Prospects for multitarget lipid-raft-coated silica beads: a remarkable online biomaterial for discovering multitarget antitumor lead compounds

Abstract

A recently discovered receptor-rich lipid raft, which is linked to numerous transmembrane signal transduction pathways, has emerged as a medically significant biomaterial for screening antitumor agents. Compounds that interact with the biomaterial might potentially inhibit cancer cell growth, and thus be valuable as antitumor agents. Two standard anticancer drugs, lestaurtinib and gefitinib, interacted with the tropomyosin-related tyrosine kinase A (TrkA) receptor-rich lipid raft. The fact that they are both well known inhibitors of TrkA receptor strengthened the observed linkage. There is now a considerable interest in developing other related biomaterials to serve a similar purpose, and more importantly, support the concept of multitarget drug discovery. It is expected that new anticancer drugs strategically act on multiple pathways to achieve optimal therapeutic efficacies and decreased stimulation of acquired resistance. However, the current conventional approaches such as chemical screening systems and in silico methods cannot fully satisfy the increasing demand for the therapeutic agents. It has therefore become imperative to explore other alternatives to increase the number of clinically important antitumor agents. Here, we report the prospect of establishing lipid raft biomaterial with well endowed multiple cancer-related receptors for screening antitumor leads that affect multiple pathways. This review also examines receptor–ligand interactions in the hunt for novel antitumor agents and the associated key receptors.

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Caleb Kesse Firempong

Caleb Kesse Firempong was awarded BSc (2000) and MPhil (2004) in Biochemistry from University of Ghana, Legon, and Kwame Nkrumah University of Science and Technology (KNUST), Kumasi-Ghana, respectively. He completed his PhD at Jiangsu University, where he focused on lipid raft biomaterials for screening novel antitumor agents from natural sources under the tutelage of Ximing Xu. His research interests include cancer drug development, high-throughput screening, formulation of active compounds and biochemical analysis. Currently, he doubles as lecturer (Department of Biochemistry and Biotechnology) and laboratory manager (Clinical Analysis Laboratory) at KNUST.

Xia Cao

Xia Cao received both her MSc (2008) and PhD (2011) in Clinical Diagnostics from Jiangsu University. Her PhD study was centred on drug/gene delivery and tissue engineering under the guidance of Ximing Xu. She is currently a lecturer at School of Pharmacy, Jiangsu University, where her research directions include the construction of tissue engineering cartilage, induced differentiation of cancer stem cells, novel nano vector for drug/gene delivery and induced pluripotent stem cell with non-viral methods.

Shanshan Tong

Shanshan Tong is currently an Associate Professor in School of Pharmacy, Jiangsu University and a visiting scholar at University of Southern California. She obtained her PhD in Clinical Diagnostics under the leadership of Jiangnan Yu and Ximing Xu at Jiangsu University in 2012. She is an expert in pharmaceutical analysis, acquisition of active natural compounds and development of high-throughput screening technology.

Jiangnan Yu

Jiangnan Yu obtained both her MSc (1993) and PhD (1996) at China Pharmaceutical University. She is currently a Professor at Department of Pharmaceutics; Deputy Director, Department of Science and Technology; and Vice-chairman, New Traditional Chinese Medicine Development Committee of Jiangsu Province, all at Jiangsu University. Her research areas include biopharmaceutical analysis, quality evaluation of novel particulate drug delivery systems and in vitro model drug metabolism via tissue engineering techniques.

Ximing Xu

Ximing Xu completed his PhD at China Pharmaceutical University in 1999. He became a visiting Associate Professor/Research Fellow at Tissue Engineering Laboratories (VA Boston Healthcare System), Harvard Medical School, in 2004. He was also a Research Fellow (2006) at Laboratory of Gene Transfer and Tissue Repair, Harvard Medical School. Presently, he is a Professor of Pharmaceutics; Dean, School of Pharmacy; and Director, Centre for Nano Drug/Gene Delivery and Tissue Engineering at Jiangsu University. His studies focus on natural biomaterials for nano-gene transfer and tissue engineering, controlled release preparations, formulation of poorly soluble drugs and high-throughput screening.

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1. Introduction

Growing evidence that several effective drugs produce their actions via interactions with multiple targets is boosting the development of research areas that can challenge the reductionist approach.¹ Consistent with this, the concepts of drug repurposing,² polypharmacology,³ chemogenomics,⁴ phenotypic screening and high-throughput in vivo testing of mixture-based libraries⁵ in an integrated manner have provided meaningful alternatives to the current paradigm of drug discovery. Thus, a shift from a single target to the more preferred multiple target approach. Although these platforms have provided clinically important multitarget lead compounds, their significant outputs are woefully inadequate in meeting the ever increasing demand for therapeutic drugs. As a result, there is an urgent need to investigate other promising approaches that could accelerate progress in this domain. To this end, establishing a biological model to effectively screen multitarget agents is a high priority for a successful anticancer drug discovery strategy.

Previous report on monotarget lipid raft biomaterial strongly signalled the possibility of developing a multitarget model for identifying antitumor components which can act on multiple pathways. The biological interactions between receptor-rich biomaterial and the ligands, associated thermodynamics and effective applications of the model was extensively investigated.⁶ The presence of overexpressed TrkA receptors on lipid raft silica beads (LRSB) was confirmed via immunofluorescence bioassay. The receptor specificity was also validated by TrkA target (lestaurtinib and gefitinib) and non-TrkA target (gemcitabine) standard anticancer drugs (Fig. 1). The chromatograms of target drugs exhibited longer retention time as compared to non-target drugs. Likewise, bioactive LRSB isolate (ether fraction) exhibited similar trend as the target drugs, and significantly inhibited cancer cell growth. However, these active components failed to exert cytotoxic activities in the presence of TrkA inhibitors (K252a and primary antibody). These findings support the interactions between effective components and sufficiently expressed TrkA receptor in the lipid raft.

Fig. 1 Development and online application of lipid raft biomaterial, with confirmatory MTT bioassay data. This figure illustrates the culture of U251 glioma cancer cells with overexpressed TrkA receptor. The extracted lipid raft was immobilized on activated silica beads to form lipid raft-coated silica beads as validated by fluorescence analysis. The biomaterial was packed into a column to serve as a stationary phase for online analysis of potential antitumor components (see chromatograms). The bioactive components, including standard anticancer drugs (gefitinib and lestaurtinib), and ether fraction revealed long retention characteristics. The ether extract exhibited significant cytotoxicity against cancer cells as observed in the MTT analysis. This cytotoxicity was comparable to the standard anticancer drug, 5-fluorouracil.

In spite of comprehensive studies on well-known cancer target receptors such as epidermal growth factor receptor (EGFR), vascular epidermal growth factor receptor (VEGFR) and Fas receptor (FasR), there is still very limited reporting regarding their bioscreening activities for potential antitumor agents.^6,7 So far the best known studies have been limited to cell membrane chromatography (CMC) with highly expressed receptors like EGFR and VEGFR. The medical benefits of employing these receptors for affinity screening cannot be overemphasized in the quest for effective chemotherapeutic drugs. Fortunately, the first TrkA receptor-rich lipid raft from U251 glioma cancer cells has set the stage for obtaining other lipid rafts with multiple receptors from related cancer cells. Several bioscreening systems including immobilized DNA chromatography,⁸ immobilized plasma protein chromatography,⁹ immobilized liposome chromatography¹⁰ and immobilized biomembrane chromatography¹¹ have been developed to detect interactions between a chemical and its target protein.¹² The yet to be established multitarget lipid raft belongs to this class of screening systems with the advantage of having enriched multiple receptors. The multitarget biomaterial can provide different sites for bioactive compounds, and more importantly, isolate compounds capable of acting on multiple pathways.

The desired multitarget lipid raft can overcome most of the problems associated with multitarget drug discovery, particularly longer processing time, high cost implications, low “hit” rates, and most significantly, the lack of correlation between in vitro and in vivo data. Technically, the feasibility of the proposed lipid raft biomaterial has been verified via its monotarget analogue, and it can be seen as an extension of the existing model.⁶ Why does a multitarget lipid raft present such unique opportunities? Perhaps, it can offer a more realistic biological system with enriched diverse cancer-related receptors for bioactive compounds to locate and dock with. More importantly, it can isolate lead compounds from complex mixtures, especially natural products that could affect multiple pathways. The system might also provide experimentally verified data and enhance the search for multitarget agents. With the development of multitarget LRSB, the effective online pharmacological studies and rapid screens for multitarget agents could be easily accessible. However, it must be noted that present reports describe the exploratory stages, and there is a need for more comprehensive studies to demonstrate the clinical value of this novel technology.

2. Prominent transmembrane activities of lipid rafts

Certain cues for cell growth, survival and other physiological processes are transmitted through specialized sub-domains in plasma membrane known as lipid rafts.¹³ The rafts contain a variety of proteins, e.g., caveolins, flotillins, GPI-linked proteins, G proteins, Src family kinases, EGF receptors, PDGF receptors, endothelin receptors, phosphotyrosine phosphatase, MAPK, protein kinase C and PI 3-kinase.¹⁴ They also have the potential to regulate signal transduction by activating or suppressing phosphorylation cascades.¹⁵ For instance, they can function as negative regulators of EGFR tyrosine phosphorylation,¹⁶ thus, upon ligand binding and receptor activation, the EGFR migrates out of the rafts.¹⁷ The membrane rafts might serve as a novel target in cancer therapy due to the ability to organize receptors and their downstream molecules. It has been reported that several growth factor signalling pathways depend on lipid raft. This is evident in the role of lipid rafts in the regulation of differentiation, apoptosis and cell migration, which is linked to invasiveness and metastasis.¹⁸ The study also produced certain synthetic and naturally occurring substances that are known to affect the lateral membrane organization in tumour cell growth which could provide potential or actual therapeutics. Even though most drugs bind to proteins and regulate their activities, some drugs act via a new therapeutic approach called membrane-lipid therapy (binding to lipids).¹⁹ Mollinedo and Gajate identified Fas/CD95 death receptor and lipid rafts as new targets for apoptosis-directed cancer therapy.²⁰ Similarly, the cannabinoid CB1 receptor antagonist rimonabant (SR141716) inhibited human breast cancer cell proliferation via a lipid raft-mediated mechanism.²¹

Many “homing devices”, including folate,²² transferrin²³ and peptides,²⁴ have been installed on liposomes or nanoparticles as a means of achieving effective receptor-target tumour therapy.²⁵ Similarly, lipid rafts have been conjugated to certain drugs to impact lipophilicity, and simultaneously, a target moiety that can be recognized by a specific transporter/receptor in cell membrane was also tethered to the rafts. The findings indicated that both the lipid rafts and target moiety acted synergistically toward cellular uptake.²⁶ Nanogel surfaces decorated with alginate or mannosylated alginate were used to target dendritic cell receptors. The uptake of nanogel was dependent on endosomal-based processes. However, inhibition of lipid raft activities impaired the uptake which suggested the involvement of more than one entry routes.²⁷ In a related study, a drug delivery mechanism for target nanoparticles that utilizes direct cytoplasmic delivery into a target cell while avoiding endosomal incorporation was observed in lipophilic delivery. This phenomenon entails the direct delivery of lipophilic substances to cell membrane via lipid mixing, and subsequent intracellular trafficking via lipid raft dependent processes.²⁸

Effective delivery of therapeutic genes to designated target cells and their action at the intracellular site are crucial requirements for successful gene therapy.²⁹ Studies have suggested that lipid rafts of microvascular dermal endothelial cells are essential to Kaposi'sarcoma-associated herpes virus (KSHV) infection and gene expression. This could be due to their potential roles in the modulation of KSHV-induced PI3-K, RhoA-GTPase and Dia-2 signal molecules in post-binding and entry stages of infection.³⁰ The data indicate a possible role for lipid rafts in gene delivery. DNA nanoparticles (DNPs) are non-viral gene vectors with excellent in vivo potential. Disruption of lipid rafts by depletion of membrane cholesterol significantly inhibits DNP transfection, while the inhibition of other endocytic pathways has little effect. These events reveal the active involvement of lipid rafts in transfection.³¹ Other reports have also shown that dendritic cell utilizes multiple endocytic routes for synthetic virus-like particle uptake which is dominated by lipid raft-mediated macropinocytosis.³² Currently, there is limited information on the application of receptor-rich lipid raft biomaterial for screening antitumor agents. However, recent developments have ignited a special interest in this biomaterial, as evidenced in reports of lipid raft bioscreening assays for antitumor components in complex natural mixtures.⁶

3. Screening of antitumor lead compounds

New antitumor agents of pharmaceutical importance, described as “hits”, could be discovered from different sources such as natural/synthetic products, computational chemistry, chemical libraries and pharmaceutical biotechnology (Fig. 2). The “hits” are pharmacologically, pharmacodynamically and pharmacokinetically enhanced via chemical or functional modification to obtain a lead compound. The leads usually have known structures with well documented mechanism of actions. They could serve as drug candidates that are safe for human clinical trials after optimization and developmental studies (Fig. 2).³³ Generally, the initial routine bioscreening of any “hits” irrespective of their origin is based on cell/target assays using established cell lines to measure the cytotoxic activities (Fig. 2). Some well-established methods commonly employed in these assays include colony formation methods, crystal violet methods, tritium-labelled thymidine uptake methods, and MTT[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] and WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] bioassays. Toxicity studies of active agents on normal cells are investigated using canine kidney (MDCK), mouse fibroblasts (L929), rhesus monkey kidney from Macaca mulatta cells (LLC-MK2) and human peripheral blood mononuclear cells.³⁴ The agents or compounds that are found to be active and non-toxic in the in vitro models are tested for in vivo antitumor efficacy. In vivo models, in which tumours are induced, using NOD-SCID, Balb C57BL/6 and athymic mice, are widely employed for this kind of investigations.³⁵ In fact, the entire process of screening for antitumor agents is characterized by low output, undue delays and high financial costs. This prevailing condition has accounted for several attempts to solve the problem.

Fig. 2 Schematic representation of anticancer drug discovery and development.

4. Lipid rafts with highly expressed cancer-related receptors

Several studies have reported overexpressed receptors on some cancer cells which are clearly absent in normal cells.³⁶ The situation can be clinically explored to discover more antitumor agents with high therapeutic efficacies. As microenvironments in the exoplasmic leaflet of cell membrane, lipid rafts might contain cancer-related receptors. Despite their reduced receptor numbers as compared to cell membrane, the rafts still demonstrate high specificity and increased receptor densities, which could lead to increased output of bioactive compounds.⁶ Previous monotarget lipid raft, with abundant TrkA receptors, and the desired multitarget LRSB models, with prominent therapeutic receptors like EGFR, VEGFR and FasR can support the molecular basis of this clinically useful receptor–ligand screening bioassay.³⁷ In Fig. 3, the bioactive isolate from lipid raft (containing multiple receptors – FasR, TNFR, VEGFR, EGFR and HER2) can interact with any of the associated receptors in its antitumor applications. A follow up study could easily ascertain the specific receptors involved in the receptor–ligand interactions. Most of these receptors are sufficiently expressed in predominant cancer cell lines (prostrate, breast and colon), and therefore, present a unique opportunity that can be therapeutically explored to provide more treatment options for patients.

Fig. 3 The possible multiple targets that can be affected by bioactive isolates from lipid raft-coated silica beads containing cancer-related receptors like epidermal growth factor receptor (EGFR), tumour necrosis factor receptor (TNFR), human epidermal growth factor receptor 2 (HER2), vascular epidermal growth factor receptor (VEGFR) and Fas receptor (Fas). Most anticancer drugs act via apoptotic (programmed cell death) induction and inhibition of angiogenesis in their antitumor activities.

TrkA receptors which can be activated to engage molecules for signal transduction are highly embedded in lipid rafts.³⁸ Therefore, TrkA-rich lipid raft could act as a linchpin from which a potent death signal could be launched to become a new promising anticancer target.^20,39 The identification of EGFR as an oncogene has led to the development of anticancer therapeutics which is directed against EGFR (called ‘EGFR inhibitors’). Similarly, the overactivation of HER2 oncogene has been linked to the progression of certain aggressive cancers. Recently, this protein has become an important biomarker and therapeutic target for approximately 30% breast cancer patients.⁴⁰ The dysregulation of TNFR pathways results in a wide range of pathological conditions, including cancer, autoimmune disease, inflammation and viral infection.⁴¹ This explains the importance of these proteins as potential drug targets.^41c,42 The membrane-anchored Fas ligand on adjacent cell causes the oligomerization of Fas receptor molecules in death-inducing signalling complex (DISC).⁴³ This event could be mimicked by the binding of agonistic lead compounds which might be unravelled by a Fas-based LRSB screening system. Additionally, the vascular endothelial growth factor (VEGF), one of the important elements in regulating angiogenesis, is now a key focus in antiangiogenic therapy.⁴⁴ Thus, the VEGFR and its respective ligands, particularly VEGF, could play prominent roles in various cancer cells where they promote the growth of tumour blood vessels nurturing malignant cells.⁴⁵

5. Receptors as tools for rapid isolation of antitumor agents

Antitumor drug discovery has become one of the most challenging and researched fields in cancer therapy due to the complex mechanism of tumour initiation and growth, as well as the difficulty in treating them.⁴⁶ Numerous screening techniques, based on target receptors or enzymes, and virtual screening system, using computer-aided drug design, have been employed for investigating lead compounds or drugs.⁴⁷ A target enzyme (DNA topoisomerase II) was employed to isolate antitumor agents from a mixture of compounds, and concurrently, the agents were analysed by high performance liquid chromatography/electrospray ionization-mass spectrometry (HPLC/ESI-MS).⁴⁸ Two combined receptors (nicotinic acetylcholine and oestrogen receptors) were used in liquid chromatography for online pharmacological studies, particularly in the identification of lead candidates from complex biological or chemical mixtures.⁴⁹ Similarly, other efficient screening methods like immobilized β₂-adrenoceptor (β₂-AR), immobilized human vitamin D3 receptor (hVDR) and TrkA receptor-rich lipid raft have been used for such purposes.^6,50 Some target-based screening models for antitumor agents are presented in Table 1. Generally, these models are used to separate active compounds from complex natural products.

Table 1 Target-based screening for antitumor agents in various mixtures^a

Molecular target	Screening model	Active compounds	Mixtures	Reference
a Abbreviations: A431, epidermoid carcinoma cell line; 17-AAG, 17-allylaminogeldanamycin; CMC, cell membrane chromatography; DNA, deoxyribonucleic acid; EGFR, epidermal growth factor receptor; HEK293, human embryonic kidney 293 cells; Hsp90, heat shock protein 90; Melan A, melanoma antigen; MU89, melanoma cell line; NA, non applicable; OBAA, 3-(4-octadecyl)benzoylacrylic acid; PMA, phorbol-12-myristate-13 acetate; TrkA, tropomyosin-related tyrosine kinase A; VEGFR-2, vascular endothelial growth factor receptor-2.
β2-Adrenoceptor	Purified receptor	Amygdalin	Semen armeniacae amarum	50a
DNA topoisomerase	Purified enzyme	Doxorubicin, daunorubicin	Doxorubicin, daunorubicin, pravastain	48
EGFR	HEK293/CMC	Asarinin	Radix et rhizoma asari	56
EGFR	HEK293/CMC	Vauquline, strychnine	Semen strychni	7
EGFR	HEK293/CMC	Resveratrol	Rhizoma polygoni cuspidati	57
EGFR	A431/CMC	Taspine, caulophine	Radix caulophylli	58
EGFR	A431/CMC	Oxymatrine, matrine	Radix sophorae flavescentis	59
Hsp90	Progesterone receptor chaperone complex	Capsaicin	Chemical library	60
Melan A	MU89	OBAA, flunarizine, 17-AAG, aphidicolin, damnacanthal, PMA, dantrolene, glyburide	Chemical library	61
TrkA receptor	Lipid raft	NA	Albizziae cortex, Galla chinensis	6a and 62
VEGFR-2	HEK293/CMC	Mesaconitine, aconitine, hypaconitine	Aconitum carmichaeli debx	54

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Currently, most studies focus on receptor-rich membranes to provide a platform for direct screening of active compounds.⁵¹ These membranes usually have receptors, like EGFR and VEGR, which work in a form of bionic affinity chromatography. It is normally coupled to HPLC/MS to study receptor–ligand interactions. EGFR is closely associated with tumour cell proliferation, angiogenesis, tumour invasion, metastasis and inhibition of apoptosis.⁵² It is therefore a strategic target for screening tumour inhibitors. As a key receptor in the progression of tumour, VEGFR-2 has been an important target for antitumor therapeutic research.⁵³ Some small molecules, such as sunitinib and sorafinib, acting against neovascularisation by blocking the signal transduction pathway of VEGFR-2 have shown potent clinical effects.⁵⁴ In order to improve receptor–ligand interaction studies and existing drug screening strategies aimed at specific receptors, some biological affinity systems with better specificity have to be designed. Currently, artificial cell cultures overexpressing VEGFR-2 on cell surface, named HEK293/VEGFR cell lines, have been constructed in HEK293 engineered cell line.⁵⁵ The established HEK293/VEGFR-based CMC was considered as a model that could underline the performance of specific interactions between VEGFR-2 and its ligands.

6. Receptor-rich lipid rafts as an online biomaterial for screening antitumor agents

The online challenges in screening antitumor agents have always been: (1) designing limited bioscreening systems, (2) difficulty in reducing costs, and (3) lack of reconciling in vitro and in vivo data.⁶³ Over the years, CMC models have been able to address some of the problems. The techniques have resulted in successful isolation of numerous bioactive components from complex samples that interact with membrane receptors. The dynamic simulation of the action of drug in vivo by the CMC presents a direct screening technique for active compounds.^37a,59,64,65 But the CMC is plagued with several issues regarding its selectivity, specificity, stability and service life span. These drawbacks are due to the use of homogenized cell membrane which contains receptors at relatively low densities and existing as different varieties.^37a,64 The situation has impeded further development of the CMC, and therefore, other alternatives are urgently required.

For the first time, a novel TrkA receptor-rich lipid raft for identifying antitumor agents has been established (Fig. 1). As a promising technology with inherent high selectivity and specificity, the monotarget lipid raft offers an efficient approach for online isolation and analysis of active compounds. Previous reports have revealed that the biomaterial was stable and able to withstand extreme conditions.^6b Fluorescent images of the biomaterial showed no obvious changes in intensity before and after packing the column. Most importantly, no fluorescence attenuation was observed in the lipid raft even after 30 days of continuous use. Further treatment with different mobile phases showed no effect on fluorescent intensity. In short, the long-lasting stability was a great improvement on the cell membrane-immobilized stationary phase (only 3 days or just 1 week under continuous usage).⁶⁶ Significantly, the lipid raft biomaterial provided unique retention behaviours for easy identification of active agents (Fig. 1).

Currently, there is only one type of receptor-rich lipid raft biomaterial, and therefore, this concept can be extended to other related cells to develop more antitumor screening tools. It is possible to discover other lipid rafts with abundant multiple receptors to provide different targets for screening multitarget agents. Studies on the monotarget model have now paved way for scientists to explore the possibility of designing a multitarget analogue of this biomaterial. Technically, both the mono- and multi-target models are likely to operate on the same principle with the latter having the benefit of providing multiple interaction sites.

7. Prospect for multiple receptor-based lipid raft biomaterial in cancer therapy

Multitarget agents have been increasingly explored⁶⁷ for enhanced therapeutic efficacies, improved safety profiles, and reduced resistance,⁶⁸ while limiting unwanted cross-reactivities via optimization of target selectivity.⁶⁹ Some clinically successful multitarget anticancer drugs include the anticancer kinase inhibitors sunitinib against PDGFR and VEGFR, dasatinib against Abl and Src, and lapatinib against EGFR and HER2.⁷⁰ However, these drugs are administered to few patients and the need to develop additional drugs could be a life-saving. For years, studies aiming at multitarget antitumor drug discovery have not been very successful, and the situation continues to greatly limit the number of validated leads. The desired multitarget biomaterial could significantly increase the true hit rate which will give rise to large number of therapeutic multitarget agents. Table 2 reveals some reported antitumor agents that act on multiple receptors with related cancer target diseases. The number of such agents could be remarkably increased if the current drug discovery protocols are improved and additional ones provided. The multitarget biomaterial will not only provide bioscreening, but also serve as a tool for understanding molecular-level actions associated with cancers. Comparatively, the specificity, stability and life span of the lipid raft will stand out among numerous antitumor bioscreening systems.^6b,66 Hence, employing the biomaterial as routine mass screening system for potential antitumor agents will increase the number of leads for further development.

Table 2 Reported antitumor agents that interact with multiple receptors^a

Agent	Targeted disease	Multitarget action	Reference
a Abbreviations: AHR, aryl hydrocarbon receptor; AR, androgen receptor; CSF1R, colony stimulating factor 1 receptor; CXCR-4, C-X-C chemokine receptor type 4; DR-4/5, death receptor-4/5; EGFR, epidermal growth factor receptor; EPCR, endothelial protein C receptor; ER-α, oestrogen receptor-alpha; FasR, Fas receptor; FGFR-1, fibroblast growth factor receptor 1; HER-2, human epidermal growth factor receptor 2; H2R, histamine (2) receptor; HUVEC, human umbilical vein endothelial cells IL-8-R, interleukin-8-receptor; IR, integrin receptor; KIT, KIT receptor; LDL-R, low density lipoprotein–receptor; RET, RET receptor; VEGFR-1/2, vascular endothelial growth factor receptor-1/2.
ABT-869	Solid malignancies, acute myeloid leukemia	VEGFR-2, CSF1R	37c and 71
AMG-706	Thyroid cancers, non-small-cell lung cancer	VEGFR-2, KIT	37c and 72
Berberine	Breast cancer, prostate cancer, cervical cancer	HER-2, AR, FasR	73
Celasterol	Prostate cancer, non-small-cell lung cancer, breast cancer, human glioma	AR, FasR, DR-4/5, VEGFR-2	74
Curcumin	Colon cancer, pancreatic cancer	ER-α, FasR, IR, EPCR, H2R, HER-2, EGFR, DR-5, IL-8-R, CXCR-4, LDLR, AHR, ITR, AR	75
Indirubin/indirubin-3′-monoxime	Prostate cancer, myeloid leukaemia	VEGFR-2, FGFR-1	76
Lapatinib	Breast cancer	EGFR, ERBB-2	37c and 77
Norcantharidin	Colorectal carcinoma, colon cancer, hepatoma cells	FasR, VEGFR-2, DR-5	78
Quercetin	Pancreatic cancer, breast cancer	EGFR, ER-α	79
Resveratrol	Prostate cancer, breast cancer	AR, ER-α	80
Sunitinib	Gastrointestinal stromal tumor, renal cell carcinoma	VEGFR-2, KIT	37c and 81
Wogonin	Breast cancer, HUVEC	EGFR, ER-α, VEGFR-1	82
ZD-6474	Non-small-cell lung cancer, small-cell lung cancer, myeloma	EGFR, VEGFR-2, RET	37c and 83

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Establishing a multitarget biomaterial at this crucial moment could be very timely. Fig. 1 shows the successful development of monotarget lipid raft for screening antitumor agents. The as yet undeveloped multitarget analogue can be seen as an improved version of the previous system. The compatibility of the proposed biomaterial to existing structures will be unquestionable as witnessed in the monotarget model. The biomaterial can also be easily constructed from a range of prominent receptor-rich cancer cell lines. Structure–activity relationship (SAR) studies can be facilitated since the system can support large scale bioscreening investigations. Preliminary information on lead compounds for different target receptors and even possible mechanistic actions prior to employing confirmatory assays could be ascertained. Most significantly, the multitarget LRSB can provide multiple sites for lead antitumor compounds which might affect multiple pathways. Now comprehensive studies in the application of lipid raft as screening biomaterial are in the exploratory stages, and additional work needs to be done to help generate such systems that will eventually enhance the number of multitarget agents employed in combating cancers.

8. Challenges and future considerations in lipid raft screening applications

Generally, the lipid raft screening system can suffer from problems relating to the search for multitarget antitumor agents. But the retrieval rate of the lead compounds could be highly enhanced with shortened processes, and even very strong correlation between in vitro and in vivo data. The capacity to effectively develop and identify sufficiently expressed receptors in cancer cells can be problematic. The use of appropriate technology to rapidly culture target cells within a reasonable time could also be a daunting task with some cost implications. Optimization of lipid raft extraction and technical modification of the biomaterial to improve the life span cannot be ignored. To further build therapeutically useful models, we need to have an in-depth molecular knowledge about the target receptors and also consider the role of co-receptors in modulating these activities. Establishing a new lipid raft model capable of screening multitarget antitumor agents will be characterized by high failures, but it is worth the effort.

More studies should be directed at developing receptor-rich multitarget chemical screening systems. In that regard, understanding the mechanistic actions of these cancer-related receptors will not only become academically prudent, but also could lead to the development of highly specific drug inhibitors for clinical applications. It may also be necessary to modify existing chemical screening approaches from single to multiple target-oriented standard operating systems. As a necessity, knowledge on the whole receptor-based screening system including ligands, stationary and mobile phases, and other accessory components requires a scientific upgrade. The efficient activities of the proposed multitarget model could also be realised by incorporating existing multitarget drug information on the current and future discovery. Now the scientific community has at its disposal a monotarget analogue of lipid raft biomaterial which is still in its infancy. In the opinion of these authors, there is a high probability of developing a multitarget analogue of the biomaterial for effective isolation of multitarget antitumor agents. The monotarget model was constructed from U251 cells; however, other cancer cells with sufficiently expressed receptors can also be employed. The final outcome could be clinically very important.

9. Conclusion

TrkA receptor-rich lipid raft biomaterial has been successfully employed to screen antitumor compounds which dramatically inhibited the growth of cancer cells. Comprehensive studies on this monotarget biomaterial in relation to its specificity, sensitivity, stability, life span and thermodynamic properties have been promising. The biomaterial has been easily adaptable to existing HPLC protocols for sample analysis. Most importantly, this monotarget model has paved way for the establishment of multitarget analogue which will operate on the same principle. This report therefore presents a promising would-be multitarget biomaterial for fast screening of antitumor lead components with the potential to interact with multiple targets.

10. Conflict of interest

No potential conflicts of interest.

Acknowledgements

We appreciate the support by National Natural Science Foundation of China (30973677, 81373371), Doctoral Fund of Ministry of Education of China (20113227110012) and Special Funds (JHB2012-37, GY2012049, GY2013055, GY2011028) in Zhenjiang, Jiangsu Province for all projects related to lipid raft biomaterial.

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