Iwona Wertel

Apoptosis induction in human glioblastoma multiforme T98G cells upon temozolomide and quercetin treatment

Glioblastoma multiforme is the most aggressive primary brain tumour. At the cellular and molecular levels, several mechanisms responsible for apoptosis or autophagy induction are blocked. Identification of molecular targets stimulating cells to initiate programmed cell death should be performed for therapeutic purposes. A promising solution is the combination of temozolomide and quercetin. The aim of our study was to evaluate the effect of both drugs, applied alone and in combinations, on apoptosis and autophagy induction in human glioblastoma multiforme T98G cells. Our results clearly indicate that quercetin and temozolomide induce apoptosis very significantly, having no effect on autophagy induction. At the molecular level, it was correlated with caspase 3 and 9 activation, cytochrome c release from the mitochondrium and a decrease in the mitochondrial membrane potential. Both drugs are also potent Hsp27 and Hsp72 inhibitors. This suggests that the apoptotic signal goes through an internal pathway. Increased expression of caspase 12 and the presence of several granules in the cytoplasm after temozolomide treatment with or without quercetin preceding appearance of apoptosis may suggest that apoptosis is initiated by ER stress. Additionally, it was accompanied by changes in the nuclear morphology from circular to ‘croissant like’.

Increased levels of oxidative stress markers in the peritoneal fluid of women with endometriosis

OBJECTIVE To evaluate 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-isoprostane levels in the peritoneal fluid (PF) of women with endometriosis. STUDY DESIGN One hundred and ten women with laparoscopically and histopathologically confirmed endometriosis and, as reference groups, 119 patients with simple serous (n=78) and dermoid (n=41) ovarian cysts were studied. Peritoneal fluid 8-OHdG and 8-isoprostane concentrations were evaluated by enzyme-linked immunosorbent assays. RESULTS 8-OHdG and 8-isoprostane levels in peritoneal fluid were significantly higher in patients with endometriosis compared with the reference groups. Higher PF 8-OHdG and 8-isoprostane concentrations were observed in patients with advanced stages of endometriosis. A statistically significant positive correlation was found between 8-OHdG and 8-isoprostane levels in peritoneal fluid. CONCLUSION Endometriosis induces greater oxidative stress and frequent DNA mutations in peritoneal fluid than nonendometriotic ovarian cysts. The most severe oxidative stress occurs in the peritoneal cavity of women with more advanced stages of the disease.

Quercetin and Sorafenib as a Novel and Effective Couple in Programmed Cell Death Induction in Human Gliomas

The aim of the present study was to investigate the effect of sorafenib and quercetin on the induction of apoptosis and autophagy in human anaplastic astrocytoma (MOGGCCM) and glioblastoma multiforme (T98G) cell lines. In MOGGCCM cells, sorafenib initiated mainly apoptosis, mediated by the mitochondrial pathway with mitochondrial membrane permeabilization, cytochrome c release to the cytoplasm, and activation of caspase 9 and 3. Additional incubation with quercetin potentiated the pro-apoptotic properties of sorafenib. In T98G cells, autophagy was observed most frequently after the sorafenib treatment. It was accompanied by increased beclin 1 and LC3II expression. Administration of quercetin after the sorafenib treatment resulted in an increased number of autophagic cells. After simultaneous drug application, the level of autophagy was lower in favour of apoptosis. Inhibition of heat shock proteins expression by specific small interfering RNA significantly increased the sensitivity of both the cell lines to induction of apoptosis, but not autophagy. We demonstrated for the first time that sorafenib and quercetin are very effective programmed cell death inducers in T98G and MOGGCCM cells, especially in cells with blocked expression of heat shock proteins.

Tumor-Associated Macrophages and Myeloid-Derived Suppressor Cells as Immunosuppressive Mechanism in Ovarian Cancer Patients: Progress and Challenges

Cancers are complex masses of malignant cells and nonmalignant cells that create the tumor microenvironment (TME). Non-transformed cells of the TME such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) have been observed in the TME of ovarian cancer (OC) patients. Although these subsets may contribute to each step of carcinogenesis and are commonly associated with poor prognosis, still little is known about creation of the protumor microenvironment in OC. In this review, we focused on the nature and prognostic significance of TAMs and MDSCs in OC patients. Moreover, we discuss the main problems and challenges that must be overcome by researchers and clinicians to enrich our knowledge about the immunosuppressive microenvironment of cancers.

Low-Density Lipoproteins Oxidation and Endometriosis

The etiopathogenesis of endometriosis still remains unknown. Recent data provide new valuable information concerning the role of oxidative stress in the pathophysiology of the disease. It has been proved that levels of different lipid peroxidation end products are increased in both peritoneal fluid (PF) and serum of endometriotic patients. We assessed the concentration of oxidized low-density lipoproteins (oxLDL) in PF of 110 women with different stages of endometriosis and 119 women with serous (n = 78) or dermoid (n = 41) ovarian cysts, as the reference groups. PF oxLDL levels were evaluated by ELISA. We found that concentrations of oxLDL in PF of endometriotic women were significantly higher compared to women with serous but not dermoid ovarian cysts. Interestingly, by analyzing concentrations of oxLDL in women with different stages of the disease, it was noted that they are significantly higher only in the subgroup of patients with stage IV endometriosis as compared to women with ovarian serous cysts. In case of minimal, mild, and moderate disease, PF oxLDL levels were similar to those noted in reference groups. Our results indicate that disrupted oxidative status in the peritoneal cavity of women with endometriosis may play a role in the pathogenesis of advanced stages of the disease.

Peritoneal Immune System in Patients with Advance Epithelial Ovarian Cancer

The development of epithelial ovarian cancer is associated with changes in the peritoneal cavity microenvironment. Tumor cells produce different factors, which impairs differentiation, maturation, and function of antigen-presenting cells. In this review, we focus on selected cell populations in the peritoneal cavity immune system and their potential role in epithelial ovarian cancer immunopathogenesis. We devote most attention to dendritic cells because they are considered to be superior in their antigen-presenting ability, compared with both macrophages and B lymphocytes. We also present a brief characterization of tumor-infiltraiting cells in epithelial ovarian cancer patients.

Investigation of glutathione concentrations in peritoneal fluid from women with and without endometriosis

UNLABELLED Changes in the peritoneal fluid (PF) environment have been implicated in the pathogenesis of endometriosis as well as in the decrease of fertility. OBJECTIVE To evaluate the concentration of glutathione in PF of women with endometriosis. PATIENTS Twenty-one patients with endometriosis (I or II rAFS stage, n=11; III or IV rAFS stage, n=10), and 29 patients with follicular or dermoid ovarian cysts (n=17 and n=12, respectively). RESULTS Mean (+/-S.D.) PF glutathione concentration was 0.22+/-0.01 micromol/ml in patients with minimal or mild endometriosis, 0.21+/-0.05 micromol/ml in women with III or IV stage of the disease, 0.24 +/- 0.03 micromol/ml in women with follicle ovarian cysts, and 0.23+/-0.05 micromol/ml in patients with dermoid tumors of ovaries. No significant difference in the peritoneal glutathione level was found between the groups. CONCLUSION These results suggest that PF glutathione is not involved in the progression of endometriosis.

Temozolomide and sorafenib as programmed cell death inducers of human glioma cells

BACKGROUND Gliomas are aggressive brain tumors with very high resistance to chemotherapy. Therefore, the aim of the present study was to investigate the effectiveness of sorafenib and Temozolomide in elimination of human glioma cells through apoptosis and autophagy. METHODS MOGGCCM (anaplastic astrocytoma) and T98G (glioblastoma multiforme) cell lines incubated with sorafenib and/or Temozolomide were used in the experiments. Cell morphology (ER stress, apoptosis, autophagy, and necrosis) was analyzed microscopically while apoptosis and mitochondrial membrane potential were assessed with flow cytometry. Beclin1, LC3, p62, Hsp27, and Hsp72 levels were analyzed by immunoblotting. The activity of caspase 3, 8, and 9 was evaluated fluorometrically. Expression of Hsps was blocked by transfection with specific siRNA. RESULTS In MOGGCCM cells, Temozolomide most frequently induced autophagy, which was accompanied by decreased p62 and increased beclin1 and LC3II levels. Sorafenib initiated mainly apoptosis. Additional incubation with Temozolomide, synergistically potentiated the pro-apoptotic properties of sorafenib, but it was mediated in a caspase-independent way. In T98G cells, the effect of the analyzed drugs on programmed cell death induction was different from that in MOGGCCM cells. Sorafenib induced autophagy, while Temozolomide initiated mainly apoptosis. After simultaneous drug application, apoptosis dominated, suggesting synergistic action of both drugs. Inhibition of Hsp27 and Hsp72 expression increased the sensitivity of both cell lines to ER stress and, to a lesser extent, to induction of apoptosis, but not autophagy. CONCLUSIONS Sorafenib and Temozolomide applied in combination are potent apoptosis inducers in T98G and MOGGCCM cells. ER stress precedes the elimination. Blocking of Hsp expression has a greater impact on ER stress rather than apoptosis induction.

Evaluation of Myeloid and Lymphoid Dendritic Cells in Peritoneal Fluid in Women with Non‐malignant Ovarian Tumors

Problem: Identification of myeloid and lymphoid dendritic cells (DCs) in peritoneal fluid (PF) and peripheral blood (PB) of patients with ovarian pathology.

Influence of ovarian cancer type I and type II microenvironment on the phenotype and function of monocyte-derived dendritic cells

PURPOSEThe aim of this study was to evaluate the influence of ovarian cancer cell lysates isolated from type I or type II ovarian cancer (OC) on the phenotype of monocyte-derived dendritic cells (Mo-DCs) and the cytokine profile. We also determined whether the Mo-DCs and tumor microenvironment, reflected by peritoneal fluid (PF) from type I or II ovarian cancer, could promote regulatory T cell (Tregs) differentiation from naive CD4+ lymphocytes in vitro.RESULTSOur results show a significant role of the ovarian cancer microenvironment reflected by PF from type I or II OC in the inhibition of the DC differentiation process. Interestingly, the percentage of cells co-expressing CD45 and CD14 antigens in the cultures stimulated with PF from both type I and type II OC was higher than in the control. Furthermore, the percentage of cells expressing CD1a, i.e., a marker of immature DCs, was significantly reduced in the cultures stimulated with PF from type I and type II OC. The results obtained show that ovarian cancer type II lysates induce differentiation of monocytes into macrophage-like cells with a CD1a+/HLA-DR+/CD83− phenotype and significantly higher CD86/HLA-DR expression. We show that ovarian cancer type II Mo-DCs are able to prevent an immune response by release of IL-10, whereas OC type I Mo-DCs can promote the generation of Tregs.CONCLUSIONS We demonstrate that each type of ovarian cancer can induce a unique phenotype of DCs and differentiation of Tregs, both associated with immune-suppressive function, which may be an obstacle while developing effective anticancer dendritic cell vaccination.