Regulation of mitochondrial dynamics in 2-methoxyestradiol-mediated osteosarcoma cell death.

Osteosarcoma (OS) is one of the most malignant tumors of childhood and adolescence. Research on mitochondrial dynamics (fusion/fission) and biogenesis has received much attention in last few years, as they are crucial for death of cancer cells. Specifically, it was shown that increased expression of the cytoplasmic dynamin-related protein 1 (Drp1) triggers mitochondrial fission (division), which activates BAX and downstream intrinsic apoptosis, effectively inhibiting OS growth. In the presented study, human OS cells (metastatic 143B OS cell line) were incubated with 2-methoxyestradiol (2-ME) at both physiologically and pharmacologically relevant concentrations. Cell viability was determined by the MTT assay. Confocal microscopy and western blot methods were applied to examine changes in Drp1 and BAX protein levels. Mitochondrial Division Inhibitor 1, MDIVI-1, was used in the study to further examine the role of Drp1 in 2-ME-mediated mechanism of action. To determine quantitative and qualitative changes in mitochondria, electron microscopy was used. 2-ME at all used concentrations increased mitochondrial fission and induced autophagy in OS cells. At the concentration of 1 µM 2-ME increased the area density of mitochondria in OS cells. Subsequent, upregulated expression of Drp1 and BAX proteins by 2-ME strongly suggests the activation of the intrinsic apoptosis pathway. We further observed 2-ME-mediated regulation of glycolytic state of OS cells. Therefore, we suggest that changes of mitochondrial dynamics may represent a novel mechanism of anticancer action of 2-ME. This finding may open new approaches to improve the efficacy of chemotherapy in the treatment of OS, however, it has to be confirmed by in vivo studies.

Impact of gold nanoparticles shape on their cytotoxicity against human osteoblast and osteosarcoma in in vitro model. Evaluation of the safety of use and anti-cancer potential.

Due to development of nanotechnology and gold nanoparticles (AuNPs) increasing use in different areas of medicine, especially in oncology, better understanding of their potential cytotoxicity is necessary to protect patients safety. Shape and size of AuNPs is an important modulator of their cytotoxicity. Therefore, we investigated the cytotoxicity of AuNPs rods (≈39 nm length, 18 nm width), AuNPs stars (≈ 215 nm) and AuNPs spheres (≈ 6.3 nm) against human fetal osteoblast (hFOB 1.19), osteosarcoma (143B, MG63) and pancreatic duct cell (hTERT-HPNE) lines by MTT and neutral-red uptake assay. Moreover, influence of AuNPs on level of proapoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) was measured by western blot. Cellular uptake of nanoparticles and ultrastructure changes were examined by transmission electron microscopy (TEM). In the present study we have proven that AuNPs stars are the most cytotoxic against human cells. We observed that cancer cells are more susceptible to AuNPs cytotoxic effect. Furthermore, AuNPs rods and AuNPs stars caused increased expression of Bax and decreased expression of Bcl-2 protein in osteosarcoma cells. We found that AuNPs penetrated through the cell membrane and caused ultrastructural changes. Our results clearly demonstrated that the cytotoxicity of AuNPs was shape-dependent. AuNPs stars with the highest anti-cancer potential were also the most cytotoxic type of tested NPs, whereas AuNPs spheres which appears to be the safest one had small anti-cancer potential.

Melanization of Bomirski hamster amelanotic melanoma cells (Ab line) depends on the type of culture medium.

INTRODUCTION: The effect of melanogenesis intensity on melanoma biology remains an open question, and the biological differences between melanotic and amelanotic melanoma cells have not yet been satisfactorily documented. As a result, the melanization of melanoma cells in in vitro cultures is not considered among experimental procedures. The aim of this study was to investigate the effect of the medium used to culture Bomirski amelanotic Ab melanoma cells on the melanogenesis process. MATERIAL AND METHODS: Amelanotic melanoma cells (Ab) were cultured in two media recommended for in vitro melanoma cell cultures, RPMI and DMEM. The melanization was evaluated by determining the melanin and tyrosinase presence in the cells using spectrophotometrical and western blot methods, respectively. Changes in Ab melanoma cells' ultrastructure were determined using electron microscopy (EM). RESULTS: The medium with higher level of tyrosine (DMEM) induced significant melanization of amelanotic melanoma cells (Ab) after only 24 h, while the RPMI medium, with a lower level of tyrosine, weakly affected melanin production. Melanization of Ab cells was paralleled by an increase in the amount of tyrosinase protein. Induced melanization was easily observed on EM-micrographs in the form of newly formed melanosomes containing melanin pigment. Melanosomes at stages from one (I) to four (IV) were observed. CONCLUSIONS: Culture medium has an important effect on the in vitro biology of amelanotic melanoma cells, since it can affect the rate of cellular melanization. The appropriate medium should be carefully selected, taking into account the known biology of the melanoma cells being used.

Role of Oxidative and Nitro-Oxidative Damage in Silver Nanoparticles Cytotoxic Effect against Human Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma is one of the most aggressive human malignancies, where the 5-year survival rate is less than 4% worldwide. Successful treatment of pancreatic cancer is a challenge for today's oncology. Several studies showed that increased levels of oxidative stress may cause cancer cells damage and death. Therefore, we hypothesized that oxidative as well as nitro-oxidative stress is one of the mechanisms inducing pancreatic cancer programmed cell death. We decided to use silver nanoparticles (AgNPs) (2.6 and 18 nm) as a key factor triggering the reactive oxygen species (ROS) and reactive nitrogen species (RNS) in pancreatic ductal adenocarcinoma cells (PANC-1). Previously, we have found that AgNPs induced PANC-1 cells death. Furthermore, it is known that AgNPs may induce an accumulation of ROS and alteration of antioxidant systems in different type of tumors, and they are indicated as promising agents for cancer therapy. Then, the aim of our study was to evaluate the implication of oxidative and nitro-oxidative stress in this cytotoxic effect of AgNPs against PANC-1 cells. We determined AgNP-induced increase of ROS level in PANC-1 cells and pancreatic noncancer cell (hTERT-HPNE) for comparison purposes. We found that the increase was lower in noncancer cells. Reduction of mitochondrial membrane potential and changes in the cell cycle were also observed. Additionally, we determined the increase in RNS level: nitric oxide (NO) and nitric dioxide (NO2) in PANC-1 cells, together with increase in family of nitric oxide synthases (iNOS, eNOS, and nNOS) at protein and mRNA level. Disturbance of antioxidant enzymes: superoxide dismutase (SOD1, SOD2, and SOD3), glutathione peroxidase (GPX-4) and catalase (CAT) were proved at protein and mRNA level. Moreover, we showed cells ultrastructural changes, characteristic for oxidative damage. Summarizing, oxidative and nitro-oxidative stress and mitochondrial disruption are implicated in AgNPs-mediated death in human pancreatic ductal adenocarcinoma cells.

Silver nanoparticles of different sizes induce a mixed type of programmed cell death in human pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma, with the high resistance to chemotherapeutic agents, remains the fourth leading cause of cancer-death in the world. Due to the wide range of biological activity and unique properties, silver nanoparticles (AgNPs) are indicated as agents with potential to overcome barriers involved in chemotherapy failure. Therefore, in our study we decided to assess the ability of AgNPs to kill pancreatic cancer cells, and then to identify the molecular mechanism underlying this effect. Moreover, we evaluated the cytotoxicity of AgNPs against non-tumor cell of the same tissue (hTERT-HPNE cells) for comparison. Our results indicated that AgNPs with size of 2.6 and 18 nm decreased viability, proliferation and caused death of pancreatic cancer cells in a size- and concentration-dependent manner. Ultrastructural analysis identified that cellular uptake of AgNPs resulted in apoptosis, autophagy, necroptosis and mitotic catastrophe. These alterations were associated with increased pro-apoptotic protein Bax and decreased level of anti-apoptotic protein Bcl-2. Moreover, AgNPs significantly elevated the level of tumor suppressor p53 protein as well as necroptosis- and autophagy-related proteins: RIP-1, RIP-3, MLKL and LC3-II, respectively. In addition, we found that PANC-1 cells were more vulnerable to AgNPs-induced cytotoxicity compared to pancreatic non-tumor cells. In conclusion, AgNPs by inducing mixed type of programmed cell death in PANC-1 cells, could provide a new therapeutic strategy to overcome chemoresistance in one of the deadliest human cancer.

Selective cytotoxicity of vanadium complexes on human pancreatic ductal adenocarcinoma cell line by inducing necroptosis, apoptosis and mitotic catastrophe process.

The pancreatic cancer is the fourth leading cause of cancer-related death and characterized by one of the lowest five-year survival rate. The current therapeutic options are demonstrating minimal effectiveness, therefore studies on new potential anticancer compounds, with non-significant side effects are highly desirable. Recently, it was demonstrated that vanadium compounds, in particular organic derivatives, exhibit anticancer properties against different type of tumor as well as favorable biodistribution from a pancreatic cancer treatment perspective. In this research, we showed selective cytotoxic effect of vanadium complexes, containing phenanthroline and quinoline as an organic ligands, against human pancreatic ductal adenocarcinoma cell line (PANC-1), compared to non-tumor human immortalized pancreas duct epithelial cells (hTERT-HPNE). Results exhibited that vanadium complexes inhibited autophagy process in selective cytotoxic concentration as well as caused the cell cycle arrest in G2/M phase associated with mitotic catastrophe and increased level of reactive oxygen species (ROS). Moreover, in higher concentration, vanadium derivatives induced a mix type of cell death in PANC-1 cells, including apoptotic and necroptotic process. Our investigation emphasizes the anticancer potential of vanadium complexes by indicating their selective cytotoxic activity, through different process posed by alternative type of cell deaths to apoptosis-resistant cancer cells. Further studies supporting the therapeutic potential of vanadium in pancreatic cancer treatment is highly recommended.

Ultrastructural characteristics of myenteric plexus in patients with colorectal cancer.

INTRODUCTION: It have been found previously that colorectal cancer (CRC) is accompanied by atrophy of myenteric plexuses (MPs) localized close to the tumor. The aim of the study was to compare ultrastructure of MPs localized in the unchanged part of the colon wall distant to CRC tumor with the ultrastructure of MPs in the vicinity of CRC tumor. MATERIAL AND METHODS: The present study was conducted using post-operative material derived from 11 patients with CRC. Samples of colon wall were taken from the margin of cancer invasion and from a macroscopically unchanged segment of the large intestine, immediately fixed and processed according to the standard protocol for transmission electron microscopy studies. RESULTS: In the MPs localized in the control part of colon wall the presence of numerous unmyelinated axons and cell bodies of neurons, interstitial cells of Cajal and enteroglial cells were observed. As compared to control samples, in the MPs located close to the tumor invasion, expansion of the extracellular matrix and myelin-like structures accompanying some nerve fibers were found. The appearance of mast and plasma cells was observed within MPs in the vicinity of CRC tumor. Sporadically, apoptotic cells were present inside the MPs. CONCLUSIONS: The presence of myelin-like structures and apoptotic cells within MPs located close to tumor invasion suggests that atrophy of MPs may be caused by factors released from CRC tumor.