Lipid–Saporin Nanoparticles for the Intracellular Delivery of Cytotoxic Protein to Overcome ABC Transporter-Mediated Multidrug Resistance in Vitro and in Vivo

cancers

Article Lipid–Saporin Nanoparticles for the Intracellular Delivery of Cytotoxic Protein to Overcome ABC Transporter-Mediated Multidrug Resistance In Vitro and In Vivo

1, 1, 2 1 1 Guan-Nan Zhang †, Pranav Gupta †, Ming Wang , Anna Maria Barbuti , Charles R. Ashby Jr. , Yun-Kai Zhang 1, Leli Zeng 1,3, Qiaobing Xu 2, Ying-Fang Fan 1,4,* and Zhe-Sheng Chen 1,*

1 Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, Queens, NY 11439, USA; [email protected] (G.-N.Z.); [email protected] (P.G.); [email protected] (A.M.B.); [email protected] (C.R.A.J.); [email protected] (Y.-K.Z.); [email protected] (L.Z.) 2 Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA; [email protected] (M.W.); [email protected] (Q.X.) 3 Tomas Lindahl Nobel Laureate Laboratory, Research Centre, The Seventh Aﬃliated Hospital, Sun Yat-sen University, Shenzhen 518107, China 4 Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China * Correspondence: [email protected] (Y.-F.F.); [email protected] (Z.-S.C.); Tel.: +86-131-8909-7816 (Y.-F.F.); +1-718-990-1432 (Z.-S.C.); Fax: +1-718-990-1877 (Z.-S.C.) These authors contributed equally to this paper. † Received: 5 January 2020; Accepted: 16 February 2020; Published: 21 February 2020

Abstract: Although the judicious use of anticancer drugs that target one or more receptor tyrosine kinases constitutes an effective strategy to attenuate tumor growth, drug resistance is commonly encountered in cancer patients. The ATP-binding cassette transporters are one of the major contributors to the development of multidrug resistance as their overexpression significantly decreases the intracellular concentration and thus, the efficacy of certain anticancer drugs. Therefore, the development of treatment strategies that would not be susceptible to efflux or excretion by specific ABC transporters could overcome resistance to treatment. Here, we investigated the anticancer efficacy of saporin, a ribosome-inactivating protein. Since saporin has poor permeability across the cell membrane, it was encapsulated in a lipid-based nanoparticle system (EC16-1) that effectively delivered the formulation (EC16-1/saporin) intracellularly and produced anti-cancer efficacy. EC16-1/saporin, at nanomolar concentrations, significantly inhibited the cellular proliferation of parental and ABCB1- and ABCG2-overexpressing cancer cells. EC16-1/saporin did not significantly alter the subcellular localization of ABCB1 and ABCG2. In addition, EC16-1/saporin induced apoptosis in parental and ABCB1- and ABCG2-overexpressing cancer cells. In a murine model system, EC16-1/saporin significantly inhibited the tumor growth in mice xenografted with parental and ABCB1- and ABCG2-overexpressing cancer cells. Our findings suggest that the EC16-1/saporin combination could potentially be a novel therapeutic treatment in patients with parental or ABCB1- and ABCG2-positive drug-resistant cancers.

Keywords: cancer; multidrug resistance; ATP-binding cassette transporter; saporin; EC16-1; nanoparticles

Cancers 2020, 12, 498; doi:10.3390/cancers12020498 www.mdpi.com/journal/cancers Cancers 2020, 12, 498 2 of 17

1. Introduction Multidrug resistance (MDR) is a phenomenon in which cancer cells develop resistance to structurally and mechanistically unrelated anticancer drugs [1]. MDR is one of the major causes of chemotherapy failure and increased cancer patient mortality. In the last decades of research into the cause of MDR, several mechanisms have been identified. However, the overexpression of the ATP-binding cassette (ABC) transporters in cancer continues to be the primary mediator of MDR. The ABC transporters are a family of proteins that utilize the energy derived from the hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) [2–4]. The hydrolysis of ATP by the ABC transporters catalyzes the efflux of the substrate drugs from the cancer cells, reducing their intracellular drug concentration and their efficacy [5,6]. In normal cells, the ABC transporters prevent the intracellular accumulation of potentially toxic endogenous and xenobiotic compounds; however, in cancer cells, the overexpression of these transporters significantly reduces the intracellular concentration of the substrate anticancer drug, producing desensitization, and eventually, resistance to drugs with distinct structures and mechanisms of action. To date, 49 human ABC transporters have been identified, which have been further subdivided into 7 subfamilies (ABC A through G). The two ABC transporters most commonly involved with the development of MDR in cancer cells are the ABC transporter subfamily B member 1 (ABCB1/P-gp/MDR1) and the ABC transporter subfamily G member 2 (ABCG2/BCRP/MXR) [7–10]. These two transporters are structurally distinct in that the ABCB1 transporter contains two nucleotide-binding domains (NBDs) and two transmembrane-binding domains (TMDs) [11] whereas the ABCG2 transporter, also known as a “half transporter”, contains only one TMD and one NBD, which requires a dimerization step for functional transport activity. [12,13] The ABCB1 transporter protein encoded by the ABCB1 gene located on chromosome 7p21 was the first identified ABC transporter [14,15]. The ABCB1 transporter is present on the apical membrane in a variety of tissues, including the blood–brain barrier, intestines, adrenal glands, placenta, and kidney. The transport function is vital for these tissues as it plays an important role in protecting cells from the accumulation of xenobiotics and cellular toxicants [16–18]. The ABCB1 transporter catalyzes the transport of a variety of classes of anticancer drugs, including taxanes (e.g., paclitaxel, doxetaxel), epipodophyllotoxins (e.g., etoposide and teniposide), vinca alkaloids (e.g., vinblastine and vincristine), and anthracyclines (e.g., doxorubicin and daunorubcin) [19–21]. The ABCG2 transporter is expressed in placental syncytiotrophoblasts, the apical surface of small intestines, colon epithelium, luminal surfaces of microvessels, endothelium of human brain, and in the veins and capillaries of blood vessels [22–24]. The expression of ABCG2 transporters in tumor tissues is associated with the development of resistance to chemotherapeutic drugs, such as nucleoside analogs, anthracyclines, camptothecin-derived topoisomerase I inhibitors, methotrexate, and flavopiridols [25,26]. Furthermore, clinical studies conducted in patients with adult and childhood leukemia indicate that the overexpression of the ABCG2 transporter is significantly correlated with a poor clinical prognosis [27,28]. Several recent studies have been conducted to investigate the potential of surmounting MDR by blocking the function of ABC transporters or designing novel anticancer compounds that can bypass the efflux function of these transporters. In the search for novel inhibitors of ABC transporters, the first- and second-generation tyrosine kinase inhibitors (TKIs) were considered unacceptable due to their pharmacokinetic interactions with other chemotherapeutic drugs. Although third-generation TKIs have less toxicity than the first two generations, the third-generation inhibitors did not produce beneficial results in clinical trials. Thus, it is pertinent to develop new strategies that are effective and safe while avoiding ABC transporter-mediated efflux, such as anticancer prodrugs, which can bypass adverse pharmacokinetic interactions as well as circumvent the ABC transporter function. Novel formulations can deliver anticancer drugs directly to the target site while safely bypassing the efflux transporters. One approach involves nanoparticle systems that encapsulate a protein-based toxin, allowing for a reduced concentration of anticancer drug compared to conventional small molecule-based drugs [29]. Certain nanoparticles can enter the cell by endocytosis across the cell membrane, and the loaded drug would be transported away from the ABC transporters [30,31]. This approach can be used to co-deliver Cancers 2020, 12, x FOR PEER REVIEW 3 of 17 Cancers 2020, 12, 498 3 of 17 can be used to co-deliver an anticancer drug in combination with an inhibitor of the ABC transporters, inan drug-resistant anticancer drug tumors, in combination avoiding withthe efflux an inhibitor effect on of thethe ABCintracellular transporters, drug inconcentration drug-resistant and tumors, thus re-sensitizingavoiding the ethefflux cancer effect cells on theto the intracellular anticancer drugdrug. concentration and thus re-sensitizing the cancer cellsSince to the the anticancer 1980s, protein drug. therapy, such as insulin-based formulations, have been used as a safe and effectiveSince the method 1980s, proteinto treat various therapy, diseases such as [32 insulin-based–34]. The majority formulations, of the protein-based have been used drugs as aexert safe theirand etherapeuticffective method efficacy to treat by binding various to diseases targets [ 32lo–cated34]. Theon the majority cell surface of the protein-basedor extracellular drugs domains. exert Althoughtheir therapeutic studies ehavefficacy shown by binding that the to therapeutic targets located benefits on theof protein-based cell surface or drugs extracellular are greater domains. than conventionalAlthough studies drug have delivery shown techniques, that the therapeutic the deliver benefitsy of ofprotein protein-based drug intracellularly drugs are greater is still than a considerableconventional challenge. drug delivery Saporin techniques, is a ribosome-inactivating the delivery of protein protein drug intracellularly (RIP) that was is stillisolated a considerable from the seedschallenge. of Saponaria Saporin officinalis is a ribosome-inactivating [35,36]. Saporin belongs protein to (RIP) type that I RIPs was and isolated consists from of thea single seeds polypeptide of Saponaria chain,officinalis with[35 a ,molecular36]. Saporin weight belongs of 30 to KDa type [37,38]. I RIPs The and high consists level of of a enzymatic single polypeptide activity and chain, resistance with a tomolecular conjugation weight procedures of 30 KDa and [37, 38proteases]. The high makes level Saponaria of enzymatic officinalis activity a andpotent resistance toxin that to conjugation produces anticancerprocedures efficacy and proteases by inducing makes apoptosisSaponaria and offi thecinalis inhiabition potent of toxinthe proliferation that produces of cancer anticancer cells e[39,40].fficacy However,by inducing the apoptosis use of saporin and the as inhibitionan anticancer of the drug proliferation is limited by of cancerits poor cells penetration [39,40]. However,into cancer the cells, use thusof saporin reducing as an its anticancer intracellular drug concentration is limited by an itsd poor efficacy. penetration EC16-1 into is a cancer lipid-based cells, thusnanoparticle reducing formulationits intracellular that concentrationloads saporin andthrough efficacy. electrostati EC16-1c and is a hydrophobic lipid-based nanoparticle interactions formulation[41]. Previously, that EC16-1loads saporin has been through shown electrostatic to deliver saporin and hydrophobic intracellularly interactions in a panel [41 ].of Previously, cancer cell EC16-1lines with has high been efficiencyshown to and deliver significant saporin anticancer intracellularly efficacy in a[42, panel43]. ofIn cancerthis study, cell lineswe determined with high the effi ciencyanticancer and efficacysignificant of an anticancer EC16-1-loaded efficacy saporin [42,43]. combination In this study, (EC16-1/saporin) we determined in the drug-resistant anticancer e fficancercacy ofcells. an SinceEC16-1-loaded the major saporinEC16-1/saporin combination diffuses (EC16-1 across/saporin) the cell in membrane drug-resistant by cancerendocytosis, cells. Sincewe conducted the major experimentsEC16-1/saporin to use diff usescytotoxic across proteins/drugs the cell membrane that byare endocytosis, too large to we be conductedeffluxed by experiments the MDR efflux to use pumps,cytotoxic thus proteins re-sensitizing/drugs that the are MD tooR largetumors to beto anticancer effluxed by drugs. the MDR efflux pumps, thus re-sensitizing the MDR tumors to anticancer drugs. 2. Results 2. Results 2.1. Assessment of the Magnitude of Resistance in ABCB1- and ABCG2-Overexpressing Cells 2.1. Assessment of the Magnitude of Resistance in ABCB1- and ABCG2-Overexpressing Cells Since our aim was to determine whether the expression of ABCB1 or ABCG2 affects the cytotoxicitySince our of aim EC16-1/saporin, was to determine we whether assessed the th expressione resistant of profiles ABCB1 orof ABCG2 our ABCB1- affects theand cytotoxicity ABCG2- overexpressingof EC16-1/saporin, cell welines. assessed The MTT the assay resistant was profiles used to of assess our ABCB1- resistance and in ABCG2-overexpressing the parental and resistant cell pairs,lines. TheSW620 MTT versus assay wasSW620/AD300, used to assess and resistance NCI-H460 in the versus parental NCI-H460/MX20 and resistant pairs, cells. SW620 Our results versus indicatedSW620/AD300, that paclitaxel and NCI-H460 (an ABCB1 versus substrate) NCI-H460 had/ anMX20 IC50 cells. value Our of 66.08 results ± 5.81 indicated nM in the that SW620 paclitaxel cells and(an ABCB1about 10,000 substrate) nM hadin the an IC50SW620/AD300 value of 66.08 cells,5.81 thus nM yielding in the SW620 a resistance cells and fold about of about 10,000 80 nM (figure in the ± 1A).SW620 Furthermore,/AD300 cells, mitoxantrone thus yielding (an a resistance ABCG2 foldsubstrate) of about had 80 an (Figure IC501 valueA). Furthermore, of 12.4 ± 1.08 mitoxantrone nM in the NCI-H460(an ABCG2 cells substrate) and 990 had ± an11.21 IC50 nM value in the of 12.4NCI-H460/MX201.08 nM in the cells, NCI-H460 thus yielding cells and a resistance 990 11.21 fold nM of in ± ± >150the NCI-H460 (Figure 1B)./MX20 cells, thus yielding a resistance fold of >150 (Figure1B).

FigureFigure 1.1. CytotoxicityCytotoxicity of paclitaxelof paclitaxel or mitoxantrone or mitoxant inrone parental in andparental ABCB1- and or ABCG2-overexpressingABCB1- or ABCG2- overexpressingcells. The MTT assaycells. The was conductedMTT assay towas determine conducted the etoﬀ ectdetermine of paclitaxel the effect on the of viability paclitaxel of parental on the viabilitySW620 and of ABCB1-overexpressingparental SW620 and SW620ABCB1-overexpressing/AD300 cells (A). TheSW620/AD300 eﬀect of mitoxantrone cells (A). onThe the effect viability of mitoxantrone on the viability of parental NCI-H460 and ABCG2-overexpressing NCI-H460/MX20

Cancers 2020, 12, 498 4 of 17

of parental NCI-H460 and ABCG2-overexpressing NCI-H460/MX20 cells (B). The points with error bars represent the mean SD for independent determinations in triplicate. The above figures are Cancers 2020, 12, x FOR PEER REVIEW± 4 of 17 representative of three independent experiments. cells (B). The points with error bars represent the mean ± SD for independent determinations in triplicate. The above figures are representative of three independent experiments. 2.2. Effect of EC16-1/Saporin on the Cellular Proliferation of Parental and Resistant ABCB1- and ABCG2-Overexpressing Cells 2.2. Effect of EC16-1/Saporin on the Cellular Proliferation of Parental and Resistant ABCB1- and ABCG2- LipidoidOverexpressing EC16-1 Cells was synthesized through using a ring-opening reaction between 1,2-epoxyhexadecaneLipidoid EC16-1 and was N,N synthesized0-Dimethyl-1,3-propanediamine through using a ring-opening [41]. The re MTTaction assay between was 1,2- used to determineepoxyhexadecane the cytotoxicity and N,N of EC16-1′-Dimethyl-1,3-propanediamine alone, saporin alone, or[41]. EC16-1 The /saporin,MTT assay in thewas cell used lines to used in thisdetermine study. As the shown cytotoxicity in Figure of EC16-12A, the alone, IC50 saporin value alone, of saporin or EC16-1/sa was notporin, significantly in the cell lines di ff usederent in in the parentalthis SW620study. As (IC50 shown= >in50 Figure nM) and2A, the ABCB1-overexpressing IC50 value of saporin was SW620 not /significantlyAD300 (IC50 different= >50 in nM) the cells. However,parental EC16-1 SW620/saporin (IC50 = produced>50 nM) and significant ABCB1-overex cytotoxicitypressing inSW620/AD300 the SW620 (IC50 and SW620= >50 nM)/AD300 cells. cells, basedHowever, on the decrease EC16-1/saporin in the IC50 produced value significant (3.44 0.57 cytotoxicity and 2.50 in 0.32the SW620 nM, respectively). and SW620/AD300 A similar cells, e ffect based on the decrease in the IC50 value (3.44± ± 0.57 and 2.50 ±± 0.32 nM, respectively). A similar effect was observed in the parental NCI-H460 and ABCG2-overexpressing NCI-H460/MX20 cells (Figure2B). was observed in the parental NCI-H460 and ABCG2-overexpressing NCI-H460/MX20 cells (Figure In these2B). cells,In these saporin cells, saporin had IC50 had IC values50 values of > of50 >50 nM nM whereas, whereas, EC16-1EC16-1/saporin/saporin had had an anIC50 IC50 value value of of 0.88 0.03 nM and 0.63 0.02 nM, respectively. ±0.88 ± 0.03 nM and 0.63± ± 0.02 nM, respectively.

FigureFigure 2. Cytotoxicity 2. Cytotoxicity of saporin of saporin or EC16-1 or /SaporinEC16-1/Sapo in parentalrin in parental and ABCB1- and orABCB1- ABCG2-overexpressing or ABCG2- cells.overexpressing The MTT assay cells. was The used MTT to assay determine was used the to e ffdetermineect of saporin the effect or EC16-1of saporin/saporin or EC16-1/saporin on the viability of parentalon the viability SW620 andof parental ABCB1-overexpressing SW620 and ABCB1- SW620overexpressing/AD300 SW620/AD300 cells after 72 cells h of after incubation 72 h of (A). Effectincubation of saporin (A or). Effect EC16-1 of /saporinsaporin or on EC16-1/saporin the viability on of parentalthe viability NCI-H460 of parental and NCI-H460 ABCG2-overexpressing and ABCG2- NCI-H460overexpressing/MX20 cells NCI-H460/MX20 (B). The lines cells represent (B). The the lines mean represent of triplicate the mean determinations of triplicate determinations (n = 3). The error (n = 3). The error bars represent the SD. bars represent the SD. We also determined the effects of EC16-1 alone on the proliferation of the cell lines used in this We also determined the effects of EC16-1 alone on the proliferation of the cell lines used in study. EC16-1 produced a slightly higher but non-significant cytotoxicity compared to saporin this study. EC16-1 produced a slightly higher but non-significant cytotoxicity compared to saporin (Tables 1 and 2). These results clearly indicate that the EC16-1/saporin combination potently inhibits (Tablesthe1 cellularand2). Theseproliferation results of clearlyparental, indicate ABCB1, that and theABCG2-overexpressing EC16-1 /saporin combination cells compared potently to saporin inhibits the cellularor EC16-1 proliferation alone. of parental, ABCB1, and ABCG2-overexpressing cells compared to saporin or EC16-1 alone. Table 1. Effect of EC16-1/saporin on the cellular proliferation of SW620 and SW620/AD300 cells. Table 1. Effect of EC16-1/saporin on the cellular proliferation of SW620 and SW620/AD300 cells. SW620 SW620/AD300 EC16-1 SaporinSW620 EC16-1/Saporin EC16-1 SW620Saporin/AD300 EC16-1/Saporin IC50 a >50 >50 3.44 ± 0.63 34.45 ± 2.92 >50 2.50 ± 0.43 RF b EC16-11.00 Saporin 1.00 EC16-1 1.00/Saporin EC16-1 0.69 Saporin 1.00 EC16-1 0.74/Saporin a IC50a Cytotoxicity>50 of EC16-1,> Saporin,50 and 3.44 EC16-1/Sa0.63porin in 34.45 parental2.92 SW620 and> 50ABCB1 overexpressing. 2.50 0.43 b ± ± ± RFSW620/AD3001.00 cell lines; 1.00 an IC50 value 1.00are represented as 0.69 the mean ± SD 1.00 of three independent 0.74 a Cytotoxicityexperiments of EC16-1, performed Saporin, in triplicate. and EC16-1 b/ SaporinValues repres in parentalent the SW620 resistance and ABCB1 fold (RF), overexpressing. which was obtained SW620/AD300 cell lines;by dividing an IC50 valueIC50 values are represented of resistant as thecell mean lines bySD those of three of the independent parental cell experiments lines. performed in triplicate. b Values represent the resistance fold (RF), which was± obtained by dividing IC50 values of resistant cell lines by those of the parental cell lines.

Cancers 2020, 12, 498 5 of 17 Cancers 2020, 12, x FOR PEER REVIEW 5 of 17

TableTable 2.2. EEffectﬀect ofof EC16-1EC16-1/saporin/saporin onon thethe cellular proliferationproliferation ofof NCI-H460 andand NCI-H460NCI-H460/MX20/MX20 cells.cells. NCI-H460 NCI-H460/MX20 NCI-H460 NCI-H460/MX20 EC16-1 Saporin EC16-1/Saporin EC16-1 Saporin EC16-1/Saporin IC50 a 19.57 ±EC16-1 0.93 >50 Saporin 0.88EC16-1 ± 0.12/Saporin 17.51EC16-1 ± 1.32 Saporin >50 EC16-1 0.63/Saporin ± 0.07 IC50 a 19.57 0.93 >50 0.88 0.12 17.51 1.32 >50 0.63 0.07 RF b 1.00 ± 1.00 1.00 ± 0.89± 1.00 ±0.71 RF b 1.00 1.00 1.00 0.89 1.00 0.71 a Cytotoxicity of EC16-1, Saporin, and EC16-1/Saporin in parental NCI-H460 and ABCG2 a overexpressingCytotoxicity of NCI-H460/MX20 EC16-1, Saporin, andcell EC16-1lines. An/Saporin IC50 invalue parental is represente NCI-H460d as and the ABCG2 mean overexpressing± SD of three NCI-H460/MX20 cell lines. An IC50 value is represented as the mean SD of three independent experiments performedindependent in triplicate. experimentsb Values performed represent thein triplicate. resistance foldb Values (RF), which represent± was obtained the resistance by dividing fold IC50 (RF), values which of resistantwas obtained cell lines by by dividing those of theIC50 parental values cell of lines.resistant cell lines by those of the parental cell lines.

2.3.2.3. EEffectffect of EC16-1EC16-1/Saporin/Saporin onon thethe IntracellularIntracellular LocalizationLocalization ofof ABCB1ABCB1 andand ABCG2ABCG2 SinceSince ABCB1ABCB1 andand ABCG2ABCG2 areare membranemembrane proteins,proteins, theirtheir translocationtranslocation toto thethe cytoplasmcytoplasm wouldwould diminishdiminish theirtheir eeffluxfflux function.function. Therefore, EC16-1EC16-1/s/saporinaporin could decrease the intracellularintracellular levels by decreasingdecreasing thethe translocationtranslocation of the ABCB1 and ABCG2ABCG2 transporter proteins into the cell membrane. WeWe used an an immunofluorescence immunofluorescence assay assay to determine to determine the presence the presence of the ofABCB1 the ABCB1 and ABCG2 and proteins ABCG2 proteinsin the parental in the parentaland ABCB1- and ABCB1-and ABCG2-overexpres and ABCG2-overexpressingsing cells incubated cells incubated with EC16-1/saporin. with EC16-1/saporin. EC16- EC16-11/saporin/saporin (5 nM) (5 did nM) not didsignificantly not significantly alter the alterlocalization the localization and internalization and internalization of ABCB1 (Figure of ABCB1 3A) (Figureor ABCG23A) or(Figure ABCG2 3B). (Figure These3 B).findings These findingssuggest suggestthat the thatcytotoxic the cytotoxic effects eofff EC16-1/saporinects of EC16-1/saporin on the onABCB1- the ABCB1- or ABCG2-overexpressing or ABCG2-overexpressing cells were cells werenot due not dueto an to alteration an alteration in the in thecellular cellular localization localization of ofthese these proteins. proteins.

FigureFigure 3.3. EEffectffect of EC16-1EC16-1/Saporin/Saporin onon thethe intracellularintracellular localizationlocalization ofof ABCB1ABCB1 oror ABCG2.ABCG2. The parental andand ABCB1-ABCB1- or or ABCG2-overexpressing ABCG2-overexpressing cells cells were were treated treated with with E16-1 E16-1/saporin,/saporin, at 5 nM, at for5 nM, 24h for and 24h stained and withstained fluorescent with fluorescent ABCB1 orABCB1 ABCG2 or antibody,ABCG2 antibody as described, as described in the material in theand material methods and andmethods observed and underobserved a fluorescent under a microscope.fluorescent The microscope. effect of EC16-1 The /saporineffect of on EC16-1/saporin the intracellular localizationon the intracellular of ABCB1 (localizationA) or ABCG2 of ( BABCB1). DAPI (A (blue)) or ABCG2 counterstains (B). DAPI the nuclei. (blue) Magnification, counterstains20 the (fluorescencenuclei. Magnification, microscopy) 20×. × (fluorescence microscopy). 2.4. Effect of EC16-1/Saporin on the Apoptosis of ABCB1- or ABCG2-Overexpressing Cells 2.4. EffectTo determine of EC16-1/Saporin if the inhibition on the ofApop thetosis growth of ABCB1- of the parental or ABCG2-Overexpressing and ABCB1- and ABCG2-overexpressing Cells cancerTo cellsdetermine by EC16-1 if the/saporin inhibition was of duethe togrow theth inductionof the parental of apoptosis, and ABCB1- the parentaland ABCG2- and ABC-overexpressingoverexpressing cancer cells cells were by EC incubated16-1/saporin with was various due concentrationsto the induction of of EC16-1 apoptosis,/saporin the forparental 24 h andand apoptosisABC-overexpressing was determined cells were as described incubated in with the methods.various concentrations The majority of of EC16-1/saporin the cells were viablefor 24 inh and the controlapoptosis group was anddetermined showed as minimal described or noin the signs meth of apoptosisods. The majority (Figure 4of, bluethe cells color were bars). viable After in incubationthe control withgroup 5 nMand ofshowed saporin, minimal there wasor no a non-significantsigns of apoptosis increase (Figure in the4, blue number color of bars). apoptotic After cellsincubation in SW620, with SW6205 nM of/AD300, saporin, H460, there was and a H460 non-si/MX20.gnificant Interestingly, increase in the EC16-1 number/saporin of apoptotic produced cells a concentration-dependentin SW620, SW620/AD300, increase H460, in theand number H460/MX20. of apoptotic Interestingly, cells in all ofEC16-1/saporin the cell lines. EC16-1 produced/saporin, a atconcentration-dependent 5 nM, produced an increase increase in the in apoptotic the number cell populationof apoptotic in thecells parental in all of SW620 the cell (Figure lines.4A) EC16- and ABCB1-overexpressing1/saporin, at 5 nM, produced SW620/AD300 an increase cells (Figure in the4 B),apoptotic compared cell to population the control in (0.98% the parental vs. 68.70% SW620 and 5.83%(Figure vs. 4A) 53.21%, and respectively).ABCB1-overexpressing Similarly,EC16-1 SW620/AD30/saporin,0 atcells 5 nM, (Figure produced 4B), an compared increase in to the the apoptotic control cell(0.98% population vs. 68.70% in parentaland 5.83% NCI-H460 vs. 53.21%, (Figure respectively).4C) and ABCG2-overexpressing Similarly, EC16-1/saporin, NCI-H460 at 5 nM,/MX20 produced cells (Figurean increase4D), compared in the apoptotic to the control cell (0.76%population vs. 61.02% in parental and 3.11% NCI-H460 vs. 66.10%, (Figure respectively). 4C) and ABCG2-

Cancers 2020, 12, x FOR PEER REVIEW 6 of 17 overexpressing NCI-H460/MX20 cells (Figure 4D), compared to the control (0.76% vs. 61.02% and 3.11% vs. 66.10%, respectively). CancersThese2020, 12 results, 498 indicated that induction of apoptosis by EC16-1/saporin inhibits cancer6 ofcell 17 proliferation in parental, ABCB1-, and ABCG2-overexpressing cancer cells.

FigureFigure 4. EffectEffect of ofEC16-1/saporin EC16-1/saporin on the on induction the induction of apoptosis of apoptosis in parental in parental and ABCB1- and ABCB1- or ABCG2- or ABCG2-overexpressingoverexpressing cells. The cells. parental The parentalSW620 ( SW620A) and (ABCB1-overexprA) and ABCB1-overexpressingessing SW620/AD300 SW620 (/AD300B) cells (wereB) cells incubated were incubated with saporin with saporin(5 nM) or (5 EC16-1/saporin. nM) or EC16-1/saporin. The parental The parentalNCI-H460 NCI-H460 (C) and (ABCG2-C) and ABCG2-overexpressingoverexpressing NCI-H460/MX20 NCI-H460 /(MX20D) cells (D were) cells incubated were incubated with saporin with saporin (5 nM) (5 or nM) EC16-1/saporin. or EC16-1/saporin. The Theapoptotic apoptotic cell population cell population was quantified was quantified using usingflow cytometry. flow cytometry. The bar The graphs bar graphsrepresent represent the average the averagecell population cell population from three from independent three independent experiments experiments and the error and thebars error represent bars representthe SD. * thep < 0.05 SD. *comparedp < 0.05 compared with control. with control. These results indicated that induction of apoptosis by EC16-1/saporin inhibits cancer cell 2.5. EC16-1/Saporin Decreases Tumor Volume and Weight in an ABCB1-Overexpressing Tumor Xenograft proliferation in parental, ABCB1-, and ABCG2-overexpressing cancer cells. Model 2.5. EC16-1Based/ Saporinon in vitro Decreases findings, Tumor we Volumesubsequently and Weight conducted in an ABCB1-Overexpressing experiments to determine Tumor the Xenograft efficacy of ModelEC16-1/saporin in an in vivo mouse tumor xenograft model. Parental (SW620) and ABCB1- overexpressingBased on drug-resistantin vitro findings, (SW620/AD300) we subsequently cells were conducted implanted experiments into athymic nude to determine mice to create the ethefficacy ABCB1-overexpressing of EC16-1/saporin tumor in an inxenograft vivo mouse model. tumor xenograft model. Parental (SW620) and ABCB1-overexpressing drug-resistant (SW620/AD300) cells were implanted into athymic nude mice to create the ABCB1-overexpressing tumor xenograft model. As shown in Figure5, there was a significant increase in the tumor volume in the SW620 (Figure5A) and SW620/AD300 (Figure5B) groups that were treated with saline. The administration of saporin Cancers 2020, 12, x FOR PEER REVIEW 7 of 17 Cancers 2020, 12, x FOR PEER REVIEW 7 of 17 Cancers As2020 shown, 12, 498 in Figure 5, there was a significant increase in the tumor volume in the SW620 (Figure7 of 17 As shown in Figure 5, there was a significant increase in the tumor volume in the SW620 (Figure 5A) and SW620/AD300 (Figure 5B) groups that were treated with saline. The administration of 5A) and SW620/AD300 (Figure 5B) groups that were treated with saline. The administration of saporin (400 μg/kg, q3d × 4, i.p.) or EC16-1 (1 mg/kg, q3d × 4, i.p.) did not significantly reduce the (400saporinµg/kg, (400 q3d μg/kg,4, i.p.)q3d or× 4, EC16-1 i.p.) or (1 EC16-1 mg/kg, (1 q3dmg/kg,4, q3d i.p.) × did 4, i.p.) not significantlydid not significantly reduce thereduce tumor the tumor volume ×compared to animals treated with saline× (Figures 5A,B and 6A,B). However, EC16-1 volumetumor volume compared compared to animals to animals treated withtreated saline with (Figure saline 5(FiguresA,B and 5A,B Figure and6A,B). 6A,B). However, However, EC16-1 EC16-1 (1 mg/kg)/saporin (400 μg/kg, q3d × 4) significantly reduced tumor growth compared to animals (1(1 mg mg/kg)/saporin/kg)/saporin (400 (400µg /μkg,g/kg, q3d q3d4) significantly× 4) significantly reduced reduce tumord tumor growth growth compared compared to animals to treatedanimals treated with saline. There was× a significant reduction in tumor volume in both SW620 and withtreated saline. with There saline. was There a significant was a reductionsignificant in reduction tumor volume in tumor in both volume SW620 in and both SW620 SW620/AD300 and SW620/AD300 tumors in animals treated with EC16-1/saporin, compared to the control, saporin, and tumorsSW620/AD300 in animals tumors treated in withanimals EC16-1 treated/saporin, with EC16-1/saporin, compared to the compared control, saporin, to the control, and EC16-1 saporin, groups. and EC16-1 groups. This effect on the tumor volume was first observed on day 3 and continued until day ThisEC16-1 effect groups. on the This tumor effect volume on the was tumor first volume observed wa ons first day observed 3 and continued on day 3 untiland continued day 12 (final until day) day 12 (final day) of the treatment regimen. Furthermore, on the final day, we measured the weight of of12 the (final treatment day) of regimen. the treatment Furthermore, regimen. on Furthermore, the final day, on we the measured final day, the we weight measured of each the tumorweight in of each tumor in the animals. As shown in Figure 6C,D, there was significant reduction in the weight of theeach animals. tumor Asin the shown animals. in Figure As shown6C,D, therein Figure was 6C,D, significant there reductionwas significant in the reduction weight of in the the tumors weight in of the tumors in the EC16-1/saporin group, compared to the control, saporin, and EC16-1 groups, for thethe EC16-1 tumors/saporin in the group,EC16-1/saporin compared group, to the compared control, saporin, to the andcontrol, EC16-1 saporin, groups, and for EC16-1 both SW620 groups, and for both SW620 and SW620/AD300 tumors. SW620both SW620/AD300 and tumors. SW620/AD300 tumors.

Figure 5. Effect of EC16-1/saporin on the parental and ABCB1 or ABCG2 tumors in nude athymic FigureFigure 5. 5.E ﬀEffectect of of EC16-1 EC16-1/saporin/saporin on on the the parental parental and an ABCB1d ABCB1 or ABCG2 or ABCG2 tumors tumors in nude in athymicnude athymic mice. mice. The images of excised SW620 (A) and SW620/AD300 (B) tumors implanted subcutaneously in Themice. images The ofimages excised of SW620excised (ASW620) and SW620(A) and/AD300 SW620/AD300 (B) tumors (B implanted) tumors implanted subcutaneously subcutaneously in athymic in athymic NCR nude mice (n = 6). Images of the excised NCI-H460 and (C) and NCI-H460/MX20 (D) NCRathymic nude NCR mice nude (n = mice6). Images (n = 6). of Images the excised of the NCI-H460 excised NCI-H460 and (C) and and NCI-H460 (C) and NCI-H460/MX20/MX20 (D) tumors (D) tumors implanted subcutaneously in athymic NCR nude mice (n = 6). The images were taken at the implantedtumors implanted subcutaneously subcutaneously in athymic in NCR athymic nude NCR mice nude (n = 6). mice The (n images = 6). The were images taken were at the taken end of at the the end of the 12-day treatment period. 12-dayend of treatment the 12-day period. treatment period.

Saline A Saline B Saline μ Saline Saporin 400 g/kg 3000A Saporin 400 μg/kg 3000B Saporin 400 μg/kg 3000 Saporin 400 μg/kg 3000 EC16-1 1mg/kg EC16-1 1mg/kg 3) 3) EC16-1 1mg/kg EC16-1 1mg/kg Saporin (400 µg/kg) / EC16-1 (1mg/kg) 3) Saporin (400 µg/kg) / EC16-1 (1mg/kg) 3) Saporin (400 µg/kg) / EC16-1 (1mg/kg) Saporin (400 µg/kg) / EC16-1 (1mg/kg) 2000 2000 2000 2000

# 1000 1000 # 1000 # 1000 # Tumor volume (mm volume Tumor (mm volume Tumor Tumor volume (mm volume Tumor (mm volume Tumor 0 0 0 03691215 0 03691215 03691215 03691215 Days of treatment Days of treatment Days of treatment C Days of treatment C Figure 6. Cont. D

CancersCancers2020 2020, 12, 12, 498, x FOR PEER REVIEW 8 of8 of 17 17 Cancers 2020, 12, x FOR PEER REVIEW 8 of 17 Days of treatment Days of treatment C Days of treatment Days of treatment 1.5C D 1.5 1.5D 1.5

1.0 1.0 1.0 # 1.0 # * # * # * * 0.5 Tumor wt (g) 0.5 0.5 Tumor wt (g) Tumor wt (g) 0.5 Tumor wt (g)

0.0 0.0 0.0 0.0 Saline Saporin EC16-1 EC16-1 (1 mg/kg) Saline Saporin EC16-1 EC16-1 (1 mg/kg) Saline (400 Saporin µg/kg) (1mg/kg)EC16-1 Saporin(400EC16-1 (1 mg/kg)µg/kg) Saline (400 Saporin µg/kg) (1mg/kg)EC16-1Saporin(400EC16-1 (1 mg/kg)µg/kg) (400 µg/kg) (1mg/kg) Saporin(400 µg/kg) (400 µg/kg) (1mg/kg) Saporin(400 µg/kg) Figure 6. Effect of EC16-1/saporin on the volume and weight of SW620 and SW620/AD300 tumors. FigureFigure 6. 6.E ffEffectect of of EC16-1 EC16-1/saporin/saporin on on the the volume volume and and weight weight of of SW620 SW620 and and SW620 SW620/AD300/AD300 tumors. tumors. The changes in tumor volume over time in SW620 (A) and SW620/AD300 (B) in xenografts following TheThe changes changes in in tumor tumor volume volume over over time time in in SW620 SW620 (A ()A and) and SW620 SW620/AD300/AD300 (B )(B in) in xenografts xenografts following following implantation. Each point on the line graph represents the mean tumor volume (mm3 3) on each day implantation.implantation. Each Each point point on on the the line line graph graph represents represents the the mean mean tumor tumor volume volume (mm (mm)3 on) on each each day day after implantation. The error bars represent the SD. The mean weight (n = 6) of the excised SW620 (C) afterafter implantation. implantation. The The error error bars bars represent represent the the SD. SD. The The mean mean weight weight (n (=n 6)= 6) of of the the excised excised SW620 SW620 (C ()C) and SW620/AD300 (D) tumors from the mice treated with saline, saporin, EC16-1, and EC16- andand SW620 SW620/AD300/AD300 (D) ( tumorsD) tumors from from the mice the treated mice treated with saline, with saporin, saline, EC16-1,saporin, and EC16-1, EC16-1 and/saporin, EC16- 1/saporin, at the end of the 12-day treatment period. The error bars represent the SD. * p < 0.05, at1/saporin, the end of theat the 12-day end treatmentof the 12-day period. treatment The error period. bars representThe error the bars SD. represent * p < 0.05, the compared SD. * p with< 0.05, compared with the saporin# alone group. # p < 0.05, compared with the control group. thecompared saporin alone with group.the saporinp < alone0.05, comparedgroup. # p < with 0.05, the compared control group. with the control group. 2.6.2.6. EC16-1 EC16-1/Saporin/Saporin Decreases Decreases Tumor Tumor Volume Volume and an Weightd Weight in ABCG2-Overexpressing in ABCG2-Overexpressing Tumor Tumor Xenograft Xenograft Models 2.6. EC16-1/Saporin Decreases Tumor Volume and Weight in ABCG2-Overexpressing Tumor Xenograft Models ModelsIn order to investigate the effect of EC16-1/saporin on the ABCG2-overexpressing tumors, the parentalIn order (NCI-H460) to investigate and the ABCG2-overexpressing effect of EC16-1/saporin (NCI-H460 on the ABCG2-overexpressing/MX20) cells were implanted tumors, into the In order to investigate the effect of EC16-1/saporin on the ABCG2-overexpressing tumors, the athymicparental nude (NCI-H460) mice to create and theABCG2-overexpressing ABCG2-overexpressing (NCI-H460/MX20) tumor xenograft model.cells were implanted into parental (NCI-H460) and ABCG2-overexpressing (NCI-H460/MX20) cells were implanted into athymicSimilar nude to themice SW620 to create and the SW620 ABCG2-/AD300overexpressing tumors, there tumor was xenograft a significant model. increase in the tumor athymic nude mice to create the ABCG2-overexpressing tumor xenograft model. volumeSimilar in the to NCI-H460 the SW620 (Figures and SW620/AD3005C and7A) and tumors, NCI-H460 there was/MX20 a significant (Figures 5increaseD and7 B)in tumorsthe tumor Similar to the SW620 and SW620/AD300 tumors, there was a significant increase in the tumor involume animals in treated the NCI-H460 with saline. (Figures There 5C wasand a7A) significant and NCI-H460/MX20 reduction in the(Figures tumor 5D volume and 7B) in tumors animals in volume in the NCI-H460 (Figures 5C and 7A) and NCI-H460/MX20 (Figures 5D and 7B) tumors in treatedanimals with treated EC16-1 with/saporin, saline. There compared was a to significant the control, reduction saporin, in or the EC16-1 tumor groups, volume in in both animals NCI-H460 treated animals treated with saline. There was a significant reduction in the tumor volume in animals treated andwith NCI-H460 EC16-1/saporin,/MX20 tumors.compared This to the eff control,ect on the saporin, tumor or volumeEC16-1 groups, was first in observedboth NCI-H460 on day and 3 andNCI- with EC16-1/saporin, compared to the control, saporin, or EC16-1 groups, in both NCI-H460 and NCI- continuedH460/MX20 until tumors. day 12 This (final effect day) on of the the tumor treatment volume regimen. was first In observed addition, on we day measured 3 and continued the effect until of H460/MX20 tumors. This effect on the tumor volume was first observed on day 3 and continued until EC16-1day 12/saporin (final day) on theof the weight treatment of the regimen. tumors. EC16-1In addition,/saporin we significantlymeasured the decreased effect of theEC16-1/saporin tumor weight on day 12 (final day) of the treatment regimen. In addition, we measured the effect of EC16-1/saporin on inthe both weight NCI-H460 of the tumors. and NCI-H460 EC16-1/saporin/MX20 tumors, significan comparedtly decreased to the control,the tumor saporin, weight or in EC16-1 both NCI-H460 groups the weight of the tumors. EC16-1/saporin significantly decreased the tumor weight in both NCI-H460 (Figureand NCI-H460/MX207C,D). tumors, compared to the control, saporin, or EC16-1 groups (Figure 7C,D). and NCI-H460/MX20 tumors, compared to the control, saporin, or EC16-1 groups (Figure 7C,D). A B 4000A B4000 Saline Saline 4000 4000 Saline μ 3) 3) Saline Saporin 400 g/kg Saporin 400 μg/kg μ 3) 3) Saporin 400 g/kg Saporin 400 μg/kg EC16-1 1mg/kg 3000 EC16-1 1mg/kg 3000 EC16-1 1mg/kg 3000 SaporinEC16-1 (4001mg/kg µg/kg) / EC16-1 (1mg/kg) 3000 Saporin (400 µg/kg) / EC16-1 (1mg/kg) Saporin (400 µg/kg) / EC16-1 (1mg/kg) Saporin (400 µg/kg) / EC16-1 (1mg/kg) 2000 2000 2000 2000

1000 1000 1000 1000 # Tumor volume (mm Tumor Tumor volume (mm volume Tumor # # Tumor volume (mm Tumor Tumor volume (mm volume Tumor # 0 0 003691215 003691215 03691215 03691215 Days of treatment Days of treatment Days of treatment Days of treatment Figure 7. Cont.DD

CancersCancers 20202020,, 1212,, 498x FOR PEER REVIEW 99 ofof 1717 Cancers 2020, 12, x FOR PEER REVIEW 9 of 17 y y y C D y 2.5C 2.5D 2.5 2.5 2.0 2.0 2.0 2.0 1.5 1.5 1.5 1.5 # # 1.0 * # 1.0 * # 1.0 * 1.0 * Tumor wt (g) Tumor wt (g) Tumor wt (g) 0.5 Tumor wt0.5 (g) 0.5 0.5 0.0 0.0 Saline Saporin EC16-1 EC16-1 (1 mg/kg) 0.0 Saline Saporin EC16-1 EC16-1 (1 mg/kg) 0.0 Saline (400 Saporinµg/kg) (1mg/kg)EC16-1Saporin(400EC16-1 (1 µg/kg) mg/kg) Saline Saporin EC16-1 EC16-1 (1 mg/kg) (400 µg/kg) (1mg/kg) Saporin(400 µg/kg) (400 µg/kg) (1mg/kg) Saporin(400 µg/kg) (400 µg/kg) (1mg/kg) Saporin(400 µg/kg) Figure 7. Effect of EC16-1/saporin on the tumor volume and weight of NCI-H460 and NCI- FigureFigure 7. E7.ff ectEffect of EC16-1 of EC16-1/saporin/saporin on the on tumor the volumetumor andvolume weight and of NCI-H460weight of andNCI-H460 NCI-H460 and/MX20 NCI- H460/MX20 tumors. The changes in tumor volume over time in NCI-H460 (A) and NCI-H460/MX20 tumors.H460/MX20 The changes tumors. in The tumor changes volume in overtumor time volume in NCI-H460 over time (A )in and NCI-H460 NCI-H460 (A/)MX20 and NCI-H460/MX20 (B) xenografts (B) xenografts following tumor implantation. Each point on the line graph represents the mean tumor following(B) xenografts tumor following implantation. tumor Eachimplantation. point on Each the linepoint graph on the represents line graph the repr meanesents tumor the mean volume tumor volume (mm3) on each day after implantation. The mean weight (n = 6) of the excised NCI-H460 (C) (mm3)volume on (mm each3) day on each after day implantation. after implantation. The mean The weight mean (weightn = 6) of(n the= 6)excised of the excised NCI-H460 NCI-H460 (C) and (C) and NCI-H460/MX20 (D) tumors from the mice treated with saline, saporin, EC16-1, and EC16- NCI-H460and NCI-H460/MX20/MX20 (D) tumors (D) tumors from the from mice the treated mice with treated saline, with saporin, saline, EC16-1, saporin, and EC16-1, EC16-1 and/saporin, EC16- 1/saporin, at the end of the 12-day treatment period. The error bars represent the SD. * p < 0.05, at1/saporin, the end of theat the 12-day end treatmentof the 12-day period. treatment The error period. bars represent The error the bars SD. represent * p < 0.05, the compared SD. * p to < the0.05, compared to the saporin# alone group. # p < 0.05, compared with the control group. saporincompared alone to group. the saporinp < 0.05,alone compared group. # p with < 0.05, the compared control group. with the control group. 2.7.2.7. Assessment of Potential Toxicity ofof EC16-1EC16-1/Saporin/Saporin 2.7. Assessment of Potential Toxicity of EC16-1/Saporin GivenGiven that toxicity is a major concern for any ch chemotherapeuticemotherapeutic regimen, we regularly measured Given that toxicity is a major concern for any chemotherapeutic regimen, we regularly measured thethe bodybody weightsweights ofof eacheach animalanimal asas aa generalgeneral indicatorindicator ofof theirtheir overalloverall healthhealth throughoutthroughout thethe coursecourse the body weights of each animal as a general indicator of their overall health throughout the course ofof the study. The The body body weight weight of of each each animal animal wa wass recorded recorded every every three three days, days, beginning beginning on day on day0 and 0 of the study. The body weight of each animal was recorded every three days, beginning on day 0 and andending ending on day on day12 (final 12 (final day). day). As shown As shown in Figure in Figure 8A, there8A, there was wasno significant no significant change change in the in body the ending on day 12 (final day). As shown in Figure 8A, there was no significant change in the body bodyweight weight of the ofanimals the animals in the inABCB1 the ABCB1 tumor tumor xenogr xenograftaft models. models. These Theseresults resultsindicated indicated that EC16- that weight of the animals in the ABCB1 tumor xenograft models. These results indicated that EC16- EC16-11/saporin/saporin produces produces anti-cancer anti-cancer efficacy efficacy in inparental parental and and ABCB1-overexpressing ABCB1-overexpressing tumors tumors without 1/saporin produces anti-cancer efficacy in parental and ABCB1-overexpressing tumors without producingproducing anyany overtovert toxicitytoxicity oror weightweight loss.loss. However, the animal body weight of thethe animalsanimals waswas producing any overt toxicity or weight loss. However, the animal body weight of the animals was significantlysignificantly decreased by the EC16-1EC16-1/saporin/saporin treatmenttreatment comparedcompared withwith dayday 1,1, inin thethe ABCG2ABCG2 tumortumor significantly decreased by the EC16-1/saporin treatment compared with day 1, in the ABCG2 tumor xenograftxenograft modelsmodels (Figure(figure 88B).B). xenograft models (figure 8B).

FigureFigure 8.8. EEffectﬀect of of EC16-1 EC16-1/saporin/saporin on on the the body body weight weight of animals of animals bearing bearing parental parental and ABCB1 and ABCB1 or ABCG2 or Figure 8. Effect of EC16-1/saporin on the body weight of animals bearing parental and ABCB1 or tumors.ABCG2 Thetumors. changes The in changes the mean in body the weight mean of body mice (weightn = 6) bearing of mice SW620 (n = and 6) SW620bearing/AD300 SW620 tumors and ABCG2 tumors. The changes in the mean body weight of mice (n = 6) bearing SW620 and (SW620/AD300A) and the changes tumors in ( theA) meanand the body changes weight in the of mice mean (n body= 6) bearingweight of NCI-H460 mice (n =and 6) bearing NCI-H460 NCI-H460/MX20 SW620/AD300 tumors (A) and the changes in the mean body weight of mice (n = 6) bearing NCI-H460 tumorsand NCI-H460/MX20 (B). The changes tumors in the (B body). The weight changes are in plottedthe body for weight day 0 are (before) plotted and for day day 12 0 (after)(before) of and the and NCI-H460/MX20 tumors (B). The changes in the body weight are plotted for day 0 (before) and treatment.day 12 (after) The of barthe graphstreatment. represent The bar the graphs mean re bodypresent weight the mean and thebody error weight bars and represent the error the bars SD. day 12 (after) of the treatment. The bar graphs represent the mean body weight and the error bars *representp < 0.05 comparedthe SD. * p with < 0.05 day compared 0. with day 0. represent the SD. * p < 0.05 compared with day 0. 3. Discussion 3. Discussion 3. Discussion Following the approval of imatinib by the United States Food and Drug Administration in Following the approval of imatinib by the United States Food and Drug Administration in 2001, 2001, severalFollowing anti-cancer the approval drugs of have imatinib been developedby the United for States the treatment Food and of Drug both benignAdministration and malignant in 2001, several anti-cancer drugs have been developed for the treatment of both benign and malignant tumorsseveral [44 anti-cancer,45]. The majority drugs have of these been drugs developed produce for cytotoxic the treatment eﬀects of on both tumor benign cells withand minimalmalignant tumors [44,45]. The majority of these drugs produce cytotoxic effects on tumor cells with minimal tumors [44,45]. The majority of these drugs produce cytotoxic effects on tumor cells with minimal adverse effects in normal cellular physiology [46,47]. The discovery of novel pathways and adverse effects in normal cellular physiology [46,47]. The discovery of novel pathways and

Cancers 2020, 12, 498 10 of 17 adverse effects in normal cellular physiology [46,47]. The discovery of novel pathways and mechanisms causing cancer has facilitated the development of targeted compounds that attenuate tumor growth with minimal toxicity. Despite the advances in pharmacotherapeutics and translational medicine, the vast majority of patients fail to respond to conventional treatment strategies. This leads to the development of drug resistance and an increase in the overall mortality associated with cancer. Novel drug delivery techniques, such as encapsulation of a chemotherapeutic drug in a nano-carrier, has yielded significant success in improving the survival of cancer patients. Nanoparticle formulations have reduced particle size (in the nanometer range) and can be designed to deliver multiple drugs simultaneously [48]. Studies have reported that nanoparticles, as small as 100 nm, can easily penetrate the cell membrane through active endocytosis and reach the tumor tissue [49]. Anticancer nanoparticle drug formulations can deliver one or more chemotherapeutic drug (such as paclitaxel or doxorubicin) by encapsulating them in a protective coating. This protective layer can be a polymer, such as polyethylene glycol (PEG), which delivers the drug to the cellular target site, without being subjected to metabolism or degradation [50]. Nanoparticles offer a number of advantages over conventional drug delivery techniques, which includes: (1) Greater retention in the blood due to the reduced particle size; (2) easier delivery of drugs that have poor solubility and stability; (3) decreased drug clearance and increased bioavailability; and (4) a multi-functional and multi-targeted drug delivering platform [51–53]. Owing to their aforementioned advantages compared to conventional drug delivery systems, nanoparticles have also been developed to treat resistance to chemotherapeutic drugs. An increasing number of synthetic nanoparticles have been designed for this purpose, such as liposomes, polymeric nanoparticles, polymeric micelles, and dendrimers. PEGylate liposome (Doxil) liposomes loaded with doxorubicin and non-PEGylated liposome (Myocet) are clinically used to treat certain types of cancers. In addition, lipoplatin is a promising liposomal platinum drug formulation that is currently undergoing clinical investigation [54–56]. Recently, Wang and colleagues reported that a tea–nanoparticle system effectively delivered doxorubicin and overcame ABC transporter-mediated drug resistance by increasing the intracellular concentration of doxorubicin [57]. This system produced a greater drug accumulation and reversal of in vitro and in vivo MDR in a drug-resistant cancer model compared to conventional doxorubicin therapy. Saporin, a potent cytotoxic compound, has been shown to have anti-cancer efficacy. However, its use in anticancer treatment regimens has been limited due to its poor diffusion across the cell membrane. A strategy to kill MDR cells is to use cytotoxic proteins/drugs that are too large to be the ABC efflux transporters. Here, we determined the in vitro and in vivo efficacy of saporin encapsulated in a lipid-based system, EC16-1. Previous studies have shown that the combination EC16-1 and saporin has an enhanced entry into various cancer cell lines as compared to saporin [41,43]. We conducted experiments to determine the anti-cancer efficacy of EC16-1/saporin and its interaction with the ABC transporters ABCB1 and ABCG2. To our knowledge, this is the first study to report the in vitro and in vivo anti-cancer efficacy of EC16-1/saporin on parental and ABCB1- and ABCG2-overexpressing cells and in mouse xenograft tumor models. Our results indicated that the ABCB1-overexpressing SW620/AD300 cells were greater than 80-fold resistant, as compared to the parental SW620 cells, whereas the ABCG2-overexpressing NCI-H460/MX20 cells were greater than 150-fold resistant, compared to the non-resistant parental NCI-H460 cells. As reported previously, the resistance fold (RF) was calculated by dividing the substrate drug IC50 values in the drug-resistant cells by the IC50 values in the parental cells [10,46,58]. The resistant profile of our cells was comparable to the previously published studies [12,59]. Our results indicated that our parental ABCB1- and ABCG2-overexpressing cells were an acceptable model to evaluate the anti-cancer and reversal efficacy of EC16-1/saporin. Cancers 2020, 12, 498 11 of 17

We also investigated the efficacy of EC16-1/saporin on the proliferation of parental and ABCB1- or ABCG2-overexpressing cells. Our results indicated that the EC16-1/saporin combination produced significantly greater cytotoxicity compared to saporin alone. Since a significant decrease in the IC50 value of EC16-1/saporin was observed in both parental and ABCB1- or ABCG2-overexpressing cells, it can be concluded that the cytotoxic efficacy of EC16-1/saporin is not significantly affected by the presence of the ABCB1 or ABCG2 transporters. However, because the localization of ABC transporters is a major factor that regulates their efflux function, we determined the effect of EC16-1/saporin on the intracellular localization of the ABCB1 or ABCG2 transporters. The incubation of cells with EC16-1/saporin, at 5 nM, for 24 h, did not significantly alter the intracellular localization of the ABCB1 or ABCG2 transporters. Indeed, the expression of the ABCB1 and ABCG2 transporters was almost exclusively localized on the cell membrane. These results are in accordance with earlier studies, where chemotherapeutic-based nanoparticle formulations were reported to have no significant effect on the in vitro intracellular localization of ABC transporters [57,60]. Since our cell proliferation studies confirmed that EC16-1/saporin is a cytotoxic compound in the cancer cell types used in the study, we subsequently determined the effect of EC16-1/saporin on the induction of apoptosis in the parental and ABCB1- and ABCG2-overexpressing cells. Our results indicated that EC16-1/saporin induced apoptosis in parental and ABCB1- or ABCG2-overexpressing cells. Moreover, a concentration-dependent induction in apoptosis was observed following incubation with EC16-1/saporin for 24 h. These findings suggested that the cytotoxic effects of EC16-1/saporin in the parental and the ABCB1- and ABCG2-overexpressing cells are due to an induction of apoptosis. This finding is similar to previous studies indicating that certain saporin can produce cytotoxicity by inducing apoptosis [61,62]. The findings based on our in vitro experiments led us to determine the effect of EC16-1/saporin in the parental and the ABCB1- and ABCG2-overexpressing cancer cells using mouse xenograft tumor models. As previously reported, we established ABCB1- or ABCG2-overexpressing tumor xenograft models, with a slight modification [13,58]. The animals treated with saline (control group) had a significant increase in tumor growth over the 12-day treatment period. The saporin or EC16-1 alone treatment groups did not significantly attenuate tumor growth compared to the control group. However, the EC16-1/saporin combination treatment significantly inhibited tumor growth. A significant reduction in tumor volume and weight occurred in the parental and ABCB1- or ABCG2-overexpressing tumors, compared to the single treatment groups. Furthermore, EC16-1/saporin produced significant weight loss during the study period. The safety profile of EC16-1/saporin has not been determined in humans.

4. Materials and Methods

4.1. Reagents Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and trypsin 0.25% were purchased from Hyclone (Pittsburgh, PA, USA). Monoclonal antibody against ABCB1 and ABCG2 were purchased from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Alexa Fluor 488 conjugated goat anti-mouse IgG secondary antibody was also purchased from Thermo Fisher Scientific Inc. Paclitaxel and mitoxantrone were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Propidium iodide (PI) and Annexin-V were purchased from BD biosciences (San Jose, CA, USA).

4.2. EC16-1/Saporin Formulation Preparation The synthetic lipid EC16-1 and lipid/saporin formulation were prepared according to our published procedure [41]. In brief, the EC16-1 was synthesized using a ring-opening reaction between 1,2-epoxyhexadecane and N,N0-Dimethyl-1,3-propanediamine. The reactants were heated at Cancers 2020, 12, 498 12 of 17 molar ratios of 1:2.4 without any solvent for 48 h, and purified using flash chromatography on silica gel. For in vitro experiment, EC16-1 was directly used for saporin complexation and delivery. For in vivo formulation, EC16-1/saporin nanoparticles were formulated by a thin film hydration method. Briefly, EC16-1, cholesterol, and DOPE were mixed at a weight ratio of 16:4:1 in chloroform, and the organic solvent was evaporated under vacuum. To formulate EC16-1/protein nanoparticles, thin layer film that had formed was re-hydrated with PBS to make a cloudy solution. The solution was then mixed with saporin at a weight/weight ratio of 2.5:1 (EC16-1:saporin). The EC16-1/protein complex was post-modified with mPEG2000-ceramide C16 (25% weight percentage in total compared to EC16-1), followed by a further 60 min of incubation at room temperature. The final EC16-1 concentration in all formulations was kept at 1 mg/mL.

4.3. Cell Lines and Cell Culture The human colorectal adenocarcinoma cell line, SW620, and the doxorubicin-induced SW620/AD300-resistant cell line (overexpressing ABCB1) were used in this study. In addition, the non-small cell lung cancer cell line NCI-H460 and mitoxantrone-induced NCI-H460/MX20-resistant cell line (overexpressing ABCG2) were also used. The cells were cultured in DMEM media supplemented with 10% FBS and 1% penicillin/streptomycin at 37 ◦C in a humidiﬁed atmosphere of 5% CO2. The cells were grown in culture ﬂasks as monolayers in drug-free medium for at least more than 2 weeks before the performed assays.

4.4. MTT Assay The anti-proliferative efficacy of EC16-1, saporin, or the EC16-1/saporin combination were determined by a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric assay. Briefly, the cells were seeded onto 96-well plates at a density of 4 103 cells × per well. Following 24 h of incubation, the cells were incubated with EC16-1, saporin, or EC16/saporin at the indicated concentrations. After 72 h, 20 µL of MTT (4 mg/mL) was added to each well and the plates were further incubated at 37 ◦C for 4 h. Subsequently, the MTT–media solution was removed from each well and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals formed by the mitochondrial reduction of MTT in proliferating cells. The absorbance values were then measured at 570 nm using an Opsys microplate reader (Dynex Technologies, Chantilly, VA, USA).

4.5. Immunofluorescence Cells (3 104) were seeded in 24-well plates and incubated overnight, followed by incubation × with 5 nM of EC16-1/saporin for 24 h. After 24 h, the cells were fixed in 4% paraformaldehyde for 15 min and permeabilized by 0.1% Triton X-100 for 10 min. Subsequently, blocking was done with 6% BSA for 1.5 h at room temperature. The cells were incubated with monoclonal antibody C219 against ABCB1 (1:400) and BXP-21 against ABCG2 (1:400) overnight, followed by Alexa Fluor 488 conjugated secondary antibody (1:1000) for 1 h. DAPI was used to counterstain the nuclei. Immunofluorescence images were collected using a Nikon TE-2000S fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA).

4.6. Apoptosis Assay The cells were incubated with either saporin alone at 5 nM or EC16-1/saporin at 0.5, 1, 2, and 5 nM for 24 h. After 24 h, the cells were collected and washed twice with PBS supplemented with 0.5% bovine serum albumin (BSA) at 4 ◦C. The cell pellet was ﬁxed overnight in ice cold 70% ethanol at 4 ◦C. The cells were subsequently stained with FITC-labeled Annexin-V (BD Pharminogen, San Diego, CA, USA) and PI, at 37 ◦C for 30 min. Flow cytometric analysis was performed to determine the apoptotic cell population. Cancers 2020, 12, 498 13 of 17

4.7. Experimental Animals Four- to six-week-old male athymic NCR (nu/nu) nude mice (homozygous, albino color), weighing 18–22 g, were used for the ABCB1 or ABCG2 xenograft models. These animals were purchased from Taconic Farms (Taconic Biosciences, New York, NY, USA). All animals were provided with sterile water, rodent chow ad libitum, and maintained on an alternating 12-h light/dark cycle. All animals were housed at the animal facility of St. John’s University. All animals were treated humanely in accordance with the guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at St. John’s University (Queens, NY, USA) and carried out in accordance with the guidelines on animal care and experiments of laboratory animals (animal study protocol number: 1842).

4.8. Methodology for Preclinical Antitumor Efficacy Briefly, matching cell line pairs, parental and resistant, were implanted into the left and right armpit region, respectively, of each mouse. The parental SW620 (6 106) and ABCB1-overexpressing × SW620/AD300 (6 106) cells were injected subcutaneously into one set of mice. The parental NCI-H460 × (5 106) and ABCG2-overexpressing NCI-H460/MX20 (5 106) cells were injected subcutaneously × × into the second set of mice. Once the tumors reached a mean diameter of 0.5 cm, the mice were randomly dived into four treatment groups (n = 6) and administered either: (a) Vehicle (normal saline, q3d 4, i.v.); (b) saporin (400 µg/kg, q3d 4, i.v.); (c) EC16-1 (1 mg/kg, q3d 4, i.v.); and (d) EC16-1 × × × (1 mg/kg)/saporin (400 µg/kg) (q3d 4, i.v.). The doses selected for saporin and EC16-1 were non-toxic × to the animals and were based on the results of our pilot study. The animals were monitored carefully, and tumor sizes were measured and recorded using a caliper. The body weight of animals was recorded every 3rd day to access any drug-related toxicities as well as disease progression. The tumor volume was calculated by using the following formula: V = π/6 ((A+B)/2)3, where A and B are the two perpendicular diameters of a tumor. At the end of the study, all the animals were euthanized using carbon dioxide. The tumor tissues were excised, weighed, and stored at 80 C until further analysis. − ◦ 4.9. Statistical Analysis All the experiments were repeated at least three times and the differences were determined using the two-tailed Student t-test. The a priori significance level was p < 0.05.

5. Conclusions Our results indicated that saporin, a potent cytotoxic compound, when encapsulated in the lipid-based EC16-1 nanoparticle, significantly inhibits the proliferation of cancer cells. The EC16-1/saporin complex induces apoptosis in the parental cancer cells and in the resistant ABCB1- and ABCG2-overexpressing cells and does not significantly alter the intracellular localization of ABCB1 or ABCG2. Finally, EC16-1/saporin significantly decreases the growth of the parental and ABC transport-mediated resistant tumors in vivo, based on our tumor xenograft mouse models. Therefore, the EC16-1/saporin nanocomplex represents a novel anticancer strategy, not only for cancer patients with non-resistant tumor but also for patients with ABCB1 or ABCG2 drug-resistant cancers.

Author Contributions: Designed the study, G.-N.Z., Y.-F.F., Q.X. and Z.-S.C.; performed the experiments, G.-N.Z., M.W., A.M.B., P.G., Y.-K.Z., L.Z.; wrote and revised the manuscript, G.-N.Z., P.G., C.R.A.J., and Q.X. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the St. John’s University Research Seed Grant (No. 579-1110-7002) for Zhe-Sheng Chen and National Institutes of Health (NIH) Grant R01 EB027170-01 for Qiaobing Xu. Acknowledgments: The authors are thankful to Susan E. Bates and Robert W. Robey (NIH, MD) for providing NCI-H460 and NCI-H460/MX20, SW620 and SW620/AD300 cell lines (NIH, MD). Conﬂicts of Interest: The authors declare no conﬂict of interest. Cancers 2020, 12, 498 14 of 17

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