Lysosomal sequestration of weak base drugs, lysosomal biogenesis, and cell cycle alteration.

Lysosomes, now known to take part in multiple cellular functions, also respond to various stress stimuli. These include biogenesis in response to nanomolar concentrations of hydrophobic weak-base anticancer drugs. However, since lysosomal stress mediated by accumulation of weak-base drugs at such concentrations has never been proven and these drugs have diverse effects on malignant cells, we investigated whether the interpretation of the data was true. We found that lysosomal accumulation of the drugs daunorubicin, doxorubicin, mitoxantrone, symadex, chloroquine, clomipramine and sunitinib alone, was insufficient to induce lysosomal alkalization i.e., lysosomal stress-mediated biogenesis at nanomolar concentrations. Instead, we found that some of the drugs used induced G2 phase arrest and lysosomal biogenesis that is associated with activation of transcription factor EB (TFEB). Similarly, cantharidin, a control compound that does not belong to the weak base drugs, induced cell cycle arrest in the G2 phase associated with TFEB-driven lysosomal biogenesis. Overall none of the tested drugs caused stress-induced lysosomal biogenesis at nanomolar concentrations. However, daunorubicin, doxorubicin, mitoxantrone, symadex and cantharidin induced a massive block in the G2 phase of the cell cycle which is naturally associated with TFEB-driven lysosomal biogenesis.

Changes in expression of lysosomal membrane proteins in leucocytes of cancer patients treated with tyrosine kinase inhibitors.

Lysosomal sequestration of weak base drugs has been identified as one of the stress-related mechanisms that trigger in vitro lysosomal biogenesis controlled by transcription factor EB (TFEB). Whether such mechanism can induce lysosomal biogenesis in vivo is unknown. In this study, we addressed the question whether prolonged treatment with sunitinib (SUN) in patients with advanced renal cell carcinoma (n = 22) and with imatinib (IM) in those with gastrointestinal stromal tumor (n = 6) could induce lysosomal biogenesis in leukocytes. Lysosomal biogenesis was monitored using immunoblotting of three lysosomal membrane proteins: lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) and vacuolar H+-ATPase, B2 subunit (ATP6V1B2). Present results indicate that prolonged treatment with SUN affects LAMP1 and LAMP2 expression only marginally in most patients. In contrast, changes in ATP6V1B2 expression were marked and resembled irregular oscillations. Very similar changes in the expression of lysosomal membrane proteins were also found in IM-treated patients. Conclusion: prolonged treatment of cancer patients with SUN and IM did not induce leucocyte lysosomal biogenesis but dramatically affected expression of ATP6V1B2.

Can image analysis provide evidence that lysosomal sequestration mediates daunorubicin resistance?

Altered intracellular distribution of weak base anticancer drugs owing to lysosomal sequestration is one purported mechanism contributing to chemotherapy resistance. This has often been demonstrated with the example of daunorubicin (DNR), chemotherapy with its characteristic red fluorescence used to trace it in cellular compartments. Here we addressed the question whether image analysis of DNR fluorescence can reflect its real intracellular distribution. We observed that the relationship between the intensity of the DNR fluorescence and its concentration in water solutions with or without proteins is far from linear. In contrast, nucleic acids, RNA and DNA in particular, dramatically diminish the DNR fluorescence, however, the intensity was proportional to the amount. Therefore, image analysis reflects the composition of different cell compartments (i.e., the presence of proteins and nucleic acids) rather than the actual concentration of DNR in these compartments. In line with these results, we observed highly fluorescent lysosomes and low fluorescent nucleus in sensitive cancer cells treated with low DNR concentrations, a fluorescence pattern thought to be found only in resistant cancer cells. Importantly, LC/MS/MS analysis of extracts from sensitive cells treated with DNR or DNR in combination with an inhibitor of vacuolar ATPase, concanamycin A, indicated that lysosomal accumulation of DNR increased with increasing extracellular concentration. However, even the highest lysosomal accumulation of DNR failed to reduce its extralysosomal concentration and thus change the cell sensitivity to the drug. In conclusion, our results strongly suggest that DNR fluorescence within cells does not indicate the real drug distribution. Further they suggested that lysosomal sequestration of DNR can hardly contribute to its resistance in cancer cells in vitro.

N-acetylcysteine dual and antagonistic effect on cadmium cytotoxicity in human leukemia cells.

Although cadmium (Cd2+) is unable to form reactive oxygen species (ROS) directly, many of its adverse effects are connected to increased ROS generation resulting in cell death. In support of this supposition, a large number of studies have shown protective effects of antioxidants such as N-acetylcysteine (NAC) against cadmium induced cytotoxicity. Here, we describe the cytotoxic effects of Cd2+ on human leukemia U937 and K562 cells that were not mediated by oxidative stress. Surprisingly, we observed that addition of low concentrations of NAC can drastically potentiate cadmium cytotoxicity solely via ROS production. However, all adverse effects of the metal were prevented by NAC at high concentrations. Detailed analysis indicated that the protective effect of NAC was mediated by its ability to form stable complex with cadmium [Cd(NAC)2]. In conclusion, NAC exhibits dual and antagonistic effects on Cd2+ cytotoxicity in human leukemia cells.

Clinically relevant interactions of anti-apoptotic Bcl-2 protein inhibitors with ABC transporters.

In this work we studied clinically relevant interactions between the BH3 mimetics and the ABCB1 and ABCG2 transporters. We observed that the intracellular levels of ABT-263 and ABT-199, but not ABT-737, might be reduced by ABCB1 or ABCG2. Importantly, this effect was proportional to the transporter expression level. High transporter expression levels decreased the intracellular levels of ABT-263 and ABT-199 substantially. Low transporter expression levels, which are clinically relevant, affected the intracellular level of ABT-263 slightly but significantly, however, they failed to decrease the intracellular level of ABT-199 below the control level in parental cells. Our results further revealed that ABT-263 did not inhibit the ABCB1 mediated transport, however, it partially inhibited the ABCG2 mediated transport at clinically relevant concentrations. In contrast, ABT-199 inhibited partially the ABCB1 mediated transport and it fully inhibited the ABCG2 mediated transport at clinically relevant concentrations. Importantly, cells expressing higher drug transporters levels required higher concentrations of ABT-263 or ABT-199 to achieve certain inhibition of substrate efflux. CONCLUSIONS: Antiproliferative effects of ABT-263 and ABT-199 might be reduced by ABCB1 or ABCG2, however, this effect depends on transporter expression levels. Since the expression levels of ABCB1 and ABCG2 are rarely high in clinical samples, their contribution to the overall resistance to ABT-263 or ABT-199 is probably low. Inhibition study revealed that ABT-199, but not ABT-263, fully inhibited low expression level of ABCG2. Our data suggest that ABT-199 should be evaluated beyond its original application as an inhibitor of the ABCG2 transporter in clinical settings.

Neutrophilic differentiation modulates the apoptotic response of HL-60 cells to sodium butyrate and sodium valproate.

Differentiation of myeloid leukemic cells may result in less sensitivity to various apoptotic stimuli. We examined whether human leukemia HL-60 cells differentiating by all-trans retinoic acid (ATRA) acquired resistance to the apoptogenic activity of two histone deacetylase (HDAC) inhibitors, butyrate and valproate. In undifferentiated cells, the cytotoxicity of both butyrate and valproate was associated with activation of the intrinsic apoptotic pathway since we observed dissipation of mitochondrial membrane potential, induction of caspase-9 and caspase-3 activities, appearance of sub-G1 DNA and loss of plasma membrane asymmetry and/or integrity. Both HDAC inhibitors were also able to induce accumulation of undifferentiated cells in the G0/G1 phase of the cell cycle. ATRA was found to enhance the apoptotic effect of both butyrate and valproate in undifferentiated cells. This aside, ATRA appeared to synergize with butyrate in the induction of the G0/G1 cell cycle arrest. In cells pretreated for 72 h with ATRA, butyrate and valproate in combination with ATRA induced lower dissipation of mitochondrial membrane potential and weaker apoptotic and/or necrotic changes in plasma membrane, whereas DNA fragmentation was not diminished compared to undifferentiated cells. Similar results were also obtained when butyrate or valproate were combined with another neutrophilic differentiation inducer, dimethyl sulfoxide. We conclude that neutrophilic differentiation modulates but does not abrogate the apoptotic response of HL-60 cells to butyrate and valproate, and nuclei are preferentially affected during apoptosis in differentiated cells.

Induction of apoptosis by A3 adenosine receptor agonist N-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide in human leukaemia cells: a possible involvement of intracellular mechanism.

AIM: The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA) was studied. METHODS: To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed. RESULTS: We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resistance phenotype because of overexpression of P-gp, were significantly more resistant. We found that the intracellular level of IB-MECA in K562/HHT cells was approx. 10 times lower than those in HL-60 or K562 cells. Inhibitors of P-gp, including cyclosporine A (CsA) and verapamil (Vpa), increased the intracellular level of IB-MECA and reversed the resistance of K562/HHT cells to this drug. Accordingly, shRNA-mediated down-regulation of P-gp significantly increased the intracellular level of IB-MECA in K562/HHT cells which simultaneously exhibited reduced resistance to this A3AR agonist. In addition, an in vitro enzyme-based assay provided evidence that IB-MECA might serve as a substrate for P-gp. CONCLUSION: Our results suggest that P-gp overexpression prevents cells from IB-MECA induced apoptosis despite the A3AR expression. Pro-apoptotic effect of IB-MECA seemed to strongly depend on its intracellular accumulation rather than on its interaction with A3AR.

Prenatal protracted irradiation at very low dose rate induces severe neuronal loss in rat hippocampus and cerebellum.

Prenatal irradiation is known to damage the developing brain. However, little is known about the consequences of very low dose rate prenatal protracted irradiation over several days on neuron numbers in the offspring brain, and on volumes of the corresponding brain regions. Pregnant Wistar rats were exposed either to a protracted gamma irradiation from embryonic day (E) 13 to E16 (0.7 mGy/min; total cumulative dose approximately 3 Gy) or were sham-irradiated. Thirty months old male and female offspring were then analyzed for alterations in hippocampal and cerebellar morphology. Using design-based stereology and the analysis of sets of sections systematically and randomly sampled to span the entire brain region of interest, a statistically significant decrease in numbers of hippocampal pyramidal and granule cells as well as of cerebellar Purkinje and granule cells (approximately 50%) was found in male and female irradiated offspring. The volumes of these brain regions were comparably altered. The analysis of only a "representative" section per animal yielded mostly non-significant trends. Evaluation of neuron densities showed no differences between prenatally irradiated and sham-irradiated offspring. Most importantly, very low dose rate prenatal protracted gamma irradiation did not result in the same morphologic alterations in the offspring brain as previously observed after prenatal single irradiation such as derangement of the laminar structure of pyramidal cells within the hippocampus or malformation of cerebellar lobules.

Modification of horse heart cytochrome c with trans-2-hexenal.

Horse heart cytochrome c reacting with trans-2-hexenal was used as a simple model of the nonspecific interactions of proteins with 2-alkenals. The reaction mixtures containing relatively high concentrations of the protein and aldehyde were characterized using visible spectrophotometry, fluorescence, and circular dichroism measurement, capillary isoelectric focusing, size-exclusion chromatography, polyacrylamide gel electrophoresis, and mass-spectrometric techniques. The mass-spectrometric data indicate that cytochrome c becomes modified with one or two molecules of hexenal as the major reaction product. The modified species with a correspondingly lowered isoelectric point were detected through capillary isoelectric focusing. The results of proteolytic studies indicate nonspecific modifications. Significant quantities of the oligomeric forms of hexenal-modified protein were also observed electrophoretically.

Antimicrobial activity of colostrum after administering killed Escherichia coli O111 vaccine orally to expectant mothers.

An attempt was made to see if it was possible to produce antimicrobial activity in colostrum after killed Escherichia coli O111 vaccine had been given orally to expectant mothers. The colostral samples were used in vitro for the inhibition test immediately after the start of lactation. The colostrum from 7 of the 47 vaccine-treated mothers inhibited the growth of E. coli O111 compared with only one colostrum from 101 controls. No complication has occurred either in the vaccine-treated mothers or their suckling babies. The association between the presence of antimicrobial activity in the colostrum and the time of vaccine application was insignificant.