Hitoshi Kusaba

Hypomethylation Status of CpG Sites at the Promoter Region and Overexpression of the Human MDR1 Gene in Acute Myeloid Leukemias

Selection of human cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance 1 gene (MDR1), which encodes the cell surface P-glycoprotein, as a result of gene amplification or transcriptional activation. Moreover, overexpression of the MDR1 gene has been shown to be associated closely with clinical outcome in various hematological malignancies, including acute myeloid leukemia (AML). However, the precise mechanism underlying overexpression of the MDR1 gene during acquisition of drug resistance remains unclear. We recently described an inverse correlation between the methylation status of CpG sites at the promoter region and expression of the MDR1 gene in malignant cell lines. In this study, we expanded this analysis to 42 clinical AML samples. We adapted a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for gene expression and a quantitative PCR after digestion by Hpa II for methylation status of the MDR1 gene. We observed a statistically significant inverse correlation between methylation and MDR1 expression in clinical samples. The hypomethylation status of the MDR1 promoter region might be a necessary condition for MDR1 gene overexpression and establishment of P-glycoprotein-mediated multidrug resistance in AML patients.

B cell activation regulates exosomal HLA production

Exosomes are nanovesicles produced constitutively and inducibly by several types of cells. They are generated as intraluminal vesicles of multivesicular bodies and express MHC and several endosomal/lysosomal proteins. In spite of their potential role in cellular immunity, the regulatory mechanisms of exosome production are largely unknown. In this study, we have established a novel ELISA system to quantify exosomal HLA using a combination of anti‐HLA class I and anti‐HLA‐DR mAb. We found that exosomal HLA production of B cells was enhanced by contact with CD4+ T cells. Neutralizing anti‐CD154 (CD40L) mAb inhibited this effect, and a soluble CD40L significantly increased production of exosomal HLA in B cells. In addition, B cell stimulation via BCR and TLR9 enhanced their production while IL‐4 stimulation alone failed to do so. Strikingly, an inhibitor of the classical NF‐κB pathway drastically inhibited exosomal HLA production in stimulated B cells, indicating that the classical NF‐κB pathway is critical for exosomal HLA production in B cells. Together, these findings suggest a pivotal role of B cell activation in exosomal HLA production in vivo.

Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells

Purpose: Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been demonstrated to exhibit its antitumor activity by the blockade of EGF receptor, the role of signaling pathways downstream of EGFR in gefitinib sensitivity remains unknown. In this study, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity. Methods: Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively. Results: Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Gefitinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-src-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially reversed the gefitinib resistance, with concomitant inhibition of Akt and Erk. Conclusion: Our results suggest that activated Ras and Src could induce gefitinib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationale for assessment of these specific signaling molecules downstream of EGFR to customize treatment.

Phase I/II trial of 2‐weekly docetaxel combined with cisplatin plus fluorouracil in metastatic esophageal cancer (JCOG0807)

We carried out a phase I/II trial of adding 2‐weekly docetaxel to cisplatin plus fluorouracil (CF) therapy (2‐weekly DCF regimen) in esophageal cancer patients to investigate its safety and antimetastatic activity. Patients received 2‐weekly docetaxel (30 mg/m2 [dose level (DL)1] or 40 mg/m2 [DL2] with a 3 + 3 design in phase I, on days 1 and 15) in combination with fixed‐dose CF (80 mg/m2 cisplatin, day 1; 800 mg/m2 fluorouracil, days 1–5) repeated every 4 weeks. The primary endpoint was dose‐limiting toxicity (DLT) in phase I and central peer review‐based response rate in phase II. At least 22 responders among 50 patients were required to satisfy the primary endpoint with a threshold of 35%. Sixty‐two patients were enrolled in phase I and II. In phase I, 10 patients were enrolled with DLT of 0/3 at DL1 and 2/7 in DL2. Considering DLT and treatment compliance, the recommended phase II dose was determined as DL1. In phase II, the response rate was 62% (P < 0.0001; 95% confidence interval, 48–75%); median overall survival and progression‐free survival were 11.1 and 5.8 months, respectively. Common grade 3/4 adverse events were neutropenia (25%), anemia (36%), hyponatremia (29%), anorexia (24%), and nausea (11%). No febrile neutropenia was observed. Pneumonitis caused treatment‐related death in one patient. The 2‐weekly DCF regimen showed promising antimetastatic activity and tolerability. A phase III study comparing this regimen with CF therapy is planned by the Japan Clinical Oncology Group. This study was registered at the UMIN Clinical Trials Registry as UMIN 000001737.

Enhanced expression of PCAF endows apoptosis resistance in cisplatin-resistant cells

Histone acetyltransferase (HAT) regulates transcription. We have previously shown that two HAT genes, Clock and Tip60, are overexpressed, and upregulate glutathione biosynthesis and the expression of DNA repair genes in cisplatin-resistant cells. To better understand the mechanism of HAT-related drug resistance, we investigated the role of another HAT gene, p300/CBP-associated factor (PCAF), and found that PCAF was also overexpressed in cisplatin-resistant cells and endowed an antiapoptotic phenotype through enhanced E2F1 expression. PCAF-overexpressing cells showed enhanced expression of E2F1 and conferred cell resistance to chemotherapeutic agents. Downregulation of PCAF decreased E2F1 expression and sensitized cells to chemotherapeutic agents. Moreover, knockdown of PCAF induced G1 arrest and apoptosis. These results suggest that PCAF is one of pleiotropic factors for drug resistance and seems to be critical for cancer cell growth. Mol Cancer Res; 8(6); 864–72. ©2010 AACR.

Non-cross resistance of ZD0473 in acquired cisplatin-resistant lung cancer cell lines

ZD0473 is a new generation platinum agent that, in preclinical studies, shows evidence of an extended spectrum of anti-tumor activity and overcomes platinum resistance mechanisms. The drug contains a bulky methylpyridine ligand at its platinum center, which is responsible for its ability to overcome platinum resistance. We examined the growth inhibitory effects of ZD0473 in human lung cancer cell lines resistant to cisplatin in vitro. Four cisplatin resistant human lung cancer cell lines (PC-14/CDDP, SBC-3/CDDP, PC-9/CDDP, H69/CDDP) showed the expected resistance to cisplatin but were non-cross, or much less, resistant to ZD0473, as determined by an MTT assay. A reduction in the intracellular accumulation of cisplatin, but not of ZD0473, was observed in the PC-14/CDDP cells compared with the levels in PC-14 parental cells. The reduction in cisplatin accumulation is considered a major mechanism of the acquired cisplatin resistance in PC-14/CDDP cells. Therefore, the increase in platinum accumulation is considered a possible mechanism underlying the activity of ZD0473 in cisplatin-resistant cells. Glutathione-mediated resistance to cisplatin was also overcome by ZD0473 in PC-14/CDDP cells. In addition, we showed that the intraperitoneal administration of ZD0473 at its maximum tolerable dose in mice produced a marked in vivo antitumor activity against cisplatin-resistant PC-14/CDDP tumors. These results suggest that ZD0473 may be a potent agent in human lung cancer cells with multifactorial cisplatin resistance.

Oxaliplatin-induced allergic reaction in patients with colorectal cancer in Japan

BackgroundOxaliplatin is a platinum compound that is clinically effective for colorectal cancer (CRC), in combination with 5-fluorouracil (5-FU) and leucovorin (LV), and it is widely used for metastatic disease and for the adjuvant treatment of stage III CRC. With the increasing use of oxaliplatin in Japan, serious adverse events have been experienced other than hematologic and neurologic toxicities.MethodsIn order to clarify the clinical features of allergic reactions to oxaliplatin, we retrospectively investigated CRC patients who had received oxaliplatin-based chemotherapies.ResultsOne hundred and twenty-five CRC patients who had been treated with FOLFOX regimens (containing oxaliplatin, 5-FU, and LV) were examined, and 21 patients (17%) were found to have developed allergic reactions. Sixteen patients (13%) had grade 1/2 adverse events, classified according to the common terminology criteria for adverse events (CTC-AE) version 3.0 and 5 (4%) had grade 3/4 adverse events. The allergic reaction appeared after a median number of nine cycles (range, 2–15 cycles). Previous chemotherapy included 5-FU/LV, CPT-11, and S-1. All of the patients with allergic reactions recovered completely when treated with antiallergy drugs. Oxaliplatin was reintroduced in 11 patients, with the use of prophylactic agents; allergic reaction to the reintroduction was not observed in 8 patients and grade 1/2 allergic reactions developed in 3 patients. No correlation was identified between allergic reaction and patients’ background characteristics such as sex, history of allergy, and profile of other adverse events.ConclusionAllergic reactions to oxaliplatin remain an important issue for patients being able to safely continue effective chemotherapies; further analysis will be needed to establish methods for the prediction and prophylaxis of such reactions.

Human Nanog pseudogene8 promotes the proliferation of gastrointestinal cancer cells

There is emerging evidence that human solid tumor cells originate from cancer stem cells (CSCs). In cancer cell lines, tumor-initiating CSCs are mainly found in the side population (SP) that has the capacity to extrude dyes such as Hoechst 33342. We found that Nanog is expressed specifically in SP cells of human gastrointestinal (GI) cancer cells. Nucleotide sequencing revealed that NanogP8 but not Nanog was expressed in GI cancer cells. Transfection of NanogP8 into GI cancer cell lines promoted cell proliferation, while its inhibition by anti-Nanog siRNA suppressed the proliferation. Immunohistochemical staining of primary GI cancer tissues revealed NanogP8 protein to be strongly expressed in 3 out of 60 cases. In these cases, NanogP8 was found especially in an infiltrative part of the tumor, in proliferating cells with Ki67 expression. These data suggest that NanogP8 is involved in GI cancer development in a fraction of patients, in whom it presumably acts by supporting CSC proliferation.

Maintenance of hypomethylation status and preferential expression of exogenous human MDR1/PGY1 gene in mouse L cells by YAC mediated transfer.

Selection of cells for resistance to vincristine or doxorubicin often induces overexpression of the multidrug resistance (MDR) genes, which encode the cell surface P-glycoproteins, as a result of gene amplification, transcriptional activation, or mRNA stabilization. The LMD1 and LMD4 cell lines were established after the transfer into mouse L cells of two independent yeast artificial chromosome clones containing 300 and 850 kb, respectively, of the human MDR locus. The human MDR1/PGY1 gene, but not the endogenous mouse mdr1a and mdr1b genes, was overexpressed as a result of gene amplification and transcriptional activation in various sublines of LMD1 and LMD4 cells selected for resistance to vincristine. Then we asked why human MDR1/PGY1 gene, but not mouse relevant gene, was expressed. Determination of the methylation status of cytosine residues at Msp I/Hap II cleavage sites (CCGG) in the promoter regions of human MDR1/PGY1 and mouse mdr1a revealed hypomethylation and hypermethylation of the human and mouse genes, respectively in LMD1, LMD4, and their vincristine-resistant derivatives. Various vincristine-resistant sublines were also established after exposure of LMD1 cells for 48 h to 5-aza-2′-deoxycytidine, an inhibitor of DNA methyltransferase. These sublines exhibited overexpression of mouse mdr1a and mdr1b, but not of human MDR1/PGY1, as well as hypomethylation of the mouse mdr1a promoter region. Thus, the selective expression of human or mouse MDR genes in this cell system appears to be related to the methylation status of the respective gene promoter regions.

Bi-cytopenia possibly induced by anti-PD-1 antibody for primary malignant melanoma of the esophagus: A case report.

Background:Anti-programmed cell death 1 antibody nivolumab is a promising agent for various cancers. Immune-related adverse events are recognized; however, bi-cytopenia with nivolumab has not been reported. Case presentation:A 73-year-old man was diagnosed with advanced primary malignant melanoma of the esophagus with liver, lung, and lymph node metastases. Previous therapies including dacarbazine and radiation of 39 Gy to the esophageal region were performed, but the liver metastases deteriorated. The patient was then administered nivolumab (2 mg/kg, every 3 weeks). After 3 cycles, the esophageal tumor and lymph nodes showed marked reductions in size, the lung metastases disappeared, and the liver metastases shrank partially. The treatment continued with 7 cycles for 4 months. However, severe anemia and thrombocytopenia appeared in the 6th cycle, and intermittent blood transfusions were required. The patient received high-dose intravenous methylprednisolone therapy for bi-cytopenia, but it was ineffective. Seven months after the initiation of nivolumab, the patient died of tumor. Although the mechanisms of bi-cytopenia were unclear, it could have been induced by nivolumab. Conclusion:The present case shows a rare but serious life-threatening bi-cytopenia possibly associated with nivolumab and suggests the importance of awareness of hematological adverse events during nivolumab therapy.