The novel amino-artemisinin derivative WHN-11 disrupts mitochondria and protein homeostasis, and induces autophagy and apoptosis in cancer cells

The novel amino-artemisinin derivative WHN-11 disrupts mitochondria and protein homeostasis, and induces autophagy and apoptosis in cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The novel amino-artemisinin derivative WHN-11 disrupts mitochondria and protein homeostasis, and induces autophagy and apoptosis in cancer cells Deborah Kajewole, Ho Ning Wong, Alexander Kriegsheim, Richard K. Haynes, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5315239/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Semi-synthetic derivatives of artemisinin exhibit anti-cancer activity in vitro and in vivo in addition to anti-malarial activity. Here, we report the anti-cancer and anti-cancer stem cell potential of novel C-10 substituted amino-artemisinin derivatives, among which the 4'-trifluoromethylarylurea piperazinyl derivative WHN-11 demonstrated consistent cytotoxic activity at high nanomolar concentrations across a range of cancer cell lines. WHN-11 reduced short- and long-term survival of triple-negative breast cancer (TNBC) cells, a highly aggressive breast cancer subtype that currently lacks standardized targeted treatments. Mechanistically, WHN-11 induced a stress response and increased proteasome-mediated turnover of ubiquitinated proteins. Significantly, WHN-11 promoted mitochondrial depolarization and fission, suppressing the expression of anti-apoptotic B-cell lymphoma extra-large (Bcl-xL) protein and ATP synthesis, thereby decreasing cellular energy production, and inducing apoptosis. WHN-11 treatment also increased autophagosomes, acidic vesicular organelles and lipid droplets, and promoted the dissociation of Bcl2-Beclin1 complexes. Activation or inhibition of autophagy synergized with the activity of WHN-11 in promoting cellular toxicity, as did increasing cellular dependence on oxidative phosphorylation. The effects of WHN-11 appear independent of substantial reactive oxygen species (ROS) production. Taken together, the data support ROS-independent mechanisms of anticancer action for WHN-11 and suggest that amino-artemisinins related to WHN-11 are promising candidates for anti-TNBC therapies targeting the mitochondria alone or in combination with autophagy modulators. Biological sciences/Cell biology Biological sciences/Cell biology/Autophagy Biological sciences/Cell biology/Cell death Biological sciences/Biochemistry/Proteins Biological sciences/Biochemistry/Proteomics Amino-artemisinin triple-negative breast cancer (TNBC) reactive oxygen species (ROS) apoptosis autophagy mitochondrial fission Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The antimalarial drug artemisinin is the active principle of the sweet wormwood Artemisia annua that has long been used in Traditional Chinese Medicine for its antipyretic properties [ 1 ]. Artemisinin and its synthetic derivatives (Supplementary Fig. 1) are promising candidates for repurposing for other clinical conditions such as autoimmune diseases and inflammation [ 2 – 5 ], parasitic infections in addition to those due to malaria [ 6 ], viral infections [ 6 – 9 ] and cancer [ 10 ]. Artemisinins are active against cancer cells in vitro and against tumours in vivo [ 11 – 18 ]. Artesunate has been submitted to clinical trials [ 19 , 20 ] although regression-free survival was not enhanced. Against tumour cells in vitro , the amino-artemisinin artemisone elicits activities superior to artemisinin [ 21 ] and in principle, because of relative lack of toxicity and enhanced pharmacokinetic properties including lack of metabolism to DHA, will be better suited for clinical use [ 22 ]. IC 50 activities of artemisinins in vitro range from 0.26–95.7 µM against a variety of tumour cell lines [ 19 , 20 , 23 – 25 ]. Combinations of artemisinins with known and developmental cancer drugs have been examined, largely with the view of enhancing activity of the latter through additivity or synergism, and thereby attenuating toxicity of the partner drug through use of lower amounts [ 26 , 27 ]. In this respect, artemisone shows additivity in its combinations with oxaliplatin and gemcitabine [ 21 ]. Activity of artemisone (IC 50 95.7 µM) towards A375 melanoma cells is markedly synergized in a 1:1 combination with the redox-active copper-(II) complex of the anticancer drug elesclomol [ 28 ]. The geranyl amino artemisinins WHN-296 and WHN-298 likewise act in synergy with the elesclomol-copper(II) complex against A375 melanoma cells [ 28 ]. The antitumour action of artemisinins may best be categorized as pleiotropic. Artemisinins are known to increase oxidative stress through generation of reactive oxygen species (ROS) that through downstream signalling effects elicited by the ROS overwhelm redox homeostasis in the cancer cell, and thereby induce cell-cycle arrest and apoptosis [ 21 , 25 , 29 – 32 ]. One potential pathway involves facile oxidation by the artemisinin of reduced flavin cofactors of disulfide reductases such as glutathione reductase (GR), thioredoxin reductase (TrxR) and others [ 32 – 34 ]. Generation of ROS is demonstrated during treatment of A375 melanoma cells with the geranyl piperazine derivative WHN-296 [ 28 ]. There is pronounced synergism of the amino-artemisinins with the redox active elesclomol-copper(II) complex, which is known to exert activity by generation of ROS [ 35 ]. However, mechanistic pathways evidently independent of ROS generation have also been reported [ 36 , 37 ]. Here, we report the anticancer activities of a distinct class of artemisinin derivative termed amino-artemisinins (Supplementary Figs. 1b and 2) against cancer cell lines including triple-negative breast cancer (TNBC) cells. TNBCs make up 15–20% of reported breast cancer cases, are highly aggressive, and lack effective standardised chemotherapeutic treatment regimens to date [ 38 – 42 ]. Amino-artemisinins, obtained from DHA by replacing the hydroxyl group at C-10 by an amino group (Supplementary Figs. 1b and 2) [ 43 , 44 ], have greatly enhanced efficacies against the malaria parasite and cancer cells [ 28 , 45 – 48 ]. These derivatives have improved pharmacokinetics [ 13 ], and generation of active metabolites with relatively long half-lives [ 47 ]. Using established artemisinins (Supplementary Fig. 1a) as controls, we identify the 4'-trifluoromethylarylurea piperazinyl derivative WHN-11 as an anticancer hit compound, with a mechanism of action targeting the mitochondria to induce autophagy and apoptosis independent of explicit ROS generation. 2. Materials and Methods 2.1. Compounds Reference compounds and the artemisinins used for screening were ≥95% pure as established previously [ 28 , 45 , 46 ]. The piperazinyl ureas WHN-10 and WHN-11, the piperazinyl amides WHN-14 and WHN-15[ 47 ] and the DHA-piperazine dimer WHN-27[ 48 ] were prepared and characterized as reported previously. Elesclomol, 98% pure, was obtained from Kaixuan Chemical Company, Changzhou, Jiangsu, China, and used as received [ 28 ]. For screening as described below, compounds were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 100 mM and stored at -20°C. 2.2. Cell Cultures The triple negative breast cancer cell lines HCC1937 (ATCC: CRL-2336), HCC70 (ATCC: CRL-2315), MDA-MB-231 (ATCC: HTB-26), the cervical carcinoma cell line HeLa (ATCC: CCL-2), and colon cancer cell line HCT116 (ATCC: CCL-247) were purchased from the ATCC. The glioblastoma U-87 (ATCC: HTB-14), 501mel melanoma (CVCL_4633) and HEK293T (ATCC: CRL-3216) cell lines were provided by Prof. Sharon Prince, Department of Human Biology, University of Cape Town, South Africa, and the MCF-12A (ATCC: CRL-10782) breast epithelial cell line was provided by Prof. Anna-Mart Engelbrecht, Department of Physiological Sciences, Stellenbosch University, South Africa. The culture conditions are reported in Supplementary Table 1. All cell lines were cultured at 37°C and 9% CO 2 and were confirmed to be mycoplasma-free by Hoechst 33342 staining. Cells used for experiments were between 10 and 30 passages. 2.3. Viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye For all cell lines other than the MDA-MB-231-line, viability was assessed based on the conversion of MTT dye into the insoluble formazan. Cells seeded overnight in 96-well plates (5 × 10 3 cells/well) were treated for 72 hours with increasing concentrations of the compounds or 0.1% (v/v) DMSO in culture medium. Thereafter, cells were incubated with 200 µg/mL MTT dye for 4 hours and an insoluble formazan was produced overnight with 10% (w/v) acidified SDS solution. Absorbance was measured at 595 nm and the half maximal inhibitory concentrations (IC 50 ) of the compounds were calculated by non-linear regression using GraphPad Prism version 4.00 (San Diego, CA, USA). 2.4. Viability assay using sulforhodamine B (SRB) dye For the cytotoxicity screening with the MDA-MB-231 cell line, SRB dye was used since the MTT assay gave poor results for this cell line. Cells seeded in 96-well plates (1 × 10 4 cells/well) overnight were treated as per the MTT assay. Subsequently, cells were fixed with cold 50% (w/v) aqueous trichloroacetic acid (TCA) for an hour at 4°C, washed in slow-running water and the plate air-dried. Fixed cells were stained with 0.04% (w/v) SRB in acetic acid for an hour, washed repeatedly with 1% (v/v) aqueous acetic acid to remove unbound dye and solubilized for 10 minutes with 10 mM Tris base solution (pH 10.5) on an orbital shaker. Absorbance was measured at 510 nm and the IC 50 values determined as described above. 2.5. Tumoursphere assay for cancer stem cell activity As previously described [ 49 ], HCC1937 cells were seeded as single cell suspensions into a 96-well Corning® Costar® Ultra-Low attachment multiple well plate (1 × 10 3 cells/well). Cultures were grown in anchorage-independent growth (AIG) medium consisting of DMEM supplemented with 1% (v/v) PSA, 2% (v/v) B-27 supplement, 1% (v/v) GlutaMAX, 20 ng/mL human epidermal growth factor (hEGF), 20 ng/mL basic fibroblast growth factor (bFGF), 4 ng/mL heparin and 10 µg/mL insulin. Cells were immediately treated with 0.1% (v/v) DMSO or 50 µM of each compound and incubated for 7 days. Treated cells were supplemented with freshly prepared AIG medium at 48-hour intervals. Tumourspheres formed were quantified at the end of the incubation period to calculate the percentage Tumoursphere Forming Efficiency (TFE): (average total number of tumoursphere formed/number of cells seeded) × 100%. 2.6. Clonogenic assay in treated HCC1937 cells Seeded cells (1.5 × 10 2 cells/well) were incubated for 6 hours, treated with 0.1% (v/v) DMSO, 5-fluorouracil (5-FU) or WHN-11 for 9 days and supplemented with fresh complete medium every 2 days. Colonies were fixed with methanol:acetic acid (3:1) and stained overnight with 1% (w/v) crystal violet in methanol. Excess stain was removed in slow-running water and the plate air-dried. Pictures of colonies were taken using a Molecular Imager ChemiDoc XRS + System (Bio-Rad, USA). To quantify the colonies formed, crystal violet-stained cells were solubilized with 1% (w/v) acidified-SDS for 4 hours at 37°C and absorbance measured at 570 nm. 2.7. SDS-PAGE and Western Blot Analysis According to the standard modifications of the Laemmli protocol [ 50 ], equal amounts of protein from treated whole cell lysates were resolved on a 12% SDS-PAGE gel and transferred to nitrocellulose membrane. Membranes were blocked for 1 hour in 1% (w/v) Tris-buffered saline (TBS) containing 1% (w/v) solution of non-fat powdered milk (Blotto). Thereafter, membranes were incubated overnight with specific primary antibodies in 0.1% (v/v) TBS-Tween-20 (TBST) containing 1% (w/v) Blotto at 4°C. Membranes were submitted to repeated washes with 0.1% TBST, prior to incubation with secondary antibodies and protein bands visualized by chemiluminescence using Clarity Western Enhanced chemiluminescence (ECL) substrate (Bio-Rad; 170–5061) on a Molecular Imager ChemiDoc XRS + System (Bio-Rad, USA). 2.8. Proteomic analysis of differentially expressed genes in WHN-11 treated HCC1937 cells by mass spectrometry HCC1937 cells treated with 0.1% (v/v) DMSO or 10 µM WHN-11 were harvested and lysed in PBS buffer (pH 7.4) containing 0.1% (v/v) Triton-X100 and 1 mM phenylmethansulfonyl fluoride (PMSF). Equal amounts of protein (~ 50 µg) were resuspended at a 5:1 ratio in digestion solution containing 5% (w/v) sodium deoxycholate (Sigma-Aldrich; D6750), 50 mM tris-(2-carboxyethyl)phosphine, hydrochloride (TCEP-HCl, Thermo Fisher Scientific; 20490) and 100 mM chloroacetamide (Sigma-Aldrich; C0267) for 5 minutes at 95°C. Lysates were digested at a 100:1 ratio with mass spectrometry (MS)-grade trypsin protease (Thermo Fisher Scientific; 90057) at 37°C overnight. Digested samples were incubated with 5% (v/v) trifluoroacetic acid (TFA, Thermo Fisher Scientific; 85183) in isopropanol and centrifuged at 12,000 ×g for 10 minutes at 4°C. The supernatants were transferred to styrene divinylbenzene polymer (SDB) spin-columns and centrifuged at 1,000 ×g for 5 minutes. Samples were first washed with isopropanol containing 1% (v/v) TFA and then with 0.2% (v/v) TFA in MS grade water. Mass spectra were collected and analysed on a Q-Exactive MS connected to an Ultimate Ultra 3000 chromatography system (Thermo Scientific, Germany) as previously described [ 51 ]. Gene set enrichment analysis (GSEA) using the c5.all.v7.5.1.symbols.gmt [Gene Ontology] gene sets database was conducted on the genes in the proteomic dataset comparing WHN-11 treated lysates to a DMSO-treated control. A thousand permutations were run, and the maximum and minimum gene set sizes set to 500 and 15, respectively [ 52 , 53 ]. 2.9. Indirect immunofluorescence staining for the detection of ubiquitinated proteins Cells grown on sterile coverslips (1 × 10 5 cells/well) were treated overnight with 0.1% (v/v) DMSO in culture medium or 10–50 µM WHN-11. Thereafter, cells were fixed briefly with ice-cold methanol and permeabilised with 0.1% (v/v) Triton-X100, blocked with 1% (w/v) bovine serum albumin (BSA) containing 1X TBS and incubated overnight with anti-ubiquitin antibody (Santa Cruz; sc-8017) at 4°C. Following incubation with Alexa-Fluor 488 anti-mouse secondary antibody (Abcam; ab15105) and Hoechst 33342, coverslips were mounted on slides with DAKO fluorescence mounting medium (DAKO; S3023). Images were captured using laser wavelengths of Ex 488nm /Em 562nm on a Zeiss LSM 780 confocal microscope and analysed using Zeiss Zen 2 (blue edition) software, version 2.0 (Carl Zeiss Microscopy, GmbH). 2.10. Chymotrypsin-like proteasome activity assay Cell pellets were lysed in proteasome lysis buffer (50 mM HEPES pH 7.8, 10 mM NaCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 250 mM sucrose and 5 mM DTT) for 20 minutes at 4°C. Lysates were sonicated, cleared by centrifugation, the supernatant collected, and equal amounts of protein (~ 16 µg) seeded into black-walled 96-well plates for proteasome assay. Lysate was incubated with compounds or DMSO control for an hour at 37°C in proteasome lysis buffer containing 2 mM ATP and 100 µM of N -succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin proteasome substrate (Suc-LLVY-AMC, Sigma-Aldrich; S6510). Fluorescence was measured at Ex 360nm /Em 460nm and the proteasome activity of each treatment was determined relative to the vehicle control. 2.11. Analysis of caspase activation for the detection of apoptosis HCC1937 cells (5 × 10 5 cells/well) seeded overnight were treated for 24 hours with 0.1% (v/v) DMSO, 5 µM KRIBB-11 or 1–50 µM WHN-11. Using Vybrant™ FAM poly caspases assay kit (Thermo Fisher Scientific, V35117), cells were pelleted, incubated in the dark at 37°C, 9% CO 2 with 1X FLICA reagent for an hour and briefly stained with 1.25 µg/mL propidium iodide. Fluorescence was detected on a BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA) using Ex 488nm and Em 530nm filters, and analysed with FlowJo v10.0.7 software (FlowJo, LLC). 2.12. Identification of Acidic Vesicular Organelles (AVOs) using Acridine Orange staining Cells were seeded (2 × 10 4 cells /well) in a 15µ-slide angiogenesis chamber (Ibidi GmbH, 81506) and treated for 4 hours with compounds. Live cells were briefly stained in the dark with 1 µg/mL Acridine Orange hemi (zinc chloride) salt (Sigma-Aldrich, A6014). Excess stain was removed by washing with PBS (pH 7.4) and coverslips mounted on slides with DAKO mounting medium. Images were captured using laser wavelengths of Ex 561nm /Em 637nm for red signals and Ex 488nm /Em 539nm for green signals on a Zeiss LSM 780 confocal microscope as described above. 2.13. Nile Red staining for lipid droplets Cells seeded in 15µ-slide angiogenesis chambers (Ibidi GmbH) were treated for 4 hours with compounds. Thereafter, cells were washed once with HBSS containing 20 mM HEPES pH 7.0 (HHBS). Live cells were stained in the dark for 30 minutes with 200 nM Nile Red dye in HHBS at room temperature. Excess stain was removed by washing with 1X HHBS and cells were mounted on slides with DAKO mounting medium. Images were captured for differential interference contrast (DIC) or Nile Red signals using laser wavelengths of Ex 488nm /Em 566nm on a Zeiss LSM 780 confocal microscope as described above. 2.14. Direct immunostaining for Bcl-xL protein expression Treated HCC1937 cells were harvested, fixed with 0.01% (v/v) formaldehyde in PBS, and resuspended in permeabilization (PERM) buffer containing 0.1% (v/v) Triton-X100 and 0.5% (w/v) BSA in PBS (pH 7.4) in the dark for 15 minutes. Cells were pelleted and subjected to immunostaining with PERM buffer containing 1 µg of mouse PE-conjugated IgG1 Kappa isotype control (eBiosciences, 12-4714-42) or PE-conjugated anti-Bcl-xL (H-5) antibody (Santa Cruz, sc-8392) for an hour at 4°C. Fluorescence was detected using a BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA) using laser wavelengths of Ex 480nm /Em 575nm to determine the expression of Bcl-xL protein, represented as the mean fluorescence intensity (MFI). 2.15. Detection of cellular ATP concentrations ATP levels in treated HCC1937 cells was assessed using the ATP Bioluminescence Assay Kit CLS II (Roche, 11-699-695-001). Cells were lysed at 4°C for 10 min in lysis buffer containing 50 mM Tris buffer, 5% (v/v) glycerol, 1 mM DTT, 60 mM MgCl 2 and 150 mM NaCl. ATP concentrations between 50 pM and 50 µM were used as standards. Equal volumes of lysate or standard samples and luciferase reagent were mixed in a 96-well plate and luminescence was measured. 2.16. JC-1 staining for mitochondrial membrane potential Treated HCC1937 cells were stained with 2.5 µg/mL JC-1 dye (Abcam; ab113850) for 30 minutes as previously described [ 54 ]. Harvested cells were thereafter analysed on a BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA). For each sample, 10,000 events were recorded using Ex 488nm /Em 529nm and Ex 488nm /Em 590nm for green and red signals, respectively. Carbonyl cyanide m -chlorophenyl hydrazone (CCCP) treatment was used as a control for loss of mitochondrial membrane potential. 2.17. Colocalization of TRAP1 with Bcl-xL in treated cells HCC1937 cells treated for 3 hours were fixed with 4% (w/v) aqueous PFA, permeabilised and incubated for an hour with 1% (w/v) BSA in 1X TBS. Following this, cells were incubated at 4°C overnight with primary anti-TRAP1 antibody (Thermo Fisher; MA1-010) in 0.1% BSA-TBST buffer (1:100) and for an hour with Alexa-Fluor 488 secondary anti-mouse antibody (Abcam; ab15105) in the dark. Stained cells were also incubated for 1.5 hours with 1 µg of PE-conjugated Bcl-xL (H-5) antibody (Santa Cruz; sc-8392) and Hoechst 33342 at 4°C and mounted on slides with DAKO mounting medium. Images were captured using laser wavelengths of Ex 488nm /Em 557nm for Alexa-Fluor 488 signals and Ex 561nm /Em 618nm for PE signals on a Zeiss LSM 780 confocal microscope as described above. 2.18. Cell survival assay for galactose-dependent mitochondrial function HCC1937 cells were seeded either in glucose-containing medium (as in the MTT assay) or in galactose-containing medium [RPMI-1640 (Thermo Fisher Scientific; 11879020) supplemented with 2 g/L galactose, 10% (v/v) FBS, 1% (v/v) 100 U/mL PSA, 1% (v/v) GlutaMAX ™ and 0.25% (v/v) sodium bicarbonate]. Thereafter, cells were treated with compounds and processed as previously described with the MTT assay to determine the IC 50 values. 2.19. Immunoprecipitation of Bcl2 proteins in treated HEK293T cells HEK293T cells were transiently co-transfected with 2 µg of purified pLV-eGFP (control; Addgene plasmid 36083) and 2 µg GFP-Bcl2 (Addgene plasmid 17999) or Beclin1-FLAG (Addgene plasmid 24388) for 48 hours. Thereafter, cells were treated for 3 hours with 0.1% (v/v) DMSO, HBSS, 5 µM colchicine or 20 µM WHN-11. Equal amount of protein from harvested lysate was incubated overnight at 4°C with 5 µg/mL magnetic anti-GFP conjugated beads (Abcam; ab193983). GFP-bound protein complexes were eluted and resolved on a 12% SDS-PAGE gel and subject to immunoblot analysis for GFP-Bcl2 and Beclin1 (FL) using anti-GFP and anti-FLAG antibodies, respectively. Densitometry analysis was conducted using ImageJ 1.51j8 NIH freeware. 2.20. Combination assay for the analysis of synergistic relationships between compounds Using the standard compound combination protocols [ 36 , 37 , 55 ], HCC1937 cells were treated at constant or non-constant ratios with the relevant compounds for 72 hours and subjected to MTT assay as previously described. Combination indexes (CI) were developed from constant ratio of combined compounds and algorithms in CompuSyn software, version 1.0 as previously described [ 56 ]. CI was defined as synergistic (CI 1) from a graph representing the CI versus Fa (fraction of cells affected). 3. Results 3.1. Amino-artemisinin derivatives exhibit anti-cancer and anti-cancer stem cell activity in vitro: selection of WHN-11 as hit compound. For the purpose of expanding the amino-artemisinin repertoire, we used our synthetic methodologies originally developed for preparing artemiside and artemisone (Supplementary Fig. 1b) from DHA[ 45 , 57 ] to provide the C-10 substituted piperazinyl derivatives (Supplementary Fig. 2) [ 28 , 47 , 48 ]. Details on preparation of these compounds is provided in the supplementary data. We established the half-maximal inhibitory concentrations (IC 50 ) of artemisinins against HCC1937, HCC70 and MDA-MB-231 TNBC cell lines, HeLa cervical carcinoma, U87 glioblastoma, 501mel melanoma and HCT116 colon cancer cell lines, as well as the non-cancerous non-transformed breast epithelial MCF-12A and human embryonic kidney HEK293T cell lines (Fig. 1 a; Supplementary Table 2). The amino-artemisinins exhibited increased toxicity to all cell lines compared to the parent compound artemisinin (Fig. 1 a; Supplementary Table 2). Both DHA and artesunate showed greater toxicities against all cell lines compared to artemisinin. Notably, compared to artemisinin, the WHN-series of amino-artemisinins exhibited IC 50 values in the low micromolar/high nanomolar range across all cell lines, of which WHN-11 was consistently the most toxic in all cell lines tested (Fig. 1 a). While all the artemisinins showed greater toxicity to the MCF-12A breast epithelial cell line and the HEK293T transformed embryonic kidney cell line, compared to the cancer cell lines, WHN-11 was substantially more toxic to all cancer cell lines except the U87 glioblastoma compared to the HEK293T cell line (SI values from 2.85–12.58; Supplementary Table 2). We next investigated the ability of artemisinins to inhibit cancer stem-like cell (CSC) activity. The ability of CSCs to both self-renew and differentiate has been linked to metastases and chemoresistance [ 58 , 59 ]. This was assessed in vitro through the formation of tumourspheres, which grow anchorage independently in serum-free, non-adherent growth conditions and are enriched in CD44 + /CD24 - stem-like cells, which express higher levels of Oct4 stem cell transcription factor [ 49 ]. DHA, artesunate, WHN-11 and WHN-298 inhibited tumoursphere formation in HCC1937 cells (Fig. 1 b). Based on these data, we selected WHN-11 as a putative hit compound for further mechanistic investigation. We next examined the ability of WHN-11 to block long-term survival in the clonogenic assay [ 60 ]. Both 5-fluorouracil (5-FU, positive control) and WHN-11 significantly decreased colony formation of HCC1937 cells in a dose-dependent manner (Fig. 1 c). We confirmed that WHN-11 was cytotoxic and non-cytostatic through increased activation of caspases (Fig. 1 d). Increasing concentrations of WHN-11 promoted the cleavage of both poly-ADP ribose polymerase-1 (PARP-1) and caspase-3 (Fig. 1 e) indicating induction of apoptosis, like the KRIBB11 and GA controls [ 61 , 62 ]. Artemisinins often exert activity through generation of reactive oxygen species (ROS) that through downstream signalling effects elicited by the ROS overwhelm redox homeostasis and induce cell-cycle arrest and apoptosis [ 20 , 21 , 29 , 32 , 38 – 41 ]. To identify and quantitate ROS production in HCC1937 cells treated with WHN-11, we used 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), which has been used previously to demonstrate ROS production by artemisinins [ 20 , 63 ]. While H 2 O 2 and artemisinin significantly induced ROS formation, WHN-11 did not induce significant levels of ROS compared to the vehicle treatment (Supplementary Fig. 3a). Using an alternative assay in which ROS detection is linked to activation of an antioxidant response element (ARE) reporter plasmid [ 64 ], we observed ROS production with the positive control elesclomol [ 35 ] but not WHN-11 (Supplementary Fig. 3). Additionally, inclusion of ROS scavenger, N -acetyl-L-cysteine (NAC) did not alter the toxicity of WHN-11, but did reverse the cytotoxic effects of elesclomol and artemisinin (Supplementary Fig. 3). Taken together, these data suggested that WHN-11 showed anticancer toxicity but did not induce significant formation of ROS in cancer cells. 3.2. WHN-11 treatment perturbs protein homeostasis in HCC1937 cells To understand the mode of action of WHN-11, we conducted an unbiased global analysis of the proteome of WHN-11 versus vehicle-treated HCC1937 cells by mass spectrometry [ 53 ]. A total of 3,629 unique proteins were identified, of which 51 proteins were significantly upregulated (> 2-fold) and 84 significantly downregulated (<-2 fold) in WHN-11-treated cells versus the vehicle treatment (Supplementary Fig. 4). Some significantly upregulated proteins included the death-associated protein 1 (DAP), ubiquitin-like modifier-activating enzyme (ATG7), interferon-stimulated gene 15 (ISG15), ubiquitin specific peptidase 24 (USP24) and E3 ubiquitin-protein ligase (HERC2). Significantly downregulated proteins included the cytochrome c oxidase assembly factor 7 (COA7), vacuolar protein sorting-associated protein 37B (VPS37B), Ras-related protein Rap-2B (RAP2B), growth arrest and DNA damage-inducible proteins-interacting protein 1 (GADD45GIP1) and cyclin-dependent kinase 4 (CDK4; Supplementary Material). Twelve of the upregulated proteins were identified only in the WHN-11-treated samples. Among these were ubiquitin carboxyl-terminal hydrolase 24 (USP24), profilin-2 (PFN2), ADP-ribosylation factor 6 (ARF6) and mas-related genes (MRG/MORF4L)-binding protein (MRGBP) (Supplementary Material). In addition, 18 downregulated proteins were detected only in DMSO-treated cells. These included the ATP-binding cassette sub-family B member 10 (ABCB10), nuclear factor 1 B-type (NF1B), growth arrest and DNA damage-inducible proteins-interacting protein 1 (GADD45GIP1) and tumour necrosis factor alpha-induced protein 2 (TNFAIP2; Supplementary Material). Gene set enrichment analysis [ 53 , 54 ] suggested that WHN-11 induced alterations in gene sets associated with development and morphogenesis, transmembrane transport, cell cycle, serine/threonine kinase activity and the Wnt signalling pathway (Fig. 2 a, Supplementary Material). Multiple gene sets involved in protein catabolism, ubiquitination, cellular responses to starvation and protein targeting to the lysosome were identified as significantly enriched (either positively or negatively) upon WHN-11 treatment (Fig. 2 a-c). Consistent with our biological assessment, we did not detect significant enrichment of gene sets associated with ROS production. The global proteomic analysis suggested WHN-11-induced imbalances in proteostasis, including ubiquitination and proteasomal degradation. Both processes occur during cellular stress induced by unfolded protein response (UPR) and/or heat shock response (HSR) [ 65 ]. To assess stress-induced protein accumulation in cancer cells as previously shown following artemisinin treatment in malaria [ 66 ], induction of protein ubiquitination by WHN-11 in HCC1937 cells was analysed in the absence or presence of the proteasome inhibitor MG132. Immunofluorescence (IF) staining of ubiquitin showed ubiquitinated proteins in the cytoplasm, which decreased in a dose dependent fashion on treatment with WHN-11, suggesting enhanced proteasome activity (Figs. 3 a-b). Inhibition of the proteasome with MG132 significantly accumulated ubiquitinated protein aggregates localized at the periphery and within the nucleus of co-treated cells. Higher concentrations of WHN-11 also increased cytoplasmic protein aggregates (Fig. 3 a). Artemisinin disrupts protein homeostasis and inhibits the malarial proteasome [ 66 ]. To test if WHN-11 inhibited human proteasomes, we assessed the ability of the compounds to directly inhibit the proteasome in whole cell lysates (Fig. 3 c). While MG132 treatment inhibited the chymotrypsin-like activity of the proteasome in a dose-dependent manner, WHN-11 did not significantly alter the proteasome activity compared to treatment with the vehicle (Fig. 3 c). However, when cells were treated overnight prior to assessment of proteasome activity in lysates, WHN-11 significantly promoted proteasome activity (Fig. 3 d). In combination experiments, WHN-11 and MG132 exhibited a strong synergistic relationship (Table 1 , Supplementary Fig. 5). Changes due to proteostatic stress were indicated by a significant increase in the stress-responsive Hsp70 mRNA levels in WHN-11-treated cells (Fig. 3 e). These data suggested that WHN-11 does not directly inhibit the proteasome, but that WHN-11 promotes protein turnover via the proteasome by a distinct mechanism [ 67 ]. Table 1 Combination of WHN-11 with inducers or activators of autophagy and proteostatic stress Compound Mechanism of action Average CI* when combined with WHN-11 Relationship MG132 Proteasome inhibitor 0.20 Strong synergism Chloroquine Inhibits lysosome acidification 0.19 Strong synergism Rapamycin mTOR inhibitor 0.34 Strong synergism Tunicamycin Glycosylation inhibitor 0.38 Strong synergism 3-Methyladenine PI3K inhibitor 0.58 Moderate synergism *Average combination index (CI) where half the population of cells are affected by the co-treatment with the compounds (Fa = 0.5). 3.4. WHN-11 induced autophagy in cancer cells Our findings that WHN-11-induced apoptosis and proteasome-independent regulation of protein turnover suggested autophagy as an alternative degradation pathway. This was supported by the gene set enrichment analysis which identified responses to starvation and protein targeting to the lysosome as enrichment processes (Supplementary Fig. 6). We previously observed the formation of vesicle-like structures in WHN-11 treated cells (data not shown), which indicated possible sites of early endosomal or autophagic vesicles, lipid, or other acidic vesicles [ 68 , 69 ]. Formation of acidic vesicular organelles (AVOs) in HCC1937 cells treated with WHN-11 were assessed using acridine orange (AO) dye and confocal microscopy with Hanks' Balanced Salt Solution (HBSS) used as a positive control for autophagy induction. WHN-11 significantly increased the formation of cytoplasmic AVOs (Figs. 4 a-b) and the number of cells with AVOs compared to treatment with DMSO (white arrows, Fig. 4 c). Although this suggested the possible induction by WHN-11 of formation of autophagosomes, other vesicle-like structures (yellow arrows) not coinciding with AVOs were visible (Fig. 4 a). Using Nile Red dye, treatment with WHN-11 and HBSS showed enlarged lipid droplets (LDs) in the cytosol and near the nuclear membranes, exhibiting substantial depth through treated cells, compared to the vehicle treatment (Fig. 4 d). LDs are specialized compartments for fatty acid products after lysosomal degradation [ 70 ] and are formed during the lipidation of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3B) [ 71 ]. We subsequently established that WHN-11 indeed promoted the lipidation of LC3B-I, which was promoted by co-treatment with chloroquine (CQ) (Fig. 5 a and b). WHN-11, like the controls HBSS and colchicine, significantly decreased the association between GFP-Bcl2 and Beclin1-FLAG in transiently transfected HEK293T cells relative to the cells treated with vehicle (Fig. 5 c) indicating autophagy induction [ 72 ]. Increased autophagic flux by WHN-11 was confirmed in HeLa cells stably expressing mCherry-EGFP-LC3 by confocal analysis. Increased yellow puncta arising via the overlap of green-EGFP and red-mCherry signals indicated the formation of autophagosomes [ 73 ] like the controls HBSS and Baf-A1 (Supplementary Fig. 6). WHN-11 also showed strong synergy with the autophagy inducers rapamycin, tunicamycin and the autophagy inhibitor CQ and moderate synergism with autophagy inhibitor 3-methyladenine (3-MA) (Table 1 ). 3.5. WHN-11 induces mitochondrial dysfunction WHN-11 induced both autophagy (as indicated by LC3B-I lipidation) and apoptosis (as indicated by caspase-3 cleavage) only after a prolonged exposure of 16 hours (Fig. 5 b), suggesting that other molecular events may precede cell death. The mitochondrial Bcl2 family of proteins drives apoptosis [ 74 ]. Prior to mitochondrial-induced (intrinsic) apoptosis, mitochondrial membrane potential ΔΨm changes, ATP levels drop and Bcl2 levels change [ 75 ]. WHN-11 significantly decreased the expression of anti-apoptotic Bcl-xL like the positive control KRIBB-11 (Fig. 6 a), which confirms the apoptotic response to WHN-11 and links its mechanism of action to the mitochondrion. Consistent with these observations, WHN-11 reduced cellular ATP in a dose-dependent manner (Fig. 6 b). The mitochondrial membrane potential ΔΨm enhances the transfer of protons between the matrix and intermembrane space (IMS) during ATP synthesis [ 76 ]. A loss in ΔΨm causes mitochondrial outer membrane permeabilization (MOMP), a non-reversible apoptotic stage involving the release of essential proteins within the mitochondrial IMS [ 77 ]. Changes in the ΔΨm were determined using the membrane permeable JC-1 dye [ 78 ]. WHN-11 mediated a significant loss in ΔΨm (Fig. 6 c), consistent with the positive control carbonyl cyanide m -chlorophenylhydrazone (CCCP). Consistent with the flow cytometric analysis (Fig. 6 a), immunofluorescence staining indicated that Bcl-xL protein levels were reduced by treatment with WHN-11 in a dose-dependent manner compared with treatment by the vehicle (Fig. 6 d). TRAP1 staining was consistent with mitochondrial localisation and appeared as a reticular network. WHN-11 did not alter TRAP1 staining intensity, but did induce distinct morphological changes in mitochondria, which appeared fragmented compared to the elongated tubular network in the control cells (Figs. 6 d i and ii). To confirm the mitotoxic role of WHN-11, we assessed WHN-11 toxicity in cells grown in either glucose- or galactose-containing medium. In the presence of high glucose concentrations, cancer cells produce ATP via glycolysis and suppress oxidative phosphorylation in a process known as the Crabtree effect. The Crabtree effect is reversed by replacing glucose with galactose to promote ATP production via oxidative phosphorylation (OXPHOS) via the mitochondria [ 79 ]. Increased sensitivity of cells to compounds in the presence of galactose indicates mitotoxicity [ 80 ]. Galactose significantly increased the toxicity of WHN-11 (GAL-IC 50 = 0.19 ± 0.07 µM, GLU-IC 50 = 0.74 ± 0.40 µM). In contrast, the toxicity of artemisinin was not significantly different (GAL-IC 50 = 218.00 ± 80.69 µM, GLU-IC 50 = 389.33 ± 92.70 µM; Table 2 ). This confirmed that WHN-11 targets the mitochondria to induce toxicity. The WHN-11 treatment-induced mitochondrial dysfunction may explain the inability to detect significant ROS production upon treatment with the compound (Supplementary Fig. 3). Table 2 IC 50 values of artemisinins in HCC1937 cells in glucose- and galactose-containing medium. Compound IC 50 (µM) ± SD (n = 3) Glucose Galactose Artemisinin 389.33 ± 92.70 218.00 ± 80.69 (ns) WHN-11 0.74 ± 0.40 0.19 ± 0.07 (*) Statistical significance of IC 50 values in + galactose samples relative to + glucose samples was determined by a two-tailed student’s t-test, where * p < 0.05 and ns - not significant. 4. Discussion Studies on the development of novel therapies and the identification of alternative agents and cellular targets to address the lack of standardised treatment for TNBC are ongoing [ 81 ]. The anticancer properties of artemisinins are well studied [ 21 – 27 , 29 , 31 – 35 ]. Many artemisinin derivatives comprising artemisinin hybrids connected to different pharmacophores [ 82 ], artemisinin dimers and others elicit enhanced activities against tumour cells [ 83 – 86 ]. The amino-artemisinin WHN-11 is structurally unique in incorporating a nitrogen atom attached directly to C-10 which is attached by a piperazine linker to a p -trifluoromethylaryl urea group (Supplementary Fig. 2). WHN-11 exhibited the highest cytotoxicity (IC 50 0.19 ± 1.52 µM) among the amino-artemisinins against TNBC cell lines compared to the parent compound artemisinin (202.90 ± 1.30 µM). To date, the mechanisms of action of artemisinin and its derivatives is still a topic of significant debate; the predominant view of the mechanism of action of artemisinins is the induction of ROS in cancer cells [ 21 , 29 , 32 ]. Indeed, the artemisinins used clinically for treatment of malaria, namely artemisinin [ 87 ], DHA [ 63 ], artemether[ 88 ] and artesunate[ 36 ] (Supplementary Fig. 1a) induce formation of ROS in a range of cancer cells, leading to growth inhibition. However, it is important to note that artemisinin has also been shown to be specific for ROS-induction by malaria and yeast mitochondria and not mammalian mitochondria [ 89 ]. Furthermore, artemisinin and its derivatives can induce cell death independent of ROS generation[ 90 ] and artemisinin derivatives have been linked to perturbations in protein homeostasis. DHA-mediated cell death in malaria parasites involves protein damage and proteasome inhibition, which activates ER-stress related cell death [ 66 ]. In contrast to the effects of DHA in malaria [ 66 ], WHN-11 did not directly inhibit the proteasome but rather promoted a modest but significant increase in proteosome activity in treated TNBC cells coinciding with increased ubiquitinated protein turnover; overall this is consistent with increased autophagy. Therefore, it is likely that artemisinin derivatives can induce cell death via multiple mechanisms dependent on the context. For WHN-11, we have no evidence of significant ROS induction using a range of biological assays, including those previously used to demonstrate ROS production by artemisinins. While there is the possibility that this is due to technical reasons in detection of ROS, it may also be explained by the observed mitochondrial dysfunction. Many artemisinins induce apoptotic cell death through mitochondrial dysfunction in cancer cells that is not exclusively associated with ROS production [ 46 , 47 ]. WHN-11 resulted in mitochondrial fission, mitochondrial outer membrane permeabilization (MOMP), loss of ATP production and down-regulation of the anti-apoptotic mitochondrial protein Bcl-XL culminating in apoptosis. The toxicity of WHN-11 was potentiated by reliance of cells on OXPHOS for ATP production, supporting mitochondrial defects induced by WHN-11 as a main cause of toxicity. Mitochondrial fission is a known response to stress and is linked to apoptosis, both of which were induced by WHN-11 treatment. The reduced Bcl-XL levels induced by treatment with WHN-11 must culminate in release of Bax from inhibitory Bcl-XL-Bax complexes, allowing free Bax to promote mitochondrial fission linked to apoptosis [ 91 , 92 ]. Indeed, artesunate induced Bax-mediated apoptosis in HepG2 cells, which was similarly independent of ROS production [ 47 ]. The role of induction of autophagy by WHN-11 as a cell death mechanism is less clear, even though other artemisinins also induce autophagy and resistance to artemisinin therapy is linked to autophagy in the malaria parasite [ 93 ]. Like WHN-11, DHA activated major apoptotic and autophagy-related proteins, and suppressed the interaction between Bcl2 and Beclin1, promoting cell death in KKU-213 cholangiocarcinoma cells [ 94 ]. In normal cells, autophagy is considered a pro-survival response which is upregulated in response to stress, particularly starvation, to provide nutrients to sustain cell survival. In cancer cells, autophagy is linked to both tumour killing and tumour survival, although the mechanisms by which this occurs are not well defined. In the case of WHN-11, it is probable that autophagy is activated as a response to mitochondrial dysfunction to circumvent or prevent apoptosis. The lipid accumulation observed upon treatment with WHN-11 can arise due to a lack of OXPHOS resulting from mitochondrial defects and which subsequently triggers autophagy [ 95 ]. Mitochondria and MOMP are important in autophagy activation [ 96 ], although this process is usually in response to oxidative stress and associated with ROS production [ 97 ]. In the case of WHN-11 however, in the absence of any substantive evidence supporting ROS production, we assume that ROS-independent changes to the mitochondria may trigger autophagy and subsequently cell death by apoptosis. There is a growing appreciation of the role of autophagy modulators, both activators and inhibitors, in potentiating the anti-cancer activity of several chemotherapeutic agents for clinical benefit [ 98 ]. Therefore, the next steps require an evaluation of drug combinations of WHN-11 and structurally related amino-artemisinin derivatives with known modulators of autophagy like temsirolimus or everolimus in the drive to develop these compounds as viable chemotherapeutic options, in particular for treatment of TNBC. 5. Conclusions We report the promising anti-cancer activity of a novel amino-artemisinin derivative WHN-11 against multiple cell lines, including the aggressive TNBC subtype. Unlike most other artemisinin derivatives, WHN-11 anti-cancer activity was independent of substantive ROS production. Rather, WHN-11 altered cellular protein homeostasis pathways to induce mitochondrial fission and dysfunction, depriving the cell of ATP, activating autophagy and subsequent cell death by apoptosis. The anticancer effect of WHN-11 was synergistic with inducers of autophagy, suggesting that future combination treatment with modulators of autophagy will have therapeutic potential. Declarations Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The views reflected are those of the authors and should not be attributed to the funders. Author Contribution Conceptualization, A.L.E, J.d.l.M, and D.K.; formal analysis, D.K, A.L.E, J.d.l.M; investigation, D.K, H.N.W and A.v.K; resources, R.K.H and A.v.K; data curation, D.K, A.v.K, and A.L.E; writing—original draft preparation, D.K, A.L.E. and R.K.H; writing—review and editing, J.d.l.M and A.v.K; visualization, D.K and A.L.E; supervision, A.L.E, J.d.l.M and R.K.H; project admin-istration, A.L.E; funding acquisition, A.L.E and R.K.H. All authors have read and agreed to the published version of the manuscript. Acknowledgement We thank our colleagues Heinrich Hoppe, Demetra Mavri-Damelin, Sharon Prince, Anna-Mart Engelbrecht and Earl Prinsloo for providing reagents and conducting useful discussions. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Hsu, E. Reflections on the discovery of the antimalarial qinghao. Br. J. Clin. 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Supplementary Files Kajewoleetalsuppdataforsubmission.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 07 Feb, 2025 Reviewers agreed at journal 07 Jan, 2025 Reviews received at journal 04 Jan, 2025 Reviewers agreed at journal 04 Jan, 2025 Reviews received at journal 27 Nov, 2024 Reviewers agreed at journal 14 Nov, 2024 Reviewers agreed at journal 11 Nov, 2024 Reviewers invited by journal 11 Nov, 2024 Editor assigned by journal 11 Nov, 2024 Editor invited by journal 07 Nov, 2024 Submission checks completed at journal 06 Nov, 2024 First submitted to journal 22 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5315239","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":399012150,"identity":"5e4383fd-3aed-42b4-9e3f-214f37fcacb1","order_by":0,"name":"Deborah Kajewole","email":"","orcid":"","institution":"Rhodes University","correspondingAuthor":false,"prefix":"","firstName":"Deborah","middleName":"","lastName":"Kajewole","suffix":""},{"id":399012151,"identity":"61c8d44b-3d30-4f0f-abdd-09155d236979","order_by":1,"name":"Ho Ning Wong","email":"","orcid":"","institution":"Rural Health Research Institute, Charles Sturt University","correspondingAuthor":false,"prefix":"","firstName":"Ho","middleName":"Ning","lastName":"Wong","suffix":""},{"id":399012152,"identity":"f5553f2d-c105-4100-a367-a8f0bcf6dba9","order_by":2,"name":"Alexander Kriegsheim","email":"","orcid":"","institution":"University of Edinburgh","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Kriegsheim","suffix":""},{"id":399012153,"identity":"1aad5f7c-8b64-47c8-8372-d5072eb022ba","order_by":3,"name":"Richard K. Haynes","email":"","orcid":"","institution":"Rural Health Research Institute, Charles Sturt University","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"K.","lastName":"Haynes","suffix":""},{"id":399012154,"identity":"b3cbc488-15d0-4e29-95a9-71cbac261f2b","order_by":4,"name":"Jo-Anne Mare","email":"","orcid":"","institution":"Rhodes University","correspondingAuthor":false,"prefix":"","firstName":"Jo-Anne","middleName":"","lastName":"Mare","suffix":""},{"id":399012155,"identity":"27b95c97-cd2a-4a94-9568-2ec4a359f0d9","order_by":5,"name":"Adrienne Lesley Edkins","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIie2QsQrCMBRFXwlkimRNQdpfeFIoFIT8isXVoR/QIeCaD+hn1MU5EqhLcS862MXJWRwUrJtb2s0hZ7i84R4uPACP508JFQLw6UqopilDoBlb54ocuqq4yOTcIjxLkNw1JwxdZzXe8v1pg4FugAjnnGFpeEW7SluGMFNAwaXEhj++ikw0w+CtgMUuBQ2jYY02qBlDMqwI5x8WliZZhTavWlrYeSNw4VKi47bv9MtKrsmuv5dLGbkUID/3UBauvsfj8XjG8AFOAjpx0QfCfgAAAABJRU5ErkJggg==","orcid":"","institution":"Rhodes University","correspondingAuthor":true,"prefix":"","firstName":"Adrienne","middleName":"Lesley","lastName":"Edkins","suffix":""}],"badges":[],"createdAt":"2024-10-23 03:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5315239/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5315239/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-05284-7","type":"published","date":"2025-07-01T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75038441,"identity":"1b115cfa-0604-4031-accd-355dbb1f1565","added_by":"auto","created_at":"2025-01-29 17:51:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2796731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel artemisinin derivatives exhibit anti-cancer and anti-cancer stem cell activity \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Heatmap showing the IC\u003csub\u003e50\u003c/sub\u003e values (in µM) of control and novel artemisinin compounds screened against non-cancerous epithelial breast cell line: MCF-12A; TNBC cell lines: HCC1937, HCC70, MDA-MB-231; non-breast cancer cell lines: HeLa, U87, 501 melanoma and HCT116 cell lines; and transformed HEK293T cell line. ART - artemisinin, DHA - dihydroartemisinin, AM - artemether, AS - artesunate, ND - not done. (\u003cstrong\u003eb\u003c/strong\u003e) Percentage tumoursphere forming efficiency (%TFE) of cells cultured in AIG media and treated at a single-point concentration of each compound (50 µM) or DMSO for 7 days; % TFE = [total number of tumoursphere formed / number of cells seeded] × 100%. Data shown as averages ± SEM (n =3), where * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 and ns - not significant using a two-tailed student’s t-test comparing treatments to the DMSO control. Representative images show tumourspheres at Day 0 or 7 for DMSO and WHN-11. (\u003cstrong\u003ec\u003c/strong\u003e) Representative images of colonies in a clonogenic assay for treated HCC1937 cells cultured over 9 days. Following solubilization, average absorbance at 570 nm (A570) ± SEM (n = 3) was taken. Statistical significance was assessed by one-way ANOVA with Tukey's multiple comparison test, *** p \u0026lt; 0.001 and ns - not significant. 5FU: 5-fluorouracil (\u003cstrong\u003ed\u003c/strong\u003e) Caspase activation in treated HCC1937 cells using Vybrant™ FAM Poly Caspase Assay Kit. Data shown are averages ± SEM of MFI (n = 3), where * p \u0026lt; 0.05, *** p \u0026lt; 0.001 and ns - not significant by a two-tailed student’s t-test. (\u003cstrong\u003ee\u003c/strong\u003e) Western blot analysis of PARP-1 and caspase-3 activation in treated cells, tubulin used as a loading control. GA: geldanamycin (see Supplementary blot figure F1 for full length blots).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/9b2c400573c581966127429d.png"},{"id":75038436,"identity":"32245ada-6fc9-433d-98c4-ccc54a11cad1","added_by":"auto","created_at":"2025-01-29 17:51:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6430083,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWHN-11 induces changes in cellular proteome associated with protein turnover. \u003c/strong\u003eGene set enrichment analysis (GSEA) of proteomic changes associated with WHN-11 treatment. (\u003cstrong\u003ea\u003c/strong\u003e) Significantly positively (top 20) and negatively (top 20) enriched gene sets in WHN-11 versus DMSO treated cells. (\u003cstrong\u003eb\u003c/strong\u003e) Gene set enrichment plots of genes associated with response to starvation, targeting to lysosome and protein catabolism. (\u003cstrong\u003ec\u003c/strong\u003e) Leading edge analysis identifying gene clusters associated with protein catabolism, ubiquitination, lysosome, mitochondrion, and cellular starvation. Red shading indicates gene sets or genes enriched in WHN-11 lysates compared to the DMSO lysates, while blue shading indicates genes enriched in the DMSO treated lysates relative to WHN-11.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/98e1a549fdf28f3f61a5ef48.png"},{"id":75038758,"identity":"c782f7b5-dc1a-452f-9ccb-01835e84785c","added_by":"auto","created_at":"2025-01-29 17:59:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6092754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWHN-11 promotes protein ubiquitination and turnover in HCC1937 cells.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Confocal analysis of ubiquitin staining in untreated cells or cells treated with 2 µM MG132. Representative images shown (n = 3). (\u003cstrong\u003eb\u003c/strong\u003e) Mean of the Corrected Total Cell Fluorescence. Data represent averages ± SD (n = 3), where ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 and ns - not significant using two-Way ANOVA, Bonferroni post-test. (\u003cstrong\u003ec\u003c/strong\u003e) Direct chymotrypsin-like proteasome inhibition activity assay in cell lysates. (\u003cstrong\u003ed\u003c/strong\u003e) Proteasome activity after overnight pre-treatment of live cells prior to lysate preparation. Data shown are averages ± SEM (n = 3), where *** p \u0026lt; 0.001 and ns - not significant by One-way ANOVA using Bonferroni's multiple comparison test. (\u003cstrong\u003ee\u003c/strong\u003e) Quantitative analysis of the mRNA copy number of Hsp70 in treated HCC1937 cells. Data shown are averages ± SEM (n = 3), where * p \u0026lt; 0.05, ** p \u0026lt; 0.01 and ns - not significant using a two-tailed student´s t-test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/c531d1c42478e400ac7af0b4.png"},{"id":75038759,"identity":"124c9bb5-41b5-4921-b088-47c14069029c","added_by":"auto","created_at":"2025-01-29 17:59:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4793451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWHN-11 promotes formation of autophagic vesicles in HCC1937 cells\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Acidic vesicular organelles (AVOs, white arrows) detected with acridine orange dye in cells treated for 4 hours with 0.1% DMSO, 10 µM WHN-11 or Hanks' Balanced Salt Solution (HBSS). Yellow arrows indicate unstained vesicles. Quantitation of (\u003cstrong\u003eb\u003c/strong\u003e) fluorescence intensity of AVOs and (\u003cstrong\u003ec\u003c/strong\u003e) number of cells with AVOs. Data represent averages ± SEM (n = 3), where * p \u0026lt; 0.05, ** p \u0026lt; 0.01 and *** p \u0026lt; 0.001 by one-way ANOVA comparing treated cells to DMSO control. (\u003cstrong\u003ed\u003c/strong\u003e) Lipid droplets (white arrows) stained with Nile Red dye in cells treated for 4 hours with 0.1% DMSO, HBSS or 10 µM WHN-11. Z depth panel shows 3D z-stack of equivalent depth. Representative images shown (n = 3).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/0878dbd91d2e98cfc0c9bfb8.png"},{"id":75038451,"identity":"b73911cc-54c1-495a-a938-02f4cddaab97","added_by":"auto","created_at":"2025-01-29 17:51:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3085103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of apoptotic and autophagic signalling pathways in WHN-11-treated HCC1937 cells\u003c/strong\u003e. (\u003cstrong\u003ea\u003c/strong\u003e) Western blot analysis of LC3B lipidation in cells treated overnight with 10 µM WHN-11 and for 4 hours with 2 µM chloroquine (CQ). Actin was used as a loading control. Ratios indicated are normalized to the untreated control. Representative blot shown (n = 2). See supplementary blot figure F2 for full length blots. (\u003cstrong\u003eb\u003c/strong\u003e) Time-dependent treatment of cells with 0.1% DMSO or WHN-11 for caspase-3 and LC3B activation. Actin was used as a loading control. Ratios of LC3B-II:LC3B-I and cCas-3:Cas-3 are normalized to the untreated control. Representative blot shown (n = 2). See supplementary blot figure F3 for full length blots. (\u003cstrong\u003ec\u003c/strong\u003e) Immunoprecipitation of GFP or GFP-Bcl2 complexes in lysates (input) for HEK293T cells transfected with pLV-eGFP (untreated control) or GFP-Bcl2 and Beclin1-FLAG for 48 hours and treated for 3 hours with 0.1% DMSO, Hanks’ Balanced Salt Solution (HBSS), 5 µM colchicine or 10 µM WHN-11. Representative blots shown. Analysis of the average fold difference ± SEM (n = 3) of Beclin1-FLAG levels in immunoprecipitates relative to GFP-Bcl2 and normalized to DMSO treatment (taken as 1), where * p \u0026lt; 0.05 and ** p \u0026lt; 0.01 by a two-tailed student’s t-test. See supplementary blot figure F4 for full length blots.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/f571fe4895599e2b2812eb1a.png"},{"id":75038444,"identity":"3450d2de-aa9c-4592-9858-4d2c21545d94","added_by":"auto","created_at":"2025-01-29 17:51:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":8976413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWHN-11 disrupts mitochondrial function.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Analysis of Bcl-xL expression in treated HCC1937 cells overnight. Data indicate the mean fluorescence intensity ± SEM (n = 3), where * p \u0026lt; 0.05 by a two-tailed student’s t-test. (\u003cstrong\u003eb\u003c/strong\u003e) Cellular ATP levels quantified in treated cells. Data shown are averages ± SEM (n = 3), where * p \u0026lt; 0.05, ** p \u0026lt; 0.01 and *** p \u0026lt; 0.001 by a two-tailed student’s t-test. (\u003cstrong\u003ec\u003c/strong\u003e) Mitochondrial membrane potential (ΔΨm) measured by JC-1 staining in treated HCC1937 cells. Data shown are averages ± SEM (n = 3) of the relative percentage ratio of red to green fluorescence, and ** p \u0026lt; 0.01, *** p \u0026lt; 0.001 and ns - not significant by a two-tailed student’s t-test. (\u003cstrong\u003ed\u003c/strong\u003e) Confocal analysis of the co-expression of TRAP1 and Bcl-xL in treated HCC1937 cells. Representative images shown (n = 2).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/f2c857b5b9537cc512e007f7.png"},{"id":86179714,"identity":"4ab729f5-6050-47f9-a72c-0f911a173bfc","added_by":"auto","created_at":"2025-07-07 16:18:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":43000146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/0493b6be-d5a0-429a-a28d-462f9ee95e45.pdf"},{"id":75038437,"identity":"e6f1c2f2-774c-4006-97cd-51304f18a57d","added_by":"auto","created_at":"2025-01-29 17:51:15","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2468481,"visible":true,"origin":"","legend":"","description":"","filename":"Kajewoleetalsuppdataforsubmission.docx","url":"https://assets-eu.researchsquare.com/files/rs-5315239/v1/04fa86913fc4fc0ba432fce9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The novel amino-artemisinin derivative WHN-11 disrupts mitochondria and protein homeostasis, and induces autophagy and apoptosis in cancer cells","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe antimalarial drug artemisinin is the active principle of the sweet wormwood \u003cem\u003eArtemisia annua\u003c/em\u003e that has long been used in Traditional Chinese Medicine for its antipyretic properties [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Artemisinin and its synthetic derivatives (Supplementary Fig.\u0026nbsp;1) are promising candidates for repurposing for other clinical conditions such as autoimmune diseases and inflammation [\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], parasitic infections in addition to those due to malaria [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], viral infections [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and cancer [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Artemisinins are active against cancer cells \u003cem\u003ein vitro\u003c/em\u003e and against tumours \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16 CR17\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Artesunate has been submitted to clinical trials [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] although regression-free survival was not enhanced. Against tumour cells \u003cem\u003ein vitro\u003c/em\u003e, the amino-artemisinin artemisone elicits activities superior to artemisinin [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and in principle, because of relative lack of toxicity and enhanced pharmacokinetic properties including lack of metabolism to DHA, will be better suited for clinical use [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. IC\u003csub\u003e50\u003c/sub\u003e activities of artemisinins \u003cem\u003ein vitro\u003c/em\u003e range from 0.26\u0026ndash;95.7 \u0026micro;M against a variety of tumour cell lines [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Combinations of artemisinins with known and developmental cancer drugs have been examined, largely with the view of enhancing activity of the latter through additivity or synergism, and thereby attenuating toxicity of the partner drug through use of lower amounts [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this respect, artemisone shows additivity in its combinations with oxaliplatin and gemcitabine [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Activity of artemisone (IC\u003csub\u003e50\u003c/sub\u003e 95.7 \u0026micro;M) towards A375 melanoma cells is markedly synergized in a 1:1 combination with the redox-active copper-(II) complex of the anticancer drug elesclomol [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The geranyl amino artemisinins WHN-296 and WHN-298 likewise act in synergy with the elesclomol-copper(II) complex against A375 melanoma cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe antitumour action of artemisinins may best be categorized as pleiotropic. Artemisinins are known to increase oxidative stress through generation of reactive oxygen species (ROS) that through downstream signalling effects elicited by the ROS overwhelm redox homeostasis in the cancer cell, and thereby induce cell-cycle arrest and apoptosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. One potential pathway involves facile oxidation by the artemisinin of reduced flavin cofactors of disulfide reductases such as glutathione reductase (GR), thioredoxin reductase (TrxR) and others [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Generation of ROS is demonstrated during treatment of A375 melanoma cells with the geranyl piperazine derivative WHN-296 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. There is pronounced synergism of the amino-artemisinins with the redox active elesclomol-copper(II) complex, which is known to exert activity by generation of ROS [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, mechanistic pathways evidently independent of ROS generation have also been reported [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we report the anticancer activities of a distinct class of artemisinin derivative termed amino-artemisinins (Supplementary Figs.\u0026nbsp;1b and 2) against cancer cell lines including triple-negative breast cancer (TNBC) cells. TNBCs make up 15\u0026ndash;20% of reported breast cancer cases, are highly aggressive, and lack effective standardised chemotherapeutic treatment regimens to date [\u003cspan additionalcitationids=\"CR39 CR40 CR41\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Amino-artemisinins, obtained from DHA by replacing the hydroxyl group at C-10 by an amino group (Supplementary Figs.\u0026nbsp;1b and 2) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], have greatly enhanced efficacies against the malaria parasite and cancer cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. These derivatives have improved pharmacokinetics [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and generation of active metabolites with relatively long half-lives [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Using established artemisinins (Supplementary Fig.\u0026nbsp;1a) as controls, we identify the 4'-trifluoromethylarylurea piperazinyl derivative WHN-11 as an anticancer hit compound, with a mechanism of action targeting the mitochondria to induce autophagy and apoptosis independent of explicit ROS generation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Compounds\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eReference compounds and the artemisinins used for screening were \u0026ge;95% pure as established previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The piperazinyl ureas WHN-10 and WHN-11, the piperazinyl amides WHN-14 and WHN-15[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and the DHA-piperazine dimer WHN-27[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] were prepared and characterized as reported previously. Elesclomol, 98% pure, was obtained from Kaixuan Chemical Company, Changzhou, Jiangsu, China, and used as received [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For screening as described below, compounds were dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 100 mM and stored at -20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell Cultures\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe triple negative breast cancer cell lines HCC1937 (ATCC: CRL-2336), HCC70 (ATCC: CRL-2315), MDA-MB-231 (ATCC: HTB-26), the cervical carcinoma cell line HeLa (ATCC: CCL-2), and colon cancer cell line HCT116 (ATCC: CCL-247) were purchased from the ATCC. The glioblastoma U-87 (ATCC: HTB-14), 501mel melanoma (CVCL_4633) and HEK293T (ATCC: CRL-3216) cell lines were provided by Prof. Sharon Prince, Department of Human Biology, University of Cape Town, South Africa, and the MCF-12A (ATCC: CRL-10782) breast epithelial cell line was provided by Prof. Anna-Mart Engelbrecht, Department of Physiological Sciences, Stellenbosch University, South Africa. The culture conditions are reported in Supplementary Table\u0026nbsp;1. All cell lines were cultured at 37\u0026deg;C and 9% CO\u003csub\u003e2\u003c/sub\u003e and were confirmed to be mycoplasma-free by Hoechst 33342 staining. Cells used for experiments were between 10 and 30 passages.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.3. Viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide\u003c/em\u003e (MTT) \u003cem\u003edye\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor all cell lines other than the MDA-MB-231-line, viability was assessed based on the conversion of MTT dye into the insoluble formazan. Cells seeded overnight in 96-well plates (5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) were treated for 72 hours with increasing concentrations of the compounds or 0.1% (v/v) DMSO in culture medium. Thereafter, cells were incubated with 200 \u0026micro;g/mL MTT dye for 4 hours and an insoluble formazan was produced overnight with 10% (w/v) acidified SDS solution. Absorbance was measured at 595 nm and the half maximal inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) of the compounds were calculated by non-linear regression using GraphPad Prism version 4.00 (San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Viability assay using sulforhodamine B (SRB) dye\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the cytotoxicity screening with the MDA-MB-231 cell line, SRB dye was used since the MTT assay gave poor results for this cell line. Cells seeded in 96-well plates (1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well) overnight were treated as per the MTT assay. Subsequently, cells were fixed with cold 50% (w/v) aqueous trichloroacetic acid (TCA) for an hour at 4\u0026deg;C, washed in slow-running water and the plate air-dried. Fixed cells were stained with 0.04% (w/v) SRB in acetic acid for an hour, washed repeatedly with 1% (v/v) aqueous acetic acid to remove unbound dye and solubilized for 10 minutes with 10 mM Tris base solution (pH 10.5) on an orbital shaker. Absorbance was measured at 510 nm and the IC\u003csub\u003e50\u003c/sub\u003e values determined as described above.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Tumoursphere assay for cancer stem cell activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAs previously described [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], HCC1937 cells were seeded as single cell suspensions into a 96-well Corning\u0026reg; Costar\u0026reg; Ultra-Low attachment multiple well plate (1 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well). Cultures were grown in anchorage-independent growth (AIG) medium consisting of DMEM supplemented with 1% (v/v) PSA, 2% (v/v) B-27 supplement, 1% (v/v) GlutaMAX, 20 ng/mL human epidermal growth factor (hEGF), 20 ng/mL basic fibroblast growth factor (bFGF), 4 ng/mL heparin and 10 \u0026micro;g/mL insulin. Cells were immediately treated with 0.1% (v/v) DMSO or 50 \u0026micro;M of each compound and incubated for 7 days. Treated cells were supplemented with freshly prepared AIG medium at 48-hour intervals. Tumourspheres formed were quantified at the end of the incubation period to calculate the percentage Tumoursphere Forming Efficiency (TFE): (average total number of tumoursphere formed/number of cells seeded) \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Clonogenic assay in treated HCC1937 cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSeeded cells (1.5 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e cells/well) were incubated for 6 hours, treated with 0.1% (v/v) DMSO, 5-fluorouracil (5-FU) or WHN-11 for 9 days and supplemented with fresh complete medium every 2 days. Colonies were fixed with methanol:acetic acid (3:1) and stained overnight with 1% (w/v) crystal violet in methanol. Excess stain was removed in slow-running water and the plate air-dried. Pictures of colonies were taken using a Molecular Imager ChemiDoc XRS\u0026thinsp;+\u0026thinsp;System (Bio-Rad, USA). To quantify the colonies formed, crystal violet-stained cells were solubilized with 1% (w/v) acidified-SDS for 4 hours at 37\u0026deg;C and absorbance measured at 570 nm.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. SDS-PAGE and Western Blot Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAccording to the standard modifications of the Laemmli protocol [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], equal amounts of protein from treated whole cell lysates were resolved on a 12% SDS-PAGE gel and transferred to nitrocellulose membrane. Membranes were blocked for 1 hour in 1% (w/v) Tris-buffered saline (TBS) containing 1% (w/v) solution of non-fat powdered milk (Blotto). Thereafter, membranes were incubated overnight with specific primary antibodies in 0.1% (v/v) TBS-Tween-20 (TBST) containing 1% (w/v) Blotto at 4\u0026deg;C. Membranes were submitted to repeated washes with 0.1% TBST, prior to incubation with secondary antibodies and protein bands visualized by chemiluminescence using Clarity Western Enhanced chemiluminescence (ECL) substrate (Bio-Rad; 170\u0026ndash;5061) on a Molecular Imager ChemiDoc XRS\u0026thinsp;+\u0026thinsp;System (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Proteomic analysis of differentially expressed genes in WHN-11 treated HCC1937 cells by mass spectrometry\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHCC1937 cells treated with 0.1% (v/v) DMSO or 10 \u0026micro;M WHN-11 were harvested and lysed in PBS buffer (pH 7.4) containing 0.1% (v/v) Triton-X100 and 1 mM phenylmethansulfonyl fluoride (PMSF). Equal amounts of protein (~\u0026thinsp;50 \u0026micro;g) were resuspended at a 5:1 ratio in digestion solution containing 5% (w/v) sodium deoxycholate (Sigma-Aldrich; D6750), 50 mM tris-(2-carboxyethyl)phosphine, hydrochloride (TCEP-HCl, Thermo Fisher Scientific; 20490) and 100 mM chloroacetamide (Sigma-Aldrich; C0267) for 5 minutes at 95\u0026deg;C. Lysates were digested at a 100:1 ratio with mass spectrometry (MS)-grade trypsin protease (Thermo Fisher Scientific; 90057) at 37\u0026deg;C overnight. Digested samples were incubated with 5% (v/v) trifluoroacetic acid (TFA, Thermo Fisher Scientific; 85183) in isopropanol and centrifuged at 12,000 \u0026times;g for 10 minutes at 4\u0026deg;C. The supernatants were transferred to styrene divinylbenzene polymer (SDB) spin-columns and centrifuged at 1,000 \u0026times;g for 5 minutes. Samples were first washed with isopropanol containing 1% (v/v) TFA and then with 0.2% (v/v) TFA in MS grade water. Mass spectra were collected and analysed on a Q-Exactive MS connected to an Ultimate Ultra 3000 chromatography system (Thermo Scientific, Germany) as previously described [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Gene set enrichment analysis (GSEA) using the c5.all.v7.5.1.symbols.gmt [Gene Ontology] gene sets database was conducted on the genes in the proteomic dataset comparing WHN-11 treated lysates to a DMSO-treated control. A thousand permutations were run, and the maximum and minimum gene set sizes set to 500 and 15, respectively [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Indirect immunofluorescence staining for the detection of ubiquitinated proteins\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCells grown on sterile coverslips (1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) were treated overnight with 0.1% (v/v) DMSO in culture medium or 10\u0026ndash;50 \u0026micro;M WHN-11. Thereafter, cells were fixed briefly with ice-cold methanol and permeabilised with 0.1% (v/v) Triton-X100, blocked with 1% (w/v) bovine serum albumin (BSA) containing 1X TBS and incubated overnight with anti-ubiquitin antibody (Santa Cruz; sc-8017) at 4\u0026deg;C. Following incubation with Alexa-Fluor 488 anti-mouse secondary antibody (Abcam; ab15105) and Hoechst 33342, coverslips were mounted on slides with DAKO fluorescence mounting medium (DAKO; S3023). Images were captured using laser wavelengths of Ex\u003csub\u003e488nm\u003c/sub\u003e/Em\u003csub\u003e562nm\u003c/sub\u003e on a Zeiss LSM 780 confocal microscope and analysed using Zeiss Zen 2 (blue edition) software, version 2.0 (Carl Zeiss Microscopy, GmbH).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Chymotrypsin-like proteasome activity assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCell pellets were lysed in proteasome lysis buffer (50 mM HEPES pH 7.8, 10 mM NaCl, 1.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose and 5 mM DTT) for 20 minutes at 4\u0026deg;C. Lysates were sonicated, cleared by centrifugation, the supernatant collected, and equal amounts of protein (~\u0026thinsp;16 \u0026micro;g) seeded into black-walled 96-well plates for proteasome assay. Lysate was incubated with compounds or DMSO control for an hour at 37\u0026deg;C in proteasome lysis buffer containing 2 mM ATP and 100 \u0026micro;M of \u003cem\u003eN\u003c/em\u003e-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methyl-coumarin proteasome substrate (Suc-LLVY-AMC, Sigma-Aldrich; S6510). Fluorescence was measured at Ex\u003csub\u003e360nm\u003c/sub\u003e/Em\u003csub\u003e460nm\u003c/sub\u003e and the proteasome activity of each treatment was determined relative to the vehicle control.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Analysis of caspase activation for the detection of apoptosis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHCC1937 cells (5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well) seeded overnight were treated for 24 hours with 0.1% (v/v) DMSO, 5 \u0026micro;M KRIBB-11 or 1\u0026ndash;50 \u0026micro;M WHN-11. Using Vybrant\u0026trade; FAM poly caspases assay kit (Thermo Fisher Scientific, V35117), cells were pelleted, incubated in the dark at 37\u0026deg;C, 9% CO\u003csub\u003e2\u003c/sub\u003e with 1X FLICA reagent for an hour and briefly stained with 1.25 \u0026micro;g/mL propidium iodide. Fluorescence was detected on a BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA) using Ex\u003csub\u003e488nm\u003c/sub\u003e and Em\u003csub\u003e530nm\u003c/sub\u003e filters, and analysed with FlowJo v10.0.7 software (FlowJo, LLC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Identification of Acidic Vesicular Organelles (AVOs) using Acridine Orange staining\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCells were seeded (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells /well) in a 15\u0026micro;-slide angiogenesis chamber (Ibidi GmbH, 81506) and treated for 4 hours with compounds. Live cells were briefly stained in the dark with 1 \u0026micro;g/mL Acridine Orange hemi (zinc chloride) salt (Sigma-Aldrich, A6014). Excess stain was removed by washing with PBS (pH 7.4) and coverslips mounted on slides with DAKO mounting medium. Images were captured using laser wavelengths of Ex\u003csub\u003e561nm\u003c/sub\u003e/Em\u003csub\u003e637nm\u003c/sub\u003e for red signals and Ex\u003csub\u003e488nm\u003c/sub\u003e/Em\u003csub\u003e539nm\u003c/sub\u003e for green signals on a Zeiss LSM 780 confocal microscope as described above.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Nile Red staining for lipid droplets\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCells seeded in 15\u0026micro;-slide angiogenesis chambers (Ibidi GmbH) were treated for 4 hours with compounds. Thereafter, cells were washed once with HBSS containing 20 mM HEPES pH 7.0 (HHBS). Live cells were stained in the dark for 30 minutes with 200 nM Nile Red dye in HHBS at room temperature. Excess stain was removed by washing with 1X HHBS and cells were mounted on slides with DAKO mounting medium. Images were captured for differential interference contrast (DIC) or Nile Red signals using laser wavelengths of Ex\u003csub\u003e488nm\u003c/sub\u003e/Em\u003csub\u003e566nm\u003c/sub\u003e on a Zeiss LSM 780 confocal microscope as described above.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Direct immunostaining for Bcl-xL protein expression\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTreated HCC1937 cells were harvested, fixed with 0.01% (v/v) formaldehyde in PBS, and resuspended in permeabilization (PERM) buffer containing 0.1% (v/v) Triton-X100 and 0.5% (w/v) BSA in PBS (pH 7.4) in the dark for 15 minutes. Cells were pelleted and subjected to immunostaining with PERM buffer containing 1 \u0026micro;g of mouse PE-conjugated IgG1 Kappa isotype control (eBiosciences, 12-4714-42) or PE-conjugated anti-Bcl-xL (H-5) antibody (Santa Cruz, sc-8392) for an hour at 4\u0026deg;C. Fluorescence was detected using a BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA) using laser wavelengths of Ex\u003csub\u003e480nm\u003c/sub\u003e/Em\u003csub\u003e575nm\u003c/sub\u003e to determine the expression of Bcl-xL protein, represented as the mean fluorescence intensity (MFI).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Detection of cellular ATP concentrations\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eATP levels in treated HCC1937 cells was assessed using the ATP Bioluminescence Assay Kit CLS II (Roche, 11-699-695-001). Cells were lysed at 4\u0026deg;C for 10 min in lysis buffer containing 50 mM Tris buffer, 5% (v/v) glycerol, 1 mM DTT, 60 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 150 mM NaCl. ATP concentrations between 50 pM and 50 \u0026micro;M were used as standards. Equal volumes of lysate or standard samples and luciferase reagent were mixed in a 96-well plate and luminescence was measured.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.16. JC-1 staining for mitochondrial membrane potential\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTreated HCC1937 cells were stained with 2.5 \u0026micro;g/mL JC-1 dye (Abcam; ab113850) for 30 minutes as previously described [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Harvested cells were thereafter analysed on a BD FACSAria II flow cytometer (BD Biosciences, San Jose, CA, USA). For each sample, 10,000 events were recorded using Ex\u003csub\u003e488nm\u003c/sub\u003e/Em\u003csub\u003e529nm\u003c/sub\u003e and Ex\u003csub\u003e488nm\u003c/sub\u003e/Em\u003csub\u003e590nm\u003c/sub\u003e for green and red signals, respectively. Carbonyl cyanide \u003cem\u003em\u003c/em\u003e-chlorophenyl hydrazone (CCCP) treatment was used as a control for loss of mitochondrial membrane potential.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.17. Colocalization of TRAP1 with Bcl-xL in treated cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHCC1937 cells treated for 3 hours were fixed with 4% (w/v) aqueous PFA, permeabilised and incubated for an hour with 1% (w/v) BSA in 1X TBS. Following this, cells were incubated at 4\u0026deg;C overnight with primary anti-TRAP1 antibody (Thermo Fisher; MA1-010) in 0.1% BSA-TBST buffer (1:100) and for an hour with Alexa-Fluor 488 secondary anti-mouse antibody (Abcam; ab15105) in the dark. Stained cells were also incubated for 1.5 hours with 1 \u0026micro;g of PE-conjugated Bcl-xL (H-5) antibody (Santa Cruz; sc-8392) and Hoechst 33342 at 4\u0026deg;C and mounted on slides with DAKO mounting medium. Images were captured using laser wavelengths of Ex\u003csub\u003e488nm\u003c/sub\u003e/Em\u003csub\u003e557nm\u003c/sub\u003e for Alexa-Fluor 488 signals and Ex\u003csub\u003e561nm\u003c/sub\u003e/Em\u003csub\u003e618nm\u003c/sub\u003e for PE signals on a Zeiss LSM 780 confocal microscope as described above.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.18. Cell survival assay for galactose-dependent mitochondrial function\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHCC1937 cells were seeded either in glucose-containing medium (as in the MTT assay) or in galactose-containing medium [RPMI-1640 (Thermo Fisher Scientific; 11879020) supplemented with 2 g/L galactose, 10% (v/v) FBS, 1% (v/v) 100 U/mL PSA, 1% (v/v) GlutaMAX\u003csup\u003e\u0026trade;\u003c/sup\u003e and 0.25% (v/v) sodium bicarbonate]. Thereafter, cells were treated with compounds and processed as previously described with the MTT assay to determine the IC\u003csub\u003e50\u003c/sub\u003e values.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.19. Immunoprecipitation of Bcl2 proteins in treated HEK293T cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHEK293T cells were transiently co-transfected with 2 \u0026micro;g of purified pLV-eGFP (control; Addgene plasmid 36083) and 2 \u0026micro;g GFP-Bcl2 (Addgene plasmid 17999) or Beclin1-FLAG (Addgene plasmid 24388) for 48 hours. Thereafter, cells were treated for 3 hours with 0.1% (v/v) DMSO, HBSS, 5 \u0026micro;M colchicine or 20 \u0026micro;M WHN-11. Equal amount of protein from harvested lysate was incubated overnight at 4\u0026deg;C with 5 \u0026micro;g/mL magnetic anti-GFP conjugated beads (Abcam; ab193983). GFP-bound protein complexes were eluted and resolved on a 12% SDS-PAGE gel and subject to immunoblot analysis for GFP-Bcl2 and Beclin1 (FL) using anti-GFP and anti-FLAG antibodies, respectively. Densitometry analysis was conducted using ImageJ 1.51j8 NIH freeware.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.20. Combination assay for the analysis of synergistic relationships between compounds\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUsing the standard compound combination protocols [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], HCC1937 cells were treated at constant or non-constant ratios with the relevant compounds for 72 hours and subjected to MTT assay as previously described. Combination indexes (CI) were developed from constant ratio of combined compounds and algorithms in CompuSyn software, version 1.0 as previously described [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. CI was defined as synergistic (CI\u0026thinsp;\u0026lt;\u0026thinsp;1), additive (CI\u0026thinsp;=\u0026thinsp;1) or antagonistic (CI\u0026thinsp;\u0026gt;\u0026thinsp;1) from a graph representing the CI versus Fa (fraction of cells affected).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003e3.1. Amino-artemisinin derivatives exhibit anti-cancer and anti-cancer stem cell activity in vitro: selection of WHN-11 as hit compound.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eFor the purpose of expanding the amino-artemisinin repertoire, we used our synthetic methodologies originally developed for preparing artemiside and artemisone (Supplementary Fig.\u0026nbsp;1b) from DHA[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] to provide the C-10 substituted piperazinyl derivatives (Supplementary Fig.\u0026nbsp;2) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Details on preparation of these compounds is provided in the supplementary data.\u003c/p\u003e \u003cp\u003eWe established the half-maximal inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) of artemisinins against HCC1937, HCC70 and MDA-MB-231 TNBC cell lines, HeLa cervical carcinoma, U87 glioblastoma, 501mel melanoma and HCT116 colon cancer cell lines, as well as the non-cancerous non-transformed breast epithelial MCF-12A and human embryonic kidney HEK293T cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea; Supplementary Table\u0026nbsp;2). The amino-artemisinins exhibited increased toxicity to all cell lines compared to the parent compound artemisinin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea; Supplementary Table\u0026nbsp;2). Both DHA and artesunate showed greater toxicities against all cell lines compared to artemisinin. Notably, compared to artemisinin, the WHN-series of amino-artemisinins exhibited IC\u003csub\u003e50\u003c/sub\u003e values in the low micromolar/high nanomolar range across all cell lines, of which WHN-11 was consistently the most toxic in all cell lines tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). While all the artemisinins showed greater toxicity to the MCF-12A breast epithelial cell line and the HEK293T transformed embryonic kidney cell line, compared to the cancer cell lines, WHN-11 was substantially more toxic to all cancer cell lines except the U87 glioblastoma compared to the HEK293T cell line (SI values from 2.85\u0026ndash;12.58; Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eWe next investigated the ability of artemisinins to inhibit cancer stem-like cell (CSC) activity. The ability of CSCs to both self-renew and differentiate has been linked to metastases and chemoresistance [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. This was assessed \u003cem\u003ein vitro\u003c/em\u003e through the formation of tumourspheres, which grow anchorage independently in serum-free, non-adherent growth conditions and are enriched in CD44\u003csup\u003e+\u003c/sup\u003e/CD24\u003csup\u003e-\u003c/sup\u003e stem-like cells, which express higher levels of Oct4 stem cell transcription factor [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. DHA, artesunate, WHN-11 and WHN-298 inhibited tumoursphere formation in HCC1937 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Based on these data, we selected WHN-11 as a putative hit compound for further mechanistic investigation. We next examined the ability of WHN-11 to block long-term survival in the clonogenic assay [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Both 5-fluorouracil (5-FU, positive control) and WHN-11 significantly decreased colony formation of HCC1937 cells in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eWe confirmed that WHN-11 was cytotoxic and non-cytostatic through increased activation of caspases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Increasing concentrations of WHN-11 promoted the cleavage of both poly-ADP ribose polymerase-1 (PARP-1) and caspase-3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) indicating induction of apoptosis, like the KRIBB11 and GA controls [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Artemisinins often exert activity through generation of reactive oxygen species (ROS) that through downstream signalling effects elicited by the ROS overwhelm redox homeostasis and induce cell-cycle arrest and apoptosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To identify and quantitate ROS production in HCC1937 cells treated with WHN-11, we used 2',7'-dichlorodihydrofluorescein diacetate (DCFDA), which has been used previously to demonstrate ROS production by artemisinins [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. While H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and artemisinin significantly induced ROS formation, WHN-11 did not induce significant levels of ROS compared to the vehicle treatment (Supplementary Fig.\u0026nbsp;3a). Using an alternative assay in which ROS detection is linked to activation of an antioxidant response element (ARE) reporter plasmid [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], we observed ROS production with the positive control elesclomol [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] but not WHN-11 (Supplementary Fig.\u0026nbsp;3). Additionally, inclusion of ROS scavenger, \u003cem\u003eN\u003c/em\u003e-acetyl-L-cysteine (NAC) did not alter the toxicity of WHN-11, but did reverse the cytotoxic effects of elesclomol and artemisinin (Supplementary Fig.\u0026nbsp;3). Taken together, these data suggested that WHN-11 showed anticancer toxicity but did not induce significant formation of ROS in cancer cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.2. WHN-11 treatment perturbs protein homeostasis in HCC1937 cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo understand the mode of action of WHN-11, we conducted an unbiased global analysis of the proteome of WHN-11 versus vehicle-treated HCC1937 cells by mass spectrometry [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. A total of 3,629 unique proteins were identified, of which 51 proteins were significantly upregulated (\u0026gt;\u0026thinsp;2-fold) and 84 significantly downregulated (\u0026lt;-2 fold) in WHN-11-treated cells versus the vehicle treatment (Supplementary Fig.\u0026nbsp;4). Some significantly upregulated proteins included the death-associated protein 1 (DAP), ubiquitin-like modifier-activating enzyme (ATG7), interferon-stimulated gene 15 (ISG15), ubiquitin specific peptidase 24 (USP24) and E3 ubiquitin-protein ligase (HERC2). Significantly downregulated proteins included the cytochrome c oxidase assembly factor 7 (COA7), vacuolar protein sorting-associated protein 37B (VPS37B), Ras-related protein Rap-2B (RAP2B), growth arrest and DNA damage-inducible proteins-interacting protein 1 (GADD45GIP1) and cyclin-dependent kinase 4 (CDK4; Supplementary Material).\u003c/p\u003e \u003cp\u003eTwelve of the upregulated proteins were identified only in the WHN-11-treated samples. Among these were ubiquitin carboxyl-terminal hydrolase 24 (USP24), profilin-2 (PFN2), ADP-ribosylation factor 6 (ARF6) and mas-related genes (MRG/MORF4L)-binding protein (MRGBP) (Supplementary Material). In addition, 18 downregulated proteins were detected only in DMSO-treated cells. These included the ATP-binding cassette sub-family B member 10 (ABCB10), nuclear factor 1 B-type (NF1B), growth arrest and DNA damage-inducible proteins-interacting protein 1 (GADD45GIP1) and tumour necrosis factor alpha-induced protein 2 (TNFAIP2; Supplementary Material).\u003c/p\u003e \u003cp\u003eGene set enrichment analysis [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] suggested that WHN-11 induced alterations in gene sets associated with development and morphogenesis, transmembrane transport, cell cycle, serine/threonine kinase activity and the Wnt signalling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Supplementary Material). Multiple gene sets involved in protein catabolism, ubiquitination, cellular responses to starvation and protein targeting to the lysosome were identified as significantly enriched (either positively or negatively) upon WHN-11 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Consistent with our biological assessment, we did not detect significant enrichment of gene sets associated with ROS production.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe global proteomic analysis suggested WHN-11-induced imbalances in proteostasis, including ubiquitination and proteasomal degradation. Both processes occur during cellular stress induced by unfolded protein response (UPR) and/or heat shock response (HSR) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. To assess stress-induced protein accumulation in cancer cells as previously shown following artemisinin treatment in malaria [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], induction of protein ubiquitination by WHN-11 in HCC1937 cells was analysed in the absence or presence of the proteasome inhibitor MG132. Immunofluorescence (IF) staining of ubiquitin showed ubiquitinated proteins in the cytoplasm, which decreased in a dose dependent fashion on treatment with WHN-11, suggesting enhanced proteasome activity (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-b). Inhibition of the proteasome with MG132 significantly accumulated ubiquitinated protein aggregates localized at the periphery and within the nucleus of co-treated cells. Higher concentrations of WHN-11 also increased cytoplasmic protein aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Artemisinin disrupts protein homeostasis and inhibits the malarial proteasome [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. To test if WHN-11 inhibited human proteasomes, we assessed the ability of the compounds to directly inhibit the proteasome in whole cell lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). While MG132 treatment inhibited the chymotrypsin-like activity of the proteasome in a dose-dependent manner, WHN-11 did not significantly alter the proteasome activity compared to treatment with the vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). However, when cells were treated overnight prior to assessment of proteasome activity in lysates, WHN-11 significantly promoted proteasome activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In combination experiments, WHN-11 and MG132 exhibited a strong synergistic relationship (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Supplementary Fig.\u0026nbsp;5). Changes due to proteostatic stress were indicated by a significant increase in the stress-responsive Hsp70 mRNA levels in WHN-11-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). These data suggested that WHN-11 does not directly inhibit the proteasome, but that WHN-11 promotes protein turnover via the proteasome by a distinct mechanism [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCombination of WHN-11 with inducers or activators of autophagy and proteostatic stress\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMechanism of action\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAverage CI* when combined with WHN-11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRelationship\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMG132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProteasome inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStrong synergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloroquine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInhibits lysosome acidification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStrong synergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRapamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emTOR inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStrong synergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTunicamycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycosylation inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eStrong synergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3-Methyladenine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePI3K inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eModerate synergism\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Average combination index (CI) where half the population of cells are affected by the co-treatment with the compounds (Fa\u0026thinsp;=\u0026thinsp;0.5).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4. WHN-11 induced autophagy in cancer cells\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eOur findings that WHN-11-induced apoptosis and proteasome-independent regulation of protein turnover suggested autophagy as an alternative degradation pathway. This was supported by the gene set enrichment analysis which identified responses to starvation and protein targeting to the lysosome as enrichment processes (Supplementary Fig.\u0026nbsp;6). We previously observed the formation of vesicle-like structures in WHN-11 treated cells (data not shown), which indicated possible sites of early endosomal or autophagic vesicles, lipid, or other acidic vesicles [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Formation of acidic vesicular organelles (AVOs) in HCC1937 cells treated with WHN-11 were assessed using acridine orange (AO) dye and confocal microscopy with Hanks' Balanced Salt Solution (HBSS) used as a positive control for autophagy induction. WHN-11 significantly increased the formation of cytoplasmic AVOs (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b) and the number of cells with AVOs compared to treatment with DMSO (white arrows, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Although this suggested the possible induction by WHN-11 of formation of autophagosomes, other vesicle-like structures (yellow arrows) not coinciding with AVOs were visible (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Using Nile Red dye, treatment with WHN-11 and HBSS showed enlarged lipid droplets (LDs) in the cytosol and near the nuclear membranes, exhibiting substantial depth through treated cells, compared to the vehicle treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). LDs are specialized compartments for fatty acid products after lysosomal degradation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] and are formed during the lipidation of the autophagy marker, microtubule-associated proteins 1A/1B light chain 3B (LC3B) [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe subsequently established that WHN-11 indeed promoted the lipidation of LC3B-I, which was promoted by co-treatment with chloroquine (CQ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b). WHN-11, like the controls HBSS and colchicine, significantly decreased the association between GFP-Bcl2 and Beclin1-FLAG in transiently transfected HEK293T cells relative to the cells treated with vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) indicating autophagy induction [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Increased autophagic flux by WHN-11 was confirmed in HeLa cells stably expressing mCherry-EGFP-LC3 by confocal analysis. Increased yellow puncta arising via the overlap of green-EGFP and red-mCherry signals indicated the formation of autophagosomes [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] like the controls HBSS and Baf-A1 (Supplementary Fig.\u0026nbsp;6). WHN-11 also showed strong synergy with the autophagy inducers rapamycin, tunicamycin and the autophagy inhibitor CQ and moderate synergism with autophagy inhibitor 3-methyladenine (3-MA) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5. WHN-11 induces mitochondrial dysfunction\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWHN-11 induced both autophagy (as indicated by LC3B-I lipidation) and apoptosis (as indicated by caspase-3 cleavage) only after a prolonged exposure of 16 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), suggesting that other molecular events may precede cell death. The mitochondrial Bcl2 family of proteins drives apoptosis [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Prior to mitochondrial-induced (intrinsic) apoptosis, mitochondrial membrane potential ΔΨm changes, ATP levels drop and Bcl2 levels change [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. WHN-11 significantly decreased the expression of anti-apoptotic Bcl-xL like the positive control KRIBB-11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), which confirms the apoptotic response to WHN-11 and links its mechanism of action to the mitochondrion. Consistent with these observations, WHN-11 reduced cellular ATP in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The mitochondrial membrane potential ΔΨm enhances the transfer of protons between the matrix and intermembrane space (IMS) during ATP synthesis [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. A loss in ΔΨm causes mitochondrial outer membrane permeabilization (MOMP), a non-reversible apoptotic stage involving the release of essential proteins within the mitochondrial IMS [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Changes in the ΔΨm were determined using the membrane permeable JC-1 dye [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. WHN-11 mediated a significant loss in ΔΨm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), consistent with the positive control carbonyl cyanide \u003cem\u003em\u003c/em\u003e-chlorophenylhydrazone (CCCP). Consistent with the flow cytometric analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), immunofluorescence staining indicated that Bcl-xL protein levels were reduced by treatment with WHN-11 in a dose-dependent manner compared with treatment by the vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). TRAP1 staining was consistent with mitochondrial localisation and appeared as a reticular network. WHN-11 did not alter TRAP1 staining intensity, but did induce distinct morphological changes in mitochondria, which appeared fragmented compared to the elongated tubular network in the control cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed i and ii).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo confirm the mitotoxic role of WHN-11, we assessed WHN-11 toxicity in cells grown in either glucose- or galactose-containing medium. In the presence of high glucose concentrations, cancer cells produce ATP via glycolysis and suppress oxidative phosphorylation in a process known as the Crabtree effect. The Crabtree effect is reversed by replacing glucose with galactose to promote ATP production via oxidative phosphorylation (OXPHOS) via the mitochondria [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Increased sensitivity of cells to compounds in the presence of galactose indicates mitotoxicity [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Galactose significantly increased the toxicity of WHN-11 (GAL-IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;M, GLU-IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 \u0026micro;M). In contrast, the toxicity of artemisinin was not significantly different (GAL-IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;218.00\u0026thinsp;\u0026plusmn;\u0026thinsp;80.69 \u0026micro;M, GLU-IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;389.33\u0026thinsp;\u0026plusmn;\u0026thinsp;92.70 \u0026micro;M; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This confirmed that WHN-11 targets the mitochondria to induce toxicity. The WHN-11 treatment-induced mitochondrial dysfunction may explain the inability to detect significant ROS production upon treatment with the compound (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e values of artemisinins in HCC1937 cells in glucose- and galactose-containing medium.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u0026nbsp;\u0026plusmn;\u0026nbsp;SD (n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArtemisinin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e389.33\u0026thinsp;\u0026plusmn;\u0026thinsp;92.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e218.00\u0026thinsp;\u0026plusmn;\u0026thinsp;80.69 (ns)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWHN-11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (*)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eStatistical significance of IC\u003csub\u003e50\u003c/sub\u003e values in +\u0026thinsp;galactose samples relative to +\u0026thinsp;glucose samples was determined by a two-tailed student\u0026rsquo;s t-test, where * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and ns - not significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStudies on the development of novel therapies and the identification of alternative agents and cellular targets to address the lack of standardised treatment for TNBC are ongoing [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. The anticancer properties of artemisinins are well studied [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Many artemisinin derivatives comprising artemisinin hybrids connected to different pharmacophores [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], artemisinin dimers and others elicit enhanced activities against tumour cells [\u003cspan additionalcitationids=\"CR84 CR85\" citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. The amino-artemisinin WHN-11 is structurally unique in incorporating a nitrogen atom attached directly to C-10 which is attached by a piperazine linker to a \u003cem\u003ep\u003c/em\u003e-trifluoromethylaryl urea group (Supplementary Fig.\u0026nbsp;2). WHN-11 exhibited the highest cytotoxicity (IC\u003csub\u003e50\u003c/sub\u003e 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52 \u0026micro;M) among the amino-artemisinins against TNBC cell lines compared to the parent compound artemisinin (202.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30 \u0026micro;M). To date, the mechanisms of action of artemisinin and its derivatives is still a topic of significant debate; the predominant view of the mechanism of action of artemisinins is the induction of ROS in cancer cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Indeed, the artemisinins used clinically for treatment of malaria, namely artemisinin [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e], DHA [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], artemether[\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e] and artesunate[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] (Supplementary Fig.\u0026nbsp;1a) induce formation of ROS in a range of cancer cells, leading to growth inhibition. However, it is important to note that artemisinin has also been shown to be specific for ROS-induction by malaria and yeast mitochondria and not mammalian mitochondria [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Furthermore, artemisinin and its derivatives can induce cell death independent of ROS generation[\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e] and artemisinin derivatives have been linked to perturbations in protein homeostasis. DHA-mediated cell death in malaria parasites involves protein damage and proteasome inhibition, which activates ER-stress related cell death [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In contrast to the effects of DHA in malaria [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], WHN-11 did not directly inhibit the proteasome but rather promoted a modest but significant increase in proteosome activity in treated TNBC cells coinciding with increased ubiquitinated protein turnover; overall this is consistent with increased autophagy.\u003c/p\u003e \u003cp\u003eTherefore, it is likely that artemisinin derivatives can induce cell death via multiple mechanisms dependent on the context. For WHN-11, we have no evidence of significant ROS induction using a range of biological assays, including those previously used to demonstrate ROS production by artemisinins. While there is the possibility that this is due to technical reasons in detection of ROS, it may also be explained by the observed mitochondrial dysfunction. Many artemisinins induce apoptotic cell death through mitochondrial dysfunction in cancer cells that is not exclusively associated with ROS production [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. WHN-11 resulted in mitochondrial fission, mitochondrial outer membrane permeabilization (MOMP), loss of ATP production and down-regulation of the anti-apoptotic mitochondrial protein Bcl-XL culminating in apoptosis. The toxicity of WHN-11 was potentiated by reliance of cells on OXPHOS for ATP production, supporting mitochondrial defects induced by WHN-11 as a main cause of toxicity. Mitochondrial fission is a known response to stress and is linked to apoptosis, both of which were induced by WHN-11 treatment. The reduced Bcl-XL levels induced by treatment with WHN-11 must culminate in release of Bax from inhibitory Bcl-XL-Bax complexes, allowing free Bax to promote mitochondrial fission linked to apoptosis [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Indeed, artesunate induced Bax-mediated apoptosis in HepG2 cells, which was similarly independent of ROS production [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of induction of autophagy by WHN-11 as a cell death mechanism is less clear, even though other artemisinins also induce autophagy and resistance to artemisinin therapy is linked to autophagy in the malaria parasite [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Like WHN-11, DHA activated major apoptotic and autophagy-related proteins, and suppressed the interaction between Bcl2 and Beclin1, promoting cell death in KKU-213 cholangiocarcinoma cells [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. In normal cells, autophagy is considered a pro-survival response which is upregulated in response to stress, particularly starvation, to provide nutrients to sustain cell survival. In cancer cells, autophagy is linked to both tumour killing and tumour survival, although the mechanisms by which this occurs are not well defined. In the case of WHN-11, it is probable that autophagy is activated as a response to mitochondrial dysfunction to circumvent or prevent apoptosis. The lipid accumulation observed upon treatment with WHN-11 can arise due to a lack of OXPHOS resulting from mitochondrial defects and which subsequently triggers autophagy [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. Mitochondria and MOMP are important in autophagy activation [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e], although this process is usually in response to oxidative stress and associated with ROS production [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. In the case of WHN-11 however, in the absence of any substantive evidence supporting ROS production, we assume that ROS-independent changes to the mitochondria may trigger autophagy and subsequently cell death by apoptosis. There is a growing appreciation of the role of autophagy modulators, both activators and inhibitors, in potentiating the anti-cancer activity of several chemotherapeutic agents for clinical benefit [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Therefore, the next steps require an evaluation of drug combinations of WHN-11 and structurally related amino-artemisinin derivatives with known modulators of autophagy like temsirolimus or everolimus in the drive to develop these compounds as viable chemotherapeutic options, in particular for treatment of TNBC.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe report the promising anti-cancer activity of a novel amino-artemisinin derivative WHN-11 against multiple cell lines, including the aggressive TNBC subtype. Unlike most other artemisinin derivatives, WHN-11 anti-cancer activity was independent of substantive ROS production. Rather, WHN-11 altered cellular protein homeostasis pathways to induce mitochondrial fission and dysfunction, depriving the cell of ATP, activating autophagy and subsequent cell death by apoptosis. The anticancer effect of WHN-11 was synergistic with inducers of autophagy, suggesting that future combination treatment with modulators of autophagy will have therapeutic potential.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. The views reflected are those of the authors and should not be attributed to the funders.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eConceptualization, A.L.E, J.d.l.M, and D.K.; formal analysis, D.K, A.L.E, J.d.l.M; investigation, D.K, H.N.W and A.v.K; resources, R.K.H and A.v.K; data curation, D.K, A.v.K, and A.L.E; writing\u0026mdash;original draft preparation, D.K, A.L.E. and R.K.H; writing\u0026mdash;review and editing, J.d.l.M and A.v.K; visualization, D.K and A.L.E; supervision, A.L.E, J.d.l.M and R.K.H; project admin-istration, A.L.E; funding acquisition, A.L.E and R.K.H. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank our colleagues Heinrich Hoppe, Demetra Mavri-Damelin, Sharon Prince, Anna-Mart Engelbrecht and Earl Prinsloo for providing reagents and conducting useful discussions.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHsu, E. Reflections on the discovery of the antimalarial qinghao. \u003cem\u003eBr. J. Clin. 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Ther.\u003c/em\u003e \u003cb\u003e131\u003c/b\u003e, 130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/J.PHARMTHERA.2011.03.009\u003c/span\u003e\u003cspan address=\"10.1016/J.PHARMTHERA.2011.03.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Amino-artemisinin, triple-negative breast cancer (TNBC), reactive oxygen species (ROS), apoptosis, autophagy, mitochondrial fission","lastPublishedDoi":"10.21203/rs.3.rs-5315239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5315239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSemi-synthetic derivatives of artemisinin exhibit anti-cancer activity \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e in addition to anti-malarial activity. Here, we report the anti-cancer and anti-cancer stem cell potential of novel C-10 substituted amino-artemisinin derivatives, among which the 4'-trifluoromethylarylurea piperazinyl derivative WHN-11 demonstrated consistent cytotoxic activity at high nanomolar concentrations across a range of cancer cell lines. WHN-11 reduced short- and long-term survival of triple-negative breast cancer (TNBC) cells, a highly aggressive breast cancer subtype that currently lacks standardized targeted treatments. Mechanistically, WHN-11 induced a stress response and increased proteasome-mediated turnover of ubiquitinated proteins. Significantly, WHN-11 promoted mitochondrial depolarization and fission, suppressing the expression of anti-apoptotic B-cell lymphoma extra-large (Bcl-xL) protein and ATP synthesis, thereby decreasing cellular energy production, and inducing apoptosis. WHN-11 treatment also increased autophagosomes, acidic vesicular organelles and lipid droplets, and promoted the dissociation of Bcl2-Beclin1 complexes. Activation or inhibition of autophagy synergized with the activity of WHN-11 in promoting cellular toxicity, as did increasing cellular dependence on oxidative phosphorylation. The effects of WHN-11 appear independent of substantial reactive oxygen species (ROS) production. Taken together, the data support ROS-independent mechanisms of anticancer action for WHN-11 and suggest that amino-artemisinins related to WHN-11 are promising candidates for anti-TNBC therapies targeting the mitochondria alone or in combination with autophagy modulators.\u003c/p\u003e","manuscriptTitle":"The novel amino-artemisinin derivative WHN-11 disrupts mitochondria and protein homeostasis, and induces autophagy and apoptosis in cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-29 17:51:10","doi":"10.21203/rs.3.rs-5315239/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-07T11:06:11+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"266871166758888286382752056890800020308","date":"2025-01-07T16:47:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-01-04T13:49:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58772389619794866148992387156644729848","date":"2025-01-04T12:51:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-27T20:00:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"49205530729699428384449263780400504264","date":"2024-11-14T16:35:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321049012962702605185670256826555943209","date":"2024-11-11T10:06:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-11T09:03:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-11T08:54:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-07T14:42:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-06T06:20:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-23T03:09:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"00322fd4-2ad0-4d3b-8d1f-f5083b9923e3","owner":[],"postedDate":"January 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42513611,"name":"Biological sciences/Cell biology"},{"id":42513612,"name":"Biological sciences/Cell biology/Autophagy"},{"id":42513613,"name":"Biological sciences/Cell biology/Cell death"},{"id":42513614,"name":"Biological sciences/Biochemistry/Proteins"},{"id":42513615,"name":"Biological sciences/Biochemistry/Proteomics"}],"tags":[],"updatedAt":"2025-07-07T16:10:05+00:00","versionOfRecord":{"articleIdentity":"rs-5315239","link":"https://doi.org/10.1038/s41598-025-05284-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:57:34","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-01-29 17:51:10","video":"","vorDoi":"10.1038/s41598-025-05284-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-05284-7","workflowStages":[]},"version":"v1","identity":"rs-5315239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5315239","identity":"rs-5315239","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}