pheophorbide-a and Breast-Neoplasms

Photodynamic therapy (PDT) has been extensively explored as a promising alternative therapeutic approach for many malignant tumors. However, the PDT system generally involves unsatisfactory tumor specificity and nonspecific accumulation of photosensitizers around the target cancer cells, leading to phototoxic damage to adjacent healthy normal cells. In this study, we developed pheophorbide a (Pheo a)/human epidermal growth factor receptor 2 (HER2) targeting peptide (epitope form, HLTV, PEG2-LTVSPWY)-co-conjugated methoxy poly(ethylene glycol)-block-poly(L-lysine hydrochloride) (PEG-PLL)/hyaluronic acid (HA) (P3H2) polymeric micelles via a self-assembly method for HER2-targeted PDT treatment for breast cancer, thereby enhancing the PDT efficacy. The synthesized P3H2 polymeric micelles were spherical, with an average diameter of 125.7 ± 21.2 nm in an aqueous solution. The results of

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chlorophyll; Female; Humans; Hyaluronic Acid; Micelles; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Polylysine; Receptor, ErbB-2

It has been reported that intestinal urate excretion is increased at chronic kidney disease (CKD) state. In this report, whether uremic toxins are involved in the upregulation of intestinal breast cancer resistance protein (BCRP), an intestinal urate exporter, was examined.. Uremic toxins that were increased at least 15-fold at CKD state were selected for investigation. Caco-2 cells were exposed to these uremic toxins at clinically relevant concentrations. mRNA was quantified by real-time PCR, and flow cytometry was utilized to measure BCRP protein and function in Caco-2 cells. Transcellular secretory transport of [(14) C]urate was determined utilizing Transwell studies after uremic toxin exposure.. Indoxyl sulfate (IS) treatment alone resulted in ∼ 3-fold increase in BCRP mRNA in Caco-2 cells. Membrane protein expression of BCRP in Caco-2 cells also was increased by 1.8-fold after treatment with IS. Intracellular accumulation of pheophorbide A, a selective BCRP substrate, was decreased by 22% after IS treatment for 3 days. Consistent with these findings, transcellular secretory transport of urate across Caco-2 cell monolayers was increased by 22%.. Intestinal urate secretion may be increased at CKD state partially by upregulation of intestinal BCRP by uremic toxins such as IS.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Breast Neoplasms; Caco-2 Cells; Chlorophyll; Humans; In Vitro Techniques; Indican; Intestinal Mucosa; Intestinal Secretions; Kidney; Neoplasm Proteins; Real-Time Polymerase Chain Reaction; Renal Insufficiency, Chronic; RNA, Messenger; Up-Regulation; Uric Acid

Photodynamic therapy (PDT) has emerged as an effective treatment for various solid tumors. The transcription factor NRF2 is known to protect against oxidative and electrophilic stress; however, its constitutive activity in cancer confers resistance to anti-cancer drugs. In the present study, we investigated NRF2 signaling as a potential molecular determinant of pheophorbide a (Pba)-based PDT by using NRF2-knockdown breast carcinoma MDA-MB-231 cells. Cells with stable NRF2 knockdown showed enhanced cytotoxicity and apoptotic/necrotic cell death following PDT along with increased levels of singlet oxygen and reactive oxygen species (ROS). A confocal microscopic visualization of fluorogenic Pba demonstrated that NRF2-knockdown cells accumulate more Pba than control cells. A subsequent analysis of the expression of membrane drug transporters showed that the basal expression of BCRP is NRF2-dependent. Among measured drug transporters, the basal expression of breast cancer resistance protein (BCRP; ABCG2) was only diminished by NRF2-knockdown. Furthermore, after incubation with the BCRP specific inhibitor, differential cellular Pba accumulation and ROS in two cell lines were abolished. In addition, NRF2-knockdown cells express low level of peroxiredoxin 3 compared to the control, which implies that diminished mitochondrial ROS defense system can be contributing to PDT sensitization. The role of the NRF2-BCRP pathway in Pba-PDT response was further confirmed in colon carcinoma HT29 cells. Specifically, NRF2 knockdown resulted in enhanced cell death and increased singlet oxygen and ROS levels following PDT through the diminished expression of BCRP. Similarly, PDT-induced ROS generation was substantially increased by treatment with NRF2 shRNA in breast carcinoma MCF-7 cells, colon carcinoma HCT116 cells, renal carcinoma A498 cells, and glioblastoma A172 cells. Taken together, these results indicate that the manipulation of NRF2 can enhance Pba-PDT sensitivity in multiple cancer cells.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Colonic Neoplasms; Female; Gene Knockdown Techniques; Gene Silencing; Genetic Vectors; Humans; Laser Therapy; Lasers; Lentivirus; Neoplasm Proteins; Neoplasms; NF-E2-Related Factor 2; Peroxiredoxin III; Photochemotherapy; Radiation-Sensitizing Agents; Reactive Oxygen Species; RNA, Small Interfering; Transduction, Genetic

Multidrug resistance (MDR), usually mediated by overexpression of efflux transporters such as P-gp, ABCG2 and/or MRP1, remains a major obstacle hindering successful cancer chemotherapy. There has been great interest in the development of inhibitors towards these transporters to circumvent resistance. However, since the inhibition of transporter is not specific to cancer cells, a decrease in the cytotoxic drug dosing may be needed to prevent excess toxicity, thus undermining the potential benefit brought about by a drug efflux inhibitor. The design of potent MDR modulators specific towards resistant cancer cells and devoid of drug-drug interactions will be needed to effect MDR reversal.. Recent evidence suggests that the PTEN/PI3K/Akt pathway may be exploited to alter ABCG2 subcellular localization, thereby circumventing MDR. Three PPARγ agonists (telmisartan, pioglitazone and rosiglitazone) that have been used in the clinics were tested for their effect on the PTEN/PI3K/Akt pathway and possible reversal of ABCG2-mediated drug resistance.. The PPARγ agonists were found to be weak ABCG2 inhibitors by drug efflux assay. They were also shown to elevate the reduced PTEN expression in a resistant and ABCG2-overexpressing cell model, which inhibit the PI3K-Akt pathway and lead to the relocalization of ABCG2 from the plasma membrane to the cytoplasma, thus apparently circumventing the ABCG2-mediated MDR.. Since this PPARγ/PTEN/PI3K/Akt pathway regulating ABCG2 is only functional in drug-resistant cancer cells with PTEN loss, the PPARγ agonists identified may represent promising agents targeting resistant cells for MDR reversal.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Benzoates; Biological Transport; Breast Neoplasms; Chlorophyll; Drug Resistance, Neoplasm; Endocytosis; Female; HEK293 Cells; Humans; Kinetics; MCF-7 Cells; Neoplasm Proteins; Phosphatidylinositol 3-Kinase; Pioglitazone; PPAR gamma; Protein Transport; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA Interference; Rosiglitazone; Signal Transduction; Telmisartan; Thiazolidinediones; Transfection; Up-Regulation

Breast cancer is conventionally treated by surgery and radiotherapy, with adjuvant chemotherapy and hormonotherapy as supplementary treatments. However, such treatments are associated with adverse side effects and drug resistance. In this study, Pheophorbide a (Pa), a photosensitizer isolated from Scutelleria barbata, was analysed for its antiproliferative effect on human breast tumour cells. The IC (inhibitory concentration)(50) of the combined treatment of Pa and photodynamic therapy (Pa-PDT) on human breast tumour MCF-7 cells was 0.5 µm. Mechanistic studies in MCF-7 cells demonstrated that Pa was localized in the mitochondria, and reactive oxygen species were found to be released after Pa-PDT. Apoptosis was the major mechanism responsible for the tumour cell death, and mitochondrial membrane depolarization and cytochrome c release highlighted the role of mitochondria in the apoptotic mechanism. Up-regulation of tumour suppressor protein p53, cleavage of caspase-9 and poly (ADP-ribose) polymerase suggested that the caspase-dependent pathway was induced, while the release of apoptosis-inducing factors demonstrated that the apoptosis was also mediated by the caspase-independent mechanism. In vivo study using the mouse xenograft model showed a significant inhibition of MCF-7 tumour growth by Pa-PDT. Together, the results of this study provide a basis for understanding and developing Pa-PDT as a cure for breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Cytochromes c; DNA Fragmentation; Female; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Photochemotherapy; Photosensitizing Agents; Plant Extracts; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Scutellaria; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

In this study, we reported on the synthesis and biological evaluation of radiolabeled fluorescent dye conjugated bovine serum albumin nanoparticles within the size range 190-210 nm. The bovine serum albumin nanoparticles (BSANPs) were prepared using a desolvation method, and chemical cross-linking was performed using gluteraldehyde. Furthermore, pheophorbide-a (PH-A) was loaded on the BSANPs. The results obtained from dynamic light scattering and electron microscopy have proved that nanoparticles are highly monodisperse and near-spherical shaped. The photo-physical properties of the PH-A-BSANPs were obtained using the spectrophotometric techniques. According to the results, PH-A and BSANPs show high non-covalent interaction. PH-A loaded nanoparticles were labeled with (99m)Tc and the radio-labeling efficiency was determined as 90 ± 1.2%. Biodistribution studies of (99m)Tc labeled PH-A-BSANPs and PH-A were carried out using female Albino Wistar rats, and (99m)Tc-PH-A-BSANPs showed a significantly higher uptake in the breast and uterus than (99m)Tc-PH-A. Cell culture study was carried out in MCF-7 cell line (human breast adenocarcinoma cell line). According to the cell culture studies, (99m)Tc-PH-A-BSANPs showed a higher uptake than (99m)Tc-PH-A. Moreover, PH-A-BSANPs demonstrated good photo-physical properties and BSANPs increased the uptake of PH-A on to the MCF-7 cell line. These results confirm that (99m)Tc labeled PH-A-BSANPs could be utilized for radioimaging.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chlorophyll; Cross-Linking Reagents; Drug Carriers; Female; Glutaral; Humans; Light; Magnetic Resonance Spectroscopy; Mass Spectrometry; Microscopy, Electron; Microscopy, Fluorescence; Molecular Imaging; Nanoparticles; Particle Size; Radiation-Sensitizing Agents; Radiopharmaceuticals; Rats; Rats, Wistar; Scattering, Radiation; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet; Technetium; Tissue Distribution

Scutellaria barbata is a traditional Chinese medicine for cancer treatments. Pheophorbide-a (Pa), one of the active components isolated from this herbal medicine has been proposed to be a potential natural photosensitizer for photodynamic therapy. The anti-tumor effect of pheophorbide-a based photodynamic therapy (Pa-PDT) has been successfully demonstrated in a wide range of human malignant cell lines. However, the effectiveness of Pa-PDT has not yet been evaluated on human breast cancer, which is documented as the second common and the fifth most lethal cancer worldwide.. The cytotoxicity of Pa-PDT was evaluated by using an estrogen receptor (ER)-negative human breast adenocarcinoma cell line MDA-MB-231. The involvement of mitochondria was revealed by the change of mitochondrial membrane potential and the increase of intracellular reactive oxygen species (ROS). The hallmarks of apoptosis, ER stress and autophagy were also assessed by DNA fragmentation, Western blotting, and immunostaining assays.. Pa-PDT showed inhibitory effect on the growth of MDA-MB-231 cells with an IC(50) value of 0.5 microM at 24h. Mitogen-activated protein kinase (MAPK) pathway was found to be triggered, where activation of c-Jun N-terminal kinase (JNK) and inhibition of extracellular signal-regulated kinase (ERK) were occurred in the Pa-PDT-treated cells. Our findings suggested that Pa-PDT exhibited its anti-tumor effects by the activation of mitochondria-mediated apoptosis and the ERK-mediated autophagy in MDA-MB-231 cells.. The present study suggested Pa-PDT is a potential protocol for the late phase human breast cancer, and it is the first study to demonstrate the Pa-PDT induced autophagy contributed to the anti-tumor effects of Pa-PDT on human cancer cells.

Topics: Adenocarcinoma; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Plant Extracts; Protein Kinase Inhibitors; Radiation-Sensitizing Agents; Receptors, Estrogen; Scutellaria

A new class of specific breast cancer resistance protein (BCRP) inhibitors was identified, showing no inhibition of the ATP binding cassette (ABC) transporters P-gp and MRP1. Some of these modulators inhibit BCRP with high potency; they are only slightly less potent than Ko143 and could serve as promising lead structures for the design of novel effective BCRP inhibitors. These inhibitors are structurally related to tariquidar (XR9576) and belong to a library of multidrug-resistance modulators synthesized by our research group. The absence of the tetrahydroisoquinoline substructure appears to play a crucial role for specificity; we found that the presence of this substructure is not essential for interaction with BCRP. To determine the type of interaction between pheophorbide A and compounds with and without the tetrahydroisoquinoline substructure, various substrate pheophorbide A concentrations were used in enzyme kinetics assays. The resulting data show that these compounds share a noncompetitive-type interaction with pheophorbide A. Experiments with imatinib and pheophorbide A revealed a mixed-type interaction. The combination of imatinib and compounds with and without the tetrahydroisoquinoline substructure resulted in a positive cooperative effect, indicating that imatinib engages a binding site distinct from that of the new compounds on one side and distinct from that of pheophorbide A on the other side as well. The results of this study suggest that the category of BCRP-specific inhibitors, which includes only fumitremorgin C, Ko143 and analogues, and novobiocin needs to be extended by this new class of inhibitors, which possess three key characteristics: specificity, potency, and low toxicity.

Topics: Adenosine; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzamides; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Diketopiperazines; Drug Resistance, Multiple; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Imatinib Mesylate; Indoles; Neoplasm Proteins; Novobiocin; Piperazines; Pyrimidines; Quinolines; Structure-Activity Relationship

The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments.. RT-PCR, western blot and fluorescent immunocytochemical analyses were performed on ABCG2-overexpressing human breast cancer (MCF-MX100) cells, human and rat brain microvessel endothelial (HBEC and RBE4, respectively), and rat glial cells.. RT-PCR analysis detected ABCG2 mRNA in all the cell culture systems. Western blot analysis with anti-ABCG2 monoclonal BXP-21 antibody detected a robust band at approximately 72 kDa in the ABCG2-overexpressing MCF-MX100 cell line, whereas low expression was found in human and rat brain cell systems. Immunofluorescence microscopy detected predominant plasma membrane localization of ABCG2 in MCF-MX100 cells but weak signal in all brain cellular compartments. In the presence of ABCG2 inhibitors, the accumulation of (3)H-mitoxantrone and pheophorbide A, two established ABCG2 substrates, was significantly increased in MCF-MX100 cells but not in the human and rodent brain cell culture systems.. Our data show low endogenous ABCG2 protein expression, localization and activity in cultures of human and rat brain microvessel endothelial and glial cells.

Topics: Animals; Animals, Newborn; Astrocytes; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Brain; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Endothelial Cells; Female; Fluorescent Antibody Technique, Indirect; Humans; Indoles; Microcirculation; Microglia; Mitoxantrone; Neoplasm Proteins; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection; Tritium

The synthesis, physico-chemical properties, cellular localization and photocytotoxicity of estradiol-pheophorbide a conjugates in estrogen-dependent cancer and vascular endothelial cells are described with the aim of increasing the photodynamic activity by targeting the nucleus of both tumor and blood vessel cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Chlorophyll; DNA Damage; Endothelium, Vascular; Estradiol; Humans; Photochemotherapy; Reactive Oxygen Species; Spectrometry, Fluorescence; Spectrum Analysis

Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2(-/-) knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649-15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 micro M of the cyclin-dependent kinase inhibitor UCN-01 or 1 micro M of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 micro M tariquidar, and ABCG2-transfected cells were 6-7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Camptothecin; Chlorophyll; Drug Resistance, Neoplasm; Humans; Irinotecan; Neoplasm Proteins; Quinolines; Staurosporine; Topotecan

Three pheophorbide-related compounds (1-3) were isolated from the leaves and stems of Clerodendrum calamitosum. The methyl ester of 3 (6) and the known (10S)-hydroxypheophytin a (7) also were isolated from leaves of the related plant Clerodendrum cyrtophyllum. Compounds 1 and 6 were isolated for the first time as naturally occurring products from a plant source. All structures were elucidated by detailed spectroscopic analysis. Biological evaluation showed that 1 and 2 exhibited strong cytotoxicity against human lung carcinoma (A549), ileocecal carcinoma (HCT-8), kidney carcinoma (CAKI-1), breast adenocarcinoma (MCF-7), malignant melanoma (SK-MEL-2), ovarian carcinoma (1A9), and epidermoid carcinoma of the nasopharynx (KB), and its etoposide- (KB-7d), vincristine- (KB-VCR), and camptothecin-resistant (KB-CPT) subclones. Compound 3 was less cytotoxic than 1 and 2. Compounds 4-6, the methyl esters of 1-3, showed strongly increased cytotoxicity compared with the parent acids. Interestingly, 6 was the most active derivative among these compounds. Compound 7 was inactive.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Camptothecin; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Drug Resistance; Etoposide; Female; Humans; Ileal Neoplasms; KB Cells; Kidney Neoplasms; Lung Neoplasms; Magnetic Resonance Spectroscopy; Melanoma; Molecular Structure; Ovarian Neoplasms; Plant Leaves; Plant Stems; Plants, Medicinal; Stereoisomerism; Structure-Activity Relationship; Taiwan; Tumor Cells, Cultured; Vincristine