ABSTRACT
Baicalein, a bioactive flavonoid, has poor water solubility, thereby limiting its use in a wide range of biological applications. In the present study, we used inclusion complexes of cysteinyl ß-cyclodextrin (ß-CD) with baicalein to enhance the stability and solubility of baicalein in aqueous solution. We examined the effects of inclusion complexes of cysteinyl ß-CD on collagen synthesis following ultraviolet (UV) irradiation, as well as the mechanisms underlying its effects. Our findings demonstrated that baicalein significantly restored collagen synthesis in the UV-exposed human fibroblast Hs68 cells. In addition, synthetic cysteine functionalized ß-CDs were found to promote baicalein-induced collagen synthesis. Inclusion complexes of cysteinyl ß-CDs with baicalein significantly upregulated the protein expression of type I collagen and activated the transcription of type I, II, and III collagen. Inclusion complexes of cysteinyl ß-CDs with baicalein also downregulated matrix metalloproteinase -1 and -3, and α-smooth muscle actin expression. In addition, inclusion complexes of cysteinyl ß-CDs with baicalein attenuated the expression of caveolin-1, but this treatment enhanced the UV-induced phosphorylation of Smad in the transforming growth factor-ß pathway. These results suggested that the newly synthesized derivative of CD can be used as a complexing agent to enhance the bioavailability of flavonoids such as baicalein, especially in restoring collagen synthesis.
Subject(s)
Collagen/biosynthesis , Flavanones/metabolism , Flavonoids/metabolism , beta-Cyclodextrins/metabolism , Actins/genetics , Caveolin 1/metabolism , Collagen/genetics , Fibroblasts/metabolism , Humans , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 3/genetics , Phosphorylation/genetics , Solubility , Transforming Growth Factor beta/genetics , Ultraviolet RaysABSTRACT
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.
Subject(s)
Chemoradiotherapy , CpG Islands/genetics , DNA Methylation , Epigenesis, Genetic/physiology , Rectal Neoplasms/genetics , Rectal Neoplasms/therapy , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Chemokines , Female , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/metabolism , Gene Expression , Gene Knockdown Techniques , Genetic Markers , Genome-Wide Association Study , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Male , Middle Aged , Neoplasm Grading , Preoperative Care , Radiation Tolerance/genetics , Rectal Neoplasms/pathology , Transcription Factors/genetics , Transcription Factors/metabolism , Treatment Outcome , Zinc Finger E-box-Binding Homeobox 1ABSTRACT
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.
Subject(s)
Biomarkers, Tumor/genetics , Chemoradiotherapy/methods , Microfilament Proteins/genetics , Radiation Tolerance/genetics , Rectal Neoplasms/genetics , Rectal Neoplasms/therapy , Alleles , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis , Capecitabine , Cell Line, Tumor , Chemoradiotherapy/adverse effects , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Female , Fluorouracil/administration & dosage , Fluorouracil/analogs & derivatives , Genetic Markers/genetics , Genome-Wide Association Study/methods , Genotyping Techniques , Humans , Leucovorin/administration & dosage , Male , Middle Aged , Preoperative Care , Rectal Neoplasms/pathology , Rectal Neoplasms/surgeryABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , DNA Methylation , Aged , Antibodies, Monoclonal, Humanized , Bevacizumab , Cetuximab , DNA-Binding Proteins/genetics , Female , Genome-Wide Association Study , Humans , Male , Middle Aged , Receptor, Galanin, Type 2/genetics , Transcription Factors/genetics , Tumor Suppressor ProteinsABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Biomarkers, Tumor/genetics , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Drug Resistance, Neoplasm/genetics , Polymorphism, Single Nucleotide , Adult , Aged , Aged, 80 and over , Alleles , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/therapeutic use , Bevacizumab , Cetuximab , Colorectal Neoplasms/pathology , Female , Genome-Wide Association Study , Genotype , Humans , Male , Middle Aged , Molecular Targeted Therapy , Neoplasm Metastasis , Prognosis , Treatment OutcomeABSTRACT
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.
Subject(s)
Antineoplastic Agents/therapeutic use , Biomarkers , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Polymorphism, Single Nucleotide , Aged , Chemotherapy, Adjuvant , Cohort Studies , Disease-Free Survival , Female , Fluorouracil/therapeutic use , Gene Frequency , Humans , Linkage Disequilibrium , Male , Middle AgedABSTRACT
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.
Subject(s)
Adenocarcinoma, Mucinous/drug therapy , Adenocarcinoma/drug therapy , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/drug therapy , Enzyme Inhibitors/therapeutic use , Histone Deacetylase Inhibitors , Adenocarcinoma/enzymology , Adenocarcinoma/secondary , Adenocarcinoma, Mucinous/enzymology , Adenocarcinoma, Mucinous/secondary , Cell Proliferation/drug effects , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Synergism , Drug Therapy, Combination , Female , Humans , In Vitro Techniques , Male , Middle Aged , Neoplasm Staging , Prognosis , Prospective Studies , Treatment OutcomeABSTRACT
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.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/genetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/genetics , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Adenocarcinoma/secondary , Adenomatous Polyposis Coli Protein/genetics , Adenomatous Polyposis Coli Protein/metabolism , Biomarkers, Tumor/metabolism , Chemotherapy, Adjuvant , Colorectal Neoplasms/pathology , DNA Mismatch Repair , Disease-Free Survival , Female , Humans , Immunoenzyme Techniques , Loss of Heterozygosity , Male , Microsatellite Instability , Middle Aged , Mutation , Neoplasm Invasiveness , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Neoplasm Staging , Palliative Care , Survival Rate , Transforming Growth Factor beta2/genetics , Transforming Growth Factor beta2/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism , raf Kinases/genetics , raf Kinases/metabolismABSTRACT
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.
Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/enzymology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/enzymology , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Histone Deacetylase Inhibitors , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Drug Synergism , Female , Humans , Male , Middle Aged , Multivariate AnalysisABSTRACT
Clinicopathologic features of sporadic colorectal adenocarcinomas were compared using integrated data from 224 [corrected] patients subjected to curative resection. Individual steps in the tumorigenesis pathway, that is, adenomatosis polyposis coli (APC), Wnt-activated, base excision repair mutations, mismatch repair defects, RAF-mediated, transforming growth factor (TGF)-beta-suppressed, bone morphogenic protein (BMP)-suppressed, and p53 alterations, were examined in terms of genetic and epigenetic changes, as well as protein expression. Genetic and molecular alterations of right colon cancers were distinct from those of left colon and rectal cancers. Rectal cancers showed the attenuated phenotype of left colon cancers. Tumors most frequently displayed either TGF-beta- or BMP-suppressed alterations (81.2%), followed by RAF-mediated alterations (78.6%), and mismatch repair defects (38.4%), constituting a total of 24 integrated pathways. Tumors lacking APC mutations or carrying the RAF alteration (V600E) were frequently associated with lymphovascular invasion and lymph node metastasis (P < 0.05). Poorly differentiated or mucinous adenocarcinomas were generally associated with high level microsatellite instability, Axin2 suppression, TGF-beta1 or BMPR1A suppression, loss of heterozygosity of D18S46 or D18S474, and absence of base excision repair mutations (P < 0.0001-0.05). Early tumor recurrence was significantly correlated with lack of APC mutations (P = 0.036). Moreover, tumors that concurrently displayed APC/Wnt-activated, TGF-beta/BMP-suppressed, and p53 alterations were significantly predisposed to early recurrence (P = 0.026). Our data clearly indicate that particular steps or pathways of colorectal tumorigenesis are closely associated with characteristic clinicopathologic features that, in turn, determine biological behavior, such as tumor growth, invasion, and recurrence.