Studies on Treatment Within the Scope of Medical Biotechnology for Pancreatic Diseases.

The pancreas is made of two compartments: the exocrine pancreas, a source of digestive enzymes, and the endocrine islets which produce vital hormones. Distinct diseases could arise in the pancreas such as diabetes, neuroendocrine tumors, pancreatitis, and pancreatic cancers. Various treatment methods are being researched against these diseases. Treatment with recombinant proteins, therapeutic antibodies, vaccination, gene therapy, tissue engineering, and stem cell treatment are treatment methods. Furthermore, biomarkers are important for both treatment and diagnosis. However, some of the treatment methods mentioned above have not yet been applied to some pancreatic diseases. This review provides insights into the latest advancements in diagnosis and treatment for pancreatic diseases within the scope of medical biotechnology. In addition, some methods that are not yet used for treatment purposes for pancreatic diseases but are used in other diseases that occur in different organs due to similar reasons have been investigated. In this context, possible diagnosis and treatment methods for pancreatic diseases are interpreted. The first aim of this review is to bring together and present the current diagnosis and treatment methods for pancreatic diseases. The second aim is to highlight methods that may have treatment potential by comparing pancreatic diseases that cannot be treated with similar diseases.

Characterization and comparison of insulinoma tumor model and pancreatic damage caused by the tumor, and identification of possible markers.

Insulinoma is a neuroendocrine tumor. It arises from the uncontrolled proliferation of pancreatic ß cells. In this study, we created an insulinoma tumor model in nude mice. INS-1 cells were injected in two different ways, subcutaneously (S.C.) or intraperitoneally (I.P.). Body weight, tumor weight, and size were measured. ELISA kits were used analyze to Glucose, insulin, and CA19-9 levels in serum, pancreas, and tumor tissues. KCNN4, KCNK1, GLUT2, IR, HSP70, HSF1, and HSP90 levels were analyzed by western blotting of membrane and/or cytosolic fractions of tumor and pancreas tissue. Tumor formation occurred in nude mice, but it did not occur in Wistar albino rats. The tumor has neuroendocrine cell morphology. Insulin and CA19-9 levels increased in pancreas tissue. In tumor tissue, KCNN4 levels were higher in both membrane and cytosolic fractions, while KCNK1 levels were lower in the membrane fraction of the S.C. group. HSP70 levels were also lower in the S.C. group. In pancreas tissue, KCNK1 levels were lower in the membrane fraction of the S.C. and I.P. groups. GLUT2 levels increased in both groups according to the control group, while IR levels decreased in the S.C. group compared to the control group. However, HSF1 levels increased in the I.P. group, while HSP90 decreased in the S.C. group in pancreatic tissues. The S.C. group is a more suitable insulinoma tumor model. KCNN4, KCNK1, and HSP70 proteins may be important biomarkers in the diagnosis and treatment of insulinoma.

Exploring dual effects of dinutuximab beta on cell death and proliferation of insulinoma.

Insulinoma INS-1 cells are pancreatic beta cell tumors. Dinutuximab beta (DB) is a monoclonal antibody used in the treatment of neuroblastoma. The aim of this study is to investigate the effects of DB on pancreatic beta cell tumors at the molecular level. DB (Qarziba®) was available from EUSA Pharma. Streptozotocin (STZ) was used induce to cell cytotoxicity. DB was applied to the cells before or after the STZ application. KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were analyzed by q-RT-PCR, and protein levels were analyzed by Western blotting. Analysis of glucose-stimulated insulin secretion was performed. Ca+2 and CA19-9 levels were determined by the ELISA kit. PERK, CHOP, HSP90, p-c-Jun, p-Atf2, and p-Elk1 protein levels were analyzed by simple WES. Decreased KCND3, KCNK1, and PTHrP protein levels and increased KCND3, KCNN4, KCNK1, and PTHrP gene expression levels were observed with DB applied after STZ application. Cell dysfunction was detected with DB applied before and after STZ application. Ca19-9 and Ca+2 levels were increased with DB applied after STZ application. PERK, CHOP, and p-Elk1 levels decreased, while HSP90 levels increased with DB applied after STZ application. CHOP, p-Akt-2, and p-c-Jun levels increased in the DB group. As a result, INS-1 cells go to cell death via the ERK signaling pathway without ER stress and release insulin with the decrease of K+ channels and an increase in Ca+2 levels with DB applied after STZ application. Moreover, the cells proliferate via JNK signaling with DB application. DB holds promise for the treatment of insulinoma. The study should be supported by in vivo studies.

The therapeutic effects of exosomes the first time isolated from pancreatic islet-derived progenitor cells in the treatment of pancreatic cancer.

Insulinoma is an excessive insulin-released beta cell tumor. Pancreas cancer is one of the deadliest malignant neoplasms. Exosomes are secreted cell membrane vesicles containing a large number of proteins, lipids, and nucleic acids. The aim of this study is to investigate the effects of exosomes on two cell lines of benign and malignant character. For the first time, exosomes were isolated from pancreatic island-derived progenitor cells (PID-PCs) and applied to INS-1 and MiaPaCa-2 cells. In addition, exosomes isolated from PID-PC, MiaPaca-2, and INS-1 cells were characterized in order to compare their sizes with other previously isolated exosomes. Alix, TSG101, CD9, and CD81 were analyzed. The size and concentration of exosomes and the cell viability were detected. The cells were marked with HSP90, HSF-1, Kaspaz-8, Active-Kaspaz-3, Beclin, and p-Bcl-2. The cell cytotoxicity and insulin levels kit were measured. Alix in all exosomes, and PID-PC, MiaPaca-2 cell lysates; TSG101 in PID-PC and MiaPaca-2 cell lysates; CD9 in INS-1 exosomes were detected. The dimensions of isolated exosomes were 103.6 ± 28.6 nm, 100.7 ± 10 nm, and 147.2 ± 12.3 nm for PID-PCs, MiaPaca-2, and INS-1 cells. The cell viability decreased and HSP90 increased in the MiaPaca-2 cells. The HSF-1 was higher in the control MiaPaca-2 cell compared to the control INS-1 cell, and the exosome-treated MiaPaca-2 cell compared to the exosome-treated INS-1 cell. Beclin and p-Bcl-2 were decreased in the exosome-treated MiaPaca-2 cells. The insulin level in the cell lysates increased compared to cell secretion in INS-1 cells. In conclusion, exosomes isolated from the PID-PC caused cell death in the MiaPaca-2 cells in a time- and dose-dependent manner. The IC50 value determined for MiaPaca-2 cells has no effect on cell viability in INS-1 cells, which best mimics pancreatic beta cells and can be used instead of healthy pancreatic beta cells. Isolated exosomes can kill cancer cells without damaging healthy cells.

BuOH fraction of Salix Babylonica L. extract increases pancreatic beta-cell tumor death at lower doses without harming their function.

Salix babylonica L. is a species of the willow tree. Insulinoma is a tumor originating from pancreatic beta cells. This study aims to research the effect of different fractions of Salix babylonica L. leaf extract on INS-1 cells for treating pancreatic tumors. Cell death occurred at lower doses in the EtOAc fraction. The cells are functional in the BuOH fraction but not in EtOAc and H2O fractions. The EtOAc fraction has a higher percentage of necrosis and ROS. INS1, INS2, and AKT gene expressions in the H2O fraction, GLUT2, IR, HSP70 gene expressions, and WNT4 protein levels increased in the BuOH fraction. HSP90 gene expression, Beta-actin, GAPDH, insulin, HSP70, HSP90, HSF1, Beta-Catenin, and WNT7A protein levels were decreased, while IR immunolabelling intensity increased in both fractions. Ca+2, K+, Na+, and CA-19-9 in the cell, Ca+2 and K+ in secretion increased. The secondary metabolites in the EtOAc fraction cause more damage in INS-1 cells. Since the water fraction also causes the cells to die in high doses, cell function is damaged. The secondary metabolites in the BuOH fraction kill INS-1 cells with less damage. This makes the BuOH fraction of Salix babylonica L. more valuable.

Epibrassinolide impaired colon tumor progression and induced autophagy in SCID mouse xenograft model via acting on cell cycle progression without affecting endoplasmic reticulum stress observed in vitro.

Epibrassinolide is a member of brassinosteroids with a polyhydroxysteroid structure similar to steroid hormones of vertebrates. It was shown that EBR decreased cell proliferation and induced apoptosis in different colon cancer cell lines without exerting a cytotoxic effect in epithelial fetal human colon cells. This finding highlighted the potential of epibrassinolide in clinical therapeutic setup. In our previous studies, we showed that epibrassinolide was able to induce apoptosis via endoplasmic reticulum stress. Recently, we also showed that endoplasmic reticulum and apoptotic stresses can be prevented via autophagic induction in non-cancerous epithelial or aggressive forms of cancer cells. Therefore, here in this study, we evaluated the anti-tumoral effect of epibrassinolide as well as the autophagy involvement in the aggressive forms of colon cancer cell lines as well as in vivo SCID mouse xenograft colon cancer model for the first time. For this purpose, SCID mouse model was used for subcutaneous injection of colon cancer cells in matrigel formulation. We found that autophagy is induced in both in vitro and in vivo models. Following tumor formation, SCID mice were treated daily with increasing concentrations of epibrassinolide for two weeks. Our findings showed that EBR inhibited the volume and diameter of the tumor in a dose-dependent manner by causing cell cycle arrest. Therefore our data suggest that epibrassinolide exerts a cytostatic effect on the agrressive form of colon cancer model in vivo, without affecting endoplasmic reticulum stress and the induction of autophagy might have role in this effect of epibrassinolide.

miR-375 induces adipogenesis through targeting Erk1 in pancreatic duct cells under the influence of sodium palmitate.

The goal of this study was to research long-term saturated fatty acid overexposure that can induce differentiation of pancreatic duct cells into adipocytes and also into ß-cells. The important findings can be summarized as follows: (i) adipogenesis and early stage ß-cell differentiation were stimulated in duct cells under lipotoxicity and glucolipotoxicity conditions, (ii) miR-375 expression was upregulated while its target Erk1 was downregulated and miR-375 inhibitor upregulated Erk1 while expression of adipogenesis markers was downregulated in duct cells under both conditions, (iii) apoptosis was induced in ß and duct cells under both conditions, (iv) lipotoxicity induced proliferation of co-cultured ß-cells. These findings suggest that long-term saturated fatty acid overexposure may cause intrapancreatic fat accumulation by inducing differentiation of duct cells into adipocytes and it may contributes to ß-cell compensation by stimulating the early stage of ß-cell differentiation in duct cells. In addition, miR-375 may have the potential to be a new target in the treatment of Type 2 diabetes, and NAFPD due to its role in the adipogenesis of duct cells.

Indomethacin decreases insulin secretion by reducing KCa3.1 as a biomarker of pancreatic tumor and causes apoptotic cell death.

Insulinomas originate from pancreatic ß cells and it is the most widely known tumor. Indomethacin is a nonsteroidal anti-inflammatory drug, which is used for blocking the production of some natural substances that cause inflammation and decrease pain. In this study, I aimed to investigate the effects of indomethacin on rat insulinoma INS-1 cells. The relationship between cell death and insulin metabolism was determined with the administration of indomethacin. The cell viability by WST-1; the apoptosis and necrosis levels by ELISA kits; malondialdehyde levels by spectrophotometer; and beclin, intracellular insulin, insulin secretion, KCa3.1, insulin receptor (IR), glucose transporter type 2 (GLUT2), activating transcription factor 2 (ATF2), Elk1, c-Jun, Akt and phosphorylated ATF2, Elk1, c-Jun, Akt, intracellular betacellulin and betacellulin secretion levels by Western blot analysis investigated. The Ins1, Ins2, IR, GLUT2, ATF2, Elk1, c-Jun, Akt, and Betacellulin gene expression levels were determined by the real-time quantitative reverse transcription-polymerase chain reaction method. Apoptotic cell death was observed with the administration of indomethacin. The insulin secretion and Ins1, Ins2 gene expression levels decreased. The insulin receptor and GLUT2 levels increased, while KCa3.1 (KCNN4) levels decreased with the administration of indomethacin to insulinoma INS-1 cells. A decrease was observed in the total c-Jun, phosphorylated ATF2, Elk1, c-Jun, and Akt levels. Betacellulin secretion levels increased. In insulinoma INS-1 cells, apoptotic cell death occurred in the following manner: (i) indomethacin might decrease insulin secretion by reducing KCa3.1, (ii) might inactivate the JNK/ERK pathway with the inactivity of transcription factors.

Investigation of cell death mechanism and activity of esculetin-loaded PLGA nanoparticles on insulinoma cells in vitro.

AIM: The purpose of this study was to prepare targeted cancer therapy formulation against insulinoma INS-1 cells and to study its effect on cell death with related mechanisms in vitro. METHODS: Polylactide-co-glycolide (PLGA) nano-micelles were used for preparation of esculetin nano-formulation (nano-esculetin). The cells were treated with nano-esculetin and free esculetin. Apoptotic and necrotic cell death percentages, cell proliferation, ATP and GTP reductions and insulin levels were investigated on insulinoma INS-1 cells for both free and nano-esculetin formulations. RESULTS: About 50 mg of PLGA was able to carry 20 mg esculetin in 20 ml of formulation. The obtained optimized formulation was 150 nm, with 92% encapsulation efficiency and a slow-release behaviour was observed during release studies. Nano-esculetin bearing 25, 50 and 100 µg esculetin and free esculetin in equivalent doses successfully decreased cell viability. The prevailing cell death mechanism was necrosis. Along with cell proliferation, intracellular insulin and the ratio of ATP and GTP were decreased even with 12.5, 25 and 50 µg esculetin bearing nano-formulation and its equivalent free esculetin. CONCLUSIONS: The results revealed that esculetin is able to show its anti-tumor afficacy after loading to PLGA nano-micelles and nano-encapsulation intensifies its cytotoxic activity in vitro. Current study shows that esculetin and its nano formulations are promising agents in treatment of insulinoma.

The effects of atorvastatin on the kidney injury in mice with pulmonary fibrosis.

OBJECTIVES: The present study investigated the effects of atorvastatin on kidney injury in mice with pulmonary fibrosis (PF). METHODS: Adult mice were divided into four groups: mice treated with intratracheal bleomycin (I) and their controls (II), and mice treated with atorvastatin for 10 days after 7 days from bleomycin treatment (III) and their controls (IV). Mice were dissected on the 21st day. KEY FINDINGS: Mononuclear cell infiltrations, injured proximal tubule epithelium and p-c-Jun level increased, while cell proliferation and the levels of p-SMAD2, ELK1, p-ELK1, p-ATF2 and c-Jun decreased in the kidney tissue of mice with PF. The atorvastatin treatments to mice with PF resulted in significant increases at the TGF-ß activation, cell proliferation and kidney damage and decreases in the levels of p-SMAD2, p-ELK1, p-ATF2 and p-c-Jun, but not change the p-SMAD3, ELK1 and ATF2 in kidneys. CONCLUSIONS: The depletion of MAPK signals, rather than SMAD signalling, is effective in kidney damage of mice with PF. Atorvastatin did not regress kidney damage in these mice, whereas it increases the kidney injury. The c-Jun-mediated JNK signals could help kidney repair through cell proliferation. The treatment time and doses of atorvastatin should be optimized for regression of kidney damage.

Vitronectin, fibronectin and epidermal growth factor induce proliferation via the JNK and ERK pathways in insulinoma INS-1 cells.

An insulinoma is a tumor formed by beta cells in the Langerhans islets of the pancreas. Vitronectin (VTN), fibronectin (FN) and epidermal growth factor (EGF) are important in cell signaling. The aim of this study was to investigate the molecular mechanism that occurs in INS-1 cells with the administration of VTN, FN and EGF in proliferative doses. We determined the proliferative doses of EGF, VTN and FN. The molecular mechanism of proliferation has been investigated alone or in the combination of these proteins. It was observed that INS-1 cells did not have VTN and FN. Cell viability increased with the administration of 0.1 µg/ml VTN, 0.1 µg/ml FN and 1 mg/ml EGF. Proliferation increased with the administration of FN + EGF, and VTN + FN + EGF together when compared to the control group. The total JNK levels did not change between the groups; however, the active JNK levels increased in the VT + FN + EGF group compared to the control group. The total ERK levels increased in the VT + FN + EGF group, and the active ERK levels increased in the VTN + FN, VTN + EGF and VTN + FN + EGF groups compared to the control group. The JNK and ERK pathways are important for proliferation. The JNK and ERK pathways were activated in VTN + FN + EGF administered group. However, it was observed that the ERK pathway was more active than the JNK pathway.

Involvement of dying beta cell originated messenger molecules in differentiation of pancreatic mesenchymal stem cells under glucotoxic and glucolipotoxic conditions.

Beta cell mass regulation represents a critical issue for understanding and treatment of diabetes. The most important process in the development of diabetes is beta cell death, generally induced by glucotoxicity or glucolipotoxicity, and the regeneration mechanism of new beta cells that will replace dead beta cells is still not fully understood. The aim of this study was to investigate the generation mechanism of new beta cells by considering the compensation phase of type 2 diabetes mellitus. In this study, pancreatic islet derived mesenchymal stem cells (PI-MSCs) were isolated from adult rats and characterized. Then, beta cells isolated from rats were co-cultured with PI-MSCs and they were exposed to glucotoxicity, lipotoxicity and glucolipotoxicity conditions for 72 hr. As the results apoptotic and necrotic cell death were increased in both PI-MSCs and beta cells especially by the exposure of glucotoxic and glucolipotoxic conditions to the co-culture systems. Glucotoxicity induced-differentiated beta cells were functional due to their capability of insulin secretion in response to rising glucose concentrations. Moreover, beta cell proliferation was induced in the glucotoxicity-treated co-culture system whereas suppressed in lipotoxicity or glucolipotoxicity-treated co-culture systems. In addition, 11 novel proteins, that may release from dead beta cells and have the ability to stimulate PI-MSCs in the direction of differentiation, were determined in media of glucotoxicity or glucolipotoxicity-treated co-culture systems. In conclusion, these molecules were considered as important for understanding cellular mechanism of beta cell differentiation and diabetes. Thus, they may be potential targets for diagnosis and cellular or therapeutic treatment of diabetes.

Necrotic cell death occur via JNK pathway with the activity of transcription factor c-Jun by 4-MC in INS-1 cell line.

In this study, it was aimed to determine the doses of 4-methylcatechol causing cell death in rat insulinoma ß-cells (INS-1), to find out the type of cellular death at these doses, and to investigate the molecular mechanism of cellular death occurring. More necrotic cells were observed than apoptosis with the administration of 350, 400, and 450 µM 4-methylcatechol. Lactate dehydrogenase levels, reactive oxygen species, mitochondrial potential loss, ATP, and GTP losses increased at these doses. The JNK and ERK cellular pathway were screened. We observed an increase in p-RAF1 activity, the active JNK amount, the total c-Jun amount, while a decrease in p-RAF1 expression, the total JNK amount, JNK expression, ATF2 expression, active ERK, and its expression and Elk1 expression. It was concluded that cells perform necrotic death by the following options: i) phosphorylated RAF1 activates the JNK pathway with the activity of transcription factor c-Jun; ii) Hsp 70 and Hsp 90 do not show a change inside the cell, rendering the JNK pathway active.