Apoptosis and cell cycle regulatory effects of adenosine by modulation of GLI-1 and ERK1/2 pathways in CD44+ and CD24- breast cancer stem cells.

OBJECTIVES: Breast cancer stem cells (CSCs) are a small population of tumour cells with the ability of self-renewal and resistance to chemotherapy. Targeting CSCs is a promising strategy for treatment of cancer. A recent study demonstrated that adenosine receptor agonists inhibit glioblastoma CSCs proliferation. At present, the effect of adenosine on breast CSCs has not been reported. Therefore, this study was designed to evaluate the effect of adenosine and its signalling pathways in breast CSCs. MATERIALS AND METHODS: Anti-proliferative effect of adenosine on breast CSCs was evaluated by mammosphere formation and MTS assay. The effect of adenosine on cell cycle progression was examined using flow cytometry. Detection of apoptosis was conducted by Annexin V-FITC. The expression levels of cell cycle and apoptosis regulatory proteins as well as ERK1/2, and GLI-1 were measured by Western blot. RESULTS: Adenosine reduced CSCs population and mammosphere formation in breast CSCs. Adenosine induced G1 cell cycle arrest in breast CSCs in conjunction with a marked down-regulation of cyclin D1 and CDK4. Adenosine also induced apoptosis by regulation of Bax/Bcl-2 ratio, mitochondrial membrane potential depletion and activation of caspase-6. Moreover, adenosine inhibited ERK1/2 phosphorylation and GLI-1 protein expression. CONCLUSIONS: These findings indicated that adenosine induces cell cycle arrest and apoptosis through inhibition of GLI-1 and ERK1/2 pathways in breast CSCs.

Molecular expression of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 human ovarian cancer cell lines.

Adenosine receptors (A1, A2a, A2b and A3) have several physiological and pathological roles in cancer cell lines. The present study was carried out to evaluate the mRNA and protein expression profile and functional role of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 ovarian cancer cell lines. The levels of mRNA and protein expression of A1, A2a, A2b and A3 adenosine receptors in the ovarian cancer cell lines were measured by Real-time PCR and western blotting. The functional roles of adenosine receptors were investigated through measurement of cAMP levels after agonist treatment. The mRNA and protein of all adenosine receptors subtypes were expressed in the ovarian cancer cell lines. Our findings demonstrated that A2b and A3 had the most mRNA and protein expression. Moreover, cAMP assay confirmed the functional role of A2b and A3 adenosine receptors. This findings demonstrated that A2b and A3 subtypes are most important adenosine receptors in humn ovarian cancer cell lines. This information provide a strong possibility into the relationship of A2b and A3 adenosine receptor and ovarian cancer.

N-acetylcysteine prevents cadmium-induced apoptosis in human breast cancer MDA-MB468 cell line.

Cadmium is a heavy metal posing severe risks and destructive effects on human health. Although cadmium was classified as a human carcinogen, it has been also shown to be a cytotoxic agent that induces cell death either by necrosis or apoptosis. In this study, we investigated the protective role of N-acetylcysteine, a free radical scavenger, on cadmium induced apoptosis in MDA-MB468 cells. The breast cancer cells were exposed to increasing concentrations of CdCl2 in the presence and absence of NAC and the cell viability was assessed using MTT assay. The microscopic studies of apoptosis were carried out with fluorescent staining. To investigate the induction of apoptosis, cellular DNA was isolated using DNA kit extraction and analyzed electrophoretically. The results showed significant decrease in cellular viability upon 48 hours exposure to CdCl2 in a dose-dependent manner (p < 0.05). Pretreatment of MDA-MB468 cells with N-acetylcysteine (1mM) reversed the cadmium cytotoxicity effects and protected cells from apoptotic death. DNA Hoechst staining showed the apoptotic bodies. The electrophoresis of extracted DNA identified a fragmentation pattern consistent with apoptosis mechanism. The results suggest that cytotoxic effects and induction of apoptosis caused by CdCl2 are mediated, by oxidative stress.

Development of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo diagnosis.

In this work, the development and applications of a fluorescence detection system using optical parametric oscillator (OPO) laser excitation for in vivo disease diagnosis including oral carcinoma are described. The optical diagnosis system was based on an OPO laser for multi-wavelength excitation and time-resolved detection. The pulsed Nd-YAG-pumped OPO laser system (6 ns, 20 Hz) is compact and has a rapid, broad, and uniform tuning range. Time-gated detection of intensified charge-coupled device (ICCD) making use of external triggering was used to effectively eliminate the laser scattering and contribute to the highly sensitive in vivo measurements. Artificial tissue-simulating phantoms consisting of polystyrene microspheres and tissue fluorophores were tested to optimize the gating parameters. 51-ns gate width and 39-ns gate delays were determined to be the optimal parameters for sensitive detection. In vivo measurements with the optical diagnosis system were applied to esophagus, stomach, and small intestine using an endoscope in canine animal studies. The rapid tuning capability of the optical diagnosis system contributed greatly to the optimization of wavelength for the observation of porphyrin in the small intestine. When the small intestine was thoroughly washed with water, the emission band which corresponds to porphyrin disappeared. Based on this observation, it was concluded that the detected signal was yielded by porphyrin-containing bile secretion. Also, multispectral analyses using multiple excitations from 415 to 480 nm at 5 nm intervals confirmed the porphyrin detection in the small intestine. The optical diagnosis system was also applied to the detection of human xenograft of oral carcinoma in mice using 5-aminolevulinic acid (5-ALA) which is a photodynamic therapy (PDT) drug. Significant differences in protoporphyrin IX fluorescence intensity between normal and tumor tissue could be obtained 2 hours after the injection of 5-ALA into mice due to the preferential accumulation of 5-ALA in tumors. Results reported herein demonstrate potential capabilities of the LIF-OPO system for in vivo disease diagnosis.

Results of photodynamic therapy for ablation of dysplasia and early cancer in Barrett's esophagus and effect of oral steroids on stricture formation.

OBJECTIVE: The primary goal of this study was to investigate whether oral steroids would reduce the incidence of stricture formation after photodynamic therapy (PDT) in Barrett's patients. The effect of balloon window length, pretreatment of nodules, retreatment of skipped areas, and subsequent PDT on the incidence of strictures was also investigated. The ultimate goal of treatment was elimination of dysplasia, early cancer, and Barrett's mucosa. METHODS: A total of 60 patients were injected with Photofrin (2 mg/kg). Patients were randomized to receive PDT (n = 30) or PDT and oral prednisone (n = 30). Two days later, 630 nm light (KTP/dye laser) was delivered using a 5- or 7-cm windowed balloon at a light dose of 200 or 175 J/cm. The majority of patients received 200 J/cm using a 7-cm balloon. Nodules were pretreated with a short diffuser at a dose of 50-75 J/cm. Additional light was delivered to skipped areas 2-3 days later. Endoscopies were conducted every 3-6 months to evaluate the response. Residual or recurrent Barrett's was treated using neodymium:aluminum-yttrium-garnet (Nd:YAG) laser (small areas) or was retreated with PDT. RESULTS: The effect of steroids on the incidence of strictures was analyzed in patients receiving a single treatment with a light dose of 200 J/cm using a 7- cm balloon. There was no reduction in the incidence of strictures in patients receiving PDT and steroids (29%) compared to those receiving PDT alone (16%). Treatment using a 7-cm balloon caused more strictures (31%) than treatments using a 5-cm balloon (7%). Pretreatment of nodules or retreatment of skipped areas did not increase strictures. Patients receiving subsequent PDT had a higher incidence of strictures. Cancer was eliminated in all patients. High-grade dysplasia was eliminated in 41 of 43 patients (96%). Barrett's mucosa was totally eliminated in 25 of 60 patients (42%). CONCLUSIONS: Oral prednisone after PDT did not reduce the incidence of strictures. Subsequent PDT and longer balloon window were associated with higher incidence of strictures. PDT followed by thermal ablation of small islands eliminated dysplasia, early cancer, and Barrett's mucosa.

Light sources and delivery devices for photodynamic therapy in the gastrointestinal tract.

This article provides discussion on different light sources and light delivery devices used for photodynamic therapy of a variety of gastrointestinal malignancies. Different laser and nonlaser sources are discussed with emphasis on most commonly used clinical units. The discussion of light delivery devices is divided into several sections, each reviewing the evolution of fiberoptic delivery devices for different gastrointestinal malignancy. A discussion is provided on power meters and wavelength calibration devices used to assure proper light dosimetry. Finally, a short discussion is provided on a technique used to improve endoscopic monitoring of photodynamic therapy procedure during the treatment.

Photodynamic therapy for Barrett's esophagus: follow-up in 100 patients.

BACKGROUND: This report presents clinical results of photodynamic therapy in patients with Barrett's esophagus and dysplasia or superficial esophageal cancer. METHODS: One hundred patients including 13 with superficial cancers were treated. Light (630 nm) was endoscopically delivered to the esophageal mucosa by a diffuser or a windowed esophageal centering balloon. Nd:YAG laser was required to ablate small residual areas of Barrett's mucosa during-long-term follow-up. Patients were maintained on omeprazole and were followed for 4 to 84 months (mean 19 months). RESULTS: Conversion of approximately 75% to 80% of treated Barrett's mucosa to normal squamous epithelium was found in all patients; complete elimination of Barrett's mucosa was noted in 43 patients. Dysplasia was eliminated in 78 patients. Dysplasia developed during follow-up in 11 of 48 patients in untreated Barrett's mucosa requiring additional therapy. Ten of the 13 malignancies were ablated. Esophageal strictures occurred in 34%. Use of longer centering balloons reduced the incidence of strictures. CONCLUSION: Photodynamic therapy alone or with Nd:YAG laser thermal ablation combined with long-term acid inhibition provides an effective endoscopic therapy to (1) eliminate Barrett's mucosal dysplasia and superficial esophageal cancer and (2) reduce the extent of and, in some cases, eliminate Barrett's mucosa.

Laser-induced fluorescence for esophageal cancer and dysplasia diagnosis.

A method using laser-induced fluorescence (LIF) for in vivo cancer diagnosis of the esophagus is described. Autofluorescence of normal and malignant tissues was measured directly using a fiberoptic probe inserted through an endoscope. The measurements were performed in vivo during routine endoscopy. Measurement of the fluorescence signal from the tissue was performed using laser excitation at 410 nm. The methodology was applied to differentiate normal and malignant tumors of the esophagus. The results of this LIF approach were compared with histopathology results of the biopsy samples and indicated excellent agreement in the classification of normal and malignant tumors for the samples investigated. The LIF procedure could lead to the development of a rapid and cost-effective technique for cancer diagnosis.

Photodynamic therapy for Barrett's esophagus.

In summary, in a group of 55 patients with Barrett's esophagus and dysplasia, the authors have reported the ablation of seven superficial cancers, the elimination of dysplasia in 42 of the 55 patients, the complete elimination of Barrett's mucosa in 16 patients, and the reduction of the extent of Barrett's mucosa in all patients who were treated with PDT and were maintained on long-term omeprazole. Repeated PDT sessions were required in some patients to accomplish elimination of dysplasia. Esophageal strictures occurred in 53% of patients, but were treated satisfactorily with dilation. Current results using 5- and 7-cm centering balloons has shown a dramatic reduction in the incidence of stricture formation. Mucosal injury with these balloons is less than with diffusers or shorter balloons that require overlapping of treated areas. The authors are carefully following the patients treated with longer PDT balloons to evaluate the long-term effects on dysplasia. Lugol's staining is an important technique to identify residual patches of Barrett's mucosa following PDT. Small residual patches of Barrett's mucosa can be successfully destroyed with Nd: YAG laser therapy. The authors' results indicate that PDT alone or in combination with thermal ablation can eliminate superficial cancers, dysplasia, and Barrett's mucosa in many patients with Barrett's esophagus.

Photodynamic therapy for Barrett's esophagus: cardiac effects.

BACKGROUND AND OBJECTIVE: Atrial fibrillation has been reported following esophageal photodynamic therapy. This study presents the results of serial cardiac testing following photodynamic therapy for patients with Barrett's esophagus and with dysplasia or early carcinoma. STUDY DESIGN/MATERIALS AND METHODS: Twelve patients were treated using photodynamic therapy. Serum creatinine phosphokinase and lactic dehydrogenase isoenzyme levels were determined pretreatment and 24, 48, and 72 hours after treatment. Electrocardiograms were obtained before and 48 hours after treatment. A rhythm strip was obtained 1 week posttreatment. Clinical assessment for cardiac arrhythmias occurred daily following therapy. RESULTS: Transient atrial fibrillation was noted in one patient during a follow-up endoscopy. However, no significant or permanent abnormality was noted in cardiac enzymes or electrocardiograms. CONCLUSION: No permanent electrocardiographic changes or significant abnormalities in cardiac enzymes were detected following esophageal photodynamic therapy in patients with or without histories of cardiac disease. Delivery of esophageal PDT is not associated with permanent adverse cardiac effects.

Photodynamic therapy in Barrett's esophagus.

OBJECTIVES: This report presents clinical results using photodynamic therapy for dysplasia and superficial esophageal cancer in Barrett's esophagus. METHODS: Forty-five (45) patients with Barrett's esophagus and dysplasia were treated with photodynamic therapy using sodium porfimer 2.0 mg/kg as the photosensitizing drug. Fifteen patients also had 16 superficial esophageal cancers (0-1.5 cm; Tis-T2, N-0, M-0). Red light (630 nm) was delivered to the esophageal mucosa by a diffuser inserted through the endoscope or via a windowed esophageal centering balloon designed to improve targeted delivery of light during photodynamic therapy. Patients were maintained on omeprazole and were followed for 6-62 months following photodynamic therapy. RESULTS: Photodynamic therapy produced mucosal damage in treated areas. Ablation of dysplastic or malignant mucosa was followed by healing and conversion of approximately 75-80% of treated Barrett's mucosa to normal squamous epithelium in all patients. Complete elimination of Barrett's epithelium was found in 16 patients. Areas of dysplasia were eliminated in 35 of the 45 patients. All 16 malignancies were ablated. No cancer recurrence was found in follow-up. Healing was associated with esophageal strictures in 58%, which were treated successfully by esophageal dilation in all patients. CONCLUSION: Photodynamic therapy combined with long-term acid inhibition provides an effective endoscopic therapy to (1) eliminate Barrett's mucosal dysplasia and superficial esophageal cancer and (2) reduce the amount of and, in some cases, eliminate Barrett's mucosa.

Photodynamic therapy for Barrett's esophagus: clinical update.

OBJECTIVES: This report presents clinical results of photodynamic therapy for dysplasia and superficial esophageal cancer in Barrett's esophagus. METHODS: Thirty-six patients with Barrett's esophagus and dysplasia were treated with photodynamic therapy, and sodium porfimer 2.0 mg/kg was used as the photosensitizing drug. Fourteen patients also had 15 superficial esophageal cancers (0-1.5 cm; ultrasound-T2,N-0,M-0). Red light (630 nm) was delivered to the esophageal mucosa by a diffuser inserted through the endoscope or via a windowed esophageal centering balloon designed to improve targeted delivery of light during photodynamic therapy. Patients were maintained on omeprazole and were followed for 6-62 months after photodynamic therapy. RESULTS: Photodynamic therapy produced extensive mucosal damage in treated areas. Ablation of dysplastic or malignant mucosa was followed by healing and conversion of approximately 75-80% of treated Barrett's mucosa to normal squamous epithelium in all patients. Complete elimination of Barrett's epithelium was achieved in 10 patients. Areas of dysplasia were eliminated in 29 patients, and all 15 malignancies were ablated. No cancer recurrence was found in follow-up. Healing was associated with esophageal strictures (four were severe) that were treated successfully by esophageal dilation in 21 patients. CONCLUSION: Photodynamic therapy combined with long-term acid inhibition provides effective endoscopic therapy that 1) eliminates Barrett's mucosal dysplasia and superficial esophageal cancer, and 2) reduces the amount of, or, in some cases, eliminates Barrett's mucosa.

Endoscopic fluorescence detection of high-grade dysplasia in Barrett's esophagus.

BACKGROUND & AIMS: Early detection and treatment of esophageal cancer in Barrett's esophagus may improve patient survival if dysplasia is effectively detected at endoscopy. Typically, four-quadrant pinch biopsy specimens are taken at 2-cm intervals. This study was conducted to determine whether laser-induced fluorescence spectroscopy could be used to detect high-grade dysplasia in patients with Barrett's esophagus. METHODS: Four hundred ten-naonometer laser light was used to induce autofluorescence of Barrett's mucosa in 36 patients. The spectra were analyzed using the differential normalized fluorescence (DNF) index technique to differentiate high-grade dysplasia from either low-grade or nondysplastic mucosa. Each spectrum was classified as either premalignant or benign using two different DNF indices. RESULTS: Analysis of the fluorescence spectra from all patients collectively using the DNF intensity at 480 nm (DNF480) index showed that 96% of nondysplastic Barrett's esophagus samples were classified as benign, all low-grade dysplasia samples as benign, 90% of high-grade dysplasia samples as premalignant, and 28% of low-grade with focal high-grade dysplasia samples as premalignant. Using the two DNF indices concurrently, all patients with any high-grade dysplasia were classified correctly. CONCLUSIONS: Laser-induced fluorescence spectroscopy has great potential to detect high-grade dysplasia in Barrett's esophagus when using the DNF technique.

Balloon photodynamic therapy of esophageal cancer: effect of increasing balloon size.

BACKGROUND AND OBJECTIVE: Photodynamic therapy is currently being used to treat various malignancies including esophageal cancer. The effect of photodynamic therapy depends upon the concentration of photosensitizing drug, light energy delivered to tissue, and the presence of oxygen in the targeted tissue. We have found that an esophageal centering balloon improves light delivery to esophageal mucosa. However, balloon pressure on esophageal mucosa could possibly reduce mucosal blood flow and oxygenation, therefore reducing the effect of photodynamic therapy. This study was conducted to investigate the effect of balloon pressure on the esophageal wall during photodynamic therapy in the canine esophageal model. STUDY DESIGN/MATERIALS AND METHODS: Studies were performed in the canine esophagus of ten animals to investigate whether increasing the size of the centering balloon, and hence the pressure on esophageal mucosa, would alter the tissue effect of PDT. Porfimer sodium 4 mg/Kg was administered and 630 nm light was delivered via a 1 cm diffuser located in the center of a 360 degrees 2 cm windowed balloon. Mucosal light measurements were made to ascertain equivalent mucosal light dosing of approximately 25 J/cm2. Endoscopic and necropsy findings obtained following photodynamic therapy with 25 mm, 33 mm, and 35 mm balloons were compared. RESULTS: In larger dogs (groups A and B), increasing the size of the esophageal centering balloon from a 25-33 mm size did not result in an overly tight fit nor was the increase associated with significant change in the PDT effect. In contrast, increasing the balloon size to 35 mm in smaller dogs (group C) resulted in a tight fit of the balloon in the esophagus and in significant reduction in the PDT effect on mucosal damage when mucosal equivalent light dose was administered during photodynamic therapy in the canine esophageal model. CONCLUSION: Increasing centering balloon size resulted in reduced tissue damage when mucosal equivalent light dose was administered during photodynamic therapy in the canine esophageal model. Proper sizing of centering balloons will be necessary for balloon PDT of esophageal mucosal dysplasia or cancer in humans.

Photodynamic therapy in Barrett's esophagus: reduction of specialized mucosa, ablation of dysplasia, and treatment of superficial esophageal cancer.

Twelve patients with Barrett's esophagus and dysplasia were treated with photodynamic therapy. Five patients also had early, superficial esophageal cancers and five had esophageal polyps. Light was delivered via a standard diffuser or a centering esophageal balloon. Patients were maintained on omeprazole and followed for 6-54 months. In patients with Barrett's esophagus, photodynamic therapy ablated dysplastic mucosa and malignant mucosa in patients with superficial cancer. Healing and partial replacement of Barrett's mucosa with normal squamous epithelium occurred in all patients and complete replacement with squamous epithelium was found in three patients. Side effects included photosensitivity and mild-moderate chest pain and dysphagia for 5-7 days. In four patients with extensive circumferential mucosal ablation in the mid or proximal esophagus, healing was associated with esophageal strictures which were treated successfully by esophageal dilation. Strictures were not found in the distal esophagus. Photodynamic therapy combined with long-term acid inhibition provides effective endoscopic therapy of Barrett's mucosal dysplasia and superficial (Tis-T1) esophageal cancer. The windowed centering balloon improves delivery of photodynamic therapy to diffusely abnormal esophageal mucosa.

Spectroscopic diagnosis of esophageal cancer: new classification model, improved measurement system.

Laser-induced fluorescence spectroscopy was used to measure fluorescence emission of normal and malignant tissue during endoscopy in patients with esophageal cancer and volunteers with normal esophagus. The spectroscopy system consisted of a nitrogen-pumped dye-laser tuned at 410 nm for excitation source, an optical multichannel analyzer for spectrum analysis, and a fiberoptic probe designed for both the delivery of excitation light and the collection of fluorescence emission from tissue. The fluorescence lineshape of each spectrum was determined and sampled at 15-nm intervals from 430 to 716 nm. A calibration set of spectra from normal and malignant spectra was selected. Using stepwise discriminate analysis, significant wavelengths that separated normal from malignant spectra were selected. The intensities at these wavelengths were used to formulate a classification model using linear discriminate analysis. The model was then used to classify additional tissue spectra from 26 malignant and 108 normal sites into either normal or malignant spectra. A sensitivity of 100% and specificity of 98% were obtained.

In vivo cancer diagnosis of the esophagus using differential normalized fluorescence (DNF) indices.

BACKGROUND AND OBJECTIVE: We report the use of new diagnostic parameters based on the differential normalized fluorescence (DNF) signals for malignant tumor diagnosis. STUDY DESIGN/MATERIALS AND METHODS: Over 200 measurements of endogenous fluorescence from normal and malignant esophageal tissues were performed during routine endoscopy in 48 patients. A pulsed nitrogen-pumped dye laser was used to provide in situ excitation at 410 nm. Direct collection of the fluorescence signal emitted by the tissue was achieved using an intensified photodiode array detector equipped with a fiberoptic probe. RESULTS: The fluorescence signals were normalized with respect to the total fluorescence signal area. The cancer diagnosis indices were defined by the difference between the normalized fluorescence signal of a tumor and the mean value of a reference set of normal tissues. The results of the DNF approach were compared with endoscopic examinations and histopathology interpretations of the biopsy samples. Excellent correlation in the classification of normal and malignant tumors for the samples was found. CONCLUSION: The data indicated that the DNF approach has a significant potential to provide a direct, real-time, and in-situ technique for cancer diagnosis of the esophagus without requiring biopsy of the tumors and time-consuming histopathology tests.

Tumor cell-enhanced sensitivity of vascular endothelial cells to photodynamic therapy.

Effective antitumor photodynamic therapy (PDT) may be related to damage of vasculature within the tumor. The purpose of this study was to determine if tumor cells secrete factors that stimulate proliferation of human umbilical vein endothelial cells (HUVEC) and result in enhanced sensitivity of HUVEC to aluminum-sulfonated phthalocyanine (AlSPc)-PDT. Three human tumor cell lines--pharyngeal squamous carcinoma, colonic carcinoma, and mammary carcinoma--were used in this study. Co-culture of HUVEC and either squamous carcinoma or colonic carcinoma, but not mammary carcinoma, significantly increased HUVEC proliferation and AlSPc-PDT mediated cell damage. In addition, supernatant from squamous carcinoma and colonic carcinoma cultures also stimulated HUVEC proliferation and sensitivity to AlSPc-PDT. Both supernatant and cell lysate from squamous carcinoma cells contained angiogenic factors consistent with basic and acidic fibroblast growth factors, as evidenced by Western blot analysis and BALB/c 3T3 fibroblast cell proliferation assays. Collectively, these results suggest that selected tumor cell lines produce angiogenic factors that induce HUVEC proliferation and subsequently enhance sensitivity to AlSPc-PDT.