The combination of daphnetin with either vemurafenib or epirubicin showed an antagonistic interaction. Moreover, additive interactions were observed for the combinations of daphnetin with cisplatin and docetaxel. The most desirable synergistic interactions for human melanoma metastatic cell lines were observed for the combination of daphnetin with mitoxantrone. (4) The obtained results suggest that daphnetin should not be combined with vemurafenib or epirubicin in the treatment of malignant melanoma due to the abolition of their anticancer effects. The combination of daphnetin with mitoxantrone is beneficial in the treatment of metastatic melanoma due to their synergistic interaction.(1) The treatment of metastatic or drug-resistant melanoma is still a significant therapeutic problem. The aim of this study was to evaluate the anticancer potential of daphnetin  and its combinations with five different cytostatic drugs . (2) The viability, proliferation and cytotoxicity of daphnetin against four human malignant melanoma cell lines were evaluated. The interactions were assessed using isobolographic analysis for the combinations of daphnetin with each of the five cytostatic drugs. (3) Daphnetin showed anticancer activity against malignant melanoma, with IC Melanoma can appear anywhere on the body where there are pigment cells—melanocytes–including in mucous membranes, eyeballs and hair follicles. However, it most often appears on the skin. Melanoma is a cancer that has a high survival rate but only when it is diagnosed early. Compared with other skin cancers, melanoma patients are more likely to have metastases. As the stage of the cancer increases, the survival rate of patients decreases ,2. For sEarly diagnosis of melanoma allows for effective treatment  in its early stages . When thNowadays, it has become popular to search for new therapeutic substances of natural origin. Active substances of plant origin may be the foundation of less invasive treatments used in the future, especially due to the lack of or mild side effects. Each of the natural compounds is characterized by a different chemical structure and various biological activities. An interesting group of compounds are coumarins; it is estimated that, so far, about 1300 substances of this group have been isolated from plants, fungi or bacteria ,7.Daphne, from which the name of a compound is derived. Daphne plants are flowering shrubs  found in Europe, Asia and North Africa [One of the simple coumarins is daphnetin , which was first isolated from plants of the genus h Africa .Daphnetin possesses a number of biological activities, including anti-inflammatory, antioxidant, antibacterial, antidiabetic, immunosuppressive and anticancer activities ,10. NeurHelicobacter pylori. The mechanism of action is based on the release of specific proteins, the binding of ions and the prevention of colonization by H. pylori. This is of great importance for preventing bacterial infection but also, due to the colonization of the stomach, preventing the development of peptic ulcers or cancer of this organ [Daphnetin has a strong antibacterial effect, which has already been used in traditional Chinese medicine. It is important that bacteria do not develop resistance to the compound after its application. Experiments have shown that daphnetin is effective in neutralizing the dangerous bacterium is organ . The useis organ .2. Culture media used: for A375—DMEM high glucose; for SK-MEL28—EMEM; and for FM55P and FM55M2—RPMI1640. Previous publications have described in more detail the growth conditions of these cell line cultures [In this experiment, we used two metastatic (FM55M2 and SK-MEL28) and two primary (FM55P and A375) melanoma cell lines. From the European Collection of Authenticated Cell Cultures , we purchased FM55P and FM55M2 cell lines, and from the American Type Culture Collection , we purchased A375 and SK-MEL28 cell lines. Of note, the A375 cell line is the original and not a CRISPR-modified version of the cell line, and all the tested cell lines in this study have the BRAF V600E mutation. Experimental cell culture conditions: 37 °C in a humidified atmosphere of 95% air and 5% COcultures .Daphnetin (DAP) and the chemotherapeutics—mitoxantrone (MTX), docetaxel (DOCX) and vemurafenib (VEM)—were dissolved in DMSO. Other test compounds such as epirubicin (EPR) were dissolved in sterile hot water, and cisplatin (CDDP) was dissolved in PBS buffer . Compounds were dissolved to the tested concentrations in growth medium before adding compounds to the microtiter (96-well) plate. DMSO was maintained at the safe concentration of 0.1% and had no effect on cell growth.4 cells/mL), HEMa-LP melanocytes (density: 5 × 103 cells/mL) and four human malignant melanoma lines . After a day (24 h) of incubation, the medium was replaced with fresh medium to which was added increasing concentrations of the tested substances . The experimental steps of the MTT assay have been previously described [The effect on cell viability of DAP and the five chemotherapeutics  was assessed using the MTT assay. All tested cell lines—the HaCaT cell line model of human keratinocyte cells   was used to assess cell proliferation. The test was carried out in accordance with the manufacturer’s instructions provided. Initially, appropriate cell densities (as above) of melanoma and normal cells were plated on microtiter plates (NEST). After 24 h, the cells were exposed to increasing concentrations of DAP 2–200 µM). The next steps of the BrdU assay have been previously described ,20.0 µM. TheThe cytotoxicity of DAP was assessed using the LDH assay  based on the measurement of lactate dehydrogenase activity released into the medium from damaged cells. Specified densities (as above) of all the studied melanoma and normal human cell lines were plated on microtiter plates (NEST). After 24 h, the medium was replaced with fresh medium supplemented with DAP (at concentrations of 2–200 µM). The LDH test was carried out after 72 h incubation with DAP according to the manufacturer’s instructions. A more detailed description of the steps of the LDH test has been previously described ,19.50 values for every tested drug. The collaborative effect of two concentration–response lines was tested for the DAP + MTX, DAP + DOCX, DAP + VEM, DAP + EPR, DAP + CDDP combinations, as previously described [50 values, SEM, N, test of parallelism between the studied drugs) were computed automatically in an MS Excel spreadsheet with formulas originally derived from the log-probit method [50 values for the tested compounds when used alone. However, if the concentration-response curves are not parallel, the additivity on the isobologram is illustrated as an area bounded by two (upper and lower) lines of additivity [50 values (based on MTT assay) for the drugs administered alone, median additive inhibitory concentrations for the mixtures (IC50 add) (at the fixed ratio of 1:1) of DAP + MTX, DAP + DOCX, DAP + VEM, DAP + EPR, DAP + CDDP were calculated, as described previously [To thoroughly characterize the pharmacodynamic interactions between DAP and the five cytostatics in the malignant melanoma cell lines , we performed an isobolographic analysis of the MTT assay. On the basis of the results of the MTT test, the percentage of inhibition of cell viability in relation to the specific concentrations of the tested substances  was determined. Based on a computer-assisted log-probit method , dose-reescribed ,22,23,24t method , but thet method ,22,23,24eviously ,26. Moreeviously ,26,27,28eviously .50 and the IC50 mix) values for DAP and the five tested cytostatics  were calculated automatically with a computer-assisted log-probit method using an MS Excel spreadsheet, as described earlier [t-test with Welch’s correction was used to statistically compare the experimentally derived IC50 mix values  with their respective, theoretically calculated and presumed to be additive IC50 add values, as recommended elsewhere [GraphPad Prism  was used for statistical analysis of the data . The analyses were performed with a one-way ANOVA test followed by Tukey’s post-hoc test. Data were presented as means ± standard errors (SEMs). The median inhibitory concentration , mitoxantrone (MTX), docetaxel (DOCX), vemurafenib (VEM), epirubicin (EPR) and cisplatin (CDDP)—inhibited melanoma cell viability in a concentration-dependent manner. The main compound tested was the simple coumarin daphnetin, and its effect on viability in four human melanoma cell lines as well as in keratinocytes and melanocytes was assessed after 72 h incubation with DAP at concentrations of 2–200 µM . The melAll the tested malignant melanoma cells and normal cells were treated with increasing concentrations of DAP, displaying a concentration-dependent decrease in DNA synthesis . MeasureThe cell cytotoxicity of DAP was assessed by the LDH assay, which measures lactate dehydrogenase release into the medium . The rel50 values ± SEM). In our previous experiments, we had determined the mean inhibitory concentration (IC50) values for MTX (0.04 to 1.7 µM) [50 values for EPR and VEM, respectively, were also determined for the following cell lines: FM55P (0.29 ± 0.07 µM and 0.76 ± 0.26 µM), A375 (0.26 ± 0.05 µM and 6.07 ± 2.06 µM), FM55M2 (0.16 ± 0.03 µM and 0.62 ± 0.27 µM) and SK-MEL28 (0.39 ± 0.08 µM and 0.25 ± 0.13 µM) [50 of DAP was determined at 40.48 ± 10.90 µM in the FM55M2 cell line, 64.41 ± 9.02 µM in the FM55P cell line, 116.59 ± 18.35 µM in the SK-MEL28 cell line and 183.97 ± 18.82 µM in the A375 cell line.The inhibitory effects of the tested cytostatics—MTX, DOCX, VEM, EPR and CDDP—in combination with DAP on the viability of malignant melanoma cells were analyzed in the MTT assay. The main purpose of this work was to determine whether DAP could enhance the anti-cancer effects of the tested chemotherapeutic drugs. Concentration-response curves (CRC) were determined according to the log-probit method . From th 1.7 µM) , CDDP (1 1.7 µM) ,19 and D 1.7 µM) . The IC50.13 µM) . The IC5Parallelism of the experimentally determined curves of the concentration-response effects for all the tested combinations—DAP + MTX, DAP + DOCX, DAP + VEM, DAP + EPR and DAP+CDDP see —was asseThe isobolographic analysis of the combination of DAP with VEM (at the fixed ratio of 1:1) showed a statistically significant antagonistic interaction in the A375 and FM55M2 cell lines c,e and aIn the isobolographic analysis, the combination of DAP with CDDP a,c,e,g aSynergy was observed for the combinations of DAP with MTX in the FM55M2 and SK-MEL28 metastatic melanoma cell lines c,d. The A polygonogram  was draw50 doses of daphnetin against the tested hepatocellular carcinoma lines Huh7 and SK-HEP-1 were 69.41 and 81.96 μM, respectively [50 values of daphnetin in this study ranged from 40.48 ± 10.90 µM to 183.97 ± 18.82 µM and are comparable to those obtained against hepatocellular carcinoma. A summary of the mechanisms of action of daphnetin in the in vitro model is presented in The results of our study confirmed that daphnetin dose-dependently inhibits the proliferation and viability of all the tested human malignant melanoma cell lines . Researchers have confirmed the inhibitory effect of daphnetin on the viability of the murine metastatic osteosarcoma LM8 cell line. Daphnetin at doses of up to 30 µM had no effect on cell viability; however, the drug at a dose of 100 µM exerted an 80% inhibition of cell viability . In laboectively . The IC5In vivo experiments on rats confirmed the anticancer effect of daphnetin in inhibiting liver cancer through antioxidant and anti-inflammatory effects. More specifically, Swiss Wistar rats were given diethylnitrosamine and phenobarbital to induce and progress hepatocellular carcinoma; then, the animals were given various doses of daphnetin to determine the effect of the compound on neoplastic lesions. The assessment of biochemical parameters showed a decrease in the levels of, among others, alkaline phosphatase, aspartate aminotransferase and total bilirubin, and an increase in the level of glutathione, catalase and superoxide dismutase. This proves that daphnetin alleviated the liver damage caused by diethylnitrosamine. In addition, daphnetin showed an anti-inflammatory effect that manifested as a decrease in the level of interleukins (IL-1β and IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 and nuclear factor kappa B (NF-κB) . The strIn the case of studies on the effectiveness of daphnetin against melanoma, the experiments conducted on murine B16F10 cells confirmed its low cytotoxicity at doses of 1–50 µM . Our exp50 value of daphnetin in B16 murine melanoma cells was 54 ± 2.8 µM [50 values obtained in our experiments. For the mouse breast adenocarcinoma cells, the IC50 value for daphnetin was 74 ± 6.4 µM and for the mouse colon cancer cells, the IC50 value amounted to 108 ± 7.3 µM [In another experiment, daphnetin at a concentration of 160 µM evoked cytotoxicity in the mouse melanoma (B16), mouse colon cancer (C26) and mouse breast adenocarcinoma (MXT) cell lines . The IC5± 2.8 µM , which i± 7.3 µM . The inh± 7.3 µM . The hig± 7.3 µM .Importantly, daphnetin is safe to normal cells, which was confirmed in studies on normal ovarian cells (IOSE80), where concentrations of daphnetin (5 to 40 µg/mL ≈ 28–224 µM) were tested . Our exp50 values we obtained are much lower than the Cmax. Only for mitoxantrone was the Cmax value exceeded for the SK-MEL28 cell line, for which the IC50 was 1.74 ± 0.51 µM. Effective cancer therapy involves the use of a number of therapeutic methods, such as surgical methods, radiotherapy, chemotherapy or immunotherapy, depending on the localization and stage of the cancer. For most of the drugs used, numerous side effects are observed, and drug resistance appears after some time of use. Multidrug resistance is also becoming more and more common, which further complicates chemotherapy [The maximum concentrations (Cmax) of the tested cytostatics in the patients’ serum are as follows: MTX (0.715 µM), DOCX 5.47 µM), VEM (127 µM), EPR (16.6 µM) and CDDP (14.4 µM) [ µM, VEM otherapy ,49,50. Totherapy . In expeotherapy . Co-admiotherapy . The conotherapy . Our expotherapy . An addiotherapy  and osthotherapy  against 50 value of 253 μM [Deng et al. tested a combination of daphnetin with doxorubicin in in vitro tests on human esophageal squamous cell carcinoma cells (YM1 line). Daphnetin arrested the cell cycle in S phase, with an ICf 253 μM . In a mof 253 μM . Our expFrom the pharmacological point of view, the most desirable interaction is synergy, which proves the mutual intensification of the therapeutic effect of drugs in combination. Daphnetin in combination with mitoxantrone showed synergy in both metastatic human melanoma cell lines (FM55M2 and SK-MEL28), while in primary melanoma cell lines, the combination of daphnetin with mitoxantrone showed an additive interaction (A375) or additivity with a tendency toward synergy (FM55P).50 values of daphnetin range from 40.48 ± 10.90 µM to 183.97 ± 18.82 µM, depending on the cell line. Isobolographic analysis showed that the interaction for the combination of daphnetin with vemurafenib or epirubicin was antagonistic. An additive interaction was observed for the combinations of daphnetin with cisplatin and docetaxel. The most desirable synergistic interaction was observed for the combination of daphnetin with mitoxantrone in metastatic human melanoma lines (FM55M2 and SK-MEL28), while in primary melanoma lines, this combination showed an additivity with a tendency toward synergy. The interactions found in this study may have a clinical significance, but further research is needed to understand the mechanisms of action of the combinations, and additional pre-clinical studies on animals should shed more light on malignant melanoma treatment with daphnetin.Daphnetin, at lower concentrations of up to 100–150 µM, is safe for keratinocytes and melanocytes and does not limit their proliferation or viability. Above these concentrations, it causes only slight inhibition of the growth of normal cell lines. However, daphnetin exhibits anticancer activity by inhibiting viability and proliferation of the tested human malignant melanoma cell lines. The IC


A pipeline for metabolomics, based on UPLC-ESI-MS, was tested on two malignant breast cancer cell lines of the sub-types ER(+), PR(+), and HER2(3+) (MCF-7 and BCC), and one non-malignant epithelial cancer cell line (MCF-10A). This allowed us to quantify 33 internal metabolites, 10 of which showed a concentration profile associated with malignancy. Whole-transcriptome RNA-seq was also carried out for the three mentioned cell lines. An integrated analysis of metabolomics and transcriptomics was carried out using a genome-scale metabolic model. Metabolomics revealed the depletion of several metabolites that have homocysteine as a precursor, which was consistent with the lower activity of the methionine cycle caused by lower expression of the AHCY gene in cancer cell lines. Increased intracellular serine pools in cancer cell lines appeared to result from the over-expression of PHGDH and PSPH, which are involved in intracellular serine biosynthesis. An increased concentration of pyroglutamic acid in malignant cells was linked to the overexpression of the gene CHAC1. The deregulation of metabolism has been known to be a characteristic of cancer cells since the work of Warburg, almost a century ago . In thei13C-labeled substrates) and metabolomics enables measurement of the concentrations of intracellular metabolites. The relationships between these three types of variables  can be represented using so called genome-scale metabolic models (GSMMs) [Metabolism is a complex system that emerges from the interplay of metabolic enzymes and the small molecules (metabolites) transformed by these enzymes. Metabolism can be studied from different angles, depending on which experimental techniques are used. Proteomics and transcriptomics enable the measurement of changes in enzyme concentrations , fluxomics enables the measurement of reaction rates  . These m (GSMMs) ,10, and, (GSMMs) .13C-labeled substrates and mass spectroscopy. These studies revealed a key role of lipid metabolism in fighting oxidative stress [However, to date, it is not possible to establish a direct relationship between enzyme levels, metabolic fluxes and metabolite concentrations. It has been shown that most of the changes in the expression levels of metabolic genes (those coding metabolic enzymes) do not result in concomitant changes in metabolic fluxes , and thee stress , and thee stress .13C labeling pattern). The same metabolic flux distribution  could be achieved at different steady state concentrations of internal metabolites.There is also no direct relationship between metabolic fluxes and internal metabolite concentrations. Indeed, the methods used to infer metabolic flux distributions in steady state  do not rInternal concentrations of metabolites could have a key role in cancer progression by interacting with signaling pathways, and could also be used as informative biomarkers to assess malignancy . The firThe objective of this work is to identify perturbed metabolic processes characteristic of breast cancer cell lines of the subtypes ER(+), PR(+), and HER2(3+), which could form the basis of the identification of new markers for diagnosis and drug targets.First of all, we assessed whether the panel of 33 internal metabolites, quantified using the described analytical pipeline, can be used to cluster different cell types. The metabolite concentration data, in relative area units, are available in Principal Component Analysis (PCA) was carried out  and showThe results of PCA indicate that 36% of the variation in metabolite concentrations observed between the three cell lines is associated with malignancy. In order to identify the metabolites that contribute the most to separating the non-malignant MCF-10A cell line from the two others, the data are represented in the form of a heat-map . The sixAs discussed later, six perturbed metabolites are related to the methionine cycle or have homocysteine (an intermediate of the methionine cycle) as a metabolic precursor. These metabolites are: methionine, taurine, hypotaurine, choline, betaine and pyroglutamic acid.Box plots showing the concentration distribution of some of the discussed metabolites are shown in Differentially expressed genes were identified using a false discovery rate of 0.01 and a fold change of 2 as cut-off values. A total of 1495 genes showed significant up-regulation in MCF-7 compared MCF-10A and 1335, in which they were significantly down-regulated. For BCC, 1926 genes were up-regulated and 1669 down-regulated. Among the up-regulated genes, 274 were common to both cancer cell lines, and 563 of the down-regulated genes were observed in both cancer cell lines. The complete list of differentially expressed genes can be found in Among the 274 genes up-regulated in both cancer cell lines, 44 were metabolic genes, involved in 131 metabolic reactions. Among the 663 consensus down-regulated genes, 130 were metabolic genes, which are associated with 832 metabolic reactions. The large number of reactions affected by the down-regulated genes comes from the fact that many of these down-regulated genes are involved in cross-membrane transport. For example, the solute carrier family 36 member 4 gene, SLC36A4 (ENSG00000180773), is involved in four transport reactions . The transporter SLC6A15, also down-regulated in both cancer cell lines, is involved in the transport of 11 amino acids. A full list of the metabolic reactions linked to up- and down-regulated genes can be found in Among the perturbed metabolites that were identified in the previous analysis, three of them  can be traced back to a common metabolic precursor (homocysteine), which is an intermediate in the methionine cycle. Pyroglutamic acid is formed from the break-down of glutathione, which also can be traced back to homocysteine; however, its carbon atoms come from glutamine, which was incorporated during the synthesis of glutathione.l-homocysteine, showed significantly lower expression in MCF-7 and BCC cell lines compared to MCF-10A. AHCY has been previously identified as a tumor suppressor gene involved in p53-induced cell cycle arrest [The gene AHCY, coding the enzyme adenosylhomocysteinase, which breaks down S-adenosylhomocysteine into adenosine and e arrest . TogetheTwo more of the perturbed metabolites are linked to the methionine cycle (choline and betaine). As shown in The RNA-seq data revealed that two genes involved in serine biosynthesis from the glycolytic intermediate 3-phosphoglycerate (PHGDH and PSPH) are more expressed in the MCF-7 and BCC cell lines compared to MCF-10A. The gene PHGDH codes phosphoglycerate dehydrogenase and has been shown to be overexpressed in several cancers . It has Pyroglutamic acid is a metabolic dead end that results from the degradation of glutathione. Our transcriptomic analysis revealed the overexpression of the gene CHAC1 in both cancer cell lines, which show increased intracellular pools of pyroglutamic acid. The function of this gene product in breaking down glutathione into cysteinyl-glycine and pyroglutamic acid was characterized in 2012 . The knoOther perturbed metabolites are metabolically unrelated to the previously discussed metabolic processes. For example, increased acetyl-carnitine points to higher activity of the carnitine shuttle, which is responsible for the transfer of fatty acids from the cytosol to the mitochondria. The relationship between the carnitine shuttle and cancer is well established . FinallyThe expression levels of genes that could be directly linked to perturbations in intracellular metabolic pools are shown in 2 into bicarbonate are also predicted in malignant cells, as a result of the down-regulation of two carbonic anhydrases (CA6 and CA2). A list of the perturbed metabolic reactions and their associated genes can be found in The obtained gene expression data were used to constrain a genome-scale metabolic model using the python library pyTARG . FBA wasThere is high metabolic heterogeneity between different breast cancer cell lines, even between those belonging to the same molecular sub-types, (ER(+), PR(+), and HER2(3+)). However, some internal metabolites can be linked to malignancy. Transcriptomic data allowed us to establish relationships between the observed perturbations in internal metabolites and changes in the expression levels of key enzymes. Two main conclusions could be obtained from the integrated analysis of transcriptomic and metabolomic data. First of all, the MCF-7 and BCC cell lines showed lower internal methionine, taurine and hypotaurine levels, which, together with the down-regulation of AHCY (adenosylhomocysteinase), reveals decreased activity of the methionine cycle in cancer cells. On the other hand, a higher concentration of serine and higher expression of the genes PSPH and PHGDH are consistent with higher serine intracellular biosynthesis (which compensates for the lower extracellular serine uptake caused by the down-regulation of SLC36A4). Both methionine and serine are closely related to one-carbon metabolism. The methionine cycle is involved in the regeneration of the methyl donor SAM, while serine is involved in the regeneration of 5-methyl-tetrahydrofolate. SAM acts within the cell as a donor of methyl groups for the methylation of histones and DNA. High SAM concentrations have been shown to have anti-cancer effects (promoting apoptosis or down-regulating oncogenes through methylation); in particular, this effect has been shown on the MCF-7 cell line . 5-methyBesides its role as methyl donor, serine is also a precursor to the synthesis of glycine and other compounds, such as glutathione, involved in cellular resistance to oxidative stress. Serine can be synthesized de novo or uptaken from the exterior, and both processes have been shown to have an impact on cancer proliferation ,31. Seri2. Cells were incubated for 24 h. Metabolite extraction was performed as described earlier by Sellick and co-workers [MCF-7  and BCC cells  were cul-workers . m/z to 250 m/z range with a Xevo TQD triple quadrupole tandem mass spectrometer  coupled with an electrospray ionization (ESI) ion source. The column was set at 40 °C. Elution was applied with a mobile phase of 0.1% formic acid water solution (solvent A) and acetonitrile (solvent B). Flow rate was set to 0.4 mL/min. The starting conditions were set to 95% of solvent A. Gradient elution was applied with the following proportions of solvent A: 0 to 0.2 min was set to 95%, 0.2 to 1.5 min was set to 10%, 1.5 min to 1.8 min was maintained at 90%, and 1.8 to 2 min was set back to the starting conditions. The settings for the mass spectrometer were as follows: capillary voltage was set to negative 2 kV, source temperature to 150 °C, desolvation gas (nitrogen) temperature to 400 °C, gas flow to 700 L/h and cone gas flow to −20 L/h. Cone voltage was set to 25 V.The samples were analyzed following the protocol published by Virgiliou and co-workers , which aBased on the retention times reported by Virgiliou and co-workers ,38 diffeTM  by mixing 1 million cells with 500 µL of Shield stabilization solution. The company constructed RNA-Seq libraries from 500 ng of total RNA using the Zymo-Seq RiboFree Total RNA Library Prep Kit  according to the manufacturer’s instructions, before sequencing on an Illumina NovaSeq  to a sequencing depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample.Three biological replicates from each of the three cell lines  were sent to Zymo Research for next-gen RNA sequencing. The samples were prepared for shipment using DNA/RNA Shieldt-test was used to identify up-regulated and down-regulated genes comparing malignant and non-malignant cell lines. The obtained values were corrected for multiple testing as described in previous work [The obtained pair-ended reads were aligned on a reference sequence (the complete list of human transcripts was obtained from Ensembl BioMart) using Bowtie2 . The resous work  . It is known that broad changes in metabolic flux distributions are typically driven by changes in the expression levels of a relatively small number of metabolic genes. In order to identify these flux-controlling genes, reactions showing a significant change in both the estimated reaction rate and the expression level of at least one of their associated genes were identified.In order to obtain meaningful relationships between metabolomics and transcriptomics, we used a GSMM  in orderThe described analytical pipeline allowed us to quantify enough metabolites to obtain a clear metabolic footprint of each of the tested cell lines. Among the identified metabolites, 10 of them appeared to be associated with malignancy. The intracellular concentration of methionine was significantly lower in both malignant cell lines compared to the non-malignant MCF-10A. Together with the down-regulation of the metabolic gene AHCY and the depletion of taurine and hypotaurine (which originate from homocysteine), this observation suggests lower activity of the methionine cycle in cancer cell lines, which would cause a lower re-methylation rate of SAM. The pro-apoptotic effects of SAM supplementation in breast cancer cells have already been shown. Our data suggest that the supplementation of methionine and betaine, leading to higher flux in the methionine cycle and the higher production of SAM, could also have the same effects. This is an interesting hypothesis that should be tested in the future. An increased intracellular pool of serine was observed, which is consistent with the increased intracellular synthesis rate caused by the over-expression of the genes PHGDH and PSPH. The targeting of serine biosynthesis could be a potential alternative for the treatment of breast cancer. Finally, the recently discovered relationship between the gene CHAC1 and the production of pyroglutamic acid was confirmed. The results of this work point to a possible role of pyroglutamic acid as a biomarker, and an oncogenic role of the gene CHAC1, which remains to be explored further. All the presented conclusions are based on two breast cancer cell lines, and whether they apply to other cell lines and cancer types remains to be tested.


There were no significant associations between klotho and telomerase activity in either group, across cell types assessed here. Our results suggest that klotho levels and telomere length may be associated through a coordinated downregulation of longevity factors occurring under higher stress caregiving conditions.Aging biomarkers may be related to each other through direct co-regulation and/or through being regulated by common processes associated with chronological aging or stress. Klotho is an aging regulator that acts as a circulating hormone with critical involvement in regulating insulin signaling, phosphate homeostasis, oxidative stress, and age-related inflammatory functioning. Both klotho and telomere length are biomarkers of biological aging and decrease with age; however, the relationship between them is not well understood. Here we test the association between klotho levels and the telomere length of specific sorted immune cells among a healthy sample of mothers caregiving for a child with autism spectrum disorder  or a child without ASD, covarying age and body mass index, in order to understand if high stress associated with caregiving for a child with an ASD may be involved in any association between these aging biomarkers. In 178 caregiving women ( Aging biomarkers are often related to each other and this could occur through many different mechanisms. First, age influences aging related biomarkers, by definition, and any study of potential co-regulation must covary chronological age. Secondly, there may be direct co-regulation of each other. Thirdly, they may be regulated by common processes not associated with age, but associated with lifestyle factors such as chronic stress. Here we examine the relationship between two important biomarkers of aging, klotho and telomere length, in a healthy sample stratified into groups based on a combination of (a) stressor exposure and (b) level of perceived stress .Klotho is an aging regulator primarily expressed in the kidneys and choroid plexus of the brain that acts as a circulating hormone once cleaved from its transmembrane form –4. Once Like klotho, telomere length is increasingly recognized as a key biomarker of biological aging. Telomeres are protective DNA-protein complexes that cap the ends of chromosomes and provide genomic stability , 22. Comin vitro work using human cortical neurons derived from human pluripotent stem cells showed an age-related association between telomere attrition and klotho mRNA expression [in vitro cultured cells, a 50% inhibition of klotho gene expression led to a shortening of telomere length [Despite evidence supporting klotho and telomere length as aging biomarkers, the relationship between them has been examined in only a handful of basic studies and is not well understood. In this regard, pression . In anote length , and in e length . In mouse length . Among ie length . When ine length . To dateChronic stress, such as the stress of caregiving for a child with a neurodevelopmental disability ), is associated with accelerated biological aging and the more rapid development of age-related conditions, including cardiovascular disease –49 and pn = 90 high-stress mothers caring for a child with ASD, n = 88 low-stress mothers caring for a neurotypical child) are shown in M = 21.90, SD = 4.69) than mothers caring for a neurotypical child  = −9.03, p < .001). For brevity moving forward, we refer to the mothers caring for a child with ASD as the “high-stress” group and the mothers caring for a neurotypical child as the “low-stress” group.Full sociodemographic characteristics of the sample  and shorter telomere length across cell types . Older age was also associated with reduced CD8+CD28+ T cell telomerase activity , but there were no other significant correlations between age and telomerase activity in any other cell type (ps: .44–.97). Higher BMI was associated with lower klotho levels  and shorter telomeres in CD19+ B cells , but not shorter telomeres (ps: .19–.52) or telomerase activity (ps: .49–.95) in any other cell types. Education and race were not associated with levels of klotho, telomerase activity, or telomere length in any cell types (ps > .11).Collapsing across groups, older age was associated with lower klotho levels , p = .025) and whole blood TL , p = .013). When broken down into specific cell types, there were no statistically significant associations observed for CD4+ T cell TL , p = .07), CD8+CD28− T cell TL , p = .06), CD8+CD28+ T cell TL , p = .21), or CD19+ B cells , p = .23). Granulocyte TL was also not significantly related to klotho levels in unadjusted models , p = .15). After covarying for age and BMI, only klotho levels and whole blood TL had any signal of an association; however, this association was not statistically significant , p = .052). There were no other statistically significant associations between klotho and TL across cell types in adjusted models (ps: .12–.67). Regarding telomerase activity (TA), there were no reliable associations between klotho levels and TA across cell types in unadjusted (ps: .14–.57) or adjusted (ps: .14–.55) models.In models unadjusted for covariates, klotho levels were significantly associated with PBMC TL , p = .006; adjusted: b = 0.17, 95% CI , p = .019; see b = 0.18, 95% CI , p = .015, pB-H = .036; adjusted: b = 0.15, 95% CI , p = .039, pB-H = .12; see b = 0.28, 95% CI , p = .014, pB-H = .036; adjusted: b = 0.23, 95% CI , p = .039, pB-H = .12; see b = 0.20, 95% CI , p = .018, pB-H = .036; adjusted: b = 0.14, 95% CI , p = .084, pB-H = .17; see b = 0.08, 95% CI , p = .30, pB-H = .30; adjusted: b = 0.07, 95% CI , p = .35, pB-H = .35. There was no evidence of a significant klotho by group interaction in whole blood or for granulocyte TL (ps > .13).We found evidence of a significant klotho by group (high- vs. low-stress) interaction for PBMC TL .There was no evidence that associations between levels of klotho and cell-specific telomerase activity differed significantly between the high- and low-stress mothers. Across each cell type assessed for telomerase activity , there was no evidence of the klotho by group interaction associated with telomerase activity , p = .017; adjusted: b = 0.14, 95% CI , p = .081). There were no overly influential observations in this regression analysis based on the DFBETAS values. There were no other overly influential observations or outliers in these analyses.There was one significant outlier based on a Bonferroni outlier test of the studentized residuals in the analyses with CD8+CD28+ T cell TL. When this observation was removed the effect of the klotho by group interaction was similar  membership, and telomerase activity across cell types assessed here. This study is the first to explore associations between sorted immune cell telomere length, telomerase activity, and circulating klotho in humans; our results indicate that klotho and telomere length may be linked through a coordinated downregulation of certain longevity factors under higher stress caregiving conditions. Because both klotho and telomere length are associated with insulin signaling, oxidative stress, and age-related inflammatory functioning, the relationship between the klotho and telomere length may be mediated through these common stress- and longevity-related pathways.Building on past findings in this sample that mothers of a child with ASD had lower klotho levels than mothers of a neurotypical child , we idenFor high-stress mothers of a child with ASD, there was preliminary evidence that klotho may be associated with telomere length in specific sorted immune cells that are important for the aging immune system, namely T cells. As people age, telomere shortening happens primarily in CD8+ T cells , 60, espn = 50), this non-significant effect is worth examining in larger sample sizes.We found no evidence of reliable associations between klotho and telomerase activity in this sample in either group. The impact of chronic stress itself on telomerase activity is not clear as some studies identify that chronic stress may suppress telomerase activity , 54, 69 These findings should be interpreted while recognizing the limitations of the current study design, which also highlight opportunities for important future directions. Although we focus on the stress of caregiving for a child with an ASD in this study, the caregiving experience encompasses far more than just stress , 73. BecThese limitations are well-balanced by several key strengths of this study, including carefully matched groups of caregiving women and examining sorted immune cell telomere length. Many studies examining telomere length rely on heterogeneous populations of leukocytes without subdividing these cells; by sorting immune cells for this study we were able to identify where there were clear associations for the high-stress group between klotho and telomere length , where there was some weak evidence of an association between klotho and telomere length following the same pattern (CD8+CD28+ T cells), and where there was no evidence of an association between klotho and telomere length .There appears to be an exciting and fruitful area for future investigations to further understand the impact of caregiving stress on aging biology. There was a wide range of power to detect an effect for telomerase activity related to group membership and klotho levels; thus, for all analyses but especially those with smaller sample sizes  future work may replicate or extend these findings with larger sample sizes. Given the importance of both the chronicity and timing of stress for its impact on aging biology , future In sum, we identified that klotho levels and telomere length of certain immune cells are linked for those experiencing higher stress caregiving conditions but not linked in lower stress caregiving conditions. This may be due to the common and powerful effects of chronic stress on stress related aging pathways. We found no evidence of this pattern of associations for klotho and telomerase activity. Future research is needed to investigate the characteristics of stress exposures that link these two markers of aging and the underlying biological mechanisms driving this connection.≥13 on the Perceived Stress Scale (PSS) were categorized as a high-stress maternal caregiver. Participants who were caring for a neurotypical child  and reported a PSS score of ≤ 19 were categorized as a low-stress maternal control. This eligibility criteria were based on prior national norms [Participants included 183 mothers recruited via schools, mailings, social media, and ads circulated through child development centers in the San Francisco Bay Area as well as direct recruitment through the University of California, San Francisco Autism Clinic; 178 of the participants had serum samples available for quantifying klotho and of those, all had DNA available for quantifying telomere length. Participants were eligible if they were non-smokers between ages 20 and 50 years, with at least one child between ages of 2 and 16 years. Participants who were caring for a child diagnosed with an autism spectrum disorder (ASD) and reported a score of al norms , 77. ThiProcedures for assaying klotho have been described for this sample . BrieflyIdentical methods for determining telomere length in this sample were detailed in earlier papers , with thN = 6, assayed in triplicate) was 8% and the inter-assay variability of PBMC samples  was 6.7%.Telomerase activity was determined identically to how it was previously described . Brieflylm function from the stats package [influence.measures function from the stats package [outlierTest function from the car package, as well as through visual examination using the boxplot function [ggpredict function from the ggeffects package [ggplot2 capabilities [describe function from the psych package [cor.test function in the stats package [All statistical analyses were completed in the R statistical environment using R Studio (v2022.07.2). Linear regressions were performed using the  package . Leverag package . We examfunction , 83. Plo package , which bbilities . Descrip package . Correla package .pB-H). Lastly, sensitivity analyses tested the robustness of any significant effects by removing any potential outliers or overly influential observations. We used a Bonferonni outlier test to examine studentized residuals of each regression. Regarding leverage statistics, observations were removed if the DFBETAS value was greater than |1.0|, which indicates that a single observation may be influencing the regression parameter estimates’ values. Klotho levels underwent log-10 transformation to approximate a normal distribution. Telomere length was non-normally distributed across all cell types apart from whole blood cells; thus, we used a log-10 transformation to approximate a normal distribution for all measures of telomere length besides whole blood cells. Each measure of telomerase activity was also non-normally distributed and we applied a log-10 transformation to better approximate a normal distribution for analyses.Of the 183 participants in the parent study, 178 had serum available (missingness across relevant variables is described in Supplementary Tables


Background: Comparative transcriptomic analysis is a powerful approach for investigating the molecular mechanisms underlying various physiological and pathological processes, including liver disease. The liver is a vital organ with diverse functions, including metabolism and detoxification. In vitro models of liver cells, such as HepG2, Huh7, and Hep3B, have been widely used to study liver biology and pathology. However, there is limited information on the heterogeneity of these cell lines at the transcriptomic level. Objective: This study aimed to conduct a comparative transcriptomic analysis of three common liver cell lines  using publicly available RNA-sequencing data. In addition, we compared these cell lines to primary hepatocytes, cells isolated directly from liver tissue and considered the gold standard for studying liver function and disease. Methods: Our study included sequencing data with the following criteria: total number of reads over 20,000,000, average read length of over 60 base pairs, Illumina sequencing, and non-treated cells. The data for the three cell lines were compiled: HepG2 (97 samples), Huh7 (39 samples), and Hep3B (16 samples). We performed differential gene expression analysis using the DESeq2 package, principal component analysis, hierarchical clustering on principal components, and correlation analysis to explore the heterogeneity within each cell line. Results: We identified numerous genes and pathways differentially expressed between HepG2, Huh7, and Hep3B, such as oxidative phosphorylation, cholesterol metabolism, and DNA damage. We report that the expression levels of important genes differ significantly between primary hepatocytes and liver cell lines. Conclusion: Our study provides new insights into the transcriptional heterogeneity of commonly used liver cell lines and highlights the importance of considering specific cell line. Consequently, transferring results without considering the heterogeneity of cell lines is impractical and may lead to inaccurate or distorted conclusions. Human cancer cell lines have been extensively employed as in vitro models for cancer research and drug development. Cell lines can be used to study cell signaling, gene expression, and cell differentiation. For research outcomes to be reliably reproduced between laboratories, it is ideal that these cell lines exhibit genetic stability and cellular profiles. Unfortunately, several studies have highlighted various issues in human cancer cell lines: cell line misidentification, cross-contamination, and poor annotation that could impair the reproducibility of results obtained from these cell lines between laboratories. To authenticate cells, the researchers proposed short tandem repeat and single nucleotide polymorphism profiles. However, genetic changes during culturing and passage can affect gene expression and eventually cellular functions. In 2018, Ben-David et al. investigated 27 strains of the breast cancer cell line MCF7 and reported genetic heterogeneity and widely varied drug responses to 321 anti-cancer compounds [Immortalized liver cell lines are more commonly used in research than liver biopsies because of their low cost, ease of use, and retention of most key enzyme activities. To date, more than 40 hepatic lines, including HepaRG, Huh7, SK-Hep-1, Hep3B, and HepG2, are frequently used [Understanding the degree of heterogeneity within and between different liver cell lines is of utmost importance for researchers using these cells as a model system to study various biological processes, including drug metabolism, gene regulation, and liver disease. Despite their widespread use, the heterogeneity of liver cell lines can present challenges that may impact the reproducibility and interpretability of experimental findings.In this study, we performed a comparative transcriptomic analysis of three commonly used liver cell lines: HepG2, Huh7, and Hep3B. These cell lines were selected due to their popularity in research and the availability of publicly accessible RNA sequencing data. We aimed to explore the heterogeneity within each cell line and compare their transcriptomic profiles with each other. To achieve these goals, we used principal component analysis (PCA), hierarchical component clustering (HCPC), and correlation analysis to identify gene expression patterns that distinguished the cell lines from each other and within samples. Additionally, we utilized the DESeq2 package  to identIn order to investigate the heterogeneity of liver cell lines, we conducted a comprehensive analysis using publicly available data for HepG2, Huh7, and Hep3B cell lines downloaded from the NCBI SRA database. In addition, we incorporated our own data for HepG2 cells sequenced using Illumina NovaSeq 6000 with a read length of 100 base pairs (bp). We focused on control samples that represented the baseline cell lines in their natural, untreated state. This data is fundamental to comprehending cells and is crucial for discerning and evaluating any alterations that may transpire due to treatments, storage, or other influences.The data processing pipeline consisted of several key steps, including quality control, read trimming, alignment to the reference transcriptome, and quantification of gene expression. We performed the final step of the analysis in R, utilizing various statistical and bioinformatics tools such as the PCA, correlation analysis, differential expression (DESeq2 package), and functional enrichment analysis. The overall workflow of the experiment and a summary of data processing are presented in As a result of two transcriptomic profilings at the Institute of Biomedical Chemistry (IBMC), expression data were obtained for the HepG2 cell line in different years . The firAfter performing the quality control of the IBMC data, we defined criteria for published data from the Huh7, Hep3B, and HepG2 cell lines to be comparable in quality. The criteria were as follows: total number of reads >20,000,000; average read length >60 bp; non-treated cells; Illumina sequencing technology.Despite the fact that more than 10,000 RNAseq samples are available for liver cell lines in the NCBI SRA database, the total number of obtained samples matching our criteria makes it possible to compare only three cell lines—HepG2, Hep3B, and Huh7 . For theA quality check of the selected public data revealed that, on average, the number of fragments in cell line samples was 31 million reads, and the average read length was 128 bp. In contrast, the length of reads in the IBMC data averaged 100 bp, and the number of reads exceeded the average value in the published data and equaled 69 million. Information regarding the average data quality for each cell line is presented in As cell lines vary between laboratories, we performed statistical analyses to assess potential “laboratory effects”. This involved conducting correlation and PCA analyses and examining differentially expressed genes between the resulting clusters.Correlation analysis indicated a strong positive correlation between the expression levels of transcripts and genes in the HepG2 cell line samples. The average Spearman correlation coefficient between samples was 0.70 at the transcript level and more than 0.86 at the gene level . This suAdditionally, to identify the distribution of samples in the HepG2 dataset, we performed hierarchical clustering on principal components (HCPC) analysis. In this approach, gene expression data are first pre-processed and normalized, followed by the calculation of the principal components representing the main sources of variation in the data. A hierarchical clustering algorithm was then applied to the principal components.The analysis revealed that the HepG2 cell line data were divided into three clusters based on their transcript  and geneFinally, we used differential gene expression analysis to identify genes that were differentially expressed in the third cluster identified by HCPC analysis. The comparison results for each cluster pair are presented in The pathways that show differences between the clusters #1 and #2 in HepG2 cells are essential for energy production and proper mitochondrial function. These pathways include “The Electron Transport Chain (ETC) Oxidative Phosphorylation (OXPHOS) system in mitochondria” ”  between clusters #2 and #3. Differential expression analysis showed that in the #3 cluster, compared to the #1 and #2 clusters, there was a reduced expression of genes associated with pathways involved in drug metabolism and detoxification, such as CYP3A5, CYP20A1, SULT1A4, and SULT1A2.As a result of comparing clusters #1 and #3, we observed differences in the pathways—“The oxidation by cytochrome P450” (FDR = 0.01) and “Metapathway biotransformation phase I and II” (FDR < 0.001). The stability of these pathways is vital for cell lines because the liver is the main site of drug metabolism, and HepG2 cells are commonly used as a model system for studying the metabolism and toxicity of drugs and other xenobiotics [AHI1 and TMEM67 expression in the cluster #3 compared with that in the #1 and #2 clusters. These genes are involved in ciliary assembly, maintenance, and signaling pathways, and mutations in these genes are known to cause Joubert syndrome.Interestingly, in the analysis of cluster #1 vs #3 (FDR = 0.009) and #2 vs #3 (FDR = 0.03), changes in the pathway associated with Joubert syndrome were observed. Differential gene expression analysis showed increased The analysis of HepG2 cell line showed heterogeneity in several critical pathways, including the electron transport chain, oxidative phosphorylation, assembly of mitochondrial complexes I, IV, and III, cytoplasmic ribosomal proteins, retinoblastoma gene, and cytochrome P450 pathways. These pathways play a critical role in ATP production, maintenance of mitochondrial function, regulation of the cell cycle and proliferation, formation of ribosomal subunits, and drug metabolism. Changes in these pathways highlight the importance of careful transferring the results from one cell line to another.We observed a significant average Spearman coefficient between samples: at the transcript level, 0.7, and higher at the gene level, 0.85 . The resHCPC analysis showed that for the Huh7 cell line, five clusters were observed at the transcript level . At the In order to examine the affected pathways between the four clusters in more detail, we performed a differential gene expression analysis. The comparison of the results for each pair of clusters is presented in CDKN2A gene encoding the p16Ink4a protein and RB, are often altered in sporadic human cancer, contributing to the dysregulation of cell proliferation [RB1 gene, which encodes the RB protein, did not change between the clusters. At the same time, CDKN2A expression increased in cluster #2 compared to cluster #1 . The cytoplasmic ribosomal protein pathway (FDR = 0.02), which is responsible for cellular protein synthesis, is involved in ribosome production. DNA damage can potentially affect this pathway [Between the clusters #1 and #2, there were changes in the DNA damage-related pathway—“DNA IR-damage and cellular response via ATR” (FDR = 0.009) as DNA damage sensors in Huh7 cells [feration . The RB1 pathway .DHCR7 , DHCR24  have been downregulated. The HMGCS1 .The “Electron transport chain OXPHOS system in mitochondria” and “Mitochondrial complex I assembly model OXPHOS system” pathways associated with energy production in mitochondria were disrupted between the clusters. The disruption of these processes can lead to mitochondrial dysfunction and cell death. Thus, in the cluster #4, compared to the cluster #1 expression of the following genes was upregulated: In conclusion, our analysis revealed significant changes in various biological pathways in Huh7 cell clusters. The pathways associated with cholesterol metabolism, synthesis, and DNA damage are affected. In addition, changes in the pathways associated with mitochondrial energy production have been observed, which can lead to mitochondrial dysfunction and cell death. These results provide important information regarding the molecular mechanisms underlying the functional diversity of Huh7 cells and highlight the importance of accurately transferring results from one study to another.A strong and significant correlation was observed between the samples in the Hep3B cell line. The Spearman values at the gene expression level had an average of 0.88, whereas at the transcript level it was on average of 0.70. The correlation matrix is presented in We identified three clusters of Hep3B cells based on their transcripts  and geneFurthermore, to gain deeper insight into the affected pathways across the three clusters, we conducted an analysis of differential gene expression. The results of the comparison for each pair of clusters are displayed in CDK4 gene expression increases in cluster #2 compared to cluster #1  gene in cancer”, both involved in the regulation of the cell cycle and the DNA damage response, and their dysregulation can contribute to the development of cancer. The TP53  and “Retinoblastoma (RB) gene in cancer pathway” (FDR = 0.001). The key genes of the latter pathway, TCN2 gene, involved in the vitamin B12 pathway, was downregulated in cluster #3 compared to cluster #2  and “Vitamin B12 metabolism” (FDR < 0.001). The Hep3B cells originate from liver tissue, which is the main site for the synthesis of blood clotting factors. Between clusters #3 and #2, the “Blood clotting cascade pathway” changed (FDR < 0.001). The Hep3B cell line was used to evaluate the effects of coagulation factors on autophagy .Changes in the “Network map of the SARS-CoV-2 signaling pathway” (FDR = 0.001) between the clusters #3 and #2 can be explained by changes in the expression of genes associated with the metabolism of folic acid, vitamin B12, or selenium in these cells since SARS-CoV-2 may influence the activities of these pathways. Therefore, folic acid may be an antagonist of SARS-CoV-2; however, its effect on viruses remains unclear ,16.As a result of transcriptomic profiling of HepG2 cell line, on average, 14,609 protein-coding genes and 37,844 transcripts (TPM > 0.1) were expressed. The Huh7 cell line has 14,879 genes and 38,662 transcripts, while Hep3B has 13,940 genes and 35,864 transcripts (TPM > 0.1). We performed a correlation analysis to study the similarity between the liver cell lines. Spearman correlation values were high for both gene and transcript expression. At the gene level, the average levels of Spearman correlation for Huh7 and HepG2 samples were 0.95, while when comparing transcript expression, it was 0.92 . Similarhttps://www.ncbi.nlm.nih.gov/sra, accessed on 5 September 2022) by criteria: total first/second fragment count >20,000,000; average read length >60 base pairs (bp); non-treated cells; Illumina sequencing technology. The accession numbers for the experiments are provided in Liver cell lines were also compared with cell lines that originated from other tissue types: A549 (lung cancer), HeLa , and HEK293T (kidney cancer) as outgroups. The transcriptomic data for these cell lines were also searched in the NCBI SRA database  are liver cells that are isolated directly from tissue. In contrast, liver cell lines such as HepG2, Huh7, and Hep3B are derived from hepatoblastoma and hepatocellular carcinoma cells, respectively. Thus, comparison of hepatic cell lines with primary hepatocytes can provide insight into similarities and differences in gene expression and pathway activation between these cell types.https://www.ncbi.nlm.nih.gov/sra, accessed on 3 May 2023) according to the criteria specified above. For the primary hepatocytes, 31 samples were found. The accession numbers for the experiments are provided in The transcriptomic data for primary hepatocytes was searched in the NCBI SRA database  compared to primary hepatocytes (PHP). Dysregulation of the RB pathway is strongly associated with the development of cancer. Notably, key genes involved in this pathway, such as PPARA, is downregulated in all liver cell lines compared to PHP [Additionally, in all cell lines, the “PPAR signaling” pathway, which is involved in the regulation of lipid metabolism and glucose homeostasis, is affected. This pathway was found to play a crucial role in liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and hepatocellular carcinoma . One of d to PHP . PPARs aFurthermore, to assess the differences between three liver cell lines and primary hepatocytes (PHP), we identified 752 common genes by combining all statistically significant differentially expressed genes between hepatic cell lines and PHP . Of notehttp://pantherdb.org/, accessed on 5 May 2023). Our analysis revealed that the genes were involved in several biological processes, including “Cell cycle”  by criteria: total first/second fragment count >20,000,000; average read length >60 base pairs (bp); non-treated cells; Illumina sequencing technology. Accession numbers for the experiments found are provided in The search of public RNAseq data was performed in the NCBI SRA database . We limited our analysis to protein-coding genes and did not include transcripts assigned to the non-standard chromosomes, resulting in 60,740 protein-coding transcripts and 19,670 genes.https://github.com/kevinblighe/EnhancedVolcano, accessed on 1 February 2023), and ClusterProfiler [The R software environment was used for computations and visualization (ver. 4.1) . We usedProfiler  for geneUpon performing PCA analysis, we observed that samples from one study were separated into distinct clusters. To address this issue, we utilized the Combat method to correct for the batch effect . HoweverOur study aimed to analyze the heterogeneity among liver cell lines—HepG2, Huh7, and Hep3B. In brief, we found that HepG2, Huh7, and Hep3B cell lines differed in oxidative phosphorylation, cholesterol metabolism, and DNA damage between samples and cell lines. In addition, despite the high correlation of gene expression between samples of the same cells lines, a batch effect was noted, which is likely due to the cultivation conditions. Thus, in order to transfer the results of experiments to other cell line samples, many factors must be considered, including growing conditions such as nutrient and oxygen concentrations, pH of the medium, and other parameters, which may lead to inaccurate or distorted data.A notable fact is that when comparing cell lines of different origins based on gene expression, the difference between them is not obvious, while the use of transcript expression gives a clear separation by cell source. Together with our previous results on the difference in the metabolomic profile between the passages of HepG2 cells, it indicates that the closer the omics layer is to the phenotype, the greater the differences between the cells will be observed.Comparative transcriptomic analysis of liver cell lines and primary hepatocytes shows that these cells have a common difference in their cell cycle processes. Interestingly, there are the most differentially expressed genes in the Hep3B cell line compared to primary hepatocytes. Thus, using liver cell lines as a model for normal hepatocytes may not be appropriate in some experiments, as key pathways involved in cancer development and liver disease are affected in HepG2, Huh7, and Hep3B cell lines.


This review summarizes the significant advancements in prophylaxis against graft-versus-host disease (GvHD) presented at the 2022 ASH Annual Meeting. The use of innovative agents and regimens, along with the conventional prophylactic approach of combining post-transplant cyclophosphamide and anti-thymocyte globulin, were discussed. The innovative agents and regimens highlighted in this review include abatacept, the first FDA-approved drug for acute GvHD prophylaxis; RGI-2001, which promotes the expansion of regulatory T cells; and cell therapies such as Orca-T and Orca-Q. These advancements provide promising strategies and options for GvHD prevention, offering hope for improved post-transplant patient outcomes in terms of survival rates. Hematopoietic stem cell transplantation (HSCT) can lead to a severe multisystem disorder known as graft-versus-host disease (GvHD), resulting in considerable morbidity and non-relapse mortality (NRM). We reviewed several latest reports on GvHD prophylaxis from the 2022 ASH Annual Meeting (ASH2022).Abatacept (ABA), a CD28:CD80/86 co-stimulation blockade, has shown potential in controlling early T cell allo-proliferative escape, a major contributor to breakthrough acute GvHD (aGvHD) following calcineurin inhibitor/methotrexate (CNI/MTX) prophylaxis . ABA + CThe augmentation of regulatory T cells (Tregs) has gained significant attention as an effective strategy for preventing GvHD while preserving the graft-versus-leukemia (GvL) effect. RGI-2001, a liposomal glycolipid, expands Tregs by activating invariant natural killer cells through CD1d receptor on antigen-presenting cells, thereby modulating GvHD pathogenesis. RGI-2001 administered intravenously with tacrolimus/MTX was effective in preventing aGvHD in individuals receiving grafts from peripheral blood stem cell (PBSC) and bone marrow sources, with a favorable safety profile. Promising results justify the planning of a phase 3 clinical trial to assess the efficacy of the regimen . Orca-T Combining anti-thymocyte globulin (ATG) and PTCy as prophylaxis against GvHD is a promising strategy, surpassing the limitations of individual administration. ATG-based regimens pose viral infection risks, while PTCy-based prophylaxis is less effective with PBSC grafts. A retrospective study  showed tSalas et al. conducted trials investigating the use of reduced-dose ATG in various transplantation settings. In Abstract 2127 , a compaLow-dose PTCy has been investigated in elderly patients and those with cardiac comorbidities undergoing haplo-PBSCT . The stuThe emergence of novel agents and regimens has expanded the donor pool and allowed the use of different graft sources, including mismatched donors and PBSC, to address patient needs while minimizing the risk of GvHD. For PTCy and ATG combination prophylaxis, future endeavors could focus on investigating lower-dose combination of these agents to achieve the dual goals of minimizing adverse events and maintaining prophylactic efficacy.


Sebaceous neoplasms describe a group of tumors with sebaceous differentiation commonly seen in lesions located primarily in the face and neck. The majority of these lesions are benign, while malignant neoplasms with sebaceous differentiation are uncommon. Sebaceous tumors present a strong association with the Muir–Torre Syndrome. Patients suspected with this syndrome should undergo neoplasm excision, followed by histopathologic and additional immunohistochemistry and genetics examinations. Clinical and dermoscopic features of the sebaceous neoplasms, as well as management procedures collected from the literature analysis regarding sebaceous carcinoma, sebaceoma/sebaceous adenoma, and sebaceous hyperplasia are described in the current review. A special note is made for describing the Muir–Torre Syndrome in patients presenting multiple sebaceous tumors. Sebaceous differentiation of neoplasms is represented by sebaceous carcinoma, sebaceoma, adenoma, and sebaceous hyperplasia. The benign sebaceous gland hyperplasia is very common among the general population, while malignant neoplasms, sebaceous carcinoma, sebaceoma, and adenoma are very rare. Sebaceous gland tumors can appear at any age, but they affect mainly elderly people. The group of sebaceous tumors have been strongly associated with Muir–Torre Syndrome (MTS)—when multiple lesions are present—while sebaceous hyperplasia has been correlated with chronic sun exposure. In this review, an effort was made to describe the clinical and dermoscopic features of sebaceous neoplasms, along with the management of such lesions, regarding tumor excision and the presence of MTS.Clinically, sebaceous tumors are often mistaken for more common malignant skin neoplasms, such as basal cell carcinoma and squamous cell carcinoma. In these cases, the diagnosis can be assisted by the use of dermoscopy and confirmed with the help of histopathology. Sebaceous tumors are mainly located on the head and neck area, but they may be traced to any region of the body. The clinical presentation can vary from small, skin-colored, umbilicated papules to ulcerated lesions, subcutaneous nodules, or exophytic tumors. The lesions are solitary except in patients with MTS. In this case, numerous sebaceous tumors may be located outside the head and neck area. Moreover, patients affected from the syndrome can present multiple keratoacanthomas, or manifest gland neoplasms at a young age.For this review, we searched the following databases: PubMed, MEDLINE, Scopus, OVID, Embase, Science Direct, Cochrane library, Research Gate, and Google Scholar. The search terms were “sebaceous neoplasms” OR “sebaceous carcinoma” OR “sebaceoma” OR “adenoma” OR “sebaceous hyperplasia” OR “Muir–Torre Syndrome”. Studies were included from inception to February 2023. Data extraction was performed individually for each neoplasm. The pertinent information obtained from each study was: the first author; year of publication; type of study; type of sebaceous neoplasm; epidemiologic data; clinical features; dermoscopic features; and management. The search included exclusively English-language academic papers. The reference list of the shortlisted articles was also examined for supplementary studies. The study has been designed, and the results have been described as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Our literature search yielded 494 studies. After excluding case reports, systematic reviews, duplicate studies, and studies that did not present any relevant data, we ended up with 52 studies.Sebaceous carcinoma is an uncommon cutaneous neoplasm. The prevalence reported in two studies, based on the US and Texas registries of skin malignancies, varies between 0.05% and 0.7% among other skin tumors ,2. AlthoIn a 9-year-study about sebaceous neoplasms, from a total of 8819 biopsies, 207 sebaceous neoplasms were isolated. Among them, 1.6% were identified as sebaceomas and 1.1% as adenomas . Wh. Wh62]. Once the diagnosis has been established, MTS patients should undergo annual surveillance for visceral and cutaneous malignancy. Gastrointestinal endoscopy should be performed annually, with colonoscopy examination beginning from the 18th year of age, and upper endoscopy from the 25th . Men shoThe management of cutaneous lesions in the context of MTS does not differ from that of solitary neoplasms and was described earlier in the current review.Neoplasms with sebaceous differentiation present an everyday challenge for the clinician. In cases of benign neoplasms that clinically present relevant difficulty regarding the differential diagnosis, the use of dermoscopy could be of a great help. In order to establish the diagnosis, histopathology should be performed as neoplasm differentiation variations could lead to misdiagnosis and mistreatment. Moreover, since the differential diagnosis should include the sebaceous carcinoma, the excision management could differ significantly. The therapeutical approach in sebaceous carcinomas should include histological margin control excision techniques, as the rate of relapse or metastasis is significantly high. When this is not applicable, wide margin excision limits should be granted. Association with Muir–Torre Syndrome should also be considered in all cases with multiple tumors with sebaceous differentiation. Immunohistochemistry may provide useful information in cases where histologic distinction is difficult, mainly between sebaceous adenoma and sebaceous hyperplasia. This dilemma should be confronted since sebaceous hyperplasia is not associated with MTS, whereas sebaceous adenoma is the most common sebaceous tumor in the syndrome. The use of genetics is the gold standard in order to detect the germline mutation and achieve the diagnosis of MTS. In MTS patients, a multidisciplinary approach is essential, while close follow-up should be performed.Understanding the nature and the behavior of sebaceous tumors, defying the overlap between the various neoplasms with sebaceous differentiation, describing and improving the clinical and dermoscopic criteria for the diagnosis, and investigating the possible presence of immunohistochemistry germline mutation markers should be subject to further studies. Since clinical or dermoscopic definitive diagnosis is not achievable, all sebaceous tumors should be treated with caution, and excised totally.


Renal proximal tubule epithelial cells (RPTECs) are a primary site for kidney injury. We created two RPTEC lines from CD-1 mice immortalized with hTERT (human telomerase reverse transcriptase) or SV40 LgT antigen (Simian Virus 40 Large T antigen). Our hypothesis was that low-level, repeated exposure to subcytotoxic levels of 0.25–2.5 μM cisplatin (CisPt) or 12.5–100 μM aflatoxin B1 (AFB1) would activate distinctive genes and pathways in these two differently immortalized cell lines. RNA-seq showed only LgT cells responded to AFB1 with 1139 differentially expressed genes (DEGs) at 72 h. The data suggested that AFB1 had direct nephrotoxic properties on the LgT cells. However, both the cell lines responded to 2.5 μM CisPt from 3 to 96 h expressing 2000–5000 total DEGs. For CisPt, the findings indicated a coordinated transcriptional program of injury signals and repair from the expression of immune receptors with cytokine and chemokine secretion for leukocyte recruitment; robust expression of synaptic and substrate adhesion molecules (SAMs) facilitating the expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and the expression of nephrogenesis transcription factors. Pathway analysis supported the concept of a renal repair transcriptome. In summary, these cell lines provide in vitro models for the improved understanding of repeated renal injury and repair mechanisms. High-throughput screening against toxicant libraries should provide a wider perspective of their capabilities in nephrotoxicity. Proximal tubule cells of the kidney are primary sites for reabsorption of ions and intermediary metabolites driven by energy-driven transporters. Renal proximal tubule epithelial cells (RPTECs) express multiple transporter systems that have broad substrate specificity and special functions including the disposition of many xenobiotics ,2. WhileCKD can occur after long-term renal injury as a comorbidity from diabetes and hypertension , by enviHuman RPTECs immortalized by hTERT (telomerase reverse transcriptase) have proven an extremely useful model for in vitro studies ,17,18. OWe have been intrigued by the experimental approaches using low-level, repeated exposure ,13,15 thp values. Using DESeq2 for differential expression analysis to CisPt and AFB1, we filtered for two-fold expression differences at an adjusted p ≤ 0.05 value to diminish false positives. However, for PCA analysis all differentially expressed genes were used to optimally visualize the separation among the treatment groups. In the absence of cell death, we observed conventional biomarkers of renal injury, such as Havcr1 (Kim-1) and Il6, were mildly increased, over concentration and time with AFB1 and CisPt. For example, Havcr1 ranged from a 2- to 4-fold increase in either cell line with either chemical treatment at 72–96 h. In addition, we often found increased expression of Mmp9, Mmp10, Mmp13, and Mmp25 that was consistent with their recognized roles in renal damage Hoxc12 an [Nlrp12 . It was  failure . Similarre [Lhx2 , Scn7a [2 [Scn7a , Negr1 [a [Negr1 , Ntrik1  [Ntrik1 , and Grmk1 [Grm1  are incrSlc22a2 (Oct2), is well recognized as highly responsible for CisPt bioaccumulation over the course of chemotherapeutic treatment resulting in nephrotoxicity. Since Slc22a2 is poorly expressed in human and rodent immortalized RPTEC lines, many studies have used Oct2-overexpression systems to study its transporter functions with various therapeutics [Slc31a1) are highly expressed in our cell lines and have a demonstrated function in CisPt uptake in other studies [Slc31a1, Slc31a2 , and Slc47a1 .Another aspect to CisPt toxicity is its pharmacokinetic entry into and removal from proximal tubule cells. Entry can occur by passive diffusion as well as by facilitated movement from specific membrane transporters ,65. Clinapeutics . In muri studies . AlthougOtof, ↑ Scn4b, ↓ Fhit, ↓ Pacrg) as well as observing some unique high-expression and low-expression transcripts seen either in the hTERT  or in the LgT cells . These observations reflect the diversity of biological responses at the individual gene level as cells adjust to repeated CisPt exposure over time. In addition, pathway-level interrogation is helpful for interpreting differential expression of large gene sets [2O2 stress from 25 µM CisPt for up to 24 h exposure in mouse proximal tubule cells [Trp53 signaling, Nrf2 and mitochondria-mediated oxidative stress, as well as mTOR, AMPK, and EIF2 signaling pathways [Fold changes of the top 20 DEGs reflect response intensity of individual transcripts to CisPt, though just a small proportion of the same DEGs were conserved in the top DEG group over 24–96 h. We also found several transcripts that were shared between the two cell lines  from Nanostring data agreed with the RNA-seq findings but were statistically significant due to reproducibility of high fluorescence counts. Similarly, the Nanostring data showed modest fold increases in Cdkn1a (p21) at 1.3X and 1.4X fold in the hTERT cells and 1.7X and 2.4X fold in the LgT cells at 72 h and 96 h, respectively. However, Gadd45 (p53-dependent gene) was down-regulated. Mdm2 levels shown by Nanostring were slightly increased in expression and ranged from 2 to 4 fold in both types. Although Mdm2 and Cdkn1a are generally controlled by Trp53 in primary cells, Trp53-independent roles for these transcripts are being increasingly recognized [Trp53-mediated cell death did not play a role in the response of our cell lines to nephrotoxicants under our exposure conditions. However, we did note large fold changes in the Trp53-related family members, Trp63 and Trp73, in both the cell types by RNA-seq. Trp63 and Trp73 have roles in the differentiation and development of squamous epithelial and neural cells, respectively [Trp63 and Trp73 in proximal tubule cell recovery after injury merits further investigation.Interestingly in our studies, we did not observe significant fold changes (≤1.1X) in the pressors . Nominalcognized . Furtherectively , and recectively ,76. An iIl6 and Il18 pathway engagement that was consistent with transcriptional increases over concentration and time in both the cell types. However, other critical immune transcripts and pathways were also highly upregulated including the S100a8/S100a9 pathway noted in AKI [Il17f, Il24, Il33, and Tnf pathways in kidney injury [Ltb4r1 and Ltb4r2 pathway [Lilrb4a,b receptor pathway, both involved in leukocyte recruitment [Cxcr4 [Hc (C5a) [Ptgs2 transcript (Cox2) pathway [Wnt11 [Fgf3 [Mmp12 [Csf2, secreted from renal proximal tubules cells during AKI, can promote the transition of proinflammatory M1 macrophages to an anti-inflammatory M2 phenotype in a dose and time-dependent manner [Csf2 pathway activation and increased expression over 24–96 h in both the cell lines. In addition, the GPCR-pathways were widely activated in this study since many GPCR ligands were likely elevated during injury that could include hormones, peptides, proteins such as chemokines, ions like Ca++, synaptic transmitters such as glutamine, and intermediary metabolites ranging from fatty acids, ATP, ADP, to many others [Pathway activation marked by the presence of DEGs are controlled by upstream regulators and compiling these cellular events could give broader insights into CisPt-induced effects. The number of activated pathways outnumbered the inhibitory pathways as they both increased over time from 24 to 96 h. We focused our attention on the upstream regulators that showed significant differential expression with the potential to activate or inhibit pathways. In our study, immune, neurogenic, and tissue remodeling and repair pathways were the primary cellular responses to subcytotoxic CisPt exposure in these two new cell lines. We observed d in AKI ; cytokiny injury ,79,80; t pathway  and the ruitment ,83; the t [Cxcr4  and pathHc (C5a) ; and the pathway . Upreguly [Wnt11 , Fgf3 [811 [Fgf3 , and Mmp3 [Mmp12  have beet manner , contriby others .The molecular pathogenesis of acute renal injury is highly complex but in vitro systems can help identify specific genes and orthologs for unraveling the multiple pathways involved for early detection, therapeutic intervention, and prevention. Instead of a single nephrotoxicant challenge, we feel advanced understanding in chronic kidney disease can be gained from repeated subcytotoxic exposures. We observed most expression changes at 72 h or 96 h but recognize some genes and pathways were activated as early as 24 or 48 h. We interpreted these temporal transcription differences in genes and pathways for each cell type as early molecular adjustments (3–48 h) based on their unique genomic structure. However, in the case of CisPt exposure we found each cell type eventually converged on similar pathways responses at later time points (72–96 h). We speculate that the two different immortalization processes could affect early transcriptional variations in the hTERT and LgT responses to CisPt. AFB1 toxicity in the LgT cells, compared to the absence of response in the hTERT cells, does suggest some important genomic differences to be further defined. We further expect that nephrotoxicant screening libraries will be valuable tools to uncover the unique capabilities of LgT and hTERT moRPTEC lines in various cellular formats including microfluidic RPTEC chips  and, 2D Tnf, Il17, and family members of CXC- and CC-motifs as found in our study.Single or repeated acute kidney injuries may lead to chronic kidney failure exacerbated by comorbidities, contributions by distal organs, therapeutics, and environmental exposures ,94,95. NCell Immortalization and Culture: Mouse renal proximal tubule epithelial cells (moRPTECs) isolated from CD-1 mice were obtained commercially  and were transduced with lentivirus vectors containing either human TERT (hTERT) or SV40 Large T Ag (LgT) with antibiotic selection modules. The Ef1a_Large T-antigen Puro lentivirus was a gift from Linzhao Cheng [g centrifugation for 5 min to form a loose pellet, followed by resuspension in culture medium and plating.RPTECs were grown on Biocoat collagen-1 coated 6-well plates  in a commercial culture medium  containing 2% FCS, Penicillin/Streptomycin antibiotics and a growth supplement. hTERT immortalized cells were selected and maintained by supplementing culture medium with 100 ng/mL G418-Geneticin™  while LgT immortalized cells were selected and maintained at a concentration of 0.5 ug/mL of puromycin . Cells that had been passaged for over 50 cell divisions under continuous antibiotic selection with consistent cobblestone appearance and contact inhibition were considered stable cell lines. We observed that moRPTECs strongly adhered to culture plates surfaces and notably possessed cell self-adhesion properties in suspension. We found that successfully passaging moRPTECs required 1–2 PBS washes and 5–10 min incubation in PBS at 37 °C prior to trypsinization and 120× Chemical Treatment: Aflatoxin B1  and Cisplatin  were selected as representative nephrotoxicants to characterize transcriptional responses. Experiments were conducted in confluent cultures in 6-well plates. AFB1 was solubilized in DMSO (control) and CisPt was solubilized in phosphate buffered saline . Concentrations were chosen for minimal cytotoxicity at the highest level of chemical exposure in commercial growth media at 2 mL/well. Immortalized LgT and hTERT moRPTEC lines were grown to near confluence in 6-well tissue culture plates before treatment. To examine concentration-dependent transcriptional responses to CisPt or AFB1, cells were treated for 72 h with 0.25–2.5 μM CisPt, or with 12.5–100 μM AFB1. A second experiment determined transcriptional changes in LgT, and hTERT cells that were exposed to 2.5 μM CisPt over a 96 h time period. Samples at each time point were conducted in triplicate for CisPt and a triplicate time-matched control. At 0 h, untreated cells were nearly confluent and were harvested for RNA. After a single exposure to 2.5 μM CisPt, cells were harvested for RNA at 3 h, 6 h, or 24 h. For the remaining time points, media was changed daily containing either CisPt treatment or PBS at each 24 h period and then harvested for RNA 24 h later. Chemical treatments are summarized in ® 3500 xl Genetic Analyzer . Authentication data are presented in Mouse Cell Authentication: hTERT and LgT moRPTEC lines were submitted for mouse cell authentication using a short tandem repeat (STR) profiling  performed according to published protocols . BrieflyRNA and DNA isolation; RNA-seq: Cell viscosity was reduced by use of cell column shredders prior to nucleic acid isolation . For RNA isolation , cells were washed with PBS prior to addition of lysis buffer and spin columns with on-column DNAase-1 digestion followed by washes, elution, and storage at −80 °C. RNA integrity was measured on an Agilent Model 5300 Fragment Analyzer  with RQN values of 9–10. RNA samples were prepared for RNA-seq by rRNA depletion, fragmentation , bar coding, and library construction as previously described . BrieflyActb; Gapdh, Hprt, Rpl32) that were determined by RNA-seq to be treatment stable. Samples were analyzed by nCounter at 150 ng RNA input. Data were analyzed by ANOVA and Newman–Keuls testing.Nanostring Analysis: NanoString nCounter  was used as a multiplexed gene expression platform to test for specific transcripts as previously described . Brieflyin vivo testing in both animal models or more complex multicellular experimental constructs such as organoids or microphysiological systems. The development of interconnected, pulsatile culture media systems carrying signaling molecules and hormones from different cell types represent promising new technologies for combining different immortalized cell types to better understand the causes of toxicity and disease.There are several considerations for future directions of the current work. Functional data for ion transporter activity and xenobiotic metabolic activation of these two cell lines would be important for placing context to the transcriptional changes that we reported. This could include functional assays for Oct  and Oat  transporters that are of clinical importance in the disposition of many drugs or xenobiotics. The creation of additional immortalized cell lines by human papilloma virus (HPV) or Epstein–Barr virus could provide additional breadth to the biological responses possible in moRPTEC lines. Further, the inclusion of different mouse strains of both sexes could add important gender representation and complexity of response to these two in vitro cell line models. A more comprehensive view of RPTEC responses to toxicant challenge could be gained by addition of other omics methodologies including proteomics, metabolomics, and epigenomics that could be strategically applied to critical early time points or more prolonged exposure paradigms to better simulate the conditions leading to chronic renal kidney disease. Finally, the value of in vitro findings and hypotheses generated from these renal cell lines would greatly benefit from This study presents two new immortalized RPTEC lines with similar and some distinct capabilities in their response to two different nephrotoxicants. We believe the findings from these two cell lines can provide in vitro models for improved understanding of renal toxicity and repair mechanisms. First, data in the LgT cells suggest AFB1 may have direct nephrotoxic properties independent of hepatic metabolism and processing. Second, expression from CisPt experiments in both the cell lines support the concept of a ‘coordinated transcriptional program’ of injury signals and repair processes represented by (1) externalization of immune receptors as well as cytokine and chemokine secretion for leukocyte recruitment; (2) a robust expression of both synaptic and SAM molecules to facilitate increased expression of neural and hormonal receptors, ion channels/transporters, and trophic factors; and (3) expression of nephrogenesis transcription factors to assist in regeneration and repair. Many of the neural and cardiac canonical pathways evoked by pathway analysis are not unexpected since these tissues share similar cellular processes to kidneys in synaptic and bioelectric transmission, and intravesicular transport. Third, we anticipate differences in the immortalization processes of the parent RPTECs affect the genomics that shape the kinetics of phenotypic responses in these two cell lines—a research area that is actively being investigated in our laboratory. High throughput screening of LgT and hTERT-derived moRPTECs against chemical toxicant libraries will provide a wider perspective of biological responses and the unique capabilities of these cell lines in nephrotoxicity research.