Protoberberine compounds extracted from Chelidonium majus L. as novel natural photosensitizers for cancer therapy.

BACKGROUND: It has been shown that secondary metabolites occur in Chelidonium majus L. (C. majus) crude extract and milky sap (alkaloids such as berberine, coptisine, chelidonine, chelerythrine, sanguinarine, and protopine) are biologically active compounds with a wide spectrum of pharmacological functions. Berberine, an isoquinoline alkaloid extracted from plants, possesses a wide range of biological activities, including inhibition of growth of a variety of cancer cell lines. PURPOSE AND STUDY DESIGN: In the present study, we investigated the potential anticancer effect of a protoberberine alkaloidal fraction (BBR-F) isolated from the medicinal plant C. majus on HeLa and C33A cervical cancer cells after light irradiation (PDT treatment). METHODS: BBR-F was prepared from an ethanolic extract of stems of C. majus. Identification of alkaloidal compounds was performed using high-performance liquid chromatography - mass spectrometry (HPLC/ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. BBR-F was then biologically evaluated for its anticancer properties. Cytotoxic activity after PDT treatment and without light irradiation (dark cytotoxicity) was determined by colorimetric WST-1 assay. The impact of the protoberberine alkaloidal fraction on the morphology and function of the cells was assessed by fluorescence and confocal microscopy as well as by flow cytometric analysis. To investigate the proinflammatory effect of the extracted natural BBR-F, nitric oxide concentration was determined using the Griess method. RESULTS: An effective reduction in HeLa and C33A cell viability was observed after PDT treatment of BBR-F treated cells. Furthermore, microscopic analysis identified various morphological changes in the studied cells that occurred during apoptosis. Apoptosis of HeLa and C33A cells was also characterized by biochemical changes in cell membrane composition, activation of intracellular caspases, disruption of the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) generation. CONCLUSION: Our results strongly suggest that the components of the natural plant protoberberine fraction (BBR-F) extracted from C. majus may represent promising novel photosensitive agents and can be applied in cancer photodynamic therapy as natural photosensitizers.

Microbiota dysbiosis is associated with HPV-induced cervical carcinogenesis.

Cervical microbial communities serve a crucial role in the persistence and development of oncogenic human papilloma virus (HPV) infections. In the present study, the authors hypothesised that disturbed heterogeneity of microbial flora was associated with HPV-induced carcinogenesis. Swabs of the cervical microbiota were collected from 250 women and the 16S ribosomal DNA was sequenced using a high throughput assay. The swabs of cervical microbiota were grouped according to the community state types (CSTs) as follows: Healthy cervical swabs; swabs taken from low-grade squamous intra-epithelial lesions (LSIL) and swabs taken from high-grade squamous intra-epithelial lesions (HSIL). Analysis of the bacterial classes revealed that the CST cervical swabs of the volunteers were characterised by Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis, however, Gardnerella vaginalis and Lactobacillus acidophilus were absent. In the CST of patients with LSIL the predominant type of bacteria was Lactobacillus acidophilus and Lactobacillus iners, however Lactobacillus crispatus was not detected. Swabs from CST women diagnosed with HSIL exhibited abundant Gardnerella vaginalis and Lactobacillus acidophilus, however, lacked Lactobacillus taiwanensis, Lactobacillus iners and Lactobacillus crispatus. The abundance of Lactobacillus acidophilus in swabs from the healthy women was compared with the swabs from the women with LSIL. The results of the present study indicated that the development of HPV-induced cancer is associated with a high diversity of vaginal microbiota, which is involved in the control of viral persistence, and is therefore indicative of disease prognosis.

Isolation and characterization of a non-specific lipid transfer protein from Chelidonium majus L. latex.

Plant non-specific lipid transfer proteins (nsLTPs) are small basic proteins, which mostly play a role in intracellular lipid transport and antimicrobial defense. Recently it was shown using shotgun proteomic approach that the whole plant extract of Chelidonium majus L. (Papaveraceae) contains relatively abundant nsLTPs. Therefore the aim of the work was to isolate and characterize nsLTP from C. majus latex. Results obtained using PCR approach with degenerate primers showed the presence of nsLTP protein in C. majus root latex, named CmLTP 9.5. The protein consists of 93 aa with a molecular weight of 9.5kDa (NCBI GenBank accession no. ALT21495, coded by KP733898). The mature form of CmLTP 9.5 has a molecular weight of 7.147kDa and contains typical eight strictly conserved cysteine residues. A 3D model of CmLTP 9.5 displays a hydrophobic cavity. The isolated protein fraction tested using diffusion method and critical dilution assay showed strong antibacterial activity towards Gram-negative Campylobacter jejuni as well as Gram-positive Listeria greyi and Clostridium perfringens. Further studies using protein expression system are required to fully understand CmLTP 9.5 mode of action.

Incidence of human papilloma virus in esophageal squamous cell carcinoma in patients from the Lublin region.

AIM: To assess the prevalence of human papilloma virus (HPV) in esophageal squamous cell carcinoma (ESCC) in the south-eastern region of Poland. METHODS: The study population consisted of 56 ESCC patients and 35 controls. The controls were patients referred to our department due to other non-esophageal and non-oncological disorders with no gross or microscopic esophageal pathology as confirmed by endoscopy and histopathology. In the ESCC patients, samples were taken from normal mucosa (56 mucosa samples) and from the tumor (56 tumor samples). Tissue samples from the controls were taken from normal mucosa of the middle esophagus (35 control samples). Quantitative determination of DNA was carried out using a spectrophotometric method. Genomic DNA was isolated using the QIAamp DNA Midi Kit. HPV infection was identified following PCR amplification of the HPV gene sequence, using primers MY09 and MY11 complementary to the genome sequence of at least 33 types of HPV. The sequencing results were computationally analyzed using the basic local alignment search tool database. RESULTS: In tumor samples, HPV DNA was identified in 28 of 56 patients (50%). High risk HPV phenotypes (16 or/and 18) were found in 5 of 56 patients (8.9%), low risk in 19 of 56 patients (33.9%) and other types of HPV (37, 81, 97, CP6108) in 4 of 56 patients (7.1%). In mucosa samples, HPV DNA was isolated in 21 of 56 patients (37.5%). High risk HPV DNA was confirmed in 3 of 56 patients (5.3%), low risk HPV DNA in 12 of 56 patients (21.4%), and other types of HPV in 6 of 56 patients (10.7%). In control samples, HPV DNA was identified in 4 of 35 patients (11.4%) with no high risk HPV. The occurrence of HPV in ESCC patients was significantly higher than in the controls [28 of 56 (50%) vs 4 of 35 (11.4%), P < 0.001]. In esophageal cancer patients, both in tumor and mucosa samples, the predominant HPV phenotypes were low risk HPV, isolated 4 times more frequently than high risk phenotypes [19 of 56 (33.9%) vs 5 of 56 (8.9%), P < 0.001]. A higher prevalence of HPV was identified in female patients (71.4% vs 46.9%). Accordingly, the high risk phenotypes were isolated more frequently in female patients and this difference reached statistical significance [3 of 7 (42.9%) vs 2 of 49 (4.1%), P < 0.05]. Of the pathological characteristics, only an infiltrative pattern of macroscopic tumor type significantly correlated with the presence of HPV DNA in ESCC samples [20 of 27 (74.1%) vs 8 of 29 (27.6%) for ulcerative or protruding macroscopic type, P < 0.05]. The occurrence of total HPV DNA and both HPV high or low risk phenotypes did not significantly differ with regard to particular grades of cellular differentiation, phases in depth of tumor infiltration, grades of nodal involvement and stages of tumor progression. CONCLUSION: Low risk HPV phenotypes could be one of the co-activators or/and co-carcinogens in complex, progressive, multifactorial and multistep esophageal carcinogenesis.

[Prevalence of Leptotrichia amnionii sp. nov. in pregnant women]. / Czestosc wyst powania leptotrichia amnionii sp. nov. u kobiet ciezarnych.

INTRODUCTION: Leptotrichia bacteria belong to the group of Gram-negative anaerobes most frequently colonizing the oral cavity and reproductive organs. Leptotrichia sanquigenes constitute one of the bacterial factors in postpartum bacteremia of mothers and newborns, whilst Leptotrichia amnionii sp. nov., described for the first time in 2002, has turned out to be the etiopathogenetic factor in missed abortion in the second trimester of pregnancy. AIM: The purpose of this study was evaluation of the frequency of occurrence of Leptotrichia amnionii sp. nov. in patients in the 1st, 2nd and 3rd trimester of pregnancy. MATERIALS AND METHODS: The study involved 69 patients. Amplification reactions detecting the presence of Leptotrichia amnionii so. RNA in the smears were performed using specific starters complementary to 16SrRNA Leptotrichia amnionii--primer 1 Lam upper and primer 2 Lsp lower--complementary to Leptotrichia 16SrRNA. After amplification, the specimens were placed on 1% agarose gel in the presence of MassRuler marker (Fermentas). Next, PCR products were ligated to pGEM-T Easy Victor and then competent E. coli DH5 alpha cells were transformed with pGEM vector with an insert. Plasmid DNA isolation was performed using Plasmid Mini kit from A&A Biotechnology. Sequencing of inserts from the purified plasmids was performer in Molecular Biology Techniques Laboratory in the Department of Biology at Adam Mickiewicz University in Poznan. RESULTS: In the group of 69 pregnant patients Leptotrichia amnionii sp. nov. was identified in 8 women, which is 11.6%. CONCLUSION: Recently identified Leptorichia amnionii sp. nov. is a bacterie, which relatively frequently occurs in pregnant women.