Combination of azacytidine and curcumin is a potential alternative in decitabine-resistant colorectal cancer cells with attenuated deoxycytidine kinase.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor is a novel anti-cancer drug regulating epigenetic mechanisms. Similar to conventional anti-cancer drugs, drug resistance to DAC also has been reported, resulting in tumor recurrence. Our previous study using colorectal cancer HCT116 cells found the decrease in deoxycytidine kinase (dCK) (activation enzyme of DAC) and the increase in cytidine deaminase (inactivation enzyme of DAC) in acquired DAC-resistant HCT116 (HCT116/DAC) cells. The aim of our study was to clarify the involvement of dCK and CDA in DAC resistance. In order to tackle DAC resistance, it was also examined whether other DNMT inhibitors such as azacytidine (AC) and polyphenols are effective in DAC-resistant cancer cells. When dCK siRNA was transfected into HCT116 cells, IC50 value of DAC increased by about 74-fold and reached that of HCT116/DAC cells with attenuated dCK. dCK siRNA to HCT116 cells also abolished DNA demethylation effects of DAC. In contrast, CDA siRNA to HCT116 cells did not influence the efficacy of DAC. In addition, CDA siRNA to HCT116/DAC cells with increased CDA did not restore the compromised effects of DAC. These results suggested that attenuated dCK but not increased CDA mainly contributed to DAC resistance. Regarding dCK in HCT116/DAC cells, a point mutation with amino acid substitution was observed while the product size and expression of mRNA coding region did not change, suggesting that dCK protein was decreased by post-transcriptional regulation. AC and polyphenols showed no cross-resistance in HCT116/DAC cells. AC but not polyphenols exerted DNA demethylation effect. Among polyphenols, curcumin (Cur) showed the most synergistic cytotoxicity in combination with AC while DNA demethylation effect of AC was partly maintained. Taken together, combination of AC and Cur would be a promising alternative to tackle DAC resistance mainly due to attenuated dCK.

Optimization of Analytical Conditions for Hydrophilic Nucleic Acids Using Mixed-Mode and Reversed-Phase Pentabromobenzyl Columns.

The aim of this study was to investigate appropriate analytical conditions for hydrophilic nucleosides and nucleotides (monophosphates and triphosphates) by HPLC methods using a mixed-mode AX-C18 column with anion-exchange and hydrophobic interactions by quaternary ammonium and C18, respectively, and a reversed-phase pentabromobenzyl (PBr) column with dispersion force and hydrophobic interactions by PBr group. The higher compound polarity led to stronger retention on AX-C18 (triphosphates > monophosphates > nucleosides). AX-C18 demonstrated feasible retention of nucleotides via anion-exchange interaction by increasing the salt and methanol concentrations. In contrast, on PBr, the lower compound polarity led to stronger retention. On PBr, feasible retention of both nucleosides and nucleotides was obtained via dispersion interactions with purine and pyrimidine rings by increasing the methanol concentration. Regarding the pH of phosphate buffer used as the mobile phase, pH 7.0 should be used in measuring nucleoside triphosphates on AX-C18, whereas pH 2.5 is better suited for measuring nucleotides on PBr. In terms of selectivity to highly hydrophilic nucleotides, the mixed-mode AX-C18 column had an advantage over the reverse-phase PBr column. In contrast, PBr column was more versatile than the AX-C18 column. Taken together, HPLC analyses of nucleosides and nucleotides should be carried out by optimizing the interactions between the stationary phase and nucleic acids.

Decitabine exerted synergistic effects with oxaliplatin in colorectal cancer cells with intrinsic resistance to decitabine.

Epigenetic modifiers such as decitabine (DAC), a DNA methyltransferase inhibitor, have the potential benefit of combination therapy with conventional anti-cancer drugs. This study was aimed to clarify whether DAC at the low concentration without cytotoxic effects exerts synergistic effects with conventional anti-cancer drugs in human colorectal cancer cell line, HT29 cells low-sensitive to DAC. DAC at very low concentration below its IC20 synergistically enhanced cytotoxicity of oxaliplatin (L-OHP), and the enhancement was the most remarkable for L-OHP among several anti-cancer drugs tested. DAC was found to be metabolized and incorporated into DNA in HT29 cells. Combination of L-OHP with DAC did not increase the protein expression of γH2A.X, the earliest indicator of DNA damage, in HT29 cells. On the other hand, although DAC alone did not produce reactive oxygen species (ROS), DAC significantly enhanced ROS production by L-OHP in HT29 cells. Furthermore, enhanced cytotoxicity of L-OHP by DAC was cancelled with the presence of N-acetyl-l-cysteine, a ROS scavenger. The mRNA expression of ROS-generating enzymes was significantly increased by combination in HT29 cells. Taken together, indirect enhancement of ROS production by DAC at the low concentration with negligible cytotoxicity should be at least involved in synergistic effects with L-OHP in HT29 cells with intrinsic resistance to DAC.

The Effect of Ethanol on the Hydrolysis of Ester-Type Drugs by Human Serum Albumin.

Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and hydrolyzes compounds at both sites. Although the hydrolytic interaction of ester-type drugs with other drugs by HSA has been reported, there are only a few studies concerning the effect of pharmaceutical excipients on the hydrolysis of ester-type drugs by HSA. In the present study, we investigated the effect of ethanol (2 vol%; 345 mM) on the hydrolysis of aspirin, p-nitrophenyl acetate, and olmesartan medoxomil, which are ester-type drugs, with 4 different lots of HSA preparations. The hydrolysis activities of HSA toward aspirin, p-nitrophenyl acetate, and olmesartan medoxomil were measured from the pseudo-first-order degradation rate constant (kobs) of salicylic acid, p-nitrophenol, and olmesartan, respectively, which are the HSA-hydrolyzed products. Ethanol inhibited hydrolysis of aspirin by HSA containing low levels of fatty acids, but not by fatty acid-free HSA. Ethanol inhibited hydrolysis of p-nitrophenyl acetate by both fatty acid-free HSA and HSA containing low levels of fatty acids. In contrast, the hydrolysis of olmesartan medoxomil by HSA was insignificantly inhibited by ethanol, but inhibited not only by warfarin and indomethacin but also by naproxen, which are site I binding drugs and a site II binding drug, respectively. These results suggest that the inhibitory action of ethanol on the hydrolysis of ester-type drugs by HSA differs between site I binding drugs and site II binding drugs.

Different Schedule-Dependent Effects of Epigenetic Modifiers on Cytotoxicity by Anticancer Drugs in Colorectal Cancer Cells.

Limited information is currently available on how to apply epigenetic modifiers to current colorectal cancer (CRC) chemotherapy. The purpose of this study is to clarify the schedule-dependent effects of combined treatment with conventional anticancer drugs and epigenetic modifiers in human CRC cells. Cytotoxicity in 4 CRC cell lines (SW480, HT29, SW48, and HCT116) was measured using the WST-8 assay. As epigenetic modifiers, 3 DNA methyltransferase (DNMT) inhibitors such as decitabine (DAC), azacytidine (AC), and zebularine (Zeb), and 3 histone deacetylase (HDAC) inhibitors including trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and valproic acid (VPA) were used. Combination effects were analyzed by the isobologram method. SW480 cells showed the lowest sensitivity to the anticancer drugs 5-fluorouracil, SN-38 (the active form of irinotecan), and oxaliplatin. In SW480 cells, epigenetic modifiers other than VPA showed the most significant synergistic effects when used before anticancer drugs, while VPA showed synergistic effects in co- or post-treatment. In the 3 other CRC cells, synergistic effects were less frequent and weaker. The dose of anticancer drugs may be reduced by combining epigenetic modifiers in SW480 cells, which are less sensitive to anticancer drugs, unlike the more sensitive HT29, SW48, and HCT116 cell lines. These results provide useful information for understanding how to incorporate epigenetic modifiers into current CRC chemotherapy.

Effects of Zebularine on Invasion Activity and Intracellular Expression Level of let-7b in Colorectal Cancer Cells.

The effects of zebularine, a DNA methyltransferase inhibitor, on the invasion activity as well as intracellular expression level of let-7b, tumor suppressor microRNA, were examined in three human colorectal cancer (CRC) cell lines: SW480, SW620, and oxaliplatin-resistant SW620 (SW620/OxR). Zebularine suppressed the invasion activity of SW620 and SW620/OxR cells. The intracellular expression level of let-7b was up-regulated by zebularine in SW620 and SW620/OxR cells. The overexpression of let-7b by the transfection of let-7b mimic suppressed invasion activity in SW620 and SW620/OxR cells. These results suggest that zebularine may inhibit invasion activity by up-regulating the intracellular expression level of let-7b in high-invasive CRC cells.

Differences in Esterase Activity to Aspirin and p-Nitrophenyl Acetate among Human Serum Albumin Preparations.

Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and also hydrolyzes some compounds at both sites. In the present study, we investigated differences in esterase activity among HSA preparations, and also the effects of warfarin, indomethacin, and naproxen on the hydrolytic activities of HSA to aspirin and p-nitrophenyl acetate. The esterase activities of HSA to aspirin or p-nitrophenyl acetate were measured from the pseudo-first-order formation rate constant (kobs) of salicylic acid or p-nitrophenol by HSA. Inter-lot variations were observed in the esterase activities of HSA to aspirin and p-nitrophenyl acetate; however, the esterase activity of HSA to aspirin did not correlate with that to p-nitrophenyl acetate. The inhibitory effects of warfarin and indomethacin on the esterase activity of HSA to aspirin were stronger than that of naproxen. In contrast, the inhibitory effect of naproxen on the esterase activity of HSA to p-nitrophenyl acetate was stronger than those of warfarin and indomethacin. These results suggest that the administration of different commercial HSA preparations and the co-administration with site I or II high-affinity binding drugs may change the pharmacokinetic profiles of drugs that are hydrolyzed by HSA.

Cellular Uptake of Decitabine by Equilibrative Nucleoside Transporters in HCT116 Cells.

DNA hypermethylation, an epigenetic change that silences gene expression without altering nucleotide sequences, plays a critical role in the formation and progression of colorectal cancers as well as in the acquisition of drug resistance. Decitabine (DAC), a DNA methyltransferase 1 inhibitor of nucleoside analogues, has been shown to restore gene expression silenced by hypermethylation. In the present study, the mechanisms underlying both uridine and DAC uptake were examined in the human colon cancer cell line HCT116. Real-time polymerase chain reaction analysis revealed that ENT1 mRNA was the most abundant among the nucleoside transporters examined in HCT116 cells. The ENT1 protein was detected in the membrane fraction, as determined by Western blotting. The uptake of uridine or DAC was time- and concentration-dependent, but also Na(+)-independent. The uptake of these agents was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENTs), and was also decreased in cells treated with ENT1 small interfering RNA. The uptake of both uridine and DAC was inhibited by uridine, cytidine, adenosine, or inosine, while that of DAC was also inhibited by thymidine. The expression of MAGEA1 mRNA, the DNA of which was methylated in HCT116 cells, was increased by DAC treatment, and this increment was attenuated by concomitant treatment with NBMPR. The IC50 value of DAC was also increased in the presence of NBMPR. These results suggest that DAC is mainly taken up by ENT1 and that this uptake is one of the key determinants of the activity of DAC in HCT116 cells.

Effects of Decitabine on Invasion and Exosomal Expression of miR-200c and miR-141 in Oxaliplatin-Resistant Colorectal Cancer Cells.

The effects of decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, on metastasis and exosomal expression of microRNAs were examined in SW620/OxR cells, a human colorectal cancer (CRC) cell line (SW620) with acquired resistance to oxaliplatin. This cell line shows an invasive phenotype by epithelial-mesenchymal transition. Two CRC cell lines, SW480, derived from primary CRC, and SW620, derived from lymph node metastasis, which were obtained from the same patient, as well as SW620/OxR, were also used in the present study. Cytarabine (Ara-C), a non-DNMT-inhibiting cytidine analog, was used as negative control of DAC. No significant difference was observed in the invasion abilities of SW480 cells treated with DAC or Ara-C. On the other hand, invasion ability was suppressed by treatment with DAC in SW620 and SW620/OxR cells. Up-regulated expression of E-cadherin, microRNA-200c (miR-200c), and miR-141 following DAC treatment indicated the acquisition of epithelial cell-like characteristics in SW620 and SW620/OxR cells. Exosomal expression levels of miR-200c and miR-141 were also up-regulated by DAC treatment in SW620 and SW620/OxR but not in SW480 cells. This increase in exosomal miRNA expression negatively correlated with invasion ability. These results suggest that DNA demethylation treatment caused acquisition of epithelial cell-like characteristics in SW620 and SW620/OxR cells. Furthermore, the observed increased exosomal expression of miR-200c and miR-141 may be an indicator or biomarker candidate for mesenchymal-epithelial transition of CRC cells.

Induction of epithelial-mesenchymal transition and down-regulation of miR-200c and miR-141 in oxaliplatin-resistant colorectal cancer cells.

Epithelial-mesenchymal transition (EMT) and changes in the expression of the microRNA-200 (miR-200) family were examined in the human colorectal cancer (CRC) cell line SW620 with acquired oxaliplatin (L-OHP) resistance. Two CRC cell lines, SW480, derived from primary CRC, and SW620, derived from lymph node metastasis, which were obtained from the same patient, were used in the present study. L-OHP-resistant SW620 cells were obtained by exposure to L-OHP for 155 d. The concentration of L-OHP was increased to 80 µM in a stepwise manner. The IC50 value of L-OHP was increased 16-fold in L-OHP-resistant SW620 cells, which also displayed mesenchymal cell-like characteristics, such as the down-regulation of E-cadherin and up-regulation of vimentin. However, L-OHP-resistant SW480 cells were not obtained when the concentration of L-OHP was increased in a similar stepwise manner. The expression levels of members of the miR-200 family (miR-200a, miR-200b, miR-429, miR-200c, and miR-141) were significantly higher in SW480 cells than in SW620 cells. The expression levels of miR-200c and miR-141 were significantly lower in L-OHP-resistant SW620 cells than in control SW620 cells. L-OHP-resistant SW620 cells did not exhibit cross-resistance to other anti-cancer drugs used to treat CRC, such as 5-fluorouracil, irinotecan, and the active metabolite of irinotecan (SN-38). These results suggest that the down-regulated expression of miR-200c and miR-141 plays a role in selective resistance to L-OHP and EMT in CRC cells during repeated treatments with L-OHP.

Different effects of epigenetic modifiers on the cytotoxicity induced by 5-fluorouracil, irinotecan or oxaliplatin in colon cancer cells.

We investigated the effects of epigenetic modifiers such as DNA methyltransferase (DNMT) or histone deacetylase (HDAC) inhibitors on the cytotoxicity induced by 3 anticancer drugs (5-fluorouracil (5-FU), irinotecan (CPT-11) or its active form SN38, and oxaliplatin (L-OHP)) in human colorectal cancer (CRC) cells. Cytotoxicity in 4 CRC cell lines (HT29, SW480, SW48 and HCT116) was examined by colorimetric assay after drug treatment for 72 h. The effects of drug combinations were analyzed by an isobologram method. SW480 cells showed the lowest sensitivity to cytotoxicity induced by the anticancer drugs among the 4 CRC cell lines. In SW480 cells, DNMT inhibitors, such as decitabine (DAC), azacytidine and zebularine (Zeb), showed synergic effects on the cytotoxicity induced by anticancer drugs except for SN-38 plus Zeb, while HDAC inhibitors, trichostatin A, suberoylanilide hydroxamic acid and valproic acid, showed antagonistic effects. DAC showed the most potent synergic effects among the epigenetic modifiers studied. Thus, we examined whether the synergic effect of DAC is observed in other different CRC cell lines, HT29, SW48 and HCT116 cells. In all 4 CRC cell lines, the cytotoxicity of L-OHP was enhanced in a synergic manner by co-treatment with DAC. However, synergic effects of DAC with 5-FU or CPT-11 (SN-38) were not observed in 4 CRC cell lines.

Different involvement of DNA methylation and histone deacetylation in the expression of solute-carrier transporters in 4 colon cancer cell lines.

The purpose of this study on the involvement of epigenetic control of the expression of solute carrier (SLC) transporters by DNA methylation and histone deacetylation in 4 colon cancer cells is to find the epigenetic control mechanisms of drug transporters in colon cancers. Human colon cancer cell lines (HCT116, HT29, SW48, SW480) were treated with 5-aza-2'-deoxycytidine (DAC), as a DNA methyltransferase inhibitor, followed by trichostatin A (TSA), as a histone deacetylase inhibitor. The mRNA expression and DNA methylation of several SLC transporters were analyzed by real-time polymerase chain reaction (PCR) and methylation-specific PCR, respectively. Among 12 SLC transporters possessing cytosine-phosphate-guanine (CpG) islands, thiamine transporter 2 (THTR2) (SLC19A3) gene showed a correlation between its mRNA expression level and DNA methylation status. TSA treatment increased histone H3 acetylation of THTR2 promoter region in all 4 colon cancer cell lines examined. HCT116 and SW48 cells showed a lack of THTR2 mRNA expression and methylation of its promoter, and DAC treatment induced its re-expression. In addition, the co-treatment with DAC and TSA increased THTR2 mRNA expression more markedly than DAC treatment in HCT116 and SW48 cells. In HT29 and SW480 cells that showed little methylation of THTR2 promoter, TSA treatment induced THTR2 mRNA expression markedly, but DAC treatment did not. In the 4 colon cancer cells examined, THTR2 mRNA expression is down-regulated by DNA methylation and/or histone deacetylation.

miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells.

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210-3p, and miR-224-5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210-3p or miR-224-5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210-3p, and miR-224-5p in sEVs was compared. The amount of miR-33a-5p and miR-210-3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224-5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210-3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.

Numerical analysis of apparent decitabine uptake in HCT116 cells: Incorporation of a bidirectional first-order kinetic parameter for ENT1 transport and Michaelis-Menten parameters for subsequent phosphorylation.

Decitabine (DAC), a DNA methylation inhibitor, is transported into cancer cells mainly via equilibrative nucleoside transporter 1 (ENT1) and subsequently phosphorylated by deoxycytidine kinase (dCK). We previously reported that apparent DAC uptake into cells may be described using a simple compartment model with clearance for facilitated diffusion (PS) and subsequent phosphorylation (CLmet). In the present study, time course of apparent intracellular [3H]-DAC uptake was analyzed numerically, and PS and CLmet values were calculated using the compartment model in human colon cancer HCT116 cells. PS at 0.1 µM [3H]-DAC was markedly decreased in the presence of 100 µM irinotecan or etoposide, while CLmet was markedly decreased in the presence of 100 µM cytarabine or gemcitabine. CLmet at 0.1-10 µM [3H]-DAC varied in a concentration-dependent manner and was described by Michaelis-Menten parameters Km,met and Vmax,met. In conclusion, DAC uptake mainly via ENT1 may be described by a bidirectional first-order kinetic parameter, while phosphorylation by dCK may be described by Michaelis-Menten parameters.

[Effect of withdrawal of 5-fluorouracil bolus administration on recovery from neutropenia in colorectal cancer patients treated with mFOLFOX6 chemotherapy-comparison with total dosage reduction].

A combination of oxaliplatin(L-OHP), folinic acid and 5-fluorouracil(5-FU)(mFOLFOX6)has been widely administered to treat advanced or recurrent colorectal cancer. In this regimen, a bolus of 5-FU is administered intravenously, followed by its 46-hr continuous intravenous infusion. For 12 patients who showed neutropenia during mFOLFOX6 chemotherapy at Itami City Hospital, we investigated neutrophil recovery by comparing a patient group treated by the withdrawal of the 5-FU bolus administration(n=6)with a patient group treated by total dose reduction of L-OHP, as well as both the bolus and continuous 5-FU administration(n=6). After two weeks, the neutrophil numbers in the bolus withdrawal group showed a relatively higher value than that in the total dose reduction group[p= 0.032]. For patients showing neutropenia related to mFOLFOX6 chemotherapy, withdrawal of 5-FU bolus administration is suggested to be an effective method of promoting the recovery of neutrophil numbers.

Effect of oil-in-water lipid emulsions prepared with fish oil or soybean oil on the growth of MCF-7 cells and HepG2 cells.

The growth of human breast cancer-derived MCF-7 cells was affected by oil-in-water lipid emulsions prepared with fish oil (FO) rich in n-3 fatty acids (FAs) and egg-yolk phosphatides (EYP) (FO-emulsions), but not by lipid emulsions prepared with soybean oil (SO) and EYP (SO-emulsions). On the other hand, the growth of human hepatocarcinoma HepG2 cells was affected by neither SO-emulsions nor FO-emulsions. The growth inhibition of MCF-7 cells in the presence of FO-emulsions was not affected by trolox, but was inhibited by alpha-lipoic acid, and was even potentiated by ebselen, which works as an antioxidant as well as a lipoxygenase inhibitor. Since prostaglandin E(3), generated from n-3 FAs by cyclooxygenases, has a suppressive effect on tumour cell growth, and increases when lipoxygenases are inhibited, these findings suggest that lipid emulsions incorporating triglycerides of n-3 FAs might be effective in suppressing the growth of MCF-7 cells, possibly via oxidative stress and through eicosanoid production with anti-proliferating activity against cancer cells.

Monophosphorylation by deoxycytidine kinase affects apparent cellular uptake of decitabine in HCT116 colon cancer cells.

Decitabine (DAC), a nucleoside-related DNA methylation inhibitor, is taken up into cancer cells via equilibrative nucleoside transporter 1 (ENT1), and is then monophosphorylated by deoxycytidine kinase (dCK). In the present study, we examined the contribution of dCK to the uptake of DAC in HCT116 colon cancer cells. Irinotecan and etoposide inhibited the uptake of [3H]-uridine and [3H]-DAC at 10 s and 5 min, while cytarabine and gemcitabine only inhibited that of [3H]-DAC at 5 min. Irinotecan and etoposide inhibited [3H]-DAC uptake in negative control small interfering RNA (siRNA)- or dCK siRNA-transfected cells at 10 s, whereas cytarabine and gemcitabine did not. Cytarabine and gemcitabine inhibited DAC monophosphate generation by the cytosolic proteins of HCT116 cells and recombinant human dCK protein, assessed using polyethylenimine cellulose thin-layered chromatography. Simulations using simple kinetic models showed that apparent DAC uptake in dCK and ENT1 siRNA-treated cells was attributed to its conversion to monophosphates or a decrease in the cellular flux, respectively, and that the apparent uptake of DAC in dCK-knockdown and ENT1-knockdown cells was similar at longer times, but differed at a very short time. These results suggest that the apparent uptake of DAC is affected by ENT1 and dCK in HCT116 cells.

Usefulness of glycated albumin in decisions regarding the discontinuation of a diabetes drug and factors associated with poor glycemic control following discontinuation in patients with type 2 diabetes mellitus.

We studied the usefulness of glycated albumin (GA) for decisions regarding the discontinuation of a diabetes drug in type 2 diabetic patients with good glycemic control, and the factors associated with increases in GA following discontinuation of a diabetes drug in patients with type 2 diabetes mellitus. Sixteen patients (12 males and 4 females) were enrolled in the present study and discontinued one diabetes drug. When the change in GA was less than 1 % at 4 weeks after the discontinuation of the diabetes drug (ΔGA4w), discontinuation was continued (the continuous group, n = 10), but when the change in GA was more than a 1 %, the discontinued diabetes drug was resumed (the resume group, n = 6). In the continuous group, HbA1c at 12 weeks after discontinuation (6.2 ± 0.6 %) remained unchanged from its value at discontinuation (6.2 ± 0.7 %) , but it was significantly elevated at 12 weeks after discontinuation in the resume group (changing from 6.3 ± 0.1 to 7.0 ± 0.6 %). Age, duration of diabetes, and GA were significantly lower while body mass index (BMI) was significantly higher in the continuous group than in the resume group, but no significant difference in HbA1c was observed between the groups. The discontinuation of a diabetes drug in patients with low insulin secretion must be performed carefully because factors such as duration of diabetes, BMI, and GA showed significant differences between the continuous group and resume group in our study, and these indicators are known to be linked with insulin secretion.

Effects of alogliptin on the ratio of glycated albumin to HbA1c in patients with type 2 diabetes mellitus.

The ratio of glycated albumin (GA) to HbA1c (the GA/HbA1c ratio) has been used as a glycemic control indicator that reflects postprandial plasma glucose levels or glycemic variability. In this study, we investigated the effects of alogliptin, a DPP-4 inhibitor, on the GA/HbA1c ratio in patients with type 2 diabetes mellitus. Thirty-eight patients with type 2 diabetes mellitus whose glycemic control was stable were enrolled, and alogliptin (12.5 or 25 mg/day) was then administered to them for 24 weeks. HbA1c and GA levels both significantly decreased after 24 weeks (P < 0.0001), whereas the GA/HbA1c ratio did not (P = 0.129). No correlation was observed between the change in the GA/HbA1c ratio (the ΔGA/HbA1c ratio) and HbA1c or GA level before the administration of alogliptin; however, a negative correlation was found between the ΔGA/HbA1c ratio and the GA/HbA1c ratio before the administration of alogliptin (R = -0.322, P = 0.049). Although the GA/HbA1c ratio in the low-value group (<2.80) was not significantly affected by the administration of alogliptin, that in the high-value group (≥2.80) significantly decreased (P = 0.008). The administration of alogliptin significantly decreased the GA/HbA1c ratio in the high-value group after 24 weeks. Alogliptin may be more useful for patients with high postprandial plasma glucose levels than in those with low postplandial plasma glucose levels.

Acquired resistance to decitabine and cross-resistance to gemcitabine during the long-term treatment of human HCT116 colorectal cancer cells with decitabine.

The aim of the present study was to determine the effects of long-term exposure of decitabine (DAC) to HCT116 colorectal cancer (CRC) cells on the acquisition of resistance to DAC as well as cross-resistance to anticancer drugs used for CRC or other epigenetic modifiers. In the present study, DAC-resistant HCT116 CRC cells were established through long-term treatment with increasing concentrations of DAC (10 to 540 nM); and the cross-resistance to other drugs was subsequently examined. DAC-resistant HCT116 cells were obtained following a 104-day treatment with DAC, including DAC-free intervals. The results demonstrated that the IC50 value of DAC was increased ~100-fold in DAC-resistant HCT116 cells. Messenger (m)RNA expression of secreted frizzed-related protein 1 (SFRP1), which is regulated by DNA methylation, was not detected in DAC-resistant cells; however, SFRP1 mRNA was present in HCT116 cells treated with DAC for 52 days. DNA methyltransferase 1 (DNMT1) protein levels were slightly decreased until day 81 and then returned to control levels in DAC-resistant cells. Further experiments using DAC-resistant HCT116 cells revealed that these cells exhibited cross-resistance to gemcitabine (Gem); however, cross-resistance was not observed for other DNMT inhibitors (azacitidine and zebularine), histone deacetylase inhibitors (trichostatin A, vorinostat and valproic acid) or anticancer drugs for CRC (5-fluorouracil, irinotecan and oxaliplatin). Furthermore, the protein expression levels of cytidine deaminase (CDA) were increased, while those of deoxycytidine kinase (dCK) were decreased in DAC-resistant HCT116 cells; by contrast, the mRNA expression levels for these proteins were not significantly altered. In conclusion, the results of the present study indicated that the long-term treatment of HCT116 cells with DAC led to the acquisition of resistance to both DAC and Gem. In addition, these results may be partly attributed to changes in CDA and/or dCK, which are involved in metabolic pathways common to these two drugs.