Figure 1.
Uptake and cytotoxicity of photofrin in human glioblastoma cells.
Human glioblastoma A172, U118MG, U87MG, SNB19, LN18, and T98G cells were incubated with 1 µg/ml photofrin. (a) Uptake of photofrin was time-dependent. (b) Photocytotoxic effect of photofrin was 670 nm light dose-dependent. (c) Photocytotoxic effect was photofrin dose-dependent. Data are presented as mean ± SEM of three independent experiments.
Figure 2.
Determination of induction of morphological and biochemical features of apoptosis in human glioblastoma cells following photofrin based PDT.
Treatments: control (CTL), 10, 20, and 50 µg/ml photofrin incubation for 4 h followed by irradiation with 670 nm light dose of 1 J/cm2. (a) In situ Wright staining to examine morphological features of apoptosis. (b) Annexin V-FITC/PI double staining and flow cytometric analysis of apoptotic populations after the treatments. Photofrin based PDT induced significant population of cells in A4 area, indicating induction of a biochemical feature of apoptotic death. (c) Determination of percentages of apoptosis based on biochemical feature revealed by Annexin V-FITC staining. Significant difference between CTL and a treatment was indicated by *P<0.05 or **P<0.01.
Figure 3.
Changes in capability of cell invasion and alterations in expression of angiogenic, invasive, and survival factors in glioblastoma cells after photofrin based PDT.
Treatments: control (CTL), 10, 20, and 50 µg/ml photofrin incubation for 4 h followed by irradiation with 670 nm light dose of 1 J/cm2. (a) Representative Matrigel invasion assay (48 h) using U87MG and U118MG cells. A significant reduction in the number of invaded cells indicated the decrease in invasive capability of the cells after dose-dependent photofrin based PDT. (b) Quantitative evaluation of Matrigel invasion. Data indicate mean ± SEM of 10 randomly selected microscopic fields from 3 independent wells. Significant difference between CTL and a treatment was indicated by *P<0.05 or **P<0.01. (c) Western blotting using the primary IgG antibodies against VEGF, b-FGF, EGFR, MMP-2, MMP-9, p-Akt, NF-κB, and PTEN, and β-actin (loading control).
Figure 4.
Association of network formation ability of HME cells with VEGF expression in gliblastoma U87MG or U118MG cells in co-cultures.
U87MG and U118MG cells were grown separately on chamber slides and treated with different doses of photofrin. Treatments: control (CTL), 10, 20, and 50 µg/ml photofrin incubation for 4 h followed by irradiation with 670 nm light dose of 1 J/cm2. After 24 h, HME cells were co-cultured with glioblastoma cells. Quantitative data are shown as means ± SEM of six independent experiments in each group. Significant difference between CTL and a treatment was indicated by *P<0.05 or **P<0.01. (a) Effect of photofrin based PDT on network formation ability of HME cells in co-cultures. The co-cultures were terminated at 72 h and immunohistochemically stained for expression of the von Willebrand factor VIII in HME cells. Then, in vitro networks were quantified. (b) Effect of photofrin based PDT on VEGF expression in glioblastoma cells in co-cultures. Following the same treatments and incubations as described above, another set of same network formation experiments were conducted for in situ immunofluorescence microscopic studies using the FITC conjugated anti-VEGF antibody to determine the levels of VEGF expression in glioblastoma cells in co-cultures.
Figure 5.
Alkaline comet assay and agarose gel electrophoresis to examine DNA fragmentation patterns in U87MG and U118MG cells after photofrin based PDT.
(a) Photomicrographs showing the DNA fragmentation patterns in U87MG cells. Two U87MG cells, one control (CTL) and another cell with highly damaged DNA due to photofrin based PDT, are shown in alkaline comet assay. Also, cells were treated with 50 µg/ml photofrin and irradiated with 670 nm light (1 J/cm2) and incubated for 3 h before isolation of total genomic DNA for DNA laddering assay. The CTL showed intact DNA whereas DNA ladder appeared due to photofrin based PDT. (b) Photomicrographs showing the DNA fragmentation patterns in U118MG cells. Two U118MG cells, one CTL and another cell with highly damaged DNA due to photofrin based PDT, are shown in alkaline comet assay. Also, cells were treated with 50 µg/ml photofrin and irradiated with 670 nm light (1 J/cm2) and incubated for 3 h before isolation of total genomic DNA for DNA laddering assay. The CTL showed intact DNA whereas DNA ladder appeared due to photofrin based PDT.
Figure 6.
Augmentation of efficacy of photofrin based PDT by miR-99a overexpression for induction of apoptosis in U87MG and U118MG cells.
Cells were seeded into 6-well plates at 5×105 cells per well in triplicate. After 24 h, cells were treated with 50 µg/ml photofrin and irradiated with 670 nm light of 1 J/cm2. Following 4 h incubation, cells were transfected with 50 nM pre-miR-99a mimic and incubated for another 24 h. (a) Cells were collected for estimation of apoptosis by Annexin V-FITC/PI double staining and flow cytometry. (b) Percentages of apoptotic cells from three independent experiments were shown in bar diagrams. Significant difference between control (CTL) and a treatment was indicated by *P<0.05 or **P<0.01. (c) Representative Western blots (n ≥3) showed expression of 97 kDa FGFR3, 185 kDa PI3K, 62 kDa Akt, 53 kDa p53, 35 kDa caspase-9 (active), 20 kDa caspase-3 (active), and 42 kD β-actin.
Figure 7.
Real-time qRT-PCR analyses of miR-99a expression in U87MG and U118MG cells after photofrin based PDT and miR transfection.
Cells were seeded, incubated, treated with photofrin, and irradiated with 670 nm light dose of 1 J/cm2. After 4 h incubation, cells were transfected with 50 nM anti-miR-99a mimic or miR-99a mimic and incubated for another 24 h. Total RNA was extracted and cDNA was synthesized using gene specific primers, and real-time qRT-PCR analysis was performed for relative expression of miR-99a after normalizing with expression of U6 RNA (control) in glioblastoma U87MG and U118MG cells. Significant difference between untreated control (CTL) and photofrin based PDT or miR-99a transfection was indicated by *P<0.05 or **P<0.01. Significant difference between a single therapy and combination therapy was indicated by #P<0.05.
Figure 8.
Regression of U87MG and U118MG tumors in nude mice and histopathological changes in tumor sections.
(a) Nude mice with U87MG and U118MG xenografts, (b) representative tumors removed surgically, (c) determination of tumor weight, and (d) evaluation of histopathological changes after the treatments. Mice with xenografts were treated for 11 days. Treatments: control (CTL) did not receive any treatment but tumor bearing mice (10th day after tumor implantation) were injected with photofrin (10 mg/kg) by tail vein, and 24 h later, 670 nm light was delivered to the tumor with fluencies of 100 J/cm2 at a fluency rate of 50 mW/cm2. We used 100 J/cm2 to expose most of the tumor cells to radiation (32). Then, the mixture of miR-99a mimic (50 µg) and 0.05% atelocollagen in 200 µl was injected (via tail vein) into each mouse on 14th, 17th, and 20th days. All animals were sacrificed on 21st day. We used 6 animals per group. Significant difference between CTL group and a treatment group was indicated by *P<0.05 or **P<0.01.
Figure 9.
Changes in expression of survival and apoptotic proteins in U87MG and U118MG xenografts.
Mice with xenografts were treated for 11 days. Treatments: control (CTL) did not receive any treatment but tumor bearing mice (10th day after tumor implantation) were injected with photofrin (10 mg/kg) by tail vein, and 24 h later, 670 nm light was delivered to the tumor with fluencies of 100 J/cm2 at a fluency rate of 50 mW/cm2. We used 100 J/cm2 to expose most of the tumor cells to radiation (32). Then, the mixture of miR-99a mimic (50 µg) and 0.05% atelocollagen in 200 µl was injected (via tail vein) into each mouse on 14th, 17th, and 20th days. All animals were sacrificed on 21st day. Representative Western blots (n≥3) showed expression of 97 kDa FGFR-3, 185 kDa PI3K, 62 kDa Akt, 53 kDa p53, 35 kDa caspase-9 (active), 20 kDa caspase-3 (active), and 42 kD β-actin.