BA‐induced diarrhea affects ⅓ of patients with chronic intestinal inflammation, but the underlying mechanisms remain to be elucidated. We previously reported a yin/yang in BA action, with dihydroxy, chenodeoxycholic acid (CDCA), disrupting TJs and its monohydroxy derivative, lithocholic acid (LCA) attenuating it. We hypothesize that structural differences account for the varied actions, namely the presence of the 7‐OH in CDCA, which can serve as a hydrogen bond donor. Recently, we used a fluorescein amine‐tagged CDCA (CDCA‐FA), to show that apical CDCA‐FA travels paracellularly to the basolateral surface (BLS), and like CDCA alters reactive oxygen species‐dependent TJ permeability (FASEB J’ 18:32, 747.19). We predict that, in contrast, LCA‐FA will not alter TJ or access BLS. We studied the structural basis for the yin/yang in BA action on TJ function, by synthesizing: 1. a fluorescein amine‐tagged LCA and 2. a 7, methoxy CDCA (CDCA‐Me).CDCA‐FA and LCA‐FA were synthesized by protecting their alcohol(s), converting‐COOH to an acid chloride, adding the fluorescein amine tag to form an amide, and removing the protecting groups. They were purified by column chromatography and the structure confirmed by NMR and mass spectrometry. We have synthesized CDCA‐Me, structurally similar to LCA, by protecting ‐ COOH of CDCA as a methyl ester, protecting 3‐OH as a silyl ether, converting 7‐OH to a–OCH3 using methyl triflate, and removing the protecting groups. The yield of pure CDCA‐Me is being optimized so its biological effects can be tested.Confluent T84 cells (TransEpithelial Resistance, TER >1000Ωcm2), were treated apically with DMSO (CTRL), 500μM CDCA‐FA, 50μM LCA‐FA ± 500μM CDCA/CDCA‐FA for 0.5–18 H. Cell viability was measured by propidium iodide staining, fluorescence microscopy and Image J analysis. TJ function was assessed by examining: a. pore function measured as TER; b. leak function measured as apparent permeability of CDCA‐FA (Papp CDCA‐FA) vs. LCA‐FA (Papp LCA‐FA) or FITC‐10kDa dextran flux across the monolayer.Exposure (18H) to 50μM LCA‐FA, like LCA, did not alter cell viability nor change CDCA‐FA's effect on cell death (% cell death, CTRL: 7±3 vs LCA‐FA: 9±4; CDCA‐FA: 17±5 vs. CDCA‐FA+ LCA‐FA: 18±6, n=5). Also similar to LCA, LCA‐FA alone or ± CDCA‐FA did not alter pore function (TER) over time (Ωcm2; t=18H; CTRL: 731 ± 10; LCA‐FA: 761 ± 157, CDCA‐FA: 96 ± 12*; CDCA‐FA+LCA‐FA: 118±24*, n=4; *p<0.05 vs CTRL). However, similar to LCA, LCA‐FA attenuated CDCA‐FA's permeability (18 H, Papp LCA‐FA cm/sec, LCA‐FA: 6±3; Papp CDCA‐FA, CDCA‐FA: 57±2; CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA‐FA: 21±5; p<0.05, n>3). Thus, addition of the fluorescent tag did not alter the function of BAs. In summary, CDCA, but not LCA, moves paracellularly to the BLS whereas LCA limits CDCA movement to protect barrier integrity. We postulate that the 7‐OH group in CDCA as a hydrogen bond donor is critical in its role in disrupting barrier function and triggering inflammation and our structure/function studies will guide new therapeutic strategies.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR, APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Artemisinin (ART), a common anti‐malarial drug, and its analogs are also useful as repurposed anti‐cancer drugs, but their properties are not fully elucidated. We previously synthesized novel trioxane (DMR) and dioxazanine (HSM) ART analogs and showed that: a. at low doses (5–10 μM), HSM > than DMR in inducing apoptosis in human colon and lung cancer cells, but not in normal lung cells; b. The actions involved reactive oxygen species (ROS) (Faseb J, ′18:32, 616.2). TfR expression is increased in cancer vs. normal cells. Thus, we hypothesize that, in cancer cells, TfR increases [Fe2+]i which acts on the endoperoxide to produce ROS and induce apoptosis. We examined the mechanism of action of DMR/HSM in inducing apoptosis in human breast, lung, and colon cancer cell lines as compared to normal cell lines. To increase the efficacy of the drug, we also designed and synthesized a 3rd novel analog with dual dioxazanine pharmacophores, PMW.Confluent normal (Breast: MCF10A, Lung: A549) and cancer (Breast: MCF7, Colon: T84, Lung: A549) cells were treated (18 H) with 1–50μM of DMR, HSM or PMW±Deferoxamine (DFO, iron chelator; 1–25 μM). Cells were stained with FITC‐Annexin V (apoptosis), propidium iodide (cell death) or CellRox Green (mitochondrial/nuclear ROS), imaged and quantified by flow cytometry and/or microscopy (Image J). Normal and cancer cell lysates (30 μg) were subjected to SDS‐PAGE and immunoblotting with polyclonal TfR antibody or anti‐GAPDH (control).As in other cancer cells, both DMR and HSM (5 – 50μM) induced apoptosis in breast cancer, but not normal, cells. In contrast to A549 lung cells, where HSM was more effective than DMR, in MCF7 breast cells, 50μM DMR was more efficient (~40%) in inducing apoptosis than HSM (10%). This dictates the development of novel tissue‐specific analogs. The new analog, PMW (1 – 50μM, 18H) did not induce apoptosis in the cancer cells. The larger size of PMW may have slowed entry, suggesting longer incubations.Image J analysis (mean pixel intensity; 18 H) of CellRox+ lung cells (n=3) showed that HSM dose‐dependently increased ROS in cancer A549: (DMSO: 1.5±0.2; HSM, 5 μM: 19±2; 10 μM: 48±4; 50 μM: 59±2); but not normal BEAS2B: (DMSO: 1.0±0.3; HSM, 5 μM: 1.8±1; 10 μM: 2.2±0.4; 50 μM: 2.9±2). Similarly, in breast cancer cells (n=3), DMR dose‐dependently increased ROS in MCF7 (DMSO: 1.0±0.3; DMR, 5 μM: 12±2; 10 μM: 51±5; 50 μM: 42±3); but not in MCF10A (DMSO: 1.0±0.2; DMR, 5 μM: 1.6±1; 10 μM: 3.1±1; 50 μM: 2.5±1).Immunoblotting detected a distinct 94 kDa TfR protein in colon, lung, and breast cancer, but not normal cells. The effect of DMR/HSM±DFO on apoptosis was examined to study the role of TfR. Albeit showing an inhibitory trend, DFO did not significantly alter ART analogs actions (% Annexin V+ cells,18 H: A549: HSM,10μM: 46±4; HSM+5μM DFO: 38±6; HSM +10μM DFO: 33±9; MCF7: DMR,10μM: 36±4; DMR+5μM DFO: 31±7; DMR+10μM DFO: 29±5; n≥3).Synthesizing novel ART‐analogs with improved pharmacokinetics to specifically target cancer cells and elucidating their mechanism of action will help develop new alternatives to treat cancer.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS; Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dysfunction of mucosal immune response and tight junctions (TJ) play an important role in the pathogenesis of inflammatory bowel diseases (IBD) and diarrhea. We reported that excess BA, namely chenodeoxycholic acid (CDCA; 500μM) induced PiC release and altered T84 TJ by increasing ROS. The effect on leak function of TJ was attenuated by CDCA's derivative, lithocholic acid (LCA, 50 μM) and ROS inhibitors (Physiol Rep, ′17, 5: e13294). In patients with IBD, PiC upregulate inducible nitric oxide synthase (iNOS). Thus, we hypothesize a role for RNS in mucosal damage and studied the involvement of oxidative/nitrosative stress in BA‐induced TJ dysfunction and cytokine release in T84 cells.We previously described the synthesis of fluorescein amine‐tagged CDCA (CDCA‐FA) and its use to track transepithelial (TE) BA movement. Confluent T84 cells (TE Resistance; TER >1000Ωcm2) were treated apically with DMSO, 500μM CDCA‐FA, 50μM LCA, CDCA+LCA, ± PiC ([ng/ml]: TNFα[10]+IL‐1β[10]+IFNγ[30]), ± 50μM L‐NAME ( L‐NG‐Nitroarginine methyl ester, NOS inhibitor), ± 1mM NAC, (N‐acetyl cysteine, ROS scavenger), for 0.5–18 H. [NO2/NO3] was measured by the Griess assay. We examined the role of RNS/ROS in BA action by studying the effect of BAs±L‐NAME± NAC on: a. Apoptosis (Annexin V, Flow cytometry); b. Paracellular permeability (Pore function as TER; Leak function as TE CDCA‐FA movement); and c. IL‐8 release (ELISA).CDCA (18 H), but not LCA, increased [NO2/NO3] 3‐fold and this was enhanced by PiC (μmol/mg protein; DMSO: 12±1; CDCA: 36±1; LCA: 16± 4, PiC: 18±1; PiC+CDCA: 55±2, n=3). LCA decreased CDCA±PiC‐induced [NO2/NO3] (CDCA+LCA: 25±1; PiC+LCA: 13±2, PiC+CDCA+LCA: 27±4; p<0.05). Inhibiting RNS did not alter CDCA‐induced apoptosis. In pore function, L‐NAME reduced the initial rate of CDCA‐FA‐induced decrease in TER (Ωcm2/sec; 1H CDCA‐FA: 12±1; CDCA‐FA+L‐NAME: 7±1, n>3; p<0.05 ), with no statistical difference at 4 and 18H. L‐NAME altered leak function, reducing CDCA‐FA flux by ~30% at 18 H, (Apparent permeability: Papp ×10−9 cm/sec: CDCA‐FA: 57±2; CDCA‐FA+L‐NAME: 30±1; p<0.05, n=3). LCA decreased CDCA‐FA flux by ~58%, and LCA+L‐NAME completely attenuated it (CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA+L‐NAME: 3±1; p<0.05, n>3). NAC+LCA caused only a ~85% decrease in CDCA‐FA flux (CDCA‐FA+LCA+NAC: 8±2).Like CDCA, CDCA‐FA±PiC stimulated IL‐8 release, which was decreased by LCA. Inhibiting RNS, but not ROS, caused ~40% reduction in CDCA‐induced IL‐8 release (ng/ml; CDCA: 4.6±0.5; CDCA+L‐NAME: 2.7±0.1; CDCA+NAC: 4.2±0.3; n=3), suggesting a role for NO in BA‐induced inflammatory process.We demonstrate a novel role for RNS in CDCA‐induced TJ dysfunction in T84 cells. Inhibiting RNS in the presence of LCA reverses CDCA action on leak function and reduces PiC release. Equally important, its roles overlap with but are distinct from ROS. Understanding the role of ROS/RNS in BA action can lead to novel therapeutic strategies for IBD.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bile acid‐associated diarrhea and epithelial barrier dysfunction are common occurrences in patients with inflammatory bowel diseases. There are very few studies that compare the effects of the dihydroxy bile acids (BAs), chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA), on barrier function, although they are known for their prosecretory properties. We have reported that CDCA induced apoptosis, released reactive oxygen species (ROS), nitrogen species (NO2/NO3) and the pro‐inflammatory cytokine IL‐8, to disrupt tight junctions, while its monohydroxy derivative, lithocholic acid (LCA) did not (FASEB J 2019 33:711.1). In this study, we examined the effects of dihydroxy BAs, DCA, CDCA, and ursodeoxycholic acid (UDCA, 7‐OH epimer of CDCA), on barrier function in human colon carcinoma cells (T84). We hypothesize that the effect of dihydroxy BAs on barrier function will be varied owing to the subtle differences in the position (3, 7, or 12) and stereochemistry (α or β) of the hydroxyl group. Confluent T84 cells (Trans Epithelial Resistance, TER >1000 Ω.cm2) were treated overnight (O/N) with 500μM of CDCA, DCA and UDCA and the effects on barrier function were studied as follows: i) apoptosis (Annexin‐V, flow cytometry), ii) mitochondrial and nuclear ROS production (CellRox, flow cytometry), iii) [NO2/NO3] (Griess Assay, Colorimetry), iv) IL‐8 release (ELISA) and v) paracellular permeability (TER and FITC‐10kD dextran fluxes (F10D)). Apical exposure of CDCA and DCA induced apoptosis while UDCA did not alter cell viability (% Annexin V+ cells, 18 hr: DMSO, 10±3; CDCA: 25±2*; DCA: 26±2*; UDCA: 13±4; n=4, *p<0.05, compared to control). CDCA and DCA increased ROS (% CellRox+ cells: DMSO: 12±0.5; CDCA: 32±0.1; DCA: 36±2, p<0.05) and [NO2/NO3] (μmol/mg protein: control: 68±6; CDCA: 170±18; DCA: 249±28). UDCA alone had no effect, but it attenuated CDCA’s action (% CellRox+ cells: UDCA: 14±0.5; CDCA+UDCA: 12±0.3; [NO2/NO3] UDCA: 65±12; CDCA+UDCA: 48±30; n=4, p<0.05). Similarly, CDCA and DCA increased IL‐8 release (ng/ml: Control: 78±4; CDCA: 578±86*; DCA: 789±98*), while UDCA decreased basal and CDCA induced IL‐8 release (UDCA: 20±5; CDCA+UDCA: 26±4; n=6, p<0.05). Time‐dependent studies (0.5–18 hr) show that CDCA and DCA decreased TER by 70% and 62%, respectively and increased F10D fluxes. In contrast, UDCA alone had no effect on paracellular permeability but restored CDCA‐ and DCA‐induced barrier dysfunction (18 hr, apparent permeability (x 10−9) cm/sec: Control: 6±1; CDCA: 96±10; DCA: 120±20; UDCA: 13±2; CDCA+UDCA: 41±12; DCA+UDCA: 18±2; n≥3, *p<0.05). Comparing the effects of hydrophobic CDCA and DCA on barrier function with that of the hydrophilic 7‐β isomer, UDCA, highlighted the importance of structure and stereochemistry of ‐OH groups in BA action. Therefore, UDCA, like LCA, could be used to ameliorate the deleterious effects of other dihydroxy BAs and serve as a target therapeutic drug for inflammatory and diarrheal diseases. Support or Funding Information APS‐STRIDE National Heart, Lung and Blood Institute (Grant #...

Lung cancer is one of the leading causes of cancer death. Most current treatments have debilitating side effects with poor selectivity and pharmacodynamic properties. To develop more effective and safer anticancer drugs, we synthesized trioxane (DMR) and dioxazinane (HSM), both novel Artemisinin (ART) analogs. These analogs induced apoptosis in cancer but not normal lung cells and was reactive oxygen species (ROS) dependent. We also showed that cancer cells have higher transferrin receptor (TfR) expression compared to normal cells. We hypothesize high levels of TfR expression and [iron]i are responsible for the cancer specific effects of analogs. To study this, we confirmed iron’s role in ART analog‐induced apoptosis. We also knocked out TfR in cancer cells and overexpressed TfR in normal cells. Confluent normal (BEAS2B) and cancer (A549) human lung cells were treated (18 H) with 10μM of DMR, HSM ±Deferoxamine (DFO, iron chelator; 10 μM). [Iron]i was assessed in cell lysates using a colorimetric assay (Biovision, CA). Cell death was assessed by i) staining with FITC‐Annexin V (AV, apoptosis), propidium iodide (PI, cell death), followed by imaging, and quantification using flow cytometry and/or microscopy (Image J) and ii) measuring Lactate dehydrogenase (LDH) release. Cells were transfected with CRISPR/Cas9 overexpression (OE) or knockout (KO) plasmid (Santa Cruz, TX) containing Green Fluorescent Protein (GFP). Co‐transfection with HDR plasmid allowed for puromycin selection. Transfection efficiency was assessed using RT‐PCR, Western blot and GFP expression. A549 cells had 7‐fold more [iron]i than BEAS2B cells. This was essential for the apoptotic effects, as chelating iron with DFO prevented ART analog‐induced cell death (% AV+ A549 cells, 18 H: HSM: 54±4; HSM+DFO: 1±0.5; DMR: 32±3; DMR+DFO: 0.3±0.1, n≥3). KO of TfR in A549 cells yielded a low transfection efficiency (10%, Image J). Western blot of cell lysates did not show a significant reduction in TfR protein expression. Co‐transfection with HDR plasmid followed by puromycin selection increased transfection efficiency. However, there was increased cell death in TfR KO cells in culture (72 H, PI+ cells; mean pixel intensity (mpi), control: 15±0.2; GFP: 19±2, TfR KO: 49±42; n=3). Overexpression of TfR in BEAS2B cells yielded a higher transfection efficiency (~25%; Image J and RT‐PCR). Similar to TfR KO in A549 cells, after 72 H, there was a marked increase in cell death in TfR OE BEAS2B (PI+ cells (mpi), control: 18±2; GFP: 13±4, TfR OE: 83±20; n=3). TfR overexpression in normal lung cells possibly resulted in iron overload and ferroptosis. Our findings demonstrate that abnormal TfR expression and the associated changes in iron uptake induces death in normal and cancer cells, highlighting its importance in cell survival and proliferation. Understanding the role of TfR and iron in carcinogenesis will help develop potent therapeutic drugs to treat cancer, a disease that accounts for ~ 9 million deaths annually. Further, synthesizing novel analogs such as DMR‐tagg...