Photoelectric measurements of s-BLM/nucleoli: a new technique for studying apoptosis

A new method based on photoelectric measurement for analyzing apoptosis of cell-free MCF-7 nucleoli is reported. Supported bilayer lipid membrane (s-BLM) was used to enclose nucleoli in biological environment. The s-BLM was self-assembled on the wall of a super-thin cell. During the apoptosis induced by Taxol, the photoelectric current of the self-assembled s-BLM/nucleoli was found decreasing with time, suggesting the degradation of nucleus DNA. Electron transfer along the DNA double helix and along nuclear skeleton is assumed in the interpretation. This novel photoelectric analytical method may provide a rapid and sensitive technique to evaluate apoptosis.     

紫杉醇诱导的乳腺癌MCF-7细胞核凋亡的光电化学表征

利用光电分析法研究了紫杉醇诱导的非细包体系中ＭＣＦ－７细胞核的凋亡。在ＭＣＦ－７细胞核凋亡过程中,光电流的降低与ＤＮＡ断裂和细胞核形态学的变化结果相一致与传统的琼脂糖凝胶电泳法和荧光显微镜观察法相比,光电分析法能快速灵敏地在早期检测到细胞核调亡的动力学信息,并适时追踪细胞核凋亡的整个过程。     

Role of Nuclear Factor-KB in Colon Cancer Cell Apoptosis Mediated by Aminopyropheophorbide Photosensitization

Aminopyropheophorbide (APP) is a second generation of photosensitizer for photodynamic therapy (PDT). We demonstrated that APP strongly absorbed red light and, after being taken up by colon cancer cells (HCT-116 cells), was localized in cytoplasmic and internal membranes but not in mitochondria. The APP-mediated photosensitization was cytotoxic for HCT-116 cells through an induction of apoptosis. Indeed, DNA fragmentation (DNA laddering and terminal deoxyuridine nick-end labeling) and chromatin condensation (4',6-diamidine-2'-phenylindole staining) could be visualized soon after photosensitization. Because nuclear factor (NF)-kappa B is involved in the response to many photosensitizers, we also demonstrated its nuclear translocation in two waves: a rapid and transient one, followed by a slow and sustained phase. The NF-kappa B turned out to be involved in an antiapoptotic response to APP-mediated photosensitization because the HCT-116 cell line expressing the dominant negative mutant of inhibitor-kappa B alpha was more sensitive to apoptosis as measured by DNA fragmentation and caspase activation. These data unambiguously show that a membrane-located photosensitizer can lead to effective apoptosis, reinforcing the idea that PDT can be an effective means to eradicate colon cancer cells.     

Capillary electrophoretic determination of apoptosis of HeLa cells induced by trichosanthin

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. The observation of apoptosis is very important in the research of cancer and cancer therapy. The traditional observation method of apoptosis was agarose gel electrophoresis, which is depending on the determination of ladder-liking DNA fragments extracted from apoptotic cells. It is time-consuming and low-sensitive. Recently, the sieving capillary electrophoresis has been used to detect apoptosis too. However, the problem of DNA fragments contamination is still existing. Here, we have developed a capillary electrophoresis method that could detect apoptosis of whole cell directly and do not need to extract DNA fragments from cells. Apoptosis of adherent cell HeLa cell of carcinoma induced by cyclophosphamide was used as the model to establish the method. The effluence of medicine concentration on apoptosis of cells was studied in detail. It was also found that the method could detect the change of cells in the early period of apoptosis. The induction of apoptosis of HeLa cell by trichosanthin was determined with the method, and the result of flow cytometry was also proved that trichosanthin could result in apoptosis of HeLa cells.     

Cytochrome C-dependent Fas-independent apoptotic pathway in HeLa cells induced by delta12-prostaglandin J2

Cyclopentenone prostaglandins (PGs) have antiproliferative activity on various tumor cell growth in vitro. Particularly, 9-deoxy-delta(9,12)-13,14-dihydro PGD(2) (delta(12)-PGJ(2)) was reported for its antineoplastic and apoptotic effects on various cancer cells, but its mechanism inducing apoptosis is still not clear. In this study, we have characterized apoptosis induced by delta(12)-PGJ(2) in HeLa cells. Treatment of delta(12)-PGJ(2) induced apoptosis as indicated by DNA fragmentation, chromatin condensation, and formation of apoptotic body. We also observed release of cytochrome c from mitochondria and activation of caspase cascade including caspase-3, -8, and -9. And the pan-caspase inhibitor z-Val-Ala-Asp (OMe) fluoromethyl-ketone (z-VAD-fmk) and Q-Val-Asp (OMe)-CH(2)-OPH (Q-VD (OMe)-OPH) prevented cell death induced by delta(12)-PGJ(2) showing participation of caspases in this process. However, protein expression level of Bcl-2 family was not altered by delta(12)-PGJ(2), seems to have no effect on HeLa cell apoptosis. And ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase 8 indicating that Fas receptor-ligand interaction was not involved in this pathway. Treatment of delta(12)-PGJ(2) also leads to suppression of nuclear factor kappaB (NF-kappaB) as indicated by nuclear translocation of p65/RelA and c-Rel and its DNA binding ability analyzed by EMSA. Taken together, our results suggest that delta(12)-PGJ(2)-induced apoptosis in HeLa cell utilized caspase cascade without Fas receptor-ligand interaction and accompanied with NF-kappaB inactivation.     

Involvement of ROS and JNK1 in selenite-induced apoptosis in Chang liver cells

Selenium is a dietary essential trace nutrient with important biological roles. Selenocompounds were reported to induce apoptosis in many types of tumor cells. In this study, we investigated the signaling pathway involved in the selenite-induced apoptosis using Chang liver cells as a non-malignant cell model. The Chang liver cell apoptosis induced by selenite (10 microM) was confirmed by DNA fragmentation and typical apoptotic nuclear changes. Treatment of selenite increased intracellular reactive oxygen species (ROS) level and c-Jun N-terminal kinase1 (JNK1) phosphorylation. The selenite-induced cell death was attenuated by SP600125, a specific inhibitor of JNK, and by dominant negative JNK1 (DN-JNK1). Antioxidants such as glutathione (GSH), N-acetyl cysteine (NAC), curcumin, epigallocatechin gallate (EGCG) and epicatechin (EC) inhibited selenite-induced intracellular ROS elevation and JNK1 phosphorylation. Our results suggest that selenite-induced apoptosis in Chang liver cells was preceded by the ROS generation and JNK1 activation.     

Purpurin-18 in combination with light leads to apoptosis or necrosis in HL60 leukemia cells

Photodynamic therapy (PDT), a cancer treatment using a photosensitizer and visible light, has been shown to induce apoptosis or necrosis. We report here that Purpurin-18 (Pu18) in combination with light induces rapid apoptotic cell death in the human leukemia cell line (HL60) at low doses and necrosis at higher concentrations. Cells treated with Pu18 and light under apoptotic conditions exhibited DNA laddering and an increase in both cellular content of subdiploid DNA and externalization of phosphatidylserine (PS), indicating DNA fragmentation and loss of membrane phospholipid asymmetry. In the absence of light activation, Pu18 at nanomolar concentrations had no detectable cytotoxic effect. Caspase-3 activity was increased even after 1 h from treatment with low doses of Pu18 and light. The PS exposure and nuclear features of apoptosis were prevented by treatment of cells before illumination with caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK). Conversely, the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) failed to suppress the apoptosis. No protective effect of the three caspase inhibitors was observed when the cells were exposed to necrotic concentrations of Pu18 and light. Our results show that caspase-3, but not caspase-1, is involved in the signaling of apoptotic events in PDT with Pu18-induced apoptosis of HL60 cells. Moreover, both the time course of PS exposure and the effect of caspase inhibitors on it indicate that it is regulated in the same manner as DNA fragmentation.     

p53‐Dependent and p53‐Independent Responses of Cells Challenged by Photosensitization

The p53 protein exerts fundamental roles in cell responses to a variety of stress stimuli. It has clear roles in controlling cell cycle, triggering apoptosis, activating autophagy and modulating DNA damage response. Little is known about the role of p53 in autophagy‐associated cell death, which can be induced by photoactivation of photosensitizers within cells. The photosensitizer 1,9‐dimethyl methylene blue (DMMB) within nanomolar concentration regimes has specific intracellular targets (mitochondria and lysosomes), photoinducing a typical scenario of cell death with autophagy. Importantly, in consequence of its subcellular localization, photoactive DMMB induces selective damage to mitochondrial DNA, saving nuclear DNA. By challenging cells having different p53 protein levels, we investigated whether p53 modulates DMMB/light‐induced phototoxicity and cell cycle dynamics. Cells lacking p53 activity were slightly more resistant to photoactivated DMMB, which was correlated with a smaller sub‐G1 population, indicative of a lower level of apoptosis. DMMB photosensitization seems to induce mostly autophagy‐associated cell death and S‐phase cell cycle arrest with replication stress. Remarkably, these responses were independent on the p53 status, indicating that p53 is not involved in either process. Despite describing some p53‐related responses in cells challenged by photosensitization, our results also provide novel information on the consequences of DMMB phototoxicity.     

DNA repair inhibition and cancer therapy.

The DNA repair process in mammalian cells is a multi-pathway mechanism that protects cells from the plethora of DNA damaging agents that are known to attack nuclear DNA. Moreover, the majority of current anticancer therapies (e.g. ionising radiation and chemotoxic therapies) rely on this ability to create DNA lesions, leading to apoptosis/cell death. A cells natural ability to repair such DNA damage is a major cause of resistance to these existing antitumour agents. It seems logical, therefore, that by modulating these repair mechanisms, greater killing effect to anticancer agents would occur. Experimental data support this, either through knockout studies or by the use of pharmacological inhibitors which target some of the key regulatory proteins involved in the DNA repair process. Several of these key DNA repair proteins which are actively under investigation as novel sites for intervention in cancer biology are discussed.     

1,4-Diselenophene-1,4-diketone triggers caspase-dependent apoptosis in human melanoma A375 cells through induction of mitochondrial dysfunction

Epidemiological, preclinical and clinical studies have supported the role of selenocompounds as potential cancer chemopreventive and chemotherapeutic agents. In this study, a novel selenophene-based compound, 1,4-diselenophene-1,4-diketone (DSeD), has been synthesized by Double Friedel-Crafts reaction and identified as a potent antiproliferative agent against a panel of six human caner cell lines. Despite this potency, DSeD was relatively nontoxic toward human normal cells, HS68 fibroblasts and HK-2 kidney cells. These results suggest that DSeD possesses great selectivity between cancer and normal cells. Induction of apoptosis in human melanoma A375 cells by DSeD was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Activation of caspase-9 and depletion of mitochondrial membrane potential indicated the initiation of the mitochondria-mediated apoptosis pathway. Pretreatment of cells with general caspase inhibitor z-VAD-fmk and caspase-9 inhibitor z-LEHD-fmk significantly suppressed the cell apoptosis, demonstrating the important roles of caspase and mitochondria in DSeD-induced apoptotic cell death. Furthermore, DSeD-induced apoptosis was found independent of reactive oxygen species generation. Taken together, our results suggest that DSeD induces caspase-dependent apoptosis in A375 cells through activation of mitochondria-mediated apoptosis pathway.     

Dose-dependent induction of apoptosis or necrosis in human cells by organotin compounds

In the present study, the mode of cell death induced by highly toxic trialkylated tin compounds has been evaluated. Treatment of undifferentiated HL-60 cells with submicromolar to micromolar concentrations of tri-n-butyltin (TBT) led only to a slight decrease in cell viability measured with trypan blue exclusion. Nevertheless, cell membrane blebbing was observed by means of light microscopy and condensation of nuclear chromatin and formation of apoptotic bodies was demonstrated in Hoechst 33342 stained cells. The nuclear chromatin condensation was associated with an extensive DNA fragmentation. Visualized by agarose gel electrophoresis, genomic DNA appeared as a characteristic ladder-like pattern of DNA fragments which is the biochemical hallmark of apoptosis. The typical internucleosomal DNA digestion was concentration-dependent and began within 2 to 3 h of incubation. During the incubation period a persistent and steady elevation of intracellular free calcium concentration ([Ca²⁺]_i) could be detected. Furthermore, the chromatin condensation and DNA fragmentation could be blocked by supplementation of the incubation medium with zinc pointing to an activation of a zinc-sensitive and calcium-dependent endogenous endonuclease. Higher concentrations of tributyltin (≥ 5 μmol/L TBT) led within hours to a cell killing with degenerative changes indicative of necrosis, demonstrated by plasma membrane disruption which was accompanied by random DNA breakdown. Furthermore, these concentrations also provoked a persistent elevation in [Ca²⁺]_i which reached, even after 10 min, higher levels in comparison with apoptosis-inducing concentrations. The loss in membrane integrity observed at these concentrations of TBT could be due to an activation of calcium-dependent phospholipases. Here it is shown that activation of cytosolic phospholipase A₂ (cPLA₂) leads to liberation of arachidonic acid (AA) out of the phospholipid membrane. The results presented here demonstrate that organometals are able to induce different cell death pathways depending on the applied concentration: low concentrations led to apoptosis whereas high concentrations stimulate necrosis. We suggest that there exists a direct correlation between the intracellular free calcium concentration and the mode of cell death. Received: 1 August 1997 / Revised: 8 October 1997 / Accepted: 10 October 1997     

Dose-dependent induction of apoptosis or necrosis in human cells by organotin compounds

In the present study, the mode of cell death induced by highly toxic trialkylated tin compounds has been evaluated. Treatment of undifferentiated HL-60 cells with submicromolar to micromolar concentrations of tri-n-butyltin (TBT) led only to a slight decrease in cell viability measured with trypan blue exclusion. Nevertheless, cell membrane blebbing was observed by means of light microscopy and condensation of nuclear chromatin and formation of apoptotic bodies was demonstrated in Hoechst 33342 stained cells. The nuclear chromatin condensation was associated with an extensive DNA fragmentation. Visualized by agarose gel electrophoresis, genomic DNA appeared as a characteristic ladder-like pattern of DNA fragments which is the biochemical hallmark of apoptosis. The typical internucleosomal DNA digestion was concentration-dependent and began within 2 to 3 h of incubation. During the incubation period a persistent and steady elevation of intracellular free calcium concentration ([Ca²⁺]_i) could be detected. Furthermore, the chromatin condensation and DNA fragmentation could be blocked by supplementation of the incubation medium with zinc pointing to an activation of a zinc-sensitive and calcium-dependent endogenous endonuclease. Higher concentrations of tributyltin (≥ 5 μmol/L TBT) led within hours to a cell killing with degenerative changes indicative of necrosis, demonstrated by plasma membrane disruption which was accompanied by random DNA breakdown. Furthermore, these concentrations also provoked a persistent elevation in [Ca²⁺]_i which reached, even after 10 min, higher levels in comparison with apoptosis-inducing concentrations. The loss in membrane integrity observed at these concentrations of TBT could be due to an activation of calcium-dependent phospholipases. Here it is shown that activation of cytosolic phospholipase A₂ (cPLA₂) leads to liberation of arachidonic acid (AA) out of the phospholipid membrane. The results presented here demonstrate that organometals are able to induce different cell death pathways depending on the applied concentration: low concentrations led to apoptosis whereas high concentrations stimulate necrosis. We suggest that there exists a direct correlation between the intracellular free calcium concentration and the mode of cell death. Received: 1 August 1997 / Revised: 8 October 1997 / Accepted: 10 October 1997     

Sensitive detection of soy (Glycine max) by real-time polymerase chain reaction targeting the mitochondrial atpA gene

Detection of trace amounts of allergens is essential for correct labeling of food products by the food industry. PCR-based detection methods currently used for this purpose are targeting sequences of DNA present in the cell nucleus. In addition to nuclear DNA, a substantial amount of mitochondrial DNA (mtDNA) copies are present in the cytoplasm of eukaryotic cells. The nuclear DNA usually consists of a set of DNA molecules present in two copies per cell, whereas mitochondrial DNA is present in a few hundred copies per cell. Thus, an increase in sensitivity can be expected when mtDNA is used as the target. In this study, we present a reporter probe-based real-time PCR method amplifying the mitochondrial gene of the alpha chain of adenosine triphosphate synthetase from soy. Increase in sensitivity was examined by determining the minimal amount of soy DNA detectable by mtDNA and nuclear DNA (nDNA) amplification. Additionally, the LOD of soy in a food matrix was determined for mtDNA amplification and compared to the LOD determined by nDNA amplification. As food matrix, a model spice spiked with soy flour was used. Sensitivity of PCR-based soy detection can be increased by using mtDNA as the target.     

Panduratin A inhibits the growth of A549 cells through induction of apoptosis and inhibition of NF-kappaB translocation

In the present study we investigated the effects of panduratin A, isolated from Boesenbergia rotunda, on proliferation and apoptosis in A549 human non-small cell lung cancer cells. Cell proliferation and induction of apoptosis was determined by the real-time cellular analyzer (RTCA), MTT assay and High Content Screening (HCS). The RTCA assay indicated that panduratin A exhibited cytotoxicity, with an IC?? value of 4.4 μg/mL (10.8 μM). Panduratin A arrested cancer cells labeled with bromodeoxyuridine (BrdU) and phospho-Histone H3 in the mitotic phase. The cytotoxic effects of panduratin A were found to be accompanied by a dose-dependent induction of apoptosis, as assessed by DNA condensation, nuclear morphology and intensity, cell permeability, mitochondrial mass/ potential, F-actin and cytochrome c. In addition, treatment with an apoptosis-inducing concentration of panduratin A resulted in significant inhibition of Nuclear Factor-kappa Beta (NF-κB) translocation from cytoplasm to nuclei activated by tumor necrosis factor-alpha (TNF-α), as illustrated by the HCS assay. Our study provides evidence for cell growth inhibition and induction of apoptosis by panduratin A in the A549 cell line, suggesting its therapeutic potential as an NF-κB inhibitor.     

Synthesis and characterization of Mannich bases of lawsone and their anticancer activity

Abstract

Natural and synthetic naphthoquinones are known for a large number of biological activities. Lawsone (2-hydroxy-1, 4-naphthoquinone) is a simplest naturally occurring compound obtained from dried henna (Lawsonia inermis) leaves. In literature, some lawsone derivatives have been reported to exhibit anticancer activity. Hence, a clean and facile one-pot protocol was developed for the synthesis of new aminonaphthoquinones derived from lawsone by three-component Mannich reaction, at room temperature for potential anti-cancer application. Herein we present a small library of Mannich bases with different amines and aromatic aldehydes with moderate to high yield. Synthesized compounds were characterized using various spectroscopic techniques. The anticancer activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay) along with nuclear morphology assessment (4′,6-diamidino-2-phenylindole or DAPI staining), apoptosis assessment (acridine orange/ ethidium bromide staining), hemolysis and DNA ladder assay evaluated on human liver carcinoma cell line HepG2 are presented.     

Cytosolic accumulation of gammaH2AX is associated with tropomyosin-related kinase A-induced cell death in U2OS cells

Tropomyosin-related kinase A (TrkA) plays an important role in cell survival, differentiation, and apoptosis in various neuronal and nonneuronal cell types. Here we show that TrkA overexpression by the Tet-On system mimics NGF-mediated activation pathways in the absence of nerve growth factor (NGF) stimulation in U2OS cells. In addition, p53 upregulation upon DNA damage was inhibited by TrkA, and p21 was upregulated by TrkA in a p53-independent manner. TrkA overexpression caused cell death by interrupting cell cycle progression, and TrkA-induced cell death was diminished in the presence of its specific inhibitor GW441756. Interestingly, TrkA-mediated cell death was strongly related to gammaH2AX production and poly (ADP-ribose) polymerase cleavage in the absence of DNA damage inducer. In this study, we also reveal that gammaH2AX production by TrkA is blocked by TrkA kinase inhibitors K-252a and GW441756, and it is also significantly inhibited by JNK inhibitor SP600125. Moreover, reduction of cell viability by TrkA was strongly suppressed by SP600125 treatment, suggesting a critical role of JNK in TrkA-induced cell death. We also found that gammaH2AX and TrkA were colocalized in cytosol in the absence of DNA damage, and the nuclear localization of gammaH2AX induced by DNA damage was partly altered to cytosol by TrkA overexpression. Our results suggest that the abnormal cytosolic accumulation of gammaH2AX is implicated in TrkA-induced cell death in the absence of DNA damage.     

Spectral properties of topical retinoids prevent DNA damage and apoptosis after acute UV-B exposure in hairless mice

We showed in a recent study that topical retinyl palmitate prevented UV-B-induced DNA damage and erythema in humans. Given that retinyl palmitate is a precursor of retinoic acid, the biological form of vitamin A that acts through nuclear receptors, we wondered whether these protective effects toward UV-B exposure were either receptor dependent or linked to other properties of the retinoid molecule such as its spectral properties. We determined the epidermal retinoid profile induced by topical retinoic acid in hairless mice and analyzed its effect on markers of DNA photodamage (thymine dimers) and apoptosis following acute UV-B exposure; we compared these effects to those induced by other natural topical retinoids (retinaldehyde, retinol and retinyl palmitate) which do not directly activate the retinoid receptors. We then analyzed the direct action of these retinoids on UV-B-induced DNA damage and apoptosis in cultured A431 keratinocytes. Topical retinoic acid significantly decreased (approximately 50%) the number of apoptotic cells, as well as the formation of thymine dimers in the epidermis of mice exposed to acute UV-B. Interestingly, the other topical retinoids decreased apoptosis and DNA damage in a similar way. On the other hand, neither retinoic acid nor the other retinoids interfered with the apoptotic process in A431 keratinocytes exposed to UV-B, whereas DNA photodamage was slightly decreased. We conclude that the decrease of apoptotic cells in hairless mouse epidermis following topical retinoids and UV-B irradiation reflects a protection of the primary targets of UV-B (DNA) by a mechanism independent of the activation of retinoid nuclear receptors, rather than a direct inhibition of apoptosis.     

Impact of metal binding on the antitumor activity and cellular imaging of a metal chelator cationic imidazopyridine derivative

A new water soluble cationic imidazopyridine species, viz. (1E)-1-((pyridin-2-yl)methyleneamino)-3-(3-(pyridin-2-yl)imidazo[1,5-a]pyridin-2(3H)-yl)propan-2-ol (1), as a metal chelator is prepared as its PF(6) salt and characterized. Compound 1 shows fluorescence at 438 nm on excitation at 342 nm in Tris-HCl buffer giving a fluorescence quantum yield (φ) of 0.105 and a life-time of 5.4 ns. Compound 1, as an avid DNA minor groove binder, shows pUC19 DNA cleavage activity in UV-A light of 365 nm forming singlet oxygen species in a type-II pathway. The photonuclease potential of 1 gets enhanced in the presence of Fe(2+), Cu(2+) or Zn(2+). Compound 1 itself displays anticancer activity in HeLa, HepG2 and Jurkat cells with an enhancement on addition of the metal ions. Photodynamic effect of 1 at 365 nm also gets enhanced in the presence of Fe(2+) and Zn(2+). Fluorescence-based cell cycle analysis shows a significant dead cell population in the sub-G1 phase of the cell cycle suggesting apoptosis via ROS generation. A significant change in the nuclear morphology is observed from Hoechst 33258 and an acridine orange/ethidium bromide (AO/EB) dual nuclear staining suggesting apoptosis in cells when treated with 1 alone or in the presence of the metal ions. Apoptosis is found to be caspase-dependent. Fluorescence imaging to monitor the distribution of 1 in cells shows that 1 in the presence of metal ions accumulates predominantly in the cytoplasm. Enhanced uptake of 1 into the cells within 12 h is observed in the presence of Fe(2+) and Zn(2+).     

Photoelectrochemistry as a novel strategy for DNA hybridization detection

The special properties of ssDNA and dsDNA molecules in structure and electric behavior, may offer us some new ideas for the fabrication of genosensors and DNA-chips. In this work, the photoelectrochemical method was firstly employed to characterize the photoelectric behavior of a ssDNA probe electrode, which was prepared with the self-assembly technique, and its resulting dsDNA electrode. The obvious decrease in the photocurrent of the dsDNA modified electrode at open potential or a bias voltage indicated that photoelectrochemistry was another useful method for DNA hybridization detection. Using the special design of ssDNA probes, we attempt to discuss further the relationship between the properties of DNA molecules and their photoelectric behaviors. In addition, the electrochemical impedance method was employed to verify the occurrence of some modifications over the electrode interface before and after the hybridization event.     

PHOTODYNAMIC THERAPY WITH PHOTOFRIN II INDUCES PROGRAMMED CELL DEATH IN CARCINOMA CELL LINES

Abstract The mode of cell death following photodynamic therapy was investigated from the perspective of programmed cell death or apoptosis. Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a) and rat mammary carcinoma (MTF7) were treated by photodynamic therapy. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder indicative of internucleosomal cleavage of DNA during apoptosis. The magnitude of the response and the photodynamic therapy dosage required to induce DNA fragmentation were different in PC3 and MTF7. The MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD₅₀). In contrast, PC3 showed only marginal response at the LD₅₀ but had a marked response at the LD₈₅. Thus, apoptosis did not ensue as quickly in PC3 as in MTF7. The H322a cells were killed by photodynamic therapy but failed to exhibit any apoptotic response. The results also suggested that apoptosis in these cell lines has a minor requirement for de novo protein synthesis and no requirement for de novo RNA synthesis. This study indicates that although apoptosis can occur during photodynamic therapy-induced cell death, this response is not universal for all cancer cell lines.