Colon Carcinoma Therapy Research Articles

Transcription Factor FOSL1 Promotes Angiogenesis of Colon Carcinoma by Regulating the VEGF Pathway Through Activating TIMP1.

Angiogenesis is the critical media for tumor growth and migration. Tissue Inhibitor Matrix Metalloproteinase-1 (TIMP1) acts as an oncogene in colon carcinoma (CC), but the biological effects of TIMP1 on angiogenesis remain an open issue. This study sought to explore the exact function and mechanism of TIMP1 in the angiogenesis of CC. Bioinformatics methods were utilized to analyze the expression of TIMP1 and its upstream transcription factor FOS-like antigen 1 (FOSL1) in the tumor tissue of CC. Meanwhile, in CC cell lines, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot were utilized to verify the expression of TIMP1 and FOSL1. Cell counting kit-8 and tube formation assays were utilized to analyze the proliferation and angiogenesis abilities of human umbilical vein endothelial cells (HUVECs). Western blot was used to detect the protein expression of VEGFA, VEGFR-2, and VEGFR-3. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were carried out to explore the specific interaction between FOSL1 and TIMP1. The present study discovered that TIMP1 and FOSL1 were evidently up-regulated in CC tissue and cells. Meanwhile, TIMP1 was found to participate in regulating the signaling pathway of vascular endothelial growth factor (VEGF). Silenced TIMP1 conspicuously suppressed the proliferation and angiogenesis of HUVECs and reduced the protein expression of VEGFA, VEGFR-2, and VEGFR-3. Moreover, FOSL1 could promote TIMP1 transcription by binding with its promoter and the inhibition of TIMP1 expression obviously reversed the promotion effects of FOSL1 overexpression on the proliferation and angiogenesis of HUVECs. FOSL1 activated VEGF pathway by up-regulating TIMP1 expression, thereby advancing CC angiogenesis. We provided theoretical basis that the FOSL1/TIMP1/VEGF pathway might be a novel option for anti-angiogenesis therapy of CC.

Hafnium (Hf)-Chelating Porphyrin-Decorated Gold Nanosensitizers for Enhanced Radio-Radiodynamic Therapy of Colon Carcinoma.

X-ray-induced radiodynamic therapy (RDT) that can significantly reduce radiation dose with an improved anticancer effect has emerged as an attractive and promising therapeutic modality for tumors. However, it is highly significant to develop safe and efficient radiosensitizing agents for tumor radiation therapy. Herein, we present a smart nanotheranostic system FA-Au-CH that consists of gold nanoradiosensitizers, photosensitizer chlorin e6 (Ce6), and folic acid (FA) as a folate-receptor-targeting ligand for improved tumor specificity. FA-Au-CH nanoparticles have been demonstrated to be able to simultaneously serve as radiosensitizers and RDT agents for enhanced computed tomography (CT) imaging-guided radiotherapy (RT) of colon carcinoma, owing to the strong X-ray attenuation capability of high-Z elements Au and Hf, as well as the characteristics of Hf that can transfer radiation energy to Ce6 to generate ROS from Ce6 under X-ray irradiation. The integration of RT and RDT in this study demonstrates great efficacy and offers a promising therapeutic modality for the treatment of malignant tumors.

Down-regulation of EZH2 genes targeting RUNX3 affects proliferation, invasion, and metastasis of human colon cancer cells by Wnt/β-catenin signaling pathway.

In order to detect the effect of EZH2 genes on proliferation, migration, invasion, and apoptosis of colon carcinoma cell strains HCT116 and HT29 by the Wnt/β-catenin signaling pathway, qRT-PCR was applied to measure relative expressions of EZH2, RUNX3, CEA, CA199, MMP-9, VEGF, β-catenin, and CyclinD1 in each group; Western-blot was employed with the intention of exploring relative expressions of these proteins in vivo and in vitro; monoclonal proliferation experiments and CCK-8 assay was adopted so as to check cell proliferation; the effect on cell migration was investigated via Transwell assay and cell scratch wound assay; flow cytometry was applied with a view to determining the effect on cell apoptosis. Transfected HCT116 cells are injected subcutaneously into nude mice. In colon cell strains HCT-116 and HT29, contrasted to the si-NC group, the RUNX3 expression was prominently up-regulated in the si-EZH2 group. Besides, expressions of CEA, CA199, MMP-9, and VEGF were significantly reduced; down-regulation of EZH2 genes remarkably inhibited cell proliferation, invasion and migration when facilitating apoptosis; down-regulation of EZH2 genes also significantly reduced expressions of essential proteins β-catenin and CyclinD1 on the Wnt pathway. The subcutaneous tumor body of nude mice was reduced. EZH2-OE is the opposite trend to si-EZH2; The EZH2 gene may target regulatory RUNX3 regulation via that Wnt/β-catenin signaling pathway, hence affecting colon carcinoma cell proliferation, invasion, migration, and apoptosis. Therefore, EZH2 may become a promising target for the clinical therapy of colon carcinoma.

HCMV-Mediated Interference of Bortezomib-Induced Apoptosis in Colon Carcinoma Cell Line Caco-2.

Human cytomegalovirus (HCMV) has been implicated in the development of human malignancies, for instance in colon cancer. Proteasome inhibitors were developed for cancer therapy and have also been shown to influence HCMV infection. The aim of this study was to investigate if proteasome inhibitors have therapeutic potential for colon carcinoma and how this is influenced by HCMV infection. We show by immunofluorescence and flow cytometry that the colon carcinoma cell line Caco-2 is susceptible to HCMV infection. Growth curve analysis as well as protein expression kinetics and quantitative genome analysis further confirm these results. HCMV has an anti-apoptotic effect on Caco-2 cells by inhibiting very early events of the apoptosis cascade. Further investigations showed that HCMV stabilizes the membrane potential of the mitochondria, which is typically lost very early during apoptosis. This stabilization is resistant to proteasome inhibitor Bortezomib treatment, allowing HCMV-infected cells to survive apoptotic signals. Our findings indicate a possible role of proteasome inhibitors in colon carcinoma therapy.

First report on the cytotoxicity of Pinus merkusii bark extract in WiDr, A human colon carcinoma cell line

Pine bark was previously known as a waste product from the lumber industry, but it is now regarded as a natural ingredient that is rich in proanthocyanidin content with potential medicinal properties. This study was aimed to investigate the cytotoxic effects of P. merkusii bark extract (PMBE) on WiDr, a human colon carcinoma cell line. The cytotoxicity test of PMBE was performed using the MTT assay. PMBE inhibited the viability of WiDr cells with an IC50 of 235.60 μg/mL. Despite the weak cytotoxic effect, the current study represents the first report of the potency of PMBE as an anticancer agent in WiDr cell lines. In addition, further observation to find the optimal dosage and mechanism action of PMBE needs to be undertaken as a strategy in human colon carcinoma therapy.

Tumor necrosis factor‑related apoptosis‑inducing ligand inhibits the growth and aggressiveness of colon carcinoma via the exogenous apoptosis signaling pathway

Colon cancer is one of the most common types of gastrointestinal tumor. Previous studies have demonstrated that tumor necrosis factor-(TNF)-related apoptosis-inducing ligand (TRAIL) reduces the aggressiveness of colon cancer tumors and promotes the apoptosis of colon carcinoma cells. In the present study, the inhibitory effects of TRAIL were investigated and the potential mechanism of TRAIL-mediated apoptosis was explored in colon cancer cells. Reverse transcription-quantitative polymerase chain reaction, western blotting, immunofluorescence, immunohistochemistry, TUNEL and flow cytometry assays were used to analyze the effects of TRAIL on the growth, migration, invasion and apoptosis of colon tumor cells. In vivo experiments were performed in mice to analyze the therapeutic effects of TRAIL. The results demonstrated that TRAIL significantly suppressed the growth of colorectal tumor cells in a dose-dependent manner (0.5–2.5 mg/ml) and also promoted colon tumor cell death. The migration and invasion of colon tumor cells were inhibited by the downregulation of fibronectin, Vimentin and E-cadherin. The apoptotic rate revealed that TRAIL (2.0 mg/ml) significantly promoted the apoptosis of colon tumor cells by regulating apoptosis-related gene expression. TRAIL administration promoted the apoptosis of colon tumor cells via the exogenous apoptosis signaling pathway due to the upregulation of caspase-3, caspase-8 and nuclear factor-κB protein expression. In vivo assays revealed that TRAIL administration significantly inhibited tumor growth and promoted apoptotic body and lymphocyte infiltration, which led to increased survival in tumor-bearing mice compared with the control group. Immunohistochemistry revealed that P53 and B-cell lymphoma-2 were downregulated in TRAIL-treated tumors. In conclusion, TRAIL treatment significantly inhibited the growth and aggressiveness of colon tumors by inducing apoptosis via the exogenous apoptosis pathway, which suggests that TRAIL may be a potential anticancer agent for colon carcinoma therapy.

Eudragit encapsulated cationic poly (lactic-co-glycolic acid) nanoparticles in targeted delivery of capecitabine for augmented colon carcinoma therapy

The present study was aimed at developing capecitabine loaded poly(lactic-co-glycolic acid) (PLGA Cap) based nanoparticles (NPs) formulation as a biodegradable polymeric drug carrier to treat colon cancer. NPs were prepared by the double emulsion solvent evaporation technique, then surface modified using chitosan and encapsulated in Eudragit® S100. Full factorial design (FFD) matrix was used to study the influence of three different independent variables on the responses particle size, Zeta potential, and encapsulation efficiency. For that, a three-level factorial design experiment was used for obtaining a mathematical model and prediction of the optimized formulation. The optimized PLGA Cap NPs were analyzed using DSC, AFM and Raman chemical spectral mapping. The particle size was found to be 386.13 ± 54.18 nm with polydispersity index 0.28 and Zeta potential of +12.3 ± 2.1 mV with an encapsulation efficiency of 63.81 ± 5.69%. In vitro drug release studies results showed a burst release followed by sustained release of Cap in the simulated colonic media with matrix-diffusion assisted drug release. The optimized NPs depicted higher in vitro mucoadhesion compared to the unmodified NPs. In vitro cell line studies showed that the PLGA Cap NPs exhibited significant anti-proliferative activity. The NPs were stable according to studies carried out following ICH Guidelines. All these outcomes signify the promising applicability of the formulated PLGA Cap NPs as a novel and targeted drug delivery system for colon carcinoma therapy with better patient compliance.

Tunicamycin inhibits colon carcinoma growth and aggressiveness via modulation of the ERK-JNK-mediated AKT/mTOR signaling pathway.

Epidemiology and evidence have demonstrated that colon carcinoma is one of the most common gastrointestinal tumors in the clinic. Reports have suggested that Tunicamycin significantly inhibits aggressiveness of colon carcinoma cells by promotion of apoptosis. In the present study, the inhibitory effect of tunicamycin on colon cancer cells and the potential underlying molecular mechanism was investigated. Western blotting, immunohistochemistry, apoptotic assays and immunofluorescence were used to analyze the therapeutic effects of tunicamycin on apoptosis, growth, aggressiveness and cell cycle of colon tumor cells, by downregulation of fibronectin, vimentin and E-cadherin expression levels. In vitro experiments demonstrated that tunicamycin significantly inhibited growth, migration and invasion of colon carcinoma cells. In addition, tunicamycin administration promoted apoptosis of colon carcinoma cells via upregulation of apoptotic protease activating factor 1 and cytochrome c expression levels, which are proteins that have a role in mitochondrial apoptosis signaling. Cell cycle assays revealed that tunicamycin suppressed proliferation and arrested S phase entry of colon carcinoma cells. Mechanistic analysis demonstrated that tunicamycin reduced expression and phosphorylation levels of extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and protein kinase B (AKT), and inhibited mammalian target of rapamycin (mTOR) expression levels in colon carcinoma cells. Endogenous overexpression of ERK inhibited tunicamycin-mediated downregulation of JNK, AKT and mTOR expression, which further blocked tunicamycin-mediated inhibition of growth and aggressiveness of colon carcinoma. In vivo assays revealed that tunicamycin treatment significantly inhibited tumor growth and promoted apoptosis, which led to long-term survival of tumor-bearing mice compared with the control group. In conclusion, these results suggested that tunicamycin may inhibit growth and aggressiveness of colon cancer via the ERK-JNK-mediated AKT/mTOR signaling pathway, and suggested that tunicamycin may be a potential anti-cancer agent for colon carcinoma therapy.

Functional Analysis of B7-H3 in Colonic Carcinoma Cells

B7-H3 is a newly discovered member of the B7/CD28 superfamily which functions as an important T-cell immune molecule. It has been reported recently that B7-H3 is highly expressed in many cancer cells, the data indicating that it may be a regulation factor contributing to tumor-resistance. In our study, we used bioinformatics to identify differentially expressed genes between colonic cancer cells and normal colonic cells, aiming to analyze mechanisms and identify sub-pathways closely related to progression, with the final aim of finding small molecule drugs which might interfere this progression. We found that ajmaline is one related factor which may enhance self-immunity in colon carcinoma therapy and B7-H3 plays important roles with regard to immunoreactions of colonic cancer cells. All the results indicate that H7-B3 is a favorable prognostic biomarker for colon carcinomas, providing novel information regarding likely targets for intervention.

A let-7/Fas double-negative feedback loop regulates human colon carcinoma cells sensitivity to Fas-related apoptosis

Interferon-γ (IFN-γ) is considered essential for the regulation of anti-tumor reactions as it sensitizes Fas-related apoptosis in HT29 cells, but the mechanism is unclear. In the current study, our data demonstrated that IFN-γ stimulation and Fas activation suppressed Dicer processing and let-7 microRNA biogenesis, while let-7 microRNA strongly inhibited Fas expression by directly targeting Fas mRNA. Accordingly, our results indicate that Fas and let-7 microRNAs form a double-negative feedback loop in IFN-γ and Fas induced apoptosis in colon carcinoma cell line HT29, which may be an important synergistic mechanism in anti-tumor immune response. We also found that a let-7 microRNA inhibitor increased Fas expression and sensitized cells to Fas-related apoptosis, which may have future implications in colon carcinoma therapy.

Gene therapy for C-26 colon cancer using heparin-polyethyleneimine nanoparticle-mediated survivin T34A.

BackgroundGene therapy provides a novel method for the prevention and treatment of cancer, but the clinical application of gene therapy is restricted, mainly because of the absence of an efficient and safe gene delivery system. Recently, we developed a novel nonviral gene carrier, ie, heparin-polyethyleneimine (HPEI) nanoparticles for this purpose.Methods and resultsHPEI nanoparticles were used to deliver plasmid-expressing mouse survivin-T34A (ms-T34A) to treat C-26 carcinoma in vitro and in vivo. According to the in vitro studies, HPEI nanoparticles could efficiently transfect the pGFP report gene into C-26 cells, with a transfection efficiency of 30.5% ± 2%. Moreover, HPEI nanoparticle-mediated ms-T34A could efficiently inhibit the proliferation of C-26 cells by induction of apoptosis in vitro. Based on the in vivo studies, HPEI nanoparticles could transfect the Lac-Z report gene into C-26 cells in vivo. Intratumoral injection of HPEI nanoparticle-mediated ms-T34A significantly inhibited growth of subcutaneous C-26 carcinoma in vivo by induction of apoptosis and inhibition of angiogenesis.ConclusionThis research suggests that HPEI nanoparticle-mediated ms-T34A may have a promising role in C-26 colon carcinoma therapy.

Antitumor activity of mutant bacterial cytosine deaminase gene for colon cancer

To evaluate bacterial cytosine deaminase (bCD) mutant D314A and 5-fluorocytosine (5-FC) for treatment of colon cancer in a mouse model. Recombinant lentivirus vectors that contained wild-type bCD gene (bCDwt), and bCD mutant D314A gene (bCD-D314A) with green fluorescence protein gene were constructed and used to infect human colon carcinoma LoVo cells, to generate stable transfected cells, LoVo/null, LoVo/bCDwt or LoVo/bCD-D314A. These were injected subcutaneously into Balb/c nude mice to establish xenograft models. Two weeks post-LoVo cell inoculation, PBS or 5-FC (500 mg/kg) was administered by intraperitoneal (i.p.) injection once daily for 14 d. On the day after LoVo cell injection, mice were monitored daily for tumor volume and survival. Sequence analyses confirmed the construction of recombinant lentiviral plasmids that contained bCDwt or bCD-D314A. The lentiviral vector had high efficacy for gene delivery, and RT-PCR showed that bCDwt or bCD-D314A gene was transferred to LoVo cells. Among these treatment groups, gene delivery or 5-FC administration alone had no effect on tumor growth. However, bCDwt/5-FC or bCD-D314A/5-FC treatment inhibited tumor growth and prolonged survival of mice significantly (P < 0.05). Importantly, the tumor volume in the bCD-D314A/5-FC-treated group was lower than that in the bCDwt/5-FC group (P < 0.05), and bCD-D314A plus 5-FC significantly prolonged survival of mice in comparison with bCDwt plus 5-FC (P < 0.05). The bCD mutant D314A enhanced significantly antitumor activity in human colon cancer xenograft models, which provides a promising approach for human colon carcinoma therapy.

The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma

Colon carcinoma is one of the common malignant tumors and has high morbidity and mortality in the world. Pigment epithelial-derived factor (PEDF) has been found to be the most potent natural inhibitor of angiogenesis and PEDF gene has been extensively used for the therapy of tumors, which suggests a potential approach to the therapy of colon carcinoma. However, the transfer of PEDF gene largely depends on the effective gene delivery systems. Poly (lactic-co-glycolic acid) nanoparticles (PLGANPs) have been extensively used for gene therapy due to its low-toxicity, biocompatibility and biodegradability, due to its potential to be an excellent carrier of the PEDF gene. We investigated the effect of PEDF gene loaded in PLGA nanoparticles (PEDF-PLGANPs) on the mouse colon carcinoma cells (CT26s) in vitro and in vivo. Blank PLGANPs (bPLGANPs) showed lower cytotoxicity than PEI to the CT26s. In vitro, PEDF-PLGANPs directly induced CT26 apoptosis and inhibit human umbilical vein endothelial cell (HUVEC) proliferation. In vivo, PEDF-PLGANPs inhibited CT26 tumors growth by inducing CT26 apoptosis, decreasing MVD and inhibiting angiogenesis. Our present study demonstrates the inhibitory effect of PEDF-PLGANPs on the growth of CT26s in vitro and in vivo for the first time. PLGANP-mediated PEDF gene could provide an innovative strategy for the therapy of colon carcinoma.

Heat-inducible in vivo gene therapy of colon carcinoma by human mdr1 promoter–regulated tumor necrosis factor-α expression

The promoter of the human multidrug resistance gene (mdr1) harbors defined heat-responsive elements, which could be exploited for construction of heat-inducible expression vectors. To analyze the hyperthermia inducibility of the mdr1 promoter in vitro and in vivo, we used the pcDNA3-mdrp-hTNF vector construct for heat-induced tumor necrosis factor alpha (TNF-alpha) expression in transfected HCT116 human colon carcinoma cells at mRNA level by quantitative real-time reverse transcription-PCR and at protein level by TNF-alpha ELISA. For the in vitro studies, the pcDNA3-mdrp-hTNF-transfected tumor cells were treated with hyperthermia at 43 degrees C for 2 h. In the animal studies, stably transfected or in vivo jet-injected tumor-bearing Ncr:nu/nu mice were treated for 60 min at 42 degrees C to induce TNF-alpha expression. Both the in vitro and in vivo experiments show that hyperthermia activates the mdr1 promoter in a temperature- and time-dependent manner, leading to an up to 4-fold increase in mdr1 promoter-driven TNF-alpha expression at mRNA and an up to 3-fold increase at protein level. The in vivo heat-induced TNF-alpha expression combined with Adriamycin (8 mg/kg) treatment leads to the inhibition of tumor growth in the animals. These experiments support the idea that heat-induced mdr1 promoter-driven expression of therapeutic genes is efficient and feasible for combined cancer gene therapy approaches.

Rapid in vitro chemosensitivity analysis of human colon tumor cell lines

In oncology, diagnostic assays have the potential to individualize treatment. Due to a large number of chemotherapeutic agents available, a chemosensitivity assay would be of great value for patients receiving chemotherapy. However, no broadly accepted test exists to date. Our work aims at establishing a chemosensitivity test using a microphysiometer, a device that measures the extracellular acidification rate of cells. The test assesses chemosensitivity by comparing the acidification rate of cells treated with cytostatic agents with that of nontreated cells. We performed experiments with two human colon tumor cell lines, HCT 116 and SW620, and cytostatic agents commonly used in therapy of colon carcinoma. We showed that a chemosensitivity test can be reproducibly performed using a microphysiometer. Suitable media and conditions for the assay were found. Test results were further correlated with the crystal violet assay and a tetrazolium salt assay. In comparison, cytostatic effects were rapidly and most clearly displayed with the microphysiometer. Our results suggest that microphysiometry is a promising platform for chemosensitivity testing.

Expression of proliferation-specific genes in the mucosa adjacent to colon carcinoma.

One of the main concerns in colon carcinoma therapy is local recurrence of the malignancy at the site of resection. Previous studies have shown that morphologically normal-appearing mucosa adjacent to colon carcinoma is different from mucosa distant from carcinoma. Mucosa adjacent to a carcinoma is characterized by crypt lengthening, cell hypertrophy, and change in production of mucopolysaccharides from sulfomucin in normal mucosa to sialomucins in carcinomas and adjacent mucosa. Recently there have been reports suggesting that there is an upward extension of the proliferative compartment in colonic crypts of this adjacent mucosa. Immunoblot analysis using antibodies to retinoblastoma, statin, c-Fos, c-Jun, and Cdc-2 proteins was used for our study on the expression of early cell cycle genes in carcinoma and its adjacent mucosa. In all, 15 tissue samples obtained from patients with colon carcinoma were analyzed. Tissue specimens were collected and immediately dissected as tumor, 0 to 1, 1 to 2, 2 to 3, 3 to 4, and 4 to 5 cm from the primary lesion. Dissected pieces were homogenized separately and subjected to immunoblot analysis. We found upregulation of c-Fos, c-Jun, and Cdc-2 expression in carcinoma and adjacent mucosa up to 4 cm from the edge of the carcinoma. The phosphorylated form of retinoblastoma is present in the carcinoma as well as in adjacent mucosa up to 4 cm from the margin of the carcinoma. Furthermore, we observed that the level of statin, a nonproliferation-specific nuclear protein, is very low in the primary lesion and in adjacent mucosa up to 3 cm. These results indicate that adjacent tissue up to 3 to 4 cm from the carcinoma has elevated levels of expression for cell cycle traverse-associated genes and down-regulation of nonproliferation-specific gene expressions such as statin. This imbalance indicates that within 3 to 4 cm from the edge of the carcinoma, colonic epithelial cells are already abnormal and may be in the hyperproliferative and preneoplastic state, susceptible to further steps leading to eventual malignant transformation.

Combination Therapy of Colon Carcinoma 26 in Mice with Recombinant Human Interleukin‐2 and Interferon‐α A/D: Occurrence of Large Granular Cells in the Tumor

The antitumor effects of recombinant human interleukin‐2 (rIL‐2), in combination with recombinant human interferon‐α A/D hybrid (rIFN‐α A/D) on colon carcinoma 26 (colon 26) in mice were examined histologically. Colon 26 was transplanted subcutaneously into female BALB/c mice on day 0. The mice bearing the tumor received intramuscular injection of rIL‐2, rIFN‐α A/D or the combination of rIL‐2 and rIFN‐α A/D for 2–10 consecutive days starting on day 7. Mice were killed on days 9, 13, 17 and 21. After day 13, growth of the tumor was significantly suppressed in the mice treated with rIL‐2 or rIFN‐α A/D alone and was stopped in the mice treated with rIL‐2 in combination with rIFN‐α A/D. Histologically, tumor necrosis developed in all treated groups, though the degree was the most severe in the group receiving combination treatment. Many large cells (about 15 30 μm in diameter) infiltrated into the tumor, and they had Thy‐1 surface antigen and many periodic acid‐Schiff‐positive round grannies in the cytoplasm. The incidence of these large granular cells was correlated well with the reduction in tumor weight. The ultrastructural features of the large granular cells were very similar to those of murine large granular lymphocyte‐like cells maintained in vitro in an IL‐2‐containing medium. The present large granular cells appear to be a kind of activated lymphoid cells.