Effects of N1, N13-diethylnorspermine (DENSPM) and X-radiation treatment on human colorectal tumor clones with varying X-radiation and drug responses

This study was designed to examine the effects of treatment with N1, N13-diethylnorspermine (DENSPM), a spermine analog, and X radiation on survival and on the polyamine and spermidine/spermine N1-acetyltransferase (SSAT) levels in closely related human colorectal tumor (HCT116) clones exhibiting a wide range of X-radiation and drug responses. After treatment with DENSPM and X radiation, clonogenic cell survival was measured. SSAT protein levels were measured by Western blot analysis and SSAT enzymatic activities by the conversion of [1-14C]acetyl-CoA into [1-14C]acetylspermidine. Polyamine [i.e. putrescine (PUT), spermine (SPM) and spermidine (SPD)] levels were measured with high-performance liquid chromatography. DENSPM enhanced the efficacy of radiation treatment in HCT116, HCT116-Clone2 (a radiation-resistant clone) and HCT116-Clone10 (a clone with similar X-radiation response as the parental HCT116 cells) but not in HCT116-CloneK (an X-radiation-sensitive but relatively drug-resistant clone). Treatment with DENSPM without X radiation caused the most significant increase in SSAT activity (approximately 22-fold) and an almost complete depletion of SPD levels in HCT116-CloneK. Our results suggest that (a) the lack of sensitization of X-radiation treatment by DENSPM in HCT116-CloneK was likely due to the prior depletion of SPD levels by DENSPM alone, (b) natural polyamine contents and/or inducibility of SSAT may be important factors influencing cellular response to combined X-radiation and DENSPM treatments, and (c) more importantly, there may be a potentially novel role for combining polyamine analogs such as DENSPM with X rays.     

Molecular cloning,genomic characterization and over-expression of a novel gene, <Emphasis Type="Italic">XRRA1</Emphasis>, identified from human colorectal cancer cell HCT116<Superscript>Clone2_XRR</Superscript> and macaque testis

Background  

As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116^Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene.     

Effects of inducible overexpression of DNp73alpha on cancer cell growth and response to treatment in vitro and in vivo

The p73 gene has a complex regulation, which leads to the expression of different isoforms, often with opposite biological effects. We have generated in the human colocarcinoma cell line HCT116, expressing a wild-type p53, an inducible DNp73alpha expressing system. Two clones (HCT116/DN3 and HCT116/DN14), upon doxycycline addition, show a strong expression of DNp73alpha. In vitro the two DNp73alpha overexpressing clones grow at similar rate of the control transfected clone (HCT116/8a) and similarly respond to DNA damage. When injected in mice, HCT116/DN3, HCT116/DN14, and HCT116/8a cells grew similarly in the absence or presence of tetracycline. In HCT116/DN3 and HCT116/DN14 tumors, tetracycline induced a strong expression of DNp73alpha both as mRNA and protein. These results indicate that in this system the overexpression of the DNp73alpha does not induce a more aggressive phenotype and does not seem to be associated with a reduced response of the cells to treatment with anticancer agents.     

Fractionated X-radiation treatment can elicit an inducible-like radioprotective response that is not dependent on the intrinsic cellular X-radiation resistance/sensitivity

Inducible responses are well documented to play a role in the radiation response of cells. However, it is not known whether clinically relevant fractionated X-radiation treatment could elicit an inducible-like radioprotective response and whether there is a direct correlation between the inducible radiation response phenomenon and the intrinsic radiation response of the cell. Therefore, the purpose of this study was to determine whether closely related human colorectal tumor (HCT116) clones treated with fractionated X rays could elicit an inducible-like radiation response to a subsequent acute (i.e. single) X-ray challenge, and whether the magnitude of the inducible-like response correlates with the intrinsic X-ray resistance of the responding clones. After fractionated X irradiation, only the radiosensitive clone showed enhanced clonogenic survival with a subsequent acute X-ray exposure. Cell cycle changes or the selection of subclones with increased intrinsic radiation resistance induced by the fractionated X rays were excluded as the basis of this enhanced tolerance, suggesting the presence of an inducible-like radioprotective response. Using the comet assay, we found similar amounts of intrinsic DNA damage among the clones after acute X irradiation. Our findings demonstrate that fractionated X-ray treatment can elicit an inducible-like radioprotective response and represent the first evidence that this response is independent of the intrinsic radiation resistance/sensitivity of the responding cells.     

Induction of accelerated senescence by gamma radiation in human solid tumor-derived cell lines expressing wild-type TP53

Recent studies have demonstrated that p21WAF1 (now known as CDKN1A)-dependent and -independent accelerated senescence responses are a major determinant of the sensitivity of cancer cells to chemotherapeutic agents. The objective of the present study was to determine whether human solid tumor-derived cell lines that express wild-type TP53 can exhibit levels of CDKN1A induction after exposure to ionizing radiation that are sufficient to activate the accelerated senescence program. Exposure to 60Co gamma radiation (< or =8 Gy) triggered accelerated senescence in all five TP53 wild-type tumor cell lines examined, albeit to differing degrees. Three of the TP53 wild-type tumor cell lines, HCT116, A172 and SKNSH, activated the TP53 signaling pathway similarly to normal human fibroblasts, as judged by the nuclear accumulation of TP53, magnitude and duration of induction of CDKN1A mRNA and CDKN1A protein, and propensity to undergo accelerated senescence after radiation exposure. In the clonogenic survival assay, the degree of radiosensitivity of these three tumor cell lines was also in the range displayed by normal human fibroblasts. On the other hand, two other TP53 wild-type tumor cell lines, A498 and A375, did not maintain high levels of CDKN1A mRNA and CDKN1A protein at late times postirradiation and exhibited only low levels of accelerated senescence after radiation exposure. Studies with a CDKN1A knockout cell line (HCT116CDKN1A-/-) confirmed that the radiation-triggered accelerated senescence is dependent on CDKN1A function. We conclude that (1) clinically achievable doses of ionizing radiation can trigger CDKN1A-dependent accelerated senescence in some human tumor cell lines that express wild-type TP53; and (2) as previously documented for normal human fibroblasts, some TP53 wild-type tumor cell lines (e.g. HCT116, A172 and SKNSH) may lose their clonogenic potential in response to radiation-inflicted injury primarily through undergoing accelerated senescence.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116R^FK866. In this study, we investigated gene mutations in parental HCT116 and HCT116R^FK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD⁺ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116R^FK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.     

Antiproliferative activity and induction of apoptosis in human colon cancer cells treated in vitro with constituents of a product derived from Pistacia lentiscus L. var. chia

In this report, we demonstrate that a 50% ethanol extract of the plant-derived product, Chios mastic gum (CMG), contains compounds which inhibit proliferation and induce death of HCT116 human colon cancer cells in vitro. CMG-treatment induces cell arrest at G(1), detachment of the cells from the substrate, activation of pro-caspases-8, -9 and -3, and causes several morphological changes typical of apoptosis in cell organelles. These events, furthermore, are time- and dose-dependent, but p53- and p21-independent. Apoptosis induction by CMG is not inhibited in HCT116 cell clones expressing high levels of the anti-apoptotic protein, Bcl-2, or dominant-negative FADD, thereby indicating that CMG induces cell death via a yet-to-be identified pathway, unrelated to the death receptor- and mitochondrion-dependent pathways. The findings presented here suggest that CMG (a) induces an anoikis form of cell death in HCT116 colon cancer cells that includes events associated with caspase-dependent pathways; and (b) might be developed into a chemotherapeutic agent for the treatment of human colon and other cancers.     

Radiation-Stimulated ERK1/2 and JNK1/2 Signaling can Promote Cell Cycle Progression in Human Colon Cancer Cells

The abilities of mutated active K-RAS and H-RAS proteins, in an isogenic human carcinoma cell system, to modulate the activity of signaling pathways and cell cycle progression following exposure to ionizing radiation is largely unknown. Loss of K-RAS D13 expression in parental HCT116 colorectal carcinoma cells blunted basal ERK1/2, AKT and JNK1/2 activity by ~70%. P38 activity was not detected. Deletion of the allele to express activated K-RAS nearly abolished radiation-induced activation of all signaling pathways. Expression of H-RAS V12 in HCT116 cells lacking an activated RAS molecule (H-RAS V12 cells) restored basal ERK1/2 and AKT activity to that observed in parental cells, but did not restore or alter basal JNK1/2 and p38 activity. In parental cells radiation (1 Gy) caused stronger ERK1/2 pathway activation compared to that of the PI3K/AKT pathway. In H-RAS V12 cells radiation caused stronger PI3K/AKT pathway activation compared to that of the ERK1/2 pathway. Radiation (1 Gy) promoted S phase entry in parental HCT116 cells within 24h, but not in either HCT116 cells lacking K-RAS D13 expression or in H-RAS V12 cells. In parental cells radiation-stimulated S phase entry correlated with ERK1/2-, JNK1/2- and PI3K-dependent increased expression of cyclin D1 and cyclin A, and to a lesser extent cyclin E, 6–24 h after exposure. Cyclin A and cyclin D1 expression were not increased by radiation in cells lacking K-RAS D13 expression or in H-RAS V12 cells. Radiation (1 Gy) modestly enhanced expression of p53, hMDM2 and p21 in parental cells 2-6h after exposure, which was abolished in cells lacking K-RAS D13 expression. Introduction of H-RAS V12 into cells lacking mutant active RAS partially restored radiation-induced expression of p21 and p53, and enhanced the induction of hMDM2 beyond that observed in parental cells. Collectively, our findings argue that the coordinated activation of multiple signaling pathways, in particular ERK1/2 and JNK1/2, by radiation is required to elevate the expression of G1 and S phase cyclin proteins and to promote S phase entry in human colon carcinoma cells expressing wild type p53. In HCT116 cells H-RAS V12 promotes hMDM2 expression after radiation exposure which correlates with reduced p53 expression and increased cell survival.     

Growth inhibition due to complementation of transforming growth factor-beta receptor type II-defect by human chromosome 3 transfer in human colorectal carcinoma cells

The transforming growth-beta receptor type II (TGF-beta RII) gene is one of the target genes of the DNA mismatch repair (MMR) defect. The human colorectal carcinoma cell line HCT116 has mutations in the hMLH1 gene and in the microsatellite region of the TGF-beta RII gene, both located on the short arm of chromosome 3. Introduction of the wild-type hMLH1 gene on transferred human chromosome 3 restores many characteristics of MMR-deficiency in HCT116. In this study, we determined whether transfer of chromosome 3 into HCT116 also complements the TGF-beta RII gene defect. We compared in vitro growth characteristics between HCT116 and HCT116 with a transferred chromosome 3 (HCT116 + ch3). The growth was suppressed in HCT116 + ch3 compared with parental HCT116. This suppression was abolished by frequent replacement with fresh medium, suggesting that the autocrine TGF-beta-TGF-beta RII system may be responsible for growth suppression. To explore this possibility, we determined several characteristics essential for the autocrine system. We found that HCT116 + ch3 expresses wild-type as well as mutated TGF-beta RII mRNA. In addition, phosphorylation of TGF-beta RI and growth inhibition were observed in HCT116 + ch3 but not in HCT116 by exposure to exogenous TGF-beta. The amount of TGF-beta1 in HCT116 + ch3 cultures was remarkably less than that in the HCT116, suggesting that TGF-beta produced by HCT116 + ch3 cells may be consumed by the cells. The conditioned medium from HCT116 cultures inhibits HCT116 + ch3 growth. This inhibition was neutralized by the anti-TGF-beta antibody. Taken together, these results strongly suggest that the TGF-beta RII gene defect in HCT116 is complemented by a wild-type gene on the transferred chromosome 3 and that HCT116 + ch3 gained the ability to respond to TGF-beta. Simultaneous complementation of defects of a responsible gene and a major target gene by the chromosome transfer is useful to prove the inactivated phenotypes acquired during colorectal tumorigenesis.     

Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.     

Difference in O6-methylguanine methyltransferase activity among transformed NIH3T3 cell clones

We examined the sensitivity to the lethal effects of methylating agents and the O6-methylguanine methyltransferase (MTR) activities of in vitro transformed NIH3T3 cell clones. The sensitivities to the lethal effects of MNNG were not different among all 49 transformed cell clones examined and do not correlate with the MTR activities. All 8 spontaneously transformed cell clones showed the same sensitivities to ACNU as the parental cell line. 2 of 20 transformants induced by UV or MNNG showed higher sensitivities to the ACNU although the MTR activity was normal. One cell clone transformed by UV was sensitive to ACNU and showed about half MTR activity. 5 of 19 cell clones transformed by oncogenes (Ha-ras or SV40 ori-) were sensitive to the lethal effects of ACNU and showed the low MTR activities, but were not as much sensitive as a Ha-MuSV transformed cell clone, Ha821.     

Expression of fragile X in human-mouse somatic cell hybrids

Fragile X expression was studied in human-mouse cell hybrids prepared from lymphocytes and fibroblasts obtained from a mentally retarded male. The patient showed a fragile X in 29-35.5% of his lymphocytes in medium 199 (M199) and in M199 plus fluorodeoxyuridine (FdU). One lymphocyte hybrid clone showed no expression in M199 and low expression in M199 + FdU. The other lymphocyte hybrid clone showed significantly increased expression in both media, comparable to levels in the parental cells. Fibroblast cultures from the patient showed no fragile X expression in M199 and 17% expression in M199 + FdU. Fragile X expression was also found in fibroblast hybrid clones in M199 and was significantly enhanced by the addition of FdU. Fragile X expression in one clone was consistently lower than in the other two clones and in the parental fibroblasts. Our results indicate that the level of fragile X expression varies in the hybrid clones, since frequencies similar to those of parental cells and suppressed frequencies were found. The presence or absence of a specific human chromosome did not correlate with the level of fragile X expression.     

Drug specific resistance to oxaliplatin is associated with apoptosis defect in a cellular model of colon carcinoma

To investigate acquired resistance to oxaliplatin, we selected two resistant clones from the HCT116 cell line. We found that the resistant phenotype was associated with resistance to oxaliplatin-induced apoptosis as demonstrated by FACS analysis and by Western blotting of caspase 3 activation. In addition, the resistant phenotype showed a concomitant resistance to lonidamine and arsenic trioxide which are inducers of mitochondrial apoptosis. Furthermore, a complete loss of Bax expression due to a frameshift mutation was observed in the most resistant clone. Taken together, these findings suggest that altered mitochondrial-mediated apoptosis could play a role in oxaliplatin resistance.     

Expression of Recombinant Carcinoembryonic-Antigen-Human-β-Defensin-2 Gene in Colon Cancer HCT116 Cells

The objective of this study was to construct a system driving high expression of human-β-denfensin-2 (HBD-2) system in eukaryotic cell. To construct recombinant retrovirus expression vector, CEA signal peptide-HBD-2 (mature peptide sequence) was cloned in the retrovirus expression vector PLNCX2. The retroviral vector was transfected into PT67 cells by DOTAP; after screening and amplifying single clone cell by G418, the virus particles were collected and infected to eukaryotic, colon cancer HCT116 cells. Furthermore, the HCT116 cells were also screened by using G418 and the resistance clones were obtained. Finally, the expression of HBD-2 was detected by Western blotting, which suggested a high level of expression of HBD-2 in HCT116 cells. In conclusion, the results indicate that high level of HBD-2 expression was obtained in HCT116 cells which will help in effective commercial production and purification of HBD-2 protein.     

Investigating the effect of radiosensitizer for Ursolic Acid and Kamolonol Acetate ‌ on HCT-116 cell line

PurposeThe aim of this study was evaluating the cytotoxic and radiosensitizing effects of Ursolic Acid (UA) and Kamolonol Acetate (KA) on HCT116 cell line and finally investigating the functional role of NF-κB and CCND1 genes in the radiosensitizing activity of UA and KA.Materials and methodThe cytotoxic effects of UA and KA by MTT assay was evaluated on HCT-116. Clonogenic assay was performed to investigate of radiosensitizing effects of UA and KA on HCT116. To assessment the expression levels of NF-κB and CCND1 genes, real-time PCR method was used.ResultsThe results of MTT assay revealed that UA and KA have cytotoxic effects on HCT116 cell line. According to clonogenic assay, survival fraction of treated cells with UA and KA has been decreased compared to the survival fraction of untreated cells. UA and KA lead to the decrease in the expression level of NF-κB. Synergistic effect of radiosensitizing agents with radiation was only approved for UA and 2 Gy of radiation.ConclusionBased on our study, UA and KA have cytotoxic effects on HCT116 cell line. Furthermore, UA may lead to radiosensitization of human colorectal tumor cells by NF-κB1 and CCND1signaling pathways.     

Three new compounds from Artemisia anomala and their antitumor activities

Three new compounds were isolated from Artemisia anomala, and their structures were determined using HR-ESI-MS, IR, UV, and NMR. The antitumor activities of the three compounds were evaluated in the human lung cancer cell line A549 and the human colorectal cancer cell line HCT116. The results showed that compound 2 significantly inhibited cell viability and proliferation and promoted apoptosis of HCT116 and A549 cells, suggesting that compound 2 may be used for colon and lung cancer treatments in clinical practice.