Nonlinear ultrasound parameter to monitor cell death in cancer cell samples.

A scaling subtraction method was proposed to analyze the radio frequency data from cancer cell samples exposed to an anti-cancer drug and to estimate a nonlinear parameter. The nonlinear parameter was found to be well correlated (R2 = 0.62) to the percentage of dead cells in apoptosis and necrosis. The origin of the nonlinearity may be related to a change in contacts between cells, since the nonlinear parameter was well correlated to the average total coordination number of binary packings (R2 ≥ 0.77). These results suggest that the scaling subtraction method may be used to early quantify chemotherapeutic treatment efficiency.

Effect of microformulation on the bioactivity of an anthocyanin-rich bilberry pomace extract ( Vaccinium myrtillus L.) in vitro.

In cell culture were compared the different release rates of anthocyanins from a bilberry pomace extract encapsulated either in food grade whey protein-based matrix capsules (WPC) or in pectin amid-based hollow spherical capsules (PHS). The impact of the formulations on typical anthocyanin-associated biological end points such as inhibition of the epidermal growth factor receptor (EGFR) and suppression of cell growth in HT29 colon carcinoma cells was assessed. The purpose was to find whether the release rates are sufficient to maintain biological activity and whether encapsulation affected EGFR inhibitory and growth suppressive properties of the extract. Even though anthocyanin release from extract-loaded capsules was proven under cell culture conditions, the inhibitory potential toward the EGFR was diminished. However, nonencapsulated extract as well as both extract-loaded encapsulation systems diminished the growth of HT29 cells to a comparable extent. The loss of EGFR inhibitory properties by encapsulation despite anthocyanin release indicates substantial contribution of other further constituents not monitored so far. Taken together, both applied encapsulation strategies allowed anthocyanin release and maintained biological activity with respect to growth inhibitory properties. However, the loss of EGFR inhibitory effects emphasizes the need for biological profiling to estimate process-induced changes of plant constituent's beneficial potencies.

Bifidobacterium breve - HT-29 cell line interaction: modulation of TNF-α induced gene expression.

To provide insight in the molecular basis for intestinal host-microbe interactions, we determined the genome-wide transcriptional response of human intestinal epithelial cells following exposure to cells of Bifidobacterium breve. To select an appropriate test system reflecting inflammatory conditions, the responsiveness to TNF-α was compared in T84, Caco-2 and HT-29 cells. The highest TNF-α response was observed in HT-29 cells and this cell line was selected for exposure to the B. breve strains M-16V, NR246 and UCC2003. After one hour of bacterial pre-incubation followed by two hours of additional TNF-α stimulation, B. breve M-16V (86%), but to a much lesser extent strains NR246 (50%) or UCC2003 (32%), showed a strain-specific reduction of the HT-29 transcriptional response to the inflammatory treatment. The most important functional groups of genes that were transcriptionally suppressed by the presence of B. breve M-16V, were found to be involved in immune regulation and apoptotic processes. About 54% of the TNF-α induced genes were solely suppressed by the presence of B. breve M-16V. These included apoptosis-related cysteine protease caspase 7 (CASP7), interferon regulatory factor 3 (IRF3), amyloid beta (A4) precursor proteinbinding family A member 1 (APBA1), NADPH oxidase (NOX5), and leukemia inhibitory factor receptor (LIFR). The extracellular IL-8 concentration was determined by an immunological assay but did not change significantly, indicating that B. breve M-16V only partially modulates the TNF-α pathway. In conclusion, this study shows that B. breve strains modulate gene expression in HT-29 cells under inflammatory conditions in a strain-specific way.

A highly selective fluorescent chemosensor for zinc ion and imaging application in living cells.

A new 2,6-bis(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol (1) serves as a highly selective and sensitive fluorescent probe for Zn(2+) in a HEPES buffer (50 mM, DMSO:water = 1:9 (v/v), pH = 7.2) at 25 °C. The increase in fluorescence in the presence of Zn(2+) is accounted for by the formation of dinuclear Zn(2+) complex [Zn(2)(C(35)H(25)N(6)O)(OH)(NO(3))(2)(H(2)O)] (2), characterized by X-ray crystallography. The fluorescence quantum yield of the chemosensor 1 is only 0.019, and it increases more than 12-fold (0.237) in the presence of 2 equiv of the zinc ion. Interestingly, the introduction of other metal ions causes the fluorescence intensity to be either unchanged or weakened. By incubation of cultured living cells (A375 and HT-29) with the chemosensor 1, intracellular Zn(2+) concentrations could be monitored through selective fluorescence chemosensing.

Deficiency in the 15 kDa selenoprotein inhibits human colon cancer cell growth.

Selenium is an essential micronutrient for humans and animals, and is thought to provide protection against some forms of cancer. These protective effects appear to be mediated, at least in part, through selenium-containing proteins (selenoproteins). Recent studies in a mouse colon cancer cell line have shown that the 15 kDa selenoprotein (Sep15) may also play a role in promoting colon cancer. The current study investigated whether the effects of reversing the cancer phenotype observed when Sep15 was removed in mouse colon cancer cells, were recapitulated in HCT116 and HT29 human colorectal carcinoma cells. Targeted down-regulation of Sep15 using RNAi technology in these human colon cancer cell lines resulted in similarly decreased growth under anchorage-dependent and anchorage-independent conditions. However, the magnitude of reduction in cell growth was much less than in the mouse colon cancer cell line investigated previously. Furthermore, changes in cell cycle distribution were observed, indicating a delayed release of Sep15 deficient cells from the G(0)/G(1) phase after synchronization. The potential mechanism by which human colon cancer cells lacking Sep15 revert their cancer phenotype will need to be explored further.

Colon cancer chemopreventive activities of pomegranate ellagitannins and urolithins.

Pomegranate juice derived ellagitannins and their intestinal bacterial metabolites, urolithins, inhibited TCDD-induced CYP1-mediated EROD activity in vitro with IC(50) values ranging from 56.7 microM for urolithin A to 74.8 microM for urolithin C. These compounds exhibited dose- and time-dependent decreases in cell proliferation and clonogenic efficiency of HT-29 cells. Inhibition of cell proliferation was mediated through cell cycle arrest in the G(0)/G(1) and G(2)/M stages of the cell cycle followed by induction of apoptosis. These results indicate that the ellagitannins and urolithins released in the colon upon consumption of pomegranate juice in considerable amounts could potentially curtail the risk of colon cancer development, by inhibiting cell proliferation and inducing apoptosis.

[Effect of 5-Aza-CdR on expression and methylation of E-cadherin gene in human colon carcinoma cells].

BACKGROUND AND OBJECTIVE: Colon cancer is one of the most common malignant tumors, and its pathogenesis is not fully understood. Transcriptional silencing by DNA methylation is believed to be an important mechanism of carcinogenesis. E-cadherin can suppress tumor cell invasion and metastasis, and is considered as an invasion/metastasis suppressor gene. Inactivation of E-cadherin gene often occurs in colon carcinoma. This study was to investigate the correlation between E-cadherin gene expression and the methylation status of E-cadherin 5' CpG islands in human colon carcinoma cell line HT-29, and to explore the mechanism of carcinogenesis of colon cancer. METHODS: Immunocytochemical dicho-step method and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect the expression of E-cadherin protein and mRNA in HT-29 cells after 5-Aza-CdR treatment; methylation specific PCR was used to analyze the methylation status at promoter of E-cadherin gene. RESULTS: The expression of E-cadherin gene could be restored by 5-Aza-CdR treatment, immunocytochemical staining showed the positive expression ratio of E-cadherin increased from (21+/-7)% (1 micromol/L) to (39+/-13)% (5 micromol/L); E-cadherin genes were methylated and not expressed in HT-29 cells in the colon carcinoma. CONCLUSIONS: E-cadherin methylation plays an important role in the carcinogenesis of colon carcinoma cells and can re-express after the treatment with 5-Aza-CdR.

Oncogenic Ras, but not (V600E)B-RAF, protects from cholesterol depletion-induced apoptosis through the PI3K/AKT pathway in colorectal cancer cells.

Cholesterol is necessary for proliferation and survival of transformed cells. Here we analyse the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in colorectal cancer cells carrying oncogenic Ras or (V600E)B-RAF mutations. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment results in a significant increase in apoptosis in HT-29 and Colo-205 cells containing the (V600E)B-RAF mutation, but not in HCT-116 and LoVo cells harbouring the (G13D)Ras mutation, or BE cells, which possess two mutations, (G13D)Ras and (G463V)B-RAF. We also demonstrate that oncogenic Ras protects from apoptosis induced by cholesterol depletion through constitutive activation of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. The specific activation of the PI3K/AKT pathway by overexpression of the (V12)RasC40 mutant or a constitutively active AKT decreases the LPDS plus 25-HC-induced apoptosis in HT-29 cells, whereas PI3K inhibition or abrogation of AKT expression renders HCT-116 sensitive to cholesterol depletion-induced apoptosis. Moreover, our data show that LPDS plus 25-HC increases the activity of c-Jun N-terminal kinase proteins only in HT-29 cells and that the inhibition of this kinase blocks the apoptosis induced by LPDS plus 25-HC. Finally, we demonstrate that AKT hyperactivation by oncogenic Ras protects from apoptosis, preventing the activation of c-Jun N-terminal kinase by cholesterol depletion. Thus, our data demonstrate that low levels of cholesterol induce apoptosis in colorectal cancer cells without oncogenic Ras mutations. These results reveal a novel molecular characteristic of colon tumours containing Ras or B-RAF mutations and should help in defining new targets for cancer therapy.

Tumor suppressor FOXO3 participates in the regulation of intestinal inflammation.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by chronic mucosal injury and the infiltration of inflammatory cells. Tumor suppressor FOXO3 regulates gene expression and its translocation to the cytosol leads to the abrogation of its transcriptional function. We have previously shown that bacterial infection regulates FOXO3 in intestinal epithelial cells and increases cytokine levels. As TNFalpha is a major contributor in intestinal inflammation, the aim of this study was to assess its effect on FOXO3 and FOXO3's contribution to intestinal inflammation in vitro and in vivo. TNFalpha induces the translocation of nuclear FOXO3 into the cytosol where it undergoes proteasomal degradation in human intestinal HT-29 cells. Proximally, the PI3K and IKK pathways mediate TNFalpha-induced FOXO3 phosphorylation. In FOXO3-silenced HT-29 cells, TNFalpha-induced IL-8 expression is increased approximately 83%. In vivo, Foxo3 is present in the nuclei and cytosol of colonic crypt epithelia. In DSS-induced colonic inflammation, Foxo3's nuclear localization is lost and it is only found in the cytosol. Consistent with a role for Foxo3 in colitis, Foxo3-deficient mice treated with DSS developed more severe colonic inflammation with an increased number of intraepithelial lymphocytes and PMNs infiltrated in the epithelia, than wild-type mice. In summary, TNFalpha inactivates FOXO3 in intestinal epithelia through the PI3K and IKK pathways and FOXO3 inactivation leads to the upregulation of IL-8 in vitro; in vivo Foxo3 is in the cytosol of inflamed colonic epithelia and Foxo3 deficiency leads to severe intestinal inflammation.

Coagulation factor Xa inhibits cancer cell migration via protease-activated receptor-1 activation.

Cell migration is critically important in (patho)physiological processes. The metastatic potential of cancer cells partly depends on activation of the coagulation cascade. The aim of the present study was to determine whether coagulation factor X (FXa) can regulate the migration and invasion of cancer cells. Quite unexpectedly, we found that FXa markedly diminished the migration of different cancer cell lines of various origins (breast, lung and colon cancer cells). We showed that FXa mediated inhibition of cancer cell migration was specific, as it was inhibited by TAP (a specific FXa inhibitor) but not by Hirudin (a specific thrombin inhibitor). Moreover, the FXa effect was dose dependent, with a maximal inhibitory effect reached at 0.75 U/ml FXa (corresponding to 130.5 nM). Next, we determined that FXa acted via protease-activated receptor (PAR)-1-dependent signaling, and PAR-1 desensitization, as well as knocking-down PAR-1 expression, abolished the FXa effects. Finally, we showed that Gialpha was not involved in FXa mediated inhibition of cell migration as its effects were not reverted by pertussis toxin. These results suggest that, beyond its role in blood coagulation, FXa plays a key role in cancer cell migration. They also shed light on an unexpected role of PAR-1, which appears to be a Janus-like receptor in cancer cell biology.

The flavonoid luteolin worsens chemical-induced colitis in NF-kappaB(EGFP) transgenic mice through blockade of NF-kappaB-dependent protective molecules.

BACKGROUND: The flavonoid luteolin has anti-inflammatory properties both in vivo and in vitro. However, the impact of luteolin on experimental models of colitis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: To address the therapeutic impact of luteolin, NF-kappaB(EGFP) transgenic mice were fed a chow diet containing 2% luteolin- or isoflavone-free control chow (AIN-76), and acute colitis was induced using 3% dextran sodium sulfate (DSS). Additionally, development of spontaneous colitis was evaluated in IL-10(-/-);NF-kappaB(EGFP) transgenic mice fed 2% luteolin chow diet or control chow diet. Interestingly, NF-kappaB(EGFP) transgenic mice exposed to luteolin showed worse DSS-induced colitis (weight loss, histological scores) compared to control-fed mice, whereas spontaneous colitis in IL-10(-/-);NF-kappaB(EGFP) mice was significantly attenuated. Macroscopic imaging of live resected colon showed enhanced EGFP expression (NF-kappaB activity) in luteolin-fed mice as compared to control-fed animals after DSS exposure, while cecal EGFP expression was attenuated in luteolin-fed IL-10(-/-) mice. Interestingly, confocal microscopy showed that EGFP positive cells were mostly located in the lamina propria and not in the epithelium. Caspase 3 activation was significantly enhanced whereas COX-2 gene expression was reduced in luteolin-fed, DSS-exposed NF-kappaB(EGFP) transgenic mice as assessed by Western blot and immunohistochemical analysis. In vitro, luteolin sensitized colonic epithelial HT29 cells to TNFalpha-induced apoptosis, caspase 3 activation, DNA fragmentation and reduced TNFalpha-induced C-IAP1, C-IAP2 and COX-2 gene expression. CONCLUSIONS/SIGNIFICANCE: We conclude that while luteolin shows beneficial effects on spontaneous colitis, it aggravates DSS-induced experimental colitis by blocking NF-kappaB-dependent protective molecules in enterocytes.

Targeting of insulin-like growth factor-I receptor with a monoclonal antibody inhibits growth of hepatic metastases from human colon carcinoma in mice.

BACKGROUND: Colorectal carcinomas (CRC) express high levels of insulin-like growth factor-I/II (IGF-I/II) and the receptor (IGF-IR). We hypothesized that selective inhibition of IGF-IR would inhibit hepatic growth of human CRC in mice. METHODS: Human CRC cells were treated in vitro with anti-IGF-IR monoclonal antibody (MoAB) with and without oxaliplatin to assess cytotoxicity. The effect of anti-IGF-IR MoAB on IGF-I-induced vascular endothelial growth factor (VEGF) production in human CRC cells was assessed by Northern blot and ELISA. We injected human CRC cells intrahepatically in nude mice, and then administered anti-IGF-IR MoAB with and without oxaliplatin. We delayed treatment in one group until large hepatic tumors were present. We assessed tumors for apoptosis, proliferation, and angiogenesis. RESULTS: Anti-IGF-IR MoAB and oxaliplatin inhibited CRC cell growth in vitro and combination treatment was even more effective. IGF-I stimulation of CRC cells resulted in significant upregulation of VEGF and this was completely inhibited by pretreatment with anti-IGF-IR MoAB. Anti-IGF-IR MoAB significantly inhibited hepatic growth of tumors in mice. Anti-IGF-IR MoAB plus oxaliplatin led to a significantly greater inhibition of tumor growth. Anti-IGF-IR MoAB plus oxaliplatin was just as effective at inhibiting growth of larger, more advanced liver tumors. Anti-IGF-IR MoAB, alone and in combination with oxaliplatin, led to a significant increase in tumor cell apoptosis, and a significant inhibition of tumor cell proliferation and angiogenesis. CONCLUSIONS: These findings suggest that IGF-IR is a potential target for therapy in patients with advanced CRC.

Polymeric formula has direct anti-inflammatory effects on enterocytes in an in vitro model of intestinal inflammation.

Exclusive enteral nutrition using polymeric formula (PF) is a well-established therapeutic option for active Crohn's disease; however, its mechanisms of action are unknown. We investigated the anti-inflammatory effects of PF in an in vitro model of epithelial cell inflammation. PF did not affect cell viability over a range of dilutions, but when PF was added to the culture medium the interleukin (IL)-8 response to proinflammatory stimuli was significantly reduced. This effect was due to PF acting directly on the cells as the IL-8 response was still reduced when PF was separated from the proinflammatory stimuli in a 2-compartment system. In the presence of PF, nuclear factor (NF)-kappaB nuclear migration was not inhibited; however, IkappaBalpha degradation was delayed. PF has direct anti-inflammatory effects upon immortalized colonic enterocytes. Therefore PF may, in part, modulate gut inflammation by directly reducing the inflammatory response of the intestinal epithelium.

Anti-cancer properties of phenolics from apple waste on colon carcinogenesis in vitro.

Colorectal cancer is one of the most common cancers in Western countries. The World Health Organisation identifies diet as a critical risk factor in the development and progression of this disease and the protective role of high levels of fruit and vegetable consumption. Several studies have shown that apples contain several phenolic compounds that are potent anti-oxidants in humans. However, little is known about other beneficial properties of apple phenolics in cancer. We have used the HT29, HT115 and CaCo-2 cell lines as in vitro models to examine the effect of apple phenolics (0.01-0.1% apple extract) on key stages of colorectal carcinogenesis, namely; DNA damage (Comet assay), colonic barrier function (TER assay), cell cycle progression (DNA content assay) and invasion (Matrigel assay). Our results indicate that a crude extract of apple phenolics can protect against DNA damage, improve barrier function and inhibit invasion (p<0.05). The anti-invasive effects of the extract were enhanced with twenty-four hour pretreatment of cells (p<0.05). We have shown that a crude apple extract from waste, rich in phenolic compounds, beneficially influences key stages of carcinogenesis in colon cells in vitro.

A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells.

Cancer develops when molecular pathways that control the fine balance between proliferation, differentiation, autophagy and cell death undergo genetic deregulation. The prospects for further substantial advances in the management of colorectal cancer reside in a systematic genetic and functional dissection of these pathways in tumor cells. In an effort to evaluate the impact of p38 signaling on colorectal cancer cell fate, we treated HT29, Caco2, Hct116, LS174T and SW480 cell lines with the inhibitor SB202190 specific for p38alpha/beta kinases. We report that p38alpha is required for colorectal cancer cell homeostasis as the inhibition of its kinase function by pharmacological blockade or genetic inactivation causes cell cycle arrest, autophagy and cell death in a cell type-specific manner. Deficiency of p38alpha activity induces a tissue-restricted upregulation of the GABARAP gene, an essential component of autophagic vacuoles and autophagosomes, whereas simultaneous inhibition of autophagy significantly increases cell death by triggering apoptosis. These data identify p38alpha as a central mediator of colorectal cancer cell homeostasis and establish a rationale for the evaluation of the pharmacological manipulation of the p38alpha pathway in the treatment of colorectal cancer.

Down-regulation of prostaglandin E2 by curcumin is correlated with inhibition of cell growth and induction of apoptosis in human colon carcinoma cell lines.

Several in vitro and in vivo studies have demonstrated an association between curcumin, a diferuloylmethane derived from the plant Curcuma longa, and colorectal cancer (CRC) prevention. Nevertheless, the molecular mechanism responsible for the chemopreventive effect of curcumin is not well understood and most probably involves several pathways. Several studies indicate that curcumin may exert its effect by specifically inhibiting the cyclooxygenase-2 (COX-2) isoenzyme, which is up-regulated in 40 to 50% of colorectal polyps and in up to 85% of CRCs. However, other studies have suggested that curcumin may also inhibit polyps formation by COX-2 independent mechanisms (eg, inhibition of ErbB-1, AkT). The aim of this study was to evaluate whether curcumin's effect on the inhibition of cell growth and induction of apoptosis in human colon carcinoma cell lines is correlated with inhibition of PGE2 synthesis and down-regulation of COX-2. HT29 cells (expressing COX-2) and SW480 (deficient of COX-2) were exposed to different concentrations (0-50 microM) of curcumin for 72 hours. Growth inhibition was assessed by Coulter counter. Cell viability was assessed by the ability of metabolically active cells to reduce tetrazolium salt to colored formazan compounds (tetrazolium salt assay). Apoptosis was measured by two independent methods: flow cyto-metric analysis and 4'-6-Diamidino-2-phenylindole (DAPI) staining. Activity of COX-2 was evaluated by measuring prostaglandin E2 (PGE2) concentration using a specific enzyme-linked immunoassay. COX-1 and COX-2 expressions were measured by Western blot analysis. There was a significant difference between curcumin effect on COX-2-expressing (HT29: inhibitory concentration 50% [IC50] = 15 microM) and COX-2-deficient (SW480: IC50 = 40 microM) cells. Similarly, induction of apoptosis was higher in cells expressing COX-2. Western blot analysis and PGE2 immunoassay showed that curcumin inhibited COX-2 protein activity and expression in a dose-dependent manner. In conclusion, inhibition of cell survival and induction of apoptosis by curcumin in colorectal adenocarcinoma cell lines is associated with the inhibition of PGE2 synthesis and down-regulation of COX-2.

Adenovirus-mediated expression of both antisense ODC and AdoMetDC inhibited colorectal cancer cell growth in vitro.

AIM: To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase ( AdoMetDC ) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth. METHODS: A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by PacI. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine. RESULTS: Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine. CONCLUSION: The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.

Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate.

The differentiation of colorectal cancer cells is associated with the arrest of tumor growth and tumor regression. However, the mechanism of such tumor cell differentiation has not yet been elucidated. Several adenocarcinoma cell lines, including HT29 which differentiates only upon stimulation with a differentiation agent, have been used for the study of colorectal cells. Since we had previously obtained variable results during analyses of these cells, we selected several clones of this cell line. In this study, four clones of the parental HT29 cells, H8, G9, G10 and A3, were characterized. All of them differentiated upon treatment with sodium butyrate as the differentiation agent but they appeared different in their response regarding some of the markers of differentiation. As revealed by ultrastructural analysis, H8 and G10 clones formed numerous intercellular cysts with microvilli whereas these structures were found only occasionally in G9 and A3 clones. An elevated level of the indicator of cell differentiation, CEACAM 1, was found in H8 and G10 clones and the activity of alkaline phosphatase, another important marker of colorectal cell differentiation, was up-regulated and highly increased upon butyrate treatment in the H8 clone. Phosphorylation of p38 MAPK was increased in H8 and A3 butyrate-treated clones. According to the levels of cleaved PARP and activated caspase-3, the apoptotic response to butyrate appeared similar in all four clones, while electronoptic analysis revealed that clones G9 and A3 were more perceptive to butyrate-induced apoptosis. In conclusion, our data showed considerable heterogeneity in morphology and some enzymatic activity of the cloned cells. This fact may contribute to the evidence that many HT29 cells possess multipotent information similar to that of stem cells of the normal intestinal crypt.

Wild-type p53 gene transfer into mutated p53 HT29 cells improves sensitivity to photodynamic therapy via induction of apoptosis.

Photodynamic therapy (PDT) is an effective local cancer treatment that induces cytotoxicity through the intracellular generation of reactive oxygen species. It is generally thought that p53 regulates chemotherapy and radiation therapy responsiveness via apoptosis induction control. The current study investigated whether cellular sensitivity to PDT is increased when a wild-type (wt) p53 status is restored by gene transfer in the established HT9blk Ala273-mutant p53 human colon cancer cell line. The photosensitizer accumulation was similar in both cell lines, and survival measurements using MTT test and clonogenic assays demonstrated that wt p53 transfected cells (HT29A4) were significantly more sensitive to chlorin e6-mediated PDT. P53 protein expression and its functionality as a transcription factor demonstrated through the induction of mdm2 transactivation, were not found to be directly involved in this differential photosensitivity. However, induction of caspase 3 activation (2.6-fold), leading to significant apoptosis induction 24-h after PDT was observed in HT29A4 cells. These results suggest that the introduction of wt p53 in HT29A4 potentiates the cell sensitivity to PDT through the induction of apoptosis in relation to p53 mutational status, but independently of p53 expression level and transcriptional activity.