Modulation of inhibitory and excitatory neurotransmissions by Zn2+ on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice.

The substantia gelatinosa of the trigeminal subnucleus caudalis has been considered to be an essential location for the transference of orofacial sensory signals. The co-localization of inhibitory and excitatory neurotransmitters in the same substantia gelatinosa (SG) neurons has demonstrated their essential part in the modification of nociceptive transmission. Zn2+ is particularly numerous in the mammalian central nervous system. There are proofs demonstrating the role of Zn2+ in the modulation of voltage- and ligand-gated ion channels. However, little is known about what roles Zn2+ may play in the modulation of signal transmission in the SG neurons of the trigeminal subnucleus caudalis (Vc). Therefore, in this study, we used the whole-cell patch clamp technique to find out the effect of Zn2+ on the responses of three main neurotransmitters (glycine, GABA, and glutamate) on SG neurons of the Vc in mice. We have proved that Zn2+ induces a big potentiation of glycine receptor-mediated response but attenuates GABA- and glutamate-induced responses at micromolar concentrations, however, enhances glutamate-induced response at nanomolar concentration. Taken together, these data demonstrated that Zn2+ can modulate glycine, GABA and glutamate-mediated actions on the SG neurons of the Vc and support an important mechanism in spinal sensory information signaling.

Genome-wide identification of chemosensitive single nucleotide polymorphism markers in colorectal cancers.

Improved methods for predicting chemoresponsiveness involving the identification of polymorphic markers is highly desirable, considering narrow therapeutic index and frequent resistance to anti-cancer regimens. The genome-wide screening of chemosensitive single nucleotide polymorphisms (SNPs) was undertaken in association with in vitro chemosensitivity assays in 104 colorectal cancer patients for the initial screening step. Allele frequency, linkage disequilibrium, potential function, and Hardy-Weinberg equilibrium of the candidate SNPs were then determined for the identifying step. Finally, clinical association analysis in the other 260 evaluable patients or cell viability assays of transfected RKO cells was used to verify candidate SNPs for the validation step. In total, 12 SNPs to six regimens were initially chosen during the screening and identifying steps. In patients receiving fluoropyrimidine-based adjuvant chemotherapy, the substitution alleles of GPC5 rs553717 (AA) correlated significantly with tumor recurrence and shorter disease-free survival (P = 0.019 and 0.023, respectively). Interestingly, RKO cells expressing mutant GPC5 showed enhanced cell death in response to 5-FU in cytotoxicity assays. Patients that were homozygous for the reference alleles SSTR4 rs2567608 (AA) and EPHA7 rs2278107 (TT) showed lower disease control rates in response to irinotecan and oxaliplatin regimens, respectively, than those with substitution alleles (P = 0.022 and 0.014, respectively). Thus, we identified chemosensitive SNP markers using a novel three step process of genome-wide analysis consisting of in vitro screening, identification, and validation. The candidate chemosensitive SNP markers identified in our study, including those identified in vitro, can now be further verified in a large cohort study.

Promoter methylation of specific genes is associated with the phenotype and progression of colorectal adenocarcinomas.

BACKGROUND: Promoter methylation of colorectal cancer-related genes were examined with respect to phenotype and tumor progression. MATERIALS AND METHODS: We assayed promoter methylation of 11 genes including established CpG island methylator phenotype (CIMP) markers (MLH1, MINT1, MINT2, MINT31, p16 ( INK4a ), p14 ( ARF ), and CACNA1G) and four genes (COX2, DAPK, MGMT, and APC) frequently methylated in colorectal cancer in 285 patients with sporadic colorectal cancer. RESULTS: CIMP+ tumors were more than two times more frequent among high-frequency microsatellite instability tumors (MSI-H) than in tumors without MSI (P < or = .0001-.002). COX2 and DAPK methylation were significantly associated with CIMP+ and MSI. KRAS showed tendency toward more frequent codon 12-13 mutations identified in tumors with APC and p16 ( INK4a ) methylation than in those with unmethylation (P = .033 and .05, respectively). Additionally, tumors with synchronous adenoma were associated with p16 ( INK4a ) methylation (P = .004). The p16 ( INK4a ) methylation was significantly associated with poor overall and disease-free survival in 131 rectal cancer patients who underwent curative operation, according to multivariate analyses (relative risk [RR] = 0.317 and 0.349; P = .033 and .024, respectively). Specifically, in 175 stage II and III patients receiving adjuvant-based fluoropyrimidine chemotherapy, p16 ( INK4a ) methylation and MINT31 unmethylation showed a significant or tendency toward an association with recurrence and DFS (P = .007-.032). CONCLUSIONS: The study suggests that specific CIMP markers, such as p16 ( INK4a ) and MINT31, should be further verified as potential epigenetic targets for the design of efficient chemotherapy regimens. We also identified a subset of colorectal cancer, possibly comprising APC methylation-KRAS mutation-p16 ( INK4a ) methylation.

Evaluation of novel histone deacetylase inhibitors as therapeutic agents for colorectal adenocarcinomas compared to established regimens with the histoculture drug response assay.

BACKGROUND AND AIMS: This study was to evaluate the efficacy of histone deacetylase (HDAC) inhibitors in colorectal cancer together with other established regimens. MATERIALS AND METHODS: Chemosensitivities of 114 colorectal cancer patients to established regimens (fluorouracil (5-FU with leucovorin (FL), capecitabine, FL with irinotecan (FLIRI), and FL with oxaliplatin (FLOX)) as well as five hydroxamic acid derivatives (suberoylanilide hydroxamic acid, PXD101, and three novel candidates of CG-1, CG-2, and CG-3) were comparatively evaluated using the histoculture drug response assay. RESULTS: The chemosensitivity with established regimens was between 34.2% and 52.6%, when the cutoff value of the inhibition ratio was set at 30%, and between 54.5% and 84.1% with HDAC inhibitors. All HDAC inhibitors displayed synergistic effects in combination with established regimens of FLOX and FLIRI (P < or = 0.0001-0.002). Advanced T- and N-category tumors and patients with synchronous adenoma displayed higher chemosensitivity to CG-3, CG-2, and CG-1, respectively, on a multivariate analysis (P = 0.023, 0.044, and 0.045, respectively). Tumors with mismatch repair defects were closely correlated with chemosensitivities to combined regimens of PDX101 with FLOX and FLIRI (P = 0.044 and 0.048, respectively). CONCLUSIONS: Our findings firstly demonstrated the chemo-responsiveness of colorectal cancers to HDAC inhibitors with therapeutic efficacy comparable to the established regimens. Additionally, tumor growth and heredity were significantly associated with specific regimens, supporting their possible role as chemosensitive predictors.

Epigenetic regulation of KLHL34 predictive of pathologic response to preoperative chemoradiation therapy in rectal cancer patients.

PURPOSE: Prediction of individual responsiveness to preoperative chemoradiation therapy (CRT) is urgently needed in patients with poorly responsive locally advanced rectal cancer (LARC). METHODS AND MATERIALS: Candidate methylation genes associated with radiosensitivity were identified using a 3-step process. In the first step, genome-wide screening of methylation genes was performed in correlation with histopathologic tumor regression grade in 45 patients with LARC. In the second step, the methylation status of selected sites was analyzed by pyrosequencing in 67 LARC patients, including 24 patients analyzed in the first step. Finally, colorectal cancer cell clones with stable KLHL34 knockdown were generated and tested for cellular sensitivity to radiation. RESULTS: Genome-wide screening identified 7 hypermethylated CpG sites (DZIP1 cg24107021, DZIP1 cg26886381, ZEB1 cg04430381, DKK3 cg041006961, STL cg00991794, KLHL34 cg01828474, and ARHGAP6 cg07828380) associated with preoperative CRT responses. Radiosensitivity in patients with hypermethylated KLHL34 cg14232291 was confirmed by pyrosequencing in additional cohorts. Knockdown of KLHL34 significantly reduced colony formation (KLHL34 sh#1: 20.1%, P=.0001 and KLHL34 sh#2: 15.8%, P=.0002), increased the cytotoxicity (KLHL34 sh#1: 14.8%, P=.019 and KLHL34 sh#2: 17.9%, P=.007) in LoVo cells, and increased radiation-induced caspase-3 activity and the sub-G1 population of cells. CONCLUSIONS: The methylation status of KLHL34 cg14232291 may be a predictive candidate of sensitivity to preoperative CRT, although further validation is needed in large cohorts using various cell types.

Novel single-nucleotide polymorphism markers predictive of pathologic response to preoperative chemoradiation therapy in rectal cancer patients.

PURPOSE: Studies aimed at predicting individual responsiveness to preoperative chemoradiation therapy (CRT) are urgently needed, especially considering the risks associated with poorly responsive patients. METHODS AND MATERIALS: A 3-step strategy for the determination of CRT sensitivity is proposed based on (1) the screening of a human genome-wide single-nucleotide polymorphism (SNP) array in correlation with histopathologic tumor regression grade (TRG); (2) clinical association analysis of 113 patients treated with preoperative CRT; and (3) a cell-based functional assay for biological validation. RESULTS: Genome-wide screening identified 9 SNPs associated with preoperative CRT responses. Positive responses (TRG 1-3) were obtained more frequently in patients carrying the reference allele (C) of the SNP CORO2A rs1985859 than in those with the substitution allele (T) (P=.01). Downregulation of CORO2A was significantly associated with reduced early apoptosis by 27% (P=.048) and 39% (P=.023) in RKO and COLO320DM colorectal cancer cells, respectively, as determined by flow cytometry. Reduced radiosensitivity was confirmed by colony-forming assays in the 2 colorectal cancer cells (P=.034 and .015, respectively). The SNP FAM101A rs7955740 was not associated with radiosensitivity in the clinical association analysis. However, downregulation of FAM101A significantly reduced early apoptosis by 29% in RKO cells (P=.047), and it enhanced colony formation in RKO cells (P=.001) and COLO320DM cells (P=.002). CONCLUSION: CRT-sensitive SNP markers were identified using a novel 3-step process. The candidate marker CORO2A rs1985859 and the putative marker FAM101A rs7955740 may be of value for the prediction of radiosensitivity to preoperative CRT, although further validation is needed in large cohorts.

Genome-wide identification of possible methylation markers chemosensitive to targeted regimens in colorectal cancers.

PURPOSE: Few efficient methylation markers of chemosensitivity have been discovered. The genome-wide analysis of methylation markers is needed to identify chemosensitive candidates to targeted therapy. METHODS: This study describes a two-step process to select chemosensitive candidates of methylation genes. A genome-wide screening of methylation genes was performed using a Beadarray and an in vitro chemosensitivity assay of 119 colorectal cancers (CRCs). Ten candidate genes identified during the initial screening were verified by biological utility assessment using cell viability assays of transfected CRC cells. RESULTS: Five methylation genes related to sensitivity to bevacizumab regimens (RASSF1, MMP25, KCNQ1, ESR1, and GALR2) or cetuximab regimens (SCL18A2, GPX7, NID2, IGFBP3, and ALX4) were chosen during the first step. A viability assay revealed that GALR2-overexpressing HCT116 cells were significantly more chemosensitive to bevacizumab regimens than control cells (P = 0.022 and 0.019 for bevacizumab with FOLFIRI and FOLFOX, respectively), concurrently verified on a caspase-3 activity assay. GPX7- or ALX4-overexpressed RKO cells were significantly less viable to cetuximab regimens compared to control cells (GPX7: P = 0.027 each for cetuximab with FOLFIRI and FOLFOX; ALX4: P = 0.049 and 0.003 for cetuximab with FOLFIRI and FOLFOX, respectively), but caspase-3 activity was not prominent in GPX7-overexpressed RKO cells. CONCLUSIONS: Two novel genes, GALR2 and ALX4, have been identified as chemosensitive methylation candidates to bevacizumab and cetuximab regimens, respectively. As our study did not include a clinical association study, the two candidates should be validated in large clinical cohorts, hopefully predicting responsive patients to targeted regimens.

Novel chemosensitive single-nucleotide polymorphism markers to targeted regimens in metastatic colorectal cancer.

PURPOSE: Methods for predicting individual responsiveness to targeted chemotherapy are urgently needed, considering the frequent resistance and extremely high cost. EXPERIMENTAL DESIGN: A chemosensitive single-nucleotide polymorphism (SNP) discovery schema is presented that utilizes (i) genome-wide SNP screening with a human SNP array and an in vitro chemosensitivity assay in 118 colorectal cancers, (ii) clinical association analysis in the other 98 patients who had received chemotherapy for metastatic cancer, and (iii) biological utility assessment using cell viability assays of transfected colorectal cancer (CRC) cells. RESULTS: Nine SNPs related to bevacizumab and cetuximab regimen sensitivity were chosen during screening. Overall responses for bevacizumab regimens revealed that patients carrying the TT genotype at ANXA11 rs1049550 or at least one G allele at LINS1 rs11247226 seemed greater chemosensitive than those carrying at least one C allele or the AA genotype, respectively (P < 0.05). For cetuximab regimens, patients carrying the GG genotype at DFNB31 rs2274159 or LIFR rs3729740 seemed greater chemosensitive than those carrying at least one A allele (P = 0.025 and P = 0.07). Cytotoxicity analyses showed that all RKO and HCT116 CRC clones transfected with the G allele at LIFR rs3729740 and the C allele at ISX rs361863 were more sensitive to cetuximab regimens than those with the A and T allele, respectively (P ≤ 0.001-0.024). CONCLUSIONS: Chemosensitive SNP markers were identified using a novel three-step process. The candidate marker LIFR rs3729740 and possibly ISX rs361863 will hopefully predict responsive patients to cetuximab regimens, although further validation is needed in large cohorts.

Chemoresponsiveness associated with canonical molecular changes in colorectal adenocarcinomas.

BACKGROUND: The canonical molecular changes in colorectal tumorigenesis were assessed for correlation with response to chemotherapy, in order to identify candidate markers. PATIENTS AND METHODS: In total, 156 patients received adjuvant postoperative fluoropyrimidine-based chemotherapy and 32 patients received oxaliplatin- or irinotecan-based chemotherapy following palliative surgery or for metastatic or recurrent colorectal tumors. Representative molecular changes in tumor tissues, including adenomatous polyposis coli (APC) gene, wingless-type MMTV integration site family (Wnt), mismatch repair (MMR), RAF, transforming growth factor (TGF)-beta, bone morphogenetic protein, and p53, had been previously determined, with an additional 42 patients included in this analysis. RESULTS: The disease-free survival period (mean+/-SEM) was significantly longer after fluoropyrimidine-based adjuvant chemotherapy in tumors with TGF-beta2 expression (42+/-1.4 vs. 21+/-4.7 months; p=0.005) and D18S46 loss of heterozygosity or microsatellite instability (45.7+/-1.5 vs. 40.5+/-1.4 months; p=0.048). In the metastatic settings, the high disease-control rate of oxaliplatin and irinotecan regimens correlated significantly with wild-type APC and intact MMR, respectively, relative to mutant APC and defective MMR (p=0.013, respectively). Interestingly, specific molecular steps of tumorigenensis were closely associated with particular toxicities. CONCLUSION: A subset of molecular changes occurring during colorectal tumorigenesis showed significant associations with therapeutic responses and toxicities to chemotherapy regimens, suggesting that these changes may be candidate predictors of chemoresponsiveness with further validation.

In vitro evaluation of histone deacetylase inhibitors as combination agents for colorectal cancer.

BACKGROUND: Our primary aim was to evaluate the additive efficacy of histone deacetylase inhibitors (HDACIs) in established treatment regimens for colorectal cancer in concurrence with identifying the clinicopathological markers significantly associated with tumor responsiveness. PATIENTS AND METHODS: The chemosensitivities of 125 colorectal carcinomas to established regimens [FLOX (5-FU + leucovorin + oxaliplatin) and FLIRI (5-FU + leucovorin + irinotecan)], two biologically targeted drugs (avastin and erbitux), and two hydroxamic acid derivatives (vorinostat, SAHA(R), and a novel candidate, CG2) were comparatively evaluated using an in vitro tumor response assay. RESULTS: The response rates of tumors (inhibition rate > or =30%) were significantly greater for FLOX and combinations (55.2-68%) compared to FLIRI and combinations (44-63.2%) (p=0.001 to 0.048), except in the case of the CG2 combination. The additive effects of HDACIs on the respective established regimens were considerably greater for non-responsive tumors (64.3-80%) than responsive tumors (32.7-45%) (p< or =0.0001 to 0.008). A number of biological parameters, including less advanced tumors and p53 overexpression, were significantly associated with additive chemosensitivity to HDACIs in combination with FLOX and FLIRI in multivariate analyses (p< or =0.001 to 0.023). Expanding tumor growth, diffuse cytoplasmic carcinoembryonic antigen (CEA) expression and synchronous adenoma were associated with combination regimens with targeted drugs (p=0.013 to 0.032). CONCLUSION: Our findings show additive chemoresponsiveness of colorectal tumors to HDAC inhibitors in combination with established regimens. The significant parameters associated with combination regimens of targeted drugs and HDACIs may be applied as effective chemosensitive markers in future clinical trials.