chlorophyll-a and Liver-Neoplasms

High-efficient vectors for the co-delivery of photosensitizers and chemotherapeutics were urgently needed for the combination therapy. In this work, a redox-responsive micelle (PCL-SS-CMC-GA) was prepared for the co-delivery of doxorubicin (DOX) and pheophorbide A (PHA). Poly-ε-caprolactone was linked to carboxymethyl chitosan through a disulfide bond, which was easily broken in the reductive solution to release the payloads. The charge conversion property and glycyrrhetinic acid (GA) targeting ligand of the micelles effectively extended the average residence time (up to 18 times) in circulation and improved their intracellular uptake by HepG2 cells. The micelles exhibited an enhanced tumor accumulation and near infrared (NIR) imaging performance. More interestingly, this nanoplatform could fully exert the synergistic effect of DOX and PHA to improve the inhibition efficiency (with an inhibitory rate of 80.5 %) in vivo. With impressive photo-chemo theranostic and NIR imaging capability, PCL-SS-CMC-GA@DOX/PHA showed great potential in image-guided treatment of liver cancer.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Survival; Chitosan; Chlorophyll; Doxorubicin; Drug Carriers; Drug Combinations; Drug Liberation; Drug Synergism; Female; Hep G2 Cells; Humans; Infrared Rays; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Micelles; Nanostructures; Optical Imaging; Oxidation-Reduction; Photochemotherapy; Radiation-Sensitizing Agents; Tumor Burden; Xenograft Model Antitumor Assays

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

Lung adenocarcinoma is a leading human malignancy with fatal prognosis. Ninety percent of the deaths, however, are caused by metastases. The model of subcutaneous tumor xenograft in nude mice was adopted to study the growth of control and photodynamically treated tumors derived from the human A549 lung adenocarcinoma cell line. As a side-result of the primary studies, observations on the metastasis of these tumors to the murine lungs were collected, and reported in the present paper. The metastasizing primary tumors were drained by a prominent number of lymphatic vessels. The metastatic tissue revealed the morphology of well-differentiated or trans-differentiated adenocarcinoma. Further histological and histochemical analyses demonstrated the presence of golden-brown granules in the metastatic tissue, similar to these found in the tumor tissue. In contrast to the primary tumors, the electron paramagnetic resonance spectroscopy revealed no nitric oxide - hemoglobin complexes (a source of intense paramagnetic signals), in the metastases. No metastases were found in other murine organs; however, white infarctions were identified in a single liver. Taken together, the A549-derived tumors growing subcutaneously in nude mice can metastasize and grow on site in the pulmonary tissue. Thus, they can represent an alternative for the model of induced metastatic nodule formation, following intravenous administration of the cancerous cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Chlorophyll; Electron Spin Resonance Spectroscopy; Humans; Injections, Subcutaneous; Light; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Photosensitizing Agents; Skin Neoplasms; Transplantation, Heterologous

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

Chemoprevention by chlorophyll (Chl) was investigated in a rat multi-organ carcinogenesis model. Twenty-one male F344 rats in three gavage groups (N = 7 rats each) received five daily doses of 250 microg/kg [(3)H]-aflatoxin B(1) ([(3)H]-AFB(1)) alone, or with 250 mg/kg chlorophyllin (CHL), or an equimolar amount (300 mg/kg) of Chl. CHL and Chl reduced hepatic DNA adduction by 42% (P = 0.031) and 55% (P = 0.008), respectively, AFB(1)-albumin adducts by 65% (P < 0.001) and 71% (P < 0.001), respectively, and the major AFB-N(7)-guanine urinary adduct by 90% (P = 0.0047) and 92% (P = 0.0029), respectively. To explore mechanisms, fluorescence quenching experiments established formation of a non-covalent complex in vitro between AFB(1) and Chl (K(d) = 1.22 +/- 0.05 microM, stoichiometry = 1Chl:1AFB(1)) as well as CHL (K(d) = 3.05 +/- 0.04 microM; stoichiometry = 1CHL:1AFB(1)). The feces of CHL and Chl co-gavaged rats contained 137% (P = 0.0003) and 412% (P = 0.0048) more AFB(1) equivalents, respectively, than control feces, indicating CHL and Chl inhibited AFB(1) uptake. However, CHL or Chl treatment in vivo did not induce hepatic quinone reductase (NAD(P)H:quinone oxidoreductase) or glutathione S-transferase (GST) above control levels. These results are consistent with a mechanism involving complex-mediated reduction of carcinogen uptake, and do not support a role for phase II enzyme induction in vivo under these conditions. In a second study, 30 rats in three experimental groups were dosed as in study 1, but for 10 days. At 18 weeks, CHL and Chl had reduced the volume percent of liver occupied by GST placental form-positive foci by 74% (P < 0.001) and 77% (P < 0.001), respectively compared with control livers. CHL and Chl reduced the mean number of aberrant crypt foci per colon by 63% (P = 0.0026) and 75% (P = 0.0004), respectively. These results show Chl and CHL provide potent chemoprotection against early biochemical and late pathophysiological biomarkers of AFB(1) carcinogenesis in the rat liver and colon.

Topics: Aflatoxin B1; Animals; Anticarcinogenic Agents; Carcinogens; Chlorophyll; Colonic Neoplasms; Liver Neoplasms; Male; Random Allocation; Rats; Rats, Inbred F344

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 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