Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading ... more Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading cause of cancer deaths worldwide. Although therapeutic advances have been made in the few last years, the prognosis remains poor. Thus, there is a dire need to develop novel therapeutic strategies. In this regard, two approaches can be considered: (1) the identification of tumor-targeted delivery systems and (2) the targeting of molecule(s) whose aberrant expression is confined to tumor cells. In this work, we focused on the second approach. Among the different kinds of possible target molecules, we discuss the potential therapeutic value of targeting non-coding RNAs (ncRNAs), which include micro interfering RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). These molecules represent the most significant RNA transcripts in cells and can regulate many HCC features, including proliferation, apoptosis, invasion and metastasis. In the first part of the review, the ma...
Background Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Mo... more Background Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Most studies have reported the safety and effectiveness of electrochemotherapy for palliative treatment of gynecological cancers and mostly vulvar squamous cell carcinoma. Some tumors, however, fail to respond to electrochemotherapy. The biological features/determinants for the nonresponsiveness are not determined yet. Patient and methods A recurrence of vulvar squamous cell carcinoma was treated by electrochemotherapy using intravenous administration of bleomycin. The treatment was performed by hexagonal electrodes according to standard operating procedures. We analyzed the factors that could determine nonresponsiveness to electrochemotherapy. Results Based on the presented case of nonresponsive vulvar recurrence to electrochemotherapy, we hypothesize that the vasculature of the tumors prior to treatment may predict the response to electrochemotherapy. The histological analysis showed min...
Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As ... more Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. Methods: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. Results: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse mod...
Background One of the new treatment options for unresectable locally advanced pancreatic cancer i... more Background One of the new treatment options for unresectable locally advanced pancreatic cancer is electrochemotherapy (ECT), a local ablative therapy that potentiates the entry of chemotherapeutic drugs into the cells, by the application of an electric field to the tumor. Its feasibility and safety were demonstrated in preclinical and clinical studies; however, there is a lack of preclinical studies assessing the actions of different drugs used in ECT, their mechanisms and interactions with other target drugs that are used in clinical practice. Materials and methods The aim of the study was to determine the cytotoxicity of two chemotherapeutic drugs usually used in ECT (bleomycin and cisplatin) in the BxPC-3 human pancreatic carcinoma cell line and evaluate the interactions of ECT with the targeted drug sunitinib. First, the cytotoxicity of ECT using both chemotherapeutics was determined. In the next part, the interactions of ECT and sunitinib were evaluated through determination o...
The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigati... more The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigation. Among the different novel drugs, small interfering RNAs (siRNAs) are particularly attractive because of their specificity of action and efficacy, as documented in many experimental setups. However, the fragility of these molecules in the biological environment necessitates the use of delivery materials able to protect them and possibly target them to the cancer cells. Among the different delivery materials, those based on polymers and lipids are considered very interesting because of their biocompatibility and ability to carry/deliver siRNAs. Despite these features, polymers and lipids need to be engineered to optimize their delivery properties for OC. In this review, we concentrated on the description of the therapeutic potential of siRNAs and polymer-/lipid-based delivery systems for OC. After a brief description of OC and siRNA features, we summarized the strategies employed to mi...
Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense ag... more Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense against infection. Despite extensive progress in the study of sensor signaling pathways during the last decade, the detailed mechanisms remain unclear. In our previous studies, we reported increased type I interferon expression and the upregulation of several proposed cytosolic DNA sensors after transfection of several tumor cell types with plasmid DNA (pDNA). In the present study, we sought to reveal the early events in the cytosolic sensing of this nucleic acid in a myoblast cell line. We demonstrated that DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) bound plasmid DNA in the cytosol within 15 minutes of transfection and at consistent levels for 4 h. Interferon activated gene 204 protein (p204) and DEAH box helicase 9 (DHX9) also bound pDNA, peaking 15 and 30 min respectively. Plasmid DNA was not detectably bound by DEAD box helicase 60 (DDX6...
Despite the advances in anticancer therapies, their effectiveness for many human tumors is still ... more Despite the advances in anticancer therapies, their effectiveness for many human tumors is still far from being optimal. Significant improvements in treatment efficacy can come from the enhancement of drug specificity. This goal may be achieved by combining the use of therapeutic molecules with tumor specific effects and delivery carriers with tumor targeting ability. In this regard, nucleic acid-based drug (NABD) and particularly small interfering RNAs (siRNAs), are attractive molecules due to the possibility to be engineered to target specific tumor genes. On the other hand, polymeric-based delivery systems are emerging as versatile carriers to generate tumor-targeted delivery systems. Here we will focus on the most recent findings in the selection of siRNA/polymeric targeted delivery systems for hepatocellular carcinoma (HCC), a human tumor for which currently available therapeutic approaches are poorly effective. In addition, we will discuss the most attracting and, in our opini...
Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane,... more Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane, allowing non-permeant anticancer drugs to enter the cell, thus augmenting their cytotoxicity by orders of magnitude. In preclinical studies, bleomycin and cisplatin proved to be the most suitable for clinical use. Intravenous administration of cisplatin for electrochemotherapy is still not widely accepted in the clinics, presumably due to its lower antitumor effectiveness, but adjuvant therapy by immunomodulatory or vascular-targeting agents could provide a way for its potentiation. Hence, the aim of the present study was to explore the possibility of adjuvant tumour necrosis factor α (TNF-α) therapy to potentiate antitumor effectiveness of electrochemotherapy with intravenous cisplatin administration in murine sarcoma. In vivo study was designed to evaluate the effect of TNF-α applied before or after the electrochemotherapy and to evaluate the effect of adjuvant TNF-α on electrochemoth...
The search for an ideal delivery system capable of targeted delivery of therapeutic molecules to ... more The search for an ideal delivery system capable of targeted delivery of therapeutic molecules to tumor cells after a systemic administration is ongoing. Recently liposomes prepared from archaeal lipids (archaeosomes) emerged as a viable alternative to traditional liposomes. They are highly stable and have low toxicity in vitro. But even once inside the cell, they still do not release their cargo. Thus, when combined with nanosecond electroporation nsEP, which permeabilizes small vesicles inside the cell, presents an attractive delivery system with spatial and temporal control of the therapeutic molecules delivery. Therefore, the aim of our study was to determine the in vivo stability of archaeosomes from Aeropyrum pernix K1 and their extravasation from tumor blood vessels. We used intravital video microscopy to determine the circulation time of archaeosomes in normal and tumor blood vessels and whether they extravasate from tumor blood vessels. Additionally, their stability after exposure to mouse blood in situ was determined. The intravenously injected archaeosomes were circulating for 6 minutes in normal and tumor blood vessels and showed a preference for increased retention in tumor blood vessels. The measured size of archaeosomes in vivo varied from 200 nm up to several thousand nm, due to aggregation of archaeosomes when in contact with blood, which was confirmed in situ. The obtained results elucidate the behavior of archaeosomes from A. pernix in vivo and indicate future steps necessary to make them a viable delivery system potentially combined with nsEP.
V zadnjih letih smo v onkologiji priča hitremu napredku na področju biopsije telesnih tekočin oz.... more V zadnjih letih smo v onkologiji priča hitremu napredku na področju biopsije telesnih tekočin oz. tekočinske biopsije. Tekočinska biopsija predstavlja analizo vzorca odvzete telesne tekočine (v onkologiji je to običajno kri), v katerem iščemo znake navzočnosti raka. V krvi lahko analiziramo cirkulirajočo prosto/cirkulirajočo tumorsko DNA (cf/ctDNA) iz tumorskih celic, eksosome, ki jih v kri sproščajo tumorske celice, in tudi cirkulirajoče tumorske celice (CTC). V pregledu predstavljamo najnovejša dognanja in raziskave na področju tekočinske biopsije pri raku in poleg uporabnosti izpostavljamo tudi njihove omejitve, ki jih bo treba natančno opredeliti, če bomo želeli to metodo uporabljati v vsakodnevni obravnavi bolnikov.Recently, new exciting developments in oncology have been made in the field of liquid biopsies. In liquid biopsy, samples of blood or other body fluids are analyzed for tumor-derived material. Tumor-derived material comprises cell-free circulating tumor DNA (cf/ctDNA...
Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To ful... more Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of exp...
Background Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Di... more Background Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different isolation tech-niques based on their biological or physical features were established. Currently, the most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology. Materials and methods The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix®) and biological (MACS®) separation technique was performed. Results In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer...
— Polymerase chain reaction (PCR) has enabled enormous progress in the field of molecular biology... more — Polymerase chain reaction (PCR) has enabled enormous progress in the field of molecular biology in the last twenty-five years. It became popular due to its high sensitivity and specificity and was introduced to numerous scientific fields. Today many different variants of PCR exist, with real-time PCR being the most common. This method is being successfully used at our institution for a variety of different applications, among which gene expression analyzes and development of protocols for clinical diagnostics currently dominate. But the extreme flexibility and customizability of real-time PCR, coupled with our team’s expertise, ensures efficient application of this technique to various new research projects.
Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading ... more Hepatocellular carcinoma (HCC) remains a global health challenge, representing the third leading cause of cancer deaths worldwide. Although therapeutic advances have been made in the few last years, the prognosis remains poor. Thus, there is a dire need to develop novel therapeutic strategies. In this regard, two approaches can be considered: (1) the identification of tumor-targeted delivery systems and (2) the targeting of molecule(s) whose aberrant expression is confined to tumor cells. In this work, we focused on the second approach. Among the different kinds of possible target molecules, we discuss the potential therapeutic value of targeting non-coding RNAs (ncRNAs), which include micro interfering RNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). These molecules represent the most significant RNA transcripts in cells and can regulate many HCC features, including proliferation, apoptosis, invasion and metastasis. In the first part of the review, the ma...
Background Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Mo... more Background Electrochemotherapy has good local effectiveness in the treatment of vulvar cancer. Most studies have reported the safety and effectiveness of electrochemotherapy for palliative treatment of gynecological cancers and mostly vulvar squamous cell carcinoma. Some tumors, however, fail to respond to electrochemotherapy. The biological features/determinants for the nonresponsiveness are not determined yet. Patient and methods A recurrence of vulvar squamous cell carcinoma was treated by electrochemotherapy using intravenous administration of bleomycin. The treatment was performed by hexagonal electrodes according to standard operating procedures. We analyzed the factors that could determine nonresponsiveness to electrochemotherapy. Results Based on the presented case of nonresponsive vulvar recurrence to electrochemotherapy, we hypothesize that the vasculature of the tumors prior to treatment may predict the response to electrochemotherapy. The histological analysis showed min...
Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As ... more Background: For hepatocellular carcinoma (HCC), effective therapeutic approaches are lacking. As aberrant gene methylation is a major contributor to HCC development, demethylating drugs such as 5-azacytidine (5-Aza) have been proposed. As most 5-Aza mechanisms of action are unknown, we investigated its phenotypic/molecular effects. Methods: 5-Aza effects were examined in the human HCC cell lines JHH-6/HuH-7 and in the rat cell-line N1-S1. We also employed a xenograft mouse model (HuH-7), a zebrafish model (JHH-6), and an orthotopic syngeneic rat model (N1-S1) of HCC. Results: 5-Aza downregulated cell viability/growth/migration/adhesion by upregulating miR-139-5p, which in turn downregulated ROCK2/cyclin D1/E2F1 and increased p27kip1, resulting in G1/G0 cell accumulation. Moreover, a decrease in cyclin B1 and an increase in p27kip1 led to G2/M accumulation. Finally, we observed a decrease in MMP-2 levels, a stimulator of HCC cell migration. Aza effects were confirmed in the mouse mod...
Background One of the new treatment options for unresectable locally advanced pancreatic cancer i... more Background One of the new treatment options for unresectable locally advanced pancreatic cancer is electrochemotherapy (ECT), a local ablative therapy that potentiates the entry of chemotherapeutic drugs into the cells, by the application of an electric field to the tumor. Its feasibility and safety were demonstrated in preclinical and clinical studies; however, there is a lack of preclinical studies assessing the actions of different drugs used in ECT, their mechanisms and interactions with other target drugs that are used in clinical practice. Materials and methods The aim of the study was to determine the cytotoxicity of two chemotherapeutic drugs usually used in ECT (bleomycin and cisplatin) in the BxPC-3 human pancreatic carcinoma cell line and evaluate the interactions of ECT with the targeted drug sunitinib. First, the cytotoxicity of ECT using both chemotherapeutics was determined. In the next part, the interactions of ECT and sunitinib were evaluated through determination o...
The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigati... more The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigation. Among the different novel drugs, small interfering RNAs (siRNAs) are particularly attractive because of their specificity of action and efficacy, as documented in many experimental setups. However, the fragility of these molecules in the biological environment necessitates the use of delivery materials able to protect them and possibly target them to the cancer cells. Among the different delivery materials, those based on polymers and lipids are considered very interesting because of their biocompatibility and ability to carry/deliver siRNAs. Despite these features, polymers and lipids need to be engineered to optimize their delivery properties for OC. In this review, we concentrated on the description of the therapeutic potential of siRNAs and polymer-/lipid-based delivery systems for OC. After a brief description of OC and siRNA features, we summarized the strategies employed to mi...
Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense ag... more Mammalian cells express a variety of nucleic acid sensors as one of the first lines of defense against infection. Despite extensive progress in the study of sensor signaling pathways during the last decade, the detailed mechanisms remain unclear. In our previous studies, we reported increased type I interferon expression and the upregulation of several proposed cytosolic DNA sensors after transfection of several tumor cell types with plasmid DNA (pDNA). In the present study, we sought to reveal the early events in the cytosolic sensing of this nucleic acid in a myoblast cell line. We demonstrated that DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) bound plasmid DNA in the cytosol within 15 minutes of transfection and at consistent levels for 4 h. Interferon activated gene 204 protein (p204) and DEAH box helicase 9 (DHX9) also bound pDNA, peaking 15 and 30 min respectively. Plasmid DNA was not detectably bound by DEAD box helicase 60 (DDX6...
Despite the advances in anticancer therapies, their effectiveness for many human tumors is still ... more Despite the advances in anticancer therapies, their effectiveness for many human tumors is still far from being optimal. Significant improvements in treatment efficacy can come from the enhancement of drug specificity. This goal may be achieved by combining the use of therapeutic molecules with tumor specific effects and delivery carriers with tumor targeting ability. In this regard, nucleic acid-based drug (NABD) and particularly small interfering RNAs (siRNAs), are attractive molecules due to the possibility to be engineered to target specific tumor genes. On the other hand, polymeric-based delivery systems are emerging as versatile carriers to generate tumor-targeted delivery systems. Here we will focus on the most recent findings in the selection of siRNA/polymeric targeted delivery systems for hepatocellular carcinoma (HCC), a human tumor for which currently available therapeutic approaches are poorly effective. In addition, we will discuss the most attracting and, in our opini...
Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane,... more Electrochemotherapy is a tumour ablation modality, based on electroporation of the cell membrane, allowing non-permeant anticancer drugs to enter the cell, thus augmenting their cytotoxicity by orders of magnitude. In preclinical studies, bleomycin and cisplatin proved to be the most suitable for clinical use. Intravenous administration of cisplatin for electrochemotherapy is still not widely accepted in the clinics, presumably due to its lower antitumor effectiveness, but adjuvant therapy by immunomodulatory or vascular-targeting agents could provide a way for its potentiation. Hence, the aim of the present study was to explore the possibility of adjuvant tumour necrosis factor α (TNF-α) therapy to potentiate antitumor effectiveness of electrochemotherapy with intravenous cisplatin administration in murine sarcoma. In vivo study was designed to evaluate the effect of TNF-α applied before or after the electrochemotherapy and to evaluate the effect of adjuvant TNF-α on electrochemoth...
The search for an ideal delivery system capable of targeted delivery of therapeutic molecules to ... more The search for an ideal delivery system capable of targeted delivery of therapeutic molecules to tumor cells after a systemic administration is ongoing. Recently liposomes prepared from archaeal lipids (archaeosomes) emerged as a viable alternative to traditional liposomes. They are highly stable and have low toxicity in vitro. But even once inside the cell, they still do not release their cargo. Thus, when combined with nanosecond electroporation nsEP, which permeabilizes small vesicles inside the cell, presents an attractive delivery system with spatial and temporal control of the therapeutic molecules delivery. Therefore, the aim of our study was to determine the in vivo stability of archaeosomes from Aeropyrum pernix K1 and their extravasation from tumor blood vessels. We used intravital video microscopy to determine the circulation time of archaeosomes in normal and tumor blood vessels and whether they extravasate from tumor blood vessels. Additionally, their stability after exposure to mouse blood in situ was determined. The intravenously injected archaeosomes were circulating for 6 minutes in normal and tumor blood vessels and showed a preference for increased retention in tumor blood vessels. The measured size of archaeosomes in vivo varied from 200 nm up to several thousand nm, due to aggregation of archaeosomes when in contact with blood, which was confirmed in situ. The obtained results elucidate the behavior of archaeosomes from A. pernix in vivo and indicate future steps necessary to make them a viable delivery system potentially combined with nsEP.
V zadnjih letih smo v onkologiji priča hitremu napredku na področju biopsije telesnih tekočin oz.... more V zadnjih letih smo v onkologiji priča hitremu napredku na področju biopsije telesnih tekočin oz. tekočinske biopsije. Tekočinska biopsija predstavlja analizo vzorca odvzete telesne tekočine (v onkologiji je to običajno kri), v katerem iščemo znake navzočnosti raka. V krvi lahko analiziramo cirkulirajočo prosto/cirkulirajočo tumorsko DNA (cf/ctDNA) iz tumorskih celic, eksosome, ki jih v kri sproščajo tumorske celice, in tudi cirkulirajoče tumorske celice (CTC). V pregledu predstavljamo najnovejša dognanja in raziskave na področju tekočinske biopsije pri raku in poleg uporabnosti izpostavljamo tudi njihove omejitve, ki jih bo treba natančno opredeliti, če bomo želeli to metodo uporabljati v vsakodnevni obravnavi bolnikov.Recently, new exciting developments in oncology have been made in the field of liquid biopsies. In liquid biopsy, samples of blood or other body fluids are analyzed for tumor-derived material. Tumor-derived material comprises cell-free circulating tumor DNA (cf/ctDNA...
Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To ful... more Interleukin 12 (IL-12) is a key cytokine that mediates antitumor activity of immune cells. To fulfill its clinical potential, the development is focused on localized delivery systems, such as gene electrotransfer, which can provide localized delivery of IL-12 to the tumor microenvironment. Gene electrotransfer of the plasmid encoding human IL-12 is already in clinical trials in USA, demonstrating positive results in the treatment of melanoma patients. To comply with EU regulatory requirements for clinical application, which recommend the use of antibiotic resistance gene-free plasmids, we constructed and developed the production process for the clinical grade quality antibiotic resistance gene-free plasmid encoding human IL-12 (p21-hIL-12-ORT) and its ortholog encoding murine IL-12 (p21-mIL-12-ORT). To demonstrate the suitability of the p21-hIL-12-ORT or p21-mIL-12-ORT plasmid for the first-in-human clinical trial, the biological activity of the expressed transgene, its level of exp...
Background Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Di... more Background Circulating tumor cells (CTCs) have become an important biomarker in breast cancer. Different isolation tech-niques based on their biological or physical features were established. Currently, the most widely used methods for visualization after their separation are based on immunofluorescent staining, which does not provide the information on the morphology. Materials and methods The aim of this study was to evaluate how two different separation techniques affect cell morphology and to analyse cell morphology with techniques used in routine cytopathological laboratory. A direct side-by-side comparison of physical (Parsortix®) and biological (MACS®) separation technique was performed. Results In the preclinical setting, both isolation techniques retained the viability and antigenic characteristics of MCF7 breast cancer cells. Some signs of degeneration such as cell swelling, cytoplasmic blebs, villous projections and vacuolization were observed. In metastatic breast cancer...
— Polymerase chain reaction (PCR) has enabled enormous progress in the field of molecular biology... more — Polymerase chain reaction (PCR) has enabled enormous progress in the field of molecular biology in the last twenty-five years. It became popular due to its high sensitivity and specificity and was introduced to numerous scientific fields. Today many different variants of PCR exist, with real-time PCR being the most common. This method is being successfully used at our institution for a variety of different applications, among which gene expression analyzes and development of protocols for clinical diagnostics currently dominate. But the extreme flexibility and customizability of real-time PCR, coupled with our team’s expertise, ensures efficient application of this technique to various new research projects.
Uploads
Papers by Maja Cemazar