Colorectal Cancer Cell Line LoVo Research Articles

Cisplatin-loaded gelatin-poly(acrylic acid) nanoparticles: Synthesis, antitumor efficiency in vivo and penetration in tumors

Cisplatin (CDDP)-loaded gelatin-poly(acrylic acid) (GEL-PAA) nanoparticles were successfully prepared by polymerizing acrylic acid in the presence of gelatin in aqueous solution followed by incorporating CDDP into the formed GEL-PAA nanoparticles through polymer–metal complex formation of CDDP with carboxylic groups in the nanoparticles. The obtained nanoparticles had a spherical shape, with a mean size of about 100 nm, and high drug payload as well as stability. It is found that CDDP can be released from the nanoparticles in a sustained manner with a small initial burst release. In vitro cytotoxicity revealed that CDDP-loaded nanoparticles had similar cytotoxicity to free CDDP after 48 h co-incubation with human colorectal cancer cell line LoVo. In vivo antitumor activity indicated that the nanoparticle formulation was superior in anticancer effect to free CDDP on murine hepatic H22 tumor-bearing mice model through intraperitoneal (i.p.) administration and displayed a dose-dependent antitumor efficacy. Further, the penetration examination of the nanoparticles through tumor tissue revealed that the CDDP-loaded GEL-PAA nanoparticles could only affect the cells near the tumor vasculature after they entered into the tumor tissue.

Effect of epigallocatechin-3-gallate on Notch1 and Notch2 expression in human colorectal cancer cell lines SW480 and LoVo

Effect of epigallocatechin-3-gallate on Notch1 and Notch2 expression in human colorectal cancer cell lines SW480 and LoVo

MicroRNA-195 promotes apoptosis and suppresses tumorigenicity of human colorectal cancer cells

Deregulated microRNAs and their roles in cancer development have attracted much attention. In the present study, we analyzed the roles of miR-195 in colorectal cancer pathogenesis, as its participation in some other types of cancer has been suggested by previous reports. By comparing miR-195 expression in 81 human colorectal cancer tissues and matched non-neoplastic mucosa tissues, we found that miR-195 was downregulated in cancer tissues. And restoration of miR-195 in colorectal cancer cell lines HT29 and LoVo could reduce cell viability, promote cell apoptosis and suppress tumorigenicity. Moreover, important antiapoptotic Bcl-2 was identified to be directly targeted by miR-195, and miR-195 was further suggested to exert its proapoptotic function mainly through targeting Bcl-2 expression. Taken together, our study provides important roles of miR-195 in colorectal cancer pathogenesis and implicates its potential application in cancer therapy.

Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo

To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad-p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was >or= 50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G(0)/G(1), while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G(0)/G(1). Therefore, p27mt can induce apoptosis in colorectal cells.

Inactivation of the UNC5C Netrin-1 Receptor Is Associated With Tumor Progression in Colorectal Malignancies

The UNC5H netrin-1 receptors (UNC5H1-3 also called UNC5A-C) belong to the functional dependence receptors family, which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer a tumor-suppressor activity. Indeed, cells harboring these receptors are thought to be dependent on ligand availability for their survival, thereby inhibiting uncontrolled tumor cell proliferation. We investigate here whether UNC5C acts as a tumor suppressor in colorectal malignancies. The level of UNC5C was analyzed in a panel of 86 primary sporadic colorectal carcinomas. Loss of heterozygosity in the UNC5C locus and epigenetic alterations in the UNC5C promoter were also analyzed. Intestinal tumor progression was monitored in mice bearing both UNC5C and APC1638N mutations, and apoptosis was measured in intestinal tumors developed in UNC5C/APC1638N mutant mice. We show here that UNC5C expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, in mice, inactivation of UNC5C is associated with increased intestinal tumor progression and a decrease in tumor cell apoptosis. The loss of UNC5C expression observed in human colorectal cancer is a selective advantage for tumor progression, in agreement with the dependence receptor hypothesis. Thus, the UNC5C dependence receptor is a tumor suppressor that regulates sporadic colorectal cancer.

β-Catenin Interacts With the FUS Proto-oncogene Product and Regulates Pre-mRNA Splicing

beta-Catenin is a downstream effector of the Wnt signaling pathway and is believed to exert its oncogenic function by activating T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcriptional factors. However, it is still uncertain whether the diverse effects of beta-catenin are caused solely by aberrant gene transactivation. In this study, we used a proteomics approach to obtain further insight into the functional properties of nuclear beta-catenin. The protein assembly of a native beta-catenin-containing complex in nuclear extracts from a colorectal cancer cell line, DLD-1, was identified using immunoprecipitation and mass spectrometry. beta-Catenin physically interacted with fusion (FUS)/translocated in liposarcoma (TLS) and various RNA-binding proteins. The expression of FUS/TLS was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. The transient transfection of FUS suppressed beta-catenin-evoked gene transactivation of TCF/LEF, and beta-catenin transfection affected the splicing pattern of the E1A minigene and induced a novel splicing variant of estrogen receptor (ER)-beta exerting a dominant-negative activity. Human cancer expresses a large variety of alternatively spliced messenger RNA (mRNA), but the precise molecular mechanisms responsible for cancer-related alternative splicing are largely unknown. In this study, we demonstrated the interaction of beta-catenin with FUS/TLS and other RNA-binding proteins involved in the regulation of pre-mRNA splicing. Certain mRNA splicing abbreviations seen in human cancers may be induced by the activation of the Wnt signaling pathway.

NF-κB and Hsp70 are involved in the phospholipase Cγ1 signaling pathway in colorectal cancer cells

The majority of deaths from colorectal cancer are due to tumor invasion and metastasis. Induced migration of tumor cell is generally considered to be one critical step in cancer progression to the invasive and metastatic stage. Phospholipase Cγ1 (PLCγ1) is a key molecular switch in the process. But, the mechanism and function of PLCγ1 in colorectal cancer motility are unclear. We showed first in this report that epidermal growth factor (EGF) stimulated the phosphorylation of PLCγ1 in human colorectal cancer cell line LoVo. Inhibition of PLCγ1 with the pharmacologic agent U73122 decreased the migration of LoVo cells in a dose-dependent manner while EGF treatment reversed it partially. PLCγ1 signaling pathway also upregulated the activity of NF-κB. Furthermore, expression of Hsp70 was increased by treatment with U73122 or pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor. These data indicated that PLCγ1 played a pivotal role in the migration of human colorectal cancer cell and first demonstrated that upregulation of NF-κB binding activity and downregulation of Hsp70 expression were PLCγ1-dependent in LoVo cells.

Lectin-mediated cytotoxicity and specificity of 5-fluorouracil conjugated with peanut agglutinin (5-Fu-PNA) in vitro

In order to take advantage of the biorecognition between lectin and carbohydrate for targeted drug delivery, the lectin of peanut (Arachis hypogaea) agglutinin (PNA) was coupled by fixing its amino groups to the carbodiimide-activated carboxylic groups of 5-fluorouracil (5-Fu) derivative (N1-substituted 5-Fu acetate) to form 5-Fu-PNA conjugate. When the coupling reaction was carried out in the presence of d-galactose (d-gal, specific sugar for PNA), the affinity of PNA was maintained after its coupling to N1-substituted 5-Fu acetate, which was confirmed by the result of the haemagglutination test. Otherwise, PNA would lose its affinity after the cross-linking reaction. The cytotoxicity, specificity and selectivity of 5-Fu-PNA were examined on the human colorectal cancer cell line LoVo and the human normal liver cell line Chang using MTT assay. Compared with free drug, the active conjugate, which maintained the affinity of lectin, had similar cytotoxic effect on LoVo cells with much lower cytotoxicity on Chang cells (p < 0.05). On the other hand, lower cytotoxic effects on LoVo cells were observed for the non-active conjugate even at higher drug concentrations. The cytotoxic effect of conjugate was specific because only the active conjugate could inhibit the growth of LoVo cells in a dose- and time-dependent manner as that of the free drug. The achieved results indicate the significance to maintain the affinity of lectin for lectin-mediated cytotoxicity. Still, the potential of 5-Fu-PNA conjugate as a targeting agent for colorectal cancer needs to be further investigated in vivo.

Cancer-specific killing by the CD suicide gene using the human telomerase reverse transcriptase promoter

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated in more than 90% of tumor cells. It may be used as a tool for driving a gene to kill tumors specifically. To test this idea, luciferase reporter gene was used and the results showed that hTERT promoter could restrict the gene expression in the telomerase-positive tumor cells. A tumor-specific expression plasmid phTERT-CD was constructed, in which the E. coli cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and a normal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug, 5-fluorocytosine (5FC), over 800-fold, while the sensitivity of WISH cells to 5FC was increased only 6-fold. Mixed cell experiments showed a strong "bystander effect" on CD-negative cells. Furthermore, a significant anti-tumor effect of the phTERT-CD/5FC system was observed in nude mice bearing mammalian carcinoma induced by s.c. inoculation of LoVo cells when the mice were given 250 mg/kg 5FC twice a day for 10 consecutive days. These results indicated that hTERT promoter could target the suicidal effect of CD gene to tumor cells, and therefore, may be a novel and promising targeting approach to the treatment of cancer.

Endothelin Receptor Expression in Colorectal Cancer

The distribution of endothelin-A- and B- (ET(A), ET(B)) receptor subtypes was compared in colorectal cancer to that in normal colon and their expression in the colorectal cancer cell lines LIM1215. HT29, SKCO1, SKCO17 and LoVo was determined, using gross and high resolution autoradiography and quantified by densitometry. ET(A) and ET(B) binding sites were expressed by all the cell lines. There was significantly (p = 0.008) higher expression of ET(A)-receptors by cancers (205.95 dpm x 1000/mm2) compared normal colon (129.19 dpm x 1000/mm2). However, for ET(B)-receptors, this was reversed, with significantly (p = 0.008) higher expression of ET(B) binding in normal colon (207.00 dpm x 1000/mm2) than in tumours (122.35 dpm x 1000/mm2).

INHIBITION OF PROLIFERATION, ACTIVATION AND FUNCTION OF HUMAN NATURAL-KILLER-CELLS IN-VITRO BY SECRETED PRODUCTS OF LOVO, A COLORECTAL-CANCER CELL-LINE

It has previously been suggested that some tumours may elaborate substances which can suppress the functional activity of lymphokine activated killer (LAK) cells. We have investigated the effects of secreted products from the colorectal cancer cell line LoVo on the pattern of expression of T lymphocyte subsets and natural killer cell phenotype and activation antigens during in vitro treatment of mixed populations of human peripheral blood mononuclear cells with recombinant human IL-2 (rhIL-2). Tumour cell line products inhibited the proliferation of cells expressing the NK cell antigen CD56 and of cells expressing the CD8 T cell subset antigen. Increased expression of the IL-2 receptor alpha chain component CD25, and the activation associated antigens CD71 and HLA-DR induced by rhIL-2 was inhibited by the presence of tumour cell products. Enhanced cytotoxic activity against standard target. cell lines Daudi and K562 induced by treatment with rhIL-2 was diminished by tumour cell supernatants. These mechanisms may contribute to the resistance of some tumours to immune response modulation treatment regimens which depend upon induction of lymphokine activation responses by rhIL-2.

Antigenic Heterogeneity of Human Colorectal Cancer Cell Lines Analyzed by a Panel of Monoclonal Antibodies. I. Heterogeneous Expression of la-Like and HLA-Like Antigenic Determinants&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

The pattern of reactivity of 10 monoclonal antibodies (MCA) developed against human lymphoid leukemia cells and tested with human T-cells, B-cells, as well T-lymphoma and B-lymphoma cell lines suggested that six of them detect la-like determinants, three detect HLA-like determinants, and the remaining one detects a non-Ia B-cell antigen. With the use of three binding assays, the six MCA that appeared to detect Ia-like determinants reacted strongly with the human colorectal cancer cell (CCC) line LoVo, and none of the three MCA that reacted with HLA-like determinants reacted with this cell line. Immunoprecipitation and polyacrylamide gel electrophoresis analysis confirmed the apparent specificities of the MCA for Ia-like and HLA molecules, demonstrated the presence of Ia-like molecules in LoVo, and failed to detect HLA in these cells. The cellular enzyme-linked immunosorbent assay was used for the testing of our anti-Ia and anti-HLA MCA with 15 other CCC lines. Marked heterogeneity was found in the expression of different Ia-like and HLA determinants defined by different or overlapping subsets of MCA, which suggested that these determinants might be present on different molecules or that different conformations of the same molecules exist in various CCC lines. Analysis of the surface phenotype of subclones of LoVo cells revealed the presence of stable variant cell subpopulations, which lost reactivity with four out of six of the anti-Ia-like MCA but retained at least one Ia-like molecule recognized by two of our MCA. All of the subclones maintained the HLA-negative phenotype. The possible immunologic and diagnostic consequences of the presence or absence of Ia-like or HLA markers on nonlymphoid tumor cells are discussed.