Search

Systemic inflammatory response syndrome and sepsis are considered to contribute to hypercytokinemia in both patients with severe infection and immunocompromised condition. Past research has demonstrated that antibiotics and antifungals not only have antimicrobial efficacy but also affect the immune system. We previously examined whether immune cells were modulated by antibiotics such as tetracyclines or macrolides. The modulation of lipopolysaccharide-stimulated cells by those agents was elucidated. However, few reports about the modulation of the immune system by antifungal agents were found. In this study, the production of pro-inflammatory cytokines and chemokines and signaling pathways involved were investigated in zymosan-activated THP-1 cells. The effects were examined using antifungal agents such as echinocandin including caspofungin (CAS) and micafungin. Pro-inflammatory cytokine and chemokine levels were determined using enzyme-linked immunosorbent assay. Protein phosphorylation was evaluated by western blot analysis. CAS significantly decreased zymosan-induced pro-inflammatory cytokine and chemokine release in THP-1 cells. CAS (30 µg/mL) also downregulated tumor necrosis factor alpha levels, as shown by enzyme-linked immunosorbent assay. In western blot analysis, inhibitor of nuclear factor-kappa-B alpha, p38, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and nuclear factor of activated T-cells phosphorylation and activation of caspase-1 and spleen tyrosine kinase (Syk) were downregulated. The major underlying mechanism of pro-inflammatory cytokine and chemokine suppression by CAS is to inhibit activation of Syk and its downstream signaling molecules. Based on the results, it can be concluded that CAS activity possibly involves Syk signaling pathways and has potential to prevent hypercytokinemia in fungal sepsis.

Background Human airway smooth muscle cells (ASMCs) contribute to bronchial contraction and airway hyperresponsiveness in patients with bronchial asthma. They also generate cytokines, chemokines, and matricellular proteins. Ovarian cancer G protein-coupled receptor 1 (OGR1) senses extracellular protons and mediates the production of interleukin-6 (IL-6) and connective tissue growth factor (CTGF) in ASMCs. Methods ASMCs were stimulated for the indicated time by pH 6.3 or pH 7.4-adjusted Dulbecco’s Modified Eagle Medium (DMEM) containing 0.1% bovine serum albumin (BSA) (0.1% BSA-DMEM). As a control stimulant, pH 7.4-adjusted 0.1% BSA-DMEM containing 10 ng/mL tumor necrosis factor-α (TNF-α) was used. Interleukin-8/C-X-C motif chemokine ligand 8 (CXCL8) mRNA expression in ASMCs was quantified by RT-PCR using real-time TaqMan technology. CXCL8 secreted from ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Phosphorylation at serine 536 of NF-κB p65 and binding of p65 to oligonucleotide containing an NF-κB consensus binding site were analyzed by Western blotting and an ELISA-based kit. Results Acidic pH induced a significant increase of CXCL8 mRNA expression and CXCL8 protein secretion in ASMCs. ASMCs transfected with small interfering RNA (siRNA) targeted for OGR1 produced less CXCL8 compared with those transfected with non-targeting siRNA. Protein kinase C (PKC) inhibitor, MEK1/2 inhibitor, and the inhibitor of IκB phosphorylation reduced acidic pH-stimulated CXCL8 production in ASMCs. Dexamethasone also inhibited acidic pH-stimulated CXCL8 production of ASMCs in a dose-dependent manner. Dexamethasone did not affect either phosphorylation or binding to the consensus DNA site of NF-κB p65. Conclusions CXCL8 released from ASMCs by extracellular acidification may play a pivotal role in airway accumulation of neutrophils. Glucocorticoids inhibit acidic pH-stimulated CXCL8 production independent of serine 536 phosphorylation and the binding to DNA of NF-κB p65, although NF-κB activity is essential for CXCL8 production in ASMCs.

Although spiral bacteria are uncommon, they cause bacteremia. We evaluated their characteristics, in particular, the time from the start of blood culture to the first report of a positive result to physicians, using the BACTEC blood culture system. In cases of spiral bacteremia, an extended treatment period should be considered.

Enterococci is one of a major cause of bloodstream infection (BSI). Because of its intrinsic drug-resistant nature, empiric antibiotic treatment tends to be inappropriate. We conducted a single-center retrospective cohort study to evaluate the impact of Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOFMS) on the improvement of early antibiotic treatment for enterococcal infection. We also investigated the 28-day mortality, length of hospitalization and duration of antibiotic treatment for enterococcal bacteremia. A total of 173 BSI episodes (172 patients) between June 2012 and June 2019 were enrolled. Patients were divided into 2 groups before (n = 82) and after (n = 91) the implementation of MALDI-TOFMS (Control group and MALDI-TOF group, respectively). Almost an equal number of Enterococcus faecalis and Enterococcus faecium cases were identified in each group (51.2% and 48.8%, and 47.3% and 52.7% in each group). By implementing MALDI-TOFMS, the time to definitive antibiotic treatment was significantly improved (median 3 vs 1 days, p 0.001). The 28-day mortality (29.3% vs 26.4%, p = 0.63) and length of hospitalization (median 16 vs 19 days, p = 0.58) were not significantly different. The duration of antibiotic treatment did not significantly differ between the two groups (median 11 vs 11 days, p = 0.78), but the duration was often shorter in older patients (74 years old) in MALDI-TOF group, excluding those in the terminal phase of malignancy. By implementing MALDI-TOFMS, the time to definitive antibiotic treatment was significantly shortened. Although associated outcomes did not significantly differ, the duration of antibiotic treatment may be shortened.

YM155, a novel small molecule inhibitor of survivin, shows broad anticancer activity. Here, we have focused on the cytotoxic activity of YM155 against multiple myeloma (MM) including cytokinetically quiescent (G0/G1) cells and bortezomib resistant cells. YM155 strongly inhibited the growth of MM cell lines with the IC50 value of below 10 nM. YM155 also showed potent anti-myeloma activity in mouse xenograft model. YM155 suppressed the expression of survivin and rapidly directed Mcl-1 protein for proteasome degradation. YM155 abrogated the interleukin-6-induced STAT3 phosphorylation, subsequently blocked Mcl-1 expression and induced apoptosis in MM cells. Triple-color flow cytometric analysis revealed that YM155 potently induced cell death of MM cells in G0 phase. Quiescent primary MM cells were also sensitive to YM155. We established bortezomib-resistant MM cell line, U266/BTZR1, which possess a point mutation G322A. YM155 exhibited similar cytotoxic potency against U266/BTZR1 compared with parental cells. Interestingly, survivin expression was markedly elevated in U266/BTZR1 cells. Treatment with YM155 significantly down-regulated this increased survivin and Mcl-1 expression in U266/BTZR1 cells. In conclusion, our data indicate that YM155 exhibits potent cytotoxicity against quiescent (G0/G1) MM cells and bortezomib-resistant cells. These unique features of YM155 may be beneficial for the development of new therapeutic strategies to eliminate quiescent MM cells and overcome bortezomib resistance.

s Background Arsenic trioxide (ATO) is reported to be an effective therapeutic agent in acute promyelocytic leukemia (APL) through inducing apoptotic cell death. Buthionine sulfoximine (BSO), an oxidative stress pathway modulator, is suggested as a potential combination therapy for ATO-insensitive leukemia. However, the precise mechanism of BSO-mediated augmentation of ATO-induced apoptosis is not fully understood. In this study we compared the difference in cell death of HL60 leukemia cells treated with ATO/BSO and ATO alone, and investigated the detailed molecular mechanism of BSO-mediated augmentation of ATO-induced cell death. Methods HL60 APL cells were used for the study. The activation and expression of a series of signal molecules were analyzed with immunoprecipitation and immunoblotting. Apoptotic cell death was detected with caspases and poly (ADP-ribose) polymerase activation. Generation of intracellular reactive oxygen species (ROS) was determined using a redox-sensitive dye. Mitochondrial outer membrane permeabilization was observed with a confocal microscopy using NIR dye and cytochrome c release was determined with immunoblotting. Small interfering (si) RNA was used for inhibition of gene expression. Results HL60 cells became more susceptible to ATO in the presence of BSO. ATO/BSO-induced mitochondrial injury was accompanied by reduced mitochondrial outer membrane permeabilization, cytochrome c release and caspase activation. ATO/BSO-induced mitochondrial injury was inhibited by antioxidants. Addition of BSO induced phosphorylation of the pro-apoptotic BCL2 protein, BIMEL, and anti-apoptotic BCL2 protein, MCL1, in treated cells. Phosphorylated BIMEL was dissociated from MCL1 and interacted with BAX, followed by conformational change of BAX. Furthermore, the knockdown of BIMEL with small interfering RNA inhibited the augmentation of ATO-induced apoptosis by BSO. Conclusions The enhancing effect of BSO on ATO-induced cell death was characterized at the molecular level for clinical use. Addition of BSO induced mitochondrial injury-mediated apoptosis via the phosphorylation of BIMEL and MCL1, resulting in their dissociation and increased the interaction between BIMEL and BAX.

Barasertib, an aurora B inhibitor, terminates cell division, introduces polyploidy, and consequently causes apoptosis. In the present study, we evaluated the effect of the combination of barasertib and cytarabine (ara-C), a key agent for leukemia chemotherapy, on leukemic cells in vitro. Human leukemia HL-60 cells and HL-60/ara-C20 cells, a 20-fold ara-C-resistant variant, were used. The 50% growth inhibitory concentrations of an active metabolite of barasertib, barasertib-hydroxyquinazoline-pyrazol-aniline (Barasertib-HQPA), and ara-C were 51 nM and 300 nM for HL-60 cells and 70 nM and 5300 nM for HL-60/ara-C20 cells, respectively. Barasertib-HQPA induced polyploidy with a subsequent induction of sub-G1 phase apoptosis, indicating the M-phase specific cytotoxicity. Cells treated with the S-phase specific ara-C accumulated in S phase and subsequently died through apoptosis. When HL-60 cells were treated with barasertib-HQPA and ara-C in combination, a greater-than-additive apoptosis was induced. This enhancement was obtained when the cells were treated with barasertib-HQPA prior to ara-C (37.9% sub-G1) or with both concurrently (31.2% sub-G1), but not with ara-C prior to barasertib-HQPA (17.8% sub-G1). The combination effects were similarly obtained in HL-60/ara-C20 cells with 19.7% sub-G1 for barasertib-HQPA→ara-C, 18.4% sub-G1 for both concurrently, and 13.8% sub-G1 for ara-C→barasertib-HQPA, and another leukemic U937 cells with 25.4% sub-G1 for barasertib-HQPA→ara-C, 28.2% sub-G1 for both concurrently, and 16.0% sub-G1 for ara-C→barasertib-HQPA. Barasertib-HQPA inhibited aurora B autophosphorylation and histone H3 phosphorylation in all the cell lines. Barasertib-HQPA did not inhibit DNA synthesis, allowing ara-C incorporation into DNA for its cytotoxicity. Thus, barasertib-HQPA and ara-C provided a greater-than-additive cytotoxicity in leukemic cells in vitro.

Clofarabine (CAFdA) is incorporated into leukemic cells by human equilibrative nucleoside transporters (hENT) 1 and 2 and human concentrative nucleoside transporter (hCNT) 3. CAFdA is then phosphorylated to the active metabolite CAFdA triphosphate (CAFdATP) by deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Two novel CAFdA-resistant variants were established and their mechanism of resistance was elucidated. The two variants (HL/CAFdA20, HL/CAFdA80) were 20-fold and 80-fold more CAFdA-resistant than HL-60, respectively. mRNA levels of hENT1, hENT2 and hCNT3 were 53.9, 41.8 and 17.7% in HL/CAFdA20, and 30.8, 13.9 and 7.9% in HL/CAFdA80, respectively, compared with HL-60. Thus, the total nucleoside transport capacity of CAFdA was reduced in both variants. dCK protein levels were 1/2 in HL/CAFdA20 and 1/8 in HL/CAFdA80 of that of HL-60. dGK protein levels were 1/2 and 1/3, respectively. CAFdATP production after 4-h incubation with 10 μM CAFdA was 20 pmol/10(7) cells in HL/CAFdA20 and 3 pmol/10(7) cells in HL/CAFdA80 compared with 63 pmol/10(7) cells in HL-60. The decreased CAFdATP production attenuated drug incorporation into both mitochondrial and nuclear DNA. In addition, the two variants were resistant to CAFdA-induced apoptosis due to Bcl2 overexpression and decreased Bim. A Bcl2 inhibitor, ABT737, acted synergistically with CAFdA to inhibit the growth with combination index values of 0.27 in HL/CAFdA20 and 0.23 in HL/CAFdA80, compared with 0.65 in HL-60. Thus, the mechanism of resistance primarily included not only reduced CAFdATP production, but also increased antiapoptosis. The combination of CAFdA and ABT737 may be effective against CAFdA resistance.

Multiple myeloma (MM) is a molecularly heterogeneous hematologic malignancy and remains mostly incurable despite the recent improvement of treatment strategies by several novel agents. Therefore, it is important to develop more efficacious drug against MM. YM155, a novel small molecule suppressant of survivin, shows anti-proliferative activities against various human cancer cells. YM155 was identified in a survivin gene promoter assay by high throughput screening of chemical libraries. In the present study, we investigated the cytotoxic mechanism of YM155 against human myeloma cells including bortezomib (BTZ) resistant cells (U266/BTZ). Three myeloma cell lines, U266, KMS-11 and KMS-12, were employed. YM155 inhibited the cell growth of these cell lines with the IC50 value of below 5 nM. YM155 suppressed the expression of mRNA and protein of survivin. We also found that YM155 inhibited the protein expression of Mcl-1, as an essential anti-apoptotic protein for survival of myeloma cells. In addition, we observed that YM155 markedly suppressed the phosphorylation of STAT3, which is known as transcription factor of Mcl-1. When KMS-12 cells were incubated with IL-6, phosphorylation of STAT3 and upregulation of Mcl-1 protein were observed. Treatment of KMS-12 with YM155 inhibited these events and eventually induced apoptosis in KMS-12 cells. Interestingly, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. RQ-PCR analysis indicated that the level of Mcl-1 mRNA was not affected after YM155 treatment. Immunoblot analysis showed that proteasome inhibitor MG-132 blocked the inhibition of Mcl-1 expression by YM155, suggesting that proteasome-mediated degradation is involved in YM155-induced Mcl-1 downregulation. MM is a low-growth-fraction disease and low proliferation of MM seems to contribute to resistance to various anticancer drugs. To determine whether YM155 shows cytotoxic effect against quiescent (G0/G1) MM cells, U266 were cultured in low-serum medium to enrich the G0/G1 population. Dual-parametric flow cytometric analysis using Hoechest33342 and the RNA specific dye pyronin Y revealed that YM155 potently induced cell death of quiescent (G0/G1) MM cells. In quiescent MM cells, inhibitory effect of YM155 on Mcl-1 protein expression was much stronger than that on survivin. We also examined whether similar effect of YM155 could be observed in primary MM cells. The majority of primary MM cells from patients was found to be in quiescent phase by cell-cycle analysis. YM155 showed similar cytotoxic activity against primary MM cells. In contrast, Ara-C, the S-phase specific anticancer drug, never killed quiescent primary MM cells. We established BTZ-resistant MM cell line (U266/BTZ). The IC50 value was 45-fold higher than its parental cell line. DNA sequencing data indicated that U266/BTZ cells possess a point mutation, G322A, in the gene encoding the proteasome beta-5 subunit. YM155 almost equally exhibited cytotoxic activity against U266/BTZ compared with parental cells. U266/BTZ displayed significantly lowered amounts of bcl-2, survivin and aurora-B kinase proteins. Interestingly, U266/BTZ overexpressed the Mcl-1 protein. Treatment with YM155 rapidly suppressed Mcl-1 protein expression and induced apoptosis. These data suggest that overexpression of Mcl-1 may contribute to bortezomib resistance and downregulation of Mcl-1 by YM155 could overcome it. In conclusion, our data indicate that YM155 may exert robust cytotoxic activity against quiescent (G0/G1) MM and bortezomib resistant cells via inhibition of Mcl-1 and survivin. Disclosures No relevant conflicts of interest to declare.

A 77-year-old man treated with prednisolone for pemphigus developed severe sepsis by Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Several antibiotics were administered. A peripheral blood smear showed growth of a large number of yeast extending pseudohyphae which could be seen both inside and outside of leucocytes. Antifungal agents were added immediately; however, he did not recover. Several days later, blood culture showed Candida albicans septicemia. The autopsy revealed microabscesses in the lung, heart, liver and kidney. A large amount of neutrophil invasion and yeast with pseudohyphae were also detected.

Current first-line treatment options for chronic myeloid leukemia (CML) include imatinib (IM) and the second-generation agents nilotinib and dasatinib. Despite the effectiveness of these tyrosine kinase inhibitors, a small percentage of chronic-phase CML patients are primarily refractory or acquire secondary resistance to these agents. Moreover, the prognosis of patients in blast crisis is still poor despite the use of the current treatment modalities because of drug resistance. The major mechanism of drug resistance is the re-activation of ABL kinase either through mutations in the BCR-ABL gene or by BCR-ABLgene amplification. A novel pan-histone deacetylase inhibitor, panobinostat (formerly LBH589), induces the acetylation of heat shock protein 90, thereby inhibiting its chaperone function in association with its client proteins BCR-ABL, leading to the degradation of BCR-ABL. A new pan–ABL tyrosine kinase inhibitor, ponatinib, is a promising therapeutic option in patients with all kinds of BCR-ABLmutation including T315I. It was thus hypothesized that the combination of panobinostat and ponatinib exerts synergistic cytotoxicity against IM-resistant cells through a mechanism of action different from that of each agent. To test this hypothesis, K562/IM-R1 and Ba/F3/T315I cell lines were evaluated for the cytotoxicity of panobinostat and ponatinib in vitro. K562/IM-R1 cells, established in our previous study, showed BCR-ABL overexpression due to BCR-ABL gene amplification. The Ba/F3/T315I cell line showed BCR-ABL with a T315I mutation. The XTT proliferation assay revealed that K562/IM-R1 cells were 12-fold more IM-resistant (50%-inhibitory concentration (IC50), 7.6 µM) than K562 cells (IC50, 0.6 µM). Ba/F3/T315I cells were refractory to IM treatment (IC50, 34.1 µM) compared with Ba/F3 cells (IC50, 3.4 µM). Panobinostat overcame IM-resistance and inhibited similarly the growth of K562, K562/IM-R1, Ba/F3, and Ba/F3/T315I cells, with IC50 values of around 50 nM (40.0 - 51.0 nM). Ponatinib inhibited the growth of both K562/IM-R1 cells (IC50, 4 nM) and Ba/F3/T315I cells (25 nM) as potently as parental K562 cells (IC50, 2 nM) and Ba/F3 cells (IC50, 5 nM). Importantly, the combination of panobinostat and ponatinib exhibited enhanced growth inhibition effects on all cell lines. The IC50 values for this combination were 0.7 nM for K562 cells, 1.3 nM for K562/IM-R1 cells, 3.7 nM for Ba/F3 cells, and 10 nM for Ba/F3/T315I cells. The combination index clearly showed synergism, with values of 0.5 for K562 cells, 0.28 for K562/IM-R1 cells, 0.9 for Ba/F3 cells, and 0.4 for Ba/F3/T315I cells. When the cells were treated with 10 nM panobinostat or 10 nM ponatinib for 48 h alone or in combination, the combination of the 2 agents led to greater-than-additive apoptotic cell death than each agent alone in all cell lines, evaluated by annexin V-positivity. Western blotting was used to evaluate the protein expression levels of BCR-ABL and phospho-BCR-ABL in cells after treatment with panobinostat or ponatinib or both in combination. BCR-ABL expression was greater in K562/IM-R1 than K562 cells. Phospho-BCR-ABL expression was not inhibited by IM in K562/IM-R1 or Ba/F3/T315I cells. However, ponatinib inhibited the autophosphorylation of BCR-ABL in these cell lines. Treatment with panobinostat reduced the BCR-ABL and phospho-BCR-ABL expression levels in K562/IM-R1 and Ba/F3/T315I cells. The combination of the 2 agents augmented inhibition of the autophosphorylation of BCR-ABL in these IM-resistant cell lines. The activity of histone deacetylase, determined using the HDAC assay Kit (Active Motif, Carlsbad, CA, USA), was inhibited by panobinostat in all cell lines regardless of IM sensitivity. In comparison, IM did not alter cellular histone deacetylase activity. Upon treatment of K562 cells with panobinostat, the protein expression levels of acetylated histone H3 and H4 were increased, suggesting the consequence of the inhibition of histone deacetylase. In conclusion, we firstly reported that panobinostat and ponatinib demonstrated synergistic cytotoxicity against IM-resistant cell lines not only due to BCR-ABL gene amplification but also BCR-ABL T315I mutation. The synergism is attributable to the greater inhibition of ABL kinase activity through a mechanism of action different from that of each agent. Disclosures: No relevant conflicts of interest to declare.

Abstract 1356 Clofarabine(2-Chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine,2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, CAFdA) is a relatively new purine nucleoside analog. Upon administration, CAFdA is incorporated into leukemic cells by human Equilibrative Nucleoside Transporters (hENT) 1 and 2, and human Concentrative Nucleoside Transporter (hCNT) 3. Inside the cell, the agent is phosphorylated to CAFdA monophosphate by cytosolic deoxycytidine kinase (dCK) and mitochondrial deoxyguanosine kinase (dGK), and then to an intracellular active metabolite CAFdA triphosphate (CAFdATP). CAFdATP inhibits ribonucleotide reductase and is incorporated into DNA, thereby terminating DNA synthesis as an antimetabolite. Moreover, CAFdA induces apoptosis via direct mitochondrial damage. Clinical studies suggest that CAFdA is effective against both acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). CAFdA therapy should be optimized based on the mechanistic understanding, because pharmacological determinants that correlate to the drug sensitivity may predict clinical efficacy of CAFdA as biological surrogate markers. Here, we have established two novel leukemic cell line variants that were resistant to CAFdA, and elucidated the mechanism of the drug resistance. The study focused on factors that were involved in the intracellular CAFdATP production and in the induction of apoptosis. To develop resistant variants, HL-60 cells were treated with escalating concentrations of CAFdA with the initial concentration at 1/100 of the concentration that inhibited 50% cell growth. After 7 months of the repeated passage, one cell line resistant to CAFdA (HL/CAFdA20) was cloned by the limiting dilution method. A part of this clone was further maintained with the drug for the subsequent 4 months to develop another variant (HL/CAFdA80). The 2 variants were 20- and 80-fold more CAFdA-resistant than HL-60 cells, respectively. They were cross-resistant to similar nucleoside analogs such as cladribine, gemcitabine, and cytarabine. Compared with HL-60 cell line, mRNA levels of the transporters (hENT1, hENT2, hCNT3) and protein levels of kinases (dCK, dGK), and the subsequent production of intracellular CAFdATP were all reduced in both CAFdA-resistant variants. Real time RT-PCR demonstrated that mRNA levels of hENT1, hENT2, and hCNT3 were 53.9%, 41.8%, 18.3% in HL/CAFdA20 cells, and 30.8%, 41.6%, 31.5% in HL/CAFdA80 cells, respectively, compared to the parental cells. The values of the initial uptake of CAFdA into the cell at 40 seconds after administration of tritiated CAFdA are 0.2 pmol/107cells in HL/CAFdA20 cells, and 0.1 pmol/107cells in HL/CAFdA80 cells, compared with 0.6 pmol/107 cells in parental HL60 cells. Western blotting revealed that protein levels of kinases were also reduced in these resistant variants with the greater reduction in HL/CAFdA80 cells. The subsequent production of CAFdATP after 4-h incubation with 10 μM CAFdA was 20 pmpl/107 cells in HL/CAFdA20 cells, 3 pmol/107cells in HL/CAFdA80 cells, and 63 pmol/107cells in HL-60 cells. The decreased CAFdATP production led to the attenuated incorporation of the drug into both mitochondrial and nuclear DNA. Concerning apoptosis, antiapoptotic Bcl2 protein overexpressed in the 2 resistant variants. The two variants were resistant to mitochondria-related apoptosis induced by CAFdA, in part due to the enhanced Bcl2 expression. A Bcl2 inhibitor ABT737 synergized the cytotoxic effect and the growth inhibition effect of CAFdA in both variants and HL-60, but the synergism was more profound in the resistant cell lines. The Combination Index values were 0.27 in HL/CAFdA20 cells, and 0.21 in HL/CAFdA80 cells, compared with 0.63 in HL-60 cells. This suggested the contribution of the enhanced Bcl2 protein to the mechanism of drug resistance. In conclusion, the mechanism of cellular resistance to CAFdA in the 2 variants was multifactorial, but primarily includes the reduced CAFdATP production and the increased antiapoptotic factor. It is noted that the decreased dGK level and Bcl2 overexpression were not reported previously in the context of CAFdA resistance. We suggest combination of CAFdA and ABT737 might be effective to CAFdA resistant and refractory leukemia. (This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. No.23501307) Disclosures: No relevant conflicts of interest to declare.

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml) were added after treatment with LPS (10 μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1β was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course., Highlights • Tetracyclines inhibit cytokine and chemokine production in LPS-induced THP-1 cells. • Tetracyclines inhibit NF-κB, p38 and activate ERK phosphorylation. • Tetracyclines regulate these pathways and NF-κB pathway has a pivotal role.

Background Nine-beta-D-arabinofuranosylguanine (ara-G), an active metabolite of nelarabine, enters leukemic cells through human Equilibrative Nucleoside Transporter 1, and is then phosphorylated to an intracellular active metabolite ara-G triphosphate (ara-GTP) by both cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. Ara-GTP is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Methods In the present study, we developed a novel ara-G-resistant variant (CEM/ara-G) of human T-lymphoblastic leukemia cell line CCRF-CEM, and elucidated its mechanism of ara-G resistance. The cytotoxicity was measured by using the growth inhibition assay and the induction of apoptosis. Intracellular triphosphate concentrations were quantitated by using HPLC. DNA synthesis was evaluated by the incorporation of tritiated thymidine into DNA. Protein expression levels were determined by using Western blotting. Results CEM/ara-G cells were 70-fold more ara-G-resistant than were CEM cells. CEM/ara-G cells were also refractory to ara-G-mediated apoptosis. The transcript level of human Equilibrative Nucleoside Transporter 1 was lowered, and the protein levels of deoxycytidine kinase and deoxyguanosine kinase were decreased in CEM/ara-G cells. The subsequent production of intracellular ara-GTP (21.3 pmol/107 cells) was one-fourth that of CEM cells (83.9 pmol/107 cells) after incubation for 6 h with 10 μM ara-G. Upon ara-G treatment, ara-G incorporation into nuclear and mitochondrial DNA resulted in the inhibition of DNA synthesis of both fractions in CEM cells. However, DNA synthesis was not inhibited in CEM/ara-G cells due to reduced ara-G incorporation into DNA. Mitochondrial DNA-depleted CEM cells, which were generated by treating CEM cells with ethidium bromide, were as sensitive to ara-G as CEM cells. Anti-apoptotic Bcl-xL was increased and pro-apoptotic Bax and Bad were reduced in CEM/ara-G cells. Conclusions An ara-G-resistant CEM variant was successfully established. The mechanisms of resistance included reduced drug incorporation into nuclear DNA and antiapoptosis.