chlorophyll-a and Carcinoma--Hepatocellular

In recent years, photodynamic therapy (PDT) was considered to be a promising cancer treatment modality, however, the therapeutic efficiency was often attenuated by the intrinsic antioxidant defense systems. Herein, a kind of novel glutathione-induced amino-activatable micelle was designed, which was expected to weaken the antioxidant capacity and in the meantime release the photosensitizer by the exhaustion of intracellular glutathione (GSH). The amphiphilic poly(ethylene glycol)-(2-((2,4-dinitro-N-(ethyl) phenyl)sulfonamido) ethyl methacrylate) copolymers were synthesized and assembled into a core-shell nano structure in aqueous media. The nano structure demonstrated high sensitivity and selectivity to bio-thiols in vitro and in vivo. Subsequently, pheophorbide a (PhA) was encapsulated as the model photosensitizer. Upon internalization by HepG2 cells, the strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl groups on the PADEE segments were readily cleaved by GSH, during which time the secondary amino groups (pKb = 11.32) were recovered and completely protonated, leading to disassembly of the micelles and rapid release of PhA. Importantly, the consumption of GSH weakened the intracellular antioxidant capacity, resulting in the synergetic accumulation of reactive oxygen species (ROS) under laser irradiation. As a result, this micellar photosensitization system could overcome the antioxidant capacity of advanced stage tumors through a simultaneous extrinsic and intrinsic strategy, facilitating therapeutic efficiency. These results demonstrate that the as-designed micelles provide a versatile photosensitization platform for on-demand PDT.

Topics: Acrylic Resins; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Chlorophyll; Drug Synergism; Female; Glutathione; Hep G2 Cells; Humans; Mice; Mice, Inbred BALB C; Micelles; Oxidation-Reduction; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols

The laver (Porphyra tenera), red seaweed, has been reported to have anticancer activity, but little is known about its molecular mechanisms of action. The objective of this study was to determine the effects of laver extract on cancer cell proliferation, invasion, and metastasis in SK-Hep1 cells using migration and invasion assays. We also investigated the relationship of MMP-2/-9 and TIMP-1/-2 expression at both the protein and gene level in SK-Hep1 human hepatoma carcinoma cells after laver extract treatment. Laver extract inhibited cancer cell growth in a dose-dependent manner. In an invasion assay conducted in Transwell chambers, laver extract showed 19.6 and 27.2% inhibition of cancer cell at 200 and 400 μg/mL, respectively, compared to the control. The mRNA levels of both MMP-2 and MMP-9 were down-regulated by laver extract treatment in a dose-dependent manner. Laver extract, at 400 μg/mL, was inhibited by MMP-2 and MMP-9 expressions by 70.1 and 77.0%, respectively. An inverse relationship in the mRNA contents of MMP-2/-9 and TIMP-1/-2 expressions in SK-Hep1 cells was found by laver extract treatment. Our results demonstrate antimetastatic properties of laver extract in inhibiting the adhesion, invasion, and migration of SK-Hep1 human hepatoma cancer cells.

Topics: Antineoplastic Agents; beta Carotene; Carcinoma, Hepatocellular; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chlorophyll; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phenol; Plant Extracts; Porphyra; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

A preparative column chromatographic method for isolation of carotenoids and chlorophylls from Gynostemma pentaphyllum, a traditional Chinese herb, was developed to evaluate their antiproliferative effects on the hepatoma cell Hep3B. An open column containing 70 g of magnesium oxide-diatomaceous earth (1:2.5, wt/wt) was used to elute carotenoid with 2% ethanol in ethyl acetate and chlorophyll with 50% ethanol in acetone. After high-performance liquid chromatography-mass spectrometry analysis, the carotenoid fraction was composed of all-trans- and cis-isomers of lutein, α-carotene, and β-carotene as well as epoxy-containing carotenoids, while the chlorophyll fraction consisted of chlorophylls a and b and their derivatives. Both carotenoid and chlorophyll fractions as well as lutein and chlorophyll a standards at 50-100 μg/mL were effective against Hep3B cells with a dose-dependent response with the following order: carotenoid fraction > chlorophyll fraction > lutein > chlorophyll a. For all treatments, the cell cycle was arrested in the G₀/G₁ phase, with Hep3B cells undergoing necrosis or apoptosis.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Hepatocellular; Carotenoids; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Chromatography, Liquid; Drugs, Chinese Herbal; G1 Phase; Gynostemma; Humans; Inhibitory Concentration 50; Liver Neoplasms; Necrosis; Plant Leaves; Resting Phase, Cell Cycle; Spectrometry, Mass, Electrospray Ionization

Photodynamic therapy (PDT) is a treatment modality that uses a combination of a photosensitizer and light to induce a photokilling process in the tumor tissue. Recently we re-considered pheophorbide a (Pba), a second-generation photosensitizer that has not yet been thoroughly investigated. Here, we report that Pba irradiated at 14 J/cm(2) induces a strong photodynamic effect in four tumor cell lines, with IC(50) values ranging between 70 and 250 nM. The mechanism of phototoxicity has been investigated in HeLa (IC(50) = 150 nM) and HepG2 (IC(50) = 95 nM) cells. In both cell lines Pba induces lipid peroxidation, as indicated by a marked increase of malonyldialdehyde and oxidized C11 BODIPY(581/591). At high doses (>IC(50)), Pba arrests cell growth completely by activating apoptosis and/or necrosis, while at low doses (

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorophyll; Drug Screening Assays, Antitumor; Drug Synergism; Glutathione Transferase; HeLa Cells; Heme Oxygenase-1; Humans; Inhibitory Concentration 50; Lipid Peroxidation; Liver Neoplasms; Neoplasm Proteins; Oxidative Stress; Photochemotherapy; Radiation-Sensitizing Agents; RNA Interference; RNA, Small Interfering

Multidrug resistance (MDR) is frequently observed after prolonged treatment in human hepatoma with conventional anti-tumor drugs, and photodynamic therapy (PDT) is a recently suggested alternative to overcome MDR. The therapeutic potential of PDT was evaluated in a multidrug resistance (MDR) human hepatoma cell line R-HepG2 with photosensitizer pheophorbide a (Pa).. Our results demonstrated that intracellular accumulation of Pa was not reduced by the overexpression of P-glycoprotein. Pa-based PDT (Pa-PDT) significantly inhibited the growth of R-HepG2 cells with an IC50 value of 0.6 microM. Mechanistic study demonstrated that genomic DNA fragmentation and phosphatidylserine externalization occurred where increase of intracellular singlet oxygen level triggers the phosphorylation of c-Jun N-terminal Kinase (JNK) and leads to activation of intrinsic apoptotic caspases cascade during the Pa-PDT treatment. The cytotoxicity of Pa-PDT, accumulation of sub-G1 population, and depolarization of mitochondrial membrane could be inhibited by JNK inhibitor in the Pa-PDT treated cells. Interestingly, the Pa-PDT induced JNK activation showed inhibitory effect on MDR by the down-regulation of P-glycoprotein in R-HepG2 cells in a dose-dependent manner. In addition, significant reduction of tumor size was obtained in Pa-PDT treated R-HepG2-bearing nude mice with no significant damages in liver and heart.. In summary, our findings provided the first evidence that PDT could inhibit the MDR activity by down-regulating the expression of P-glycoprotein via JNK activation using pheophorbide a as the photosensitizer, and our work proved that Pa-PDT inhibited the growth of MDR hepatoma cells by mitochondrial-mediated apoptosis induction.

Topics: Analysis of Variance; Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Activation; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Photochemotherapy; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays

Scutellaria barbata, a Traditional Chinese Medicine native in southern China, has been widely used for treating liver diseases. In this study, the anti-proliferative effect of Pheophorbide a (Pa), an active component from S. barbata, was examined on a multi-drug resistant (MDR) human hepatoma cell line R-HepG2. Our study showed that Pa could significantly inhibit the growth of R-HepG2 cells with an IC50 value at 25.0 microM after 48 hours treatment. When compared with the parental HepG2 cells, Pa showed weak resistance to R-HepG2. Efflux of Pa out of R-HepG2 cells was not observed as its cellular uptake level showed no significant difference comparing with HepG2 cells. Interestingly, significant reduction of P-glycoprotein expression on Pa-treated R-HepG2 cells was found at both transcriptional and translational levels, leading to reduction of P-glycoprotein activity. In addition, mechanistic study elucidated that Pa induced cell cycle arrest at G2/M phase and inhibited the expressions of G2/M phase cell cycle regulatory proteins, cyclin-A1 and cdc2 in a dose-dependent manner.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; Scutellaria

Photodynamic therapy (PDT) is an alternative anticancer treatment in which direct tumor-cell killing results from selective accumulation of photosensitizers in the tumor sites and phototoxicity occurs when light-activated photosensitizers transfer the energy to oxygen nearby to produce singlet oxygen. The objective of this study was to investigate the effects of PDT using chlorophyll derivatives such as pheophytin a (phe a), pheophytin b (phe b), pheophorbide a (pho a) and pheophorbide b (pho b) as the photosensitizers, and the 660 nm light-emitting diodes (LEDs) irradiation on human hepatocellular carcinoma cells (HuH-7). The drug concentration-dependent inhibition of HuH-7 cell viability was studied under LEDs irradiation (10 mW cm(-2)) at radiant exposure of 5.1 and 10.2 J cm(-2) by MTT assay. Significant inhibition of the survival of HuH-7 cells (<10%) was observed when an irradiation dose of 10.2 J cm(-2) combined with the concentration of 0.5 microg ml(-1) of phe a, 0.125 microg ml(-1) of pho a, 0.25 microg ml(-1) of phe b, and 0.125 microg ml(-1) of pho b were applied. The results from Annexin V-propidium iodide staining revealed that phe a, phe b, pho a and pho b could induce cell death in HuH-7 cells predominantly via a necrotic process. The results from immunoblot analyses exhibited that chlorophyll derivative-mediated PDT initiated cytochrome c release, caspase-9 and caspase-3 activation, followed by poly ADP-ribose polymerase (PARP) cleavage. Thus, apoptosis also occurred in HuH-7 cells after PDT treatment, and the execution of the apoptotic process may be initiated from the loss of mitochondrial function. Our findings demonstrate that both apoptosis and necrosis can be induced in HuH-7 cells after PDT using phe a, phe b, pho a and pho b and LEDs.

Topics: Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Cell Line, Tumor; Chlorophyll; Collagen Type XI; Cytochromes c; Humans; Photochemotherapy; Photosensitizing Agents

Scutellaria barbata has long been used as a Chinese medicine for the treatment of liver diseases such as hepatitis and hepatocellular carcinoma. In the present study, a bioassay-guided method was used to isolate the most active components from Scutellaria barbata. The anti-proliferative effects on human hepatoma HepG2 and Hep3B cells of each fraction at every stage of the purification were monitored. An active component, which is 97% pure by high performance liquid chromatographic analysis, was isolated. Based on nuclear magnetic resonance (NMR) and mass spectrophotometric (MS) analysis, this active component was identified to be pheophorbide a (C35H36N4O5). Mechanistic studies showed that pheophorbide a induced apoptosis in Hep3B cells, a viral-induced hepatoma cell line. However, it was found to be non-toxic in normal human liver cells WRL-68. DNA fragmentation, sub-G1 cell cycle arrest, as well as suppression of the anti-apoptotic protein Bcl-2, release of cytochrome c to the cytosol, and activation of pro-caspase 3 and pro-caspase 9 were observed when Hep3B cells were treated with 40 microg/mL (i. e., 67.5 microM) pheophorbide a for 48 hours. In conclusion, this is the first report describing the isolation of pheophorbide a from Scutellaria barbata using a bioassay-guided isolation method. The anti-proliferative activity and possible mechanisms of action of pheophorbide a on hepatoma Hep3B cells are also discussed.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chemical Fractionation; Chlorophyll; DNA Fragmentation; Flow Cytometry; Humans; Molecular Structure; Plant Extracts; Scutellaria

Photodynamic therapy (PDT) is an effective treatment for cancer by inducing apoptosis or necrosis in the target cells. Pheophorbide a (Pa), a chlorophyll derivative, is a photosensitzier which can induce significant anti-proliferative effects in a number of human cancer cell lines. This study investigated the action mechanism of Pa-mediated photodynamic therapy (Pa-PDT) on the human hepatocellular carcinoma, Hep3B cells. Pa-PDT significantly inhibited the growth of Hep3B cells with an IC50 value of 1.5 microM. Intracellular ROS level was increased in Pa-PDT treated cells and the cytotoxic effect could be reversed when ascorbic acid was applied. Pa was found to be localized in the mitochondria and then induced the target cells to undergo apoptosis, which was confirmed by propidium iodide staining and DNA fragmentation assay. Pa-PDT treatment also led to the depolarization of mitochondrial membrane potential (Deltapim) and a release of cytochrome c from mitochondria to the cytosol. The caspase cascade was activated as shown by a significant decrease of procaspase-3 and -9 in Pa-PDT treated cells in a dose-dependent manner. Furthermore, in nude mice model, Pa-PDT treatment could reduce the tumor size by 57% after 14 days treatment.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Chlorophyll; Humans; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Photochemotherapy; Radiation-Sensitizing Agents; Reactive Oxygen Species; Scutellaria; Xenograft Model Antitumor Assays

Hepatocellular carcinoma (HCC) ranks the sixth among the most common malignancies, with chronic HBV infection being the most common cause. HCC is more common in Africa, China and south-east Asia, but its incidence in the USA, Canada and Australia is rising. Current treatment modalities for HCC are not effective, and only a small percentage of patients are suitable for surgical resection and liver transplantation. Thus other treatment options and improvement of available modalities are badly in need. Photodynamic therapy (PDT) may have some therapeutic benefit for patients with HCC. The study by Tang et al. has implicated that coupled with Pheophorbide a (Pa), PDT may offer therapeutic benefit for patients with HCC. Inhibition of cell proliferation and induction of apoptosis by Pa may be mechanistically responsible for Pa-PDT. As Pa is an extract from a Chinese herbal medicine Scutellaria Barbata, which is widely available, less toxic and less expensive, such a combination may find a better clinical usage in the treatment of HCC patients. More studies are mandatory to fully elucidate the efficacy and mechanisms of Pa-mediated PDT.

Topics: Carcinoma, Hepatocellular; Chlorophyll; Humans; Liver Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents; Scutellaria

Plant chlorophylls and carotenoids are highly colored, conjugated polyenes that play central roles in photosynthesis. Other porphyrins (tetrapyrroles), such as cytochromes, which are structurally related to chlorophyll, participate in redox reactions in many living systems. An unexpected new property of tetrapyrroles, including tetramethyl coproporphyrin III, tetrabenzoporphine, copper chlorin e4 ethyl ester, and of carotenoids including zeaxanthin and alpha-cryptoxanthin is their ability to induce mammalian phase 2 proteins that protect cells against oxidants and electrophiles. The capacity of these compounds to induce the phase 2 response depends upon their ability or that of their metabolites to react with thiol groups, a property shared with all other classes of phase 2 inducers, which show few other structural similarities. Pseudo second-order rate constants of these inducers are correlated with their potency in inducing the phase 2 enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine hepatoma cells. One of the most potent inducers was isolated from chlorophyllin, a semisynthetic water-soluble chlorophyll derivative. Although chlorophyll itself is low in inducer potency, it may nevertheless account for some of the disease-protective effects attributed to diets rich in green vegetables because it occurs in much higher concentrations in those plants than the widely studied 'phytochemicals'.

Topics: Animals; Antioxidants; Carcinoma, Hepatocellular; Chlorophyll; Chlorophyllides; Cytoprotection; Enzyme Induction; Gene Expression Regulation; Liver Neoplasms; Mice; Tetrapyrroles; Tumor Cells, Cultured

The effect on photodynamic therapy (PDT) of using pheophorbide-a as a photosensitizer and Nd: YAG laser (Q-switch) was evaluated. Two hundred micrograms (0.1 ml) of pheophorbide-a was injected into tumors that had been subcutaneously implanted into the backs of nude mice (BALB/c-nu). Interstitial Nd: YAG laser irradiation (Q-switch; mean power 0.5 W, duration 10 min.) was performed 72 hours after pheophorbide-a injection. Forty-eight hours after laser irradiation, the areas of tumor necrosis were measured; these were larger in the group with the injection of pheophorbide-a than in those without. The areas of tumor necrosis after Nd: YAG laser irradiation at a higher mean power (Q-switch; mean power 2 W, control temperature 43-43.5 degrees C, duration 10 min.) were also measured. Again, the areas were larger in the group injected with pheophorbide-a. These results show that the injection of pheophorbide-a and interstitial irradiation using Nd: YAG laser induce a photodynamic reaction, and that this combination is useful in the treatment of deep-seated tumors.

Topics: Animals; Carcinoma, Hepatocellular; Chlorophyll; Liver Neoplasms; Mice; Photochemotherapy; Rabbits

Intra-tumoral concentrations of pheophorbide-a, a new photosensitizer, were measured by high-performance liquid chromatography after administration of pheophorbide-a to nude mice (BALB/c-nu) that had been implanted with human hepatocellular carcinoma. The results were as follows: 1) 0.009 +/- 0.007 microgram/g tissue in 1 mg/kg body p.o. administration group, 2) 0.22 +/- 0.06 microgram/g tissue in 250 mg/kg body p.o. administration group, 3) 0.85 +/- 0.14 microgram/g tissue in 5 mg/kg body intra-peritoneal administration group, 4) 3.42 +/- 2.84 microgram/g tissue in not-injected side, and 116 +/- 24 microgram/g tissue in injected side of 200 micrograms intra-tumoral administration group. Tumors in each group were irradiated using an Nd:YAG laser (Q-switch; mean power 0.5 W, duration 10 min.). Areas of tumor necrosis were larger than in the control group only in the intra-tumoral administration group. These results suggest that no photodynamic reaction occurs if the intra-tumoral pheophorbide-a concentration is less than 0.85 microgram/g tissue.

Topics: Animals; Carcinoma, Hepatocellular; Chemical Phenomena; Chemistry; Chlorophyll; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Photochemotherapy; Tissue Distribution