Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

E-cadherin in canine mast cell tumors: decreased expression and altered subcellular localization in Grade 3 tumors.

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells.

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARß by 2.0-fold (quantitative real-time PCR). Our data show that RARß may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARß is epigenetically altered.

In vitro chemosensitivity of canine mast cell tumors grades II and III to all-trans-retinoic acid (ATRA).

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect the skin and soft tissue of dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. While retinoids are well recognized as promising antitumor agents, there have been only a few reports about retinoids' effect on canine cancers. The aim of this study was to investigate the chemosensitivity of MCT grades II and III to all-trans retinoic acid (ATRA). Immediately after surgical resection, MCT were prepared for primary culture. Samples of MCTs were also fixed in formalin for histopathology and grading according to the classification of Patnaik et al. (Veterinary Pathology 21(5):469-474, 1984). The best results were obtained when neoplastic mast cells were co-cultivated with fibroblasts. Cultured mast cells were, then, treated with concentrations of 10(-4) to 10(-7) M of ATRA, in order to evaluate their chemosensitivity to this retinoid. MTT assay was performed to estimate cell growth and death. The highest level of mast cell chemosensivity was obtained at the dose of 10(-4) M (p < 0,002). MCT of grades II or III were equally susceptible to the treatment with ATRA. Cell death was observed on the first 24 h until 48 h. According to these results, ATRA may be a potential chemotherapeutic agent for the treatment of canine MCT.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice.

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6% reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 +/- 0.26, N = 6; GUA: 0.27 +/- 0.24, N = 6; P = 0.0043), a 57.9% reduction in tumor proliferation index (CO: 23.75 +/- 20.54, N = 6; GUA: 9.99 +/- 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 +/- 22.95, N = 6; GUA: 324.37 +/- 266.74 AB/mm(2), N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

Paullinia cupana Mart var. sorbilis, guaraná, reduces cell proliferation and increases apoptosis of B16/F10 melanoma lung metastases in mice

We showed that guaraná (Paullinia cupana Mart var. sorbilis) had a chemopreventive effect on mouse hepatocarcinogenesis and reduced diethylnitrosamine-induced DNA damage. In the present experiment, we evaluated the effects of guaraná in an experimental metastasis model. Cultured B16/F10 melanoma cells (5 x 10(5) cells/animal) were injected into the tail vein of mice on the 7th day of guaraná treatment (2.0 mg P. cupana/g body weight, per gavage) and the animals were treated with guaraná daily up to 14 days until euthanasia (total treatment time: 21 days). Lung sections were obtained for morphometric analysis, apoptotic bodies were counted to calculate the apoptotic index and proliferating cell nuclear antigen-positive cells were counted to determine the proliferation index. Guaraná-treated (GUA) animals presented a 68.6 percent reduction in tumor burden area compared to control (CO) animals which were not treated with guaraná (CO: 0.84 ± 0.26, N = 6; GUA: 0.27 ± 0.24, N = 6; P = 0.0043), a 57.9 percent reduction in tumor proliferation index (CO: 23.75 ± 20.54, N = 6; GUA: 9.99 ± 3.93, N = 6; P = 0.026) and a 4.85-fold increase in apoptotic index (CO: 66.95 ± 22.95, N = 6; GUA: 324.37 ± 266.74 AB/mm², N = 6; P = 0.0152). In this mouse model, guaraná treatment decreased proliferation and increased apoptosis of tumor cells, consequently reducing the tumor burden area. We are currently investigating the molecular pathways of the effects of guaraná in cultured melanoma cells, regarding principally the cell cycle inhibitors and cyclins.

[The role of daunorubicin in induction therapy for adult acute myeloid leukemia].

The relationship between the total dose of daunorubicin (DNR) in induction therapy and the treatment outcome were evaluated based upon individualized doses of DNR during induction therapy for patients with acute myeloid leukemia(AML). Ninety-two previously untreated adult AML patients admitted to our hospital were analyzed for the dose of DNR required for complete remission (CR), the CR rate, disease-free survival (DFS) and overall survival (OS). The induction therapy consisted of DNR (40 mg/m2/d, i.v., from D 1 until the marrow was hypoplastic), Ara-C, prednisolone, and/or 6-thioguanine. Eighty-three out of 92 patients were assessable. Sixty-three patients entered CR (76%), of whom 52 attained CR with the first course of induction therapy. The 10-year DFS and OS rates were 31.2% and 42.3%, respectively. The median total dose of DNR in the induction therapy was 280 mg/m2 (120-480 mg/m2), which was not influenced by initial WBC count, or FAB type. These results indicate that when the dose is linked to the observed tumor response, the optimal dose of DNR in the induction therapy is around 280 mg/m2 (40 mg/m2 x 7 times), which is higher than the conventional dose of 40-60 mg/m2 for 3 days. The higher dose of DNR in the induction therapy for adult AML should be selected when the feasibility of a new drug is evaluated in a randomized trial.

Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection.

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.

[Two cases of functioning parathyroid cysts].

Two cases of functioning cysts are reported. The first patient was a 48-year-old man who underwent percutaneous cyst puncture for a palpable mass in the neck at another hospital. Hypercalcemic crisis brought the patient to Koga General Hospital. The second patient was a 69-year-old woman with the complaint of discomfort in the right neck of several years duration and with a palpable mass identified on physical examination at a local hospital. Both patients had high serum calcium and parathyroid hormone (PTH) levels and were diagnosed as having functioning parathyroid cysts by imaging studies including ultrasonography, computed tomographic scan, magnetic resonance imaging and scintigraphy. After parathyroidectomy, their serum calcium and PTH levels became normal, but calcium supplement was necessary in the first patient. To our knowledge, these are the 40th and 41st cases reported in the Japanese literature.

[Prognostic significance of CD7 expression in adult acute myeloid leukemia].

To evaluate the prognostic significance of CD7 expression in de novo acute myeloid leukemia (AML), we studied 63 patients with AML who had been admitted to our hospital between September 1989 and January 1996. Even of the patients were later eliminated from the study (9 due to insufficient surface marker analyses, and 2 due to early death). The remaining 52 patients (median age: 42.5 years) were evaluated for morphologic subtype, immunophenotypic classification, complete remission (CR), disease-free survival (DFS) and overall survival (OS). All 52 patients were grouped by the French-American-British classification system: 10 as M1, 16 as M2, 11 as M3, 8 as M4, 5 as M5, and 2 as M6. Ten of the patients expressed CD7 on their leukemia cells (positive rate > or = 25) and were classified as CD7(+)AML, with morphological subtypes as follows: 3 as M1, 6 as M2, and 1 as M3. Thirty-three of the 42 patients with CD7 + AML (78.6%) and 6 of the 10 patients with CD7 + AML (40%) achieved CR. DFS and OS rates for the patients with CD7(+)AML were 22.1% and 35.4%, respectively; those for the CD7(+)AML patients were 53.3% and 44.4%, respectively. No significant differences in gender hematological findings, clinical manifestations such as hepatosplenomegaly, lymphadenopathy, or incidence of central nervous system involvement, CR rate, and DFS distinguished patients with CD7(+)AML from those with CD7(+)AML. These suggest that CD7 expression is unlikely to be a prognostic factor in AML.

Role of daunorubicin in the induction therapy for adult acute myeloid leukemia.

PURPOSE: To evaluate the relationship of total-dose of daunorubicin (DNR) to the induction therapy and treatment outcome, we have administered individualized doses of DNR during induction treatment to patients with acute myelogenous leukemia (AML). PATIENTS AND METHODS: Ninety-two previously untreated adult patients with AML who entered our hospital were analyzed for the dose of DNR required to achieve complete remission (CR), the CR rate, disease-free survival (DFS), and overall survival (OS). Induction therapy consisted of DNR 40 mg/m2 daily intravenously from day 1 until the marrow was hypoplastic, cytarabine (Ara-C), prednisolone (PRD), and/or 6-thioguanine (6-TG). RESULTS: Eighty-three of 92 patients with adult AML were assessable for this study. Sixty-three (76%) patients achieved CR. Fifty-two of 63 CR patients achieved the CR in the first course of induction therapy, and 11 patients required the second course of induction therapy. The 5-year and 10-year DFS rates were 31.2% and 5-year and 10-year OS rates were 45.1% and 42.3%, respectively. The median total dose of DNR in the induction therapy was 280 mg/m2 (120 to 480 mg/m2). DNR dose did not influence the response to therapy and was not influenced by the initial WBC count or French-American-British (FAB) system classification. CONCLUSION: These results indicated that when the dose was linked to observed tumor response, the optimal dose of DNR in the induction therapy was approximately 280 mg/m2 (40 mg/m2 for 7 days), which is greater than the conventional dose of 40 to 60 mg/m2 for 3 days.

[EPOCH therapy for relapsed/refractory lymphoid malignancies].

Patients with refractory or relapsed non-Hodgkin's lymphoma (NHL), acute T-cell leukemia (ATL), ATL lymphoma and acute lymphocytic leukemia (ALL) received EPOCH therapy. All were previously treated with doxorubicin (DOX), vincristine (VCR) and other drugs. The EPOCH treatment schedule is consisted with DOX (10 mg/M2/day, 5 days c.i.v.), VCR (0.4 mg/M2/day, 4 days c.i.v.), etoposide (50 mg/M2/day, 4 days c.i.v.), cyclophosphamide (750 mg/M2/day, day 6 i.v.) and prednisolone (60 mg/M2/day, 5 days p.o.). Twenty-one patients (ALL:10, NHL:8, ATLL:2, ATL:1) were assessable for response and toxicity. Two patients with ALL and NHL, respectively, achieved a complete remission and 3 patients obtained partial remission (NHL:2, ATLL:1). The hematological toxicity (grade > 1) included neutoropenia, anemia and thrombocytopenia, which were observed in 83.3%, 76.7% and 76.7% respectively, of total 30 EPOCH courses. The major non-hematological toxicities were nausea/vomiting, constipation and infection, but most of the toxicity were tolerable with sufficient clinical supportive care. These results indicate that continuous infusion of DOX, VCR and ETP might be effective in patients who were treated with, and presumed to be resistant to the same drugs administrated by bolus infusion.

In vitro and in vivo characterization of the dopamine D4 receptor, serotonin 5-HT2A receptor and alpha-1 adrenoceptor antagonist (R)-(+)-2-amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)-4-oxobutyl] pyrrolidin-3-yl]thiazole (NRA0045).

(R)-(+)-2-Amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)-4-oxobutyl]+ ++pyrrolidin-3-yl]thiazole (NRA0045), a novel thiazole derivative, has high affinities for the human cloned dopamine D4.2, D4.4 and D4.7 receptors, with Ki values of 2.54, 0.55 and 0.54 nM, respectively. NRA0045 is approximately 91-fold more potent at the dopamine D4.2 receptor, compared with human cloned dopamine D2L receptor. NRA0045 also has high affinities for the serotonin (5-HT)2A receptor (Ki = 1.92 nM) and alpha-1 adrenoceptor (Ki = 1.40 nM) but weak affinities (IC50 values are approximately 1 microM) for six other neurotransmitter receptors (adenosine1, 5-HT1A, 5-HT1C, dopamine transporter, alpha2A and alpha2A) and negligible affinities (IC50 values are over 10(-5) M) for 42 other receptors, including neurotransmitters and hormones, ion channels and second messenger systems. Locomotor hyperactivity induced by methamphetamine (1 mg/kg i.p.) in mice was dose-dependently antagonized by NRA0045 (ED50 = 0.5 mg/kg i.p. and 1.9 mg/kg p.o., respectively). Methamphetamine (10 mg/kg i.p.)-induced stereotyped behavior in mice was dose-dependently antagonized by NRA0045, whereas NRA0045 did not exceed 50% inhibition even at the highest dose given (30 mg/kg i.p.). Catalepsy was dose-dependently and significantly induced by NRA0045 in rats, whereas NRA0045 did not exceed 50% induction even at the highest dose given (30 mg/kg i.p.). Thus NRA0045 blocks behaviors associated with activation of the mesolimbic/mesocortical dopaminergic neurons more selectively than behaviors associated with nigrostriatal dopaminergic neurons. In rats, tryptamine-induced clonic seizure, a 5-HT2 receptor-mediated behavior, was also dose-dependently inhibited by NRA0045 (ED50 = 1.7 mg/kg i.p.). Norepinephrine-induced lethality is regarded as being induced through the alpha-1 adrenoceptor. NRA0045 dose-dependently antagonized norepinephrine-induced lethality in rats (ED50 = 0.2 mg/kg i.p.). Thus NRA0045 may have a unique antipsychotic activity with regard to dopamine D4 and 5-HT2A receptors and alpha-1 adrenoceptor antagonistic activities, without producing the extrapyramidal side effects.

Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions.

NF1 was first identified as the gene responsible for the pathogenesis of the human genetic disorder neurofibromatosis type 1. cDNA cloning revealed that its putative protein product has a domain showing significant sequence homology with the mammalian Ras GTPase activating protein and two yeast Saccharomyces cerevisiae proteins, Ira1 and Ira2. The Ras GTPase activating protein-related domain of the NF1 gene product (NF1-GRD) stimulates GTPase activity of normal Ras proteins but not of oncogenic mutant Ras from both mammalian and yeast cells. Thus, in yeast, NF1-GRD can suppress the heat-shock-sensitive phenotype of ira- cells but not the same phenotype of activated RAS such as RAS2Val19 and RAS2Leu68. We have screened a pool of mutagenized NF1 expression plasmids and obtained two mutant NF1 cDNA clones that can suppress the heat-shock-sensitive phenotype of RAS2Val19 cells. One clone (NF201) suppressed RAS2Leu68, RAS2Ser41, and RAS2Val19, whereas another clone (NF204) preferentially suppressed RAS2Val19. When expressed in mammalian cells, these mutant NF1-GRDs were able to induce the morphological reversion of v-ras-transformed NIH 3T3 cells. Both wild-type and mutant NF1-GRDs can stimulate the GTPase activity of normal but not transforming Ras. We suggest that mutant NF1-GRDs may bind tightly to transforming Ras, which stays in GTP-bound conformation, thus preventing the interaction with the putative effector molecule. On the other hand, normal Ras cannot be sequestered since the bound GTP is rapidly hydrolyzed upon interaction with mutant NF1-GRD to yield Ras-GDP, which is readily released from the NF1-GRD and recycled.

Chromosomal translocation t(13;16) in a patient with idiopathic hypogonadotropic hypogonadism.

A patient with idiopathic hypogonadotropic hypogonadism (IHH) had an apparently balanced reciprocal translocation involving chromosomes 13 and 16 [t(13;16)(q14.11;q24)]. The patient's father has the same chromosomal translocation with no apparent physical abnormalities. The role of the chromosomal translocation in this patient is discussed.

[Studies on factor affecting mother-to-child HTLV-I transmission].

Epidemiological data indicate that breast feeding is the primary route of mother-to-child transmission of HTLV-I. However, serological studies show that 60 to 90% of infants who are fed breast milk from HTLV-I carriers avoid HTLV-I infection. To understand the mechanism of the virus transmission, the titer of HTLV-I antibodies and p40tax antibody and the copy number of the HTLV-I proviral DNA in peripheral lymphocytes in the HTLV-I carrier-mothers were surveyed. The seroprevalence of HTLV-I in the children born to carrier mothers was 6.1% in a normal breast feeding group, 7.7% in a group fed with freeze-thawed breast milk and none in a bottle-fed group. The difference between the seroconversion rate for the normal breast feeding group and others was statistically insignificant. Our studies also indicated that neither the titer of HTLV-I antibodies in carriers nor the duration of breast feeding was relevant to seroconversion in the children. The seroconversion rate in children was higher in p40tax antibody-positive mothers than in a seronegative group, but the difference was statistically insignificant. In conclusion, the copy number of the HTLV-I provirus in the lymphocytes of the carrier mothers was the most relevant factor in the seroconversion of infants who received breast milk from HTLV-I carriers.

A case of Graves' disease associated with Klinefelter's syndrome.

Graves' disease was found in a 41-year-old, married male patient with Klinefelter's syndrome. The patient began having finger tremor 5 years previously, and developed palpitation and weight loss 3 months prior to examination. He had a diffuse goiter, exophthalmos, and atrial fibrillation. Plasma levels of T3, T4 and free T4 were 2.8 ng/ml, 16.6 micrograms/dl and 4.5 ng/dl respectively. [123I] uptake was 53%, and TSH receptor antibody was 75%. Although he had no gynecomastia, his general physical appearance was that of typical eunuchoism. Chromosome studies showed a karyotype of 47,XXY. A diagnosis of Graves' disease associated with Klinefelter's syndrome was made.