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…     

超声激活血卟啉对S180 肿瘤细胞表面EGFR 表达量的影响

目的：探讨超声激活血卟啉处理S180肿瘤细胞后膜表面EGFR表达量的变化。方法：将腹水瘤细胞随机分为四组,U组和UH组细胞分别于频率1．8MHz、2．0W／cm^2的超声装置中照射3min,并分别在1h3h5h后取材,应用免疫组化方法在光镜下观察EGFR的表达情况。结果：1h、3h取材,U组和UH组平均光密度值明显低于Cr组和H组,UH组最低。而5h取材时,UH组平均光密度值显著下降,其它组基本无变化。结论：提示在高频低强度处理下,随着时间的延长,超声激活HpD对EGFR的抑制作用增加,显示可能是基因调控使EGFR表达下调,从而使肿瘤细胞增殖减慢。     

Sonodynamic effects of hematoporphyrin monomethyl ether on CNE-2 cells detected by atomic force microscopy

Hematoporphyrin monomethyl ether (HMME) has been effectively used to treat solid tumors of some types. However, its application in nasopharyngeal carcinoma has not been studied yet. In this paper, the detailed sonodynamic effects of HMME‐SDT (sonodynamic therapy) on CNE‐2 cells including cell growth inhibition, apoptosis induction, and membrane toxicity were investigated. It was found that HMME alone had less cytotoxicity whereas HMME‐SDT could suppress the cell proliferation in a dose‐dependent manner as detected by MTT assay. The annexin V‐based flow cytometric data indicated that upon SDT, different concentrations of HMME induce distinct types of cell death, apoptosis by low concentration (60 µg/ml) of HMME and necrosis by higher concentration (120 µg/ml). The immunofluorescence of cytoskeleton and nuclei morphology showed that upon HMME‐SDT, the cells became rounding and the cytoskeletal network disappeared, and, the nuclei represented a total fragmented morphology of nuclear bodies. These alternations showed the apoptosis induction by HMME‐SDT. Further AFM study showed that the cell membrane structure and cytoskeleton networks were destroyed, and, the Young's modulus, tip‐cell‐surface adhesion force decreased to 0.22 ± 0.11 Mpa, 35.4 ± 12.8 pN of cells with 120 µg/ml HMME‐SDT from 0.48 ± 0.21 Mpa, 69.6 ± 22.3 pN of native cells, respectively. These membrane changes caused the collapse of mitochondrial transmembrane potential and disturbance of intracellular calcium homeostasis, which was consistent with the results detected by flow cytometry. Therefore, membrane toxicity and cytoskeleton disrupture induced by HMME‐SDT maybe important factors to induce cell apoptosis, and, the disturbance of mitochondrial transmembrane potential and calcium channels might be the apoptosis mechanisms. J. Cell. Biochem. 112: 169–178, 2011. © 2010 Wiley‐Liss, Inc.     

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.     

Interferon gamma (IFNgamma ) and tumor necrosis factor alpha synergism in ME-180 cervical cancer cell apoptosis and necrosis. IFNgamma inhibits cytoprotective NF-kappa B through STAT1/IRF-1 pathways

We investigated the molecular mechanism of the synergism between interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNgamma/TNFalpha synergistically induced apoptosis of ME-180 cervical cancer cells. IFNgamma induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNgamma/TNFalpha-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFalpha. Dominant-negative IkappaBalpha transfection sensitized ME-180 cells to TNFalpha. IFNgamma pretreatment attenuated TNFalpha- or p65-induced NF-kappaB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-kappaB. IRF-1 transfection alone inhibited TNFalpha-induced NF-kappaB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNgamma/TNFalpha combination; however, caspase inhibition did not abrogate IFNgamma/TNFalpha-induced cell death. Instead, caspase inhibitors directed IFNgamma/TNFalpha-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNgamma and TNFalpha synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNgamma play a critical role in IFNgamma/TNFalpha synergism by inhibiting cytoprotective NF-kappaB. IFNgamma/TNFalpha synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death.     

电磁辐射对原代培养海马神经元的损伤效应及其机制探讨

研究X带高功率微波、S带高功率微波及电磁脉冲辐射对原代培养海马神经元的损伤效应并探讨其机制。通过体外培养原代海马神经元,建立电磁波辐照细胞模型。采用Annexin V-PI双标记、流式细胞术检测细胞凋亡与坏死,原子力显微镜检测细胞膜表面形态,Fluo-3-AM荧光探针负载、激光扫描共聚焦显微镜测定胞内[Ca2 ]i。结果表明,辐射后海马神经元凋亡与坏死均增加,其中坏死增加明显;细胞膜表面粗糙度加大,膜穿孔增多;胞内[Ca2 ]i明显升高。且以上变化均以X带高功率微波组最重,S带高功率微波组次之,电磁脉冲组最轻。提示细胞膜穿孔增多,膜通透性增加,导致胞外Ca2 内流增加,甚至胞内钙超载是辐射致海马神经元凋亡与坏死的机制之一;三种电磁辐射对海马神经元的损伤程度与照射频率呈正相关。     

瘤体内注射凋亡素基因诱导人骨肉瘤S180细胞株凋亡研究

观察瘤体内注射凋亡素基因诱导人骨肉瘤S180细胞株凋亡的效果。利用脂体介导将重组质粒pCDNAVP3转染至荷瘤裸鼠肿瘤结节内。PCR和RT PCR检测结果表明 ,质粒可存在和持续表达 14d以上。抑瘤试验结果证实 ,凋亡素可抑制S180细胞的生长 ,抑瘤率达 2 7.2 %。TUNFL染色法检测结果表明 ,试验组的TUNEL阳性细胞率明显高于对照组。上述结果显示 ,所注射的凋亡素基因可诱导人骨肉瘤S180细胞株凋亡。     

Photofrin--factor of photodynamic therapy induces apoptosis and necrosis

Photodynamic therapy (PDT) causes irreversible photodamage of tumor and other malignant tissues. The effect of reactive oxygen species generation in the presence of photofrin (HpD) was studied. The studies were performed on endothelial cell line from foetal aorta of calves and on normal fibroblasts cell line (3T3 -Balb) and also on malignant line (A431). The cells were grown in presence of photofrin at different time intervals. Time of interaction of photosensitiser with cells was very important. Short time of exposure of the cells to photofrin induced mostly apoptosis in normal cells and apoptotic or necrotic changes in malignant cells. Longer effect of these factors on cells provoked necrosis. The factors of PDT influence dynamic changes of SOD and CT activity. It was dependent on the intensity of factors. These results strongly suggest that HpD has an effect on generation of ROS, which are a signal for development of morphological changes (apoptosis or necrosis) in normal and malignant cells.     

Role of isothiocyanate conjugate of pterostilbene on the inhibition of MCF-7 cell proliferation and tumor growth in Ehrlich ascitic cell induced tumor bearing mice

Naturally occurring pterostilbene (PTER) and isothiocyanate (ITC) attract great attention due to their wide range of biological properties, including anti-cancer, anti-leukemic, anti-bacterial and anti-inflammatory activities. A novel class of hybrid compound synthesized by introducing an ITC moiety on PTER backbone was evaluated for its anti-cancer efficacy in hormone-dependent breast cancer cell line (MCF-7) in vitro and Ehrlich ascitic tumor bearing mice model in vivo. The novel hybrid molecule showed significant in vitro anti-cancer activity (IC₅₀=25±0.38) when compared to reference compound PTER (IC₅₀=65±0.42). The conjugate molecule induced both S and G2/M phase cell cycle arrest as indicated by flow cytometry analysis. In addition, the conjugate induced cell death was characterized by changes in cell morphology, DNA fragmentation, activation of caspase-9, release of cytochrome-c into cytosol and increased Bax: Bcl-2 ratio. The conjugate also suppressed the phosphorylation of Akt and ERK. The conjugate induced cell death was significantly increased in presence of A6730 (a potent Akt1/2 kinase inhibitor) and PD98059 (a specific ERK inhibitor). Moreover, the conjugated PTER inhibited tumor growth in Ehrlich ascitic cell induced tumor bearing mice as observed by reduction in tumor volume compared to untreated animals. Collectively, the pro-apoptotic effect of conjugate is mediated through the activation of caspases, and is correlated with the blockade of the Akt and ERK signaling pathways in MCF-7 cells.     

红托竹荪多糖诱导肿瘤细胞凋亡的作用初探

探讨红托竹荪多糖对小鼠腹水瘤S180细胞凋亡的作用。分别使用不同浓度的红托竹荪多糖处理S180细胞24h,Western blot法检测Bcl-xl、Bax、caspase-9和Caspase-3等蛋白表达,使用流式细胞仪检测细胞凋亡。Western blot 法检测结果显示Bcl-xl表达量随竹荪多糖剂量增加而降低,Bax表达量则相反,每组Bax/Bcl-xl的比值增加,Caspase-9、Caspase-3表达量增加;流式细胞仪检测结果显示：0、25、50和100mg/L组细胞凋亡率分别为5.12%±0.11%、9.61%±0.61%、16.39%±0.19%和17.05%±0.13%,与对照组相比细胞凋亡率增高,差异具有统计学意义（P<0.05）,推断红托竹荪多糖具有诱导S180细胞凋亡的功能。     

荧光显微镜观察大蒜油对腹水癌细胞的影响

用吖啶橙染色,荧光显微镜观察大蒜油对—S180,昆明小鼠,U14 C57BL/6J小鼠和L1210DBA小鼠的癌细胞作用的影响,结果表明大蒜油有直接破坏癌细胞的DNA,RNA和分裂中期染色体的作用。给药后2—6小时作用最强,12小时后癌细胞数逐渐恢复,因此给药间隔不应超过12小时。我们研究证实大蒜油有明显的抗癌作用,为临床应用提供有力的依据。     

Microbubbles Enhance the Antitumor Effects of Sinoporphyrin Sodium Mediated Sonodynamic Therapy both In Vitro and In Vivo

Objectives: To evaluate the anti-cancer effect of sonodynamic therapy combined with microbubbles both in vitro and in vivo.Methods: Cell viability was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide and guava viacount assays. Annexin V-FITC/PI staining was adopted to analyze cell apoptosis rate. FD500 uptake assay was performed to assess cell membrane permeability changes. Tumor weight, mice weight and the visual image of tumor size were used to reflect the anti-tumor effect of this combined method. Histological change of tumor tissue after different treatments was measured through hematoxylin and eosin (H&E) staining.Results: Microbubbles can significantly enhance the cytotoxicity and necrocytosis rate induced by SDT treatment. Increased cell membrane permeability and more uptake of DVDMS were founded in SDT combined with microbubbles group. For in vivo experiments, SDT with microbubbles can significantly reduce tumor weight and size with pimping difference of mice weight compare with other treatment groups. In addition, microbubbles notably improved tumor tissue destruction caused by ultrasound and SDT treatment.Conclusion: The results suggest that microbubbles can markedly improve the anti-cancer effect of DVDMS mediate sonodynamic therapy both in vitro and in vivo.     

电磁脉冲辐照大鼠海马区细胞凋亡与形态学变化

以体外原代培养的大鼠海马神经元和Wistar大鼠为研究对象,探讨电磁脉冲(场强为6× 104 V/m)辐照后早期海马区细胞凋亡和病理形态学的变化.在照射后1h、6h、12h、24h和48h分别采用MTT法和流式细胞仪测定死亡细胞和凋亡细胞的比例,用光镜和电镜分别进行形态学观察.结果显示在电磁脉冲辐照后,海马神经细胞不仅发生快速的坏死,而且还发生凋亡,同时在早期即可见到血管、胶质细胞和神经元等组织的形态学异常.表明大鼠大脑受电磁脉冲辐照后早期海马区可发生神经细胞坏死和凋亡,以及各组织成分的病理形态学改变,上述变化可能与电磁脉冲致细胞DNA损伤有关.     

Apoptosis-Promoting Effects of Hematoporphyrin Monomethyl Ether-Sonodynamic Therapy (HMME-SDT) on Endometrial Cancer

Objective

The aim of the present study was to examine the apoptosis-promoting effects and mechanisms of hematoporphyrin monomethyl ether (HMME)-sonodynamic therapy (SDT) on endometrial cancer cells in vitro.

Methods

Endometrial cancer cell samples were divided into four groups: 1) untreated control group, 2) HMME group, 3) pure ultrasound group, and 4) HMME combined with ultrasound, i.e. SDT group. CCK-8 method was utilized to assess the inhibiting effect of SDT on the proliferation of endometrial cancer cells. Optical microscope and field emission transmission electron microscopy were used to characterize the morphology changes of the cancer cells induced by the treatments. Apoptosis rate, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were examined by flow cytometer. Fluorescence intensity measured by laser scanning confocal microscopy was used to explore the variation of intracellular calcium ion (Ca²⁺) concentration. Apoptosis-related proteins involved in both intrinsic and extrinsic apoptosis signallings were analyzed by western blot.

Results

SDT can effectively induce the apoptosis of endometrial cancer cells. Compared with ultrasound which is known as an effective anti-tumor method, SDT leads to a significant improvement on suppression of cell viability and induction of apoptosis, together with more remarkable modifications on the morphology and substructure in both ultrasound sensitive and resistant endometrial cancer cells. Further studies reveals that SDT promotes ROS production, induces loss of MMP and increases intracellular Ca²⁺ concentration more efficiently than HMME or ultrasound alone. SDT groups also show a rather high expression of apoptosis-promoting proteins, including Bax, Fas and Fas-L, and a significant low expression of apoptosis-suspending proteins including Bcl-2 and Survivin. Meanwhile, both cleaved caspse-3 and caspase-8 are dramatically enhanced in SDT groups. Multiple pathways has been proposed in the process, including the intrinsic activation by excessive ROS and overloaded Ca²⁺, silencing survivin gene, and the extrinsic pathway mediated by the death receptor.

Conclusion

Given its considerable effectivity in both ultrasound sensitive and resistant cells, SDT may therefore be a promising therapeutic method for treating endometrial cancers.     

Allosensitization induced suppression of various murine tumors: role of non-H-2 antigens in antitumor immunity

Presence of alloantigens on various murine tumors was tested by tumor rejection in allosensitized Swiss mice. The results indicated the presence of alloantigen on immunogenic tumors like chemically induced fibrosarcoma (FS), ascitic sarcoma 180 (S 180) and immunogenic variant of lymphosarcoma (LS-A) in Swiss mice, while these antigens could not be detected by this procedure on spontaneous lymphosarcoma (LS). Allosensitization with skin graft was found to offer quantitatively higher antitumor resistance than the allosensitization achieved by allogeneic lymphocytes. Antitumor effect was not seen when tumor cells were inoculated earlier than day 3 of grafting. Further, host immunosuppression with whole body irradiation up to day of 3 of skin grafting abrogated the antitumor effect. H-2 compatible and non-H-2 incompatible skin graft sensitization of host could offer resistance against both S 180 and LS-A. Further, tumor immune mice rejected H-2 compatible, non-H-2 incompatible skin graft significantly earlier.     

Apocynum venetum leaf extract protects rat cortical neurons from injury induced by oxygen and glucose deprivation in vitro

This study aimed to investigate the protective effect of Apocynum venetum leaf extract (AVLE) on an in vitro model of ischemia-reperfusion induced by oxygen and glucose deprivation (OGD) and further explored the possible mechanisms underlying protection. Cell injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage; cell viability was measured by XTT reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3,?-8, and?-9 activation and Bcl-2/Bax protein expression were determined by Western blot analysis. We report that treatment with AVLE (5 and 50?μg/mL) effectively reduced neuronal cell death and relieved cell injury induced by OGD. Moreover, AVLE decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspase-3 and?-8 and Bax, and inhibited the reduction of Bcl-2 expression. These findings indicate that AVLE protects against OGD-induced injury by inhibiting apoptosis in rat cortical neurons by down-regulating caspase-3 activation and modulating the Bcl-2/Bax ratio.     

Hydrogen peroxide produced by Aplysia ink toxin kills tumor cells independent of apoptosis via peroxiredoxin I sensitive pathways

Marine snails of the genus Aplysia possess numerous bioactive substances. We have purified a 60 kDa protein, APIT (Aplysia punctata ink toxin), from the defensive ink of A. punctata that triggers cell death with profound tumor specificity. Tumor cell death induced by APIT is independent of apoptosis but is characterized by the rapid loss of metabolic activity, membrane permeabilization, and shrinkage of nuclei. Proteome analysis of APIT-treated tumor cells indicated a modification of peroxiredoxin I, a cytoplasmic peroxidase involved in the detoxification of peroxides. Interestingly, knockdown of peroxiredoxin I expression by RNA interference sensitized cells for APIT-induced cell death. APIT induced the death of tumor cells via the enzymatic production of H2O2 and catalase completely blocked APITs' activity. Our data suggest that H2O2 induced stress and the modulation of peroxiredoxins might be a promising approach for tumor therapy.     

藻红蛋白亚基光敏剂对小鼠移植瘤作用的超微结构研究

目的：从形态学角度探讨藻红蛋白(R-PE)β亚基光动力学抗肿瘤效果及其作用机理。方法：用不同密度的波长为496nm的氩离子激光对S180小鼠移植瘤进行β亚基光动力学治疗,并对治疗后的瘤体进行透射电镜的形态学观察。结果：用100μg／m1的β亚基,在200J/cm2激光照射剂量条件下治愈了瘤体直径为0．5cm-0．7cm大小的小鼠移植瘤,发现瘤组织中引起细胞死亡的途经有差异,被PDT抑制的肿瘤内部细胞表现出典型的凋亡细胞特征。结论：R-PE β亚基具较强的光动力学抗肿瘤效果,光动力治疗机理可能涉及肿瘤内部细胞死亡主要是凋亡途径而瘤周为坏死,且与血管系统破坏及白细胞参与的抗炎症反应相关。     

曲格列酮增加Hela细胞的自噬和混合型细胞死亡

[目的]为了研究Troglitazone(Trog)对HeLa细胞自噬和程序性细胞死亡的影响.[方法]利用电镜、荧光显微镜、免疫杂交、流式细胞计数等对经Trog处理后的HeLa细胞进行了细胞发生自噬和死亡情况的观察.[结果] 电镜、荧光显微镜的结果均提示,Trog能够诱导HeLa细胞自噬活动的增加;免疫杂交显示, 该药物能增加自噬相关基因LC3的表达,并于刺激后4 h达到高峰;除了能使细胞凋亡外,Trog也可以造成细胞的坏死.[结论]上述结果表明,曲格列酮可以引起混合型细胞死亡.     

Degradation of focal adhesion proteins during nocodazole-induced apoptosis in rat-1 cells

Nocodazole, a microtubule-disrupting agent, induced apoptosis in Rat-1 cells, as indicated by changes in cell morphology, DNA fragmentation, and eventual cell death. During nocodazole-induced apoptosis, normally flat cells became rounded in shape and detached from the extracellular matrix. These morphological changes appeared to be closely associated with degradation of focal adhesion proteins, including p130cas, p125(FAK) and paxillin. p130cas was also degraded in cells treated with staurosporine or etoposide, suggesting that degradation of focal adhesion proteins is a characteristic feature of apoptosis. Nocodazole-induced apoptosis was antagonized by Bcl-2: degradation of focal adhesion proteins was suppressed and cell viability was enhanced in bcl-2 over-expressing cells, even in the presence of nocodazole. Further study of the molecular mechanism of Bcl-2 activation should provide an understanding of the apoptosis induced by disruption of the microtubule network.