Novel DNA methyltransferase-1 (DNMT1) depleting anticancer nucleosides, 4′-thio-2′-deoxycytidine and 5-aza-4′-thio-2′-deoxycytidine

Purpose

Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2′-deoxycytidine (dCyd) analogs, 4′-thio-2′-deoxycytidine (T-dCyd) and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth.

Methods

DNMT1 protein levels in in vitro and in vivo cancer models were determined by Western blotting and antitumor efficacy was evaluated using xenografts. Effects on CpG methylation were evaluated using methylation-specific PCR. T-dCyd metabolism was evaluated using radiolabeled substrate.

Results

T-dCyd markedly depleted DNMT1 in CCRF-CEM and KG1a leukemia and NCI-H23 lung carcinoma cell lines, while it was ineffective in the HCT-116 colon or IGROV-1 ovarian tumor lines. On the other hand, aza-T-dCyd potently depleted DNMT1 in all of these lines indicating that dCyd analogs with minor structural dissimilarities induce different DNMT1 turnover mechanisms. Although T-dCyd was deaminated to 4′-thio-2′-deoxyuridine, very little was converted to 4′-thio-thymidine nucleotides, suggesting that inhibition of thymidylate synthase would be minimal with 4′-thio dCyd analogs. Both T-dCyd and aza-T-dCyd also depleted DNMT1 in human tumor xenografts and markedly reduced in vivo tumor growth. Interestingly, the selectivity index of aza-T-dCyd was at least tenfold greater than that of decitabine.

Conclusions

Collectively, these data show that 4′-thio modified dCyd analogs, such as T-dCyd or aza-T-dCyd, could be a new source of clinically effective DNMT1 depleting anticancer compounds with less toxicity.     

Expression of Hyaluronan Synthases (HAS1–3) and Hyaluronidases (HYAL1–2) in Serous Ovarian Carcinomas: Inverse Correlation between HYAL1 and Hyaluronan Content

Background  

Hyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and hyaluronidases (HYAL1 and HYAL2), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1–3 immunoreactivity.     

Biochemical mechanisms for the scheduled synergism of (αS, 5S)-2 amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid and 5-fluorouracil in P388 leukemia

Summary A study was made of the in vivo effects of equitoxic doses of AT-125 and 5-FU combination, being administered either simultaneously (% ILS 152) or with a 6-h pretreatment with AT-125 (% ILS 184). To examine the biochemical basis for the scheduled synergism, measurements were made of the concentration of PRPP, the specific activities of CPS II, cytidine, thymidine, uridine, deoxyuridine kinases, and fluorinated nucleotide formation in P388 tumors and the small intestine. Two hours after in vivo simultaneous treatment of mice bearing tumors the concentration of PRPP increased 9- and 6-fold above baseline in the tumor and the small intestine, respectively. In the AT-125 pretreatment arm the concentration of PRPP increased 18- and 7-fold above baseline in the tumor and the small intestine, respectively. CPS II activity was reduced to 28%–18% of control in the tumors in the simultaneous and pretreatment groups, respectively, whereas it remained unchanged in the small intestine. Specific activities of cytidine kinase (5.5±1), thymidine kinase (4.0±1.6), uridine kinase (35.6±6.5), and deoxyuridine kinase (2.4±1.1) nmol/mg protein/h remained unchanged with treatment. In concert with the increased intratumor concentration of PRPP, fluorinated nucleotide formation was proportionally increased in the treatment arms. These results indicate the importance of drug scheduling of the above two agents in treating P388 leukemia.Abbreviation AT 125 - S,5S -amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid - 5-FU 5-fluorouracil - 5-FdUMP 5-fluorodeoxyuridine monophosphate - PRPP phosphoribosyl pyrophosphate - 5-FUMP 5-fluorouridine monophosphate - 5-FUDP 5-fluorouridine diphosphate - 5-FUTP 5-fluorouridine triphosphate - UMP uridine monophosphate - UDP uridine diphosphate - UTP uridine triphosphate - ATP adenosine triphosphate - CPS II carbamylphosphate synthetase II - PCA perchloric acid Presented in part at the Seventy-fourth Annual Meeting of the American Association for Cancer Research, San Diego, California, May 1983     

Relationship between CCL5 and transforming growth factor-β1 (TGFβ1) in breast cancer

Purpose

Investigate circulating CCL5 in breast cancer patients and healthy controls, along with gene expression levels in corresponding tumour tissue and isolated primary stromal cells. Hormonal control of CCL5, and a potential relationship with TGFβ1, was also investigated.

Methods

Circulating levels of CCL5 and TGFβ1 were measured in 102 breast cancer patients and 66 controls using ELISA. Gene expression levels (CCL5, CCR5, TGFβ1, TGFβRII) were quantified in corresponding tumour tissue (n = 43), normal tissue (n = 16), and isolated tumour (n = 22) and normal (n = 3) stromal cells using RQ-PCR. CCL5 and circulating menstrual hormones (LH, FSH, Oestradiol, Progesterone) were analysed in serum samples from healthy, premenopausal volunteers (n = 60).

Results

TGFβ1 was significantly higher in breast cancer patients (Mean(SEM) 27.4(0.9) ng/ml) compared to controls (14.9(0.9) ng/ml). CCL5 levels decreased in the transition from node negative (59.6(3.7) ng/ml) to node positive disease (40.5(6.3) ng/ml) and increased again as the number of positive lymph nodes increased (?3 positive 50.95(9.8) ng/ml). A significant positive correlation between circulating CCL5 and TGFβ1 (r = 0.423, p < 0.0001) was observed, and mirrored at the gene expression level in tumour tissue from the same patients (r = 0.44, p < 0.001). CCL5, CCR5 and TGFβ1 expression was significantly higher in tumour compared to normal breast tissue (p < 0.001). A significant negative correlation was observed between circulating CCL5, Oestradiol and Progesterone (r = −0.50, r = −0.39, respectively, p < 0.05).

Conclusion

CCL5 expression is elevated in the tumour microenvironment. The data support a role for hormonal control of circulating CCL5 and also highlight a potentially important relationship between CCL5 and TGFβ1 in breast cancer.     

Effect of (E)-5-(2-bromovinyl)-2′-deoxyuridine on life-span and 5-fluorouracil metabolism in mice with hepatic metastases

5-Fluorouracil (5FU) is rapidly metabolised in the liver by dihydrouracil dehydrogenase. Bromovinyluracil is formed in the liver from (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) by pyrimidine nucleoside phosphorylase and is a potent inhibitor of dihydrouracil dehydrogenase. The co-administration of 5FU (intravenously) and BVDU (orally) was investigated in normal BDF₁ mice and in those bearing liver metastases of Lewis lung carcinoma. 5FU alone rapidly disappeared from plasma and liver within 60 min of dosing. Administered with BVDU, 5FU persisted in plasma and liver for 60–180 min. The combination also significantly enhanced the life-span of tumour-bearing mice. 5FU plus BVDU may have therapeutic potential in the treatment of primary and secondary liver tumours.     

Cordycepin (3′-deoxyadenosine) suppressed HMGA2, Twist1 and ZEB1-dependent melanoma invasion and metastasis by targeting miR-33b

Malignant melanoma, the most deadly form of skin cancer, has a high propensity for metastatic spread and is notoriously chemotherapy-resistant. Cordycepin, the active component of Cordyceps spp., has been identified to have anti-metastatic effect on tumor progression and thus possesses pharmacological and therapeutic potentials. However, the mechanisms of anti-metastatic effects of cordycepin at cellular levels remain elusive. We analyzed the effect of cordycepin on human melanoma miRNA expression profiles by miRNAarray and found that miR-33b was upregulated in highly-metastatic melanoma cell lines following cordycepin exposure. Cordycepin-mediated miR-33b expression was dependent on LXR-RXR heterodimer activation. miR-33b directly binds to HMGA2, Twist1 and ZEB1 3′-UTR to suppress their expression. The negative correlations between miR-33b levels and HMGA2, Twist1 or ZEB1 expression were detected in 72 patient melanoma tissue samples. By targeting HMGA2 and Twist1, miR-33b attenuated melanoma migration and invasiveness upon cordycepin exposure. miR-33b knockdown or ZEB1 overexpression reverted cordycepin-mediated mesenchymal-epithelial transition (MET), triggering the expression of N-cadherin. In spontaneous metastasis models, cordycepin suppressed tumor metastasis without altering primary tumor growth. We showed for the first time that targeting miRNA by cordycepin indicates a new mechanism of cordycepin-induced suppression of tumor metastasis and miR-33b/HMGA2/Twist1/ZEB1 axis plays critical roles in regulating melanoma dissemination.     

The Differential Effects of Cyclophosphamide, Epirubicin and 5-Fluorouracil on Apoptotic Marker (CPP-32), Pro-Apoptotic Protein (p21 WAF−1) and Anti-Apoptotic Protein (bcl-2) in Breast Cancer Cells

Cyclophosphamide (CYC), epirubicin (EPI) and 5-fluorouracil (5FU) are commonly used cytotoxic drugs for the treatment of breast cancer. The efficacy of these drugs in the induction of caspases (CPP-32), pro-apoptotic (p21 ^WAF–1) and anti-apoptotic (bcl-2) proteins is tested in vitro on breast cancer cells lines MDA-MB-231 and MCF-7. The cell proliferation rate and the levels of CPP-32, p21 ^WAF–1 and bcl-2 are measured at 3, 6, 12 and 24 h. For MDA-MB-231 all three drugs caused significant inhibition in cell growth. CYC produces significant induction of CPP-32 at 3–6 h for MCF-7 only. For MDA-MB-231 and MCF-7, respectively, EPI induces CPP-32 at significant levels at 12–24 h and 6–12 h while 5FU creates induction for MDA-MB-231 at 3 h and for MCF-7 at 3–12 h. The levels of expression of p21 ^WAF–1 and bcl-2 for all test groups were significantly different from their respective control groups. In the case of MDA-MB-231, regression analysis reveals that changes in CPP-32 levels and p21 ^WAF–1 levels have a significant positive relationship. In all likelihood, other mechanisms of cell death are implicated in the antitumor effect of these drugs, beyond the activation of CPP-32 and p21 ^WAF–1 as described in this paper.     

Molecular Evaluation of DNMT3A and IDH1/2 Gene Mutation:Frequency,Distribution Pattern and Associations with Additional Molecular Markers in Normal Karyotype Indian Acute Myeloid Leukemia Patients

Mutations in the DNMT3A and IDH genes represent the most common genetic alteration after FLT3/NPM1in acute myeloid leukemia (AML). We here analyzed the frequency and distribution pattern of DNMT3A andIDH mutations and their associations with other molecular markers in normal karyotype AML patients. Fortyfivepatients were screened for mutations in DNMT3A (R882), IDH1 (R132) and IDH2 (R140 and R172) genesby direct sequencing. Of the 45 patients screened, DNMT3A and IDH mutations were observed in 6 (13.3%)and 7 (15.4%), respectively. Patients with isolated DNMT3A mutations were seen in 4 cases (9%), isolatedIDH mutations in 5 (11.1%), while interestingly, two cases showed both DNMT3A and IDH mutations (4.3%).Nucleotide sequencing of DNMT3A revealed missense mutations (R882H and R882C), while that of IDH revealedR172K, R140Q, R132H and R132S. Both DNMT3A and IDH mutations were observed only in adults, with ahigher frequency in males. DNMT3A and IDH mutations were significantly associated with NPM1, while trendstowards higher coexistence with FLT3 mutations were observed. This is the first study to evaluate DNMT3A/IDH mutations in Indian patients. Significant associations among the various molecular markers was observed,that highlights cooperation between them and possible roles in improved risk stratification.     

Antitumor mechanisms and metabolism of the novel antitumor nucleoside analogues, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine and 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)uracil

The antitumor ribonucleoside analogues 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)uracil (EUrd), first synthesized in 1995, have strong antitumor activity against human cancer xenografts without severe side effects. Here, we studied the antitumor mechanisms of ECyd and EUrd using mouse mammary tumor FM3A cells in vitro and the mechanism of selective cytotoxicity of ECyd using human tumor xenografts in nude rats in vivo. In FM3A cells, ECyd and EUrd were rapidly phosphorylated to ECyd 5′-triphosphate (ECTP) and EUrd 5′-triphosphate (EUTP), which strongly inhibiting RNA synthesis. Cells treated with EUrd were later found to contain both EUTP and ECTP, and ECTP accumulated as the final product. Probably the uracil moieties of EUrd derivatives were efficiently converted to cytosine moieties in the cells. EUrd and its derivatives were minor metabolites in the cells treated with ECyd, so cytidine forms probably were not converted to uridine forms at the nucleoside or nucleotide stage. The ultimate metabolite of ECyd and EUrd, ECTP, is stable in cultured cells with a half-life of at least 3 days, so ECyd and EUrd are on a “closed” metabolic pathway to ECTP. These characteristics of ECyd and EUrd may be important for their antitumor activity. ECyd had strong and selective antitumor activity against the human tumor xenografts. ECyd-phosphorylating activity (uridine/cytidine kinase) in the xenografts was higher than that in the organs of the rats. This finding may account for the strong activity with mild side effects. ECyd and EUrd may be a new kind of antitumor nucleoside analogue for clinical use. Received: 8 June 1998 / Accepted: 5 November 1998     

A new nitrosourea derivative TA-077, 1-(2-Chloroethyl)-3-isobutyl-3-(β-maltosyl)-1-nitrosourea

Summary A new water-soluble nitrosourea derivative, 1-(2-chloroethyl)-3-isobutyl-3-(-maltosyl)-1-nitrosourea (TA-077), was tested for antitumor activity against murine tumors and a human mammary carcinoma (MX-1) implanted in athymic mice, and the results were compared with those obtained with five other nitrosourea derivatives currently in clinical use: 1-(2-chloroethyl)-3-(methyl--d-glucopoyranos-6-yl)-1-nitrosourea (MCNU), 1-(2-chloroethyl)-3-(-d-glucopyranosyl)-1-nitrosourea (GANU), 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU), chlorozotocin, and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (Me-CCNU).The results indicated that TA-077 had a unique optimal treatment schedule different from other nitrosoureas. With daily IV treatments for 5 days, TA-077 showed the highest antitumor activity of all against the advanced Lewis lung carcinoma, defined by tumor weight and the survival of tumor-bearing mice. Furthermore, TA-077 given according to this treatment schedule was successful in inducing an apparent cure (complete regression and no recurrence) in all the athymic mice bearing MX-1, which the other five nitrosoureas could not. In addition, TA-077 possessed higher therapeutic indices (optimal dose/ILS₂₅) against L1210 and P388 leukemias than MCNU, which possessed the highest therapeutic index against L1210 leukemia among the five nitrosourea derivatives.     

Disposition and metabolism in mice of the potential antitumor and anti-human immunodeficiency virus-1 agent, 2-chloro-2′,3′-dideoxyadenosine

Summary A high-performance liquid chromatographic (HPLC) procedure was developed to examine the preclinical pharmacology and pharmacokinetics of 2-chloro-2,3-dideoxyadenosine (ClddAd). The HPLC assay for ClddAd in human plasma was linear from 0.25 to 500 g ClddAd/ml. Coefficients of variation for the measurement of ClddAd in human plasma were 9.7%, 4.1%, and 2.7% at 2.5, 25, and 250 g/ml, respectively. Binding of ClddAd to human and mouse plasma proteins was determined by filtration to be 26,9% and 34.4%, respectively. ClddAd concentrations decreased by <5% when ClddAd was stored for 126 h at 37° C in 0.9% NaCl or 0.1m NaH₂PO₄ (pH 7.4) or when ClddAd was stored for 24 h at 37° C in citrate-buffered human blood or plasma. Estimates of the lethal dose for 50% (LD₅₀) and 10% (LD₁₀) of male CD2F₁ mice that received a single i.v. dose of ClddAd were 27 and 24 mg/kg, respectively. Elimination of a 24-mg/kg i.v. bolus dose of ClddAd from mouse plasma was biphasic, with half-lives of 0.73 and 14.7 min. The apparent volume of distribution of ClddAd was 215 ml/kg and total body clearance was 20 ml min^–1 kg^–1. No ClddAd metabolites were detected in mouse plasma after in vivo exposure or in human whole blood or plasma after in vitro incubation. ClddAd was detected in the urine of mice within 2 min after exposure, and the total urinary excretion of unchanged ClddAd for 24 h after exposure to 24 mg/kg was 3.4% of the delivered dose. At least two possible ClddAd metabolites were detected in mouse urine; they did not co-elute with 2-chloro-2,3-dideoxy-inosine, 2-chloroadenine, or 2-chlorohypoxanthine.Portions of this work have previously been presented (Proc Am Assoc Cancer Res (1989) 30: 589)     

Pharmacokinetic and pharmacodynamic comparison of fluoropyrimidine derivatives, capecitabine and 5′-deoxy-5-fluorouridine (5′-DFUR)

Purpose Capecitabine is a three-step prodrug that was rationally designed to be a more effective and safer alternative to its intermediate metabolite, 5-deoxy-5-fluorouridine (5-DFUR). We compared the pharmacokinetics/pharmacodynamics of these drugs in metastatic breast cancer patients.Methods Six patients received oral capecitabine at 1657 mg/m² twice daily and 17 received 5-DFUR at 400 mg three times daily. Both drugs were administered for 21 days followed by a 7-day rest.Results Median daily 5-DFUR AUC was significantly higher for capecitabine than for 5-DFUR (81.1 vs 32.6 mmol h/l; P=0.01). Following treatment with 5-DFUR, the median AUC and C_max of 5-DFUR tended to be higher in patients with a partial response (3.83 g h/ml and 4.88 g/ml) and stable disease (6.46 g h/ml and 4.96 g/ml) than in those with disease progression (2.53 g h/ml and 1.36 g/ml). The AUC and C_max of 5-DFUR was significantly related to overall survival.Conclusions These results support the superiority of capecitabine over 5-DFUR.     

Activity and expression of progesterone metabolizing 5α-reductase, 20α-hydroxysteroid oxidoreductase and 3α(β)-hydroxysteroid oxidoreductases in tumorigenic (MCF-7, MDA-MB-231, T-47D) and nontumorigenic (MCF-10A) human breast cancer cells

Background  

Recent observations indicate that human tumorous breast tissue metabolizes progesterone differently than nontumorous breast tissue. Specifically, 5α-reduced metabolites (5α-pregnanes, shown to stimulate cell proliferation and detachment) are produced at a significantly higher rate in tumorous tissue, indicating increased 5α-reductase (5αR) activity. Conversely, the activities of 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO enzymes appeared to be higher in normal tissues. The elevated conversion to 5α-pregnanes occurred regardless of estrogen (ER) or progesterone (PR) receptor levels. To gain insight into these differences, the activities and expression of these progesterone converting enzymes were investigated in a nontumorigenic cell line, MCF-10A (ER- and PR-negative), and the three tumorigenic cell lines, MDA-MB-231 (ER- and PR-negative), MCF-7 and T-47D (ER- and PR-positive).     

PAI-1在鼻咽癌细胞系(CNE-2Z)及其克隆株(CNE-2Z-H5,CNE-2Z-H5-9)中表达和意义

目的:探讨尿激酶激活剂抑制剂-1(Plasminogenactivatorinhibitor-1,PAI-1)在不同淋巴道转移能力的鼻咽癌细胞系(CNE-2Z)及其克隆株(CNE-2Z-H5,CNE-2Z-H5-9)中的表达和意义。方法:应用逆转录聚合酶反应(RT-PCR)方法检测PAI-1在基因转录水平的表达;使用异质粘附、侵袭抑制实验观察PAI-1在鼻咽癌细胞侵袭和转移过程中的作用。结果:PAI-1在CNE-2Z,CNE-2Z-H5中均有转录,但无显著性差异;PAI-1在CNE-2Z-H5-9中无转录。抗PAI-1抗体对CNE-2Z-H5-9异质粘附、侵袭无影响。结论:PAI-1可能抑制鼻咽癌的侵袭和转移。