Osteogenic Potential of Human Dental Pulp Stem Cells (hDPSCs) Growing on Poly L-Lactide-Co-Caprolactone and Hyaluronic Acid (HYAFF-11TM) Scaffolds.

Bone tissue engineering using different scaffolds is a new therapeutic approach in regenerative medicine. This study explored the osteogenic potential of human dental pulp stem cells (hDPSCs) grown on a hydrolytically modified poly(L-lactide-co-caprolactone) (PLCL) electrospun scaffold and a non-woven hyaluronic acid (HYAFF-11™) mesh. The adhesion, immunophenotype, and osteogenic differentiation of hDPSCs seeded on PLCL and HYAFF-11™ scaffolds were analyzed. The results showed that PLCL and HYAFF-11™ scaffolds significantly supported hDPSCs adhesion; however, hDPSCs' adhesion rate was significantly higher on PLCL than on HYAFF-11™. SEM analysis confirmed good adhesion of hDPSCs on both scaffolds before and after osteogenesis. Alizarin red S staining showed mineral deposits on both scaffolds after hDPSCs osteogenesis. The mRNA levels of runt-related transcription factor 2 (Runx2), collagen type I (Coll-I), osterix (Osx), osteocalcin (Ocn), osteopontin (Opn), bone sialoprotein (Bsp), and dentin sialophosphoprotein (Dspp) gene expression and their proteins were higher in hDPSCs after osteogenic differentiation on both scaffolds compared to undifferentiated hDPSCs on PLCL and HYAFF-11™. These results showed that PLCL scaffolds provide a better environment that supports hDPSCs attachment and osteogenic differentiation than HYAFF-11™. The high mRNA of early osteogenic gene expression and mineral deposits observed after hDPSCs osteogenesis on a PLCL mat indicated its better impact on hDPSCs' osteogenic potential than that of HYAFF-11™, and hDPSC/PLCL constructs might be considered in the future as an innovative approach to bone defect repair.

Characterization of Biological Properties of Dental Pulp Stem Cells Grown on an Electrospun Poly(l-lactide-co-caprolactone) Scaffold.

Poly(l-lactide-co-caprolactone) (PLCL) electrospun scaffolds with seeded stem cells have drawn great interest in tissue engineering. This study investigated the biological behavior of human dental pulp stem cells (hDPSCs) grown on a hydrolytically-modified PLCL nanofiber scaffold. The hDPSCs were seeded on PLCL, and their biological features such as viability, proliferation, adhesion, population doubling time, the immunophenotype of hDPSCs and osteogenic differentiation capacity were evaluated on scaffolds. The results showed that the PLCL scaffold significantly supported hDPSC viability/proliferation. The hDPSCs adhesion rate and spreading onto PLCL increased with time of culture. hDPSCs were able to migrate inside the PLCL electrospun scaffold after 7 days of seeding. No differences in morphology and immunophenotype of hDPSCs grown on PLCL and in flasks were observed. The mRNA levels of bone-related genes and their proteins were significantly higher in hDPSCs after osteogenic differentiation on PLCL compared with undifferentiated hDPSCs on PLCL. These results showed that the mechanical properties of a modified PLCL mat provide an appropriate environment that supports hDPSCs attachment, proliferation, migration and their osteogenic differentiation on the PLCL scaffold. The good PLCL biocompatibility with dental pulp stem cells indicates that this mat may be applied in designing a bioactive hDPSCs/PLCL construct for bone tissue engineering.

Comparison of p53, HSP90, E-cadherin and HPV in oral lichen planus and oral squamous cell carcinoma.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease. There are no markers that can be used to identify the risk of a malignant transformation of OLP to oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemical staining was performed among 56 patients with OLP and 66 patients with OSCC for p53, HSP90 and E-cadherin expression and presence of HPV16/18. RESULTS: Significant differences in p53 and HSP90 expression between OLP and OSCC were found (p = 0.01 and p = 0.006, respectively). A positive correlation between HSP90 and p53 expression was seen in OLP (p = 0.017). Univariate analysis identified HSP90 expression and HPV16/18 presence as prognostic factors for overall survival time (OS) (p < 0.05). In multivariate analysis, only HSP90 expression was an independent prediction factor for shorter OS of OSCC patients (p = 0.016). CONCLUSIONS: The present study suggests that cooperation between p53 and HSP90 as well as between HPV16/18 and HSP90 exists in OLP and may affect the biological behaviour of OLP. The observed expression of HSP90 and p53 in OLP and their increase in OSCC suggests that these proteins participate in the malignant transformation of OLP. HSP90 may be a potential independent prognostic biomarker that can predict poor prognosis in OSCC.

Dental Pulp Stem Cell-Derived Secretome and Its Regenerative Potential.

The therapeutic potential of the dental pulp stem (DSC) cell-derived secretome, consisting of various biomolecules, is undergoing intense research. Despite promising in vitro and in vivo studies, most DSC secretome-based therapies have not been implemented in human medicine because the paracrine effect of the bioactive factors secreted by human dental pulp stem cells (hDPSCs) and human exfoliated deciduous teeth (SHEDs) is not completely understood. In this review, we outline the current data on the hDPSC- and SHED-derived secretome as a potential candidate in the regeneration of bone, cartilage, and nerve tissue. Published reports demonstrate that the dental MSC-derived secretome/conditional medium may be effective in treating neurodegenerative diseases, neural injuries, cartilage defects, and repairing bone by regulating neuroprotective, anti-inflammatory, antiapoptotic, and angiogenic processes through secretome paracrine mechanisms. Dental MSC-secretomes, similarly to the bone marrow MSC-secretome activate molecular and cellular mechanisms, which determine the effectiveness of cell-free therapy. Many reports emphasize that dental MSC-derived secretomes have potential application in tissue-regenerating therapy due to their multidirectional paracrine effect observed in the therapy of many different injured tissues.

The association between p53 protein phosphorylation at serine 15, serine 20 and sensitivity of cells isolated from patients with ovarian cancer and cell lines to chemotherapy in in vitro study.

BACKGROUND: The association between p53 protein phosphorylated at serine 15 (Ser15), serine 20 (Ser20) and ovarian tumor cell sensitivity after chemotherapy was analyzed in order to define the influence of p53 activation on tumor cell sensitivity to chemotherapy. METHODS: The study was performed on ovarian cancer cell line (OvBH-1), colon adenocarcinoma metastasis to ovary (SW626) and on cells isolated from ascitic fluids from patients with ovarian cancer: with (p53+) or without (p53-) p53 nuclear protein accumulation. p53 protein, Ser15, Ser20, Bax, Noxa and PgP protein expression was evaluated by means of immunocytochemical staining before and after chemotherapy. Cell viability after treatment was estimated using MTT assay. RESULTS: Cell lines and tumor cells p53+, p53- revealed a significant decrease in cell survival after camptothecin, paclitaxel, cisplatin treatment, compared to the control group (p < 0.01). In p53+ group, the expression of Ser20 significantly increased after camptothecin and paclitaxel (p < 0.05). Ser15, Ser20, Bax, Noxa expression correlated with MTT and depended on p53+, p53- tumor cell and the drug used (p < 0.05). Expression of Bax and Noxa were dependent on the type of tumor cells and drug used. The correlation between Ser15, Ser20 and Bax, Noxa expression was found in cell lines and tumor cells (p < 0.05). CONCLUSIONS: Our study suggests that the relation between Ser15 or Ser20 and tumor cell viability might reflect their role in tumor sensitivity on chemotherapy in dependent p53 protein status. Revealed association between p53 protein phosphorylated at Ser15, Ser20 and Bax, Noxa protein expression determined the apoptotic activity of tumor cells.

The Association Between HSP90/topoisomerase I Immunophenotype and the Clinical Features of Colorectal Cancers in Respect to KRAS Gene Status.

AIM: The aim of this study was to investigate heat shock protein 90 (HSP90) and topoisomerase I (Topo I) expression and the association between both proteins and clinicopathological parameters of colorectal cancer (CRC), in order to describe their role in tumor biology regarding to Kirsten Ras (KRAS) - positive/negative cases. MATERIALS AND METHODS: Expression of HSP90 and Topo I, and KRAS gene mutations were estimated in primary CRCs. RESULTS: HSP90/Topo I immunophenotype correlated with gender, Duke staging, tumor grade and lymph node metastasis (p<0.01). Positive correlation was found between KRAS mutation and HSP90 expression (p=0.02). HSP90, Topo I expression, and co-expression of HSP90/Topo I correlated with unfavorable parameters of CRCs in respect to KRAS gene status (p<0.001). CONCLUSION: Our results revealed that cooperation between HSP90 and Topo I expression exists in CRCs, independently of KRAS gene status, suggesting that co-expression of both proteins might be considered as a double target on individual tumor cells.

[The role of cancer stem cells in progressive growth and resistance of ovarian cancer: true or fiction?]. / Udzial nowotworowych komórek macierzystych w progresywnym wzroscie i opornosci raka jajnika: prawda czy mit?

Growing evidence indicates that biological heterogeneity of ovarian cancer is associated with a small subpopulation of cancer cells existing within tumor tissue and defined as cancer stem cells (CSCs). This small group of ovarian cells possesses the capacity of self-renewal. Recent data revealed that progression, metastasis and relapse of ovarian cancers are related to the behavior of cancer stem cells. However, how ovarian CSCs maintain their migration properties is still unclear. The clinical relevance of CSCs has been supported by emerging evidence, showing that CSCs are resistant to conventional chemotherapy of ovarian cancer. Identification of biomarkers of ovarian cancer stem cells seems to be important for target therapy. Therapeutic strategies aimed at eliminating CSCs in ovarian cancers might extend disease survival and limit recurrence. This review will describe the current knowledge of ovarian CSCs biology and contribution of these cells to metastasis and chemoresistance of ovarian cancer as well as the possibility to use target therapy of ovarian CSCs.

Expression and Interactions Between Cell Adhesion Molecules CD44v6 and E-Cadherin in Human Gliomas.

BACKGROUND: Gliomas are a heterogenous group of tumors that show the same histological features but differ in their behavior. Gliomas are characterized by biological aggressiveness and extensive infiltrative growth into surrounding healthy brain tissue. OBJECTIVES: In this study we estimated CD44v6 and E-cadherin expression and correlation between CD44v6 and E-cadherin in relation to glioma malignancy. We also analyzed simultaneous expression of CD44v6 and E-cadherin in the same tumor sample in order to determine the biological tumor behavior. MATERIAL AND METHODS: Expression of CD44v6 and E-cadherin was evaluated on ninety-two formalin-fixed paraffin-embedded glioma tissue blocks using immunohistochemistry (IHC). RESULTS: CD44v6 expression was found in 71.6% of gliomas. There was a statistically significant difference between the frequency of positive cases for CD44v6 expression in low (grade I) vs. high (grade IV) as well as in grade I vs. grade II of glioma malignancy (p = 0.001). E-cadherin membrane staining was observed in 28.8% of gliomas. No significant differences were observed between E-cadherin expression and grade of gliomas (p > 0.05). However, re-expression of E-cadherin was found in grade II gliomas. In this group, E-cadherin expression was revealed in 43.3% of the cases. In order to define the relationship between CD44v6 expression and E-cadherin, we analyzed the simultaneous expression of CD44v6 and E-cadherin in the same glioma sample in the whole group and in respect to the degree of glioma malignancy. A positive correlation between studied biomarkers was observed in the analyzed gliomas (p = 0.004) but a simultaneous expression of CD44v6 and E-cadherin revealed no significant differences in respect to glioma malignancy. CONCLUSIONS: Our results showed that the level of E-cadherin might reflect different biological features of gliomas, whereas CD44v6 is associated with tumor cell malignancy. The simultaneous presence of CD44v6 and E-cadherin in a set of low-grade gliomas indicates that both these molecules might strengthen cell migration and may be a hallmark of glioma invasive growth.

The association between the p53/topoisomerase I and p53/ topoisomerase IIalpha immunophenotypes and the progression of ovarian carcinomas.

BACKGROUND: In in vitro studies it has been revealed that p53 protein expression might regulate topoisomerase I (topo I) and topoisomerase IIalpha (topo IIalpha) levels in tumor cells. So far, the association between the p53 protein and topo I and topo IIalpha expression and its impact on ovarian carcinoma progression has not been analyzed. OBJECTIVES: The aim of the study was to examine the association between topo I and topo IIalpha expression and p53 protein overexpression with respect to the morphological features and progressive growth of ovarian tumors. MATERIAL AND METHODS: The expression of the studied biomarkers was estimated by immunohistochemical staining in tumor sections from 136 malignant and 30 benign ovarian neoplasms. RESULTS: Significant differences for topo I, topo IIalpha and p53 expression between malignant and benign tumors were observed (p < 0.01). The expression of topo IIalpha and p53 protein was associated with advanced stages of ovarian carcinomas (p < 0.01). Differences between topo I-positive cases and low (G1) and high (G3) tumor grade had only borderline significance (p = 0.07). In ovarian carcinomas, positive correlations between topo I and topo IIalpha, topo I and p53 and topo Ilalpha and p53 protein expression were revealed (p = 0.001). No relationship between the studied biomarkers was found in benign ovarian tumors (p > 0.05). p53/topo I and p53/topo IIalpha immunophenotypes were associated with advanced stages of ovarian carcinoma (p = 0.045 and p = 0.009, respectively), p53/topo IIalpha positive ovarian carcinomas were more frequently observed in high than in low tumor grades and the differences were only of borderline significance (p = 0.07). CONCLUSIONS: Current findings suggest that on the one hand, cooperation between topo I, topo IIalpha and p53 protein participates in the progressive growth of ovarian tumors. On the other hand, simultaneous expression of the studied proteins identifies the subgroup of ovarian cancers with aggressive biological features which might be considered in therapy.

Expression of p53 protein phosphorylated at serine 20 and serine 392 in malignant and benign ovarian neoplasms: correlation with clinicopathological parameters of tumors.

INTRODUCTION: The modification of p53 protein by phosphorylation plays an important role in its stabilization and the regulation of its biological properties. The study investigated the expression of p53 protein phosphorylated at serine 20 (Ser20) and Ser392 and the association between clinicopathological parameters of ovarian neoplasms with respect to p53 protein overexpression. METHODS: p53 protein expression was evaluated on tissues from malignant and benign ovarian tumors. Protein expression was measured in a subset of the specimens using immunohistochemistry. RESULTS: The correlation between p53 protein overexpression and p53-Ser392 phosphorylation was found in ovarian carcinomas (P = 0.001, r = +0.27). In the total group of ovarian carcinomas, significant differences were observed in p53 protein overexpression between well (G1) and poor (G3) tumor grades (P = 0.005) and between serous and endometrioid types of tumor (P = 0.04), whereas p53-Ser20 phosphorylation was associated with advanced International Federation of Gynecology and Obstetrics stage (P = 0.004) and high tumor grade (P = 0.02). In p53-positive ovarian carcinomas, p53-Ser392 phosphorylation was associated with advanced tumor stage (P = 0.02) and high tumor grade (P = 0.049). p53-Ser20 phosphorylation was associated with low tumor grade of p53-positive ovarian carcinomas (P = 0.02) and with high tumor grade of p53-negative ovarian carcinomas (P = 0.02). CONCLUSIONS: These results revealed that p53 phosphorylation at Ser20 and Ser392 is an early event in ovarian tumor development. The authors suggest that the expression of p53 protein phosphorylated at Ser20 and Ser392 in ovarian carcinomas determines their individual clinical features depending on p53 protein status and may be useful biological biomarkers characterizing their behavior.

Cytogenetic and molecular cytogenetic characterization of the stable ovarian carcinoma cell line (OvBH-1).

Detailed characterization and identification of cancer cell lines is the basis for the credibility of experimental studies. Therefore, chromosomal analysis should be routinely included in the protocol of cell line characterization and in the protocols of experimental studies performed on cell lines. In 2000, our group established and characterized cytomorphologically and immunophenotypically a new cell line, OvBH-1, which was derived from the ascitic fluid cells of an untreated patient with ovarian clear-cell adenocarcinoma. The aim of the current study was to characterize OvBH-1 cytogenetically and to monitor its stability by comparison of morphologic, immunohistochemical, and cytogenetic features between the early (135) and late (385) passages. Conventional and molecular cytogenetic analyses (fluorescence in situ hybridization and spectral karyotyping) of OvBH-1 revealed the following hypotriploid karyotype with random translocations: der(2)t(2;13),der(4)t(4;22), der(5)t(2;5). Complex rearrangements involving chromosomes 3, 15, and 20 were also found. FISH analysis with a p53 probe indicated the deletion of this region in two out of three copies of chromosome 17. The morphologic and immunophenotypic features, as well as the karyotypes observed in OvBH-1 in passages 135 and 385, were comparable. The monoclonality of the cell line was confirmed in a single cell cloning experiment. Our study indicated that OvBH-1 is characterized by a distinct karyotype and remains stable over 250 passages. Taking into account its thermosensitivity, its unusual karyotype, and its stability, this line can be considered as a valuable model for various experimental studies.

Type IV collagen and CD44v6 expression in benign, malignant primary and metastatic ovarian tumors: correlation with Ki-67 and p53 immunoreactivity.

OBJECTIVE: Loss of basement membrane (BM) components, such as type IV collagen has been demonstrated in ovarian cancer, but the associations with other molecules like CD44v6, involved in metastatic process of ovarian carcinoma, have not been fully analyzed. This study investigates the expression of type IV collagen, CD44v6 molecule in correlation with p53 and Ki-67 presence in primary and metastatic lesion of ovarian carcinoma to define their role in metastases of ovarian carcinoma. METHODS: The expression of type IV collagen, CD44v6, p53, and Ki-67 was evaluated on frozen tissue sections from primary ovarian tumors (malignant n = 37, benign n = 16), metastatic lesions (n = 29) and ascitic fluid cells (n = 28). Protein expression of all studied biomarkers was evaluated in a subset of specimens using immunohistochemistry (IHC). RESULTS: Type IV collagen expression in the primary ovarian carcinoma was positively correlated with International Federation of Gynecology and Obstetrics (FIGO) stage and tumor grade. Significant difference was observed for type IV collagen immunoreactivity in carcinoma cells in effusions when compared to corresponding primary tumors (P < 0.001) and metastatic lesions (P < 0.001). Likewise down-regulation of type IV collagen expression was seen in primary ovarian carcinomas (P = 0.01), ascitic fluid cells (P < 0.001), and metastases (P = 0.003) when compared to benign ovarian neoplasms. CD44v6 expression was detected in a comparable percentage of primary carcinomas (51%) and metastatic lesions (52%). In cells isolated from ascitic fluid, CD44v6 immunopositivity was observed in 43% of cases. A comparative analysis of primary and metastatic tumors and carcinoma cells in effusion did not reveal differences in expression of CD44v6. Positivity of CD44v6 was found in 2/16 (12%) of benign ovarian neoplasms. There were no significant differences between CD44v6 expression in benign neoplasms compared to primary malignant tumors and metastases (P > 0.05). CD44v6 expression in primary ovarian carcinomas was associated with higher tumor grade (P = 0.01) and histological type of tumors (P = 0.01). An inverse relationship of type IV collagen expression with p53 and CD44v6 positivity in benign and malignant ovarian tumors was found (P > 0.01). Type IV collagen expression was inversely correlated with p53 status (P = 0.03) in metastatic lesions. A slight trend toward an inverse correlation between Ki-67 and type IV collagen expression was observed in both benign and malignant ovarian tumors and metastases. CONCLUSIONS: Our data suggest that observed inverse correlation of type IV collagen expression with p53, CD44v6, and slight with Ki-67 positivity in primary benign and malignant tumors indicates that these molecules may cooperate in the invasion and progression of ovarian carcinomas.

Temperature-sensitive ovarian carcinoma cell line (OvBH-1).

OvBH-1 cells from a patient with ovarian clear cell carcinoma were established and their biochemical status was analyzed. Cells grown at 37 degrees C exhibited normal cell cycle distribution, whereas the cells shifted to 31 degrees C were arrested in the G(2) / M phase of the cell cycle. Immunochemical analysis using anti-p53 antibodies (DO-1, PAb240, PAb421, and PAb1620) revealed that only the DO-1 antibody reacted with p53 with a high and similar percentage at both temperatures. PAb240 reacted with a low percentage of cells at 37 degrees C and no reaction was observed at 31 degrees C. PAb421 antibody stained a significantly lower percentage of cells at 37 degrees C than at 31 degrees C. Cells were not stained with PAb1620 antibody and were negative for antibodies against p21(WAF1) and MDM2 proteins independently of the temperature. Sequencing of all coding exons of the p53 gene demonstrated only a neutral genetic polymorphism, i.e. a G-to-A substitution (GAG to GAA) at nucleotide position 13 432. Thus, the observed temperature sensitivity of OvBH-1 cells cannot be ascribed to a p53 primary structure mutation. Based upon immunochemical analyses, we consider, however, that p53 in nuclei of OvBH-1 cells is in a highly unstable conformation. Furthermore, the N-terminal portion of the p53 protein at Ser20 has not been modified, and Lys373 and / or Ser378 of the C-terminus is acetylated and / or phosphorylated. The nuclear location signal of p53 is preserved. Induction of MDM2 protein is uncoupled from the cell regulatory machinery and the induction of p21(WAF1) by p53 is impaired in OvBH-1 cells.

Relations between immunologically different p53 forms, p21(WAF1) and PCNA expression in ovarian carcinomas.

The role of tumor suppressor p21WAF1 expression in epithelial ovarian cancer has not been definitely explained and the clarification of mutual p53 and p21WAF1 relations considering proliferative activity seems to be very important for understanding of a functional link between p53 and cell-cycle control. Therefore the expression of p53 and p21WAF1 was assessed immunohistochemically in a series of 50 ovarian carcinomas considering clinicopathological variables. The reactivity of three anti-p53 monoclonal antibodies (DO-7, PAb240, PAb1620) recognizing immunologically distinct forms of p53 were analysed in relation to p21WAF1 level in individual patients. p21WAF1 was expressed in 24 (48%) of all cases. The detection of p53 protein was related to the antibody applied and DO-7 antibody appears to be better than both PAb240 and PAb1620. However, independently of antibody used significant inter- and intratumoral heterogeneity in p53 and p21WAF1 expression was revealed. The identification of different p53/p21WAF1 phenotypes reflect the complex and multiple relations between these two cell-cycle regulators indicating that in ovarian carcinomas p21WAF1 activation may be both p53-dependent and p53-independent. High cell proliferation was usually accompanied by undetectable or weak p21WAF1 staining. There was no significant correlation between p53 and p21WAF1 expression and histology, stage and grade of ovarian carcinomas (p>0.05).

[Expression of p-glycoprotein in malignant solid human tumors]. / Wystepowanie p-glikoproteiny w zlosliwych, litych nowotworach ludzkich.

One of the main causes of cancer resistance to chemotherapy is multidrug resistance. The role of P-glyco-protein in different solid human cancers was assessed by taking into account clinico-pathological parameters. The application of inhibitors when multidrug resistance appeared was discussed. The participation of other proteins and enzymes in adding resistance to the cancer cells was stressed.