chlorophyll-a and Mouth-Neoplasms

The primary objective was to evaluate safety of 3-(1'-hexyloxyethyl)pyropheophorbide-a (HPPH) photodynamic therapy (HPPH-PDT) for dysplasia and early squamous cell carcinoma of the head and neck (HNSCC). Secondary objectives were the assessment of treatment response and reporters for an effective PDT reaction.. Patients with histologically proven oral dysplasia, carcinoma in situ, or early-stage HNSCC were enrolled in two sequentially conducted dose escalation studies with an expanded cohort at the highest dose level. These studies used an HPPH dose of 4 mg/m(2) and light doses from 50 to 140 J/cm(2). Pathologic tumor responses were assessed at 3 months. Clinical follow up range was 5 to 40 months. PDT induced cross-linking of STAT3 were assessed as potential indicators of PDT effective reaction.. Forty patients received HPPH-PDT. Common adverse events were pain and treatment site edema. Biopsy proven complete response rates were 46% for dysplasia and carcinoma in situ and 82% for squamous cell carcinomas (SCC) lesions at 140 J/cm(2). The responses in the carcinoma in situ/dysplasia cohort are not durable. The PDT-induced STAT3 cross-links is significantly higher (P = 0.0033) in SCC than in carcinoma in situ/dysplasia for all light doses.. HPPH-PDT is safe for the treatment of carcinoma in situ/dysplasia and early-stage cancer of the oral cavity. Early-stage oral HNSCC seems to respond better to HPPH-PDT in comparison with premalignant lesions. The degree of STAT3 cross-linking is a significant reporter to evaluate HPPH-PDT-mediated photoreaction.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Chlorophyll; Disease-Free Survival; Female; Humans; Male; Middle Aged; Mouth Neoplasms; Photochemotherapy; Photosensitizing Agents; STAT3 Transcription Factor; Tissue Distribution; Treatment Outcome

MicroRNAs (miRNAs) regulate key biological processes, and their aberrant expression has been related to cancer development. Photodynamic therapy (PDT) has emerged as one of the most promising modalities for cancer treatment. However, the application of PDT has been limited to superficially localized human cancerous and precancerous lesions. To increase the usefulness of both PDT and miRNAs in cancer therapy, this study investigated whether apoptosis-related miRNA expression is influenced by PDT in oral cancer and whether miRNAs can enhance PDT efficacy. To achieve this goal, we performed a miRNA array-based comparison of apoptosis-related miRNA expression patterns following PDT using pheophorbide a (Pa) as a photosensitizer. After Pa-PDT, 13.1% of the miRNAs were down-regulated, and 16.7% of the miRNAs were up-regulated. Representative miRNAs were selected according to expression difference: miR-9-5p, miR-32-5p, miR-143-3p, miR-145-5p, miR-192-5p, miR-193a-5p, miR-204-5p, miR-212-3p, miR-338-3p, and miR-451a. Among them, only miR-145-5p showed the consistent reduction repeatedly in all cell lines after Pa-PDT. Further, the combined treatment of a miR-145-5p mimic and Pa-PDT increased phototoxicity, reactive oxygen species generation, and apoptotic cell death, suggesting that miRNAs expression could be a useful marker for enhancing the therapeutic effect of Pa-PDT. This study will provide a promising strategy for introducing miRNA as cancer therapy.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Mouth Neoplasms; Oligonucleotide Array Sequence Analysis; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

Photodynamic therapy (PDT) is a therapeutic alternative for malignant tumors that uses a photosensitizer. Our group recently synthesized photosensitizer pheophorbide a (Pa) from chlorophyll-a. The present study investigated the therapeutic effect of PDT using intratumoral administration of the synthetic photosensitizer Pa in an in vivo murine oral squamous cell carcinoma (OSCC) animal model. Pa accumulation was measured using the fluorescence spectrum and imaging in living C3H mice. Intratumoral treatment of Pa-PDT (IT Pa-PDT) significantly inhibited the growth of transplanted OSCC cells. Histopathological examination of tumor tissues showed that PCNA expression was significantly decreased, while TUNEL-stained cells were markedly increased in the IT Pa-PDT group compared to controls. IT Pa-PDT-induced apoptosis was confirmed by immunoblot. Reduction of Bcl-2 and cleavage of caspase 3 and PARP were observed in IT Pa-PDT. These data demonstrate that IT Pa-PDT inhibited tumor cell proliferation and induced apoptosis, which is correlated with the anticancer activity of IT Pa-PDT. These potent antitumor activities of IT Pa-PDT were observed in both the immunohistochemistry and Western blot experiments. Our findings suggest the intratumoral therapeutic potential of Pa-PDT on OSCC. Additionally, demonstrated detection of Pa using a fluorescence spectroscopy system or molecular imaging system provides a means for simultaneous diagnosis and treatment of OSCC.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Dose-Response Relationship, Drug; Mice; Mice, Inbred C3H; Mouth Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents

Photodynamic therapy (PDT) is a highly localized and minimally invasive cancer treatment modality with many important advantages, but the lack of ideal photosensitizers (PSs) greatly restricts its clinical utility. To develop new PSs with highly efficient singlet oxygen production and high tumor-localizing ability to reduce damage to healthy adjacent tissues, we conjugated folic acid (FA) with pyropheophorbide a (Pyro), a potent PS with a very high singlet oxygen quantum yield and a high extinction coefficient. In the present work, we describe the synthesis and PDT evaluation of this FA-Pyro conjugate both in vitro and in vivo. This conjugation increased the accumulation of Pyro inside the tumors and improved the efficiency of PDT, resulting in eradication of subcutaneous xenograft KB (human mouth epidermal carcinoma) tumors after only 1 or 2 applications of external near infrared light irradiation. This outstanding PDT outcome in a tumor-bearing mouse model and the simple synthesis of the conjugate should have very good practical potential for clinical application.

Topics: Animals; Cell Line, Tumor; Cell Survival; Chlorophyll; Female; Folic Acid; Humans; Mice, Inbred BALB C; Mouth Neoplasms; Photochemotherapy; Photosensitizing Agents

Photodynamic therapy (PDT) with photosensitizer is one of the promising modalities for cancer treatment. For clinical use of PDT, screening process should be preceded to enhance sensitivity to PDT. Thus, we investigated a molecular biomarker to determine the sensitivity to pheophorbide a (Pa)-PDT in immortalized human oral keratinocytes (IHOK) and oral squamous cell carcinoma (OSCC) cell lines. Two IHOK and several OSCC cell lines were used. After Pa-PDT, cell viability was reduced by more than 50%, and reactive oxygen species were generated in IHOK and OSCC cell lines. Additionally, apoptosis occurred in PDT-treated cells. IHOK(S) and IHOK(P), the two IHOK cell lines derived from the same source, showed a difference in cytotoxicity after Pa-PDT. To explain this difference in cytotoxicity, we looked at the expression of Wnt signaling-related genes in these two cell lines, for the morphology of IHOK(S) which was spindle like and elongated and distinct from IHOK(P) and the parent cell. Among the relevant genes, runt-related transcription factor 3 (RUNX3), an apoptosis-related gene, was selected as a potential marker that confers sensitivity to PDT. We found that the cytotoxicity by Pa-PDT was proportional to RUNX3 expression in OSCC cell lines. Additionally, knockdown of RUNX3 expression reduced cytotoxicity by Pa-PDT, suggesting that RUNX3 might be a biomarker to determine sensitivity to Pa-PDT. This was the first study to find a new target molecule that enhances Pa-PDT effects in IHOK and OSCC cell lines. Hence, the development of a PDT-dependent biomarker could provide a novel approach to improve the effects of PDT on oral precancerous and cancerous lesions.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Chlorophyll; Core Binding Factor Alpha 3 Subunit; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Mouth Neoplasms; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Signal Transduction; Wnt Proteins

Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.5,6,24,48]  h incubation time. We observed a tissue-dependent maximum of 35% variation in the total correction factor over the visible wavelength range. Significant differences in spectral shape over time between sensitizers were observed. Although the wavelength position of the fluorescence intensity maximum for ce6 shifted to the red, Bremachlorin showed a blue shift. Furthermore, the Bremachlorin peak appeared to be broader than the ce6 fluorescence peak. Intrinsic fluorescence intensity, which can be related to photosensitizer concentration, was decreasing for all time points but showed significantly more Bremachlorin present compared to ce6 at long incubation times. Results from this study can be used to define an optimal treatment protocol for Bremachlorin-based photodynamic therapy.

Topics: Animals; Carcinoma, Squamous Cell; Chlorophyll; Chlorophyllides; Female; Fluorescence; Green Fluorescent Proteins; Liver; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Mouth Neoplasms; Normal Distribution; Optics and Photonics; Photochemotherapy; Photosensitizing Agents; Porphyrins; Skin; Spectrometry, Fluorescence; Spectrophotometry; Tongue

Pheophorbide a (Pa) is a chlorine-based photosensitizer derived from an ethnopharmacological herb, and our group recently synthesized Pa by the removal of a magnesium ion and a phytyl group from chlorophyll-a. In this study, the effect of photodynamic therapy (PDT) with synthesized Pa was examined in a human oral squamous cell carcinoma (OSCC) cells.. Cells were treated with PDT with Pa, and reactive oxygen species (ROS) and mitochondrial membrane potential [ΔΨ (m)] were examined. Apoptosis was measured using annexin V staining and immunoblot. Autophagy was characterized by the increase in LC3B-II and the formation of autophagosome and acidic vesicular organelles (AVOs).. Pa-PDT inhibited the proliferation of OSCC cells in a dose-dependent manner. Pa-PDT increased the number of apoptotic cells by inactivating ERK pathway. Pa-PDT also induced autophagy in OSCC cells evidenced by the increased levels of LC3 type II expression and the accumulation of AVOs. The inhibition of autophagy enhanced Pa-PDT-mediated cytotoxicity through an increase in necrosis.. These results suggest that synthesized Pa-PDT exerts anti-tumor effects by inducing apoptosis and autophagy and provide novel evidence that Pa-PDT induces autophagy, and autophagy inhibition enhances Pa-PDT-mediated necrosis in OSCC cells.

Topics: Apoptosis; Autophagy; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Humans; MAP Kinase Signaling System; Microtubule-Associated Proteins; Mouth Neoplasms; Necrosis; Phagosomes; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

Photodynamic therapy (PDT) with several photosensitizers is a promising modality for the treatment of cancer. In this study, the therapeutic effect of PDT using the synthetic photosensitizer pheophorbide a (Pa-PDT) was examined in AT-84 murine oral squamous cell carcinoma (OSCC) cells. The MTT assay revealed that Pa-PDT induced cell growth inhibition in a dose- and time-dependent manner. Pa-PDT treatment significantly induced intracellular ROS generation, which is critical for cell death induced by Pa-PDT. Cell cycle analysis showed the increased sub-G1 proportion of cells in Pa-PDT-treated cells. Induction of apoptotic cell death was confirmed by DAPI staining and the reduction of mitochondrial membrane potential (ΔΨm) on Pa-PDT-treated cells. The changes in apoptosis-related molecules were next examined using western blotting. Cytochrome c release and cleavage of caspase-3 and PAPR were observed in AT-84 cells, whereas Bcl-2 protein levels were decreased. To determine the therapeutic effect of Pa-PDT in vivo, a murine OSCC animal model was used. Treatment of mice with Pa-PDT significantly inhibited tumor growth, especially PDT with Pa intravenous administration (i.v. Pa-PDT), and increased proliferative cell nuclear antigen (PCNA) levels and TUNEL-stained apoptotic cells compared to vehicle-treated controls. The data demonstrate that the in vitro effects of Pa-PDT on the inhibition of tumor cell proliferation and induction of apoptosis correlate to the anticancer activity of Pa-PDT in vivo. Our findings suggest the therapeutic potential of Pa-PDT in OSCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Cytochromes c; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C3H; Mitochondria; Mouth Neoplasms; Photochemotherapy; Photosensitizing Agents; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species