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Background: Platinum-based therapy represents the main pharmacological treatment for ovarian carcinoma. Since molecular targeting of receptor tyrosine kinases (RTK) affects factors that may modulate drug response, the aim of this study was to examine whether downstream targets of AXL RTK could be exploited to improve cell response to cisplatin., Materials and Methods: Inhibitors of p38 (SB203580) and of signal transducer and activator of transcription 3 (stattic) were employed in combination with cisplatin in ovarian carcinoma cell lines. Apoptosis assay and western blot analysis were performed to evaluate cell response after treatment., Results: SB203580 produced a synergistic effect in combination with cisplatin in cisplatin-resistant IGROV-1/Pt1 cells. In addition, a favorable drug interaction was observed in A2780 cells when pre-incubated with cisplatin prior to stattic. The analysis of cell response after combined treatment showed down-regulation of the pro-apoptotic protein BCL2-associated agonist of cell death (BAD)., Conclusion: Our results support the notion that downstream targets of AXL in ovarian carcinoma cells can be exploited to increase cisplatin activity in ovarian carcinoma models., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASCs) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs)., Method: We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigated for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells., Results: The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells., Conclusion: Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would use less resource and time and it could possibly contribute to simplification of GMP procedures., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Epilepsy is a frequent symptom in patients with glioma. Although treatment with antiepileptic drugs is generally effective in controlling seizures, drug-resistant patients are not uncommon. Multidrug resistance proteins (MRPs) and P-gp are over-represented in brain tissue of patients with drug-resistant epilepsy, suggesting their involvement in the clearance of antiepileptic medications. In addition to their anticonvulsant action, some drugs have been documented for cytotoxic effects. Aim of this study was to evaluate possible in vitro cytotoxic effects of two new-generation antiepileptic drugs on a human glioma cell line U87MG., Methods: Cytotoxicity of brivaracetam and lacosamide was tested on U87MG, SW1783 and T98G by MTS assay. Expression of chemoresistance molecules was evaluated using flow cytometry in U87MG and human umbilical vein endothelial cells (HUVECs). To investigate the putative anti-proliferative effect, apoptosis assay, microRNA expression profile and study of cell cycle were performed., Results: Brivaracetam and lacosamide showed a dose-dependent cytotoxic and anti-migratory effects. Cytotoxicity was not related to apoptosis. The exposure of glioma cells to brivaracetam and lacosamide resulted in the modulation of several microRNAs; particularly, the effect of miR-195-5p modulation seemed to affect cell cycle, while miR-107 seemed to be implicated in the inhibition of cells migration. Moreover, brivaracetam and lacosamide treatment did not modulate the expression of chemoresistance-related molecules MRPs1-3-5, GSTπ, P-gp on U87MG and HUVECs., Conclusion: Based on antineoplastic effect of brivaracetam and lacosamide on glioma cells, we assume that patients with glioma could benefit by the treatment with these two molecules, in addition to standard therapeutic options.

Background Aims: Pancreatic cancer (pCa) is a tumor characterized by a fibrotic state and associated with a poor prognosis. The observation that mesenchymal stromal cells (MSCs) migrate toward inflammatory micro-environments and engraft into tumor stroma after systemic administration suggested new therapeutic approaches with the use of engineered MSCs to deliver and produce anti-cancer molecules directly within the tumor. Previously, we demonstrated that without any genetic modifications, MSCs are able to deliver anti-cancer drugs. MSCs loaded with paclitaxel by exposure to high concentrations release the drug both in vitro and in vivo, inhibiting tumor proliferation. On the basis of these observations, we evaluated the ability of MSCs (from bone marrow and pancreas) to uptake and release gemcitabine (GCB), a drug widely used in pCa treatment., Methods: MSCs were primed by 24-h exposure to 2000 ng/mL of GCB. The anti-tumor potential of primed MSCs was then investigated by in vitro anti-proliferation assays with the use of CFPAC-1, a pancreatic tumor cell line sensitive to GCB. The uptake/release ability was confirmed by means of high-performance liquid chromatography analysis. A cell-cycle study and secretome evaluation were also conducted to better understand the characteristics of primed MSCs., Results: GCB-releasing MSCs inhibit the growth of a human pCa cell line in vitro., Conclusions: The use of MSCs as a "trojan horse" can open the way to a new pCa therapeutic approach; GCB-loaded MSCs that integrate into the tumor mass could deliver much higher concentrations of the drug in situ than can be achieved by intravenous injection., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: In attempting to develop new strategies to circumvent the immunosuppression associated with glioblastoma (GB), novel approaches have been designed using dendritic cell (DC)-based vaccination, which is considered a promising strategy to attack high-grade glioma. In previous studies, we demonstrated that human mesenchymal stromal cells without genetic manipulation but primed with Paclitaxel (PTX) acquire a potent anti-tumor activity, providing an interesting new biological approach for drug delivery. On the basis of these results, we here investigated whether both CD14+ and their derived DCs may behave like mesenchymal stromal cells acquiring anti-tumor activity on priming with PTX., Methods: Human CD14+ cells were isolated from peripheral blood. Fluorescence-activated cell sorter analysis was performed to determine the purity of CD14+ and their differentiation into mature DCs. Cells were primed by incubation with 1 μg/mL of PTX for 24 h, and the PTX released by cells was assessed by mass spectrometry analysis. Anti-tumor activity was checked by testing the conditioned medium (CM) on the proliferation of U87 MG, a GB cell line., Results: Both CD14+ and DCs were able to incorporate PTX and release the drug in the CM in a time-dependent manner (maximal release over 24 h). The addition of CM from CD14+ and DCs loaded with PTX strongly inhibits proliferation of U87 MG cells., Conclusions: Our results are the first demonstration that peripheral blood-derived CD14+ and DCs, in addition to their application for immunotherapy for GB, could also be used to delivery anti-cancer drugs, such as PTX, to kill GB cells., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stromal cells (MSCs) have been proposed for delivering anticancer agents because of their ability to home in on tumor microenvironment. We found that MSCs can acquire strong anti-tumor activity after priming with Paclitaxel (PTX) through their capacity to uptake and then release the drug. Because MSCs secrete a high amount of membrane microvesicles (MVs), we here investigated the role of MVs in the releasing mechanism of PTX. The murine SR4987 line was used as MSC model. The release of PTX from SR4987 in the conditioned medium (CM) was checked by HPLC and the anti-tumor activity of both CM and MVs was tested on the human pancreatic cell line CFPAC-1. MVs were isolated by ultracentrifugation, analyzed by transmission (TEM) and scanning electron microscopy (SEM), and the presence of PTX by the Fourier transformed infrared (FTIR) microspectroscopy. SR4987 loaded with PTX (SR4987PTX) secreted a significant amount of PTX and their CM possessed strong anti-proliferative activity on CFPAC-1. At TEM and SEM, SR4987PTX showed an increased number of "vacuole-like" structures and shed a relevant number of MVs, but did not differ from untreated SR4987. However, SR4987PTX-derived-MVs (SR4987PTX-MVs) demonstrated a strong anti proliferative activity on CFPAC-1. FTIR analysis of SR4987PTX-MVs showed the presence of an absorption spectrum in the corresponding regions of the PTX marker, absent in MVs from SR4987. Our work is the first demonstration that MSCs are able to package and deliver active drugs through their MVs, suggesting the possibility of using MSCs as a factory to develop drugs with a higher cell-target specificity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Treatment of ovarian carcinoma often fails to be curative because of drug resistance, and many efforts are directed to overcome tumor cell resistance by increasing apoptosis induction. The potential of second mitochondria-derived activator of caspases (SMAC) mimetics (SMACm) has appeared in preclinical studies, but novel proapoptotic agents of this class with improved pharmacological profile are needed. To identify novel treatment options for ovarian carcinoma by interfering with antiapoptotic factors, in the present study a novel homodimeric SMACm (SM83) was employed in preclinical models both in vitro and in vivo. An investigation of the structural features of dimeric SM83 as compared to a closely related reference compound indicated slight differences, likely because of the interaction between one of the terminal phenyl groups and triazole rings of SM83 with the BIR2 domain. Although SM83 per se did not inhibit cell proliferation, it displayed a synergistic effect in combination with TNF-related apoptosis inducing ligand (TRAIL) in cell sensitivity assays. Because the tumor microenvironment is a reservoir of cytokines that may act in conjunction with SMACm to affect tumor growth, the activity of the novel compound was tested in vivo in ovarian carcinoma cells subcutaneously xenografted into immunodeficient mice. A significant tumor volume inhibition was observed together with activation of caspase 3 and apoptotic cell death. A biochemical analysis of tumor necrosis factor (TNF) and TRAIL content in specimens from xenografted mice indicated that SM83 downmodulated the levels of human TNF in plasma samples and tended to upmodulate human TRAIL levels in tumors. Thus, TRAIL appears to contribute to the antitumor activity of novel SMACm SM83 in subcutaneously grown ovarian carcinoma. Overall, our results indicate that SM83 is an attractive candidate for further development.

The main goal in cancer chemotherapy is to drive the drug into the tumor microenvironment to kill as many cancer cells as possible while producing the lowest collateral toxicity. Previously, we have shown that human bone marrow derived mesenchymal stromal cells (hBM-MSCs) exposed to Paclitaxel (PTX) were able to uptake and subsequently release the drug in the culture medium. PTX primed hBM-MSCs (hBM-MSCsPTX) located in the vicinity of cancer cells produced a strong inhibition of tumor cell growth both in vitro and in vivo. To expand these observations, in the present study we exposed human skin derived fibroblasts (hSDFs) to 2,000 ng/ml of PTX and then tested both cells and their conditioned medium (CM) in vitro for their capacity to inhibit the proliferation of human tumor cell lines (MOLT-4, DU-145, U87-MG, SH-SY5Y(+) and LAN-5). We found that hSDFs primed with PTX (hSDFsPTX) were able to uptake and subsequently release PTX in a time dependent manner. hSDFsPTX-derived CM(hSDFsPTX-CM) from 1:4 to 1:10 dilutions produced a significant (p < 0.05) in vitro tumor growth inhibition. hSDFsPTX co-cultured with leukemia cells at 1:1 to 1:10 ratio, completely inhibited cells growth whereas no inhibition was induced by normal hSDFs cells. Our results demonstrate for the first time that hSDFs can be loaded in vitro with PTX and thus can acquire a potent anti-tumor activity. Since hSDFs can be easily isolated from skin biopsies without any particular pain and discomfort to donor patients, we conclude that hSDFs may represent a valid cell type option for carrying and delivering anti-cancer drugs.

Background: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation., Methods and Principal Findings: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth., Conclusions: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy.

The survival of patients with malignant gliomas is still unsatisfactory despite multimodality treatment, therefore new therapeutic strategies are required. Tumor necrosis factor apoptosis related ligand (TRAIL/Apo2L), a member of the tumor necrosis factor superfamily, may induce apoptotic cell death in several tumors, but not in normal cells, upon binding with specific receptors. In the present study, the expression and function of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) has been investigated in five human glioma cell lines (U87, U138, U373, A172, SW1783) in ex vivo tumors and in primary cultures obtained from the tumors. Our data show that gliomas preferentially express TRAIL R2 and that treatment with topotecan, a topoisomerase I inhibitor, significantly up-regulates its expression as detected by flow cytometry and western blotting. Moreover, in most cases, treatment with topotecan resulted in an increased sensitivity to TRAIL-dependent apoptosis, although cyclohexymide had to be added to induce apoptosis. On glioma cell lines, the effects of irradiation on TRAIL receptors were also analysed. In our experimental conditions, irradiation with 2 Gy had a modest additive effect on TRAIL-dependent apoptosis and was not able to modulate TRAIL receptor expression.

The aims of this phase I study in patients with recurrent malignant gliomas were to determine the maximum tolerated dose (MTD) and toxicity profile of fotemustine when combined with a fixed dose of procarbazine (PCZ), and to evaluate the extent of O6-alkylguanine-DNA alkyltransferase (ATase) depletion in circulating lymphocytes during treatment. Sixteen patients received an induction cycle consisting of 100 mg/day oral PCZ for 12 consecutive days and a 1-h intravenous infusion of fotemustine given 4 h after PCZ on days 5 and 12 at escalated doses (50, 75, 100 and 125 mg/m2/day). After a 6-week rest period, a maximum of 4 maintenance cycles (PCZ 300 mg/day, 4 days; fotemustine, day 4) was given every 4 weeks. ATase activity was measured on days 1, 5 and 12 over 4 h after PCZ intake. Fifteen patients had previously received at least one nitrosourea-based chemotherapy, associated with PCZ in 12 cases. The MTD of fotemustine was 125 mg/m2 (days 5 and 12) with myelosuppression as the dose limiting toxicity (DLT). At this dose level, half of patients experienced grade 3 anemia, neutropenia or thrombopenia. No extra-hematological DLT was observed. No significant depletion of ATase activity by PCZ was evidenced. One partial response and 7 stable diseases were obtained leading to a disease control rate of 50%. The median times to progression and survival were 2.6 and 9.7 months, respectively. This combined regimen of PCZ and fotemustine was well tolerated with a good disease control rate in heavily pretreated glioma patients and merits further investigation in phase II studies.