Cytotoxic Drug Gemcitabine Research Articles

Smart Modification on Magnetic Nanoparticles Dramatically Enhances Their Therapeutic Properties.

Simple SummaryIn this work, a smart gemcitabine delivery system based on magnetic nanoparticles (MNP) is proposed. Gemcitabine (GEM) is a chemotherapeutic agent usually employed as monotherapy for the treatment of pancreatic cancer. Unfortunately, this drug presents short half-life and high toxicity in non-tumoral tissues. Thus, new efficient drug delivery systems are needed. In this regard, we modified MNP to attach this drug via disulfide bonds (MNP-GEM) to promote the selective release of GEM in pancreatic cancer cells, and the great potential of our proposed nanocarrier for biomedical applications is broadly assessed. Remarkably, this modification has proved to prevent the unspecific binding of proteins, reduced the cytotoxic effect of the drug in non-cancerous cells, improved the internalization in pancreatic cancer cells, and its activity was synergistically enhanced in combination with magnetic hyperthermia.Magnetic nanoparticles (MNP) are employed as nanocarriers and in magnetic hyperthermia (MH) for the treatment of cancers. Herein, a smart drug delivery system composed of MNP functionalized with the cytotoxic drug gemcitabine (MNP-GEM) has been thoroughly evaluated. The linker employed is based on a disulfide bond and allows the controlled release of GEM under a highly reducing environment, which is frequently present in the cytoplasm of tumor cells. The stability, MH, and the interaction with plasma proteins of the nanoparticles are evaluated, highlighting their great potential for biological applications. Their cytotoxicity is assessed in three pancreatic cancer cell lines with different sensitivity to GEM, including the generation of reactive oxygen species (ROS), the effects on the cell cycle, and the mechanisms of cell death involved. Remarkably, the proposed nanocarrier is better internalized than unmodified nanoparticles, and it is particularly effective in PANC-1 cells, resistant to GEM, but not in non-tumoral keratinocytes. Additionally, its combination with MH produces a synergistic cytotoxic effect in all cancer cell lines tested. In conclusion, MNP-GEM presents a promising potential for treating pancreatic cancer, due to multiple parameters, such as reduced binding to plasma proteins, increased internalization, and synergistic activity when combined with MH.

Advances in oncolytic adenovirus therapy for pancreatic cancer

Survival rates for pancreatic cancer patients have remained unchanged for the last four decades. The most aggressive, and most common, type of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), which has the lowest 5-year survival rate of all cancers globally. The poor prognosis is typically due to late presentation of often non-specific symptoms and rapid development of resistance to all current therapeutics, including the standard-of-care cytotoxic drug gemcitabine. While early surgical intervention can significantly prolong patient survival, there are few treatment options for late-stage non-resectable metastatic disease, resulting in mostly palliative care. In addition, a defining feature of pancreatic cancer is the immunosuppressive and impenetrable desmoplastic stroma that blocks access to tumour cells by therapeutic drugs. The limited effectiveness of conventional chemotherapeutics reveals an urgent need to develop novel therapies with different mechanisms of action for this malignancy.An emerging alternative to current therapeutics is oncolytic adenoviruses; these engineered biological agents have proven efficacy and tumour-selectivity in preclinical pancreatic cancer models, including models of drug-resistant cancer. Safety of oncolytic adenoviral mutants has been extensively assessed in clinical trials with only limited toxicity to normal healthy tissue being reported. Promising efficacy in combination with gemcitabine was demonstrated in preclinical and clinical studies. A recent surge in novel adenoviral mutants entering clinical trials for pancreatic cancer indicates improved efficacy through activation of the host anti-tumour responses. The potential for adenoviruses to synergise with chemotherapeutics, activate anti-tumour immune responses, and contribute to stromal dissemination render these mutants highly attractive candidates for improved patient outcomes.Currently, momentum is gathering towards the development of systemically-deliverable mutants that are able to overcome anti-viral host immune responses, erythrocyte binding and hepatic uptake, to promote elimination of primary and metastatic lesions. This review will cover the key components of pancreatic cancer oncogenesis; novel oncolytic adenoviruses; clinical trials; and the current progress in overcoming the challenges of systemic delivery.

Development of a peptide-based bifunctional chelator conjugated to a cytotoxic drug for the treatment of melanotic melanoma.

The cytotoxic drug gemcitabine (GEM) has been conjugated to receptor-binding peptides to target melanoma tumors. A hexapeptide having a Lys-Gly-His-Lys sequence (pep-1), an octapeptide with an Arg-Gly-Asp-Lys-Gly-His-Lys sequence (pep-2), a GEM-conjugated Lys-Gly-His-Lys peptide (GEM-pep-3) and a GEM-conjugated Asp-Gly-Arg peptide (GEM-pep-4) were synthesized and characterized. In vitro uptake of fluorescently labeled GEM-pep-3 and GEM-pep-4 on B16F10 cells was investigated. Fluorescence microscopy studies demonstrated significant uptake of GEM-pep-3 in the B16F10 mouse melanoma cell line. The peptides and GEM-coupled peptides were radiolabeled with [99mTc(CO)3(H2O)3]+ and examined for in vitro cell binding in the B16F10 melanoma cell line and in vivo biodistribution and scintigraphic studies in a B16F10 melanoma tumor-bearing mice model. In vitro cellular uptake studies and biological evaluation confirmed significant deposition of GEM-pep-3 at the melanoma tumor site. The MTT assay depicted higher cytotoxic behaviour of GEM-pep-3 than free GEM. A considerable amount of cell apoptosis was also observed in B16F10 cells. Finally, the in vivo therapeutic efficacy study revealed a significant decrease in tumor growth in the GEM-pep-3-treated animal model. These studies reveal enough potentiality of GEM-pep-3 to treat melanoma and underline the need for further evaluation.

Cell cycle specific radiosensitisation by the disulfiram and copper complex

The disulfiram and copper complex (DSF:Cu) has emerged as a potent radiosensitising anti-cancer agent. The ability of copper to stabilise DSF in a planar conformation and to inhibit DNA replication enzymes stimulated our investigation of the effect of DSF:Cu on cell cycle regulation. Flow cytometry and immunoblotting were used to assess the effect of DSF:Cu on cell cycle progression of the neuroblastoma cell line SK-N-BE(2c) and the glioma cell line UVW. Treatment with 0.1 and 0.3 μM DSF:Cu inhibited DNA synthesis in SK-N-BE(2c) and UVW cells, respectively. The increased potency of ionising radiation treatment induced by DSF:Cu and/or gemcitabine was determined by clonogenic assay. Treatment with 0.3 μM DSF:Cu resulted in greater radiation kill, exemplified by dose enhancement factor values of 2.64 and 2.84 in SK-N-BE(2c) and UVW cells, respectively. Although DSF:Cu failed to sensitise S phase cells to irradiation, we observed that DSF:Cu radiosensitisation was potentiated by the S phase-specific cytotoxic drug gemcitabine. The efficacy of the combination treatment consisting of DSF:Cu, gemcitabine and ionising radiation was schedule-dependent. Together, these results describe cell cycle specific radiosensitisation by DSF:Cu. The well-established toxicity profiles of DSF and gemcitabine should facilitate their evaluation as a combination treatment in patients undergoing radiotherapy.

The E1B19K-deleted oncolytic adenovirus mutant AdΔ19K sensitizes pancreatic cancer cells to drug-induced DNA-damage by down-regulating Claspin and Mre11

Adenovirus-mediated sensitization of cancer cells to cytotoxic drugs depends on simultaneous interactions of early viral genes with cell death and survival pathways. It is unclear what cellular factors mediate these interactions in the presence of DNA-damaging drugs. We found that adenovirus prevents Chk1-mediated checkpoint activation through inactivation of Mre11 and downregulation of the pChk1 adaptor-protein, Claspin, in cells with high levels of DNA-damage induced by the cytotoxic drugs gemcitabine and irinotecan. The mechanisms for Claspin downregulation involve decreased transcription and increased degradation, further attenuating pChk1-mediated signalling. Live cell imaging demonstrated that low doses of gemcitabine caused multiple mitotic aberrations including multipolar spindles, micro- and multi-nucleation and cytokinesis failure. A mutant virus with the anti-apoptotic E1B19K-gene deleted (AdΔ19K) further enhanced cell killing, Claspin downregulation, and potentiated drug-induced DNA damage and mitotic aberrations. Decreased Claspin expression and inactivation of Mre11 contributed to the enhanced cell killing in combination with DNA-damaging drugs. These results reveal novel mechanisms that are utilised by adenovirus to ensure completion of its life cycle in the presence of cellular DNA damage. Taken together, our findings reveal novel cellular targets that may be exploited when developing improved anti-cancer therapeutics.

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells.

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.

Noninvasive Quantification of Solid Tumor Microstructure Using VERDICT MRI

There is a need for biomarkers that are useful for noninvasive imaging of tumor pathophysiology and drug efficacy. Through its use of endogenous water, diffusion-weighted MRI (DW-MRI) can be used to probe local tissue architecture and structure. However, most DW-MRI studies of cancer tissues have relied on simplistic mathematical models, such as apparent diffusion coefficient (ADC) or intravoxel incoherent motion (IVIM) models, which produce equivocal results on the relation of the model parameter estimate with the underlying tissue microstructure. Here, we present a novel technique called VERDICT (Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumors) to quantify and map histologic features of tumors in vivo. VERDICT couples DW-MRI to a mathematical model of tumor tissue to access features such as cell size, vascular volume fraction, intra- and extracellular volume fractions, and pseudo-diffusivity associated with blood flow. To illustrate VERDICT, we used two tumor xenograft models of colorectal cancer with different cellular and vascular phenotypes. Our experiments visualized known differences in the tissue microstructure of each model and the significant decrease in cell volume resulting from administration of the cytotoxic drug gemcitabine, reflecting the apoptotic volume decrease. In contrast, the standard ADC and IVIM models failed to detect either of these differences. Our results illustrate the superior features of VERDICT for cancer imaging, establishing it as a noninvasive method to monitor and stratify treatment responses.

Abstract 2671: Study of the role of functional HIF-1 in the in vitro radiosensitizing effect of gemcitabine in normoxic and anoxic breast adenocarcinoma cells

Abstract Introduction: It has been well documented that the efficacy of both chemotherapy and radiotherapy is directly linked with an adequate oxygen tension, and that hypoxic regions in solid tumors often contain viable cells that are intrinsically more resistant to anticancer treatment. Recently, it has been demonstrated that the cytotoxic drug gemcitabine retains its radiosensitizing potential under low oxygen conditions in lung adenocarcinoma cells. As the transcription factor ‘hypoxia inducible factor 1’ (HIF-1) plays a crucial role in regulating the adaptive responses of tumor cells to survive under hypoxic conditions, the present study investigated the potential influence of HIF-1 status on radiosensitization by gemcitabine. Materials & methods: Three isogenic human breast adenocarcinoma cell lines with different HIF-1 status were included in this study: MDA-MB-231 (breast adenocarcinoma cell line, wt HIF-1), MDA-MB-231 DN-HIF (transfected with dominant-negative HIF-1alpha, abrogating HIF-1 function) and MDA-MB-231 EV (empty vector transfected control, functional HIF-1). Anoxic conditions (<0.1% O2) were achieved in a Bactron IV anaerobic chamber. To analyze the radiosensitizing effect of gemcitabine under normoxic versus anoxic conditions, the clonogenic assay was performed. Cells were treated with 0-8 nM gemcitabine for 24h directly before radiation (0-8 Gy). Using radiosensitizing conditions, cell cycle distribution was monitored flow cytometrically according to the Vindelov method. Results: A clear concentration-dependent radiosensitizing effect of gemcitabine was observed under both normoxic and anoxic conditions (dose enhancement factor (DEF) under normoxia: 1.02-1.70; DEF under anoxia: 1.11-2.04). Combination index (CI) analysis showed an additive interaction between gemcitabine and radiation under normal oxygen conditions (CI 0.902-1.148), and a synergistic interaction under reduced oxygen conditions (CI 0.455-0.889). Statistical analysis using two-way ANOVA revealed no significant differences in radiobiological parameters (DEF, ID10, ID50, mean inactivation dose, surviving fraction at 2 Gy, oxygen enhancement ratio) between MDA-MB-231 EV and MDA-MB-231 DN-HIF cells, suggesting no influence of HIF-1 functionality on radiosensitivity. Considering the cell cycle distribution after treatment with gemcitabine, 24h treatment with 4 or 8 nM gemcitabine established a significant S-phase block in both normoxic and anoxic MDA-MB-231 wt and DN-HIF cells. Conclusion: This study showed that the retained radiosensitizing effect of gemcitabine under anoxic conditions was not tumor tissue specific and could be observed in MDA-MB-231 breast cancer cells. No major role for functional HIF-1 protein in radiosensitization by gemcitabine could be demonstrated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2011-2671

Targeting IL‐13Rα2 in human pancreatic ductal adenocarcinoma with combination therapy of IL‐13‐PE and gemcitabine

Pancreatic cancer is an aggressive disease with only limited therapeutic options available. We have identified that 71% pancreatic ductal adenocarcinoma (PDA) express high levels of IL-13Rα2, a high-affinity receptor for IL-13. To target IL-13Rα2, we have developed a recombinant immunotoxin, which is a fusion of IL-13 and Pseudomonas exotoxin (IL-13-PE). Since IL-13-PE and a commonly used cytotoxic drug gemcitabine act by a different mechanism, we hypothesized that they synergize in mediating antitumor response. Both IL-13-PE and gemcitabine-mediated cytotoxicity to two pancreatic cancer cell lines and when combined synergistic cytotoxicity was observed. This synergism was also demonstrated in vivo in an orthotopic mouse model of human PDA. IL-13-PE and gemcitabine showed complete eradiation of tumors as assessed by whole body imaging of GFP-transfected tumors in 57% of mice in an early cancer model resulting into prolongation of survival. In contrast, monotherapy with either agent did not produce complete eradiation, but tumor volumes were significantly decreased. In advanced PDA model, combination therapy also produced dramatic reduction in tumor growth and enhanced survival compared to animals treated with either agent alone. When IL-13Rα2 was knocked-down by RNAi prior to tumor implantation, IL-13-PE and gemcitabine did not synergize indicating that IL-13Rα2 is essential. Mechanistically, gemcitabine increased IL-13Rα2 expression in vitro and in vivo, which resulted in a synergism of combination therapy. Interestingly, PDA cancer stem cells were resistant to gemcitabine, but not to IL-13-PE. These results suggest that combination therapy with IL-13-PE and gemcitabine may be a useful strategy for PDA therapy.

Concurrent inhibition of NF‐κB, cyclooxygenase‐2, and epidermal growth factor receptor leads to greater anti‐tumor activity in pancreatic cancer

Inactivation of survival pathways such as NF-kappaB, cyclooxygenase (COX-2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3'-Diindolylmethane (B-DIM) is an inhibitor of NF-kappaB and COX-2 and is a well-known chemopreventive agent. We hypothesized that the inhibition of NF-kappaB and COX-2 by B-DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti-tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B-DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX-2, EGFR, and NF-kappaB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down-regulation in the expression of COX-2, NF-kappaB, and EGFR in BxPC-3, COLO-357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF-kappaB, and COX-2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B-DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi-targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX-2, EGFR, and NF-kappaB.

Establishment of a novel orthotopic xenograft model of human gallbladder carcinoma

Gallbladder cancer is characterized by high morbidity and mortality. An appropriate human xenograft animal model could serve as a research tool to investigate new therapeutic strategies. To date, the few reports describing a xenograft animal model showed significant limitations. We improved a murine orthotopic human xenotransplantation model by implanting human gallbladder carcinoma cells directly into the lumen of the gallbladder. Mz-ChA-1 cells were orthotopically injected into the gallbladder of Severe Combined Immune Deficiency (SCID) beige mice inducing the growth of solid tumors. The natural course of the disease, tumor growth, and metastases were analyzed. The cytotoxic drug gemcitabine was tested in vitro and in vitro. All animals revealed solid tumors in the inoculated area with liver infiltration. The median tumor volume in the untreated group was significantly higher than in the gemcitabine-treated group. Immunohistochemical staining revealed expression of human cytokeratin 7 and cytokeratin 8. To analyze tumor cell proliferation, the tumors were stained for the antigen Ki-67, and labeling indices were calculated for both groups. Animals receiving gemcitabine treatment showed significantly lower mean labeling indices. In vitro investigation revealed a significant reduction of DNA synthesis. DNA fragmentation, as a measure of apoptosis, was elevated by roughly 20% within 24 h of treatment. With this, we successfully established an orthotopic xenotransplant animal model and investigated the in vitro and in vivo effects of gemcitabine in human xenografted Mz-ChA-1 gallbladder adenocarcinoma. This model resembles the clinical situation as closely as possible and offers a relevant option for the preclinical testing of new therapeutic strategies.