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Summary Background 5-Aminolevulinic acid (ALA) is a natural heme precursor metabolized into protoporphyrin IX (PpIX). PpIX preferentially accumulates in tumor cells resulting in the formation of singlet oxygen upon exposure to visible light. Doxil®, an active agent against breast and ovarian cancer, is a nano-formulation of doxorubicin. This study aimed to investigate in vitro synergistic cytotoxic effect of low doses of combined chemotherapy and ALA/PDT to human breast adenocarcinoma cells (MCF-7) compared to high doses of each individual therapy. Methods MCF-7 cells were pretreated with Doxil® (48 h) followed by ALA/PDT (4 h). The cell viability was evaluated by trypan blue assay and PpIX production was measured spectrofluorometrically. Alkaline phosphatase was determined as a marker for cellular differentiation. Apoptosis and necrosis were evaluated by fluorescence stains. The apoptosis cell death pathways were investigated: detection of mitochondrial membrane potential (ΔΨm) and percent of DNA fragmentation, malondialdehyde, histone deacetylase (HDAC) activity, caspase-3 and death receptors (DR4 and DR5). Vascular endothelial growth factor (VEGF) was determined by ELISA, as an angiogenic mediator. Results There was a higher reduction in cell viability in Doxil® + ALA/PDT-treated cells compared with their individual effect. The combined therapy showed enhanced apoptosis with a significant increase in the loss of ΔΨm, DNA fragmentation %, caspase-3, DR4, DR5 and lipid peroxides and inhibited HDAC. Pretreatment with Doxil® resulted in a twofold increase in the intracellular PpIX, by increasing the PDT killing of MCF-7 cells. Conclusion The combined therapy using 50% of IC50 of ALA/PDT and Doxil® possessed a synergistic apoptotic effect on MCF-7 cells compared to 100% of IC50 of each therapy through enhancing both intrinsic and extrinsic apoptotic pathways, thus may minimize side effects of Doxil® and ALA.

Summary Background Photodynamic therapy (PDT) is used for the treatment of many types of predominantly epithelial cancers. Photosensitizer is taken up by fast growing tumor cells more actively than by other body cells and is activated by light, generating reactive oxygen species that cause cell death by necrosis or apoptosis. This study aimed to evaluate the efficacy of PDT with indocyanine green (ICG) through the investigation of TP53, HER-2 and TOP2A genes signals as breast cancer gene markers by interphase fluorescence in situ hybridization (nuc-FISH). Methods The photosynthetizer ICG (200 μM) was applied to breast cancer cell line MCF-7 cells (adenocarcinoma) in combination with laser irradiation (807 nm) exposure for 20 min and then incubated for 12, 24 and 48 h. Cell viability was evaluated using trypan blue. The signals for nuc-FISH was investigated and counted for probes specific for the genes TP53 (17p13), HER-2 (17q11.2-q12), and TOP2A (17q21-q22), and BAC-probes RP11-746M1 in 17p11.2 and RP11-403E9 in 17q11.2. Results The cell viability of MCF-7 did not reduced significantly when the cells were treated with ICG (200 μM) or exposed to laser irradiation for 20 min followed by incubation for 24 h. ICG/PDT treatment with laser irradiation exposure for 20 min reduced the cell viability after incubating cells for 12, 24 and 48 h highly significantly in a time dependent manner. For nuc-FISH analysis, TP53, HER-2, TOP2A, RP11-746M1 and RP11-403E9 signals did not reduce or increase in a significant manner when the cells were treated with ICG or exposed to laser irradiation for 20 min then incubated for 24 h. PDT enhanced amplification of TP53 signals from nuc ish 17p13(TP53×2) to nuc ish 17p13(TP53×3) or nuc ish 17p13(TP53×4). However, the signals of HER-2 gene, TOP2A gene and BAC probes were reduced highly significantly when MCF-7 cells were treated with PDT with all time intervals. Conclusion ICG/PDT and laser induced cytotoxic effect in MCF-7 cells. Also, PDT enhanced TP53 gene amplification, and reduced HER-2, TOP2A, and BAC probes RP11-746M1 and RP11-403E9 signals. Therefore ICG/PDT can be used for breast cancer treatment. It has the potential to induce apoptotic effect and reduce HER-2 and TOP2A genes propagation. Further in vivo studies are needed to evaluate ICG/PDT as a promising therapeutic approach for breast cancer.

Summary Background Indocyanine green (ICG) is a promising water-soluble photosensitizer for photodynamic therapy (PDT) of tumors. It was reported to have promising phototoxic effect on different cell lines. This study aimed to evaluate the efficacy of ICG as an efficient PS agent for skin cancer induced in mice. Methods Skin squamous cell carcinoma was induced in female CD-1 mice by 7,12-dimethylbenzanthracene and 12- O -tetradecanoyl-phorbol-13-acetate followed by an ICG/PDT treatment. The laser irradiation for PDT was adjusted to cover the whole body of the mice to make sure that the treatment protocol will be delivered to multiple tumors. Results The treatment of skin cancer by ICG/PDT using intravenously injected ICG initiated tumor cell death and significantly decreased cell proliferation as indicated by the reduction in proliferating cell nuclear antigen positivity. A significant reduction in the inflammatory mediators; tumor necrosis factor-α, nitric oxide and 5-lipoxygenase was reported, however the level of cyclooxygenase-2 (COX-2) was significantly elevated after ICG/PDT treatment. Conclusion The proposed ICG/PDT treatment modality showed a significant anti-tumor and anti-inflammatory activity against skin cancer accompanied with COX-2 elevation.

Summary Background Photodynamic therapy (PDT) is a therapeutic modality involving the use of a photosensitizer agent activated by light of appropriate wavelength to selectively destroy tumor cells. Indocyanine green (ICG) is a promising photosensitive agent for PDT of tumor cells. The main disadvantage of using ICG in PDT is the instability of ICG in aqueous solutions. Encapsulating ICG dye in a biocompatible matrix based on PEBBLE technology showed an improvement of aqueous stability comparing with free ICG dye. The main objective of this study is to investigate the photodynamic effect of ICG-ormosil PEBBLEs on two different cell lines: human breast adenocarcinoma cells (MCF-7) and hepatocellular carcinoma cells (HepG2). Methods ICG-embedded ormosil PEBBLEs were synthesized based on a sol–gel process, and characterized by transmission electron microscopy and other fluorescence tests. The cell viability was evaluated by MTT and trypan blue assays. Apoptosis, necrosis, and DNA damage (comet assay), were evaluated by fluorescence microscopic tests. Results The results declared that ICG-ormosil PEBBLEs and free ICG both have the same cytotoxic and phototoxic effect on MCF-7 and HepG2 cell lines, where the apoptotic mode of cell death is preferentially occurred in case of PDT using ICG-ormosil PEBBLEs. Both ICG and ICG-ormosil PEBBLEs induced DNA damage after laser exposure. These results would suggest that entrapping ICG in Polymeric nanoparticles forming ICG-ormosil PEBBLEs improve the aqueous stability of the photosensitizer and in the same time retain its photodynamic activity, suggesting that it is preferred to use ICG-ormosil PEBBLEs instead of free ICG dye.