Impact of Truncated Oxidized Phosphatidylcholines on Phospholipase A2 Activity in Mono- and Polyunsaturated Biomimetic Vesicles.

The interplay between inflammatory and redox processes is a ubiquitous and critical phenomenon in cell biology that involves numerous biological factors. Among them, secretory phospholipases A2 (sPLA2) that catalyze the hydrolysis of the sn-2 ester bond of phospholipids are key players. They can interact or be modulated by the presence of truncated oxidized phosphatidylcholines (OxPCs) produced under oxidative stress from phosphatidylcholine (PC) species. The present study examined this important, but rarely considered, sPLA2 modulation induced by the changes in biophysical properties of PC vesicles comprising various OxPC ratios in mono- or poly-unsaturated PCs. Being the most physiologically active OxPCs, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) have been selected for our study. Using fluorescence spectroscopy methods, we compared the effect of OxPCs on the lipid order as well as sPLA2 activity in large unilamellar vesicles (LUVs) made of the heteroacid PC, either monounsaturated [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)], or polyunsaturated [1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC)] at a physiological temperature. The effect of OxPCs on vesicle size was also assessed in both the mono- and polyunsaturated PC matrices. Results: OxPCs decrease the membrane lipid order of POPC and PDPC mixtures with PGPC inducing a much larger decrease in comparison with POVPC, indicative that the difference takes place at the glycerol level. Compared with POPC, PDPC was able to inhibit sPLA2 activity showing a protective effect of PDPC against enzyme hydrolysis. Furthermore, sPLA2 activity on its PC substrates was modulated by the OxPC membrane content. POVPC down-regulated sPLA2 activity, suggesting anti-inflammatory properties of this truncated oxidized lipid. Interestingly, PGPC had a dual and opposite effect, either inhibitory or enhancing on sPLA2 activity, depending on the protocol of lipid mixing. This difference may result from the chemical properties of the shortened sn-2-acyl chain residues (aldehyde group for POVPC, and carboxyl for PGPC), being, respectively, zwitterionic or anionic under hydration at physiological conditions.

Antitumor Effect of Iscador on Breast Cancer Cell Lines with Different Metastatic Potential.

Studies were performed for the first time on the effect of Iscador Qu and Iscador M on phototoxicity, cytotoxicity, antiproliferative activity, changes in ξ-potential of cells, membrane lipid order, actin cytoskeleton organization and migration on three breast cancer lines with different metastatic potential: MCF10A (control), MCF-7 (low metastatic) and MDA-MB231 (high metastatic) cells. The tested Iscador Qu and M did not show any phototoxicity. The antiproliferative effect of Iscador species appeared to be dose-dependent and was related to the metastatic potential of the tested cell lines. A higher selectivity index was obtained for Iscador Qu and M towards the low metastatic MCF-7 cell line compared to the high metastatic MDA-MB-231. Iscador Qu demonstrated higher selectivity for both cancer cell lines compared to Iscador M. The malignant cell lines exhibited a decrease in fibril number and thickness regardless of the type of Iscador used. The strongest effect on migration potential was observed for the low metastatic cancer cell line MCF-7 after Iscador treatment. Both Iscador species induced a slight increase in the percentage of cells in early apoptosis for the low and high metastatic cell lines, MCF-7 and MDA-MB-231, unlike control cells. Changes in the zeta potential and membrane lipid order were observed for the low metastatic MCF-7 cell line in contrast to the high metastatic MDA-MB-231 cells. The presented results reveal a higher potential of Iscador as an antitumor agent for the low metastatic cancer cell line MCF-7 compared to the high metastatic one. Iscador Qu appears to be more potent compared to Iscador M, but at this point, the exact mechanism of action is still unclear and needs further investigations.

Effect of Quercetin and Fingolimod, Alone or in Combination, on the Sphingolipid Metabolism in HepG2 Cells.

Combinations of anti-cancer drugs can overcome resistance to therapy and provide new more effective treatments. In this work we have analyzed the effect of the polyphenol quercetin and the anti-cancer sphingosine analog fingolimod on the sphingolipid metabolism in HepG2 cells, since sphingolipids are recognized as mediators of cell proliferation and apoptosis in cancer cells. Treatment of hepatocellular carcinoma HepG2 cells with quercetin and fingolimod, alone or in combination, induced different degrees of sphingomyelin (SM) reduction and a corresponding activation of neutral sphingomyelinase (nSMase). Western blot analysis showed that only treatments containing quercetin induced up-regulation of nSMase expression. The same treatment caused elevation of ceramide (CER) levels, whereas the observed alterations in sphingosine (SPH) content were not statistically significant. The two tested drugs induced a reduction of the pro-proliferative sphingolipid, sphingosine 1 phosphate (S1P), in the following order: quercetin, fingolimod, quercetin + fingolimod. The activity of the enzyme responsible for CER hydrolysis, alkaline ceramidase (ALCER) was down-regulated only in the incubations involving quercetin and fingolimod did not affect this activity. The enzyme, maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was down-regulated by incubations in the following order: quercetin, fingolimod, quercetin + fingolimod. Western blot analysis showed down-regulation in SK1 expression upon quercetin but not upon fingolimod treatment. Studies on the effect of quercetin and fingolimod on the two proteins associated with apoptotic events, AKT and Bcl-2, showed that only quercetin, alone or in combination, down-regulated the activity of the two proteins. The reported observations provide information which can be useful in the search of novel anti-tumor approaches, aiming at optimization of the therapeutic effect and maximal preservation of healthy tissues.

Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells.

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity-apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)-in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies.

SPECT-CT Imaging with [99mTc]PSMA-T4 in patients with Recurrent Prostate Cancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein with a large extracellular domain with overexpression of the prostatic tumour cells. Several small molecules of PSMA ligands of inhibitors binding to the active site of PSMA were developed. [99mTc]Tc-PSMA-T4 is a new radiopharmaceutical (Polatom) for imaging loco-regional metastases and/or local relapse in patients with prostate cancer. The purpose of this work was to evaluate the clinical application of SPECT-CT imaging with [99mTc]Tc-PSMA-T4 in patients with recurrent prostate cancer. MATERIAL AND METHODS: Thirty-six patients with prostate cancer, aged 60-80 years with biochemical relapse of PSA (ranged from 0.1 to 73 ng/mL) were included. Three patients were studied after tru-cut biopsy, hormonal and cytoreductive radiotherapy and 33 patients out of 36 - after radical treatment (total prostatectomy or definitive radiotherapy of the tumour). All of them underwent whole-body imaging examinations with subsequent target SPECT-CT studies of the pelvis, abdomen and/or chest, 1-3 hrs post i.v. administration of [99mTc]Tc-PSMA-T4. The average activity dose was 6.3 MBq/kg in a man of 70 kg. A Dual-head SPECT-CT gamma camera with a low dose CT scan (Symbia T2, Siemens) was used. The images were interpreted based on all other clinical and radiological data. Follow-up could be conducted in 11/36 patients during that period. RESULTS: Normal biodistribution of the radiopharmaceutical with high activity background was observed in the liver, spleen, kidneys, lacrimal and salivary glands, bowels and urinary bladder. Positive imaging for local relapse in the prostate bad was imaged in 21 patients, lymph node metastases - in 16 cases, bone lesions - in 10 cases, pulmonary metastases - in 2 cases, hepatic lesions were visualised in one of them and in another - adrenal suprarenal metastasis with intensive tracer uptake significant for overexpression of PSMA. There was a suspicion for local recurrences in 4 patients with negative MRT studies who were followed up. In 3 cases, previously treated bone metastases were partially negative without tracer uptake, only some progressive bone lesions were positive. Five patients were with negative results. Sensitivity was 84.37% (27/32), specificity - 100% (4/4) and accuracy - 86.11% (31/36). CONCLUSIONS: In conclusion SPECT-CT imaging with [99mTc]Tc-PSMA-T4 could be applied in patients with prostate cancer for the diagnosis of recurrent disease to determine personalized treatment for each patient. Specific uptake of this tracer, depicted by SPECT-CT images has clinical importance of identifying and assessing PSMA expression before consideration of Radio Ligand Therapy (RLT) with [¹77Lu]Lu-PSMA. SPECT-CT imaging with [99mTc]PSMA is promising and reliable nuclear medicine approach to monitoring therapeutic effect after treatment and for restaging of the disease.

Clinical application of SPECT-CT with 99mTc-Tektrotyd in bronchial and thymic neuroendocrine tumors (NETs).

Neuroendocrine tumors (NETs) of the thorax including bronchial and thymic tumors belong to foregut NETs. Limited loco-regional thoracic NETs can be resected with surgery, but in extensive metastatic disease the treatment is mainly palliative. A high incidence and density of somatostatin receptors (SSTR2, SSTR3, and SSTR5) are found in thoracic NETs. The purpose of this study was to evaluate the role of SPECT-CT somatostatin receptor scintigraphy (SRS) with 99mTc-Tektrotyd for imaging, staging and follow up of patients with bronchial and thymic neuroendocrine tumors. Forty-one patients with thoracic tumors with neuroendocrine differentiation were studied. Sixty-eight examinations including SPECT-CT studies of the neck and chest and/or abdomen and pelvis were carried out 2-4 hrs. post i.v. administration of aver-age 740 MBq activity dose of 99mTc-EDDA/HYNIC-TOC (Tektrotyd, Polatom). In all 41 investigated patients we obtained 81.25% (13/16), 88% (22/25) and 85.36% (35/41) of sensitivity, specificity and accuracy of this diagnostic approach, respectively. Somatostatin-receptor scintigraphy correctly identified all primary NETs located in the lungs and thymus. SPECT-CT studies with 99mTc-EDDA/HYNIC-TOC resulted in exact pre-surgical and pre-treatment N/M staging of bronchial and thymic NETs, except 2 cases with multiple hepatic metastases and 1 with massive suprarenal metastasis. It can be concluded that SPECT-CT with 99mTc-EDDA/HYNIC-TOC is a valuable tool for staging and follow-up of patients with thoracic NETs.