A family of monomodified bovine serum albumin (BSA) linked to methotrexate (MTX) through a variety of spacers was prepared. All analogues were found to be prodrugs having low MTX-inhibitory potencies toward dihydrofolate reductase in a cell-free system. The optimal conjugates regenerated their antiproliferative efficacies following entrance into cancerous glioma cell lines and were significantly superior to MTX in an insensitive glioma cell line. A BSA–MTX conjugate linked through a simple ethylene chain spacer, containing a single peptide bond located 8.7 Å distal to the protein back bone, and apart from the covalently linked MTX by about 12 Å, was most effective. The inclusion of an additional disulfide bond in the spacer neither enhanced nor reduced the killing potency of this analogue. Disrupting the native structure of the carrier protein in the conjugates significantly reduced their antiproliferative activity. In conclusion, we have engineered BSA–MTX prodrug analogues which undergo intracellular reactivation and facilitate antiproliferative activities following their entrance into glioma cells. Methotrexate (MTX) is an antiproliferative and immunosuppressive agent widely and effectively used against a broad spectrum of diseases, primarily cancerous tumors but also certain autoimmune diseases such as psoriasis and rheumatoid arthritis . It faciAs with most antimetabolites, MTX is only partially selective for tumor cells and is toxic to all rapidly dividing normal cells, such as those of the intestinal epithelium and bone marrow . AlthougOne of the approaches undertaken in recent years as an attempt to increase selectivity of chemotherapeutic agents was to link them covalently to macromolecules such as albumin. Extravasation of albumin is upregulated about six-fold in inflamed, diseased, and malignant tissues ,7. If thWe thus initially searched for a chemical procedure to covalently link MTX to amino-containing molecules through the γ-carboxylate moiety of MTX. The linkage of such molecules to the α-carboxylate of MTX was documented to yield inactive analogues ,10. SecoIn this study, we describe our synthetic procedures for obtaining a variety of BSA–MTX conjugates, their cell-free DHFR-inhibitory potencies in the absence and in the presence of glutathione or cysteine, and their antiproliferative efficacies in several lines of cancerous cells relative to that of methotrexate. Two glioma cell lines are investigated here, for the first time, in the context of albumin conjugates to align with other research in our lab, in which new compounds and methods that enable blood–brain barrier (BBB) opening, and thus, the entrance of large therapeutic molecules such as albumin are being developed ,16,17,18N,N′dicyclohexylcarbodiimide (DCC), β-nicotinamide adenosine dinucleotide 2′ phosphate (NADPH), reduced glutathione (GSH) oxidized glutathione (GSSG), and l-cysteine were purchased from Sigma . Methotrexate linked to a peptide (MTX–AYGRKKRRQRRR) was synthesized by the manual conventional solid-phase synthesis. An Fmoc (N-9-flurenylmethoxy carbonyl) strategy was employed through the peptide chain assembly. All other materials used in this study were of analytical grade.Methotrexate, bovine serum albumin, cystamine dihydrochloride, hexamethyl diamine-dihydrochloride, dihydrofolate (DHF), Methotrexate  dissolved in 0.7 mL dimethylformamide (DMF) and 95 μL from a solution of 1 M DCC in DMF (95 μM) was added. The reaction was carried out for 3 h at 25 °C. Dicyclohexylurea was filtered out. The MTX–anhydride formed was kept at 4 °C until used.2O and centrifuged. This procedure, which removes residual dicystamine and unreacted MTX, was repeated five times. The solid material was then lyophilized. MTX–COγNH–(CH2)3–S–S–(CH2)2–NH2 was obtained in 60% yield. It has a molar extinction coefficient similar to that of MTX . Calculated ESMS = 588 Da, found ESMS for M − H = 587.26 Da.Dicystamine di-HCl (100 μM) was dissolved in 0.7 mL DMF, neutralized with DIPEA, and combined with the solution of MTX–anhydride (150 μM in 0.7 mL DMF). The reaction mixture was stirred for 40 min, precipitated with ether, washed three times with ether, and desiccated. The solid material was suspended in HγNH(CH2)6–NH2] and MTX–glutathione [MTX–COγNH–GSSG] were obtained in 28 and 37% yields, respectively. Calculated ESMS for MTX–hexamethylamine is 552 Da, found for M + H = 553.41 Da, and M − H = 551.38 Da. Calculated ESMS for MTX–GSSG is 1048 Da, found M + H = 1049.4, and M − H = 1047.51.These derivatives were synthesized by the same procedure applied for MTX–dicystamine except that dicystamine was replaced with hexamethyldiamine-di-HCl or with GSSG. MTX–hexamethyl-amine , BSA–GSSG–MTX; [BSA–S–MAL–(CH2)3–CO–NH–GSSG–NHγCO–MTX] and BSA–(CH2)6–NHγCO–MTX; [BSA–S–MAL–(CH2)3–CONH–(CH2)6–NHγCO–MTX] , enabling introduction of MTX in a monomodified fashion, through a spacer maleimide (MAL) molecule linked to MTX through its γ-carboxylate moiety . Three such analogues were prepared: BSA–S–S–MTX; [BSA–S–MAL– at concentrations of 100 μM or lower 3–CONH–(CH2)6–NHCO–MTX, we incubated this derivative with 100 molar excess of GSH for a period of 7 h at pH 7.3, 37 °C. The conjugate was then dialyzed, lyophilized, and examined for its absorbance at 372 nm. As can be seen in 2)3–CONH–MTX did not undergo significant maleimide–thiol exchange following prolonged incubation with 100-fold molar excess of GSH. This suggests that the maleimide linked to cysteine 34 of BSA is not exposed to the external medium.Shen et al.  have dem50 of 2.4 µM with a large variation among different lines [50 (the effective concentration needed to obtain 50% of viable cells relative to untreated cells) of ~0.07 µM and 64% maximal effect  within 48 h. The second cell line, the human glioma U251, is relatively insensitive to MTX. It has EC50 of ~10 µM which is also the concentration needed to obtain a 49% maximum effect within 48 h of treatment (The documented inhibitory effects of MTX in a variety of glioma cell lines has an average ECnt lines . Followireatment .2)6–MTX, were found to be significantly more potent in the MTX-insensitive glioma cell line . Both analogues had negligible potency to inhibit DHFR in the cell-free system (2)6–MTX are highly potent. Thus, it appears that preservation of the native structure of the carrier protein is required for either entry into those cell lines and/or obtaining the appropriate fragmentation requiring to turn those “silent” analogues into cytotoxic-active species.We prepared two analogues of albumin–MTX in which the native structure of the carrier protein was disrupted, either by cleaving it at four sites with cyanogen bromide (CNBr-cleaved BSA–S–S–MTX) or derivatizing several of its amino side chains with MTX (HSA–MTXe system . We nexte system . This wa2)6–MTX, both in the sensitive and the insensitive glioma cell lines . Those were found to be inactive antiproliferative prodrugs which efficiently regained their cytotoxic efficacy following entrance into cancerous cells. The antiproliferative efficacy of these conjugates was studied in cancerous cell lines of brain glioma origin, to complement another activity of our laboratory aiming to transfer chemotherapeutically active macromolecules from the blood to the brain via the blood–brain barrier (BBB) for treating major brain disorders such as glioblastoma multiforme ,15,16,17The steps involved in preparing the appropriate BSA–MTX conjugates included the application of a chemical procedure enabling linkage of a variety of amino-containing nucleophiles (the spacers) to MTX with the preservation of a significant level of the DHFR-inhibitory potencies ; the linOf equal interest was to determine the efficacy of those glioma cell lines to regenerate the antiproliferative potencies of albumin–MTX analogues which lost their native three-dimensional configurations A,B.The superiority of the BSA–MTX conjugates over that of MTX in the insensitive human glioma cell line A is of p2)6–MTX showed essentially equipotent antiproliferative activity, both in the MTX-sensitive and the MTX-insensitive glioma cell lines  the inhibitory potency of MTX toward DHFR (2)6–MTX, which lacks this disulfide bond, shows equipotent potency as BSA–S–S–MTX (γNH–(CH2)6–NH2, an MTX derivative which preserves 69% (IC50 = 0.08 μM) of the DHFR-inhibitory potency of the native folate antagonist 3–CONH–MTX, which was incubated for a prolonged period at pH 7.3, 37 °C with high concentrations of reduced glutathione (GSH). We found that such exchange does not occur with this conjugate 6–NH2, covalently linked to carboxylate moieties, such as PEG40–COOH, undergo unusual MTX-dependent acid-dependent peptide bond cleavage, catalyzed by the α-carboxylate moiety of MTX [2)6–MTX at neutral pH value  MTX should be linked to the amino-containing spacer via its γ carboxylate moiety; (ii) the spacer should contain a readily cleavable bond in close proximity to the MTX; (iii) in agreement with previous studies ,41, the


This paper studied characterized the plasmonic effects of silver nanoparticles (Ag-NPs), the luminescent down-shifting of Eu-doped phosphor particles, and the metal-enhanced fluorescence (MEF) achieved by combining the two processes to enhance the conversion efficiency of silicon solar cells. We obtained measurements of photoluminescence (PL) and external quantum efficiency (EQE) at room temperature to determine whether the fluorescence emissions intensity of Eu-doped phosphor was enhanced or quenched by excitation induced via surface plasmon resonance (SPR). Overall, fluorescence intensity was enhanced when the fluorescence emission band was strongly coupled to the SPR band of Ag-NPs and the two particles were separated by a suitable distance. We observed a 1.125× increase in PL fluorescence intensity at a wavelength of 514 nm and a 7.05% improvement in EQE (from 57.96% to 62.05%) attributable to MEF effects. The combined effects led to a 26.02% increase in conversion efficiency (from 10.23% to 12.89%) in the cell with spacer/NPs/SOG-phosphors and a 22.09% increase (from 10.23% to 12.48%) in the cell with spacer/SOG-phosphors, compared to the bare solar cell. This corresponds to an impressive 0.85% increase in absolute efficiency (from 12.04% to 12.89%), compared to the cell with only spacer/SOG. Most efforts to further the development of silicon-based solar cells have focused on enhancing conversion efficiency and reducing overall costs ,2,3,4. Tg) of 1.1 eV. Photons with energy below Eg are not absorbed (transmission loss) and photons with energy exceeding Eg are absorbed; however, much of the excess energy is lost via thermalization loss by generated carriers [However, note that the theoretical maximum conversion efficiency of single-junction crystalline silicon solar cells is 31% ,15 due tcarriers ,17. Matecarriers ,19, downcarriers ,21, up ccarriers ,23, and carriers ,25. DC icarriers ,27. The carriers ,29,30.This paper systematically characterizes the effects of Ag-NPs, Eu-doped phosphor particles, and their combination in terms of absorbance, photoluminescence (PL), and optical reflectance spectra. These effects were also shown to enhance the efficiency of silicon solar cells, as confirmed by external quantum efficiency (EQE) response and photovoltaic current density–voltage (J-V) measurements under one-sun AM 1.5 G. MEF effects in this study achieved a 26.02% increase in efficiency and 0.85% increase in absolute efficiency.2 spacer layer was deposited on six clean quartz and silicon substrates. Films of Ag were deposited to a thickness of 3, 5, and 7 nm over the spacer layer using E-beam evaporation. Then, the samples were annealed at 200 °C for 30 min under ambient H2 to form Ag-NPs of various diameters on the spacer layer. Group 2: Three duplicate samples from Group 1 were coated (over the Ag-NP layer) with a SiO2 capping layer to a thickness of 74 nm using e-beam evaporation. The diameter and surface coverage of Ag-NPs of all samples in Group 1 were examined via scanning electron microscopy  in conjunction with Image-J software. We also examined the plasmonic effects ) produced by the Ag-NPs by measuring the absorbance spectra of test samples on a quartz substrate using a spectrometer  as well as the reflectance spectra using a UV/VIS/NIR spectrophotometer .We first characterized the plasmonic effects induced by Ag-NPs of various diameters by preparing two groups of test samples. Group 1: A 20-nm-thick SiO2 spacer layer was deposited to a thickness of 20 nm on the front surface of the bare solar cells. Ag films were deposited over the spacer layer to a thickness of 3, 5, or 7 nm and then subjected to annealing at 200 °C for 30 min under ambient H2 to form Ag-NPs of various diameters. The NP layer was coated with a SiO2 capping layer to a thickness of 74 nm using e-beam evaporation. The total thickness of the coatings on the substrate (spacer and capping layers) was 94 nm. Note that the Ag-NPs embedded in the coatings formed a plasmonic anti-reflection coating (ARC) for the silicon solar cells. 2 layer. The optical reflectance, EQE, and photovoltaic J-V of the cells were measured at each processing stage to confirm the occurrence of plasmonic effects and their influence on solar cell performance.The degree to which the plasmonic effects affected photovoltaic performance was assessed by coating planar silicon solar cells with Ag-NPs of various diameters. Bare planar solar cells were fabricated using the methods reported in a previous study ,27. A Si2  containing Eu-doped phosphor species  at a concentration of 3 wt %. Note that the coating solution was prepared by mixing 1.94 g of silicate solution with 0.06 g of various species of Eu-doped phosphors . The resulting SOG:Eu-doped phosphor solutions were spin-coated onto clean silicon substrates at 3000 rpm for 60 s and then baked at 200 °C for 30 min under a clean dry air atmosphere. The average diameter of the phosphor particles was 15 μm, and the coverage on the test samples was 8% [Next, we characterized the fluorescence performance of Eu-doped phosphors by coating clean silicon substrates with a silicate solution of SiOs was 8% . We exams was 8% .2 spacer layer was first deposited to a thickness of 20 nm on the front surface of bare silicon solar cells using E-beam evaporation. Then, SOG solutions containing Eu-doped phosphors  were spin-coated to a thickness of 200 nm over the SiO2 spacer layer. Then, the cells were baked at 200 °C for 30 min under a clean dry air atmosphere to remove organic solvents. The degree to which the luminescent down shifting affected photovoltaic performance was assessed by spin-coating planar silicon solar cells with an SOG layer containing Eu-doped phosphors (SOG:Eu-doped phosphor). Note that a SiO2 spacer layer was deposited on silicon substrates, over which we deposited a layer of Ag to a thickness of 3, 5, or 7 nm via E-beam evaporation. Then, the samples were annealed at 200 °C for 30 min under ambient H2 to form Ag-NPs. Finally, SOG solution diluted with ethanol at a ratio of 2:8 was mixed with Eu-doped phosphors  powder (3 wt %) and spin-coated over the Ag-NP layer at 3000 rpm for 60 s and baked at 200 °C for 30 min under an air atmosphere to remove organic solvent.We also characterized the effects of Eu-doped phosphor incorporated with Ag-NPs in terms of enhanced or quenched fluorescence emissions. A 20-nm-thick SiOEQE), and photovoltaic J-V of the cells at each processing stages were measured to confirm the effects of metal-enhanced fluorescence emissions on solar cell performance. Note that all reported data were averaged from three measurements. Overall, the diameter of the Ag-NPs was proportional to the thickness of the Ag films, and the distance between the Ag-NPs and Eu-doped phosphor particles was shown to affect (enhance or quench) the fluorescence emission characteristics, as estimated using PL measurements at room temperature. Our results guided the formulation of a model describing metal-enhanced fluorescence emissions.The LSPR induced by metallic nano-size particles provided impressive near-field light concentration and far-field scattering. The plasmonic resonance of NPs can be modified by adjusting the size, shape, constituent materials, and surrounding medium. 2 spacer/quartz-substrate and (b) SiO2-cap/Ag-NPs/SiO2-spacer/quartz-substrate. The broad absorption range (350 to 500 nm) can be attributed to the broad range of NP diameters (5 to 60 nm). Note that NPs of larger diameter produced peaks of higher intensity with a distinct red-shift in the peak wavelength. The absorption range and intensity were further enhanced by the SiO2 capping layer, which altered the refractive index of the medium surrounding the Ag-NPs from 1.0 (air) to 1.45 (SiO2).Excitation by light at a specific wavelength was shown to induce the collective oscillation of conduction electrons on Ag-NP surfaces, in a phenomenon known as LSPR. 2 spacer layer, cells with Ag-NPs of various diameter on a SiO2 spacer layer, and cells capped with an NP layer and SiO2 capping layer. For the sake of comparison, we also included a cell coated with a 94-nm-thick SiO2 spacer + capping layer . wR) of all cells calculated at wavelengths from 350 to 1000 nm. The reflectance of the cell coated with a SiO2 spacer layer was lower than that of the bare cell due to the anti-reflective property of the SiO2. The plasmonic effects of the Ag-NPs further reduced reflectance to below that of the cells with only a spacer layer. Note that the sharp drop in reflectance (at wavelengths of 350 to 500 nm) can be attributed to the SPR absorption of Ag-NPs. The drop in reflectance (at wavelengths of 550 to 1000 nm) can be attributed to the plasmonic forward scattering of incident photons by Ag-NPs. Our results revealed that samples with Ag-NPs of larger diameter presented lower reflectance due to an increase in SPR absorption. Increasing the diameter of the Ag-NPs was also shown to red-shift the trough in the reflectance spectrum, which is in good agreement with the absorbance spectra.2). For the sake of comparison, we also included a cell coated with a 94-nm-thick sample comprising a SiO2 spacer + capping layer . wEQE) of all cells calculated at wavelengths from 350 to 1100 nm. At wavelengths of 300 to 375 nm, the EQE values of the cell with only a spacer layer and the cell with a spacer/NPs exceeded those of the bare cell, due to the passivation effects of SiO2 and the lower reflectance of SiO2 capping layer. Cells with NPs without the capping layer presented EQE values slightly lower than those of the bare cell at wavelengths from 375 to 425 nm, due to the SPR absorption of the NPs. However, note that the EQE values were higher for incident photons with a wavelength exceeding 450 nm, due to an increase in the optical length of incident photons in the silicon active layer resulting from plasmonic forward scattering induced by Ag-NPs. The EQE values of cells with a spacer/NPs/capping layer were even higher due to the anti-reflection effects of the SiO2 capping layer. The EQE response values are in good agreement with the optical reflectance results. Taken together, it appears that the EQE response of solar cells can be enhanced by plasmonic effects induced by Ag-NPs of various diameters.2). oc) values of the cell with only the spacer and cells with spacer/NPs were 3 to 11 mV higher than that of the bare solar cell due to the passivation effects of SiO2 on the Si surface. The short-circuit current–density (Jsc) and conversion efficiency (η) values were as follows: bare solar cell , spacer only , spacer/NPs  , and spacer/NPs/capping layer  . The Jsc values corresponded to the EQE response values. The Jsc and η values of cells with spacer/NPs were higher than those of the cell with only a spacer layer, due to the plasmonic effects of the Ag-NPs. The Jsc and η values of cells with spacer/NPs/capping layer  were also higher than those of the cell with only a SiO2 ARC , which was due to the plasmonic effects of the Ag-NPs embedded in SiO2. The insets in sc was dependent on the plasmonic effect and diameter of Ag-NPs. Our results demonstrate that applying Ag-NPs (Ag: 7 nm) to induce plasmonic effects improves the conversion efficiency by 21.02% (from 10.23% to 12.38%) compared to bare solar cells.SC and η of the silicon solar cells. The wavelength of photons emitted from species-600 exceeded the wavelengths from species-550 and species-500. The responsivity of the silicon p-n diodes was approximately 0.15, 0.20, and 0.26 A/W at the peak emission wavelength of species-500, species-550, and species-600, respectively. Therefore, it is reasonable to expect that the EQE and η of solar cells containing phosphors of a longer emission-wavelength (species-600) would exceed those coated with phosphors of a shorter emission-wavelength .wR of all cells calculated at wavelengths from 350 to 1100 nm. The reflectance of a solar cell with spacer/SOG displayed antireflective characteristics, with the lowest reflectance at a wavelength of approximately 575 nm, due to destructive interference. The reflectance of the solar cells spacer/SOG-phosphors was lower than that of the bare solar cell across the entire range of wavelengths. The reflectance of cells spacer/SOG-phosphors was lower than that of the cell with spacer/SOG across wavelength ranges of 350 to 500 nm and 650 to 1000 nm. This can be attributed to the absorption of high energy photons (350 to 500 nm) by the Eu-doped phosphor particles, due to LDS effects and the forward scattering of low energy incident photons (at wavelengths of 650 to 1000 nm). Across a wavelength range of 515 to 650 nm, the reflectance of the cells with spacer/SOG-phosphors was higher than that of the cell with a spacer/SOG, due to weaker destructive interference. We expected that LDS effects and forward light scattering would further enhance the EQE response and photovoltaic performance of solar cells.2. The EQE responses of cells with spacer/SOG-phosphors exceeded that of the cell without phosphors. The EQE responses of all of the cells corresponded to the observed changes in optical reflectance. The EQE response of the cell with longer emission-wavelength phosphors (species-600) slightly exceeded that of the cells with shorter emission-wavelength phosphors (species-550 > species-500), which is in agreement with the responsivity of silicon as a function of incident photon wavelength. wEQE of all cells calculated at wavelengths from 350 to 1100 nm. These results indicate that the spacer/SOG-phosphors enhanced the EQE response of the solar cells while providing a corresponding increase in photovoltaic performance.sc values of the cells with spacer/SOG-phosphors exceeded those of the cell with spacer/SOG and the bare cell, due to LDS effects and forward light scattering. As shown in the inset of sc values increased following the inclusion of phosphor particles in the SOG layer as a function of phosphor species. The Jsc of the cell with longer emission-wavelength phosphors (species-600) exceeded that of cells with shorter emission-wavelength phosphors (species-550 > species-500), which is in good agreement with EQE response. Overall, η was proportional to Jsc. The highest η (12.54%) and Jsc (32.32 mA/cm2) values were obtained from the cell with species-600 phosphors, exceeding those of the cell with SOG  by 0.5% and the bare cell  by 2.3%. These results demonstrate that the inclusion of phosphors (species-600) increased conversion efficiency by 23.08% (from 10.23% to 12.54%), compared to the bare cell.2 spacer layer. Thus, if the plane of the SiO2 spacer layer was horizontal, then the height of larger Ag-NPs would exceed that of smaller NPs. Under these conditions, the distance between the NPs and phosphor in the SOG layer varied as a function of NP size, as follows: d1 (Ag: 3 nm) > d2 (Ag: 5 nm) > d3 (Ag: 7 nm). Note that an excessively small distance between particles in the two layers would cause the transmission of fluorescence energy loss from Eu-doped phosphor particles to Ag-NPs. wR of all cells calculated at wavelengths from 350 to 1000 nm. The wR values of cells with spacer/NPs/SOG-phosphors were lower than those of the cells with spacer/SOG-phosphors and spacer/SOG only. Note that this difference was particularly pronounced in samples with NPs of larger diameter.wEQE of all cells calculated at wavelengths from 350 to 1000 nm. The EQE of the cell with spacer/SOG was higher than that of the bare cell across the entire range of wavelengths, due to the antireflection effects of SiO2. The wEQE of the cell with spacer/NPs (3 nm) was higher than that of the cell with spacer/SOG-phosphors followed by the cell with spacer/SOG. EQE) spectra of cells with spacer/SOG-phosphors vs. the bare cell (ΔEQE1) as well as cells with spacer/NPs/SOG-phosphors vs. the cell with spacer/SOG-phosphors (ΔEQE2). The positive values for ΔEQE1 and ΔEQE2 over a wavelength range of 400 to 520 nm can be attributed to MEF effects, whereas negative values can be attributed to the quenching of fluorescence emissions. At wavelengths beyond 600 nm, the high ΔEQE1 and ΔEQE2 values can be attributed to the scattering of incident light by Ag-NPs and phosphor particles.sc of all evaluated cells illustrating LDS and MEF effects. The Jsc and η of cells with spacer/SOG  and spacer/SOG-phosphors  were higher than those of the bare cell , which was due to the anti-reflection effects of SOG and the combined LDS and light scattering effects of phosphor particles. The Jsc and η of cells with spacer/NPs/SOG-phosphors (NP: 3 nm)  were far higher than those of the cell with spacer/SOG-phosphors (without NPs), which was due to the MEF effects of phosphors induced by the SPR effects of Ag-NPs. sc and η values are in good agreement with the WEQE values, which was due to the fact that Jsc and η values are generally proportional to the EQE response of a solar cell. Note that the Jsc and η of cells with phosphors and larger NPs (5 and 7 nm) were less than those of similar cells with phosphors and smaller NPs (3 nm). This can be attributed to the quenching of fluorescence emissions due to the loss of fluorescence energy when the distance between phosphor particles and NPs was too small. The combined effects led to a 26.02% increase in η (from 10.23% to 12.89%) in the cell with spacer/NPs/SOG-phosphors and a 22.09% increase (from 10.23% to 12.48%) in the cell with spacer/SOG-phosphors, compared to the bare solar cell. This corresponds to an impressive 0.85% increase in absolute efficiency (from 12.04% to 12.89%), compared to the cell with only spacer/SOG.This study systematically analyzed the plasmonic effects of Ag-NPs, the LDS effects of Eu-doped phosphor particles, and the MEF effects obtained when the two are combined. We also examined the degree to which the various layers affect photovoltaic performance. Finally, we established an MEF model of Ag-NPs and Eu-doped phosphor particles based on measurements of PL spectra and EQE responses. A combination of Ag-NPs (Ag: 3 nm) and Eu-doped phosphor particles (species-500) produced the most pronounced MEF effects with a corresponding increase in photovoltaic performance. Compared to bare cells, we observed significant improvements in the conversion efficiency of silicon solar cells: plasmonic effects from Ag-NPs , LDS effects of SOG-phosphors , and MEF effects obtained by combining NPs with SOG-phosphors . This corresponds to an impressive 0.85% increase in absolute efficiency (from 12.04% to 12.89%) compared to the cell with only spacer/SOG.


Interestingly, only OS cells with low nicotinic acid phosphoribosyltransferase domain containing 1 (NAPRT1) RNA expression were sensitive to NAMPT inhibition. Using a publicly available ) and a previously published dataset, it was shown that in OS cell lines and primary tumors, low NAPRT1 RNA expression correlated with NAPRT1 methylation around the transcription start site. These results suggest that targeting NAMPT in osteosarcoma could be considered as a novel therapeutic strategy, where low NAPRT expression can serve as a biomarker for the selection of eligible patients.For osteosarcoma (OS), the most common primary malignant bone tumor, overall survival has hardly improved over the last four decades. Especially for metastatic OS, novel therapeutic targets are urgently needed. A hallmark of cancer is aberrant metabolism, which justifies targeting metabolic pathways as a promising therapeutic strategy. One of these metabolic pathways, the NAD+ synthesis pathway, can be considered as a potential target for OS treatment. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the classical salvage pathway for NAD+ synthesis, and NAMPT is overexpressed in OS. In this study, five OS cell lines were treated with the NAMPT inhibitor FK866, which was shown to decrease nuclei count in a 2D in vitro model without inducing caspase-driven apoptosis. The reduction in cell viability by FK866 was confirmed in a 3D model of OS cell lines ( Osteosarcoma is the most common high-grade malignant tumor of the bone, primarily diagnosed in children and adolescents . HistoloIn a recent study, a connection between NAMPT and osteogenic differentiation was discovered , in whicIn this study, it was discovered that cell viability of osteosarcoma cells decreased after treatment with the NAMPT inhibitor FK866, in both 2D cultured and 3D cultured cells. More importantly, only OS cells with a low expression of NAPRT1, and as such depending more on the classical salvage pathway for NAD+ synthesis, were sensitive to NAMPT inhibition. Low NAPRT1 expression in primary tumor samples of osteosarcoma correlated with the methylation of the NAPRT1 promotor. Therefore, low NAPRT expression could serve as a potential biomarker for the selection of osteosarcoma patients who could benefit from treatments that inhibit NAMPT.50 values ranging from 3.0 to 26.7 nM, except for MHM which showed very limited sensitivity to NAMPT inhibition. There was marked variability in sensitivity to NAMPT inhibition: in order to define a biomarker for the NAMPT inhibitor sensitivity of OS cell lines, RNA expression levels of NAMPT and NAPRT1 as well as the NAMPT/NAPRT1 ratio were analyzed in a panel of seven OS cell lines. NAMPT and NAPRT1 expressions were highly variable in OS cell lines, as were NAMPT/NAPRT1 expression ratios , medium (ZK58), or low (MHM) sensitivity to FK866. Although NAD+ itself reduced nuclei counts, NAD+ treatment rescued the inhibitory effect of the NAMPT inhibitor FK866 partially in OS cell lines 143B and ZK58 C. MHM ce50 of 292 nM  of the OS cell lines MHM, MG63, SAOS2 were generated and treated for 72 h with the NAMPT inhibitor FK866 to determine cell viability. 3D cultured OS cell lines were sensitive to the NAMPT inhibitor treatment A. The MHf 292 nM B. In ourf 292 nM C. Immunof 292 nM D,E, whicf 292 nM E. Howevef 292 nM D,E, wherA positive link between osteogenic differentiation in osteoblasts and NAMPT expression has recently been suggested . TherefoOS cell lines with low NAPRT1 expression exhibited the highest sensitivity to NAMPT inhibition. In addition, the low expression of NAPRT1 was previously found to be correlated with NAPRT1 promotor hypermethylation in chondrosarcomas . Thus, thttps://ocg.cancer.gov/programs/target (accessed on 22 February 2021).) initiative, comprising 86 osteosarcomas and includes data from whole genome RNA expression deep sequencing analyses and DNA methylation arrays. Methylation levels around the NAPRT1 transcription start site were determined; osteosarcomas showed variable levels of methylation around the transcription start site (2 = 0.317), but there was no correlation with parameters such as sex and the presence of metastasis (2 = 0.03)  (art site B. These art site . The mettastasis C. In con = 0.03) B,C.Despite the large number of next-generation sequencing studies identifying targets in osteosarcoma ,23,24 anWe investigated whether NAMPT inhibition affected nuclear count and cell viability in osteosarcoma cell lines by inhibiting apoptosis, or by attenuating cell proliferation. In the current study, it was shown that apoptosis was not induced in OS cells after treatment with FK866. To investigate cell proliferation, cell cycle analysis and Ki67 staining was performed on FK866-treated OS cells. Ki67 staining showed that the number of proliferating cells was reduced by FK866 in two out of three OS cell lines, suggesting that FK866 inhibited cell proliferation. Cell cycle analysis showed that the percentage of MG63 cells in the G2 phase decreased after FK866 treatment, which could suggest a G2 arrest. The observed increased percentage of cells in the S-phase suggests an arrest in the S-phase in the middle of DNA duplication, given the decrease of nuclei count observed after FK866 treatment. Interestingly, only MG63 showed changes in cell cycle progression; the two other FK866-sensitive OS cell lines, SAOS2 and ZK58, did not exhibit such changes. So far, it remains unclear whether the changes in the cell cycle observed in MG63 are an exception. The effect of FK866 is variable among OS cell lines, which is in line with previous studies in literature that have investigated the mechanism of how FK866 reduces cell viability. Previous studies have shown that FK866 does not have an effect on cell cycle distribution , or on tOsteosarcoma is characterized by tumor cells that produce an osteogenic matrix. As earlier studies suggested that NAMPT inhibits osteogenic differentiation in murine osteoblast and bone marrow-derived mesenchymal stem cells ,29, we iWe showed here for the first time that osteosarcoma cell lines are sensitive to NAD+ depletion by using NAMPT inhibition. In the sarcoma field, previous studies suggest that NAMPT inhibition could also be a potential novel therapeutic strategy in chondrosarcoma and Ewing sarcoma ,25. In cIn the current study NAMPT inhibition was tested in both 2D and 3D cultured OS cells. It was demonstrated that multi-cellular tumor spheroids have a lower sensitivity to FK866 as compared to 2D cultured cells. It was previously shown that tight intercellular contacts can cause MCTS to have poor drug penetration , which cDespite promising results from this study, early clinical trials that have used FK866 in the same concentration range as what we have used (1–10 ng/mL) , showed The status of NAPRT1 expression could be a good biomarker to predict sensitivity to NAMPT inhibitor treatment. We identified that low NAPRT1 expression correlated with high promotor methylation. Our results are in line with a previous study, which concluded that low NAPRT1 expression by promotor methylation could act as a predictive biomarker for NAMPT inhibition . The curw/v)) dissolved in DMEM, and were seeded in 1% agarose coated 96-well plates for seven days before the start of an experiment. All cells were cultured in a humidified incubator, with 5% CO2 and at 37 °C. Cell lines were regularly STR-profiled using the GenePrint 10 system kit  and tested for mycoplasm.For 2D cell culture, the osteosarcoma cell lines KPD, MHM, MG63, ZK58, SAOS2, U2OS, and 143B were cultured in RPMI 1640 medium , supplemented with 10% Fetal Bovine Serum (Gibco). Cell lines were retrieved from the EuroBoNet consortium . For 3D Cells were seeded and treated after 24 h (2D cell culture) or after seven days (3D cell culture) with 0.1% DMSO as a non-treated sample or FK866  in concentrations ranging from 0.1–1000 nM. For NAD+ rescue experiments, NAD+  was added at the same time as FK866 , with a concentration of 10, 50, or 100 µM. Dose range was determined based on a previous study . After 7MG63, SAOS2, and ZK58 cells were seeded in 6-well plates and treated for 48 h the next day with 5 (MG63 and SAOS2) or 60 nM (ZK58) of FK866, or 0.1% DMSO. Drug dose was defined as the IC75 determined in a 72 h exposure in 2D. Adherent cells were trypsinized and collected with supernatant. Cells were centrifuged, washed with PBS, and fixed in cold methanol for at least 20 min, as described in . Cells wWhole cell Hot-SDS lysates were obtained as described previously  for MG63Sections (4µm) of paraffin-embedded multicellular tumor spheroids were made; after deparaffinization and rehydration, these were used for immunohistochemical staining for Ki67  and cleaved caspase 3 (Cleaved caspase 3 (Asp175), 1:800, Cell Signaling). Antigen retrieval was performed by incubating sections in citrate buffer  for 10 min and cooling down for 2 h. Sections were incubated with primary antibody overnight at 4 °C. The next day, sections were incubated with the BrightVision one step detection system poly-HRP anti-mouse/rabbit  for 30 min at room temperature. Sections were washed with PBS and DAB+ Chromogen  was added to each slide for 10 min. Slides were counterstained with hematoxylin, dehydrated, and mounted. For quantification of the stained sections, the percentage of Ki67 or cleaved caspase 3 positive cells was determined using QuPath Software v.0.2.3 on three different sections on each slide .2 for osteogenic differentiation. One day after seeding, αMEM medium supplemented with 10% FBS was added, containing either 0.1% DMSO or FK866 (25 nM), with or without osteogenic differentiation compounds , dexamethasone , and ascorbate-2-phosphate ). Four or seven days after the addition of the osteogenic differentiation medium, cell viability was determined with Presto Blue. ALP activity was determined after four days of differentiation by lysing of cells with PBS/Triton 0.1% and incubating with pNPP  for 4 min. Absorption at 405 nm was measured using a microplate reader . ALP activity in mU was corrected for number of cells of each well. To assess mineralization, osteogenic differentiation was terminated after 21 days, and cells were stained with Alizarin Red S solution  in 60 mL water, pH 4.2) for 5 min. To determine the gene expression of osteogenic markers, RNA was harvested after four or seven days of differentiation using Trizol  according to the manufacturer’s instructions, followed by cDNA synthesis and reverse transcriptase quantitative PCR, described in detail in the next section.The OS cell line ZK58 was seeded at 5000 cells/cmALPL, COL1A1, SPARC, RUNX2, SPP1), NAMPT, NAPRT, or housekeeping genes that were found to show stable expression in mesenchymal tumors  .RNA was isolated from OS cell lines KPD, MHM, MG63, ZK58, SAOS2, U2OS, and 143B using Trizol according to the manufacturer’s instructions. cDNA synthesis was performed using iScript cDNA Synthesis Kit  according to the manufacturer’s instructions. For the RT-qPCR, iQ SYBR Green Supermix  and a Thermal Cycler (Bio-rad) were used with primers for osteogenic markers   48]. Ge. GeALPL,https://ocg.cancer.gov/programs/target (accessed on 22 February 2021)) initiative, phs000468, which include 86 osteosarcoma samples, were used in this study. Methylation data was obtained from osteosarcomas using Infinium 450 K Arrays. Probes within the complete genes, or 2000 bases around the transcription start site, of NAMPT and NAPRT1 were selected. For gene expression analysis, log2 (TPM) values of RNAseq data were used. All data used for this analysis are available on https://portal.gdc.cancer.gov/projects (accessed on 22 February 2021). For OS cell line methylation and gene expression analysis, methylation β-values and VSN-normalized gene expression data were obtained from a previously published dataset (GEO accession number GSE36002) [Methylation and gene expression data generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) (t-test was performed. For multiple comparisons between groups, a Kruskal–Wallis test was used. All statistical tests were performed on the GraphPad Prism 8.For statistical comparisons between two groups, a Student


In vitro cell culture practices are valuable techniques to understand the mechanisms behind vital in vivo biological processes. In vitro cells have helped us to attain a deeper understanding of functions and mechanisms conserved in the course of evolution. Toxicology studies are inevitable in drug discovery, pesticide development, and many other fields that directly interact with human beings. The proper involvement and regulatory steps that have been taken by animal ethical societies in different parts of the world resulted in the reduced in vivo use of mammals in toxicological studies. Nevertheless, experimental animals are being killed where no replacement is available. The use of mammals could be reduced by using the in vitro systems. Nowadays, invertebrate cell lines are also play important role in toxicology testing. This review analyzes the cause and consequence of insect in vitro models in toxicology studies. Unlike mammalian cells, insect cell lines utilize a phosphate buffering system Lynn, . The mosDrosophila (Baker and Thummel, D. melanogaster is one of the well-studied organisms in the animal kingdom. Cytogenetic research has led to the complete mapping and sequencing of its chromosomes, leading to their continuous use in biological and biomedical investigations (Gilbert, Drosophila led to the hypothesis that each gene controls a single enzyme—a concept that has proved fundamental to modern molecular biology (Ephrussi, in vivo alternatives to mammals. The insect models, D. melanogaster, Galleria mellonella, Bombyx mori, and Manduca sexta provided similar results that can be obtained with mammalian systems (Fuchs and Mylonakis, Among invertebrates, insects have been used as model organisms in biomedical research for a long time. The cornerstone insect widely used is Gilbert, . For insTrichoplusiani (Wang et al., G. mellonella has a great future in the field of toxicology and as a model for antibacterial drug testing (Cutuli et al., Oecophylla smaragdina. Likewise, environmental monitoring studies and screening of nanoparticles were conducted on many insect species such as D. melanogaster (Armstrong et al., Blaberus discoidalis (Zhou et al., B. mori (Pandiarajan and Krishnan, in vitro approaches, there are evident limitations. N- glycosylation in insect cell lines is not alike as seen in mammalian systems. Many insect species lack capabilities to elongate the core pentasaccharide Man3GlcNac2. It will be difficult if the recombinant protein require glycans for its maintenance. Likewise, “the cell density effect,” a sudden decline in the productivity and cell concentration is prominent in insect cell lines. Insect cell lines should be modified in a way that it meets all the requirements that a mammalian cell lines generally performs. Generating adequately modified insect cell lines may replace the existing mammalian cell lines.Established protocols for testing chemicals in insects are already available (Perry et al., in vivo models for toxicological studies. That could greatly replace many mammalian systems too. Insect in vitro models started gaining attention considering its less maintenance time and cost-effectiveness. Many insect cell lines offer several advantages over mammalian cell lines. Detailed studies are necessary to further reveal the great potential of invertebrate, especially insect in vitro system.The invertebrate models especially insects are established as successful SC, DA, and LD: wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with several of the authors DA and LD.


KRAS mutant cell lines were more resistant to MCL-1 inhibition. Conclusively, we find that CRC cell lines presented with distinct responses to BH3 mimetics that can in part be predicted by their CMS profile and KRAS/BRAF mutations. Overall, almost all CRC lines share sensitivity in the nanomolar range to combined MCL-1 and BCL-XL targeting suggesting that this would be the preferred approach to target these cancers. Colorectal cancer (CRC) is a heterogeneous disease, which in part explains the differential response to chemotherapy observed in the clinic. BH3 mimetics, which target anti-apoptotic BCL-2 family members, have shown potential in the treatment of hematological malignancies and offer promise for the treatment of solid tumors as well. To gain a comprehensive understanding of the response to BH3 mimetics in CRC and the underlying molecular factors predicting sensitivity, we screened a panel of CRC cell lines with four BH3 mimetics targeting distinct anti-apoptotic BCL-2 proteins. Treatment with compounds alone and in combination revealed potent efficacy of combined MCL-1 and BCL-XL inhibition in inducing CRC cell death, irrespective of molecular features. Importantly, expression of the anti-apoptotic protein target of BH3 mimetics on its own did not predict sensitivity. However, the analysis did identify consensus molecular subtype (CMS) specific response patterns, such as higher resistance to single and combined BCL-2 and MCL-1 inhibition in CMS2 cell lines. Furthermore, analysis of mutation status revealed that  WNT/MYC activation. CMS3 tumors exhibit metabolic activation and are enriched for KRAS mutations, whereas CMS4 is a poor-prognosis mesenchymal subtype, which is characterized by an increased stromal infiltrate and chemotherapy resistance.Despite significant advances in therapy, colorectal cancer (CRC) is still one of the leading causes of cancer-related deaths worldwide. CRC is characterized by high heterogeneity and unfavorable overall survival in late stage disease . Due to ClinicalTrials.gov Identifier: NCT03218683). ABT-263 (Navitoclax), which targets BCL-2, BCL-XL, and BCL-W, is FDA approved as well for combination therapy in hematological malignancies and is being studied in multiple clinical trials for several solid tumors . Of note, despite their remarkable efficacy in pre-clinical models, only six BH3 mimetics have entered clinical trials, partly due to on-target side-effects on platelets and hematopoietic cells. However, appropriate dosing strategies as well as targeted delivery in antibody-drug conjugate configurations that can overcome these toxicities are under investigation. Additionally, response to BH3 mimetics both in normal and cancer cells is also dependent on tissue origin and distinct molecular properties. Therefore, a better understanding of the anti-apoptotic dependencies in the context of the diverse molecular subtypes of CRC is needed to define the optimal strategy [The BCL-2 protein family is a cluster of proteins regulating the intrinsic apoptosis pathway, which can be sub-classified into three subgroups according to their corresponding structure and function. Firstly, BCL-2-associated X protein (BAX), Bcl-2 homologous antagonist/killer (BAK), and Bcl-2-related ovarian killer protein (BOK), the effectors of apoptosis, are able to form pore-like structures on the outer membrane of mitochondria and facilitate the release of cytochrome c to unleash apoptotic cell death . In the strategy ,13. KRAS/BRAF mutation status and sensitivity to BH3 mimetics. Overall, we observe that targeting BCL-XL and MCL-1 simultaneously has a dramatic synergy in CRC cell lines irrespective of CMS. This investigation provides more insight into the relationship between molecular phenotypes of CRCs and their sensitivity to BH3 mimetics.Herein, we tested four different BH3 mimetics targeting the different anti-apoptotic BCL-2 proteins in 19 CRC lines, which have been classified into four CMSs . Our datTo assess the sensitivity of CRC to BH3 mimetics, a panel of 19 CRC cell lines were treated with a titration of four BH3 mimetics. The cell lines represent the heterogeneity present in CRC and have previously been classified based on their molecular profile. First, we titrated single BH3 mimetics to determine the individual IC50 values per cell line . SubsequGenerally, in single treatments, ABT-263 exhibited a relatively higher efficiency in impairing the viability of the majority of CRC lines compared to the other three BH3 mimetics . This isNext, the relation between molecular features and BH3 mimetic sensitivity was analyzed. This revealed that CMS2 cell lines displayed increased resistance to ABT-199 and this was statistically significant when compared to CMS4 lines, while this trend was also observed in comparison to CMS1 and 3 F. IntereThe increased efficacy of ABT-263 in comparison to single inhibitors A-1155643 and ABT-199 indicated that CRCs utilize multiple anti-apoptotic family members to prevent the induction of apoptosis. Indeed, the analysis of distinct BH3 mimetic combinations revealed a dramatic synergy between combined BCL-XL and MCL-1 targeting with A-1155463/AZD5991 and/or ABT-263/AZD5991 . In agreBCL-XL and MCL1 mRNA expression was more abundant than BCL-2, which was consistent with the drug screening data showing higher dependency on BCL-XL and MCL-1 in CRC cell lines , a combination of BH3 mimetics is sufficient to activate caspase-3 and their efficacy is dampened by the pan-caspase inhibitor Q-VD-OPh, which suggests that caspase-dependent apoptotic cell death is the predominant pathway induced upon the BH3 mimetic treatment. However, as reported by Tait et al. [-1/BCL-2 . Moreove-1/BCL-2 . A simil-1/BCL-2  and mela-1/BCL-2 . Taken tKRAS, while the majority of CMS2 (4/6) cell lines have a KRAS mutation, which could explain the higher resistance to MCL-1 inhibition as suggested by the correlation analysis. Nevertheless, regardless of this basal resistance to MCL-1 inhibition, the combined inhibition of MCL-1 and BCL-XL also induces potent cell death in this subtype, as well.We have previously shown that CRC cell lines vary in their response to chemotherapy in vitro and in vivo, with CMS4 tumors showing increased resistance . In thisThe underlying reason for the observed difference between BH3 mimetic sensitivity of CMS4 lines in comparison to the reported resistance towards chemotherapy remains to be established. This is especially important to understand when related to the higher sensitivity of CMS2 tumors towards chemotherapy, while they present with lower sensitivity towards BH3 mimetics. This suggests that anti-apoptotic protein reliance on its own does not completely explain the difference in chemotherapy response between these subtypes. Obviously, chemotherapy sensitivity is defined by multiple aspects and does not only involve cell cycle speed, which is reportedly lower for CMS2 lines , but alsTo further address if the differential sensitivity to BH3 mimetics could be defined by the corresponding target expression, BCL-2, BCL-XL, and MCL-1 protein levels were defined, which revealed that the sensitivity is neither determined by the relative expression of the corresponding targets of BH3 mimetics nor the overall expression of all three anti-apoptotic BCL-2 members. Consistently, Smith et al. also indicated that the co-expression of related anti-apoptotic BCL-2 family proteins may limit the activity of ABT-199 in diffuse large B cell lymphoma despite having a high BCL-2 expression . On the In addition to CMS and BH3 mimetic target expression, we also assessed if the mutation status influences the BH3 mimetic response. This analysis revealed that CRC cell lines harboring KRAS mutations are less responsive to MCL-1 inhibition than the wild type lines, indicating that KRAS activation might be involved in resistance to apoptosis mediated by MCL-1. It has been reported by Okamoto et al. that CRC tumors with mutant KRAS are able to upregulate BCL-XL via the ERK pathway, which confers resistance to the proteasome inhibitor, Carfilzomib . HoweverAltogether, we show that CRC cell lines display differential response to BH3 mimetics, which in part relates to CMS and KRAS or BRAF mutation status. Our findings provide an in-depth overview of BCL-2 family expression and sensitivity in the context of several molecular features, which can guide future investigations to optimize the application of BH3 mimetics in CRC. Furthermore, our results emphasize the potent efficacy of combined MCL1 and BCL-XL inhibition for CRC therapy, regardless of pre-existing molecular features. 2.Nineteen colorectal cancer cell lines were kindly provided by Sanger Institute  and authenticated by STR profiling. Cell lines RKO, SW48, HT55, SW948, T84, CL-40, LS-180, CaR-1, HUTU-80, and OUMS-23 were maintained in Dulbecco’s modified Eagle’s medium/F-12 medium with L-glutamine, 15 mM HEPES  supplemented with 8% fetal bovine serum  and 50 units/mL of penicillin and streptomycin. HCT-116, KM12, RCM-1, SNU-C1, LS-1034, LS-513, COLO-320-HSR, MDST8, and NCI-H716 were maintained in RPMI 1640 with L-glutamine, 25 mM HEPES  supplemented with 8% fetal bovine serum, 50 units/mL of penicillin and streptomycin, 1% D-glucose solution plus , and 100 μM sodium pyruvate . All cells were cultured in a humidified atmosphere at 37 °C 5% COAll 19 CRC lines were classified into four CMSs by our previous work based on the consensus molecular pattern described in . The genABT-199, ABT-263, and Q-VD-OPh were purchased from Selleckchem, Houston, TX, US. A-1155463 and AZD5991 were purchased from Chemietek, Indianapolis, IN, US. All compounds were dissolved in dimethyl sulfoxide (DMSO) at a 10 mM stock concentration.® cell viability assay  was used to determine the cell viability by measuring mitochondrial respiration according to the manufacturer’s instruction. The relative viability was calculated by normalizing to the untreated control. Dose-response curves and IC50 values were generated and calculated on GraphPad Prism 8.0  with log(inhibitor) vs. normalized response—variable slope.To assess the sensitivity to four BH3 mimetics, CRC cells were plated into 96-well plates and treated with two BH3 mimetics in a matrix dilution at a time. After 48 h of treatment, the CellTiter-BlueFifty thousand cells were plated and treated with different BH3 mimetics. After 48 h, cells were trypsinized and harvested. CaspaTag caspase-3/7 in situ assay kit, Sulforhodamine  or propidium iodide  were used to label cells to detect activated caspase-3 or compromised cytoplasmic membrane, respectively. The percentage of positive cells were measured by flow cytometry on the CytoFLEX . According to , the BliThe RNA expression analysis of the CRC cell lines was performed as described in . BrieflyFor the Western blot analysis of BCL-2 family members, cells were lysed using the 1× RIPA lysis and extraction buffer  containing Halt protease and phosphatase inhibitor cocktail . Protein samples were quantified using the Pierce BCA protein assay kit  as per the manufacturer’s instructions. In addition, 20 µg of protein was loaded per well into 4–15% precast gels  and then transferred to PVDF membranes using the Trans-Blot Turbo transfer system  according to the manufacturer’s instructions using the mixed molecular weight transfer settings. Membranes were blocked for 1 h in 5% bovine serum albumin (BSA) in Tris-buffered saline and Tween-20  and stained with a primary antibody overnight at 4 °C. The following primary antibodies were tested: BCL-2 , BCL-XL , and MCL-1 , all diluted in 5% BSA in TBS-T. After washing the blots four times for 20 min each with TBS-T, the secondary antibody anti-rabbit-horseradish peroxidase  or anti-mouse-horseradish peroxidase  was added for 2 h at room temperature. Following another round of 4 × 20 min washes, the membranes were developed using the LumiLight Western blotting substrate  and imaged on the ImageQuant LAS4000 . Before proceeding with blotting for protein expression, the polyacrylamide gel was incubated for 5 min in an electrophoresis buffer containing 1% 2,2,2-Trichloroethanol  to allow for tryptophan visualization and thereby a comparison of the amount of protein loaded between the cell lines . The gelp < 0.05 was considered statistically significant. The correlation between IC50 values and target expression were analyzed by Spearman’s rank correlation. To analyze the correlation between the drug sensitivity and the phenotype of CRC cell lines, IC50 values were plotted and grouped based on their CMSs or mutation status. The original tables of IC50 values and corresponding mutation status can be found in


The poor ability of recognition and penetration of chemotherapeutic agents to tumor cells are still great challenges for targeted breast cancer treatment. Herein, we established a tumor-targeted nanostructured lipid carrier encapsulating gambogic acid (GA) and paclitaxel (PTX), which was co-modified with acid-cleavable folic acid (cFA) and a human-derived cell penetrating peptide dNP2 (CKIKKVKKKGRKKIKKVKKKGRK). The multi-functional nano-platform exhibited an enhanced targeting and penetrability to tumor tissues, which was accomplished by the combined action of cFA and dNP2. After intravenous injection, firstly, cFA could actively target the breast cancer tissues by the selective recognition of folate receptor (FR); then, upon arrival at the tumor microenvironment, the acid-cleavable FA and dNP2 dual modified nanostructured lipid carrier (cFA/dNP2-GA/PTX-NLC) exhibited sensitive cleavage of folic acid (FA), which could reduce the hindrance effect of FA to maximize the dNP2 cell-penetrating properties. The effect of different modification on cellular uptake, in vivo bio-distribution, and anticancer activity of NLCs proved our hypothesis that compared with NLCs modified by non-cleavable FA or a single ligand, cFA/dNP2-GA/PTX-NLC displayed more efficient intracellular delivery, stronger targeting ability in vivo, improved cytotoxicity on 4T1 cells, and produced the better therapeutic efficacy of GA and PTX. The strategy affords a feasible way to overcome the poor recognition and permeability of medicines in cancer treatment. Cancer is the world’s leading cause of death , especiaAs the second generation of a new lipid nano-carrier system, nanostructured lipid carriers (NLCs) have excellent biocompatibility compared with polymer-based nano carriers . MeanwhiCell-penetrating peptides (CPPs) can be cationic, amphiphilic, or hydrophobic, consisting of 30 or fewer amino acid . CPPs caTherefore, the dual modification of FA and dNP2 may show good targeting and penetrability to tumor tissues of breast cancer. In the tumor microenvironment (TME), FA could target the breast cancer cells by the specific binding FR. Nevertheless, for cell uptake, CPPs-mediated cell penetration is more effective than receptor-mediated endocytosis by the previous reports . So, theBased on this idea, we developed acid cleavage FA and dNP2 peptide co-modified NLCs (cFA/dNP2-GA/PTX-NLC) for the efficient delivery of GA and PTX into breast cancer cells, so as to achieve the therapeutic effect of the multifunctionality of GA and PTX.® 812) was purchased from Beijing Feng Li Jing Qi Trading Co., Ltd. China; LIPOID S-100 (Soybean Lecithin) was obtained from Shanghai Dong Shang Industrial Co., Ltd., China; DSPE-PEG2K-dNP2 ; Coumarin-6 (Cou-6) was acquired from Sigma ; 1,10-dioctadecyl-3,3,3,3-tetramethyl indotricarbocyanine iodide (DiR) was obtained from Biotium Inc. ; 0.25% trypsin + 0.02% EDTA, double antibiotics (10000U Penicillin streptomycin) and Fetal Bovine Serum (FBS) were acquired from Gibco; Cell Counting Kit 8 (CCK-8) was obtained from Dojindo Laboratories ; PBS, RMPI-1640 medium and 96-well white basal cell culture plate were obtained from Corning, NY; Hoechst 33342 fluorescent probes were acquired from Sigma-Aldrich ; Dulbecco’s modified eagle medium (DMEM) was acquired from Gibco . All the other chemicals with analytical reagent grades were acquired from Tianjin Guangfu Fine Chemical Research Institute .PTX with a purity of more than 98% was acquired from Shanghai yuanye Bio-Technology Co., Ltd. ; GA with a purity of more than 98% was purchased from Sichuan Weikeqi Bio-Tech Co. Ltd. ; Compritol 888 ATO (Glycerol behenate) was obtained from Gattefossé ; Myrj 52 (Polyethylene Glycol (40) Monostearate) was obtained from Saint-Priest Cedex, France; MCT 812 , cultivated in DMEM  including 1% penicillin–streptomycin and 10% FBS.MDA-MB-231 cells, 4T1 cells, and MCF7 cells were acquired from ATCC , cultivated in RPMI-1640 medium (89%) including 1% penicillin–streptomycin , and 10% FBS in a humidified atmosphere containing 5% COBALB/c nude mice  were purchased from Beijing huafukang biotechnology co., LTD and housed in a specific pathogen-free (SPF) environment with access to water and food ad libitum. Animals were used for the experiments after a weeklong acclimation period. All animal experiments were carried out according to the protocol, which was approved by the Ethics Committee of Tianjin University of traditional Chinese medicine (Ethics Code: TCM-LAEC2020024).The emulsification and solvent evaporation method was used to prepare NLCs as before ,23. To gZetasizer, Nano ZS  was used to determine the particle size, polydispersity index (PDI), and zeta potential (ZP) of the unmodified and modified GA/PTX-NLC. Transmission electron microscopy  was used to measure the particle morphology of the unmodified and modified GA/PTX-NLC.The ultrafiltration method and a validated ultra performance liquid chromatography (UPLC) method  were useWtotal and Wfree are the total weight of GA and PTX in the NLC and the weight of free GA and PTX, respectively.The EE (%) calculation formula is as follows:Total and (PTX + GA)Encapsulated are the total lipid weight of NLC and the total weight of GA and PTX in the NLC.The DL (%) calculation formula is as follows:The physical state of GA and PTX inside the different formulations (modified and unmodified NLC) was investigated by differential scanning calorimetry (DSC). DSC measurements were conducted in a thermal analyzer . The different samples were heated from 30 to 250 °C with a constant heating rate of 10 °C/min under a 50 mL/min nitrogen flow.The unmodified and modified GA/PTX-NLC were prepared in triplicate, stored in a refrigerator at 4 °C, and protected from light. Samples were taken at 0, 5, 10, and 15 days after the start, the appearance of the preparation was observed, and the particle size and distribution, the ZP, the EE, and the DL were measured to evaluate its stability.The stability of the unmodified and modified NLCs in the absence or presence of FBS was measured using a multi-functional microplate reader. The operation was as follows: the unmodified and modified NLCs were mixed with the same volume of FBS, oscillated slowly at 30 RPM, and incubated at 37 °C. At 0, 2, 4, 6, 8, 12, 24, and 48 h after incubation, 200 μL of the mixture of NLCs and FBS was placed in a 96-well plate, and each well transmittance was measured at 750 nm.Uptake of the unmodified and modified NLCs by 4T1 cells (FR-positive) , MDA-MB-4T1 cells, MDA-MB-231 cells, and MCF7 cells were seeded in 96-well flat clear-bottom black-wall microplates; after incubation for 24 h, the previous medium was replaced with fresh cell culture medium containing Cou-6-Sol, Cou-6-NLC, FA-Cou-6-NLC, dNP2-Cou-6-NLC, FA/dNP2-Cou-6-NLC, and cFA/dNP2-Cou-6-NLC with an equivalent concentration of Cou-6 at pH 7.4 for 4 h, 8 h. To evaluate the pH-cleavable FA, which could reduce the hindrance effect of FA to maximize the dNP2 cell-penetrating properties, the above cells were also incubated with cFA/dNP2-Cou-6-NLC for 4 h and 8 h at pH 6.8. Then, 50 μL RMPI-1640 medium containing Hoechst 33342 fluorescent probes (with a concentration of 1.67 μg/mL) was added. Cells were incubated for 30 min at 37 °C in the dark. The fluorescent images were acquired in each confocal scan. Columbus (PerkinElmer) was used to calculate the mean fluorescence intensity values of each image.To investigate the competitive inhibition of FA-Cou-6-NLC uptake via FR-mediated endocytosis, the 4T1 cells, MDA-MB-231 cells, and MCF7 cells were first incubated with free FA (1 mM) to block FR binding and then further treated with FA-Cou-6-NLC nanoparticles for 4 h and 8 h. In addition, the A549 cells (FR-negative) were also used in cell uptake studies.7 cells/mL. The mice were randomly divided into seven groups , while the tumor volume of mice grew up to 200 mm3 . The different NLC formulations were administered at a dose of 0.5 mg/kg of DiR. At 4, 8, 24, and 36 h after administration, mice were anesthetized with 4% chloral hydrate and then placed in an IVIS to observe the fluorescence intensity of each organ with excitation wavelength of 710 nm and emission wavelength of 790 nm. After 24 h, three mice in each group were sacrificed; the heart, lung, liver, spleen, kidney, and tumor were collected, and then, the fluorescence intensities of them were measured.The tumor targeting and dynamic distribution of different NLC formulations in BALB/c nude mice with 4T1 cells were studied using DiR as a fluorescence probe by an In Vivo Imaging System (IVIS). Briefly, the female BALB/c nude mice were subcutaneously injected with 0.1 mL of 4T1 cell suspension with a cell density of 2 × 104 cells/well and incubated for 24 h. GA/PTX-Sol, the unmodified and modified GA/PTX-NLC  were added and incubated for 24 h (pH 7.4). In addition, cells were treated with cFA/dNP2-GA/PTX-NLC at pH 6.8 to test whether the cytotoxicity of GA/PTX-NLC was promoted by the acid-cleavable FA. After incubation for 24 h, the previous culture medium was removed. Then, 100 μL 10% CCK-8 diluent was added and incubated for 3 h at 37 °C. A multi-functional microplate reader  was used to measure the absorbance of 450 nm wavelength. We used the following formula to calculate the cell inhibition rate .c, As, and Ab refer to the control wells, the experimental wells, and the absorbance of the blank wells, respectively.A CCK-8 kit was used to measure the cytotoxicity of different NLC formulations. 4T1 cells were seeded onto 96-well culture plates with a density of 0.8 × 10® assay kit  was conducted to measure the caspase-3/7 activity by the manufacturer’s instructions. First, 4T1 cells were seeded in 96-well microplates and incubated for 24 h. Then, GA/PTX-Sol, the unmodified and modified GA/PTX-NLC  were added and incubated for 24 h at pH 7.4. In addition, 4T1 cells were also treated with cFA/dNP2-GA/PTX-NLC at pH 6.8. Afterwards, the caspase-3/7 assay reagent of 100 μL was added into each well and incubated for 1 h in the dark. A microplate reader (SpectraMax M5) was used to measure the luminescence of each well. Caspase activity was expressed as fold of the untreated control.The Caspase-Glop value < 0.05 or p value < 0.01 or p value < 0.001 indicating significance.All values were represented as mean ± standard deviation (SD). IBM SPSS Statistics 22.0 software was used to analyze the statistical significance with the The sizes of the unmodified and modified GA/PTX-NLC were approximately 20 nm with PDI ≤ 0.25, and the ZP ranges −2.78 to −4.01 mV  as described previously [The cell uptake of various Cou-6-loaded NLCs and Cou-6-Sol using equivalent Cou-6 concentrations is shown in The cellular uptake was also evaluated in A549 cells using Cou-6-NLC and FA- Cou-6-NLC. The results showed that no significant difference was observed in cell intake between Cou-6-NLC and FA-Cou-6-NLC A, which p < 0.01, When cFA is scissored by the lower pH of the TME and activated to dNP2, the cellular uptake of cFA/dNP2-Cou-6-NLC is expected to be enhanced due to the significant penetration effect of dNP2. The 4T1 cells, MDA-MB-231 cells, and MCF7 cells were selected to investigate the uptake of cFA/dNP2-Cou-6-NLCs after lower the pH trigger cleavage. At pH 7.4, non-cleavable and cleavable Cou-6-NLC showed similar internalization, while the internalization ability of cFA/dNP2-Cou-6-NLC (pH 6.8) was significantly higher than that of cFA/dNP2-Cou-6-NLC (pH 7.4) and FA/dNP2-Cou-6-NLC (As shown in p < 0.05), probably owing to the tumor targeting effect of FA ligand. The cellular uptake results in vitro were consistent with the imaging results in vivo, showing that cFA/dNP2-DiR-NLC was superior to non-cleavable FA/dNP2-DiR-6-NLC and single ligand modified NLCs  was superior to dNP2-DiR-NLC (d) in tumor targeting  and PTX (0.25 μmol/L) exhibited the stronger synergistic anti-tumor efficacy . In thisp < 0.05). However, no significant difference was observed in other modified NLCs groups. The possible reason is that NLCs groups already have a high inhibition rate on cancer cells [The cytotoxicity of the unmodified and modified NLCs was also evaluated. The cytotoxicity of FA and/or dNP2 modified NLC were significantly higher than that of the unmodified NLC. The above result may be caused by the following reasons: Binding FA and/or dNP2 to the surface of NLC can enhance uptake by 4T1 cells, thereby increasing the concentration of GA and PTX in the cells, which will help to improve the therapeutic efficiency of tumor. The cytotoxicity of cFA/dNP2-GA/PTX-NLC was higher than FA-GA/PTX-NLC (er cells .The results of previous studies have shown that the combination of GA and PTX exhibited more apoptotic cells than the single drug in tumor tissues of mice, and the induction effect of GA and PTX encapsulated with NLC on the apoptotic death of cancer cells was significantly greater than that of solution . As caspTaken together, these results suggested that NLC enhances GA/PTX-induced 4T1 cell apoptosis, and the lower pH value could cleave cFA/dNP2 to form dNP2, which could further enhance cell uptake, thus increasing the concentration of GA and PTX in cells, eventually leading to increased apoptotic cells, which is consistent with the cellular uptake study and cytotoxicity study.We reported a multi-functional nano-platform co-modified by acid-cleavable FA ligand and dNP2 peptide to assist the delivery of GA and PTX to breast cancer. The cFA/dNP2 decoration could increase the cellular uptake and cytotoxicity of GA/PTX-NLC in breast cancer cells at pH 6.8. cFA/dNP2-GA/PTX-NLC displayed sensitive cleavage of FA in acidic TME and maximized the function of FA ligand and dNP2 peptide. Compared with NLCs modified by non-cleavable FA/dNP2 or a single ligand, cFA/dNP2-GA/PTX-NLC exhibited higher accumulation in breast cancer in vivo bio-distribution study, induced an enhancement of apoptosis, and achieved the maximal anticancer efficiency of GA and PTX by retaining the synergistic tumor targeting and penetration of FA and dNP2. This multi-target strategy has proven to be effective for targeted breast cancer therapy.


The schweinfurthin family of natural compounds exhibit a unique and potent differential cytotoxicity against a number of cancer cell lines and may reduce tumor growth in vivo. In some cell lines, such as SF-295 glioma cells, schweinfurthins elicit cytotoxicity at nanomolar concentrations. However, other cell lines, like A549 lung cancer cells, are resistant to schweinfurthin treatment up to micromolar concentrations. At this time, the precise mechanism of action and target for these compounds is unknown. Here, we employ RNA sequencing of cells treated with 50 nM schweinfurthin analog TTI-3066 for 6 and 24 h to elucidate potential mechanisms and pathways which may contribute to schweinfurthin sensitivity and resistance. The data was analyzed via an interaction model to observe differential behaviors between sensitive SF-295 and resistant A549 cell lines. We show that metabolic and stress-response pathways were differentially regulated in the sensitive SF-295 cell line as compared with the resistant A549 cell line. In contrast, A549 cell had significant alterations in response genes involved in translation and protein metabolism. Overall, there was a significant interaction effect for translational proteins, RNA metabolism, protein metabolism, and metabolic genes. Members of the Hedgehog pathway were differentially regulated in the resistant A549 cell line at both early and late time points, suggesting a potential mechanism of resistance. Indeed, when cotreated with the Smoothened inhibitor cyclopamine, A549 cells became more sensitive to schweinfurthin treatment. This study therefore identifies a key interplay with the Hedgehog pathway that modulates sensitivity to the schweinfurthin class of compounds. Macaranga schweinfurthii plant in 1987, members of the schweinfurthin family are prenylated stilbenes that show some activity as anticancer agents1. Many natural schweinfurthin family members have since been isolated and characterized, including schweinfurthins A-Q5. Because their extraction is difficult, with characteristically low yields and relative chemical instability, many synthetic schweinfurthin analogs have also been created10. These have been used to probe structure–activity relationships, improve cytotoxicity, increase stability, and provide fluorescent moieties to enable imaging studies12.Initially isolated from the 2. Notably, some cell lines are insensitive to schweinfurthins even at high (micromolar) concentrations, whereas others experience profound cytotoxicity at low nanomolar concentrations2. This highly differential activity suggests that schweinfurthins may exploit certain sensitivities to elicit cell death in cancer cells, while sparing other cells, possibly including normal tissue. Importantly, sensitive cell lines in the NCI-60 panel represent difficult-to-treat cancers, including triple negative breast cancers, melanomas, and gliomas2. Such cancers respond inadequately to current treatments and have poor prognoses for patients, necessitating the development of new therapeutics and novel treatment modalities. Understanding the anticancer mechanism of action of these compounds could therefore inform further drug development and may contribute to improved patient outcomes.Both natural and synthetic schweinfurthins exhibit a unique cytotoxicity profile in the NCI-60 panel of cell lines, suggesting a novel anticancer mechanism of action18. Schweinfurthins have been repeatedly shown to be highly toxic to some cell lines, such as SF-295 glioma cells, but well tolerated by others, including the A549 lung cancer cell line. Although little data is available in vivo, schweinfurthins reduce tumor growth in a Swarm rat chondrosarcoma model19 and cooperate with anti-PD-1 therapy to induce tumor regression in a murine B16.F10 xenograft model15. However, the mechanisms linking schweinfurthins to ultimate cell death are not known. The low concentrations required in sensitive cell lines suggest that their effects are mediated by a distinct molecular target(s) rather than a general biophysical disruption. Generally, the consensus of the data is that lipid membrane structures may be compromised by schweinfurthin treatment, though the cause of this is not yet clear.Despite many previous studies, the mechanism of action by which schweinfurthins elicit cytotoxicity is poorly understood, and a molecular target has not yet been identified. Thus far, these compounds have been implicated in: oxysterol binding protein activity, lipid profiles, disruption of the Golgi and endoplasmic reticulum, and cholesterol trafficking, among other processesHere, we have considered that analyses of the differential sensitivities of cell lines, such as the well-studied sensitive SF-295 line and the resistant A549 cell line, may provide insights into mechanisms of schweinfurthin action. We hypothesized that genetic or regulatory factors in cell lines contribute to differential schweinfurthin responses in sensitive and resistant cell lines, ultimately leading to survival or cell death outcomes. We therefore analyzed mRNA expression data from the NCI-60 panel of cell lines using sensitivity data from the schweinfurthin analog, methyl-schweinfurthin G (MG) and carried out RNA sequencing experiments to elucidate transcriptional responses to treatment with a novel synthetic schweinfurthin analog, TTI-3066. We carried out experiments at 6 and 24 h timepoints in both the sensitive SF-295 glioma cell line and the resistant A549 lung cancer cell line and compared responses to elucidate early and late transcriptional responses within these representative cell lines, providing key insights into potential mechanisms which contribute to resistance.MBD3.1, NOL8, and UBQLN1.2 were associated with schweinfurthin sensitivity, while TMEM185B, SYS1-DBNDD2, ZCHC1C, DMKN, SLC16A5, and SLC12A6 were associated with resistance. Importantly, all gene lists coincided well with each other, with each model including genes from the more parsimonious models before it.NCI-60 sensitivity data are available for cell lines treated with MG Fig. A, where OXTR and AMPD2, are most highly expressed in brain and secretory tissues, which tend to be highly sensitive to schweinfurthin treatment , data were available for both mutation status and GI50 for MG, and the mutated gene was identified in at least two tumor types. These requirements allowed for a reduction in the potential for confounding by tissue-type effects or insufficient cell line representation and resulted in the inclusion of 8 genes with driver mutations in our analysis . Cells were treated with 50 nM TTI-3066 . For the most parsimonious model , 6 of 9 genes were significantly different between SF-295 and A549, with one not profiled and one approaching significance , complex II (SDHD), complex III , and ATP synthase , suggesting that SF-295 cells are more reliant on these pathways in the absence of applied stressors. However, A549 cells had higher expression of a number of key genes in these pathways. These include IDH1 and IDH2, which may provide a method for the cells to balance NADP+/NADPH levels between the mitochondrial and cytosolic compartments. Similarly, A549 cells have a higher basal expression of SLC25A23, which encodes for a mitochondrial membrane solute carrier which may facilitate the transfer of metabolites between compartments. A number of complex IV genes  are also more highly expressed in A549.To develop an understanding of mRNA responses in sensitive and resistant cell lines, we employed RNAseq to compare treated and untreated cell lines after 6 or 24 h of incubation with TTI-3066, a synthetic schweinfurthin analog , alternative carbon source pathways , and p38 MAPK signaling ; bZIP transcriptional regulators were also significantly altered , and DHCR24, generally increased in response to TTI-3066 treatment, leading to significant changes in GO biological process pathway enrichment for those involved in metabolism, biosynthesis, and phosphate-containing organic substance pathways , exoribonuclease 1 (ERI1), and SLC12A2, which modulates sodium and chloride transport. Others, such as PDE4A, LIFR, PRKAA1, PLAA, MNT, and STAB2, had reduced expression in treated cells. By 24 h  and 24 h , indicating that SF-295 cells were attempting to increase metabolic activity to provide energy. On the other hand, negative interaction effects were observed for NDUFV1 and NDUFS4, suggesting a possible increased need for complex I activity in A549 cells. Such findings suggest that TTI-3066 differentially affects metabolism in these two cell lines, indicating a metabolic crisis and/or possible mitochondrial dysfunction. Importantly, the amyloid secretase and presenilin pathways, as well as phosphatidylinositol phosphorylation, are closely related to calcium signaling and/or inflammatory signals, which are themselves associated with mitochondrial dysfunction27.We analyzed interaction effects between SF-295 and A549 responses to TTI-3066 to illuminate potential genes involved in sensitivity and resistance to schweinfurthin treatment at both 6 h and 24 h  panel29, where they identified PLEKHO1, a negative regulator of PI3K/AKT signaling, as well as THEM4 and OSBP genes, as potential determinants of schweinfurthin response. Importantly, problems have been noted with the use of data sets such as the GDSC and CCLE (Cancer Cell Line Encyclopedia) due to variability between compound sensitivities and, to a lesser degree, gene expression results30. This may be due to differences in timing, cell handling, assay type, and other experimental factors between cell lines and treatment paradigms for these large-scale databases which may be attributed to the large scale of these studies. In contrast, the NCI-60 data presented herein is of a reasonable size for comparable treatment, and the compound response data was generated at the same time in all cell lines tested via the same method, enhancing reproducible conditions between cell lines.Since their initial isolation from extracts of the . Future studies will be necessary to further define the relationships between Hedgehog pathway signaling and schweinfurthin responses.Although gene expression may impact compound sensitivity, cancer cell responses to therapy may have more of an effect on sensitivity and resistance than baseline gene expression levels. Therefore, to elucidate the importance of cellular responses to treatment and sensitivity, we generated unbiased gene expression data via RNAseq for representative sensitive (SF-295) and resistant (A549) cell lines at 0, 6, and 24 h of treatment with a highly potent, novel schweinfurthin analog, TTI-3066. Comparisons between the two cell lines suggest that while they both exhibit extensive changes in gene expression, SF-295 cells were generally characterized by salvage and metabolic pathway responses, whereas A549 cells were more associated with changes in growth and differentiation associated pathways. Significant interaction effects were observed in a number of metabolic pathways at 24 h. Importantly, a sustained change in Hedgehog pathway gene expression was seen in TTI-3066 treated resistant cells at both 6 h and 24 h, suggesting a potential mechanism of resistance. Further experiments suggested that inhibition of this pathway with the Smoothened inhibitor, cyclopamine, significantly sensitized A549 cells to TTI-3066. Such findings were generalizable to sensitive cell lines and across multiple schweinfurthin analogs. These results are highly suggestive that Hedgehog pathway signaling provides a resistance mechanism for cancer cells under treatment with schweinfurthins, and thus cotreatment could enhance compound response rates in vitro and in vivoThis work underscores the validity of using large-scale genetic expression data as a method to identify sensitive cancer cells, mechanisms of drug action, and compound resistance behaviors in an in vitro context, with the potential to expand into in vivo models. Such methods enable unbiased isolation of potential contributor pathways which might not otherwise be selected for study. Such experiments are particularly useful in two areas: (1) the natural products field, where mechanisms are often unclear, and (2) compounds which generate unforeseen resistance responses in cell lines that are not associated with mutational changes. Additionally, gene expression responses may suggest repurposing of other compounds for the treatment of unrelated disorders or predict/explain unforeseen side effect profiles in in vivo models. Accordingly, we expect the availability, scope, and usage of such data sets to increase.2 and used within 20 passages.SF-295 cells were obtained from Addex Bio  and cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium  with 10% fetal bovine serum (FBS) added . A549 cells were obtained from ATCC  and were cultured in F-12 medium  with 10% FBS. All cells were maintained at 37 °C and 5% COMG and TTI-3066 was obtained from Terpenoid Therapeutics, Incorporated . They were dissolved in dimethylsulfoxide (DMSO) to 100 mM and stored in the dark at − 20 °C before use. This stock was then diluted to 10 mM immediately before dilution to final concentration in complete cell culture media, as above. Cyclopamine . RNAseq was performed by the Penn State Hershey Genome Sciences Core  to at least 25 million reads per sample on an Illumina NovaSeq 6000 . Experiments were carried out in triplicate.31. Aligned counts were normalized and formatted into log(counts per million (CPM)) and genes were filtered so that only genes with sufficiently large counts remained using the edgeR package as described previously32. Once the logCPM was calculated, principal component (PC) analysis was conducted using the limma package34 to investigate technical batch noise. This technique evaluates the overall agreeableness between replicates to ensure similar expression patterns between replicates. From the PC plots, we determined that one sample in each timepoint was sufficiently different from its technical counterparts to justify its exclusion from the analysis.Raw data was aligned to the human genome build 37 using the Rsubread package35 was used to evaluate differences in expression between conditions. We investigated baseline differences between SF-295 and A549, differences upon treatment in expression for each cell line, and the differences in differential expression between the cell lines upon treatment, also considered the interaction as seen in Eq. (1). Moderated t-statistics, moderated F-statistic, and log-odds of differential expression was calculated by the empirical Bayes moderation of standard errors as described by others37.The limma package39.To visualize the differences in gene expression between our cell lines, we utilized the circlize package[citation] in R to create heatmaps. Higher gene expression in SF-295 cells was visualized in red, while higher gene expression in A549 cells was shown in blue where absolute value affected intensity of color40. Briefly, cells were harvested in RIPA buffer with protease and phosphatase inhibitors and agitated for an hour at 4 °C before protein quantification with a Pierce BCA (bicinchoninic acid) assay. Equal amounts of protein were loaded on precast Bolt 4–12% Bis/Tris gels (Invitrogen) and run at 150 V for 45–60 min in Bolt MOPS(3-(N-morpholino)propanesulfonic acid) buffer before transfer onto a polyvinylidene fluoride membrane (Immobilon-P) in Bolt transfer buffer for 1.5 h at 25 V. Membranes were cut horizontally to enable high quality imaging with multiple antibodies per blot, blocked with 1 × ScanLater buffer, probed with primary antibodies overnight, rinsed with ScanLater wash buffer, and then probed with Eu-labeled (europium labeled) secondary antibodies  for 1 h before rinsing membranes and drying them. Dry membranes were imaged with a SpectraMax i3x with a Western blot cartridge (Molecular Devices). Running and transfer buffers were obtained from ThermoFisher Scientific , and blots were normalized to loading control, vinculin, and quantified with Licor ImageStudio Lite Version 5.2 software .Western blotting was carried out with the Molecular Devices ScanLater system , as described previouslyCells were plated in T175 flasks and allowed to grow overnight to 80% confluence before treatment. Cells were then treated with 50 nM TTI-3066 in standard growth media, as above, for 6 or 24 h. RNA was harvested on ice using RNeasy Mini Kit (Qiagen). Synthesis of cDNA from total RNA was performed following manufacturer’s instruction using superscript III reverse transcriptase (Invitrogen).−ΔCt method, whereas the mean fold change = 2 − (average ∆∆Ct) was assessed using the mean difference in the ∆Ct between the gene and the internal control. Genes assayed include: E2F1, STIL, SMO, GLI1, SUFU, CSNK1G2, PRKACB, SHH, PTCH1, BTRC, PTCH2, ZIC3, LRP2, IFT88, MTSS1, IFT52, RAB23, CSNK1G1, BNC1, CREBBP, FOXA2, GLI2, CSNK1E, GAS1, MYF5, GSK3B, CSNK1G3, DHH, HHIP, CSNK1D, STK36, DISP2, DISP1, PRKACA, ZIC2, ZIC1, FBXW11, BCL2, GLI3, CSNK1A1, PRKX, PRKY, PRKACG, and IHH. GAPDH was used as an internal control and was not significantly different for 6 and 24 h DMSO and TTI-3066 treatment across three replicates. Standard deviation of GAPDH for 6-h treatment was 0.4371 and 1.558 for DMSO and TTI-3066 respectively. Standard deviation of GAPDH for 24-h treatment was 0.3747 and 0.4102 for DMSO and TTI-3066 respectively. Statistical significance was assessed for all genes assayed using ΔCt values via two-way ANOVA with Bonferroni’s correction with GraphPad Prism 9 .Quantitative RT-PCR was performed to confirm expression of hedgehog pathway related genes using the Taqman Array, Human Hedgehog Fast 96-well (Applied Biosystems) system. Briefly the cDNA samples were combined with Taqman Gene Expression Master Mix (Applied Biosystems) and added to the appropriate wells of the plate. The thermal protocols followed were provided by Applied Biosystems and are available upon request. Relative gene expression was calculated using the 241. This analysis employed LASSO (least absolute shrinkage and selection operator) variable selection regression methodologies with cross-validation in the context of publicly available untreated log2 expression data from the NCI-60 panel and our results from initial NCI-60 screening. Due to changes in panel composition over time, only 57 cell lines were able to be compared. This regression varies a penalization term, lambda, to filter variables and discerns the performance of models across lambda. LASSO regression was employed because it is highly capable of identifying predictive genes and their relative contributions to the model. Results are presented in Fig. The National Cancer Institute (NCI) provides a service wherein potential anticancer compounds may be screened against a broad panel of cancer cell lines, known as the NCI-60 panel, to observe anticancer activity in a variety of paradigms. This panel represents a medley of well characterized cell lines of numerous tissue origins, including breast, lung, skin, central nervous system (CNS), renal, prostate, and non-solid tumors. These lines have been genetically characterized by DNA and RNA sequencing. We compared this publicly available RNA expression data to logGI50 (50% growth inhibitory) data from NCI-60 screening Fig. A to obse44 with the Fisher’s exact test. GO biological function, PANTHER pathway, and Reactome Pathways were queried. Interaction, glioma only, lung only, and baseline differences were examined at 6 h and 24 h with an adjusted p-value threshold of 0.05. Full pathway analysis results are included in Supplementary Table Pathway analysis was carried out using the Gene Ontology Consortium webtoolSupplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Legends.Supplementary Figures.