the highest amount of FdUrd in the culture medium, and it was subjected to further in vitro and in vivo assays. Although OFU106 administered alone showed no cytotoxicity up to a concentration of 0.2 mM, it produced an enhanced cytotoxic effect when administered before hypoxic irradiation and kept with the cells for 24 h. The enhancement ratios calculated at the surviving fraction of 1% were 1.35-1.4 at 0.04 mM and 1.45-1.5 at 0.2 mM. In vivo, however, administration of OFU106 (100 or 300 mg/kg) before 20 Gy of irradiation did not produce marked growth delays compared with 20 Gy of radiation alone. CONCLUSION: On hypoxic irradiation in vitro, the prodrugs of FdUrd were activated as efficiently as were the prodrugs of 5-FU, but marked in vivo effects could not be detected. This strategy of prodrug design should be used in further development of radiation-activated prodrugs of more potent anticancer agents.     

Irinotecan and radiation in vitro and in vivo

Loss of chromosome sequences at 13q14 (Rbl) and 17p13 (p53) associated with squamous cell carcinoma of head and neck (SCCHN) was evaluated in 12 recurrent tumors and 51 primary tumors from 63 patients. The incidence of LOH at 17p13 was 19 of 50 (38%) tumors, and at 13q14 was 21 of 57 (37%). LOH affecting Rbl and/or p53 was observed in 30 of 63 (48%) SCCHN. Coincident LOH at Rbl and p53 was detected in 10 of 46 (22%) tumors. There were nine cases in which primary and metastatic tumors were obtained from the same patient. Of these, seven were informative and five of these (71%) manifested LOH at p53 in both primary and metastatic sites. Examination of Rbl in these same tumors showed LOH in six of the nine metastases, and of these six, only three revealed LOH in the primary tumor. LOH at p53 or Rbl alone showed no correlation with clinical outcome. However, tumors that manifested LOH at both loci were associated with poorer patient outcome and poorer histological differentiation.     

In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles

Background

Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs.

Methods

In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model.

Results

Nano-HAPs enhanced each cell line''s radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G₂ cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation.

Conclusions

These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe.     

In vitro and in vivo potentiating the cytotoxic effect of radiation on human U251 gliomas by the c-Met antisense oligodeoxynucleotides

C-Met, a receptor tyrosine kinase, and its ligand, hepatocyte growth factor (HGF), are critical in cellular proliferation, motility, and invasion, and are known to be overexpressed in gliomas, which are related to the repair of damaged DNA. In this study, we investigated both in vitro and in vivo whether inhibition of the c-Met gene by antisense oligonucleotides (ODNs) enhances the cytotoxic effect of radiation on human U251 gliomas. A volume of 100 nM of c-Met antisense ODNs inhibited the level of mRNA by more than 95% and reduced the protein expression by about 70%. Treatment of human U251 glioma cells with 100 nM of c-Met antisense ODNs significantly enhanced the radiation-induced cell kill compared to control cells, and cells treated with nonsense ODNs. When the glioma cells were implanted in the cisterna magna of nude mice followed by treatment with c-Met antisense ODNs, the survival time of the nude mice was markedly prolonged compared to that of the untreated group (P < 0.001, logrank test). In addition, the combination of antisense ODNs and irradiation extended the survival time of the glioma-bearing nude mice much longer than could be achieved with radiation alone (P < 0.0001, logrank test). These results suggest that inhibition of c-Met can be expected to serve as a novel potentiator for radiation therapy in human U251 gliomas.     

Induction of MAGE Genes in Lymphoid Cells by the Demethylating Agent 5-Aza-2'-deoxycytidine

MAGE genes encoding tumor antigens recognized by cytotoxic T lymphocytes are appropriate target molecules for specific immunotherapy of cancer. We have investigated whether the demethylating agent 5-aza-2'-deoxycytidine (DAC) induces MAGE-1, -2, -3 , and - 6 in normal and malignant lymphoid cells. DAC induced these MAGE genes in both PHA/interleukin-2 (IL-2)-activated T cells from healthy donors and MAGE -negative T and B cell leukemias in most cases. It also induced MAGE-1 in IL-2-dependent T cell clones and all MAGE genes tested in Epstein-Barr virus-transformed B cell lines. Expression of MAGE-1 protein in the cells was confirmed by western blot analysis with anti-MAGE-1 polyclonal antibody. Therefore, demethylation is a potent stimulus to induce MAGE genes in both normal and malignant lymphoid cells.     

Radiosensitization produced in vivo by once- vs. twice-weekly 2'2'-difluoro-2'-deoxycytidine (gemcitabine)

PURPOSE: Gemcitabine (2'2'-difluoro-2'-deoxycytidine, dFdCyd) is a potent radiosensitizer of rodent and human tumor cells. Our Phase I clinical trial using once-weekly dFdCyd as a radiosensitizer in the treatment of patients with Stage IV squamous cell head and neck cancer has produced a high rate of tumor response and significant normal mucosal toxicity. These findings raised the question of whether we are using dFdCyd in the optimal dose and schedule. In vitro studies suggest that twice-weekly dFdCyd has the potential to be more effective than once-weekly dFdCyd when administered in combination with radiation (RT) given 5 days per week. Therefore, we have used a mouse model to assess whether the therapeutic ratio of combined modality therapy may be improved by using a twice-weekly drug regimen. We asked two questions: 1) Does a once-weekly or twice-weekly dFdCyd regimen cause more normal tissue radiosensitization? 2) Does a once-weekly or twice-weekly dFdCyd + RT regimen produce a better therapeutic index? METHODS AND MATERIALS: To assess normal tissue toxicity, C3H mice underwent mouth (60)Co RT (27.5 Gy in 5 daily fractions) +/- dFdCyd delivered intraperitoneally (IP) either once or twice weekly 6 hours prior to irradiation. Acute lip reactions were quantified according to a standard scoring system, and weight loss was measured. We measured tumor control using squamous cell carcinoma (SCC) VII murine squamous cell flank tumors (50-125 mm(3)) treated with the same regimens used in the mouth irradiation model. RESULTS: We found that dFdCyd delivered 800 mg/kg once weekly or 150 mg/kg twice weekly caused similar (and maximal tolerable) weight loss; therefore these regimens were chosen to test which schedule produced more acute lip radiosensitization. Twice-weekly dFdCyd + RT was somewhat more toxic by weight loss (800 mg/kg once weekly: 11.9%; 150 mg/kg twice weekly: 17.7%; p = 0.09). To assess therapeutic index, we treated SCC VII flank tumors with RT combined with isotoxic drug/RT regimens (dFdCyd 800 mg/kg once weekly or 100 mg/kg twice weekly). Tumors treated with twice-weekly dFdCyd + RT were significantly smaller than tumors treated with once-weekly drug + RT at 28 days from the start of treatment (p < 0.03). CONCLUSIONS: These findings demonstrate that equitoxic once- versus twice-weekly dFdCyd regimens cause differing levels of oral mucosal radiosensitization. This would suggest that each radiation-dFdCyd schedule will require its own dFdCyd dose escalation trial (which cannot be determined by the maximum tolerated dose (MTD) for dFdCyd alone using that schedule). In addition, our findings suggest that for head and neck cancers twice-weekly dFdCyd may have a higher therapeutic index compared with once-weekly dFdCyd when combined with daily RT.     

5-氮杂脱氧胞苷增强苯丁酸钠对Kasumi-1细胞的诱导分化和凋亡作用

目的 探讨组蛋白脱乙酰化酶抑制剂苯丁酸钠(PB)联合DNA甲基化抑制剂5-氮杂脱氧胞苷(5-Aza-CdR)阻断AML1-ETO的生物学功能．消除AML1-ETO的转录抑制,诱导t(8;21)急性髓系白血病细胞系Kasumi-1细胞的分化和凋亡作用。方法 应用四甲基偶氮唑蓝(MTT)比色法观察PB单药或联合5-Aza-CdR对细胞生长的抑制作用;流式细胞术分析细胞周期和检测髓系分化抗原CD11b和CD13的表达,判断细胞分化;Annexin V和碘化丙啶(PI)双标记,经流式细胞术分析细胞早期凋亡。结果 (1)5-Aza-CdR可增强PB对Kasumi-1细胞的生长抑制作用,半数抑制浓度(IC50)下降为1．95mmol/L。(2)PB处理使Kasumi-1细胞阻滞于G0／G1期,5-Aza-CdR与PB联合则使细胞阻滞于G2／M期。(3)低剂量5-Aza-CdR增强PB诱导：Kasumi-1细胞髓系分化抗原CD11b和CD13的表达。(4)低剂量5-Aza-CAR不能增强PB诱导的Kasumi-1细胞凋亡,而高剂量5-Aza-CdR增强PB诱导的Kasumi-1细胞早期凋亡。结论 DNA甲基化抑制剂5-Aza-CdR增强脱乙酰化酶抑制剂PB抑制Kasumi-1细胞生长、诱导分化和凋亡的作用。     

脂质体介导的体内外细胞因子基因转移效果的初步研究

本研究用反相蒸发技术制备出包裹IL-2 DNA或IL-6 DNA的脂质体，将其与体外培养的靶细胞共育，或将其直接注射至小鼠腹腔内及荷瘤小鼠的瘤体内．研究其介导体内外基因转移的效果，结果表明我们研制的脂质体不但能将目的基因转移至体外培养的靶细胞中，并可通过腹腔内注射及瘤体内注射后，在腹腔内及肿瘤原位直接将IL-2和IL-6基因转移至腹腔细胞及肿瘤细胞中，稳定表达IL-2和IL-6。     

In vivo and in vitro metabolism of 5-deazaacyclotetrahydrofolate, an acyclic tetrahydrofolate analogue

5-Deazaacyclotetrahydrofolate is a cytotoxic tetrahydrofolate analogue which inhibits glycinamide ribonucleotide transformylase (Kelley et al., J. Med. Chem., 33: 561-567, 1990). Cultured mouse L-cells and human MCF-7 and MOLT-4 cells concentrated the drug several hundred-fold after 24 h of continuous exposure to a cytotoxic level (100-200 nM) of radiolabeled drug. High performance liquid chromatography analysis revealed that each cell type metabolized greater than or equal to 80% of the internalized drug to polyglutamated forms, which are more potent glycinamide ribonucleotide transformylase inhibitors. In L-cells, 45% of the polyglutamated metabolites were also N-formylated. The pharmacokinetics and distribution of [14C]-deazaacyclotetrahydrofolate were studied in C57BL/6 male mice. Its plasma half-life was 2.15 h. Radiolabel was concentrated to well above plasma level in the kidney, pancreas, and liver. Metabolism was examined in tumor-bearing and in normal mice. Twenty-four h after a single i.p. injection (50 mg/kg), drug equivalents were 0.6 nmol/g (83% polyglutamated) in colon-38 adenocarcinoma carried s.c., 2.4 nmol/g (100% polyglutamated) in ascitic P388 cells, and 3.7 nmol/g (76% polyglutamated and approximately 20% formylated) in mouse liver. Elimination was mostly in the urine as unmetabolized drug. Feces contained 5-deazaacyclotetrahydropteroate (parent compound less glutamate). In conclusion, 5-deazaacyclotetrahydrofolate was shown to be concentrated to well above the extracellular level and metabolized to more active polyglutamated forms by transformed cells grown in culture and in mice.