Mouse Colon Carcinoma Cell Line Research Articles

Cytotoxic activity of paclitaxel incorporated into polyelectrolyte nanocapsules

Nanoencapsulation is a promising solution for the delivery of chemotherapeutics to tumors. A method of preparation of drug-loaded nanocapsules based on the liquid core encapsulation by a sequential adsorption of a polyelectrolyte is described. An easily evaporative solvent, chloroform, was used as an oil phase. An interfacial complex formed with an oil-soluble, Food and Drug Administration-approved surfactant, and polycation poly-l-lysine (PLL) was used as a microemulsion stabilizer. A polyelectrolyte multilayer shell was constructed by a sequential adsorption of polyelectrolytes using biocompatible polyelectrolytes (PLL as a polycation and poly-l-glutamic acid as a polyanion). A hydrophobic anticancer agent, paclitaxel, was successfully encapsulated in the nanocarriers with the average size of 100 nm. In vitro analysis of the effects of nanoformulations was performed using a mouse colon carcinoma cell line CT26-CEA. Biocompatibility of the nanocapsules was evaluated using various biochemical assays. The results indicate that the cell viability was diminished by positively but not by negatively charged nanocarriers. Analysis of the cellular uptake of nanocapsules determined by flow cytometry and confocal microscopy confirmed their accumulation inside the cells. Encapsulated paclitaxel retains its cytotoxic/cytostatic activity; although its effects were weaker than those of the corresponding concentrations of the free drug. The generated nanocapsules seem to be a valuable vehicle for tumor drug delivery; although further work is needed to increase their overall activity.

Novel siRNA delivery system using a ternary polymer complex with strong silencing effect and no cytotoxicity.

We developed a novel small interfering RNA (siRNA) delivery system using a ternary complex with polyethyleneimine (PEI) and γ-polyglutamic acid (γ-PGA), which showed silencing effect and no cytotoxicity. The binary complexes of siRNA with PEI were approximately 73-102 nm in particle size and 45-52 mV in ζ-potential. The silencing effect of siRNA/PEI complexes increased with an increase of PEI, and siRNA/PEI complexes with a charge ratio greater than 16 showed significant luciferase knockdown in a mouse colon carcinoma cell line regularly expressing luciferase (Colon26/Luc cells). However, strong cytotoxicity and blood agglutination were observed in the siRNA/Lipofectamine complex and siRNA/PEI16 complex. Recharging cationic complexes with an anionic compound was reported to be a promising method for overcoming these toxicities. We therefore prepared ternary complexes of siRNA with PEI (charge ratio 16) by the addition of γ-PGA to reduce cytotoxicity and deliver siRNA. As expected, the cytotoxicity of the ternary complexes decreased with an increase of γ-PGA content, which decreased the ζ-potential of the complexes. A strong silencing effect comparable to siRNA/Lipofectamine complex was discovered in ternary complexes including γ-PGA with an anionic surface charge. The high incorporation of ternary complexes into Colon26/Luc cells was confirmed with fluorescence microcopy. Having achieved knockdown of an exogenously transfected gene, the ability of the complex to mediate knockdown of an endogenous housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was assessed in B16-F10 cells. The ternary complex (siRNA/PEI16/γ-PGA12 complex) exhibited a significant GAPDH knockdown effect. Thus, we developed a useful siRNA delivery system.

Augmenting Antitumor T-Cell Responses to Mimotope Vaccination by Boosting with Native Tumor Antigens

Vaccination with antigens expressed by tumors is one strategy for stimulating enhanced T-cell responses against tumors. However, these peptide vaccines rarely result in efficient expansion of tumor-specific T cells or responses that protect against tumor growth. Mimotopes, or peptide mimics of tumor antigens, elicit increased numbers of T cells that crossreact with the native tumor antigen, resulting in potent antitumor responses. Unfortunately, mimotopes may also elicit cells that do not crossreact or have low affinity for tumor antigen. We previously showed that one such mimotope of the dominant MHC class I tumor antigen of a mouse colon carcinoma cell line stimulates a tumor-specific T-cell clone and elicits antigen-specific cells in vivo, yet protects poorly against tumor growth. We hypothesized that boosting the mimotope vaccine with the native tumor antigen would focus the T-cell response elicited by the mimotope toward high affinity, tumor-specific T cells. We show that priming T cells with the mimotope, followed by a native tumor-antigen boost, improves tumor immunity compared with T cells elicited by the same prime with a mimotope boost. Our data suggest that the improved tumor immunity results from the expansion of mimotope-elicited tumor-specific T cells that have increased avidity for the tumor antigen. The enhanced T cells are phenotypically distinct and enriched for T-cell receptors previously correlated with improved antitumor immunity. These results suggest that incorporation of native antigen into clinical mimotope vaccine regimens may improve the efficacy of antitumor T-cell responses.

Safe and Effective Delivery of Small Interfering RNA with Polymer- and Liposomes-Based Complexes

We developed binary and ternary complexes based on polymers and liposomes for safe and effective delivery of small interfering RNA (siRNA). Anti-luciferase siRNA was used as a model of nucleic acid medicine. The binary complexes of siRNA were prepared with cationic polymers and cationic liposomes such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-arginine (PLA), trimethyl[2,3-(dioleoxy)-propyl]ammonium chloride (DOTMA), and cholesteryl 3β-N-(dimetylaminnoethyl)carbamate hydrochloride (DC-Chol). The ternary complexes were constructed by the addition of γ-polyglutamic acid (γ-PGA) to the binary complexes. The complexes were approximately 54-153 nm in particle size. The binary complexes showed a cationic surface charge although an anionic surface charge was observed in the ternary complexes. The polymer-based complexes did not show a silencing effect in the mouse colon carcinoma cell line expressing luciferase regularly (Colon26/Luc cells). The binary complexes based on liposomes and their ternary complexes coated by γ-PGA showed a significant silencing effect. The binary complexes showed significant cytotoxicity although the ternary complexes coated by γ-PGA did not show significant cytotoxicity. The ternary complexes coated by γ-PGA suppressed luciferase activity in the tumor after their direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, we have newly identified safe and efficient ternary complexes of siRNA for clinical use.

Immunotherapy using slow-cycling tumor cells prolonged overall survival of tumor-bearing mice

BackgroundDespite considerable progress in the development of anticancer therapies, there is still a high mortality rate caused by cancer relapse and metastasis. Dormant or slow-cycling residual tumor cells are thought to be a source of tumor relapse and metastasis, and are therefore an obstacle to therapy. In this study, we assessed the drug resistance of tumor cells in mice, and investigated whether vaccination could promote survival.MethodsThe mouse colon carcinoma cell line CT-26 was treated with 5-fluorouracil to assess its sensitivity to drug treatment. Mice with colon tumors were immunized with inactivated slow-cycling CT-26 cells to estimate the efficacy of this vaccine.ResultsWe identified a small population of slow-cycling tumor cells in the mouse colon carcinoma CT-26 cell line, which was resistant to conventional chemotherapy. To inhibit tumor recurrence and metastasis more effectively, treatments that selectively target the slow-cycling tumor cells should be developed to complement conventional therapies. We found that drug-treated, slow-cycling tumor cells induced a more intense immune response in vitro. Moreover, vaccination with inactivated slow-cycling tumor cells caused a reduction in tumor volume and prolonged the overall survival of tumor-bearing mice.ConclusionsThese findings suggest that targeting of slow-cycling tumor cells application using immunotherapy is a possible treatment to complement traditional antitumor therapy.

Effects of CXCR4 siRNA/dextran-spermine nanoparticles on CXCR4 expression and serum LDH levels in a mouse model of colorectal cancer metastasis to the liver

Effects of CXCR4 siRNA/dextran-spermine nanoparticles on CXCR4 expression and serum LDH levels in a mouse model of colorectal cancer metastasis to the liver Fatemeh Abedini1, Maznah Ismail1,4, Hossein Hosseinkhani2, Tengku Azmi Tengku Ibrahim1,3, Abdul Rahman Omar1,3, Pei Pei Chong4, Mohd Hair Bejo3, Abraham J Domb51Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 2Graduate Institute of Biomedical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan; 3Faculty of Veterinary Medicine, 4Faculty of Medicine and Health Science, Universiti Putra Malaysia, Selangor Darul Ehsan, Malaysia; 5Department of Medicinal Chemistry and Natural Products, School of Pharmacy, Hebrew University-Hadassah Medical School, Jerusalem, IsraelAbstract: Liver metastasis is the main cause of mortality related to colorectal cancer. CXCR4 is necessary for the outgrowth of colon cancer micrometastases. In oncology, it has been demonstrated that several human tumors release lactate dehydrogenase (LDH) into the circulation. CXCR4 gene expression and serum LDH levels are often increased in patients with colorectal cancer. Despite technological advances in cancer therapy, five-year overall survival is still around 50%. Therefore, better treatment needs to be developed. RNA interference (RNAi) is a modern and powerful tool for inhibition of gene expression. However, the rate-limiting step in this technology is effective delivery of RNAi agents. We have investigated a novel strategy of CXCR4 siRNA therapy and its effect on serum LDH levels in a BALB/C mouse model of colorectal cancer metastasis to the liver. Hepatic metastasis was established by injecting a CT26.WT mouse colon carcinoma cell line via the tail vein. Our results demonstrated that CXCR4 siRNA/dextran-spermine nanoparticles achieved high silencing efficiency with low toxicity. Favorable localization of the nanoparticles was confirmed with CXCR4 gene expression in the liver, that was correlated with serum LDH levels. More research will be needed to determine the effect of CXCR4 silencing on serum LDH levels, which may be a useful marker for predicting liver metastasis in colorectal cancer.Keywords: colorectal cancer, CXCR4 siRNA, serum LDH, liver, dextran-spermine

Effects of CXCR4 siRNA/dextran-spermine nanoparticles on CXCR4 expression and serum LDH levels in a mouse model of colorectal cancer metastasis to the liver

Liver metastasis is the main cause of mortality related to colorectal cancer. CXCR4 is necessary for the outgrowth of colon cancer micrometastases. In oncology, it has been demonstrated that several human tumors release lactate dehydrogenase (LDH) into the circulation. CXCR4 gene expression and serum LDH levels are often increased in patients with colorectal cancer. Despite technological advances in cancer therapy, five-year overall survival is still around 50%. Therefore, better treatment needs to be developed. RNA interference (RNAi) is a modern and powerful tool for inhibition of gene expression. However, the rate-limiting step in this technology is effective delivery of RNAi agents. We have investigated a novel strategy of CXCR4 siRNA therapy and its effect on serum LDH levels in a BALB/C mouse model of colorectal cancer metastasis to the liver. Hepatic metastasis was established by injecting a CT26.WT mouse colon carcinoma cell line via the tail vein. Our results demonstrated that CXCR4 siRNA/ dextran-spermine nanoparticles achieved high silencing efficiency with low toxicity. Favorable localization of the nanoparticles was confirmed with CXCR4 gene expression in the liver, that was correlated with serum LDH levels. More research will be needed to determine the effect of CXCR4 silencing on serum LDH levels, which may be a useful marker for predicting liver metastasis in colorectal cancer.

Hypoestoxide inhibits tumor growth in the mouse CT26 colon tumor model

To evaluate the effect of the natural diterpenoid, hypoestoxide (HE) on the growth of established colon cancer in mice. The CT26.WT mouse colon carcinoma cell line was grown and expanded in vitro. Following the expansion, BALB/c mice were inoculated s.c. with viable tumor cells. After the tumors had established and developed to about 80-90 mm(3), the mice were started on chemotherapy by oral administration of HE, 5-fluorouracil (5-FU) or combination. The antiangiogenic HE has previously been shown to inhibit the growth of melanoma in the B16F(1) tumor model in C57BL/6 mice. Our results demonstrate that mean volume of tumors in mice treated with oral HE as a single agent or in combination with 5-FU, were significantly smaller (> 60%) than those in vehicle control mice (471.2 mm(3) vs 1542.8 mm(3), P < 0.01). The significant reductions in tumor burden resulted in pronounced mean survival times (MST) and increased life spans (ILS) in the treated mice. These results indicate that HE is an effective chemotherapeutic agent for colorectal cancer in mice and that HE may be used alone or in combination with 5-FU.

Low Surface Expression of B7-1 (CD80) Is an Immunoescape Mechanism of Colon Carcinoma

Artificially enforced expression of CD80 (B7-1) and CD86 (B7-2) on tumor cells renders them more immunogenic by triggering the CD28 receptor on T cells. The enigma is that such B7s interact with much higher affinity with CTLA-4 (CD152), an inhibitory receptor expressed by activated T cells. We show that unmutated CD80 is spontaneously expressed at low levels by mouse colon carcinoma cell lines and other transplantable tumor cell lines of various tissue origins. Silencing of CD80 by interfering RNA led to loss of tumorigenicity of CT26 colon carcinoma in immunocompetent mice, but not in immunodeficient Rag-/- mice. CT26 tumor cells bind CTLA-4Ig, but much more faintly with a similar CD28Ig chimeric protein, thus providing an explanation for the dominant inhibitory effects on tumor immunity displayed by CD80 at that expression level. Interestingly, CD80-negative tumor cell lines such as MC38 colon carcinoma and B16 melanoma express CD80 at dim levels during in vivo growth in syngeneic mice. Therefore, low CD80 surface expression seems to give an advantage to cancer cells against the immune system. Our findings are similar with the inhibitory role described for the dim CD80 expression on immature dendritic cells, providing an explanation for the low levels of CD80 expression described in various human malignancies.

Sequence-Specific Suppression of mdr1a/1b Expression in Mice via RNA Interference

RNA interference (RNAi) is a powerful tool for silencing gene expression posttranscriptionally. The purpose of this study was to examine whether in vivo RNAi can be induced against endogenous mdr1a/1b in adult mice and to assess the feasibility of generating P-glycoprotein (P-gp) knockdown mice based on RNAi by a very simple intravenous injection of synthetic small interfering RNA (siRNAs) or siRNA-expressing plasmid DNAs. The targeted sequences for silencing mdr1a specifically or mdr1a/1b simultaneously were examined in an in vitro study using a mouse colon carcinoma cell line, colon26 cells, in culture. Mice were repeatedly treated with intravenous synthetic siRNAs or siRNA-expressing plasmid DNAs in naked form administered via a large-volume and high-speed injection, i.e., the hydrodynamics-based procedure. The amount of targeted mRNA and P-gp in the liver were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Among several targeted sequences, two and one optimized sequences were selected for mdr1a and mdr1a/1b, respectively, in the in vitro study. Following administration of synthetic siRNAs or siRNA-expressing plasmid DNAs directed against mdr1a, the mRNA level in the liver was significantly reduced to approximately 50-60% of that in control mice. Furthermore, a slight reduction was observed at the protein level. Similar results were obtained in the experiments using siRNA-expressing pDNA directed against mdr1a/1b. Our results demonstrate that sequence-specific suppression of mdr1 gene expression is possible at the mRNA level as well as the protein level in mice following intravenous delivery of siRNA effectors.

Fibroblast migratory factor derived from mouse colon carcinoma cells: Potential roles of fibronectin in tumor stroma formation

Mouse colon carcinoma cell line colon 38 was used to investigate migratory factor for fibroblasts because marked fibrotic tissue was associated with these cells growing at transplanted sites and liver metastases in vivo. Migration-inducing activity was detected in the serum-free culture supernatants of colon 38 cells, as shown by the Boyden chamber assays using NIH3T3 cells as target cells. The active substance was partially purified by a combination of anion-exchange, hydrophobic, and gel-permeation chromatography. An approximate relative molecular mass of the active substance was estimated to be between 100,000 and 400,000, judging from the eluting position in the gel-permeation chromatography. The migratory activity in the partially purified preparation was removed by incubation with beads coated with an anti-mouse fibronectin antibody. NIH3T3 cells incubated in the presence of culture supernatants of colon 38 cells exhibited higher growth rate, organized actin filaments, and increased chondroitin sulfate and hyaluronan. Fibronectin did not elicit such effects and partially purified migratory factor showed relatively low activity in these regards. Thus, colon 38 cells seem to secrete fibronectin and other soluble substances, which induce tumor stroma formation through migration and activation of host fibroblasts.

Highly metastatic variant of a mouse colon carcinoma cell line, LM17 and its response to GM-CSF gene therapy.

In order to establish a highly metastatic variant of a mouse colon carcinoma cell line (CT26), BALB/c mice were first subcutaneously injected with CT26 cells. Several weeks later, metastatic tumors in lungs were resected, mechanically dispersed into a single cell suspension and cultured in vitro until cells reached confluency. These tumor cells were then subcutaneously injected into new mice. After repeating this procedure five times, a highly lung metastatic cell line, denoted as LM17, has been established. The LM17 cells grow in vitro with or without serum, whereas parental CT26 cells require serum for their growth. The LM17 cells adhere to type I collagen or fibronectin stronger than CT26 cells do. The LM17 cells invade through Matrigel-coated basement membrane in greater number than CT26 cells. By gelatin zymography, LM17 cells showed higher proteinase activity than CT26. Furthermore, subcutaneous injection of irradiated LM17 cells infected with adenovirus harboring mouse GM-CSF gene prevents the growth and lung metastasis of pre-existing subcutaneous tumor. The injection of irradiated GM-CSF-producing LM17 cells after the surgical removal of pre-existing tumor also protected the occurrence of lung metastasis. These results suggest that this highly metastatic LM17 cell line could be useful for analysis of the lung metastatic mechanism and as the mouse GM-CSF gene therapy model.

Co-inoculation of human and murine carcinoma cells induces reciprocal suppression of metastasis by both cell lines.

The interactions of two cell lines having different metastatic properties, and the subsequent effects on dissemination were investigated using the chicken embryo metastasis assay. The highly aggressive human epidermoid cell line HEp-3 was tested alone or mixed with the mouse colon carcinoma cell line CL26 in this assay. When inoculated individually, each cell line forms experimental metastases in the chicken embryo, but only the HEp-3 cells give rise to spontaneous metastases. In embryos co-inoculated with both cell lines there was an overall reduction in metastatic burden in both the spontaneous and experimental metastasis assays. Furthers studies revealed that CL26 cells, when co-inoculated with HEp-3 cells did not acquire the ability to spontaneously metastasize. However, in the presence of CL26 cells, spontaneous HEp-3 metastasis was reduced. Intravenous co-inoculation of HEp-3 and CL26 cells also resulted in a reciprocal suppression of experimental metastasis by both cell lines. These studies demonstrate that the interactions of adjacent, phenotypically different tumor cells can have a suppressive effect on dissemination of one or both cell types.