Dano fotoinduzido no sistema endolisossomal: modulação da autofagia e metabolismo de lipídios em células de câncer colorretal / Endo-lysosomal system photodamage: modulation of autophagy and lipid metabolism in colorectal cancer cells

O câncer colorretal (CCR) é o terceiro câncer mais diagnosticado em humanos. O CCR causou mais de 900.000 mortes em 2020 e foi estimado, para o período entre 2020 - 2025, um incremento de 13.5 % no número de casos novos de acordo com a plataforma Web Global Cancer Observatory. A Terapia Fotodinâmica (PDT) é uma alternativa terapêutica promissora. Conhecer as vias de sinalização de morte celular, assim como, as respostas associadas com a resistência ao dano foto-oxidativo, são relevantes para incrementar a eficiência da PDT. Neste trabalho, investigamos como as células de adenocarcinoma colorretal HT 29 respondem ao dano fotoinduzido gerado pelo fotossensibilizador (FS) meso-tetrafenilporfirina dissulfônado (TPPS2a), uma molécula que é ativada pela irradiação com luz em 522 nm. Como esperado, após irradiação (2.1 J cm-2) foi verificado que com o incremento do TPPS2a houve diminuição da viabilidade celular. A concentração do FS escolhida para darmos seguimento ao estudo foi a necessária para reduzir em 30 % a sobrevida celular (DL30; 148 nM). Abordagens moleculares nos permitiram identificar que nas células fotossensibilizadas a redução na maturação da catepsina D (CTSD, 55 %) e da catepsina B (CTSB, 52 %) contribuem com a disfunção endolisossomal. Além disso, comprovamos que as células fotossensibilizadas tiveram, pela menor quantidade de CTSD ativa, o processamento da prosaposina (PSAP) significativamente afetado. Células coletadas após 24 horas de irradiação expressaram 7 vezes mais PSAP do que as amostras dos grupos controle, sugerindo que as reações de oxidação causadas pelo TPPS2a podem ocasionar o acúmulo de glicoesfingolipídios nos endossomos e nos lisossomos, mimetizando o fenótipo observado em doenças de armazenamento lisossomal. Imagens de células HT 29 com expressão estável da proteína LGALS3 fusionada ao marcador EFGP mostraram que, após 24 horas de irradiação, as células não ativaram a lisofagia para remover os endossomos e os lisossomos danificados. A ausência do recrutamento da LGALS3 também apontou que as membranas dos endossomos e dos lisossomos não apresentam rupturas permanentes que permitam a passagem de uma molécula de 26 kDa. Experimentos complementares de análise da expressão proteica dos marcadores autofágicos LC3-II e p62/SQSTM1 (referida como p62) confirmaram o bloqueio do fluxo autofágico nas células fotosenssibilizadas. Pelo envolvimento do sistema endolisossomal no tráfego de membranas e no fluxo de lipídios, o aumento da transcrição da Hidroximetilglutaril-CoA reductase (HMGCR) (≈ 1.6 vezes) uma enzima envolvida na síntese de novo do colesterol - sugeriu que a disfunção dos endossomos e dos lisossomos altera a distribuição de colesterol. Não obstante, para manter a homeostase lipídica nas células fotossensibilizadas este não foi o único mecanismocompensatório acionado, uma vez que houve um incremento sutil; porém, significativo (1.2 vezes) na transcrição da ceramidase ácida (ASAH1). Em conjunto, nossos dados apontam que a fotossensibilização com TPPS2a constitui uma ferramenta promissora para causar dano no sistema endolisossomal, inibindo a autofagia e permitindo o estudo das respostas metabólicas em células expostas a estresse oxidativo

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in humans. CRC caused more than 900,000 deaths in 2020 and it was estimated for the period 2020 - 2025, an increase of 13.5 % in the number of new cases according to the Global Cancer Observatory Web platform. Photodynamic Therapy (PDT) is a promising therapeutic alternative. Understandings of cell death signaling pathways as well as the adaptive responses associated with resistance to photo-oxidative damage are relevant to optimize the effectiveness of PDT. For this purpose, in this research, we investigated how HT-29 colorectal adenocarcinoma cells respond to photosensitization reactions generated by TPPS2a, a molecule activated by irradiation with light at 522 nm. PS concentrations displayed increased inhibitory effect on cell viability after irradiation (2.1 J cm-2). The lethal dose selected to photosensibilize cells was the TPPS2a concentration able to reduce 30 % of cell survival (LD30; 148 nM). By molecular methods, we observed a reduction in cathepsin D (CTSD, 55 %) and cathepsin B (CTSB, 52 %) maturation, depletion that may contribute to endo-lysosomal dysfunction in photosensitized cells. It is widely known that endo-lysosomal cathepsins are crucial in protein turnover and degradation. Thus, we focused on the consequence of CTSD reduction. Literature data indicate that CTSD plays a key role in prosaposin (PSAP) processing to the four saposins (SAPs) that are required in glycosphingolipids breakdown. In fact, our results in photosensitized cells showed that, due to the lower amount of active CTSD, PSAP processing was significantly affected. Cells collected after irradiation expressed 7 times more PSAP than cells from the control groups. This data suggest that oxidative photodamage induced by TPPS2a may result in glycosphingolipid-accumulating endosomes and lysosomes, phenotype which mimics lysosomal storage diseases. Furthermore, we monitored by fluorescence microscopy a form of selective autophagy which detects and removes damaged endosomes and lysosomes known as lysophagy. Images of HT-29 cells expressing Galectin 3/LGALS3 fused to EFGP showed that photosensitized cells did not activate lysophagy. The absence of LGALS3 recruitment also indicated that the membranes of endosomes and lysosomes do not present ruptures which allow the passage of proteins with a molecular weight up to at least 26 kDa. Protein expression analysis of the autophagic markers LC3-II and p62/SQSTM1 (referred as p62) confirmed autophagic flux blockade in cells challenged with photoactivated TPPS2a. The endo-lysosomal system plays a key role in membrane trafficking and lipid flux. At the transcriptional level, 1.6-fold increase in gene expression of Hydroxymethylglutaryl-CoA reductase (HMGCR) - an enzyme involved in the synthesis de novo of cholesterol - indicated that endosomes and lysosomes dysfunction alters the distribution of cholesterol in cellschallenged with photoactivated TPPS2a. However, to maintain lipid homeostasis in photosensitized cells, this was not the only compensatory mechanism triggered, since there was a slightly increase (1.2-fold) in the transcription of acid ceramidase (ASAH1). Taken together, our data showed that photosensitization with TPPS2a constitutes a promising tool to damage the endolysosomal system, to inhibit autophagy and to study metabolic responses in cells exposed to oxidative stress

High Concentrations of Aspartame Induce Pro-Angiogenic Effects in Ovo and Cytotoxic Effects in HT-29 Human Colorectal Carcinoma Cells.

Aspartame (ASP), an artificial sweetener abundantly consumed in recent years in an array of dietary products, has raised some concerns in terms of toxicity, and it was even suggested a link with the risk of carcinogenesis (colorectal cancer), though the present scientific data are rather inconclusive. This study aims at investigating the potential role of aspartame in colorectal cancer by suggesting two experimental approaches: (i) an in vitro cytotoxicity screening in HT-29 human colorectal carcinoma cells based on cell viability (Alamar blue assay), cell morphology and cell migration (scratch assay) assessment and (ii) an in ovo evaluation in terms of angiogenic and irritant potential by means of the chorioallantoic membrane method (CAM). The in vitro results showed a dose-dependent cytotoxic effect, with a significant decrease of viable cells at the highest concentrations tested (15, 30 and 50 mM) and morphological cellular changes. In ovo, aspartame (15 and 30 mM) proved to have a pro-angiogenic effect and a weak irritant potential at the vascular level. These data suggest new directions of research regarding aspartame's role in colorectal cancer.

Mucin pre-cultivated Lactobacillus reuteri E shows enhanced adhesion and increases mucin expression in HT-29 cells.

Adhesion of probiotic bacteria to the mucus layer lining the gastrointestinal tract is necessary for its effective colonisation and specific therapeutic effects. Enrichment of growth medium with mucin might stimulate bacterial adhesion, probably by increasing the expression of surface structures responsible for bacteria-gut epithelia and/or mucus interactions. The aim of this study was to determine if pre-cultivation of potentially probiotic strain Lactobacillus reuteri E (LRE) with mucin stimulates its adherence to colon cell line HT-29 and if the increased adhesion modulates mucin expression in these cells. The mucin-producing HT-29 cell line was co-cultivated for 2 h with LRE grown in MRS broth or MRS broth enriched with pig gastric mucin (LRE + M). The adherence ability of LRE was evaluated microscopically and by plate counting. The relative gene expression was measured by qPCR. Pre-cultivation of LRE in mucin enriched medium significantly increased its adhesion to 14 days HT-29 in comparison with LRE by both methods (28.64% vs. 23.83%, evaluated microscopically, and 14.31 ± 3.95 × 106 CFU ml-1 vs. 8.54 ± 0.43 × 106 CFU ml-1, evaluated by plate counting). MUC2, MUC5AC, and IL-10 were significantly upregulated after co-cultivation with LRE + M in comparison to LRE and control group (lactobacilli-free HT-29). Obtained results suggest that pre-cultivation of lactobacilli with mucin may not only stimulate their adhesion abilities but also promote their effectiveness to modulate the pathways involved in the pathophysiology of some diseases, e.g., with defective mucin synthesis in ulcerative colitis or colorectal cancer.

Predominantly Antibiotic-resistant Intestinal Microbiome Persists in Patients With Pouchitis Who Respond to Antibiotic Therapy.

BACKGROUND & AIMS: Pouchitis that develops in patients with ulcerative colitis after total proctocolectomy and ileal pouch anal anastomosis is usually treated with antibiotics. Some patients have recurrence of flares, or become antibiotic-dependent, and require repeated courses or prolonged periods of antibiotic therapy. We investigated microbial factors associated with response to antibiotic treatment and development of antibiotic dependence in patients with pouchitis. METHODS: We performed a prospective study of 49 patients who had undergone pouch surgery at a tertiary center. Disease activity was determined based on clinical, endoscopic, and histologic criteria. Pouch phenotype was defined as recurrent-acute pouchitis (n = 6), chronic pouchitis and Crohn's-like disease of the pouch (n = 27), normal pouch from patient with ulcerative colitis (n = 10), and normal pouch from patient with familial adenomatous polyposis (n = 6). Fecal samples (n = 234) were collected over time during or in the absence of antibiotic treatment (ciprofloxacin and/or metronidazole). Thirty-three patients were treated with antibiotics, for a median of 425 days of cumulative antibiotic therapy, during follow-up. Calprotectin was measured and fecal DNA was sequenced using shotgun metagenomics and analyzed with specifically designed bioinformatic pipelines. Bacterial strains were isolated from fecal samples. We assessed their ciprofloxacin resistance and ability to induce secretion of inflammatory cytokines by HT-29 intestinal epithelial cells. RESULTS: Most antibiotic-treated patients (79%) had a clinical response to each course of antibiotics; however, 89% of those who completed a 4-week course relapsed within 3 months. Median calprotectin levels decreased by 40% in response to antibiotics. Antibiotic treatment reduced disease-associated bacteria such as Clostridium perfringens, Ruminococcus gnavus, and Klebsiella pneumoniae, but also beneficial species, such as Faecalibacterium prausnitzii. The microbiomes of antibiotic-responsive patients were dominated by facultative anaerobic genera (Escherichia, Enterococcus, and Streptococcus), with multiple ciprofloxacin-resistance mutations in drug target genes and confirmed drug resistance. However, these strains had lower potential for virulence and did not induce secretion of inflammatory cytokines by epithelial cells. After antibiotic cessation, patients had an abrupt shift in microbiome composition, with blooms of oral and disease-associated bacteria. In addition, antibiotic treatment enriched for strains that acquired multidrug resistance loci, encoding enzymes that confer resistance to nonrelated antibiotics, including extended-spectrum beta-lactamases. CONCLUSIONS: The efficacy of antibiotic treatment of pouchitis might be attributed to the establishment of an antibiotic-resistant microbiome with low inflammatory potential. This microbiome might provide resistance against colonization by bacteria that promote inflammation. To avoid progression to antibiotic-dependent disease and its consequences, strategies such as short-term alternating antibiotics and nutrition- and microbiome-based interventions should be considered.

An Optimal Orthotopic Mouse Model for Human Colorectal Cancer Primary Tumor Growth and Spontaneous Metastasis.

BACKGROUND: Colorectal cancer is a leading cause of cancer-related death. Small animal models allow for the study of different metastatic patterns, but an optimal model for metastatic colorectal cancer has not been established. OBJECTIVE: The purpose of this study was to determine which orthotopic model most accurately emulates the patterns of primary tumor growth and spontaneous liver and lung metastases seen in patients with colorectal cancer. DESIGN: Using luciferase-tagged HT-29 cells coinoculated with lymph node stromal analog HK cells, 3 tumor cell delivery models were compared: intrarectal injection, intracecal injection, and acid enema followed by cancer cell instillation. Tumor growth was monitored weekly by bioluminescent imaging, and mice were sacrificed based on primary tumor size or signs of systemic decline. Liver and lungs were evaluated for metastases via bioluminescent imaging and histology. SETTINGS: The study was conducted at a single university center. MAIN OUTCOME MEASURES: Primary tumor and metastasis bioluminescent imaging were measured. RESULTS: Intrarectal injection had the lowest mortality at 4.0% (1/25) compared with the intracecal group at 17.4% (4/23) and the acid enema followed by cancer cell instillation group at 15.0% (3/20).The primary tumors in intrarectal mice had the highest average bioluminescence (3.78 × 10 ± 4.94 × 10 photons) compared with the mice in the intracecal (9.52 × 10 ± 1.92 × 10 photons; p = 0.012) and acid enema followed by cancer cell instillation groups (6.23 × 10 ± 1.23 × 10 photons; p = 0.0016). A total of 100% of intrarectal and intracecal mice but only 35% of mice in the acid enema followed by cancer cell instillation group had positive bioluminescent imaging before necropsy. Sixty percent of intrarectal mice had liver metastases, and 56% had lung metastases. In the intracecal group, 39% of mice had liver metastases, and 35% had lung metastases. Only 2 acid enema followed by cancer cell instillation mice developed metastases. LIMITATIONS: Tumor injections were performed by multiple investigators. Distant metastases were confirmed, but local lymph node status was not evaluated. CONCLUSIONS: Intrarectal injection is the safest, most reproducible, and successful orthotopic mouse model for human colorectal cancer primary tumor growth and spontaneous metastasis.

Differential Regulation of TLR Signaling on the Induction of Antiviral Interferons in Human Intestinal Epithelial Cells Infected with Enterovirus 71.

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease, which can lead to fatal neurological complications in young children and infants. Few gastrointestinal symptoms are observed clinically, suggesting the presence of a unique immunity to EV71 in the gut. We reported a robust induction of interferons (IFNs) in human intestinal epithelial cells (HT-29), which was suppressed in other types such as RD and HeLa cells. The underlying mechanism for the apparent difference remains obscure. In this study we report that in EV71-infected HT-29 cells, TLR/TRIF signaling was essential to IFN induction; viral replication increased and the induction of IFN-α, -ß, -ω, -κ, and -ε decreased markedly in TRIF-silenced HT-29 cells. Importantly, TRIF was degraded by viral 3Cpro in RD cells, but resisted cleavage, and IRF3 was activated and translocated into the nucleus in HT-29 cells. Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection.

Hydroxytyrosol, a product from olive oil, reduces colon cancer growth by enhancing epidermal growth factor receptor degradation.

SCOPE: We studied the effects and mechanism of 2-(3,4-dihydroxyphenil)ethanol (or hydroxytyrosol, HT), a polyphenol from extra virgin olive oil, investigating the regulation of epidermal growth factor receptor (EGFR) expression in colon tumour cells. METHODS AND RESULTS: We demonstrate that HT significantly downregulates EGFR expression in human colorectal adenocarcinoma cells HT-29, CaCo2, and WiDr, and in HT-29 xenografts. HT accelerates EGFR degradation by reducing its half-life. Specifically, HT induces EGFR ubiquitination that is mediated by phosphorylation at pY1045, the docking site for Cbl, thereby enabling receptor ubiquitination and degradation. Pretreatment with either the lysosomal inhibitor chloroquine, or the proteasomal inhibitor MG132 blocks HT-induced EGFR downregulation. In colon cancer cells, EGFR downregulation by HT is associated with reduced cell proliferation. Tumour growth and EGFR expression levels are also decreased by HT treatment in HT-29 xenograft. CONCLUSION: We conclude that HT downregulates EGFR expression via lysosomal and proteasomal degradation, activated by HT-induced EGFR phosphorylation at pY1045 and increased Cbl activity. Cbl activation induces, in turn, EGFR ubiquitination. Our results reveal a new mechanism for HT's antitumour effects that may be important for colon tumour prevention and treatment.

Synthetic Bichalcone TSWU-BR23 Induces Apoptosis of Human Colon Cancer HT-29 Cells by p53-Mediated Mitochondrial Oligomerization of BAX/BAK and Lipid Raft Localization of CD95/FADD.

A synthetic bichalcone analog, (E)-1-(3-((4-(4-acetylphenyl)piperazin-1-yl)methyl)-4-hydroxy-5-methoxyphenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR23), has been shown to induce apoptosis in human colon cancer HT-29 cells involving the induction of CD95 and FAS-associated protein death domain (FADD), but its precise mechanism of action has not been fully elucidated. Using cell-surface biotinylation and sucrose density-gradient-based membrane flotation techniques, we showed that the disruption of TSWU-BR23-induced lipid raft localization of CD95/FADD by cholesterol-depleting agent (methyl-ß-cyclodextrin) was reversed by cholesterol replenishment. Blockade of p53 expression by short-hairpin RNA (shRNA) suppressed oligomeric Bcl-2-associated x protein (BAX)/Bcl-2 antagonist killer 1 (BAK)-mediated mitochondrial apoptosis but did not inhibit lipid raft localization of CD95/FADD and pro-caspase-8 cleavage induced by TSWU-BR23. Co-expression of p53 shRNA and dominant-negative mutant of FADD completely inhibited TSWU-BR32-induced mitochondrial apoptotic cell death. Collectively, these data demonstrate that TSWU-BR23 leads to HT-29 cell apoptosis by inducing p53-mediated mitochondrial oligomerization of BAX/BAK and the localization of CD95/FADD with lipid rafts at the cell surface.

Colocalization of ß-catenin with Notch intracellular domain in colon cancer: a possible role of Notch1 signaling in activation of CyclinD1-mediated cell proliferation.

The Wnt and Notch1 signaling pathways play major roles in intestinal development and tumorigenesis. Sub-cellular localization of ß-catenin has been implicated in colorectal carcinogenesis. However, the ß-catenin and Notch intracellular domain (NICD) interaction has to be addressed. Immunohistochemistries of ß-catenin, NICD, and dual immunofluorescence of ß-catenin and NICD were analyzed in colorectal tissues and HT29 cell line. Moreover, real-time PCR analysis of CyclinD1, Hes1 and MUC2 was done in HT29 cells upon N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment. Dual staining emphasized the strong interaction of ß-catenin and NICD in adenoma and adenocarcinoma than in normal tissues. Hes1 transcript levels were decreased 1.5- and 7.1-fold in 12.5 and 25 µM DAPT-treated HT29 cells. CyclinD1 transcript levels decreased 1.2- and 1.6-fold, and MUC2 transcript level increased 4.3- and 7.5-fold in 12.5 and 25 µM DAPT-treated HT29 cells. The results of this study showed that the sub-cellular localization of ß-catenin converges with NICD inducing proliferation through the activation of CyclinD1 and Hes1. Moreover, the inhibition of Notch1 signaling by DAPT leads to the arrest of cell proliferation and induces apoptosis leading to the upregulation of MUC2, a secretory cell lineage marker.

Complex forming competition and in-vitro toxicity studies on the applicability of di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) as a metal chelator.

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is a potential candidate in chelation therapy as an iron chelator. This study showed that a combined treatment with 2µM easily available Fe(II), Cu(II) and Zn(II) each and 5µM Dp44mT on eight different cancer cell lines resulted in a 10-40-fold increase in the intracellular Cu content compared to control samples. The uptake of Cu and Cu-dependent cytotoxicity strictly depend on the Cu concentration of the culture medium. Even as low concentration of Dp44mT as 0.1µM can transport high amounts of copper inside the cells. The Cu accumulation and toxicity through Dp44mT can hardly be influenced by Fe. Copper uptake and toxicity triggered by 2µM extracellular Cu(II) and 5µM Dp44mT could not be influenced by Fe(II) extracellular concentrations even 50-times higher than that of Cu(II). A 50-times higher Co(II) extracellular concentration hindered the Cu(II) uptake almost completely and a 10-times higher Co(II) concentration already decreased the Dp44mT-mediated Cu toxicity. Conditional complex stability constant determinations for Dp44mT with Cu(II), Co(II), Fe(II), Ni(II) and Zn(II) revealed that the metal-to-ligand ratio is 1:1 in [Cu(II)Dp44mT] complex, while for Co(II), Fe(II) and Ni(II) is 1:2. The highest stability constant was obtained for Cu(II) (lg ß=7.08±0.05) and Co(II) (lg ß2=12.47±0.07). According to our results, Dp44mT in combination with Cu is highly toxic in vitro. Therefore, the use of Dp44mT as an iron chelator is limited if biologically available Cu is also present even at low concentrations.

The Australian fruit Illawarra plum (Podocarpus elatus Endl., Podocarpaceae) inhibits telomerase, increases histone deacetylase activity and decreases proliferation of colon cancer cells.

Fruit antioxidants have many health benefits including prevention of cancer development. The native Australian bush fruit Illawarra plum (Podocarpus elatus Endl., Podocarpaceae) has a high content of anthocyanin-rich phenolics, with an antioxidant capacity at levels higher than most fruits. In the present study the molecular mechanisms of the anti-proliferative activity of Illawarra plum on colorectal cancer cells were investigated. Non-tumorigenic young adult mouse colonic (YAMC) cells and tumorigenic human colonic (HT-29) cells were treated with a polyphenolic-rich Illawarra plum extract (0-1000 microg/ml). Illawarra plum had anti-proliferative properties in only the cancer cells, with growth suppressed in a dose- and time-dependent manner. Treatment of HT-29 cells with Illawarra plum extract (500 mg/ml; 24 h) was also associated with a 2-fold increase in apoptosis, and a cell cycle delay in the S phase (P < 0.01). Assessment of biomarkers for DNA damage revealed that plum treatment caused a 93% down-regulation of telomerase activity (P < 0.001) and a decrease in telomere length (up to 75%; P < 0.01). Treatment with Illawarra plum extract also induced morphological alterations to HT-29 cells that were suggestive of induction of autophagy, as the formation of cytoplasmic vacuoles was observed in many cells. This could be induced by the increased (6-fold) histone deacetylase (HDAC) activity (P < 0.001) and the trend for increased expression of the class III HDAC sirtuin 1. The present study has shown that Illawarra plum extract is able to reduce the proliferation of colon cancer cells by altering the cell cycle, increasing apoptosis and possibly inducing autophagy. The active ingredients in Illawarra plum may provide an alternative chemoprevention strategy to conventional chemotherapy.

Mechanism of cellular uptake of genotoxic silica nanoparticles.

Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations ≥ 1 µg/ml, but DNA damage was evident at 0.1 µg/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 µg/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4°C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal microbalance experiments on tethered bilayer lipid membrane systems show that the nanoparticles strongly bind to lipid membranes, forming an adherent monolayer on the membrane. Leakage assays on large unilamellar vesicles (400 nm diameter) indicate that binding of the silica NPs transiently disrupts the vesicles which rapidly self-seal. We suggest that an adhesive interaction between silica nanoparticles and lipid membranes could cause passive cellular uptake of the particles.

Influence of CXCR4/SDF-1 axis on E-cadherin/ß-catenin complex expression in HT29 colon cancer cells.

AIM: To study the influence of CXCR4/stromal cell-derived factor-1 (SDF-1) axis on E-cadherin/ß-catenin complex expression in HT29 colon cancer cells and its underlying mechanisms. METHODS: Effect of SDF-1 on E-cadherin/ß-catenin expression was detected by immunocytochemistry. E-cadherin and ß-catenin mRNA expression levels were measured by reverse transcriptase-polymerase chain reaction. SDF-1-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K)/AKT and ß-catenin was detected by Western blotting. RESULTS: The E-cadherin and ß-catenin mRNA expression levels in HT29 cells were lower 48 h after incubated with SDF-1 at the concentrations of 20 and 40 ng/mL (P<0.05). SDF-1-induced significant phosphorylation of PI3K/AKT and ß-catenin. AMD3100 and LY294002 inhibited the phosphorylation of PI3K/AKT and ß-catenin. CONCLUSION: SDF-1 down-regulates the E-cadherin/ß-catenin complex expression in HT29 cells by decreasing mRNA synthesis and increasing ß-catenin phosphorylation.

Nitric oxide and P-glycoprotein modulate the phagocytosis of colon cancer cells.

The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P-glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug-sensitive cells, not in drug-resistant ones, which are indeed chemo-immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo-immunoresistance. In the drug-sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton-dependent way. CRT translocation did not occur in drug-resistant HT29-dx cells, where the doxorubicin-induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29-dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug-resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29-dx cells CRT was bound to Pgp and only by silencing the latter the CRT-operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo-immunoresistant tumours.

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.

Optical imaging of CCK2/gastrin receptor-positive tumors with a minigastrin near-infrared probe.

PURPOSE: A variety of tumors in different organs with good accessibility to near-infrared light express the cholecystokinin-2 (CCK2)/gastrin receptor. Therefore, the applicability of fluorescence optical imaging was assessed using a novel peptide probe. MATERIALS AND METHODS: This study was approved by the regional animal committee. Our optical peptide probe (DY-minigastrin) was synthesized by coupling a hemicyanine dye to a gastrin derivative peptide (minigastrin). In vitro CCK2/gastrin receptor identification was performed in receptor-positive HT-29 and negative A-375 cells using flow cytometry, laser scanning microscopy, and macroscopic near-infrared fluorescent (NIRF) imaging. For in vivo studies, tumor cells were implanted into mice, and DY-minigastrin in presence or absence of nonlabeled minigastrin (control of signaling specificity) was applied intravenously. Fluorescence signals in tumors and organs were recorded and statistically analyzed. RESULTS: Flow cytometry, laser scanning microscopy, and in vitro macroscopic imaging of cell pellets revealed a distinct accumulation of our minigastrin probe in HT-29 cells, showing distinct probe internalization. In vivo NIRF whole-body animal imaging, again, demonstrated a clear depiction of HT-29 tumors, which was reversed by blocking with nonlabeled minigastrin. Semi-quantitative fluorescence analysis and histologic observations were in agreement with these observations. A distinct probe organ distribution was observed. CONCLUSIONS: Our observations indicate that DY-minigastrin-based NIRF optical imaging of CCK2/gastrin receptor protein is feasible. Because of its widespread occurrence in different tumor types, endoscopic, laparoscopic, and tomographic receptor imaging could be accomplished in the near future.

Study of 5HT3 and HT4 receptor expression in HT29 cell line and human colon adenocarcinoma tissues.

BACKGROUND: Serotonin (5HT) has been shown to be a mitogenic factor in several carcinomas. Its mitogenic effect is elicited through a wide range of 5HT receptor subtypes. In this study, the effects of 5HT, 5HT3 (1-phenylbiguanide hydrochloride) and 5HT4 (cisapride) agonists in promoting the growth of the HT29 cell line and the growth-inhibition effect of the 5HT3 receptor antagonist (Y-25130 hydrochloride) and 5HT4 receptor antagonist (RS 23597-190) were investigated. The expressions of 5HT3 and 5HT4 receptors in human colon cancer tissues and the HT29 cell line were studied. METHODS: The growth-promoting and growth-inhibition effects of 5-HT, 5HT3 and 5HT4 agonists and antagonists on the HT29 cell line were studied using MTT assay. Receptor expression has been demonstrated by western blotting. RESULTS: The results showed that 5HT, 5HT3, and 5HT4 agonists caused significant proliferation of HT29 cells. 5HT3 and 5HT4 receptor antagonists had an inhibitory effect on the growth of these cells. Western blot analysis gave bands from colon tissue extracts and the HT29 cell line. CONCLUSION: The results indicate which 5HT3 and 5HT4 receptors are significantly expressed in both colon cancer tissue and the HT29 cell line. Expression for the 5HT3 receptor is more potent. Furthermore, 5HT plays a mitogenic role in colon cancer cells and antagonists of 5HT3, and 5HT4 receptors can inhibit cancer cell growth.

Phthalocyanine-polyamine conjugates as pH-controlled photosensitizers for photodynamic therapy.

A series of aryl hydroxyamines prepared by reductive amination were treated with silicon(IV) phthalocyanine dichloride in the presence of pyridine to give the diaxially substituted phthalocyanine-polyamine conjugates 1-5. The electronic absorption, fluorescence emission, and efficiency at generating reactive oxygen species of these compounds were all sensitive to the pH environment. Under acidic conditions, the fluorescence quantum yields and the singlet oxygen quantum yields of these compounds were greatly enhanced in DMF as a result of protonation of the amino moieties, which inhibited the photoinduced electron-transfer deactivation pathway. The Q band was diminished and broadened, and the fluorescence intensity decreased as the pH increased in citrate buffer solutions. The rate of superoxide radical formation was also reduced in a higher pH environment. Compound 3, containing a terminal 4-chlorophenyl group at the axial substituent, showed the most desirable pH-responsive properties, which makes it a promising tumor-selective fluorescence probe and photosensitizer for photodynamic therapy. All of the phthalocyanines 1-5 were highly photocytotoxic against HT29 and HepG2 cells with IC(50) values as low as 0.03 microM. Compound 3 was highly selective toward lysosomes, but not mitochondria of HT29 cells.

[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.

Influence of paeonol on expression of COX-2 and p27 in HT-29 cells.

AIM: To investigate the effect of paeonol on controlling the proliferation of colorectal cancer cell line HT-29 and to discuss its possible mechanism. METHODS: The inhibitory effect of paeonol on proliferation of HT-29 cells was detected by MTT assay. The results of apoptosis were measured by flow cytometry. Immunocytochemical staining was performed to detect the expression of cyclooxygenase-2 (COX-2) and protein p27 in HT-29 cells treated with paeonol at different concentrations. Reverse transcription-polymerase chain reaction (RT-PCR) was used for mRNA analysis. RESULTS: From the data of both MTT and flow cytometry, we observed that cell proliferation was inhibited by different concentrations of paeonol. By immunocytochemical staining, we found that HT-29 cells treated with paeonol (0.024-1.504 mmol/L) reflected reduced expression of COX-2 and increased expression of p27 in a dose-dependent manner. RT-PCR showed that paeonol down-regulated COX-2 and up-regulated p27 in a dose- and time-dependent manner in HT-29 cells. CONCLUSION: One of the apoptotic mechanisms of paeonol is down-regulation of COX-2. p27 is up-regulated simultaneously and plays an important part in controlling cell proliferation and is a crucial factor in the Fas/FasL apoptosis pathway.