[Show abstract][Hide abstract] ABSTRACT: Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies.
PLoS ONE 03/2014; 9(3):e90675. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bortezomib therapy is now indispensable for multiple myeloma, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. The development of orally active proteasome inhibitors with distinct mechanisms of action is therefore eagerly awaited. Previously, we identified homopiperazine derivatives as a novel class of proteasome inhibitors with a different mode of proteasome binding from bortezomib. In this study, we show that K-7174, one of proteasome-inhibitory homopiperazine derivatives, exhibits a therapeutic effect, which is stronger when administered orally than intravenously, without obvious side effects in a murine myeloma model. Moreover, K-7174 kills bortezomib-resistant myeloma cells carrying a β5-subunit mutation in vivo and primary cells from a patient resistant to bortezomib. K-7174 induces transcriptional repression of class I histone deacetylases (HDAC1, 2 and 3) via caspase-8-dependent degradation of Sp1, the most potent transactivator of class I HDAC genes. HDAC1 overexpression ameliorates the cytotoxic effect of K-7174 and abrogates histone hyperacetylation without affecting the accumulation of ubiquitinated proteins in K-7174-treated myeloma cells. Conversely, HDAC inhibitors enhance the activity of K-7174 coincided with an increase in histone acetylation. These results suggest that class I HDACs are critical targets of K-7174-induced cytotoxicity. It is highly anticipated that K-7174 increases the tolerability and convenience of patients by oral administration and has the clinical utility in overcoming bortezomib resistance as a single agent or in combination with HDAC inhibitors.
Journal of Biological Chemistry 07/2013; · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.
PLoS ONE 01/2013; 8(4):e60649. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The definition of primary splenic lymphoma is controversial, but it has been reported to be a rare disease that comprises less than 1% of all malignant lymphomas. Three cases of primary splenic diffuse large B-cell lymphoma treated at our institution are described here. Median follow-up was 34.6 months (range 8.7∼39.2) and median age at diagnosis was 72 years old (range 65∼73). In all three cases, the diagnosis was definitively established not by splenectomy but by ultrasonically guided percutaneous splenic tissue core biopsy. Using the Hans classifier, one of the cases was subclassified as the germinal center B-cell like (GCB) subtype and two as non-GCB subtype. One case was CD5-positive diffuse large B-cell lymphoma. Two patients were in Ann Arbor stage II and one was in stage III. Using the International Prognostic Index, one was categorized as Low/intermediate risk, one as high/intermediate risk, and one as high risk. All patients underwent eight cycles of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisolone followed by irradiation therapy. These three patients attained complete response. Although the follow-up period to date has been short, all patients have maintained a complete response and are currently alive. To determine whether our management protocol is valid, further observations are needed.
[Rinshō ketsueki] The Japanese journal of clinical hematology 08/2011; 52(8):703-7.
[Show abstract][Hide abstract] ABSTRACT: Anti-CD20 antibody rituximab is now essential for the treatment of CD20-positive B-cell lymphomas. Decreased expression of CD20 is one of the major mechanisms underlying both innate and acquired resistance to rituximab. In this study, we show that histone deacetylase (HDAC) inhibitors augment the cytotoxic activity of rituximab by enhancing the surface expression of CD20 antigen on lymphoma cells. HDAC inhibitors, valproic acid (VPA) and romidepsin, increased CD20 expression at protein and mRNA levels in B-cell lymphoma cell lines with relatively low CD20 expression levels. The VPA-mediated increase in CD20 expression occurred at 1 m, which is clinically achievable and safe, but insufficient for inducing cell death. Chromatin immunoprecipitation assays revealed that HDAC inhibitors transactivated the CD20 gene through promoter hyperacetylation and Sp1 recruitment. HDAC inhibitors potentiated the activity of rituximab in complement-dependent cytotoxic assays. In mouse lymphoma models, HDAC inhibitors enhanced CD20 expression along with histone hyperacetylation in transplanted cells, and acted synergistically with rituximab to retard their growth. The combination with HDAC inhibitors may serve as an effective strategy to overcome rituximab resistance in B-cell lymphomas.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 10/2010; 24(10):1760-8. · 10.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background / Purpose:
High-dose ara-C is a basic regimen for refractory or relapsed acute myeloid leukemia (AML) patients. This prospective study was undertaken to evaluate the efficacy and toxicity of FLAGM regimen in these patients. We also attempted to predict the response by determining the intracellular ara-C triphosphate (ara-CTP) concentration in vitro before the start of treatment.
The study confirms that FLAGM yielded a 75% response rate in either relapsed or refractory AML patients. Although randomized studies are still needed, FLAGM appears to be a good option for the treatment of such patients regardless of the duration of the 1st CR or previous treatment. However, this study failed to demonstrate that the fludarabine-mediated augmentation of ara-CTP production in vitro is a predictor of the response to FLAGM.
51st American Society of Hematology Annual Meeting 2009; 03/2010
[Show abstract][Hide abstract] ABSTRACT: Bortezomib is now widely used for the treatment of multiple myeloma (MM); however, its action mechanisms are not fully understood. Despite the initial results, recent investigations have indicated that bortezomib does not inactivate nuclear factor-kappaB activity in MM cells, suggesting the presence of other critical pathways leading to cytotoxicity. In this study, we show that histone deacetylases (HDACs) are critical targets of bortezomib, which specifically down-regulated the expression of class I HDACs (HDAC1, HDAC2, and HDAC3) in MM cell lines and primary MM cells at the transcriptional level, accompanied by reciprocal histone hyperacetylation. Transcriptional repression of HDACs was mediated by caspase-8-dependent degradation of Sp1 protein, the most potent transactivator of class I HDAC genes. Short-interfering RNA-mediated knockdown of HDAC1 enhanced bortezomib-induced apoptosis and histone hyperacetylation, whereas HDAC1 overexpression inhibited them. HDAC1 overexpression conferred resistance to bortezomib in MM cells, and administration of the HDAC inhibitor romidepsin restored sensitivity to bortezomib in HDAC1-overexpressing cells both in vitro and in vivo. These results suggest that bortezomib targets HDACs via distinct mechanisms from conventional HDAC inhibitors. Our findings provide a novel molecular basis and rationale for the use of bortezomib in MM treatment.
[Show abstract][Hide abstract] ABSTRACT: The CD33 antigen is expressed on leukemia cells in most patients with acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL), and in 20% of patients with acute lymphoblastic leukemia (ALL), while it is absent from pluripotent hematopoietic stem cells and nonhematopoietic cells. Gemtuzumab ozogamicin (GO) is an immunoconjugate of an anti-CD33 antibody linked to calicheamicin, which is a potent cytotoxic agent that causes double-strand DNA breaks, resulting in cell death. GO was developed against CD33 antigen-positive leukemias. The aim of this study was to investigate the cytotoxic effects of this agent in combination with conventional antileukemic agents.
The cytotoxic effects of GO in combination with antileukemic agents were studied against human CD33 antigen-positive leukemia HL-60, U937, TCC-S and NALM20 cells. The leukemia cells were exposed simultaneously to GO and to the other agents for 4 days. Cell growth inhibition was determined using a MTT reduction assay. The isobologram method was used to evaluate the cytotoxic interaction.
GO produced synergistic effects with mitoxantrone, additive effects with cytarabine, daunorubicin, idarubicin, doxorubicin, etoposide and 6-mercaptopurine, and antagonistic effects with methotrexate and vincristine.
Our findings suggest that the simultaneous administration of GO with most agents studied would be advantageous for antileukemic activity. The simultaneous administration of GO with methotrexate or vincristine would have little cytotoxic effect, and this combination may be inappropriate. These findings may be useful in clinical trials of combination chemotherapy including GO or other monoclonal antibodies linked to calicheamicin.
Anticancer research 11/2009; 29(11):4589-96. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 38 consecutive, previously untreated adult patients with acute non-lymphocytic leukaemia (ANLL) were treated with BHAC-AMP (N4-behenoyl-1-β-D-arabinofuranosyl-cytosine, aclacinomycin A, 6-mercaptopurine, and prednisolone) therapy between March 1980 and February 1985. 25 patients (65.8%) achieved complete remission (CR). Median CR duration and median survival of patients who achieved CR were 14, and 24 months, respectively. The Kaplan-Meier analysis revealed a probability for remaining in CR of 18.0% at 5 years. Analysis of failure cases revealed that most of them were due to resistant disease. Major toxicities were infection, diarrhoea, liver dysfunction, nausea and vomiting but these were acceptable. The results indicate that BHAC-AMP therapy is comparable to the regimen with daunorubicin and cytosine arabinoside and a further clinical trial is necessary for previously untreated adult patients with ANNL.
European Journal Of Haematology 04/2009; 43(1):1 - 6. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pemetrexed and docetaxel show clinical activities against a variety of solid tumors including lung cancers. To identify the optimal schedule for combination, cytotoxic interactions between pemetrexed and docetaxel were studied at various schedules using three human lung cancer cell lines A-549, Lu-99, and SBC-5 in vitro.
Cells were incubated with pemetrexed and docetaxel simultaneously for 24 or 120 h. Cells were also incubated with pemetrexed for 24 h, followed by a 24 h exposure to docetaxel, and vice versa. Growth inhibition was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis. Cytotoxic interactions were evaluated by the isobologram method.
Simultaneous exposure to pemetrexed and docetaxel for 24 and 120 h produced antagonistic effects in all three cell lines. Pemetrexed (24 h) followed by docetaxel (24 h) produced additive effects in A-549 cells and synergistic effects in Lu-99 and SBC-5 cells. Docetaxel followed by pemetrexed produced additive effects in A-549 and Lu-99 cells and antagonistic effects in SBC-5 cells. The results of cell cycle analysis were fully consistent with those of isobologram analysis, and provide the molecular basis of the sequence-dependent difference in cytotoxic interactions between the two agents.
Sequential administration of pemetrexed followed by docetaxel may provide the greatest anti-tumor effects for this combination in the treatment of lung cancer.
Cancer Chemotherapy and Pharmacology 04/2009; 64(6):1129-37. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to define the molecule(s) responsible for CAM-DR of MM. Using four bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta1-integrin), CD44, CD49d (alpha4-integrin, a subunit of VLA-4), CD54 (intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically downregulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.
[Show abstract][Hide abstract] ABSTRACT: The efficacy and toxicity of combined paclitaxel (PTX) and gemcitabine (GEM) was evaluated as a protocol for first-line chemotherapy in 40 patients with advanced non-small-cell lung cancer (NSCLC).
Paclitaxel, 100 mg/m(2), was administered intravenously (IV) as a 1-h infusion, followed by GEM, 1,000 mg/m(2), IV over 30 min on days 1 and 8 of a 21-day cycle. The median age of patients was 66 years with a range of 33-75 years. Nearly all patients (39/40) had an ECOG performance status of 0 or 1. Thirteen patients (32%) had initial stage IIIB disease and 27 patients (68%) had stage IV disease. Histological subtypes were adenocarcinoma (73%) and squamous cell carcinoma (25%).
Twenty-two patients (55%) achieved a partial response and none achieved a complete response, giving an overall response rate of 55% (95% confidence interval: 38.2-71.8%). Disease stability was achieved in 14 patients (35%), and 4 patients (10%) had progressive disease. The median survival time was 11.9 months (95% CI: 10.3-14 months), with a 1-year survival rate of 47.5%. Grade 3 or 4 hematological toxicities observed included neutropenia in 37.5%, anemia in 2.5%, and thrombocytopenia in 5.0% of these patients. Non-hematologic toxicities were mild, with the exception of grade 3 and 4 pneumonitis. There were no deaths due to toxicity.
Weekly chemotherapy with PTX plus GEM is effective and is acceptable for the first line treatment of advanced NSCLC.
Cancer Chemotherapy and Pharmacology 11/2008; 64(1):73-8. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: PKC412 is a staurosporine derivative that inhibits several protein kinases including FLT3, and is highly anticipated as a novel therapeutic agent for acute myeloblastic leukemia (AML) carrying FLT3 mutations. In this study, we show that PKC412 exerts differential cell cycle effects on AML cells depending on the presence of FLT3 mutations. PKC412 elicits massive apoptosis without markedly affecting cell cycle patterns in AML cell lines with FLT3 mutations (MV4-11 and MOLM13), whereas it induces G2 arrest but not apoptosis in AML cell lines without FLT3 mutations (THP-1 and U937). In MV4-11 and MOLM13 cells, PKC412 inactivates Myt-1 and activates CDC25c, leading to the activation of CDC2. Activated CDC2 phosphorylates Bad at serine-128 and facilitates its translocation to the mitochondria, where Bad triggers apoptosis. In contrast, PKC412 inactivates CDC2 by inducing serine-216 phosphorylation and subsequent cytoplasmic sequestration of CDC25c in THP-1 and U937 cells. As a result, cells are arrested in the G2 phase of the cell cycle, but do not undergo apoptosis because Bad is not activated. The FLT3 mutation-dependent differential cell cycle effect of PKC412 is considered an important factor when PKC412 is combined with cell cycle-specific anticancer drugs in the treatment of cancer and leukemia.
[Show abstract][Hide abstract] ABSTRACT: With melanoma, as with many other malignancies, aberrant transcriptional repression is a hallmark of refractory cancer. To restore gene expression, use of a histone deacetylase inhibitor (HDACi) is expected to be effective. Our recent DNA micro-array analysis showed that the HDACi depsipeptide (FK228) significantly enhances gp100 antigen expression. Herein, we demonstrate that depsipeptide promotes tumor-specific T-cell-mediated killing of B16/F10 murine melanoma cells. First, by a quantitative assay of caspase-3/7 activity, a sublethal dose of depsipeptide was determined (ED50: 5 nM), in which p21(Waf1/Cip1) and Fas were sufficiently evoked concomitantly with histone H3 acetylation. Second, the sublethal dose of depsipeptide treatment with either a recombinant Fas ligand or tumor-specific T cells synergistically enhanced apoptotic cell death in B16/F10 cells in vitro. Furthermore, we found that depsipeptide increased levels of perforin in T cells. Finally, in vivo metastatic growth of B16/F10 in the lung was significantly inhibited by a combination of depsipeptide treatment and immune cell adoptive transfer from immunized mice using irradiated B16 cells and gp100-specific (Pmel-1) TCR transgenic mice (P<0.05, vs cell transfer alone). Consequently, employment of a transcriptional modulation strategy using HDACis might prove to be a useful pretreatment for human melanoma immunotherapy.
Journal of Investigative Dermatology 06/2008; 128(6):1506-16. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated the long-term outcome of very dose-intensive chemotherapy (TCC-NHL-91) for advanced intermediate-grade lymphoma, in which an eight-cycle regimen with 11 drugs was given with granulocyte colony-stimulating factor (G-CSF) support (total 18 weeks). Fifty-nine patients were treated during February 1, 1991 and March 31, 2001 (median age: 48 years). Forty-three patients (73%) were in a high-intermediate risk or high-risk group (HI/H) according to the age-adjusted International Prognostic Index (aa-IPI). Forty-six patients received 7 or 8 cycles of therapy. Ten of 15 patients over age 60 stopped before 7 cycles. Forty-three patients with an initial bulky mass or a residual mass received involved-field radiation. Overall, 56 patients (95%) achieved complete remission (CR). Grade 4 hematotoxicity was observed in all patients. With a median follow-up of 128 months, the 10-year overall survival (OS) and progression-free survival (PFS) rates were 76% and 61%, respectively. Neither aa-IPI risk factors nor the index itself was associated with response, OS, or PFS. One patient died of sepsis during the therapy and one died of secondary leukemia. This retrospective study suggests that the TCC-NHL-91 regimen achieves high CR, OS, and PFS in patients with advanced intermediate-grade lymphoma up to 60 years old and may be a valuable asset in the management of this disease. Further evaluation and prospective studies of the TCC-NHL-91 are warranted.
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics 02/2008; 17(3):137-49. · 0.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was designed to determine the optimal high dose for cytosine arabinoside (ara-C) in combination with fludarabine, granulocyte colony-stimulating factor, and mitoxantrone (FLAGM) in adult patients with relapsed or refractory acute myeloid leukemia. Nine patients were enrolled at increasing dosage levels of ara-C (8, 12, and 16 g/m2 per dose level). Ara-C and fludarabine were administered once a day at level 1, once or twice a day at level 2, and twice a day at level 3. All patients had grade 4 hematologic toxicity. The most common adverse events were of grade 2 or less, with nausea and vomiting being the most common (6 events), followed by diarrhea (5 events), and rash (5 events). Of the 13 grade 3 nonhematologic toxicities reported, the 2 most common were febrile neutropenia (6 events) and disseminated intravascular coagulation (3 events). No early deaths were observed. FLAGM with high-dose ara-C was considered safe for patients, and the recommended dosage of ara-C in this study was 2 g/m2 every 12 hours for a total dose of 16 g/m2.
International Journal of Hematology 12/2007; 86(4):343-7. · 1.68 Impact Factor