Attenuation of Pancreatic Cancer In Vitro and In Vivo via Modulation of Nrf2 and NF-κB Signaling Pathways by Natural Compounds.

Pancreatic cancer is a disease in which deregulation of signaling pathways plays a key role, thus searching for their novel modulators is a promising therapeutic strategy. Hence, in this study, the effect of phytochemical combinations on the canonical and non-canonical activation of Nrf2 and its interaction with the NF-κB pathway was evaluated in extensively proliferating pancreatic cancer cell line, PSN-1, in comparison to non-cancerous MS1 cells. The activation of Nrf2 and NF-κB, expression of their target genes, and effect on cell survival were assessed in PSN-1 cells. The tumor burden was evaluated in mice carrying xenografts. PSN-1 cells were more sensitive to the tested compounds as compared to the MS1 cell line. Combination of xanthohumol and phenethyl isothiocyanate was more effective than single compounds at decreasing the canonical and non-canonical activation of Nrf2 in PSN-1 cancer cells. Decreased activation of NF-κB, and subsequent reduced cytosolic COX-2 and nuclear STAT3 level indicated their anti-inflammatory and pro-apoptotic activities. In vivo studies showed the partial response in groups treated with xanthohumol or the combination of xanthohumol and phenethyl isothiocyanate. Overall, these results suggest that the combination of xanthohumol and phenethyl isothiocyanate may be a promising therapeutic candidate against pancreatic cancer.

Comparison of the Impact of Xanthohumol and Phenethyl Isothiocyanate and Their Combination on Nrf2 and NF-κB Pathways in HepG2 Cells In Vitro and Tumor Burden In Vivo.

Background: Increasing evidence suggests that combinations of phytochemicals are more efficient than single components in the modulation of signaling pathways involved in cancer development. In this study, the impact of phenethyl isothiocyanate (PEITC), indole-3-carbinol (I3C), xanthohumol, (X), and resveratrol (RES) and their combinations on the activation and expression of Nrf2 and NF-κB in human hepatocytes and HCC cells were evaluated. Methods: THLE-2 and HepG2 cells were exposed to single phytochemicals and their combinations for 24 h. The activation of Nrf2 and NF-κB, expression of their target genes, and effect on cells survival were assessed. The tumor burden was evaluated in mice carrying xenografts. Results: All phytochemicals enhanced the activation and expression of Nrf2 and its target genes SOD and NQO1 in HepG2 cells. The increased expression of NQO1 (~90%) was associated with increased ROS generation. X + PEITC downregulated NF-κB activation reducing binding of its active subunits to DNA resulting in diminished COX-2 expression. In contrast to single phytochemicals, X + PEITC induced apoptosis. Moderate reduction of tumor burden in mice carrying xenografts following X and PEITC or their combination was observed. Conclusions: Since Nrf2 is overexpressed in HCC its reduced activation together with diminished level of NF-κB by X + PEITC may be considered as a strategy to support conventional HCC therapy.

Conjugation of Diclofenac with Novel Oleanolic Acid Derivatives Modulate Nrf2 and NF-κB Activity in Hepatic Cancer Cells and Normal Hepatocytes Leading to Enhancement of Its Therapeutic and Chemopreventive Potential.

Combining NSAIDs with conventional therapeutics was recently explored as a new strategy in cancer therapy. Our earlier studies showed that novel oleanolic acid oximes (OAO) conjugated with aspirin or indomethacin may enhance their anti-cancer potential through modulation of the Nrf2 and NF-κB signaling pathways. This study focused on the synthesis and biological evaluation of four diclofenac (DCL)-OAO derivative conjugates in the context of these pathways' modification and hepatic cells survival. Treatment with the conjugates 4d, 3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide, and 4c, 3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester significantly reduced cell viability in comparison to the DCL alone. In THLE-2, immortalized normal hepatocytes treated with these conjugates resulted in the activation of Nrf2 and increased expression in SOD-1 and NQO1, while the opposite effect was observed in the HepG2 hepatoma cells. In both cell lines, reduced activation of the NF-κB and COX-2 expression was observed. In HepG2 cells, conjugates increased ROS production resulting from a reduced antioxidant defense, induced apoptosis, and inhibited cell proliferation. In addition, the OAO morpholide derivative and its DCL hybrid reduced the tumor volume in mice bearing xenografts. In conclusion, our study demonstrated that conjugating diclofenac with the OAO morpholide and a benzyl ester might enhance its anti-cancer activity in HCC.

Modeling the photodynamic effect in 2D versus 3D cell culture under normoxic and hypoxic conditions.

BACKGROUND: Photodynamic therapy (PDT), mainly as a combined therapy, can still be considered a promising technology for targeted cancer treatment. Besides the several and essential benefits of PDT, there are some concerns and limitations, such as complex dosimetry, tumor hypoxia, and other mechanisms of resistance. In this study, we present how the cell culture model and cell culture conditions may affect the response to PDT treatment. It was studied by applying two different 3D cell culture, non-scaffold, and hydrogel-based models under normoxic and hypoxic conditions. In parallel, a detailed mechanism of the action of zinc phthalocyanine M2TG3 was presented. METHODS: Hydrogel-based and tumor spheroids consisting of LNCaP cells, were used as 3D cell culture models in experiments performed under normoxic and hypoxic (1% of oxygen) conditions. Several analyses were performed to compare the activity of M2TG3 under different conditions, such as cytotoxicity, the level of proapoptotic and stress-related proteins, caspase activity, and antioxidant gene expression status. Additionally, we tested bioluminescence and fluorescence assays as a useful approach for a hydrogel-based 3D cell culture. RESULTS: We found that M2TG3 might lead to apoptotic cancer cell death and is strongly dependent on the model and oxygen availability. Moreover, the expression of the genes modulated in the antioxidative system in 2D and 3D cell culture models were presented. The tested bioluminescence assay revealed several advantages, such as repetitive measurements on the same sample and simultaneous analysis of different parameters due to the non-lysing nature of this assay. CONCLUSIONS: It was shown that M2TG3 can effectively cause cancer cell death via a different mechanism, depending on cell culture conditions such as the model and oxygen availability.

Multiharmonic electron paramagnetic resonance imaging as an innovative approach for in vivo studies.

This work is the first report when multiharmonic analysis (MHA) was applied for electron paramagnetic resonance imaging (EPRI) for in vivo applications. Phantom studies were performed for established methodology, and in vivo imaging was conduct as a proof-of-concept. Phantom studies showed at least six times improvement of the signal - to - noise (S/N) ratio. Application MHA for 3D EPR in vivo imaging provides images of spin probe distribution in mouse head. The EPRI, in combination with nitroxide and trityl spin probe, was performed to obtained 3D EPR in vivo images using MHA. For both used spin probes, MHA provided images with better S/N ratio, especially in the case of nitroxide, where projections obtained using conventional CW did not allow for reconstructing reliable data. Trityl radical exhibited high resolution and quality of obtained images after MHA. The MHA methodology allows the selection of a second modulation amplitude even 40 times higher than the natural EPR linewidth of the spin probe without line shape distortion, which highly improves the sensitivity of the acquired signal and allowing for imaging mice regardless of their size in a routine animal experiment.

Parenteral-Oral Immunization with Plant-Derived HBcAg as a Potential Therapeutic Vaccine against Chronic Hepatitis B.

Chronic hepatitis B (CHB) is the cause of severe liver damage, cirrhosis, and hepatocellular carcinoma for over 240 million people worldwide. Nowadays, several types of treatment are being investigated, including immunotherapy using hepatitis B core antigen (HBcAg) assembled into highly immunogenic capsid-like particles (CLPs). Immunogenicity of plant-produced and purified HBcAg, administered parenterally or intranasally, was previously reported. In this study, a novel parenteral-oral vaccination scheme is proposed using plant-derived HBcAg preparations. The antigen for injection was obtained via transient expression in Nicotiana benthamiana. HBcAg-producing transgenic lettuce was lyophilized and used as an orally delivered booster. The intracellular location of plant-produced HBcAg CLPs implies additional protection in the digestive tract during oral immunization. BALB/c mice were intramuscularly primed with 10 µg of the purified antigen and orally boosted twice with 5 or 200 ng of HBcAg. A long-lasting and significant systemic response after boosting with 200 ng HBcAg was induced, with anti-HBc titer of 25,000. Concomitantly, an insignificant mucosal response was observed, with an S-IgA titer of only 500. The profile of IgG isotypes indicates a predominant Th1 type of immune response, supplemented by Th2, after injection-oral vaccination. The results demonstrate that a low dose of parenteral-oral immunization with plant-derived HBcAg can elicit a specific and efficient response. This study presents a potential new pathway of CHB treatment.

EPR Oximetry Sensor-Developing a TAM Derivative for In Vivo Studies.

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1 mM PBS, pH = 7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH = 7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250 mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.