Photofrin Based Photodynamic Therapy and miR-99a Transfection Inhibited FGFR3 and PI3K/Akt Signaling Mechanisms to Control Growth of Human Glioblastoma In Vitro and In Vivo

Glioblastoma is the most common malignant brain tumor in humans. We explored the molecular mechanisms how the efficacy of photofrin based photodynamic therapy (PDT) was enhanced by miR-99a transfection in human glioblastoma cells. Our results showed almost similar uptake of photofrin after 24 h in different glioblastoma cells, but p53 wild-type cells were more sensitive to radiation and photofrin doses than p53 mutant cells. Photofrin based PDT induced apoptosis, inhibited cell invasion, prevented angiogenic network formation, and promoted DNA fragmentation and laddering in U87MG and U118MG cells harvoring p53 wild-type. Western blotting showed that photofrin based PDT was efficient to block the angiogenesis and cell survival pathways. Further, photofrin based PDT followed by miR-99a transfection dramatically increased miR-99a expression and also increased apoptosis in glioblastoma cell cultures and drastically reduced tumor growth in athymic nude mice, due to down regulation of fibroblast growth factor receptor 3 (FGFR3) and PI3K/Akt signaling mechanisms leading to inhibition of cell proliferation and induction of molecular mechanisms of apoptosis. Therefore, our results indicated that the anti-tumor effects of photofrin based PDT was strongly augmented by miR-99a overexpression and this novel combination therapeutic strategy could be used for controlling growth of human p53 wild-type glioblastomas both in vitro and in vivo.     

Role of apoptosis in photodynamic sensitivity of human tumour cell lines

Photodynamic therapy (PDT) using a photosensitizer, such as haematoporphyrin derivative (HpD), in conjunction with visible light is a promising new modality to treat localized cancer. Cell death caused by PDT (through the generation of reactive oxygen species) can occur either by apoptosis (interphase death or as a secondary event following mitosis) and/or necrosis depending on the cell type, concentration and intracellular localization of the sensitizer, and the light dose. Since, apoptosis induced by PDT treatment plays an important role in determining the photodynamic efficacy, in the present work we have investigated the role of apoptotic cell death in relation to the observed differences in sensitivity to HpD-PDT between a human glioma cell line (BMG-1) carrying wild-type tumour suppressor gene p53 and a human squamous carcinoma cell line (4451) with mutated p53. HpD (photosan-3; PS-3) -PDT induced apoptosis was studied by: [A] flow-cytometric analysis of DNA content (sub G0/G1 population); [B] phosphatidylserine externalization (Annexin-V +ve cells); [C] cell size and cytoskeleton reorganization (light-scatter analysis); and [D] fluorescence microscopy (morphological features). PS-3-PDT induced a significantly higher level of apoptosis in BMG-1 cells as compared to 4451 cells. This was dependent on the concentration of PS-3 as well as post-irradiation time in both the cell lines. At 2.5 microg/ml of PS-3 the fraction of BMG-1 cells undergoing apoptosis (60%) was nearly 6 folds higher than 4451 cells (10%). In BMG-1 cells the induction of apoptosis increased with PS-3 concentration up to 5 microg/ml (>80%). However, a decrease was observed at a concentration of 10 microg/ml, possibly due to a shift in the mode of cell death from apoptosis to necrosis. In 4451 cells, on the other hand, the increase in apoptosis could be observed even up to 10 microg/ml of PS-3 (60%). Present results show that the higher sensitivity to PS-3-PDT in glioma cells arise on account of a higher level of apoptosis and suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in certain cell types.     

Uptake of photofrin II,a photosensitizer used in photodynamic therapy,by tumour cells in vitro

Photosensitizing dyes are used in fluorescence diagnostics and photodynamic therapy (PDT). These usually hematoporphyrin derivatives (HpD) accumulate preferentially within neoplastic tissues. HpD is a mixture of ether and ester linked porphyrins. Its partially purified form is known under the commercial name of photofrin II (PII). PII emission spectra were studied in a hydrophilic (PBS) and a lipophilic (PC liposomes) environment. Red shift was observed in their emission maxima from 615 nm in buffer solution to 635 nm in lipid. Identical red shift was obtained when the intracellular fluorescence of two cancer cell lines, MCF 7 and Jurkat, incubated with PII was investigated. Thus, intramembrane localization of PII may be suggested. As determined from the total fluorescence intensity, the uptake of PII was only slightly higher for Jurkat than for MCF 7 cells. Nevertheless the kinetics of the uptake was found to be different for both cell lines.     

Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

Sonodynamic therapy (SDT) is one of antitumor strategies that kill tumor cells through the synergistic effects caused by the combined use of HpD and US[1]. SDT is based on the following principle. Ultrasound used in SDT can penetrate the deep tissue and activate HpD, which accu- mulated in tumor cell, and produce highly active oxygen species[2] such as singlet oxygen (1O2), which can destroy the structure of tumor cells. So far the studies on the SDT have focused mainly on the mechan…     

Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

The inhibition of ascitic S180 and induced sarcoma 180 in vivo was studied with the combination of hematoporphyrin derivatives (HpD) and ultrasound (US) at the frequency of 1.1 MHz and different intensities by light microscopy observation, electronic microscopy observation, cytochemical analysis and fluorescence labeling. The present study indicated that the injury of ascitic S180 increased as time passed and the inhibitory effect was stronger in US plus HpD group than that in other groups. Our results also indicated that the changes in cell structure, cytochrome C oxidase activity, the degradation and missing of DNA were the important factors that inhibited the tumor cell growth and even induced celldeath. The phenomenon of apoptosis of tumor cells indicated that cell death andinduced apoptosis exist in the treatment of sonodynamic therapy (SDT). Our study investigated the mechanism underlying the killing effect of S180 induced by USactivating HpD by the observation of cell morphology and dynamic changes from seminal injury to succeeded injury even to death. It would provide rich referencefor the study of SDT.     

Photodynamic treatment with anionic nanoclays containing curcumin on human triple-negative breast cancer cells: Cellular and biochemical studies

Photodynamic treatment is a minimally invasive and clinically approved procedure for eliminating selected malignant cells with activation of a photosensitizer agent at a specific light. Little is known, however, about the phototoxic properties of curcumin, as a natural phenolic compound, against different types of cancers. It is generally accepted that cellular damage occurs during photo treatment. There is a limitation in using of curcumin as a drug due to its low solubility, but nanoparticles such as anionic nanoclays or layered double hydroxide (LDH) could overcome it. The aim of this study was to investigate cellular responses to curcumin-LDH nanoparticles after photodynamic treatment of MDA-MB-231 human breast cancer cells. For this purpose, the MDA-MB-231 human breast cancer cell line treated with curcumin-LDH nanoparticle and then irradiated (photodynamic treatment). After irradiation, lactate dehydrogenase assay, clonogenic cell survival, cell death mechanisms such as autophagy and apoptosis were determined. Cell cycle distribution after photodynamic therapy (PDT) and also intracellular reactive oxygen species (ROS) generation were measured. The result showed that curcumin-LDH–PDT has a cytotoxic and antiprolifrative effect on MDA-MB-231 human breast cancer cells. Curcumin-LDH–PDT induced autophagy, apoptosis, and G0/G1 cell cycle arrest in human breast cancer cell line. Intracellular ROS increased in MDA-MB-231 cancer cell line after treatment with curcumin-LDH along with irradiation. The results suggest that curcumin-LDH nanoparticle could be considered as a novel approach in the photodynamic treatment of breast cancer.     

The influence of photodynamic therapy on apoptosis in human melanoma cell line

Melanoma is the most severe of all skin cancers as it may grow rapidly and metastasize. The application of photodynamic therapy (PDT) opens new perspectives in treatment of this cancer. Numerous studies suggest that the exposure of tumor cells to PDT can lead to cell death via two separate processes: apoptosis or necrosis. The aim of this study was to assess in vitro photodynamic therapy which induces apoptosis in the human Beidegr?m Melanoma (BM) cell line, using neutral comet assay. The cells were incubated with Photofrin II (15 microg/ml and 30 microg/ml) 4 h before and 3 h after irradiation for 5 or 10 min with the light intensity of 10 mW/cm2, using a lamp with red filter (632.8 nm). The percentage of apoptotic cells was significantly higher after PDT comparing to control cells. We observed 25% and 70% of apoptotic cells after shorter irradiation and treatment with 15 microg/ml and 30 microg/ml of Ph II, respectively. After longer irradiation, the respective values were 71.9% and 90%. The results suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in the studied cell line and that some types of DNA damage are dependent on photosensitizer concentration and time of irradiation.     

Effects of BAPTA-AM, Forskolin, DSF and Z.VAD.fmk on PDT-induced apoptosis and m-THPC phototoxicity on B16 cells

As many types of cells exposed to photodynamic therapy (PDT) appear to undergo apoptosis, various apoptosis inhibitors have already been used in studies of PDT-induced apoptosis. Although these inhibitors decrease apoptosis, their real effect on the phototoxic efficacy of photosensitisers is unclear. The good phototoxicity of m-THPC was confirmed on murine melanoma B16-A45 cells. Toxicity and phototoxicity studies were then carried out using four apoptosis inhibitors: BAPTA-AM, Forskolin, DSF, and Z.VAD.fmk. Although all inhibitors tested blocked PDT-induced apoptosis, none produced a significant modification of the phototoxic effect of m-THPC on B16 cells. It has been suggested that apoptosis and necrosis share common initiation pathways and that the final outcome is determined by the presence of an active caspase. This implies that apoptosis inhibition reorients cells to necrosis, i.e. those cells sufficiently damaged by PDT appear to be killed, regardless of the mechanism involved.     

In vitro studies on erythrosine-based photodynamic therapy of malignant and pre-malignant oral epithelial cells

Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37 °C the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ~80% of DOK cell killing was achieved as compared to ~60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans-membrane potential (ΔΨ(m)) studies showed that the H357 cells were far more resistant to the changes in ΔΨ(m) when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT's usage for cancer cell killing.     

The influence of static magnetic fields on canine and equine mesenchymal stem cells derived from adipose tissue

The aim of this study was to evaluate the proliferation rate and morphological changes of adipose-derived mesenchymal stem cells of canine and equine origin (Eq- and CaAdMSC). Investigated cells were exposed to a static magnetic field (MF) with the intensity of 0.5 T. Proliferation activity of cells was determined with the Alamar Blue assay. Obtained results, normalized in respect to the control culture, showed that EqAdMSC exposed to MF maintained a high proliferation status, whereas proliferation activity of CaAdMSC cultured in the presence of MF was decreased. Estimations of population doubling time (PDT) also revealed that EqAdMSCs exposed to static MF achieved a twofold increase in the total number of cells in a shorter amount of time than the control culture. The PDT value obtained for investigated CaAdMSCs indicated that MF exposure resulted in the prolongation of population doubling time. Morphology of cells and cellular composition was investigated using a light inverted microscope and a fluorescent microscope. A scanning electron microscope was used for microvesicles (MVs) imaging. Obtained results showed that both cell types maintained fibroblastic morphology and did not reveal signs of apoptosis or necrosis. However, the MF had an influence on the MVs secretion. While EqAdMSCs propagated in the presence of MF were characterized by the abundant MVs presence, CaAdMSCs revealed poor secretory activity. The approach presented provides complex analysis, which enables one to determine changes in equine and canine cytophysiology.     

Zinc-(II) 2,9,16,23-tetrakis (methoxy) phthalocyanine: potential photosensitizer for use in photodynamic therapy in vitro

Photodynamic therapy (PDT) is an innovative treatment for several types of malignant and non-malignant disease. In the present study, ZnPcOCH(3) was investigated on a human larynx-carcinoma cell line (Hep-2) for its use in PDT. This drug exhibited favourable properties as a photosensitizer in vitro because ZnPcOCH(3) is able to penetrate efficiently in the cytoplasm of cultured cancer cells and is partially localized in lysosomes. The results show that ZnPcOCH(3)-PDT-induced apoptosis by caspase dependent pathway. The new compound shows a good photosensitizing efficiency in vitro on Hep-2 cells, encouraging further in vivo studies.     

Protection by Bcl-2 against apoptotic but not autophagic cell death after photodynamic therapy

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.     

Necrosis predominates in the cell death of human colon adenocarcinoma HT-29 cells treated under variable conditions of photodynamic therapy with hypericin.

Photodynamic therapy (PDT) represents a new rapidly-developing anticancer approach based on administration of a non- or weakly-toxic photosensitizer and its activation with light of appropriate wavelength. Hypericin, one of the promising photosensitizers, is known to induce apoptosis with high efficiency in various cell line models. However, here we report the prevalence of necrosis accompanied by suppression of caspase-3 activation in colon adenocarcinoma HT-29 cells exposed to an extensive range of PDT doses evoked by variations in two variables -- hypericin concentration and light dose. Necrosis was the principal mode of cell death despite different PDT doses and the absence of anti-apoptotic Bcl-2 expression, even if the same condition induced caspase-3 activity at similar toxicity in HeLa cells. Introduction of Bcl-2 into HT-29 cells invoked caspase-3 activation, changed the Bcl-X(L) expression pattern, increased the apoptosis ratio with no effect on overall toxicity, and supported arrest in the G(2)/M-phase of cell cycle. Since it is known that Bcl-2 suppression in HT-29 is reversible and linked to the over-expression of mutated p53 and also considering our data, we suggest that the mutation in p53 and events linked to this feature may play a role in cell death signalling in HT-29 colon cancer cells.     

Effects of hematoporphyrin-derivative on mouse erythroleukemia cells in the absence of light irradiation

This paper concerns a general study on the effects of hematoporphyrin-derivative (HpD) on mouse erythroleukemia (MEL) cells, in the absence of light irradiation. In particular, HpD intrinsic cytotoxicity was evaluated at different doses and the results correlated with those referring to membrane functional and morphological changes. HpD uptake and release processes were also studied and compared with the above-mentioned results. In order to have an overall picture of HpD-cell interactions, time-resolved fluorescence measurements were performed on both undifferentiated and differentiated MEL cells. The results obtained indicate that, even at HpD doses exhibiting neither any cytotoxicity nor any morphological damage (1-10 micrograms/ml), membrane permeability alterations are observed. Thirty minutes of treatment are sufficient for HpD to develop its toxic effect: indeed, no differences in HpD influence on cell viability can be observed after 30 min, 60 min or 5 days of treatment. HpD cytotoxicity is reduced by high protein content in the incubation culture medium. The presence of both monomeric species and 580 nm emitting species was observed at cellular level. The latter is likelier in undifferentiated MEL cells, which also exhibit higher overall HpD uptake, as compared with differentiated MEL cells.     

The effect of laser activation of 5,10,15,20-tetra-sulphophenyl-porphyrin loaded in K562 cells and human normal mononuclear cells

Photodynamic therapy (PDT) is a relatively new type of treatment in cancer, based on a photosensitizer, visible light and molecular oxygen. Reactive oxygen species are generated, causing tumor cells death by apoptosis or necrosis. Significant nowadays research efforts are focused on finding new photosensitizers with antineoplastic activity and an acceptable toxicological profile. Although consistent information exists regarding PDT in solid tumors, relatively few data are available for PDT of blood cancers. Therefore, we carried out a comparative study on lymphoblastic K562 cells and human normal peripheral blood mononuclear cells (PBMC) treated at a density of 2 x 10(5) cells/mL with 5,10,15,20-tetra-sulphophenyl-porphyrin (TSPP) and then irradiated with He-Ne laser light (lamda = 632.8 nm). The following cell functions were investigated: viability, multiplication, RNA synthesis, total RNA levels and apoptosis. After irradiation, the viability of TSPP-loaded tumor cells decrease, the multiplication rate and the total RNA level are drastically reduced and cells undergo apoptosis. TSPP alone loaded into cells but not activated by irradiation, does not affect these cell parameters. Human normal PBMC subjected to TSPP loading and laser-irradiation develop a different cellular response, their viability and proliferative capacity not being altered by experimental PDT. Accordingly, it appears that TSPP is a non-aggressive compound for cellular physiology and becomes cytotoxic only by irradiation; moreover laser-activated TSPP affects only cells that have a tumoral pattern.     

Hypericin-photodynamic therapy leads to interleukin-6 secretion by HepG2 cells and their apoptosis via recruitment of BH3 interacting-domain death agonist and caspases

Photodynamic therapy (PDT) has emerged as a capable therapeutic modality for the treatment of cancer. PDT is a targeted cancer therapy that reportedly leads to tumor cell apoptosis and/or necrosis by facilitating the secretion of certain pro-inflammatory cytokines and expression of multiple apoptotic mediators in the tumor microenvironment. In addition, PDT also triggers oxidative stress that directs tumor cell killing and activation of inflammatory responses. However, the cellular and molecular mechanisms underlying the role of PDT in facilitating tumor cell apoptosis remain ambiguous. Here, we investigated the ability of PDT in association with hypericin (HY) to induce tumor cell apoptosis by facilitating the induction of reactive oxygen species (ROS) and secretion of Th1/Th2/Th17 cytokines in human hepatocellular liver carcinoma cell line (HepG2) cells. To discover if any apoptotic mediators were implicated in the enhancement of cell death of HY-PDT-treated tumor cells, selected gene profiling in response to HY-PDT treatment was implemented. Experimental results showed that interleukin (IL)-6 was significantly increased in all HY-PDT-treated cells, especially in 1 μg/ml HY-PDT, resulting in cell death. In addition, quantitative real-time PCR analysis revealed that the expression of apoptotic genes, such as BH3-interacting-domain death agonist (BID), cytochrome complex (CYT-C) and caspases (CASP3, 6, 7, 8 and 9) was remarkably higher in HY-PDT-treated HepG2 cells than the untreated HepG2 cells, entailing that tumor destruction of immune-mediated cell death occurs only in PDT-treated tumor cells. Hence, we showed that HY-PDT treatment induces apoptosis in HepG2 cells by facilitating cytotoxic ROS, and potentially recruits IL-6 and apoptosis mediators, providing additional hints for the existence of alternative mechanisms of anti-tumor immunity in hepatocellular carcinoma, which contribute to long-term suppression of tumor growth following PDT.     

NAG11和NAG12基因转染对鼻咽癌细胞生长的影响

为了考察鼻咽癌表达下调／缺失基因NAG11和NAG12对鼻咽癌细胞系HNE1生长的影响,构建了NAG11和NAG12基因真核表达载体pcDNA3.1（＋）／NAG11和pcDNA3.1（＋）／NAG12,采用脂质体转染技术将真核重组质粒和空载体质粒分别导入HNE1细胞,观察转染后HNE1细胞生物学特性的变化,结果显示,NAG11重表达对HNE1细胞生长和细胞周期没有明显的影响,而NAG12重表达对HNE1细胞有生长抑制作用,与空载体转染组相比,倍增时间由24.1h延长至31.1h,停滞于G0－G1期细胞数由51.42%增加至68.14%。以上实验进一步说明鼻咽癌是多基因改变的疾病,NAG12的重表达有助于处国咽癌恶性表型的逆转。     

肿瘤坏死因子α和β对电离辐射诱导细胞凋亡的效应

为探讨肿瘤坏死因子（tumor necrosis foctor)α和β（TNFα和β）对电离辐射诱发细胞凋亡的效应及其机理,采用DNA琼脂糖凝胶电泳和FACS分析等方法,观察了人肿瘤坏死因子α（hTNFα)和β（hTNFβ)对^60Co-γ射线诱发细胞凋亡的形态学,生化学变化。结果显示：hTNFα或hTNFβ均可明显抑制^60Co-γ射线诱发正常人胚肺二倍体细胞（2BS）的凋亡,而相同剂量的hTNFα能促进^60Co-γ射线诱发的人体肺腺癌细胞系A549细胞凋亡,而对另一株人体肺癌SPC细胞的效应比A549降低1倍;hTNFβ能分别增强A549和SPC的细胞凋亡频率。由此认为,hTNFα和hTNFβ均可通过调节细胞的生理生化反应来改变细胞对电离辐射的敏感性,可保护正常细胞免受辐射损伤,而增加某些肿瘤细胞对辐射的敏感性。