Investigation of the intramembrane distribution of chlorin e6 derivatives by using fluorescence quenching

Belarusian State University, 4, F. Skorina Ave., Minsk, 220050, Belarus. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 62, No. 2, pp. 179–187, March–April, 1995     

A comparative study of the processes of generation of singlet oxygen upon irradiation of aqueous preparations on the basis of chlorin e6 and coproporphyrin III

The photosensitizing ability of an agent based on chlorin e6 (Photoditazin), which is used for photodynamic diagnosis and therapy, is compared with that of a new preparation on the basis of coproporphyrin III in the environment of a phosphate buffer and a simulated biological environment (albumin solution). The efficiency of singlet-oxygen production was estimated by EPR spectroscopy and spectroscopy in the UV and visible ranges with the use of “chemical traps” of singlet oxygen. By irradiating drugs with LED emission centered at λ_max = 520 nm, we determined the quantum yield of singlet-oxygen production in a buffer solution; the obtained values are 0.60 and 0.37 for chlorine and coproporphyrin, respectively. The steady-state concentration of singlet oxygen upon irradiation of solutions of the studied photosensitizers with concentrations of 12–43 μМ and the density of radiation power within the 6–96 W/cm² region was found to be in the region of 10¹⁰–10¹¹ molecules/cm³. It is shown that the introduction into the solution of egg albumin (0.1%) reduces the sensitizing properties of the two drugs by two to three times, while the efficiencies of the preparations with respect to singlet-oxygen production become almost identical (0.19 and 0.17).     

Anti-metastatic and pro-apoptotic effects elicited by combination photodynamic therapy with sonodynamic therapy on breast cancer both in vitro and in vivo

Sono-Photodynamic therapy (SPDT), a new modality for cancer treatment, is aimed at enhancing anticancer effects by the combination of sonodynamic therapy (SDT) and photodynamic therapy (PDT). In this study, we investigated the antitumor effect and possible mechanisms of Chlorin e6 (Ce6) mediated SPDT (Ce6-SPDT) on breast cancer both in vitro and in vivo. MTT assay revealed that the combined therapy markedly enhanced cell viability loss of breast cancer cell lines (MDA-MB-231, MCF-7 and 4T1) compared with SDT and PDT alone. Propidium iodide/hoechst33342 double staining reflected that 4T1 cells with apoptotic morphological characteristics were significantly increased in groups given combined therapy. Besides, the combined therapy caused obvious mitochondrial membrane potential (MMP) loss at early 1 h post SPDT treatment. The generation of intracellular reactive oxygen species (ROS) detected by flow cytometry was greatly increased in 4T1 cells treated with the combination therapy, and the loss of cell viability and MMP could be effectively rescued by pre-treatment with the ROS scavenger N-acetylcysteine (NAC). Further, Ce6-SPDT markedly inhibited the tumor growth (volume and weight) and lung metastasis in 4T1 tumor-bearing mice, but had no effect on the body weight. Hematoxylin and eosin staining revealed obvious tissue destruction with large spaces in the Ce6-SPDT groups, and TUNEL staining indicated tumor cell apoptosis after treatment. Immunohistochemistry analysis showed that the expression level of VEGF and MMP were significantly decreased in the combined groups. These results indicated that Ce6-mediated SPDT enhanced the antitumor efficacy on 4T1 cells compared with SDT and PDT alone, loss of MMP and generation of ROS might be involved. In addition, Ce6-mediated SPDT significantly inhibited tumor growth and metastasis in mouse breast cancer 4T1 xenograft model, in which MMP-9 and VEGF may play a crucial role.     

The role of p53 in the response of tumor cells to sonodynamic therapy in vitro

p53 plays a pivotal role in apoptosis. In addition, p53 is currently extensively investigated as a promising strategy for highly specific anticancer therapy in chemotherapeutics and photodynamic therapy. However, the role of p53 in the response of tumor cells to sonodynamic therapy treatment is still unclear. In this study, we aim to investigate the activation of p53 in sonodynamic therapy. Three murine tumor models with distinct aggressiveness (S180, H-22 and EAC) were treated with 1.75 MHz continuous ultrasound at an acoustic intensity (I_SATA) of 1.4 W for 3 min in the presence of 20 μg/ml hematoporphyrin. The DNA fragment and nuclear damage were observed by TUNEL and single cell gel electrophoresis. Western blotting and RT-PCR were used to analyze the expression of p53, PUMA, Bax and Fas. Then we checked the translocation of p53 by confocal microscopy. DNA sequencing was used to determine the status of p53 gene in three tumor cell lines. Our results indicated that the level of p53 protein and mRNA increased significantly, and p53 activated the expression of its downstream pro-apoptosis gene PUMA, Bax and Fas in the S180 and H-22 cells. Meanwhile, p53 protein translocated onto mitochondria. In the EAC cells, expression and translocation of p53 was not found; the level of PUMA, Bax and Fas remained unaltered. The S180 cells showed most serious DNA fragment and nuclear damage with 77.43% TDNA; H-22 cells in the middle with 58.85% TDNA; whereas EAC cells appeared less nuclear material lost with just 15.82% TDNA. The results of DNA sequencing showed that the sequences of exons 5-8 of the p53 gene of S180, H-22 and EAC cells were the same with the sequences of wild-type p53 provided by NCBI. These results primarily demonstrated that: (1) p53 was activated to promote SDT-induced apoptosis through extrinsic and intrinsic signaling pathways in the S180 and H-22 cells; (2) cellular responses of different cells to SDT were distinct, the aggressive S180 cells were much more sensitive than H-22, whereas EAC cells were relatively less sensitive. The discrepancy among the cell lines may be due to different activation time of p53 protein.     

一种检测减影(DSA)图像中肺癌肿瘤大小的新方法

当肺癌被胸水淹没时 ,无法用常规X胸片测量肺癌肿瘤的大小。提出了采用形态算子滤波和广义模糊算子 (GFO)检测边缘 ,从而获得支气管动脉减影图中的肺癌肿瘤的边缘轮廓 ,进而计算得到肿瘤的大小。该方法为肺癌介入病人提供了一种新的肿瘤检测手段 ,且拓展了数学形态学和广义模糊算子的应用领域 ,具有一定的的实用价值和理论价值。     

Combretastatin A-4 derivatives: synthesis and evaluation of 2,4,5-triaryl-1H-imidazoles as potential agents against H1299 (non-small cell lung cancer cell)

A number of 2,4,5-triaryl-1H-imidazole derivatives were synthesized and evaluated for their antiproliferative activities against the growth of five cell lines including three non-small cell lung cancers (H460, H1299, and A549), one breast cancer (MCF-7), and one normal diploid embryonic lung cell line (MRC-5). Preliminary results indicated that both 2-(5-bromofuran-2-yl)-4,5-bis{4-[3-(dimethylamino) propoxy] phenyl}-1H-imidazole (10f) and 4,5-bis{4-[3-(dimethylamino)propoxy]phenyl}-2-(5-nitrofuran-2-yl)-1H -imidazole (10g) were selectively active against the growth of H1229 with an IC₅₀ of less than 0.1???M, thus were more active than topotecan (IC₅₀ >?10.0??? M). However, both 10f and 10g exhibited only marginal cytotoxicity against H460, A549, MCF-7, and MRC-5 requiring an IC ₅₀ of at least 4.16???M. Our results also indicated that 10f induced H1299 cell cycle arrest at G0/G1 through the inactivation of p38 MAPK, JNK, ERK, as well as the expression of SIRT1 and survivin. These results suggested that 10f might have therapeutic potential against H1299 (non-small cell lung cancer cell).     

Comparison between sonodynamic effect and photodynamic effect with photosensitizers on free radical formation and cell killing

Although enhancement of ultrasound-induced cell killing by photodynamic reagents has been shown, the sonochemical mechanism in detail is still not clear. Here, comparison between sonodynamic effect and photodynamic effect with photosensitizers at a concentration of 10 μM on free radical formation and cell killing was made. When electron paramagnetic-resonance spectroscopy (EPR) was used to detect 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) after photo-irradiation or sonication with 2,2,6,6-tetramethyl-4-piperidone (TMPD), the order of TAN formation in the photo-irradiated samples was as follows: rhodamine 6G (R6) > sulforhodamine B (SR) > hematoporphyrin (Hp) > rhodamine 123 (R123) > rose bengal (RB) > erythrosine B (Er) = 0; although there was time-dependent TAN formation when the samples were sonicated, no significant difference among these agents were observed. All these agents suppressed ultrasound-induced OH radical formation detected by EPR-spin trapping. Sensitizer-derived free radicals were markedly observed in SR, RB and Er, while trace level of radicals derived from R6 and R123 were observed. Enhancement of ultrasound-induced decrease of survival in human lymphoma U937 cells was observed at 1.5 W/cm² (less than inertial cavitation threshold) for R6, R123, SR and Er, and at 2.3 W/cm² for R6, R123, Er, RB and SR. On the other hand, photo-induced decrease of survival was observed for R6, Hp and RB at the same concentration (10 μM). These comparative results suggest that (1) ¹O₂ is not involved in the enhancement of ultrasound-induced loss of cell survival, (2) OH radicals and sensitizer-derived free radicals do not take part in the enhancement, and (3) the mechanism is mainly due to certain mechanical stress such as augmentation of physical disruption of cellular membrane by sensitizers in the close vicinity of cells and/or cavitation bubbles.     

Oxygen and Indocyanine Green loaded microparticles for dual-mode imaging and sonodynamic treatment of cancer cells

Oxygen and Indocyanine Green (ICG) loaded microparticles (OI-MPs) were fabricated by a gas-driven coaxial flow focusing (CFF) process for dual-mode imaging and sonodynamic therapy (SDT). The produced OI-MPs agent showed stable optical properties, superior imaging depth in near infrared (NIR) fluorescence imaging, and enhanced acoustic contrast after ultrasound mediation. We hypothesized that encapsulating ICG and oxygen in microparticles would enhance reactive oxygen species (ROS) production in SDT. This hypothesis was validated in a cell-free environment. We further hypothesized that ultrasound mediated fragmentation of the OI-MPs would induce cytotoxicity and apoptosis of cancer cells. This hypothesis was validated in SKOV3 ovarian cancer cells. Our research demonstrated that OI-MPs can be potentially used as a dual-mode theranostic agent for image guided SDT with enhanced efficacy. Further study is needed to delineate the mechanism of ROS-induced cell apoptosis and optimize the OI-MPs formulation for the maximal anti-cancer potency.     

The 6Li(e,e′α) and 6Li(e,e′d) reactions at 520 MeV

The reactions ⁶Li(e,e′α) and ⁶Li(e,e′d) have been studied at T₀ ≈ 520 MeV and q ≈ 1.4 fm^?1, by coincidence measurements. The angular dependence of the cross sections is interpreted in the plane wave impulse approximation to obtain the momentum distribution of α-particle and deuteron clusters in ⁶Li. The amount of clustering of ⁶Li is also discussed.     

Damage effects of protoporphyrin IX - sonodynamic therapy on the cytoskeletal F-actin of Ehrlich ascites carcinoma cells

In this study, we report evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, that has great importance for cancer treatment. Ehrlich ascites carcinoma (EAC) cells suspended in PBS were exposed to ultrasound at 1.34MHz for up to 60s in the presence and absence of protoporphyrin IX (PPIX). To evaluate the polymeric state and distribution of actin filaments (AF) we employed FITC-Phalloidin staining. The percentage of cells with intact AF was decreased with 10-80muMu PPIX after ultrasonic exposure, while only few cells with disturbed F-actin were observed with 80muMu PPIX alone. The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry. The morphological changes of EAC cells were observed by scanning electron microscope (SEM). The nuclei were stained with Hoechst 33258 to determine apoptosis. Cytoskeletal F-actin and cell morphological changes were dependent on the time after SDT. Some cells suffered deformations of plasma membrane as blebs that reacted positively to FITC-Phalloidin at 2h after SDT treatment. Many of the cells showed the typically apoptotic chromatin fragmentation. The alterations were more significant 4h later. Our results showed that cytoskeletal F-actin might represent an important target for the SDT treatment and the observed effect on F-actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment.     

Modeling cancer immunotherapy: Assessing the effects of lymphocytes on cancer cell growth and motility

A mesoscopic model is used to describe the effects of lymphocyte activity on a growing tumor. The model yields novel insights into the tumor–immune system interaction. In particular, we found that the presence of a putative chemotactic messenger that helps guide the lymphocytes towards the tumor is not critical to elicit the anti-tumor effects of the immune system, while lymphocytes that block tumor cell migration contribute to limit cancer expansion and thus have a more significant therapeutic impact.     

The effect of an NH tautomerism on the structure and vibrational states of chlorin

A random sequence (path) of counts of an ideal detector, selective for energy states of an atom, at the output of a one-atom maser is simulated numerically by the Monte Carlo method. Fragments of a path for the cases in which a micromaser undergoes quantum jumps with an increase (jump up) or a decrease in the average number of photons in a mode are obtained. An idealized dynamic model of a quantum jump is formulated on the basis of the concept of a critical fluctuation, when several tens of atoms in the same state are detected successively. Approximate formulas for the dependence of the average number of photons and the rate of its change on the number of atoms passing through a cavity along a jump path are obtained. The probability of a quantum jump in the idealized model is estimated. It is shown that accounting for the nonideality of a jump increases its probability by several orders of magnitude. The high rate of a quantum jump up (the superpump effect) is attributed to the possibility of redistribution of photons between subensembles of the mode states. The probability of a nonideal quantum jump and the most likely number of interruptions (opposite escapes) in an ideal jump path are estimated.     

The effects of Ce6-mediated sono-photodynamic therapy on cell migration,apoptosis and autophagy in mouse mammary 4T1 cell line

Purpose

Sono-Photodynamic therapy (SPDT) is an alternative therapy which claims to enhance the anti-cancer effects by combining sonodynamic therapy (SDT) with photodynamic therapy (PDT). In the present study, we investigated the effects of chlorin e6 (Ce6) mediated SPDT on migration, apoptosis and autophagy in mouse mammary 4T1 cancer cells, and its underlying mechanisms.

Materials

Cell migration was determined by wound healing assay. Apoptosis was analyzed using annexin V-PE/7-ADD staining as well as Hoechst 33342 staining. Changes of mitochondria membrane potential (MMP) was evaluated by flow cytometry. Formation of acidic vesicular organelles (AVOs) during autophagy was observed with fluorescence microscope by MDC staining. Immunofluorescence assays were performed to detect the co-localization of LC3 and Lamp2. Western blotting was employed to analyze the activity of the apoptosis related proteins Caspase-3, PARP, Bax and Bcl-2, as well as the autophagy associated processing of LC3-I to LC3-II and Beclin-1 expression.

Results

Ce6 mediated SPDT further enhanced cell migration inhibition, significantly triggered cell apoptosis, nuclear condensation and MMP drop. Cleaved Caspase-3 and PARP increased dramatically after Ce6-SPDT, accompanied by decreased Bcl-2 expression, while the expression of Bax remained stable. Additionally, AVOs formation, co-localization of LC3 and Lamp2 occurred following Ce6-SPDT and simultaneously accompanied by LC3-II processing and increased Beclin-1 expression.

Conclusions

Ce6-SPDT could enhance cell migration inhibition, and induce mitochondria-dependent apoptosis as well as autophagy in 4T1 cells.     

Digital differentiation of non-small cell carcinomas of the lung by the fractal dimension of their epithelial architecture

IntroductionIn recent years, differences have emerged in the treatment of squamous and non-squamous non-small cell lung carcinomas (NSCLCs). This highlights the importance of accurate histopathologic classification. However, there remains inter-observer disagreement when making diagnoses based on histology. Fractal dimension (FD) is a mathematical measure of irregularity and complexity of shape. We hypothesize that the FD of carcinoma epithelial architecture can assist in differentiating adenocarcinoma (ADC) from squamous cell carcinoma (SCC) of the lung.Methods134 resected (88 ADC and 46 SCC) cases of resected early-stage NSCLC were analyzed. Tissue micro arrays were generated from formalin-fixed paraffin-embedded tissue, stained with pan-cytokeratin, and digitally imaged and the FD of the epithelial structure calculated. Mean FD of ADC and SCC were compared using the independent t-test, partial correlations, and receiver operating characteristic (ROC) analyses.ResultsA statistically significant difference (p < 0.001) between the mean FD of ADC (M = 1.70, SD = 0.07) and SCC (M = 1.78, SD = 0.07) was found. Significance remained (p < 0.001) when controlling for several possible confounders. ROC analysis demonstrated an area-under-the-curve of 0.81 (p < 0.001).ConclusionsThe epithelial structure FD of NSCLC has potential as a reproducible and automated measure to help subtype NSCLCs into ADC and SCC. With further image analysis algorithm improvements, fractal analysis may be a component in computerized histomorphological assessments of lung cancer and may provide an adjunct test in differentiating NSCLCs.     

Pre-irradiation with low-dose ~(12)C~(6+) beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOI of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in 12C6+ beam induced AdCMV-p53 infected cells were more than 90%, which were signifi-cantly more than those in γ-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G0/G1 arrest and activated G2/M checkpoints. The pre-exposure to 12C6+ beam significantly improved cell to apoptosis. RBEs for the 12C6+ + AdCMV-p53 infection groups were 30%-60%, 20%-130% and 30%-70% more than those for the 12C6+-irradiated only, AdCMV-p53 infected only, and γ-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose 12C6+ beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.     

Interference effects on (e, 2e) electron momentum profiles of CF4

Interference effects on electron momentum profiles have been studied using binary (e, 2e) spectroscopy for the three outermost molecular orbitals of CF(4), which are composed of the F 2p nonbonding atomic orbitals. An analysis of the measured spherically averaged electron momentum densities has clearly shown the presence of oscillatory structures having direct information about the internuclear distance between the F atoms. Furthermore, it is demonstrated that the phase of the oscillatory structures depends upon the orientation in space of the constituent atomic orbitals.