Effects of 5-ALA mediated photodynamic therapy in oral cancer stem cells.

The aim of the present study was to investigate the effects of PDT using the photosensitizer 5-aminoulevulinic acid (5-ALA) in oral squamous cell carcinoma (OSCC) behavior, mainly regarding its role on the cancer stem cell (CSC) phenotypes and in maintenance of the stem cell properties. Two OSCC cell lines were used and divided in the groups: Control, 5-ALA, LED 6 J/cm2 and PDT. MTT and Neutral red assays were used to access cellular viability, cell migration was evaluated by the wound healing assay. The stem cell phenotype was analyzed by flow cytometry to evaluate the CD44high/ESAhigh, CD44high/ESAlow and CD44low populations, by the clonogenic and tumor sphere formation assays as well as by RT-qPCR. The presence of Protoporphyrin IX in each CSC fraction was evaluated by flow cytometry. The OSCC cell lines showed a significant decrease in cell viability and migration after PDT. The percentage of CD44high/ESAhigh cells decreased after PDT, which was associated with an increase in the CD44low cells and with a functional decrease in the colony and sphere formation capacity. CD44high/ESAhigh cells showed increased PpIX, which contributed for their greater sensitivity to PDT. INV gene increased significantly after PDT, indicating cellular differentiation. Altogether, our results demonstrate that 5-ALA mediated PDT decreases not only the fraction of oral CSC but also their functional capabilities, inducing their differentiation.

Preventive potential of Bacillus sonorensis exopolysaccharide upon hepatocellular carcinoma and quantitation of tumor suppressor protein p53.

BACKGROUND: Exopolysaccharide, a carbohydrate polymer, is known to possess several biological activities. This approach was designed to clarify the cytotoxic mechanism of Bacillus sonorensis exopolysaccharide (EPS-1) on Huh7, HepG2 and BNL cells besides exploring its influence on the expression of the tumor suppressor protein p53. p53 is the biomarker of the prognosis and occurrence of severe stages of the tumor and activation of both cell-cycle arrest and apoptosis in cancer cells which are the most targeted cellular processes for the therapy of tumor patients. METHODS: The cytotoxic impact of EPS-1 was quantified via neutral red uptake assay and the results were confirmed by a morphology study. The expression level of p53 was analyzed using quantitative real-time PCR. RESULTS: The outcomes of the present study explicated that EPS-1 with IC 50 = 164 and 398 µg ml -1 exhibited an inhibitory influence on Huh7 and HepG2 cells growth after 48 h incubation time respectively. EPS-1 showed no influence on normal BNL cells. Furthermore, the molecular genetic analysis revealed that EPS-1 provoked significant upregulation in the expression level of the p53 gene in the treated Huh7 cell line more than that in HepG2, whereas no significant gene expression was noticed in BNL cells ( P = 0.006, 0.65 and 0.83), respectively. CONCLUSION: The antitumor activity displayed by this compound may be of interest for further studies of its structure-activity relationship. Before application in phase 1 of the clinical study, in-vivo studies would be needed to confirm the results obtained in the hope of finding more active and selective anticancer agents for drug development in the future.

A Selective Inhibitor of Ubiquitin-Specific Protease 4 Suppresses Colorectal Cancer Progression by Regulating ß-Catenin Signaling.

BACKGROUND/AIMS: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of ß-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities. METHODS: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of ß-catenin, as shown by immunoblotting, and it affected the target gene expression of ß-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model. RESULTS: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased ß-catenin stability and reduced expression of ß-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume. CONCLUSION: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.

Symptomatic relief of herpetic skin lesions utilizing an energy-based approach to healing.

Herpes simplex virus induced oral and genital ulcerating lesions will fluoresce brightly yellow and yellow-orange, respectively, if treated with a chlorinated solution of neutral red and exposed to ultraviolet-A light. An orange to red fluorescence is seen with similarly treated and illuminated Herpes zoster virus induced shingles; while treated human papillomavirus induced genital warts display more of a purplish fluorescence. Pain and discomfort commonly disappear soon after the treatment and all lesions undergo expedited healing that is readily observable within 24 h. The mechanism of healing is thought to involve an interaction between neutral red and alternative cellular energy pigments (ACE pigments) present within the viral lesions that enhances responsiveness to ultraviolet light energy. The healing effects are not restricted to the treated lesions and may involve transmission of a biological energy throughout the body. Beyond its obvious clinical and diagnostic utility, this model system may help usher in a new era of energy-based medicine.

KTP laser and neutral red phototherapy of human squamous cell carcinoma.

Neutral red (NR) is a cationic, nontoxic vital dye employed as a histologic stain for proliferating cells; it has been used clinically for photodynamic treatment of herpes simplex virus lesions. NR is selectively taken up and concentrated by mitotic cells, an important characteristic for more effective antineoplastic agents. In the present study, UCLA-SO-P3 human squamous carcinoma cells displayed minimal toxicity when incubated with up to 50 microg/ml NR in the absence of light. However, cells incubated with greater than 0.5 microg/ml NR followed by exposure to KTP laser light at 532 nm exhibited nearly 100% tumor cell death. The degree of cell toxicity was proportional to NR dose and laser light fluence. This study demonstrates that NR is an excellent cancer cell photosensitizer in vitro, and, after adding additional in vivo preclinical testing, may prove to be a useful agent in photodynamic destruction of head and neck tumors.

Interaction with hyperthermia of tetrachloroplatinum(II)(Nile blue)2 and tetrachloroplatinum(II)(neutral red)2 in EMT6 murine cells and the murine FSaIIC fibrosarcoma.

Complexes of the tetrachoroplatinum(II) dianion with positively charged nuclear dyes were prepared in an effort to produce agents which gain ready access into the nucleus and become very cytotoxic at clinically relevant hyperthermia temperatures. Pt(Nile blue)2 and Pt(neutral red)2 are complexes of tetrachloroplatinum(II) with two closely related p-quinonediamine dyes. Pt(Nile blue)2 and Pt(neutral red)2 were only moderately cytotoxic to exponentially growing normally oxygenated or hypoxic EMT6 cells in vitro at pH 7.40 and 37 degrees C. At pH 7.40 and 42 degrees C and especially at 43 degrees C, however, Pt(Nile blue)2 became far more cytotoxic. At pH 6.45 Pt(Nile blue)2 became more toxic toward hypoxic cells (cell kill of 3.5 logs at 500 microM, 42 degrees C for 1 h). Pt(neutral red)2 became much more cytotoxic at pH 6.45 and 42 degrees C or 43 degrees C compared to pH 7.4, and the cell kill observed was similar in both euoxic and hypoxic cells (3 logs at pH 6.45, 43 degrees C with only 100 microM). Tumor cell survival studies in the FSaIIC murine fibrosarcoma demonstrated that both drugs killed in a dose-dependent log-linear manner. Hyperthermia treatment (43 degrees C, 30 min) immediately after either drug resulted in a dose modifying effect. The tumor growth delay produced by Pt(Nile blue)2 (100 mg/kg) was 4.6 days and by Pt(neutral red)2 (100 mg/kg) was 3.8 days. Both drugs were markedly improved by hyperthermia (tumor growth delay 1.4 days for hyperthermia; tumor growth delay 10.9 days for Pt(Nile blue)2 and 8.0 days for Pt(neutral red)2. Intracellular platinum levels were approximately 200 times higher after exposure of EMT6 cells to 25 microM of Pt(Nile blue)2 or Pt(neutral red)2 for 1 h at 37 degrees C than after exposure to the same concentration of cis-diamminedichloroplatinum(II). Treatment of cells with the drugs at 42 degrees C (1 h) resulted in no change in platinum levels with cis-diamminedichloroplatinum(II), but with Pt(Nile blue)2 and Pt(neutral red)2 an increase of 2- to 3-fold was found. Since previous work has shown that both of these complexes are active radiosensitizing agents, these new drugs seem quite well suited for further development as antitumor agents for use against solid tumors alone and in conjunction with hyperthermia and/or radiation therapy.

Vulvar histology after neutral red photoinactivation of herpes simplex virus.

The use of photodynamic dye and light inactivation for the treatment of genital herpes simplex virus infections has been associated with the risk of potential oncogenesis. Sixteen patients treated with neutral red and fluorescent light for documented herpetic infections were studied at intervals ranging from 9 to 52 months following treatment. Four patients treated with other modalities were included in the study. Biopsies of the treated areas were obtained, and 3925 tissue sections were examined. Mild atypical epithelial changes were focally present in most specimens regardless of therapy. Histologically identifiable premalignant change could not be demonstrated.

Photodynamic inactivation in recurrent infections with herpes simplex virus.

Photodynamic inactivation with neutral red (a heterotricyclic dye) and light was evaluated in a placebo-controlled study of 170 episodes of recurrent infection with herpes simplex virus in 96 patients. The technique inactivated herpes simplex virus in vitro. However, no beneficial effect, either on the rate of resolution of herpetic lesions or on the interval to subsequent recurrences, was observed in treated patients. These negative results should not deter further investigation of photodynamic inactivation as a potential treatment for herpes simplex virus infections, but the clinical application of this form of therapy should be limited to properly controlled studies until its efficacy is proven.

[The fate of corneal infiltrations in cases of epidemic keratoconjunctivitis. A follow-up study over two and a half years (author's transl)]. / Das Schicksal der Hornhautinfiltrate bei Keratoconjunctivitis epidemica. Eine Verlaufsstudie über 2 1/2 Jahre

34 out of 72 patients (47.2%) with epidemic keratoconjunctivitis due to an infection with the adeno-virus type 8 still showed, on average, corneal opacities after 2.3 years. The gamma2-test proved the statistical significance of the correlation between the prevention of persistent corneal changes and the abstention from topical treatment with corticosteroids in the acute phase of the disease. Yet, steroids mitigate the subjective discomfort, delay the development of keratitis by several days, or even prevent its manifestation as long as corticosteroids are applied. However, in one third of the cases, the keratitis recurs as soon as topical treatment with corticosteroids is stopped. Recurrent attacks of keratitis may flare up over years and the visual acuity may decrease to 6/12 causing transient incapability of working.

Photodynamic inactivation in experimental herpetic keratitis.

The effect of photodynamic inactivation on experimental herpes simplex keratitis in rabbits was investigated using neutral red as a photosensitizing dye followed by exposure to light at 425nm. Combined dye application and light exposure early in the disease (two days following infection) reduced to a minimal extent the severity and duration of the acute epithelial infection. The effect on well-established keratitis (three days postinfection) was negligible as evaluated by clinical grading, viral recovery, and histopathological study. In initial experiments, it was found that the dye and light did not have any observable deleterious effect on intact corneas or cause any noticeable delay in healing of injured cornias. Further, when light or dye were utilized alone, neither changed the severity or duration of the keratitis. In vitro treatment of the virus with light and dye destroys its ability to produce experimental keratitis.