Facilitating the Cellular Accumulation of Pt-Based Chemotherapeutic Drugs

Cisplatin, carboplatin, and oxaliplatin are Pt-based drugs used in the chemotherapeutic eradication of cancer cells. Although most cancer patient cells initially respond well to the treatment, the clinical effectiveness declines over time as the cancer cells develop resistance to the drugs. The Pt-based drugs are accumulated via membrane-bound transporters, translocated to the nucleus, where they trigger various intracellular cell death programs through DNA interaction. Here we illustrate how resistance to Pt-based drugs, acquired through limitation in the activity/subcellular localization of canonical drug transporters, might be circumvented by the facilitated uptake of Pt-based drug complexes via nanocarriers/endocytosis or lipophilic drugs by diffusion

Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21^Waf1/Cip1 and Caspase-9/-3 activation

The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21(Waf1/Cip1), Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21(Waf1/Cip1) as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21(Waf1/Cip1) and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A

Integrin β₁, osmosensing, and chemoresistance in mouse ehrlich carcinoma cells

Background/Aims: Altered expression of the integrin family of cell adhesion receptors has been associated with initiation, progression, and metastasis of solid tumors as well as in the development of chemoresistance. Here, we investigated the role of integrins, in particular integrin β1, in cell volume regulation and drug-induced apoptosis in adherent and non-adherent Ehrlich ascites cell lines. Methods: Adhesion phenotypes were verified by colorimetric cell-adhesion-assay. Quantitative real-time PCR and western blot were used to compare expression levels of integrin subunits. Small interfering RNA was used to silence integrin β1 expression. Regulatory volume decrease (RVD) after cell swelling was studied with calcein-fluorescence-self-quenching and Coulter counter analysis. Taurine efflux was estimated with tracer technique. Caspase assay was used to determine apoptosis. Results: We show that adherent cells have stronger fibronectin binding and a significantly increased expression of integrin α5, αv, and β1 at mRNA and protein level, compared to non-adherent cells. Knockdown of integrin β1 reduced RVD of the adherent but not of the non-adherent cells. Efflux of taurine was unaffected. In contrast to non-adherent, adherent cells exhibited chemoresistance to chemotherapeutic drugs (cisplatin and gemcitabine). However, knockdown of integrin β1 promoted cisplatin-induced caspase activity in adherent cells. Conclusion: Our data identifies integrin β1 as a part of the osmosensing machinery and regulator of cisplatin resistance in adherent Ehrlich cells

Volatile profile of matured Tronchón cheese affected by oxytetracycline in raw goat milk

[EN] The presence of antibiotics in milk destined for cheese production may affect the biological processes responsible for the formation of volatile compounds, leading to alterations in the characteristic cheese flavor expected by consumers. The aim of this study was to evaluate the effect of the presence of oxytetracycline in goat milk on the volatile profile of ripened cheeses. Traditional mature Tronchón cheeses were manufactured from raw goat milk spiked with different concentrations of oxytetracycline (50, 100, and 200 ug/kg). Cheese made from antibiotic-free goat milk was used as control. We analyzed the residual amounts of the antibiotic and the volatile profile of the experimental cheeses on a fortnightly basis during maturation using liquid chromatography tandem mass spectrometry and then solid-phase microextraction followed by gas chromatography-mass spectrometry. Our results suggested that oxytetracycline was widely transferred from milk to cheese: drug concentrations in the cheeses were 3.5 to 4.3 times higher than those in raw milk. Although the residual amounts of oxytetracycline significantly decreased during maturation (88.8 to 96.5%), variable amounts of residues remained in cheese matured for 60 d (<10 to 79 ug/kg). In general, the presence of oxytetracycline in goat milk did not affect the volatile profile of Tronchón cheeses; volatile profile was significantly modified by ripening time. Still, the presence of oxytetracycline residues in cheeses ripened for 60 d could be of great concern for public health.This work is part of the AGL-2013-45147-R funded by the Ministry of Science and Innovation (Madrid. Spain). The authors thank the Research and Development Support Program, "Ayudas para movilidad dentro del Programa para la Formación de Personal Investigador" (2.016) of Universitat Politècnica de València (Spain), allowing Paloma Quintanilla to perform a predoctoral stay at the Food Quality and Design Group, Wageningen University and Research (Wageningen, the Netherlands). The authors state that they have no conflicts of interest.Quintanilla-Vázquez, PG.; Hettinga, K.; Beltrán Martínez, MC.; Escriche Roberto, MI.; Molina Pons, MP. (2020). Volatile profile of matured Tronchón cheese affected by oxytetracycline in raw goat milk. Journal of Dairy Science. 103(7):6015-6021. https://doi.org/10.3168/jds.2019-16510S601560211037Attaie, R., Bsharat, M., Mora-Gutierrez, A., & Woldesenbet, S. (2015). Short communication: Determination of withdrawal time for oxytetracycline in different types of goats for milk consumption. Journal of Dairy Science, 98(7), 4370-4376. doi:10.3168/jds.2014-8616Beltrán, M. C., Althaus, R. L., Molina, A., Berruga, M. I., & Molina, M. P. (2015). Analytical strategy for the detection of antibiotic residues in sheep and goat’s milk. Spanish Journal of Agricultural Research, 13(1), e0501. doi:10.5424/sjar/2015131-6522Cabizza, R., Rubattu, N., Salis, S., Pes, M., Comunian, R., Paba, A., … Urgeghe, P. P. (2017). Transfer of oxytetracycline from ovine spiked milk to whey and cheese. International Dairy Journal, 70, 12-17. doi:10.1016/j.idairyj.2016.12.002Cabizza, R., Rubattu, N., Salis, S., Pes, M., Comunian, R., Paba, A., … Urgeghe, P. P. (2018). Impact of a thermisation treatment on oxytetracycline spiked ovine milk: Fate of the molecule and technological implications. LWT, 96, 236-243. doi:10.1016/j.lwt.2018.05.026Castillo, I., Calvo, M. V., Alonso, L., Juárez, M., & Fontecha, J. (2007). Changes in lipolysis and volatile fraction of a goat cheese manufactured employing a hygienized rennet paste and a defined strain starter. Food Chemistry, 100(2), 590-598. doi:10.1016/j.foodchem.2005.09.081Curioni, P. M. G., & Bosset, J. O. (2002). Key odorants in various cheese types as determined by gas chromatography-olfactometry. International Dairy Journal, 12(12), 959-984. doi:10.1016/s0958-6946(02)00124-3Delgado, F. J., González-Crespo, J., Cava, R., García-Parra, J., & Ramírez, R. (2010). Characterisation by SPME–GC–MS of the volatile profile of a Spanish soft cheese P.D.O. Torta del Casar during ripening. Food Chemistry, 118(1), 182-189. doi:10.1016/j.foodchem.2009.04.081Delgado, F. J., González-Crespo, J., Cava, R., & Ramírez, R. (2011). Formation of the aroma of a raw goat milk cheese during maturation analysed by SPME–GC–MS. Food Chemistry, 129(3), 1156-1163. doi:10.1016/j.foodchem.2011.05.096Gajda, A., Nowacka - Kozak, E., Gbylik - Sikorska, M., & Posyniak, A. (2017). Tetracycline antibiotics transfer from contaminated milk to dairy products and the effect of the skimming step and pasteurisation process on residue concentrations. Food Additives & Contaminants: Part A, 35(1), 66-76. doi:10.1080/19440049.2017.1397773Halling-Sørensen, B., Lykkeberg, A., Ingerslev, F., Blackwell, P., & Tjørnelund, J. (2003). Characterisation of the abiotic degradation pathways of oxytetracyclines in soil interstitial water using LC–MS–MS. Chemosphere, 50(10), 1331-1342. doi:10.1016/s0045-6535(02)00766-xHettinga, K. A., van Valenberg, H. J. F., & van Hooijdonk, A. C. M. (2008). Quality control of raw cows’ milk by headspace analysis. International Dairy Journal, 18(5), 506-513. doi:10.1016/j.idairyj.2007.10.005Katla, A.-K., Kruse, H., Johnsen, G., & Herikstad, H. (2001). Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. International Journal of Food Microbiology, 67(1-2), 147-152. doi:10.1016/s0168-1605(00)00522-5Loftin, K. A., Adams, C. D., Meyer, M. T., & Surampalli, R. (2008). Effects of Ionic Strength, Temperature, and pH on Degradation of Selected Antibiotics. Journal of Environmental Quality, 37(2), 378-386. doi:10.2134/jeq2007.0230McSweeney, P. L. H., & Sousa, M. J. (2000). Biochemical pathways for the production of flavour compounds in cheeses during ripening: A review. Le Lait, 80(3), 293-324. doi:10.1051/lait:2000127Padilla, B., Belloch, C., López-Díez, J. J., Flores, M., & Manzanares, P. (2014). Potential impact of dairy yeasts on the typical flavour of traditional ewes’ and goats’ cheeses. International Dairy Journal, 35(2), 122-129. doi:10.1016/j.idairyj.2013.11.002Quintanilla, P., Beltrán, M. C., Molina, A., Escriche, I., & Molina, M. P. (2019). Characteristics of ripened Tronchón cheese from raw goat milk containing legally admissible amounts of antibiotics. Journal of Dairy Science, 102(4), 2941-2953. doi:10.3168/jds.2018-15532Sierra, D., Contreras, A., Sánchez, A., Luengo, C., Corrales, J. C., Morales, C. T., … Gonzalo, C. (2009). Short communication: Detection limits of non-β-lactam antibiotics in goat’s milk by microbiological residues screening tests. Journal of Dairy Science, 92(9), 4200-4206. doi:10.3168/jds.2009-2101Souza, C. F. V. de, Dalla  Rosa, T., & Ayub, M. A. Z. (2003). Changes in the microbiological and physicochemical characteristics of Serrano cheese during manufacture and ripening. Brazilian Journal of Microbiology, 34(3). doi:10.1590/s1517-8382200300030001

Bacterio-plankton transformation of diazepam and 2-amino-5-chlorobenzophenone in river waters.

Benzodiazepines are a large class of commonly-prescribed drugs used to treat a variety of clinical disorders. They have been shown to produce ecological effects at environmental concentrations, making understanding their fate in aquatic environments very important. In this study, uptake and biotransformations by riverine bacterio-plankton of the benzodiazepine, diazepam, and 2-amino-5-chlorobenzophenone, ACB (a photo-degradation product of diazepam and several other benzodiazepines), were investigated using batch microcosm incubations. These were conducted using water and bacterio-plankton populations from contrasting river catchments (Tamar and Mersey, UK), both in the presence and absence of a peptide, added as an alternative organic substrate. Incubations lasted 21 days, reflecting the expected water residence time in the catchments. In River Tamar water, 36% of diazepam (p < 0.001) was removed when the peptide was absent. In contrast, there was no removal of diazepam when the peptide was added, although the peptide itself was consumed. For ACB, 61% was removed in the absence of the peptide, and 84% in its presence (p < 0.001 in both cases). In River Mersey water, diazepam removal did not occur in the presence or absence of the peptide, with the latter again consumed, while ACB removal decreased from 44 to 22% with the peptide present. This suggests that bacterio-plankton from the Mersey water degraded the peptide in preference to both diazepam and ACB. Biotransformation products were not detected in any of the samples analysed but a significant increase in ammonium concentration (p < 0.038) was measured in incubations with ACB, confirming mineralization of the amine substituent. Sequential inoculation and incubation of Mersey and Tamar microcosms, for 5 periods of 21 days each, did not produce any evidence of increased ability of the microbial community to remove ACB, suggesting that an indigenous consortium was probably responsible for its metabolism. As ACB degradation was consistent, we propose that the aquatic photo-degradation of diazepam to ACB, followed by mineralization of ACB, is a primary removal pathway for these emerging contaminants. As ACB is photo-produced by several benzodiazepines, this pathway should be relevant for the removal of other benzodiazepines that enter the freshwater environment