Magdalena Chmielewska

In vitro effect of quercetin on human gastric carcinoma: targeting cancer cells death and MDR.

The benefits of plant polyphenols as chemotherapeutic agents are of great interest due to their possible anti-cancerogenic activities. Results available up to now suggest that flavonoid quercetin induces lethal effect in many types of tumours and may sensitize resistant cells to drugs. The aim of our study was to examine the effect of quercetin on human gastric carcinoma cells and to determine mode of its action. Parental EPG85-257P cell line and its daunorubicin-resistant variant EPG85-257RDB were used as cell models. Our data revealed that quercetin exerted antiproliferative impact on studied cells (with IC(50) value of 12 μM after 72 h), mainly through induction of apoptosis. In sensitive cells cytostatic drug and flavonoid had synergistic effects, in EPG85-257RDB cells quercetin acted as a chemosensitizer. Its impact on resistance mechanism involved decrease of P-glycoprotein expression, inhibition of drug transport and downregulation of ABCB1 gene expression. The results demonstrate that quercetin may be considered as a prospective drug to overcome classical resistance in gastric cancer cells.

Quercetin as a potential modulator of P-glycoprotein expression and function in cells of human pancreatic carcinoma line resistant to daunorubicin.

P-glycoprotein (P-gp) is one of the ABC transporters responsible for the resistance of several tumours to successful chemotherapy. Numerous agents are capable of interfering with the P-gp-mediated export of drugs but unfortunately most of them produce serious side effects. Some plant polyphenols, including the flavonol quercetin (Q), manifest anti-neoplastic activity mainly due to their influence on cell cycle control and apoptosis. Reports are also available which show that Q may intensify action of cytostatic drugs and suppress the multidrug resistance (MDR) phenomenon. The study aimed at determination if Q sensitizes cells resistant to daunorubicin (DB) through its effect on P-gp expression and action. The experiments were conducted on two cell lines of human pancreatic carcinoma, resistant to DB EPP85-181RDB and sensitive EPP85-181P as a comparison. Cells of both lines were exposed to selected concentrations of Q and DB, and then membranous expression of P-gp and its transport function were examined. The influence on expression of gene for P-gp (ABCB1) was also investigated. Results of the studies confirmed that Q affects expression and function of P-gp in a concentration-dependent manner. Moreover it decreased expression of ABCB1. Thus, Q may be considered as a potential modulator of P-gp.

Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2γ, which was downregulated during development similarly to XLAP2ω. Embryonic isoforms XLAP2ω and XLAP2γ were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2β were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2β was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2β was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains.

Correlation between levels of expression of minichromosome maintenance proteins, Ki-67 proliferation antigen and metallothionein I/II in laryngeal squamous cell cancer

MCM2, MCM3 and MCM7 are minichromosome maintenance proteins found in dividing cells and they play a role in DNA synthesis. Increased MCM expression level is observed in cells of different cancer types. Additionally, metallothioneins (MT-I/II) are involved in control of cell proliferation and differentiation and changes of their expression are observed in many types of cancer. Ki-67 is known cancer cell proliferation antigen currently used in prognostic evaluation. The study material consisted of 83 laryngeal squamous cell cancer (LSCC) cases and 10 benign hypertrophic lesions of larynx epithelium as a control group. For the present study, laryngeal cancer cell line HEp-2 and human keratinocytes were employed, and to evaluate expression of all the markers, immunohistochemical method (IHC), immunofluorescence (IF) and western blot analysis were used. Statistical analysis showed strong positive correlation between expression of MCM2, MCM3, MCM7 and Ki-67 antigen in LSCC. Additionally, moderate positive correlation was observed between MCM3 and MT-I/II expression. In cancer cells, the level of expression of MCM3, MCM2, MCM7 and Ki-67 markers was increasing with the grade of LSCC malignancy. IF and western blot analysis showed higher MCM2, MCM3, MCM7 expression in HEp-2 cells in comparison to their expression in keratinocytes. MCM proteins might be useful markers of cell proliferation in LSCC.

Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability

Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

Expression of metallothioneins I and II in kidney of doxorubicin-treated rats

Metallothioneins I/II (MT) are commonly expressed in mammalian tissues and are highly inducible in the response to stress conditions. Doxorubicin (DOX) intoxication promotes oxidative stress and subsequent apoptosis leading to kidney damage. The present study investigates a correlation between endogenous MT expression and DOX-induced apoptosis in renal tubular cells. Experiments were conducted on Buffalo rats receiving DOX (8 mg/kg b.w. for 3 weeks) versus control rats injected with saline. The histopathological alterations and apoptosis (TUNEL) were evaluated in tissue sections. MT expression and tissue localization was examined using immunohistochemical method (IHC). Western blot (WB) was used to evaluate pro-caspase-3, active caspase-3 and MT expression level in tissue homogenates. Examination of renal tissue revealed severe nephrotoxicity in DOX-treated animals. Apoptosis was observed in distal convoluted tubular cells, whereas MT was detected in proximal tubular cells. A significant increase in pro-caspase-3, active caspase-3 and MT expression levels (WB) were seen in DOX group. Positive correlations between histopathological lesions, apoptosis and MT expression were observed. The results obtained in this study could suggest the protective and antiapoptotic effect of MT expression in renal proximal tubular cells under DOX intoxication.

The frequency of degenerating germ cells in the ovaries of water frogs ( Pelophylax esculentus complex)

Pelophylax esculentus is the fertile hybrid of P. ridibundus and P. lessonae. During gametogenesis, one of the parental genomes is removed from the germ line cells, whereas the other one is clonally transmitted to the gametes. In hybrids, development of gonads is delayed in comparison with parental species. This may result from complex processes of genome elimination in female tadpoles at Gosner stages 28–46, potentially responsible for increased degeneration of germ cells in developing gonads from the very beginning of sexual differentiation to ovaries with diplotene oocytes, respectively. In this work, we revealed that germ cells died by apoptosis, as detected by expression of active caspase-3 using immunohistochemical method. The main group of degenerating germ cells was primary oogonia, however, in P. lessonae and P. ridibundus also secondary oogonia and diplotene oocytes were found. The number of degenerating germ cells was significantly higher in ovaries of P. esculentus. In hybrids, positive correlations were demonstrated between Gosner stage and gonadal volume, Gosner stage and the number of degenerating germ cells, gonadal volume and number of degenerating germ cells. These observations suggest that increased rate of apoptosis in germ cells, probably as the result of improper genome elimination, may be responsible for delayed maturation of ovaries in P. esculentus.

The programmed DNA elimination and formation of micronuclei in germ line cells of the natural hybridogenetic water frog Pelophylax esculentus

DNA elimination is a radical form of gene silencing and occurs both in somatic and germ cells. The programmed DNA elimination occurs during gametogenesis in interspecies hybrids that reproduce by hybridogenesis (stick insects, fishes, and amphibians) and concerns removal of whole genomes of one of the parental species and production of clonal gametes propagating the genome of the other species. The cellular mechanisms differ considerably in hybridogenetic insects and fishes but remains unknown in edible frogs Pelophylax esculentus, natural hybrids between Pelophylax lessonae and Pelophylax ridibundus. Here we report DNA elimination mechanism in early developing gonads of diploid and triploid hybrid frogs, studied by TEM, immunofluorescence, and cytochemistry. In gonocytes of both sexes (primary oogonia and prespermatogonia), micronuclei emerge as detached nuclear buds formed during interphase. We found depletion of nuclear pore complexes in micronuclear membrane and chromatin inactivation via heterochromatinization followed by degradation of micronuclei by autophagy. Micronuclei formation does not lead to apoptotic cell death showing that genome elimination is a physiological process. Chromatin elimination via micronuclei in P. esculentus is unique among hybridogenetic animals and contributes to broadening the knowledge about reproductive modes in animals.