Circulating Tumor Cells in Colorectal Cancer: Detection Systems and Clinical Utility.

Colorectal cancer (CRC) is the third most common cancer worldwide. The high mortality from CRC is mainly related to metastasis affecting distant organs and their function. Dissemination of tumor cells from the primary tumor and hematogeneous spread are considered crucial in the formation of tumor metastases. The analysis of circulating tumor cells (CTCs) and CTC clusters in the blood can be used for the early detection of invasive cancer. Moreover, CTCs have a prognostic significance in the monitoring of a malignant disease or the response to chemotherapy. This work presents an overview of the research conducted on CTCs with the aim of finding suitable detection systems and assessing the possibility of clinical applications in patients with CRC.

Profiling Colorectal Cancer in the Landscape Personalized Testing-Advantages of Liquid Biopsy.

Drug-specific therapeutic approaches for colorectal cancer (CRC) have contributed to significant improvements in patient health. Nevertheless, there is still a great need to improve the personalization of treatments based on genetic and epigenetic tumor profiles to maximize the quality and efficacy while limiting cytotoxicity. Currently, CEA and CA 19-9 are the only validated blood biomarkers in clinical practice. For this reason, laboratories are trying to identify new specific prognostics and, more importantly, predictive biomarkers for CRC patient profiling. Thus, the unique landscape of personalized biomarker data should have a clinical impact on CRC treatment strategies and molecular genetic screening tests should become the standard method for diagnosing CRC. This review concentrates on recent molecular testing in CRC and discusses the potential modifications in CRC assay methodology with the upcoming clinical application of novel genomic approaches. While mechanisms for analyzing circulating tumor DNA have been proven too inaccurate, detecting and analyzing circulating tumor cells and protein analysis of exosomes represent more promising options. Blood liquid biopsy offers good prospects for the future if the results align with pathologists' tissue analyses. Overall, early detection, accurate diagnosis and treatment monitoring for CRC with specific markers and targeted molecular testing may benefit many patients.

Fluorescence spectroscopic investigation of competitive interactions between ochratoxin A and 13 drug molecules for binding to human serum albumin.

Ochratoxin A (OTA) is a highly toxic mycotoxin found worldwide in cereals, foods, animal feeds and different drinks. Based on previous studies, OTA is one of the major causes of the chronic tubulointerstitial nephropathy known as Balkan endemic nephropathy (BEN) and exerts several other adverse effects shown by cell and/or animal models. It is a well-known fact that OTA binds to various albumins with very high affinity. Recently, a few studies suggested that reducing the bound fraction of OTA might reduce its toxicity. Hypothetically, certain drugs can be effective competitors displacing OTA from its albumin complex. Therefore, we examined 13 different drug molecules to determine their competing abilities to displace OTA from human serum albumin (HSA). Competitors and ineffective chemicals were identified with a steady-state fluorescence polarization-based method. After characterization the competitive abilities of individual drugs, drug pairs were formed and their displacing activity were tested in OTA-HSA system. Indometacin, phenylbutazone, warfarin and furosemide showed the highest competing capacity but ibuprofen, glipizide and simvastatin represented detectable interaction too. Investigations of drug pairs raised the possibility of the presence of diverse binding sites of competing drugs. Apart from the chemical information obtained in our model, this explorative research might initiate future designs for epidemiologic studies to gain further in vivo evidence of long-term (potentially protective) effects of competing drugs administered to human patients.

Antioxidant activities and polyphenolic contents of three selected Micromeria species from Croatia.

Antioxidant activities of three selected Micromeria species growing in Croatia (M. croatica, M. juliana and M. thymifolia) were evaluated using five different antioxidant assays, in comparison with plant polyphenolic constituents and reference antioxidants. All studied ethanolic extracts exhibited considerable activity to scavenge DPPH and hydroxyl free radicals, reducing power, iron chelating ability and total antioxidant capacity in the order: M. croatica > M. juliana > M. thymifolia. Total polyphenol (9.69-13.66%), phenolic acid (5.26-6.84%), flavonoid (0.01-0.09%) and tannin (3.07-6.48%) contents in dried plant samples were determined spectrophotometrically. A strong positive correlation between antioxidant activities and contents of phenolic acids and tannins was found, indicating their responsibility for effectiveness of tested plants. Our findings established Micromeria species as a rich source of antioxidant polyphenols, especially the endemic M. croatica.

The screening and evaluation of potential clinically significant HIV drug combinations against the SARS-CoV-2 virus.

Spike glycoprotein is essential for the reproduction of the SARS-CoV-2 virus, and its inhibition using already approved antiviral drugs may open new avenues for treatment of patients with the COVID-19 disease. Because of that we analyzed the inhibition of SARS-CoV-2 spike glycoprotein with FDA-approved antiviral drugs and their double and triple combinations. We used the VINI in silico model of cancer to perform this virtual drug screening, showing HIV drugs to be the most effective. Besides, the combination of cobicistat-abacavir-rilpivirine HIV drugs demonstrated the highest in silico efficacy of inhibiting SARS-CoV-2 spike glycoprotein. Therefore, a clinical trial of cobicistat-abacavir-rilpivirine on a limited number of COVID-19 patients in moderately severe and severe condition is warranted.

Apoptosis of leukemic cells: a case report.

Transformation of leukemic cells is associated with delay in maturation and in apoptosis, and to altered responsiveness to growth factors. However, some studies have revealed that Fas (CD95/APO1) which mediates apoptotic signal and decrease of anti-apoptotic Bcl-2 are frequently observed in acute myeloid leukemia (AML) M4/M5 leukemic cells. The aim of the study was to compare cytomorphology and cytochemistry of bone marrow (BM) apoptotic leukemic cells to preserved peripheral blood (PB) leukemic cells in our patient, a 76-year-old man with AML-M5b treated at Zagreb University Hospital Center. BM and PB of the AL patient were analyzed after Pappenheim and cytochemical stainings, and leukemic cells were classified according to FAB and WHO classification. Analysis of PB revealed leukocytosis and 80-90% monocytic cells (46% monoblasts, 29% promonocytes and 11% monocytes). Only a few preserved monoblasts and promonocytes were found in BM, together with numerous morphologically altered cells with characteristic chromatin condensation and pyknosis of nucleus, as well as nuclear fragmentation and formation of apoptotic bodies. Thus, cytomorphology of PB leukemic cells pointed to proliferation of immature monocytic cells, and cytomorphology of BM to cell apoptosis. Cytochemistry of PB monocytic cells and BM apoptotic cells confirmed monocytic cell lineage because esterase was strongly positive in almost all BM apoptotic leukemic cells and PB leukemic cells, and esterase was completely inhibited with sodium fluoride. On the basis of these findings, AML-M5b was diagnosed in our patient. There are many possible explanations for our observation of BM leukemic cell apoptosis in a patient with AML-M5. The most reliable one is that apoptosis was induced ex vivo after BM aspiration in course of the air drying of BM specimen before staining. Mass BM leukemic cell apoptosis that was recorded in contrast to numerous preserved leukemic cells in PK could be probably connected to unfavorable ratio of relatively low concentration of cytokines in relation to high leukemic cell number in BM aspirated cytologic specimen.

Endothelin-1, big endothelin-1, and nitric oxide in patients with chronic renal disease and hypertension.

The complex pathogenesis of chronic renal disease (CRD) depends on endothelin (ET) axis (ETs and ET receptors) and nitric oxide (NO) because of their vasoactive effects and their role in general modulation of vascular homeostasis. Various renal cells synthesize ETs and NO that play a significant role in renal hemodynamics as well as in water and salt excretion via urine. ET-1 is a strong vasoconstrictor. Besides its vasoactive effects, ET-1 modulates mitosis and apoptosis in a cell type-dependent manner, and may play an important role in CRD pathogenesis. The aims of this study were to emphasize the role and interactions of ET-1, Big ET-1, and NO in CRD. Concentrations of these vasoactive molecules were measured in plasma/serum and/or urine of 57 patients with diabetic nephropathy (subgroup 1), arterial hypertension (subgroup 2) or CRD with chronic renal insufficiency (subgroup 3), and in healthy control subjects (n=18). In comparison with control group, urine concentration of Big ET-1 was significantly increased (13.13 pmol/L vs. 11.34 pmol/L; P<0.001) in CRD patients, whereas plasma and urine concentrations of ET-1 did not differ significantly. NO concentrations were also significantly increased in CRD patients (serum, 72.55 micromol/L; P<0.001, and urine 141.74 micromol/L; P<0.05) as compared to control group. Study results indicated that Big ET-1 and NO could be useful diagnostic parameters in CRD for their diagnostic sensitivity and diagnostic specificity (Big ET-1 in urine: 56.1 and 88.9%, and NO in serum: 66.7 and 83.3%, respectively). In addition, Big ET-1 may prove useful in the differential diagnosis of diabetic nephropathy (78.6% diagnostic sensitivity and 88.9% diagnostic specificity).

Lipid peroxidation and glutathione levels in porcine kidney PK15 cells after individual and combined treatment with fumonisin B(1), beauvericin and ochratoxin A.

Individual and combined effects of the mycotoxins fumonisin B(1), beauvericin and ochratoxin A on cell viability, lipid peroxidation (TBARS) and intracellular glutathione (GSH) were studied on porcine kidney epithelial cells (PK15). Cells were treated with 0.05, 0.5 and 5 microg/ml of each mycotoxin or the combinations of two or all three applied in equal concentrations for 24 and 48 hr. Changes in cell viability, GSH and TBARS levels showed that the cytotoxic effects of these mycotoxins were concentration- and time-dependent. After 24 hr, cell viability was significantly decreased by the exposure to 5 microg/ml of fumonisin B(1) (25%), beauvericin (30%) and ochratoxin A (35%), as compared to controls. Only ochratoxin A (5 microg/ml) increased TBARS (56%), with further significant increase (85%) after 48 hr exposure. Fumonisin B(1) and beauvericin significantly increased TBARS (57% and 80%, respectively) only when the highest dose was applied for 48 hr. After 24 hr, GSH was significantly decreased (18%) by ochratoxin A (0.05 microg/ml), whereas fumonisin B(1) and beauvericin significantly decreased GSH at the concentration of 0.5 microg/ml. Combined treatment with fumonisin B(1), beauvericin and ochratoxin A resulted mostly in additive effects especially after a 24-hr exposure, although synergistic as well as antagonistic interactions could not be excluded depending on toxin concentrations and time of exposure. This is the first report on beauvericin-induced effects on lipid peroxidation and GSH in animal cells.

Advances and Pitfalls in Using Laboratory Biomarkers for the Diagnosis and Management of Sepsis.

Sepsis is a critical patient condition with high mortality rate caused by a complex and inadequate host response to infection. Since early identification and start of antibiotic therapy in the first few hours after sepsis development dramatically improves outcomes, it is of utter importance to offer fast, reliable and specific early laboratory biomarkers to help clinicians in sepsis recognition. On the other hand, the biomarkers should also be helpful in excluding sepsis and/or confirming therapy effectiveness, and thus prevent overprescribing of antibiotics. In this paper, we discuss the significance and relative merits of three currently available protein biomarkers: C-reactive protein, procalcitonin and presepsin. Although useful, none of these biomarkers has been shown to completely fulfill the roles mentioned above.

Toxic effects of Ustilago maydis and fumonisin B1 in rats.

The toxicity of Ustilago maydis and the possible synergism with fumonisin B1 (FB1) were studied in Fischer rats by evaluating pathological changes and biochemical parameters in blood serum (LDH, ALT, GGT, ChE) and tissue homogenate of brain and liver (AChE, ChE, GGT, ALP). One experimental group (US) consumed diet with 70% of U. maydis galls and the other group (US+FB1) was fed pellets containing 70% of U. maydis galls and 1 mg of FB1 per kg of diet for 17 days. Control group (C) consumed standard pellets. During the trial, experimental animals were more excited, showing hyperactivity. Body mass gains slightly increased in both groups compared to the control. Gross pathological changes in liver, lungs, uterus and ovaries were more pronounced in the US+FB1 than in the US group. Specific catalytic activities of AChE decreased by 61% and by 63% in the liver and brain homogenate of the US group (p<0.05) compared to the control, indicating neurotoxic activity of U. maydis. Also, specific catalytic concentration of AChE and ALP was significantly decreased in the liver of the US+FB(1) group (p<0.05). Activity of LDH in the blood serum was increased up to 166% and 165% in the US+FB1 group (p<0.05) compared to the control and US group values, respectively, which indicates that FB1 was responsible for the disruption of cell membrane integrity. These findings suggest that Ustilago maydis and FB1 showed neurotoxicity in Fischer rats, which could be related to the alkaloids of U. maydis and disruption of sphingolipid metabolism by FB1 activity.

Biochemistry of apoptotic cell death.

Apoptosis is a physiological cell suicide program that is critical for the development and maintenance of healthy tissues. Regulation of programmed cell death allows the organism to control the cell number and the tissue size, and to protect itself from rogue cells that threaten homeostasis. The changed activity of numerous genes influences switching of cells to a self-destruction program. Apoptosis requires co-ordinated action and fine tuning of a set of proteins that are either regulators or executors of the process. Cancer, autoimmune diseases, immunodeficiency disease, reperfusion injury and neurodegenerative disorders are characterised by disregulation of apoptosis. Modulation of the expression and activation of the key molecular components of the apoptotic process has emerged as an attractive therapeutic strategy for many diseases.

Flavonoid diosmetin increases ATP levels in kidney cells and relieves ATP depleting effect of ochratoxin A.

Diosmetin (DIOS) is a flavone aglycone commonly occurring in citrus species and olive leaves, in addition it is one of the active ingredients of some medications. Based on both in vitro and in vivo studies several beneficial effects are attributed to DIOS but the biochemical background of its action seems to be complex and it has not been completely explored yet. Previous investigations suggest that most of the flavonoid aglycones have negative effect on ATP synthesis in a dose dependent manner. In our study 17 flavonoids were tested and interestingly DIOS caused a significant elevation of intracellular ATP levels after 6- and 12-h incubation in MDCK kidney cells. In order to understand the mechanism of action, intracellular ATP and protein levels, ATP/ADP ratio, cell viability and ROS levels were determined after DIOS treatment. In addition, impacts of different enzyme inhibitors and effect of DIOS on isolated rat liver mitochondria were also tested. Finally, the influence of DIOS on the ATP depleting effect of the mycotoxin, ochratoxin A was also investigated. Our major conclusions are the followings: DIOS increases intracellular ATP levels both in kidney and in liver cells. Inhibition of glycolysis or citric acid cycle does not decrease the observed effect. DIOS-induced elevation of ATP levels is completely abolished by the inhibition of ATP synthase. DIOS is able to completely reverse the ATP-depleting effect of the mycotoxin, ochratoxin A. Most probably the DIOS-induced impact on ATP system does not originate from the antioxidant property of DIOS. Based on our findings DIOS may be promising agent to positively influence ATP depletion caused by some metabolic poisons.

Synergism between propolis and hyperthermal intraperitoneal chemotherapy with cisplatin on ehrlich ascites tumor in mice.

We investigated antitumor, genotoxic, chemopreventive, and immunostimulative effects of local chemoimmunotherapy and hyperthermal intraperitoneal chemotherapy (HIPEC) in a mouse-bearing Ehrlich ascites tumor (EAT). Mice were treated with water-soluble derivative of propolis (WSDP) at a dose of 50 mg kg(-1) , 7 and 3 days before implantation of EAT cells, whereas cisplatin (5 or 10 mg kg(-1) ) was injected 3 days after implantation of EAT cells at 37°C and 43°C. The following variables were analyzed: the total number of cells, differential count of the cells present in the peritoneal cavity, functional activity of macrophages, comet assay, and micronucleus assay. The combination of WSDP + CIS 5 mg kg(-1) at 37°C resulted in tumor growth inhibition and increased the survival of mice by additional 115.25%. WSDP with HIPEC increased the survival of mice by additional 160.3% as compared with HIPEC. WSDP reduced cisplatin toxic and genotoxic effect to normal cells without affecting cisplatin cytotoxicity on EAT cells. In addition, WSDP with HIPEC increased the cytotoxic actions of macrophages to tumor cells. Water-soluble derivative of propolis increases macrophage activity and sensitivity of tumor cells to HIPEC and reduces cisplatin toxicity to normal cells.

Flavonoid aglycones can compete with Ochratoxin A for human serum albumin: a new possible mode of action.

The mycotoxin Ochratoxin A (OTA) appears worldwide in cereals, plant products, different foods and drinks. Ochratoxin A binds to plasma albumin with a very high affinity. However, it is well known that natural flavonoids can also bind to human serum albumin (HSA) at the same binding site as OTA does (site I, subdomain IIA). A few experimental literature data suggest that reducing the bound fraction of OTA speeds up its elimination rate with a potential decrease in its toxicity. In our experimental model competitive binding properties of flavonoid aglycones were examined with a fluorescence polarization based approach. Our data show that some of the flavonoids are able to remove the toxin from HSA. We conclude that among the 13 studied flavonoid aglycones galangin and quercetin were the most effective competitors for OTA.

Co-determination of ATP and proteins in Triton X 100 non-ionic detergent-opened monolayer cultured cells.

Human monolayer cells (HEp-2 and Hep G2) were cultured in 96-well plates. A modified Triton X 100 nonionic detergent extraction method was used for releasing intracellular ATP and protein in one step. The detergent technique was compared to perchloric acid (PCA) extraction. ATP was determined by the firefly bioluminescence method and ATP values were referred to cell protein (ATP:protein ratio). There was no significant difference in ATP data between detergent and PCA treatments. The ATP:protein ratio seems to be a sensitive tool for characterizing the metabolic activity of monolayer tissue culture cells. The protein-mobilizing capability of Triton X 100 depends on the type of cell culture used. Our modified extraction gives reliable ATP:protein values with one simple extraction step.

Expression of Hsp70 in kidney cells exposed to ochratoxin A.

Ochratoxin A (OTA) is a possible etiological agent of endemic nephropathy, a chronic renal disease with high prevalence in limited geographic areas. Ochratoxicosis has many characteristics of different pathological states in which heat shock proteins (Hsps) are usually induced. The most inducible heat shock proteins belong to the Hsp70 family. We determined the level of expression of Hsp70 by the Western blot analysis in kidneys of rats treated with low doses of OTA and in LLC-PK1 and MDCK cells exposed to OTA. Estimation of cell viability and release of lactate dehydrogenase (LDH) confirmed the toxic effects of OTA on cultured cells. OTA affects the relative distribution of two Hsp70 isoforms (68-kDa and 74-kDa isoforms), but does not change total amount of Hsp70 in rat kidney. No changes in the Hsp70 level were detected in LLC-PK1 and MDCK cells treated with OTA, although the cells were seriously injured, as was seen from the reduced cell viability and increased release of LDH. Both cell lines were capable of having Hsp70 induced following a heat shock. However, exposure of the cells to OTA before the heat shock challenge prevented Hsp70 induction. Results of the study show that OTA does not induce Hsp70 in rat kidney or in cultured kidney cells. The absence of Hsp70 protective effects in the cells and tissues might be a possible explanation for the cumulative destructive effects of OTA and a silent onset of endemic nephropathy in humans and of OTA-induced experimental nephrotoxicity in animals.

Apoptosis and oxidative stress induced by ochratoxin A in rat kidney.

Ochratoxin A (OTA) is a widespread mycotoxin produced by several species of fungi. OTA induces a tubular-interstitial nephropathy in humans and in animals. It has been implicated as one of the aetiological agents involved in the development of endemic nephropathy. OTA-induced oxidative stress and apoptosis may play key roles in the development of chronic tubulointerstitial nephritis connected to the long-term exposure to this food contaminant. We studied the effects of low doses of OTA on kidney cells. Wistar rats were treated with 120 microg OTA/kg bodyweight daily, for 10, 30 or 60 days. Toxin concentration in kidney was proportional to the time of exposure, and amounted to 547.2, 752.5 and 930.3 ng OTA/g kidney tissue after 10, 30 and 60 days, respectively. OTA treatment caused an increased number of cells undergoing apoptosis in both proximal and distal epithelial kidney cells. The apoptotic cells were visualised using the TUNEL assay and staining with haematoxylin and eosin in situ. The number of apoptotic cells in rats treated for 10, 30 and 60 days increased by 5-, 6.4- and 12.7-fold, respectively, compared with the control cells. However, DNA electrophoresis did not show characteristic fragmentation (DNA laddering). The oxidative stress was evident via increased malondialdehyde formation. The concentration of lipid peroxides showed an increase (36%), but the activity of superoxide dismutase decreased (26%) in 60-day treated rats. In spite of the observed biochemical and morphological changes in the kidney cells, renal functional status was preserved to the end of experiment. This study demonstrates that a combination of morphologic and biochemical markers can be used to monitor early cell death in OTA-induced renal injury. We have shown that the exposure to the relatively low OTA concentrations has activated apoptotic processes and oxidative damage in kidney cells.