thymosin and Colorectal-Neoplasms

Colorectal carcinoma (CRC) is a complicated and often fatal genetic disease. Fortunately, owing to rapid expansion of knowledge and technology development in oncology, much progress has been made regarding the diagnosis, understanding of the molecular genetics and malignant progression, as well as the novel regimens of CRC. In this review, we summarize the staging system, the most critical genetic and epigenetic alterations, the pleiotropic effects of MMP-7, the controversial roles of Hedgehog signaling, the intriguing involvement of thymosin beta-4, and the possible contribution of the putative colon (cancer) stem cells in CRC tumorigenesis. Current treatments as well as several potentially applicable therapeutic strategies for CRC are also discussed.

Topics: Animals; Carcinoma; Colorectal Neoplasms; Hedgehog Proteins; Humans; Matrix Metalloproteinase 7; Neoplasm Staging; Stem Cells; Thymosin; Trans-Activators

Colorectal cancer (CRC) is a complicated tumor, involving several oncogenic signaling pathways, and with a molecular mechanism not fully understood yet. The implication of thymosin β4 (Tβ4) with tumor insurgence and in migration of CRC cells was evidenced in the past with different methodologies, while Tβ10 connection with CRC has been sporadically investigated. This study focused on the implication of both types of thymosin in CRC progression and invasion by analyzing the changes in their levels according to different zones of the tumor, and to Dukes stage and budding index.. Tβ4 and Tβ10 were analyzed in deep and superficial tumor samples, and normal mucosa from 18 patients. Concentrations of Tβ4 and Tβ10 have been measured by high-pressure liquid chromatography (HPLC) coupled to electrospray-ion trap mass spectrometry (ESI-IT-MS). MS data were compared by t-test and ANOVA statistical analysis. Identification of thymosin and their proteoforms has been performed by HPLC-high resolution-ESI-IT-MSMS.. Both Tβ4 and Tβ10, exhibited intra-tumoral quantitative differences, being upregulated in the deep part of the CRC. They exhibited, moreover, strong association with the Dukes stage and the budding grade, being more concentrated in patients at Dukes stage B and with budding index "2".. The results obtained in the present investigation encouraged the hypothesis that the two thymosin are involved in colorectal cancer progression, and in promoting cancer invasion. Thus, they are good candidates to be diagnostic/prognostic biomarkers and therapy targets.

Topics: Aged; Aged, 80 and over; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Disease Progression; Female; Humans; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Prognosis; Signal Transduction; Spectrometry, Mass, Electrospray Ionization; Thymosin

DNA methyltransferases (DNMTs) take on a relevant role in epigenetic control of cancer proliferation and cell survival. However, the molecular mechanisms underlying the establishment and maintenance of DNA methylation in human cancer remain to be fully elucidated. This study was to investigate that how DNMT1 affected the biological characteristics of colorectal cancer (CRC) cells via modulating methylation of microRNA (miR)-152-3p and thymosin β 10 (TMSB10) expression.. DNMT1, miR-152-3p, and TMSB10 expression, and the methylation of miR-152-3p in CRC tissues and cells were detected. SW-480 and HCT-116 CRC cells were transfected with DNMT1 or miR-152-3p-related sequences or plasmids to explore their characters in biological functions of CRC cells. The binding relationship between DNMT1 and miR-152-3p and the targeting relationship between miR-152-3p and TMSB10 were analyzed. The tumor growth was also detected in vivo.. Upregulated DNMT1, TMSB10, reduced miR-152-3p, and methylated miR-152-3p were detected in CRC tissues and cells. Silenced DNMT1 or upregulated miR-152-3p reduced TMSB10 expression and suppressed CRC progression and tumor growth. Moreover, elevated DNMT1 could reverse the effect of miR-152-3p upregulation on CRC development and tumor growth. DNMT1 maintained methylation of miR-152-3p. TMSB10 was the direct target gene of miR-152-3p.. The study highlights that silenced DNMT1 results in non-methylated miR-152-3p to depress TMSB10 expression, thereby inhibiting CRC development, which provides a new approach for CRC therapy.

Topics: Adult; Aged; Apoptosis; Biomarkers, Tumor; Cell Cycle; Cell Movement; Cell Proliferation; Colorectal Neoplasms; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Prognosis; Survival Rate; Thymosin; Tumor Cells, Cultured

DNA aneuploidy has been identified as a prognostic factor for epithelial malignancies. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for direct analysis of multiple proteins in tissue sections while maintaining the cellular and molecular integrity. We compared diploid and aneuploid colon cancer tissues against normal mucosa of the colon by means of IMS. DNA image cytometry determined the ploidy status of tissue samples that were subsequently subjected to MALDI-IMS. After obtaining protein profiles through direct analysis of tissue sections, a discovery and independent validation set were used to predict ploidy status by applying proteomic classification algorithms [Supervised Neural Network (SNN) and Receiver Operating Characteristic (ROC)]. Five peaks (m/z 2,395 and 4,977 for diploid vs. aneuploid comparison as well as m/z 3,376, 6,663, and 8,581 for normal mucosa vs. carcinoma comparison) were significant in both SNN and ROC analysis. Among these, m/z 4,977 was identified as thymosin beta 4 (Tβ-4). Tβ-4 was subsequently validated in clinical samples using a tissue microarray to predict overall survival in colon cancer patients.

Topics: Aged; Algorithms; Area Under Curve; Biomarkers, Tumor; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Male; Middle Aged; Ploidies; Prognosis; ROC Curve; Sensitivity and Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thymosin; Tissue Array Analysis

Topics: AC133 Antigen; Adult; Aged; Antigens, CD; Biomarkers, Tumor; Colorectal Neoplasms; Female; Glycoproteins; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Recurrence, Local; Peptides; Thymosin; Tissue Array Analysis; Up-Regulation

Thymosin beta-4 (Tβ4) is known to be involved in tumorigenesis. Overexpression of this polypeptide has been observed in a wide variety of cancers, including colorectal carcinoma (CRC). Accordingly, Tβ4 has been proposed to be a novel therapeutic target for CRC, especially in its metastatic form. Although in vitro tumor-suppressive effects of Tβ4 gene silencing mediated by small hairpin RNA (shRNA) have already been demonstrated, the in vivo efficacy of such an approach has not yet been reported. Herein, we demonstrated that infection with recombinant adenovirus expressing an shRNA targeting Tβ4 markedly reduced the growth of and robustly induced apoptosis in CT-26 mouse CRC cells in culture. Additionally, tumors grown in nude mice from the CT-26 cells whose Tβ4 expression already been downregulated by virus infection were also drastically reduced. Most importantly, significant growth arrest of tumors derived from the parental CT-26 cells was observed after multiple intratumoral injections of these viruses. Together, our results show for the first time that in vivo silencing of Tβ4 expression by its shRNA generated after adenoviral infection can suppress CRC growth. These results further demonstrate the feasibility of treating CRC by a Tβ4 knockdown gene therapeutic approach.

Topics: Actins; Adenoviridae; Animals; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Colorectal Neoplasms; Disease Models, Animal; Gene Knockout Techniques; Genetic Vectors; Humans; Mice; RNA Interference; RNA, Messenger; RNA, Small Interfering; Thymosin; Transduction, Genetic; Tumor Burden; Xenograft Model Antitumor Assays

Human prothymosin-α (PTMA) plays an important role in tumorigenesis, and its overexpression triggers a TP53 response. In this study, we identified that PTMA expression was up-regulated at both the transcriptional and translational level in tumor tissue compared to that in adjacent normal tissue. PTMA overexpression was significantly associated with the depth of tumor invasion, lymph node metastasis (LNM), distant metastasis, advanced AJCC stage, and tumor differentiation. There was also a significant association between PTMA over-expression and mutant TP53 expression (r=0.515, P < 0.001). Survival analysis revealed that the disease-free survival (DFS) and overall survival (OS) rates were significantly lower among patients with PTMA- and TP53-positive tumors. Hence, PTMA might play an important role in the progression of CRC, and the assessment of both PTMA and mutant TP53 expression can help predict colon cancer prognosis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Blotting, Western; Colorectal Neoplasms; Disease-Free Survival; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Prognosis; Proportional Hazards Models; Protein Precursors; Real-Time Polymerase Chain Reaction; Thymosin; Tumor Suppressor Protein p53; Young Adult

Thymosin β4 (Tβ4) is a 43-amino-acid peptide involved in many biological processes. However, the precise molecular signaling mechanism(s) of Tβ4 in cell invasion and migration remain unclear. In this study, we show that Tβ4 was significantly overexpressed in colorectal cancer tissues compared to adjacent normal tissues and high levels of Tβ4 were correlated with stage of colorectal cancer, and that Tβ4 expression was associated with morphogenesis and EMT. Tβ4-upregulated cancer cells showed increased adhesion, invasion and migration activity, whereas Tβ4-downregulated cells showed decreased activities. We also demonstrated that Tβ4 interacts with ILK, which promoted the phosphorylation and activation of AKT, the phosphorylation and inactivation of GSK3β, the expression and nuclear localization of β-catenin, and integrin receptor activation. These results suggest that Tβ4 is an important regulator of the ILK/AKT/β-catenin/Integrin signaling cascade to induce cell invasion and migration in colorectal cancer cells, and is a potential target for cancer treatment.

Topics: Aged; beta Catenin; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Integrins; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Thymosin; Tumor Microenvironment

Thymosin β 4 (Tβ(4)) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation during embryogenesis. Recently, a role for Tβ(4) has been proposed in experimental and human carcinogenesis. This study was aimed at evaluating the correlation between Tβ(4) immunoractivity and colorectal cancer, with particular attemption to tumor cells undergoing epithelial-mesenchymal transition.. 86 intestinal biopsies were retrospectively analyzed including 76 colorectal adenocarcinomas with evident features of epithelial-mesenchymal transition, and 10 samples of normal colorectal mucosa. Paraffin sections were immunostained for Tβ(4) and for E-cadherin. Total RNA was isolated from frozen specimens obtained, at surgery, from the normal colon mucosa, the deeper regions and the superficial tumor regions in four cases of colon cancer. Tβ(4) immunoreactivity was detected in the vast majority (59/76) of colon carcinomas, showing a patchy distribution, with well differentiated areas significantly more reactive than the less differentiated tumor zones. We also noted a zonal pattern in the majority of tumors, characterized by a progressive increase in immunostaining for Tβ(4) from the superficial toward the deepest tumor regions. The strongest expression for Tβ(4) was frequently detected in invading tumor cells with features of epithelial-mesenchymal transition. The increase in reactivity for Tβ(4) matched with a progressive decrease in E-cadherin expression in invading cancer cells. At mRNA level, the differences in Tβ(4) expression between the surrounding colon mucosa and the tumors samples were not significant.. Our data show that Tβ(4) is expressed in the majority of colon cancers, with preferential immunoreactivity in deep tumor regions. The preferential expression of the peptide and the increase in intensity of the immunostaining at the invasion front suggests a possible link between the peptide and the process of epithelial mesenchymal transition, suggesting a role for Tβ(4) in colorectal cancer invasion and metastasis.

Topics: Adenocarcinoma; Cadherins; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cohort Studies; Colorectal Neoplasms; Epithelial-Mesenchymal Transition; Humans; Immunohistochemistry; Thymosin

Thymosin β4 (Tβ4) is highly expressed in saliva of human newborns but not in adults. Here preliminary immunohistochemical analyses on different human tissues are reported. Immunoreactivity for Tβ4 in human salivary glands show high quantities of Tβ4 before birth, followed by downregulation of expression in adulthood. In contrast, Tβ4 is detected in tumors of salivary glands, suggesting that tumor cells might utilize fetal programs, including Tβ4 synthesis. Immunohistochemical analyses in the gastrointestinal tract showed strong reactivity for Tβ4 in enterocytes during development, but weak immunostaining in mature enterocytes. In colorectal cancer, the association of a high expression of Tβ4 with epithelial-mesenchymal transition was observed. On the basis of these data, the process of epithelial-mesenchymal transition could represent the unifying process that explains the role of Tβ4 during fetal development and in cancer progression.

Topics: Colorectal Neoplasms; Enterocytes; Epithelial-Mesenchymal Transition; Gastrointestinal Tract; Humans; Liver; Salivary Glands; Thymosin

Thymosin β4 has been reported to play the key roles in tumor growth, metastasis, and angiogenesis. Although the importance of thymosin β4 in angiogenesis and metastasis is known, few studies to show the expression patterns of thymosin β4 in human tissues including tumors have been conducted. The comparisons of the expression of thymosin β4 between the normal and tumor tissues are also needed to study the role of thymosin β4 in tumor formation. Using tissue microarray analysis, we compared the expression patterns of thymosin β4 in the normal human tissues and in the tumors to screen certain tumors and upregulated the expression of thymosin β4 by tumorigenesis. Thymosin β4 was highly expressed in the hepatic cells in the normal adult liver, duct, and acinar cells in pancreas, and muscle cells in the heart and also expressed highly in certain tumor cells, including osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. Comparing the thymosin β4 expression between normal and tumors, thymosin β4 was upregulated specifically in osteosarcoma, colorectal carcinoma, and esophageal cancer. To confirm the over-expression of thymosin β4 in these tumors, we analyzed expression of thymosin β4 with each additional microarray of osteosarcoma, colorectal carcinoma, and esophageal cancer. The significant increased expression of thymosin β4 was observed in osteosarcoma and in colorectal cancer. These results suggest that the expression of thymosin β4 is highly related with tumorigenesis of certain tumors including the osteosarcoma and colorectal cancers.

Topics: Adult; Cell Line, Tumor; Colorectal Neoplasms; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Organ Specificity; Osteosarcoma; Thymosin; Tissue Array Analysis; Up-Regulation

The epithelial-mesenchymal transition (EMT) is crucial for the invasion and metastasis of many epithelial tumors including colorectal carcinoma (CRC). In the present study, a scattering and fibroblastic morphology with reduced intercellular contacts was found in the SW480 colon cancer cells overexpressing the gene encoding thymosin beta4 (Tbeta4), which was accompanied by a loss of E-cadherin as well as a cytosolic accumulation of beta-catenin, two most prominent markers of EMT. Whereas E-cadherin downregulation was likely to be accounted by a ZEB1-mediated transcriptional repression, the accumulation of beta-catenin was a result of glycogen synthase kinase-3beta inactivation mediated by integrin-linked kinase (ILK) and/or its downstream effector, Akt. Intriguingly, ILK upregulation in Tbeta4-overexpressing SW480 cells seemed to be attributed mainly to a stabilization of this kinase by complexing with particularly interesting new Cys-His protein (PINCH) more efficiently. In the meantime, a strong correlation between the expression levels of Tbeta4, ILK and E-cadherin in CRC patients was also revealed by immunohistochemical analysis. Taken together, these data suggest a novel role of Tbeta4 in promoting CRC progression by inducing an EMT in tumor cells via upregulating ILK and consequentially its signal transduction.

Topics: Adaptor Proteins, Signal Transducing; Animals; beta Catenin; Cadherins; Casein Kinase I; Colonic Neoplasms; Colorectal Neoplasms; DNA-Binding Proteins; Enzyme Inhibitors; Epithelial Cells; Fibroblasts; Glycogen Synthase Kinase 3; Homeodomain Proteins; Humans; Immunoenzyme Techniques; LIM Domain Proteins; Membrane Proteins; Mesoderm; Mice; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Antisense; RNA, Messenger; Signal Transduction; Thymosin; Transcription Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

Overexpression of thymosin beta-4 has been linked to malignant progression but the localization of this polypeptide within tumors is incompletely known. We therefore examined breast cancers for thymosin beta-4 using immunofluorescence. Reactive cells were identified with monoclonal cell marker antibodies. A very heterogeneous staining pattern for thymosin beta-4 was observed. Thus, while leukocytes and macrophages showed intense reactivity for this polypeptide, cancer cells, and endothelial cells showed a much more variable reactivity. A similar heterogeneous staining was observed also in colorectal carcinomas. The degree of staining of breast cancer cells for thymosin beta-4 correlated neither to histological grade nor to endothelial cell staining. However, there was a tendency toward correlation (P = 0.07) between staining of endothelial cells and histological grade. Treatment of cultured breast cancer cells (SK-BR-3) with 1-4 microg thymosin beta-4/mL significantly increased cell numbers, as determined by MTT-assays. These data reveal an unexpected cellular heterogeneity of thymosin beta-4 expression in breast and colonic carcinomas and suggest that local release of this polypeptide in the tumor microenvironment may modulate tumor behavior.

Topics: Breast Neoplasms; Cell Nucleus; Colorectal Neoplasms; Cytoplasm; Female; Fluorescent Antibody Technique; Humans; Keratins; Leukocytes; Thymosin

Our aim was to determine whether the expression levels of specific genes could predict clinical radiosensitivity in human colorectal cancer.. Radioresistant colorectal cancer cell lines were established by repeated X-ray exposure (total, 100 Gy), and the gene expressions of the parent and radioresistant cell lines were compared in a microarray analysis. To verify the microarray data, we carried out a reverse transcriptase-polymerase chain reaction analysis of identified genes in clinical samples from 30 irradiated rectal cancer patients.. A comparison of the intensity data for the parent and three radioresistant cell lines revealed 17 upregulated and 142 downregulated genes in all radioresistant cell lines. Next, we focused on two upregulated genes, PTMA (prothymosin alpha) and EIF5a2 (eukaryotic translation initiation factor 5A), in the radioresistant cell lines. In clinical samples, the expression of PTMA was significantly higher in the minor effect group than in the major effect group (P = 0.004), but there were no significant differences in EIF5a2 expression between the two groups.. We identified radiation-related genes in colorectal cancer and demonstrated that PTMA may play an important role in radiosensitivity. Our findings suggest that PTMA may be a novel marker for predicting the effectiveness of radiotherapy in clinical cases.

Topics: Aged; Biomarkers, Tumor; Biopsy; Colectomy; Colonoscopy; Colorectal Neoplasms; Eukaryotic Translation Initiation Factor 5A; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Peptide Initiation Factors; Preoperative Care; Prognosis; Protein Precursors; Radiotherapy, Adjuvant; Retrospective Studies; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; RNA, Neoplasm; Thymosin; Tumor Cells, Cultured

For hMLH1, a key enzyme of DNA mismatch repair and frequently mutated in human cancers, several additional functions have been suggested. We now identified Thymosin beta4 (Tbeta4), an actin-binding and cell motility regulating protein, by bacterial two-hybrid screening. Interaction was confirmed by coimmunoprecipitation. Tbeta4 was weakly expressed in the hMLH1-deficient cell lines 293T and HCT-116. Reconstitution of hMLH1 resulted in strong expression of Tbeta4. Confocal laser microscopy revealed nuclear colocalization of both proteins. Reconstitution with hMLH1 mutants lacking a functional nuclear localization sequence resulted in cytoplasmatic retention of both proteins. After Tbeta4- or hMLH1-siRNA treatment, cell migration of hMLH1-proficient cells was markedly decreased. Our results show that hMLH1 interacts with Tbeta4 and regulates its expression and nuclear transport. Moreover, loss of hMLH1 causes Tbeta4 deprivation and results in reduced migratory activity in vitro. These data give insight into novel functions of hMLH1 and probably disease related dysregulated mechanisms.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Cell Line; Colorectal Neoplasms; DNA Repair; Gene Expression Regulation; Humans; Kidney; MutL Protein Homolog 1; Nuclear Proteins; Thymosin

Cell-matrix and cell-cell adhesive interactions play important roles in the normal organization and stabilization of the cell layer in epithelial tissue. Alterations in the expression and function of these adhesion systems that cause a switch to a migratory phenotype in tumor invasion and metastasis are critical for the malignant conversion of epithelial cells. Thymosin beta-4 (Tbeta-4) is the major actin-sequestering protein that has been shown to be upregulated in a wide variety of human carcinomas and has been implicated to be involved in altering the motility of certain tumors. We have recently demonstrated that the growth rate, colony formation in soft agar, and motility, all good indicators for malignant progression, of SW480 colon carcinoma cells are dramatically increased by enforced Tbeta-4 expression. To test the hypothesis that overexpression of this G-actin sequestering peptide also promotes tumor invasion, we examined not only the invasion capability of Tbeta-4-overexpressing SW480 cells, but also the expression levels of Tbeta-4 as well as several proteins that participate in different stages of tumor progression in matched samples of human primary colorectal adenocarcinoma and liver metastases from several patients. A marked increase on the invasiveness in Tbeta-4-overexpressing SW480 cells with increased levels and activity of matrix metalloproteinase-7 (MMP-7) was observed. Furthermore, the levels of Fas as well as the susceptibility to Fas ligand-mediated apoptosis in Tbeta-4-overexpressing cells were significantly decreased. Interestingly, the levels of Tbeta-4 mRNA, beta-catenin, c-Myc, and MMP-7 in metastatic liver lesions were relatively higher, whereas the levels of E-cadherin and Fas were significantly lower than those in the matched primary colorectal tumors. These results suggest that upregulation of Tbeta-4, by promoting the disruption of cell-cell adhesion and a consequential activation of the beta-catenin signaling, could be a key event in the acquisition of growth advantages as well as invasive phenotypes in human colorectal carcinomas.

Topics: Cell Line, Tumor; Colorectal Neoplasms; Gene Expression; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; RNA, Messenger; Thymosin

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for their anti-inflammatory effects and have been shown to have chemopreventive effects as well. NSAIDs inhibit cyclooxygenase (COX) activity to exert their anti-inflammatory effects, but it is not clear whether their antitumorigenic ability is through COX inhibition. Using subtractive hybridization, we previously identified a novel member of the transforming growth factor-beta superfamily that has antitumorigenic activity from indomethacin-treated HCT-116 human colorectal cancer cells. On further investigation of this library, we now report the identification of a new cDNA corresponding to the thymosin beta-4 gene. Thymosin beta-4 is a small peptide that is known for its actin-sequestering function, and it is associated with the induction of angiogenesis, accelerated wound healing, and metastatic potential of tumor cells. However, only selective NSAIDs induce thymosin beta-4 expression in a time- and concentration-dependent manner. For example, indomethacin and SC-560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole] induce thymosin beta-4 expression whereas sulindac sulfide does not. We show that selective NSAIDs induce actin cytoskeletal reorganization, a precursory step to many dynamic processes regulating growth and motility including tumorigenesis. This is the first report to link thymosin beta-4 induction with NSAIDs. These data suggest that NSAIDs alter the expression of a diverse number of genes and provide new insights into the chemopreventive and biological activity of these drugs.

Topics: Actins; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Northern; Cell Line, Tumor; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Cytokines; Cytoskeleton; Dinoprostone; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Growth Differentiation Factor 15; Humans; Indicators and Reagents; Indomethacin; Prostaglandin-Endoperoxide Synthases; Pyrazoles; RNA, Neoplasm; Thymosin

We have used chemo-immunotherapy with 5-fluorouracil (5-FU), thymosin alpha1 (T alpha1) and interleukin-2 (IL-2) to treat multiple liver metastases from colorectal cancer induced by DHD/K12 cells in syngeneic BDIX rats, comparing one and two cycles of treatment, and different treatment combinations. 5-FU was delivered loco-regionally as a continuous infusion via an intraperitoneal (i.p.) catheter from a subcutaneously implanted mini-pump, a method we developed for this study. We show here that two cycles of a triple chemo-immunotherapy regimen significantly increased the average survival time compared to one cycle, and compared to untreated controls or those treated with two cycles of 5-FU alone. At 150 days, two rats treated with two cycles of triple therapy were cured, showing no signs of cancer at autopsy; all the other rats died before this time. Triple chemo-immunotherapy resulted in significantly fewer extra-hepatic metastases than in the controls and in those treated with 5-FU only. Further, we found that two cycles of triple treatment significantly increased the absolute number of peripheral T cells expressing IL-2 receptor, CD4 and CD8 compared to controls. We conclude that two cycles of chemo-immunotherapy with 5-FU, T alpha1 and IL-2 were superior to one cycle of treatment and to other treatments tested. Our results suggest that the triple therapy acts by increasing numbers of effector T cells. This method shows promise for the use of multi-cycle chemo-immunotherapy in the treatment of unresectable metastases of colorectal cancer in humans.

Topics: Animals; CD4 Lymphocyte Count; CD8-Positive T-Lymphocytes; Colorectal Neoplasms; Fluorouracil; Injections, Intraperitoneal; Interleukin-2; Male; Rats; Rats, Inbred Strains; Receptors, Interleukin-2; Thymalfasin; Thymosin

The effects of prothymosin alpha1 (Pro alpha1) in combination with interleukin-2 (IL-2) on peripheral blood lymphocytes from 50 colorectal tumor patients at different stages were studied with respect to immunocytotoxicity, adhesion to cultured SW620 colon carcinoma cells, secretion of cytokines and expression of adhesion and surface marker molecules. On average, the patients showed lower natural killer (NK) cell activity than healthy donors, which was associated with a lower adhesion capacity to the tumor target cells. The NK cell activity of the patients was inversely related to the tumor stage. The generation of lymphokine(IL-2)-activated killer (LAK) cell activity was found to be comparable on lymphocytes from healthy individuals and patients and was not correlated to tumor stage. Pro alpha1 stimulated patients' LAK cell activity only, primarily at the early stage (Dukes A/B). The Pro alpha1 effect was associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of the deficient IL-2-induced IFN gamma secretion. No significant effects on the low level of TNF alpha secretion was noted. By flow cytometry, Pro alpha1 in combination with IL-2 augmented the expression of the NK cell markers CD56, CD16/56, the subset CD3/16/56 and CD25 on lymphocytes of the patients. In contrast, Pro alpha1 was equally effective by increasing the expression of CD18 and CD11a on lymphocytes from the patients and from normal controls. In conclusion, Pro alpha1, in combination with IL-2, can partially normalize lymphocyte deficiencies of colon cancer patients in vitro. This potential might provide an experimental basis for applying Pro alpha1 or related thymic peptides in selected immunotherapies against colorectal tumors.

Topics: Adult; Aged; Aged, 80 and over; Cell Adhesion; Colonic Neoplasms; Colorectal Neoplasms; Cytotoxicity, Immunologic; Female; Humans; Immunotherapy, Adoptive; Interleukin-2; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Lymphocytes; Male; Middle Aged; Neoplasm Staging; Protein Precursors; Thymosin; Tumor Cells, Cultured

Recent animal studies demonstrate that prothymosin alpha 1 (ProT alpha) enhances the antitumor response by stimulation of mononuclear phagocyte functions. The present study was aimed at characterizing the in vitro effects by ProT alpha on blood monocytes from human colon cancer patients. Purified peripheral blood monocytes were studied in terms of tumor cytostatic ability and cytokine production after incubation with ProT alpha or interferon (rIFN-gamma) and transforming growth factor-beta (TGF beta), used as reference substances. SW620 colon carcinoma cells were used as tumor target cells in growth inhibition experiments. The level of baseline growth inhibitory activity of unstimulated patient's monocytes was significantly lower than that of normal monocytes. The defective antitumor activity of patient monocytes was associated with a higher production of the inhibitory monokines prostaglandin E2 (PGE2) and TGF beta. The stimulation of monocytes by ProT alpha and/or rIFN-gamma elevated the average antitumor activity in all donor groups. The ProT alpha-induced increase was associated with a significantly higher monocytic secretion of IL-1 beta and TNF-alpha. Moreover, the concentrations of TGF beta and PGE2 in the culture supernatants decreased significantly, when patient's monocytes were treated with ProT alpha and/or rIFN-gamma. Additionally, ProT alpha enhanced the diminished antitumor activity of TGF beta-treated normal monocytes. These results suggest that ProT alpha selectively regulates distinct functions of blood monocytes, the effect of this cytokine varying with the parameter and donor population examined. These data provide a rational and biological endpoint for further studies with ProT alpha as an activator of mononuclear function in colon cancer.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoembryonic Antigen; Colorectal Neoplasms; Cytokines; Dinoprostone; Humans; In Vitro Techniques; Interleukin-1; Middle Aged; Monocytes; Protein Precursors; Thymosin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We studied the effect of combined chemo-immunotherapy, 5-FU followed by thymosin alpha 1 (T alpha 1) and interleukin-2 (IL-2) at low doses, on liver metastases from colorectal cancer, induced by splenic injection of DHD/K12 cells (1,2-dimethylhydrazine-induced colon adenocarcinoma) in syngeneic BDIX rats. The presence of liver metastases was checked by laparotomy 14 days after tumor-cell injection. Evaluable rats were assigned randomly to 5 experimental groups designated as control, 5-FU, IL-2, 5-FU/IL-2 and 5-FU/T alpha 1/IL-2. 5-FU was administered i.v. as a continuous infusion for 7 days by an osmotic device implanted surgically. T alpha 1 and IL-2 were administered for 4 days and repeated after 11 days. Combined chemo-immunotherapy was shown both to significantly reduce the growth of liver metastases and to prevent extra-hepatic spread. 5-FU/T alpha 1/IL-2 also improved survival rate. Combined immunotherapy after 5-FU restored NK activity of the peripheral-blood-mononuclear-cell (PBMC) in tumor and/or 5-FU immunodepressed rats and enhanced PBMC cytotoxic activity against the DHD/K12 autologous cell line. This model was devised to mimic the clinical situation of unresectable liver metastases.

Topics: Animals; Carcinoma; Colorectal Neoplasms; Combined Modality Therapy; Cytotoxicity, Immunologic; Fluorouracil; Immunotherapy; Interleukin-2; Killer Cells, Natural; Liver Neoplasms; Male; Rats; Rats, Inbred Strains; Survival Analysis; T-Lymphocytes, Cytotoxic; Thymalfasin; Thymosin

We constructed a "non-metastatic cell (SW837)--metastatic cell (PMCO1)" subtraction library and identified one cDNA that was strongly expressed in SW837 but weakly expressed in PMCO1. The nucleotide sequence of the cDNA revealed that it encoded thymosin beta-4. Four non-metastatic cell lines, which produced no experimental liver metastasis in nude mice, showed high expression of thymosin beta-4. However three of four metastatic cell lines showed weak expression of thymosin beta-4. Among surgical materials, thymosin beta-4 expression was high in tumors without metastasis in comparison with non-tumorous mucosa, but one case with liver metastasis showed decreased expression in both the primary and metastatic tumors. We suspect that down-regulation of thymosin beta-4 expression is correlated with the metastatic capacity of colorectal carcinomas.

Topics: Animals; Colonic Neoplasms; Colorectal Neoplasms; DNA, Neoplasm; Gene Expression; Gene Library; Humans; Mice; Mice, Nude; Neoplasm Transplantation; RNA, Messenger; Thymosin; Transplantation, Heterologous; Tumor Cells, Cultured