Apoptosis induced by hematoporphyrin monomethyl ether combined with He-Ne laser irradiation in vitro on canine breast cancer cells
ABSTRACT Hematoporphyrin monomethyl ether (HMME) is a novel and promising porphyrin-related photosensitizer for photodynamic therapy (PDT). The aim of this study was to investigate HMME-induced apoptosis in CHMm cells, a canine breast cancer cell line. CHMm cells were treated with HMME and a He-Ne laser at a wavelength of 632.8 nm. Cell viability was determined using the trypan blue exclusion assay. Apoptosis was analyzed using Hoechst 33258, AO/EB, Annexin V/PI staining and single-cell gel electrophoresis (comet assay). Apoptotic morphology was further confirmed by Giemsa staining and transmission electron microscopy. Rates of apoptosis increased following PDT-HMME treatment in a time-dependent manner. Taken together, these results demonstrated that apoptosis plays a major role in PDT-HMME-induced reduction in the viability of CHMm cells.
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ABSTRACT: It is well established that both hematoporphyrin monomethyl ether-sonodynamic therapy (HMME-SDT) and doxorubicin (DOX) can induce cell apoptosis but each alone has its own limitations. To date, the combined effects of HMME-SDT and DOX on inducing cell apoptosis are little known and the mechanism for the combined effects remains poorly understood. In the present study, we reported the synergistic effects of HMME-SDT and DOX on inhibiting the proliferation of human cholangiocarcinoma QBC939 cells and investigated the mechanism of this synergy. The data from MTT assay, flow cytometer, Hoechst staining and cell arrest analysis showed that the combination of HMME-SDT and DOX exhibited higher inhibiting effects on proliferation of QBC939 cells than the sole application of HMME-SDT or DOX. In addition, the synergistic effects were shown to result from the DNA damage as demonstrated by single cell gel electrophoresis and DNA fragmentation. Furthermore, the expression of p53, Fas, Bax and activated caspase-3 protein was significantly upregulated in cells treated with HMME-SDT and DOX, whereas Bcl-2 protein was downregulated. Taken together, our data suggested that the application of HMME-SDT combined with DOX had better inhibiting effects on QBC939 cells and the effects were caused mainly by DNA damage.Ultrasound in medicine & biology 01/2013; 39(1):146-60. DOI:10.1016/j.ultrasmedbio.2012.08.017 · 2.10 Impact Factor
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ABSTRACT: Abstract Purpose: The present study aims to investigate apoptosis of U937 cells induced by hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy (SDT). Materials: HMME concentration was kept constant at 10 μg/mL. Tumor cells suspended in serum-free RPM1640 were exposed to ultrasound at 1.1 MHz for up to 60 seconds with an intensity of 1 W/cm(2) in the presence and absence of HMME. The viability of cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) test. Apoptosis was analyzed using a flow cytometer with Annexin V-PE/7-ADD staining as well as fluorescence microscopy with 4'-6-diamidino-2-phenylindole (DAPI) staining. The DNA damage of U937 cells, intracellular reactive oxygen species (ROS), and mitochondria membrane potential (MMP) were also analyzed by a flow cytometer after exposures. Western blotting and reverse transcriptase-polymerase chain reaction were used to analyze the protein and mRNA expression level of caspase-3 and poly(ADP-ribose) polymerase (PARP). Results: Fluorescent imaging revealed that HMME mainly localized in the mitochondria. MTT assay showed 55.6% of cell survival at 4 hours post-SDT. Flow cytometric analysis displayed a significant increase in the early- and late-apoptotic cell populations (35.6%) of U937 cells by HMME-mediated SDT. Compared with the control, ultrasound-alone, and HMME-alone groups, the intracellular ROS and the MMP loss were greatly increased in the combined SDT group. Obvious nuclear condensation was also found with DAPI staining, and the DNA fragment increased to 33.9% at 2 hours post-SDT treatment. Immunofluorescent staining indicated obvious Bax translocation after SDT. Western blot showed visible enhancement of caspase-3 and PARP cleavage. In addition, caspase-3 and PARP mRNA expression of U937 cells increased remarkably after SDT treatment. Conclusions: The findings demonstrated that HMME-mediated sonodynamic action (HMME-SDT) significantly induced apoptosis of U937 cells, suggesting that HMME may be a good sonosensitizer, and HMME-SDT might be a potential therapeutic strategy for cancer treatment.Cancer Biotherapy & Radiopharmaceuticals 03/2013; 28(3). DOI:10.1089/cbr.2012.1190 · 1.38 Impact Factor
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ABSTRACT: We investigated the antitumor effect and mechanism of hematoporphyrin monomethyl ether-mediated photodynamic therapy (HMME-PDT) in sarcomas. Intracellular uptake of HMME by osteosarcoma cells (LM8 and K7) was time- and dose-dependent, while this was not observed for myoblast cells (C2C12) and fibroblast cells (NIH/3T3). HMME-PDT markedly inhibited the proliferation of sarcoma cell lines (LM8, MG63, Saos-2, SW1353, TC71, and RD) (P<0.05), and the killing effect was improved with increased HMME concentration and energy intensity. Flow cytometry analysis revealed that LM8, MG63, and Saos-2 cells underwent apoptosis after treatment with HMME-PDT. Additionally, apoptosis was induced after HMME-PDT in a three-dimensional culture of osteosarcoma cells. Hoechst 33342 staining confirmed apoptosis. Cell death caused by PDT was rescued by an irreversible inhibitor (Z-VAD-FMK) of caspase. However, cell viability was not markedly decreased compared with the HMME-PDT group. Expression levels of caspase-1, caspase-3, caspase-6, caspase-9, and poly (ADP-ribose) polymerase (PARP) proteins were markedly up-regulated in the treatment groups and increased with HMME concentration as determined by western blot analysis. In vivo, tumor volume markedly decreased at 7-16 days post-PDT. Hematoxylin and eosin staining revealed widespread necrotic and infiltrative inflammatory cells in the HMME-PDT group. Immunohistochemistry analysis also showed that caspase-1, caspase-3, caspase-6, caspase-9, and PARP proteins were significantly increased in the HMME-PDT group. These results indicate that HMME-PDT has a potent killing effect on osteosarcoma cells in vitro and significantly inhibits tumor growth in vivo, which is associated with the caspase-dependent pathway.PLoS ONE 10/2013; 8(10):e77727. DOI:10.1371/journal.pone.0077727 · 3.53 Impact Factor