Owumi, S. Andrologia , 51 9 , e Owumi SE, et al. Visit free Relief Central. Prime PubMed is provided free to individuals by: Unbound Medicine. Related Citations Impact of prepubertal exposure to dietary protocatechuic acid on the hypothalamic-pituitary-testicular axis in rats.
Hepatorenal protective effects of protocatechuic acid in rats administered with anticancer drug methotrexate. Protective effect of sodium selenite and zinc sulfate on intensive swimming-induced testicular gamatogenic and steroidogenic disorders in mature male rats. Keeping in mind their origin and the potential risk of cancer cell contamination, wildtype status of the organoids was confirmed by whole exome sequencing.
Using normal epithelial tissue as a reference, a low number of mutations was detected in these cultures two in N1, none in N2 , with no mutations found in common cancer driver genes S2A Fig , S3 Table. Morhpology of oral mucosa organoids, brightfield microscopy. The cells form round structures with keratinized centers.
Expression of genes involved in MTX transport, metabolism and toxicity was quantified in low folate medium using quantitative qPCR. Allgenes tested were expressed at detectable levels in oral mucosa organoids.
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MTX exposure induces toxicity cell death in oral mucosa organoids grown in low folate medium, but not in normal oral mucosa medium that was previously described in Driehuis et al [ 38 ]. Organoids were exposed for five days to MTX and viability was quantified relative to untreated organoids. Reproducible killing at physiological doses of MTX was observed in low folate medium only dashed lines, circle data points , not in normal organoid medium continuous lines, square data points.
Oral mucosa organoids were subsequently exposed to MTX. Most likely, when exposed to MTX in this media, these high concentrations prevent MTX toxicity in vitro as previously described [ 59 ]. In this low-folate medium, organoids grew at similar speed and showed similar morphology when compared to the previously defined culture medium S3A and S3B Fig. Therefore, all subsequent drug screens were performed in low folate medium. To assess the role of LV on MTX toxicity, oral mucosa organoids were exposed to a clinically relevant at levels detected in patient plasma concentration range of MTX in in vitro drug screens Fig 2A.
The drug screen assays showed high technical quality as measured by Z-scores median 0. To model LV rescue therapy in vitro , organoids were exposed to different concentrations of LV at different timepoints after the start of MTX treatment. Schematic outline of the experimental set-up applied to assess the effect of LV rescue initiated post MTX exposure toxicity in vitro. Organoids were split on day 0, left to recover for two days, subsequently filtered, counted and plated in well format to be exposed to MTX for five days with or without LV added on variable timepoints after the start of MTX exposure.
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On day 7, viability readout was performed. As such, organoids were exposed to MTX for a total of five days. Each IC50 value is obtained for a 9-point dose titration of MTX, where the effect of each dose is tested in technical triplicate. Symbols indicate the different replicate experiments in which the IC50 were defined for different timepoints of LV rescue.
As such, IC50 values indicated with the same symbol at different timepoints, were determined in the same experiment. Blue symbols indicate N1 organoids, turquoise symbols indicate N2 organoids. Here, MTX kill curves are shown when 0. MTX IC50 values are shown on the y-axis.
For each timepoint, three IC50 values are show, obtained in three independent experiments. Administration of LV resulted in a decrease of MTX-induced cell death in a concentration-dependent manner, that was statistically significant Fig 2B. Considering the timing of LV administration varies that can vary per treatment protocol, but is usually initiated at timepoints ranging from 24 to 48 hours after MTX infusion, these results are relevant. As in patients, MTX plasma levels have dropped by 54 hours post infusion, LV administration is rarely continued after this timepoint.
Here we observe that, in vitro , LV administration still decreases MTX-induced toxicity beyond this timepoint. This has resulted in the hypothesis that intracellular LV from previous courses prevents toxicity during subsequent MTX courses. However, in organoid line N2, a clear rescue effect of the pre-incubation with LV was observed Fig 3D and 3E , which was also found to be statisctically significant AUC no pre-treatment: 0.
When pre-treated, the rescue effect of LV rescue administered at 72 hours—later than currently applied in the clinic—resulted in a cell survival similar to a LV rescue that would have been given at 0 hours without this pre-treatment. This suggests that pre-treatment might increase the timeframe in which LV rescue rescues MTX toxicity in oral mucosa cells.
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Deviations from the original experimental set-up used to determine MTX toxicity see Fig 2A are shown in red. In short, one day prior to the start of MTX exposure, cells were treated with 0. From there one, experimental set-up was identical to that previously described for MTX toxicity. Organoids were exposed to MTX for 5 days, either in the presence or absence of LV rescue, that was commenced at variable times after the start of MTX exposure.
Effect of a 24 hour 0. PT effect was investigated either with dashed lines or without continuous lines a 0. Toxicity was determined using a 9-point dose titration of MTX, where the effect of each dose is tested in technical triplicates. Squares indicate PT conditions, circles indicate cells that did not receive PT.
Here, IC50 values are shown when no LV rescue is performed. For N1 and N2, experimetns were not only performed in technical triplicate, but also biological triplicate. As variable responses to LV pre-treatment were observed in N1 and N2, the effect of pre-treatment was tested in organoids established from three additional donors.
In all three cultures, pre-treatment increased oral mucosa cell survival upon exposure to MTX Fig 3F. This implies that LV pre-treatment may reduce the risk of oral mucositis. Regardless, the effect of such a pre-treatment on leukemia cells much be investigated before any claims can be made on clinical testing of such an intervention. Jurkat, dark red. MOLT16, fuchsia. HSB-2, purple. REH, magenta. Nalm-6, violet. For each cell line, three IC50 values are show, obtained in three independent experiments.
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Different colors indicate the different cell lines, colors are similar to those used in A-E. Viability of cells exposed for 5 days to 0. Viability was tested in technical triplicate, where the average viability is shown for three independent experiments. To estimate the effect of LV pre-treatment when administered systemically, we compared the effect of this treatment on both oral mucosa cells and leukemia cells at a concentration of 0.
Taken together, we conclude that LV pre-treatment decreases MTX-induced cell death of leukemia cells in vitro. Regardless, the effect of LV pre-treatment should be explored with caution, but might prove to be an effective approach to decrease the severity and frequency of MTX-indicued mucositis. A local LV application is a feasible alternative, since it would likely not interfere with the systemic MTX effect on leukemia cells and may therefore be a safer approach to reduce the risk of mucositis.
Using a 3D in vitro model of primary human oral mucosa organoids, we show donor-dependent MTX-induced cell death in wildtype human oral mucosa cells. To our knowledge, this is the first in vitro model based on the model described by Driehuis et al [ 38 ] with modification in media composition to assess the effect of MTX on proliferating, wildtype oral mucosa epithelial cells. Using this system, we show that administration of LV at dosages detected in patient plasma, reduces MTX-induced cell death in a concentration- and time-dependent manner and that MTX-induced toxicity shows variability between cells derived from different individuals.
LV rescue therapy is often not administered beyond the timepoint of 54 hours after start of MTX. However, since the model presented here only studies the effect of such interventions on either mucosal cells or leukemia cell lines, these results need to be validated for example in mouse models before they can be clinically tested. Alternatively, a local application of LV one day prior to the start of MTX administration might be an alternative approach to reduce mucositis in pediatric leukemia patients.
Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation.
To assess the effect of LV pre-treatment on leukemia cells, leukemia-derived cell lines were exposed to the same pre-treatment to study the effect of this intervention on MTX toxicity. Although present, the effect of pre-treatment and LV rescue was less pronounced in leukemia cells than in oral mucosa cells.
Differences in response to LV between leukemia cells and healthy cells have been observed before. Several pre-clinical studies showed this selective mechanism of action for MTX and LV might be due to the fact that high MTX-PG levels accumulate in leukemia cell lines compared to normal intestinal and bone marrow precursor cells [ 14 — 22 ].
However, it is important to test clinical safety of the described interventions in order not to decrease anti-leukemic activity of MTX. In clinics, only a subset of patients presents with mucositis, suggesting some patients are more sensitive to MTX treatment than others, or might respond better to LV rescue treatment. This is in line with our findings, where organoid cultures derived from different patients, show variable responses to both MTX and LV exposure in vitro.