Aneta Grabarska

Betulin Elicits Anti‐Cancer Effects in Tumour Primary Cultures and Cell Lines In Vitro

Betulin is a pentacyclic triterpene found in many plant species, among others, in white birch bark. The aim of the study was in vitro characterization of the anticancer activity of betulin in a range of human tumour cell lines (neuroblastoma, rhabdomyosarcoma-medulloblastoma, glioma, thyroid, breast, lung and colon carcinoma, leukaemia and multiple myeloma), and in primary tumour cultures isolated from patients (ovarian carcinoma, cervical carcinoma and glioblastoma multiforme). In this study, we demonstrated a remarkable anti-proliferative effect of betulin in all tested tumour cell cultures. Neuroblastoma (SK-N-AS) and colon carcinoma (HT-29) were the most sensitive to the anti-proliferative effect of betulin. Furthermore, betulin altered tumour cells morphology, decreased their motility and induced apoptotic cell death. These findings demonstrate the anti-cancer potential of betulin and suggest that they may be applied as an adjunctive measure in cancer treatment.

Inhibition of mitochondrial 2-oxoglutarate dehydrogenase impairs viability of cancer cells in a cell-specific metabolism-dependent manner

2-Oxoglutarate dehydrogenase (OGDH) of the tricarboxylic acid (TCA) cycle is often implied to be inactive in cancer, but this was not experimentally tested. We addressed the question through specific inhibition of OGDH by succinyl phosphonate (SP). SP action on different cancer cells was investigated using indicators of cellular viability and reactive oxygen species (ROS), metabolic profiling and transcriptomics. Relative sensitivity of various cancer cells to SP changed with increasing SP exposure and could differ in the ATP- and NAD(P)H-based assays. Glioblastoma responses to SP revealed metabolic sub-types increasing or decreasing cellular ATP/NAD(P)H ratio under OGDH inhibition. Cancer cell homeostasis was perturbed also when viability indicators were SP-resistant, e.g. in U87 and N2A cells. The transcriptomics database analysis showed that the SP-sensitive cells, such as A549 and T98G, exhibit the lowest expression of OGDH compared to other TCA cycle enzymes, associated with higher expression of affiliated pathways utilizing 2-oxoglutarate. Metabolic profiling confirmed the dependence of cellular SP reactivity on cell-specific expression of the pathways. Thus, oxidative decarboxylation of 2-oxoglutarate is significant for the interdependent homeostasis of NAD(P)H, ATP, ROS and key metabolites in various cancer cells. Assessment of cell-specific responses to OGDH inhibition is of diagnostic value for anticancer strategies.

Assessment of Interactions between Cisplatin and Two Histone Deacetylase Inhibitors in MCF7, T47D and MDA-MB-231 Human Breast Cancer Cell Lines - An Isobolographic Analysis

Histone deacetylase inhibitors (HDIs) are promising anticancer drugs, which inhibit proliferation of a wide variety of cancer cells including breast carcinoma cells. In the present study, we investigated the influence of valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), alone or in combination with cisplatin (CDDP) on proliferation, induction of apoptosis and cell cycle progression in MCF7, T47D and MDA-MB-231 human breast carcinoma cell lines. The type of interaction between HDIs and CDDP was determined by an isobolographic analysis. The isobolographic analysis is a very precise and rigorous pharmacodynamic method, to determine the presence of synergism, addition or antagonism between different drugs with using variety of fixed dose ratios. Our experiments show that the combinations of CDDP with SAHA or VPA at a fixed-ratio of 1:1 exerted additive interaction in the viability of MCF7 cells, while in T47D cells there was a tendency to synergy. In contrast, sub-additive (antagonistic) interaction was observed for the combination of CDDP with VPA in MDA-MB-231 “triple-negative” (i.e. estrogen receptor negative, progesterone receptor negative, and HER-2 negative) human breast cancer cells, whereas combination of CDDP with SAHA in the same MDA-MB-231 cell line yielded additive interaction. Additionally, combined HDIs/CDDP treatment resulted in increase in apoptosis and cell cycle arrest in all tested breast cancer cell lines in comparison with a single therapy. In conclusion, the additive interaction of CDDP with SAHA or VPA suggests that HDIs could be combined with CDDP in order to optimize treatment regimen in some human breast cancers.

Humoral response against glial derived antigens in Parkinson's disease

To check whether glial cells have the ability to elicit adaptive immune response in Parkinsons disease and whether a change in this immune response can be observed over time. There is an increasing evidence that glial cells are involved in the neurodegenerative process in PD, in addition to neuronal structures. Measurement of autoantibodies against proteins of oligodendrocytes may serve as an indirect method to assess the level of glial cells activation or degeneration under in vivo conditions. Serum samples from 26 PD patients were collected twice, at baseline and after mean of 13 months. In addition, serum samples from 13 healthy controls matched for age and gender were assessed at one time point. IgG and IgM autoantibodies against myelin-oligodendrocyticglycoprotein (MOG), myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and proteolipoprotein (PLP) were measured in all investigated subjects by a commercially available ELISA system (Mediagnost, Germany). In a group of PD significant decrease of IgG titers was observed for anti-MAG autoantibodies over the investigated time period (p<0.05). For IgM antibodies, we observed statistically significant decrease in anti-MAG autoantibodies in the follow-up period (p<0.05) and increase in anti-MBP and anti-PLP autoantibodies (p<0.05). All antibody titers differed significantly between healthy control subjects and PD patients. Our study provides the evidence for the presence of humoral response against some glial derived antigens in PD. The increasing levels of anti MBP IgG and IgM might point to the value of this marker for monitoring disease progression.

Matrix metalloproteinase-9 contributes to the increase of tau protein in serum during acute ischemic stroke

Previous studies indicate that tau protein, a marker of damage to neurons, is present in the serum of healthy patients at a concentration approximately 40 percent that of patients with ischemic stroke We assumed that increased serum activity of gelatinases (matrix metalloproteinase [MMP]-2 and MMP-9) can influence the level of tau protein in serum, probably due to disruption of the blood-brain barrier. We obtained blood sera from 31 patients admitted within the first 24 hours of ischemic stroke on days 1, 5 and 10, following the onset of stroke. Tau protein was detected in the serum of 12 patients (38.7 percent). The highest MMP-9 activity was recorded on day 5 (p < 0.05). Serum gelatinase activity did not differ between tau protein-positive or -negative individuals. However, a high degree of correlation between mean MMP-9 activity and the maximum tau protein level was observed for patients with detectable tau protein (r = 0.71, p = 0.009). Our study suggests that MMP-9 can increase the tau protein level in the sera of patients during acute ischemic stroke.

Histone Deacetylase Inhibitor SAHA as Potential Targeted Therapy Agent for Larynx Cancer Cells

Objective: Laryngeal squamous cell carcinoma is one of the most common malignant tumors in the head and neck region. Due to the poor response to chemotherapeutics in patients and low survival rate, successful treatment of larynx cancer still remains a challenge. Therefore, the identification of novel treatment options is needed. We investigated the anticancer effects of suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, on two different laryngeal cancer cell lines RK33 and RK45. We also studied the antiproliferative action of SAHA in combination with cisplatin and defined the type of pharmacological interaction between these drugs. Materials and Methods: Viability and proliferation of larynx cancer cell lines were studied by methylthiazolyldiphenyl-tetrazolium bromide method and 5-bromo-2-deoxyuridine incorporation assay, respectively. The type of interaction between SAHA and cisplatin was determined by an isobolographic analysis. Western blotting, flow cytometry and quantitative polymerase chain reaction method were used to determine acetylation of histone H3, cell cycle progression and genes expression, respectively. Apoptosis was assessed by means of nucleosomes released to cytosol. Results: SAHA alone or in combination with cisplatin inhibited larynx cancer cells proliferation, whereas displayed relatively low toxicity against normal cells - primary cultures of human skin fibroblasts. The mixture of SAHA with cisplatin exerted additive and synergistic interaction in RK33 and RK45 cells, respectively. We showed that SAHA induced hyperacetylation of histone H3 K9, K14 and K23 and triggered apoptosis. SAHA also caused cell cycle arrest by upregulation of CDKN1A and downregulation of CCND1 encoding p21WAF1/CIP1 and cyclin D1 proteins, respectively. Conclusion: Our studies demonstrated that SAHA may be considered as a potential therapeutic agent against larynx tumors.

[Histone deacetylase inhibitors - molecular mechanisms of actions and clinical applications].

Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression implicated in cancer pathogenesis. Inhibitors of HDACs (HDI) are under investigation as novel anti-cancer drugs, which induce histone hyperacetylation. These agents modulate chromatin structure leading to transcriptional changes of a very large number of genes, which affect signaling pathways, inhibit cell cycle progression and angiogenesis, and induce apoptosis in cancer cells. Currently, several HDI are in clinical trials used in monotherapy or in combination with other cytostatics, showing promising anticancer effects. To date, more than 15 HDIs have been found as potential drugs. This paper reviews the molecular mechanisms of HDI action on cancer cells and summarizes clinical trials of the most promising HDIs in the treatment of patients with hematologic malignancies and solid tumors.

CT volume/density ratio as the marker of ischaemic brain injury

Kurzepa J, Bielewicz J, Czekajska‐Chehab E, Kurzepa J, Bartosik‐Psujek H, Grabarska A, Stelmasiak Z. CT volume/density ratio as the marker of ischaemic brain injury.
Acta Neurol Scand: 2011: 123: 310–315.
© 2010 John Wiley & Sons A/S.

Superior anticancer activity is demonstrated by total extract of Curcuma longa L. as opposed to individual curcuminoids separated by centrifugal partition chromatography

Three curcuminoids: bisdemethoxycurcumin, demethoxycurcumin, and curcumin from turmeric were successfully separated by a high capacity solvent system composed of heptane: chloroform: methanol: water mixture (5: 6: 3: 2 v/v/v/v) tailored for centrifugal partition chromatographs at K‐values of 0.504, 1.057, 1.644, respectively. These three ferulic acid derivatives obtained at a purity rate exceeding 95% were analysed by an HPLC‐MS spectrometer. Turmeric extract inhibited the proliferation/viability of A549 human lung cancer, HT29 colon cancer, and T98G glioblastoma cell lines in (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide) tetrazolium reduction assay (MTT). Single curcuminoids significantly decreased the viability/proliferation of lung cancer cells in a dose‐dependent manner. However, total extract displayed the superior anticancer activity in the investigated cell lines. Crude extract in combination with cisplatin augmented the decrease in the viability of cancer cells compared with single compound treatment in A549 lung cancer cells. Total extract of Curcuma longa could be regarded as being more effective against lung cancer cells in vitro than its separated compounds.

Pharmacological features of osthole

Coumarins are a group of naturally occurring compounds common in the plant world. These substances and their derivatives exhibit a broad range of biological activities. One of the naturally occurring coumarins is osthole, which can most frequently be found in plants of the Apiaceae family. Cnidium monnieri (L.) Cusson ex Juss. Angelica pubescens Maxim. and Peucedanum ostruthium (L.). It has anti-proliferative, anti-inflammatory, anti-convulsant, and antiallergic properties; apart from that, inhibition of platelet aggregation has also been proved. The impact of osthole on bone metabolism has been demonstrated; also its hepatoprotective and neuroprotective properties have been confirmed. The inhibitory effect of this metokcompound on the development of neurodegenerative diseases has been proved in experimental models. Anticancer features of osthole have been also demonstrated both in vitro on different cell lines, and in vivo using animals xenografts. Osthole inhibited proliferation, motility and invasiveness of tumor cells, which may be associated with the induction of apoptosis and cell cycle slowdown. The exact molecular mechanism of osthole anti-cancer mode of action has not been fully elucidated. A synergistic effect of osthole with other anti-tumor substances has been also reported. Modification of its chemical structure led to the synthesis of many derivatives with significant anticancer effects. To sum up, osthole is an interesting therapeutic option, due to both its direct effect on tumor cells, as well as its neuroprotective or anti-inflammatory properties. Thus, there is a chance to use osthole or its synthetic derivatives in the treatment of cancer.