Synergistic Anti-Tumor Effects of Combination of Photodynamic Therapy and Arsenic Compound in Cervical Cancer Cells: In Vivo and In Vitro Studies

The effects of As4O6 as adjuvant on photodynamic therapy (PDT) were studied. As4O6 is considered to have anticancer activity via several biological actions, such as free radical production and inhibition of VEGF expression. PDT or As4O6 significantly inhibited TC-1 cell proliferation in a dose-dependent manner (P<0.05) by MTT assay. The anti-proliferative effect of the combination treatment was significantly higher than in TC-1 cells treated with either photodynamic therapy or As4O6 alone (62.4 and 52.5% decrease compared to vehicle-only treated TC-1 cells, respectively, P<0.05). In addition, cell proliferation in combination of photodynamic therapy and As4O6 treatment significantly decreased by 77.4% (P<0.05). Cell survival pathway (Naip1, Tert and Aip1) and p53-dependent pathway (Bax, p21Cip1, Fas, Gadd45, IGFBP-3 and Mdm-2) were markedly increased by combination treatment of photodynamic therapy and As4O6. In addition, the immune response in the NEAT pathway (Ly-12, CD178 and IL-2) was also modulated after combination treatment, suggesting improved antitumor effects by controlling unwanted growth-stimulatory pathways. The combination effect apparently reflected concordance with in vitro data, in restricting tumor growth in vivo and in relation to some common signaling pathways to those observed in vitro. These findings suggest the benefit of combinatory treatment with photodynamic therapy and As4O6 for inhibition of cervical cancer cell growth

Role of ER Stress Response in Photodynamic Therapy: ROS Generated in Different Subcellular Compartments Trigger Diverse Cell Death Pathways

We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG) chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS). Para derivatives pTPP(EG)4 and pTPPF(EG)4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG)4 localized in the endoplasmic reticulum (ER) induced dramatic changes in Ca2+ homeostasis manifested by Ca2+ rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway

КЛИНИЧЕСКИЙ ОПЫТ ПРИМЕНЕНИЯ ВНУТРИПРОСВЕТНОЙ БРАХИТЕРАПИИ НА АППАРАТЕ «MULTISOURCE» У БОЛЬНЫХ РАКОМ ЛЕГКИХ И ТРАХЕИ

Treatment of primary and recurrent cancer of the trachea and main bronchi is a complex clinical problem. The article presents own experience of contact intraluminal radiation therapy in patients of this group. Efficacy and toxicity of this treatment was evaluated.Лечение первичного и рецидивного рака трахеи и главных бронхов является сложной клинической задачей. В статье представлен собственный опыт применения внутрипросветной контактной лучевой терапии улиц данной группы. Дана оценка эффективности и токсичности данного вида лечения

Effect of PERK siRNA on the demise of PDT-treated cells.

<p>PERK deficiency protects against mPTT(EG)4-mediated cell death. (<b>A</b>) Western blot analysis of PERK, ATF4 and CHOP in 4T1 cells after PERK siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. As a positive control were used cells treated with 1 µM thapsigargin (TG) for 6 h. (<b>B</b>) Viability of cells transfected with PERK siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 7–14). **P<0.01 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells. (<b>C</b>) Immunostaining of CHOP after EG-porphyrin derivative-mediated PDT in PERK knockdown cells. Cells were fixed 2 h post PDT, immunostained and observed with a fluorescence microscope. The left panel includes untransfected cells, the middle panel cells transfected with control siRNA and the right panel cells transfected with PERK siRNA. (<b>D</b>) Cell viability of MEF-wt and MEF-KO evaluated 24 h post-EG-porphyrin-PDT (350 nM mTPP(EG)4, 3.5 µM pTPP(EG)4, 700 nM pTPPF(EG)4). Verification of the PERK−/− phenotype in MEFs-KO cells by Western blot. The percentage of apoptotic cells was expressed as the mean ± SD (n = 6). ***P<0.001 represents statistical differences between PDT-treated MEF-wt cells vs. PDT-treated MEF-KO cells. (<b>E</b>) Fluorescence microscopy demonstrates similar cellular uptake of EG-porphyrin derivatives in MEFs-wt and MEF-KO [800 nM mTPP(EG)4, 5 µM pTPP(EG)4, and 2 µM pTPPF(EG)4].</p

The role of Ca²⁺ in photoinduced apoptosis by EG-porphyrin derivatives.

<p>(<b>A</b>) Intracellular calcium levels in HL60 and 4T1 cells. The effect EG-porphyrin derivatives on cytosolic Ca²⁺ levels was monitored by flow cytometry of laser-illuminated cells containing fluorescent Ca²⁺ indicator Fluo-4-AM (4 µM) (Molecular Probes). Cells treated in the same way but without porphyrin were used as controls. (<b>B</b>) Effect of BAPTA and L-histidine on the intracellular Ca²⁺ level. HL60 cells were loaded with EG-porphyrin derivatives for 16 h, washed and then preincubated for 1 h with ROS scavenger L-histidine (20 mM) or 2 h with calcium chelator BAPTA-AM (5 µM). The cytosolic Ca²⁺ level was monitored by flow cytometry using fluorescent Ca²⁺ indicator Fluo-4-AM (4 µM). (<b>C</b>) Effect of BAPTA-AM preincubation on photoinduced apoptosis. Cells loaded with EG-porphyrin derivatives were pre-incubated with the membrane-permeable intracellular Ca²⁺ chelator BAPTA-AM (5 µM HL60, 10 µM 4T1) for 2 h to inactivate released Ca²⁺ and then irradiated with 2.5 Jcm⁻² of light at 500±20 nm. This corresponds to an LD₇₀ PDT dose. Cell viability was determined 24 h post-photodynamic therapy by the trypan blue exclusion method. The percentage of dead cells was expressed as mean ± SD (n = 4). **P<0.01, ***P<0.001 represents statistical differences between PDT-treated cells vs. PDT-treated cells in the presence of BAPTA-AM.</p

Effect of Capn4 siRNA on the demise of PDT-treated cells.

<p>(<b>A</b>) Western blot analysis of capn4/capns1 and fodrin in 4T1 cells analyzed on the 3^rd day after Capn4 siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. (<b>B</b>) Viability of cells transfected with Capn4 siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.05 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells.</p