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[Antitransforming activity of chlorophyllin against trans-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide].

OBJECTIVE: To study the inhibitory effect of chlorophyllin (CHL) on trans-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) induced malignant transformation in human bronchial epithelial cell line (16HBE). METHODS: 10, 50 or 100 micro mol/L CHL were added into the media during the cells transformation induced by BPDE, and the malignant degree of transformed cells were identified by the ConA agglutination test and the assay for anchorage-independent growth and tumorigenicity. RESULTS: After the cells were cultured for 25 times, the time of cells agglutination in groups treated with both CHL and BPDE was increased significantly; the colony formation efficiency in soft agar in groups treated with both CHL and BPDE (7.4 per thousand, 11.4 per thousand and 14.4 per thousand ) showed significant decrease (P < 0.05) in dose-dependent manner, as compared with that in group treated with BPDE alone (19.6 per thousand ). Cells treated with both CHL and BPDE or BPDE alone developed tumor in nude mice, a squamous carcinoma confirmed by histopathological examination. The volume of tumor in groups treated with both CHL and BPDE (0.43 +/- 0.13) cm(2), (0.22 +/- 0.04) cm(2) and (0.10 +/- 0.06) cm(3) was significantly smaller (P < 0.05) and dose-dependent, as compared with that in the group treated with BPDE alone (1.71 +/- 0.37) cm(3). CONCLUSION: CHL showed significant antitransforming ability in human bronchial epithelial cell line induced by BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

Wild-type p53 gene transfer into mutated p53 HT29 cells improves sensitivity to photodynamic therapy via induction of apoptosis.

Photodynamic therapy (PDT) is an effective local cancer treatment that induces cytotoxicity through the intracellular generation of reactive oxygen species. It is generally thought that p53 regulates chemotherapy and radiation therapy responsiveness via apoptosis induction control. The current study investigated whether cellular sensitivity to PDT is increased when a wild-type (wt) p53 status is restored by gene transfer in the established HT9blk Ala273-mutant p53 human colon cancer cell line. The photosensitizer accumulation was similar in both cell lines, and survival measurements using MTT test and clonogenic assays demonstrated that wt p53 transfected cells (HT29A4) were significantly more sensitive to chlorin e6-mediated PDT. P53 protein expression and its functionality as a transcription factor demonstrated through the induction of mdm2 transactivation, were not found to be directly involved in this differential photosensitivity. However, induction of caspase 3 activation (2.6-fold), leading to significant apoptosis induction 24-h after PDT was observed in HT29A4 cells. These results suggest that the introduction of wt p53 in HT29A4 potentiates the cell sensitivity to PDT through the induction of apoptosis in relation to p53 mutational status, but independently of p53 expression level and transcriptional activity.

Apoptosis↗

[Protection of Saccharomyces cerevisiae against oxidative and radiation-caused damage by alkyl hydroxybenzenes].

The effects of C7-alkylhydroxybenzene (C7-AHB) and p-hydroxyethylphenol (tyrosol), chemical analogs of microbial anabiosis autoregulators, on the viability of yeast cells under oxidative stress were investigated. The stress was caused by reactive oxygen species (ROS) produced under gamma irradiation of cell suspensions using doses of 10-150 krad at an intensity of 194 rad/s or by singlet oxygen generated in cells photosensibilized with chlorin e6 (10 micrograms/l). C7-AHB was found to exert a protective effect. The addition of 0.05-0.16 vol% of C7-AHB to cell suspensions 30 min before irradiation protected yeast cells from gamma radiation (50 krad). The protective effect of C7-AHB manifested itself both in the preservation of cell viability during irradiation and in the recovery of their capacity to proliferate after irradiation. In our studies on photodynamic cell inactivation, the fact that the phenolic antioxidant C7-AHB protects cells from intracellular singlet oxygen was revealed for the first time. The analysis of difference absorption spectra of oxidized derivatives of C7-AHB demonstrated that the protective mechanism of C7-AHB involves the scavenging of ROS resulting from oxidative stress. The fact that tyrosol failed to perform a photoprotective function suggests that the antioxidant properties of microbial C7-AHB are not related to their chaperon functions. The results obtained make an important addition to the spectrum of known antioxidant and antistress effects of phenolic compounds.

Antioxidants↗

[CHL prevent colon neoplasms in mice and its selective inhibition on COX-2].

BACKGROUND & OBJECTIVE: Chlorophyllin (CHL) was proved to have strong anti-inducement effect toward many mutagens and epicarcinogens. This study was to explore effect of CHL in preventing colon neoplasms in mice induced by dimethylhydrazine (DMH), and the selective inhibition on cyclooxygenase 2(COX-2). METHODS: The colorectal neoplasms were induced with DMH in mice and the different dose of CHL were administered in different phases, then the prevention of colorectal neoplasms by CHL was examined; The IC50 and growth curve of HT29 cells were measured with MTT method after treated with CHL; The effect of CHL on the expression of COX-1 mRNA and COX-2 mRNA in HT29 cells were measured with RT-PCR method; The effect of CHL on the expression of COX-2 protein and NF-kappaB protein were measured with western blot and immunohistochemistry methods. RESULTS: The incidence of colon cancer, average tumor amount, and percentage of carcinoma in CHL group were significantly lower than those in DMH group (P< .05); CHL could inhibit the growth of HT29 cells. The effects were dose dependent; CHL could selectively inhibit the expression of COX-2mRNA in HT29 cells,the expression of COX-2 protein in colon neoplasms and HT29 cells, and the expression of NF-kappaB protein in colon neoplasms. CONCLUSIONS: CHL could prevent colon neoplasms in mice induced by DMH and the preventive effect related to selective inhibition on COX-2, furthermore, the inhibition of CHL on COX-2 was realized by inhibiting NF-kappaB protein.

Animals↗

[Effect of sodium iron chlorophyllin (SIC) on CFU-E and CFU-GM yields of normal and anemic animal models].

OBJECTIVE: To observe the effect of sodium iron chlorophyllin (SIC) on hematopoiesis and its therapeutic efficacy in anemic mice. METHODS: The effects of SIC on CFU-E and CFU-GM yields of normal and anemic animal models were studied. RESULTS AND CONCLUSIONS: (1) SIC significantly enhanced the CFU-E and CFU-GM yields of mouse bone marrow. (2) SIC also increased the percentage of reticulocyte in mouse. (3) In hemolytic anemia mice induced by i.p. N-acetphenylhydrazine, SIC markedly prevented the decrease of RBC and Hb. (4) In hemorrhagic anemia rats induced by bleedletting, SIC accelerated the normalization of RBC, Hb and elevated the serum iron level and transferrin saturation.

Anemia↗

ABCG2-mediated transport of photosensitizers: potential impact on photodynamic therapy.

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered followed by activation of the photosensitizer by exposure to a light source of a given wavelength. This, in turn, generates reactive oxygen species that induce cellular apoptosis and necrosis in tumor tissue. Based on our earlier finding that the photosensitizer pheophorbide a is an ABCG2 substrate, we explored the ability of ABCG2 to transport photosensitizers with a structure similar to that of pheophorbide a. ABCG2-overexpressing NCI-H1650 MX50 bronchoalveolar carcinoma cells were found to have reduced intracellular accumulation of pyropheophorbide a methyl ester and chlorin e6 compared to parental cells as measured by flow cytometry. The ABCG2 inhibitor fumitremorgin C was found to abrogate ABCG2-mediated transport. Intracellular fluorescence of hematoporphyrin IX, meso-tetra(3-hydroxyphenyl)porphyrin, and meso-tetra(3-hydroxyphenyl)chlorin was not substantially affected by ABCG2. ABCG2-overexpressing cells also displayed decreased intracellular fluorescence of protoporphyrin IX generated by exogenous application of 5-aminolevulinic acid. Mutations at amino acid 482 in the ABCG2 protein known to affect substrate specificity were not found to impact transport of the photosensitizers. In cytotoxicity assays, ABCG2-transfected HEK-293 cells were 11-fold, 30-fold, 4-fold, and >7-fold resistant to PDT with pheophorbide a, pyropheophorbide a methyl ester, chlorin e6, and 5-aminolevulinic acid, respectively. ABCG2-transfected cells were not resistant to PDT with meso-tetra(3-hydroxyphenyl) chlorin. Neither multidrug resistance-associated protein 1 expression nor P-glycoprotein expression appreciably decreased the intracellular fluorescence of any of the photosensitizers examined as determined by flow cytometry. The results presented here implicate ABCG2 as a possible cause for cellular resistance to photodynamic therapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Accumulation of chlorine e6 derivatives in cells with different level of expression and function activity of multidrug resistance protein P-gp 170.

AIM: This work deals with studying the influence of overexpression and function activity of multidrug transporter protein P-gp 170 on the intracellular accumulation of several porphyrin sensitizers, including chlorin e6 (Chl e6), di- (DME) and trimethyl esters (TME) of Chl e6. METHODS: A parental IM9 cell line and two IM9 drug-resistant IM9 sublines were used. With flow cytometry technique the rates of chlorines accumulation in different IM9 cells and values were related to the extent of multidrug resistance (MDR) phenotype. RESULTS: Using P-gp 170-specific antibodies and fluorescent probe JC-1 an increased expression and function activity of P-gp 170 was detected for drug-resistant cells. It was obtained that drug-resistant IM9 cells accumulated chlorines to a lesser extent than the respective wild type, however the differences did not exceed 30%. Verapamil, cyclosporine, known to reverse the MDR phenotype affects equally IM9-Vinc IM9-Tax and Im9 cells. CONCLUSION: Our results demonstrate that the P-gp 170 does not appear to play a role in the intracellular accumulation of chlorines. Since the enhanced activity of P-gp 170 in tumor cells is a factor of their resistance to the action of various antitumor drugs, we conclude that photodynamic therapy could be useful in killing cells exhibiting the MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗