α‐Mangostin‐encapsulated PLGA nanoparticles inhibit colorectal cancer growth by inhibiting Notch pathway

Abstract Colorectal cancer (CRC) is the fourth leading cause of cancer‐related mortality. Recent studies have stated that Notch signalling is highly activated in cancer stem cells (CSCs) and plays an important role in the development and progression of CRC. Like normal colorectal epithelium, CRCs are organized hierarchically and include populations of CSCs. In order to enhance the biological activity of α‐mangostin, we formulated α‐mangostin‐encapsulated PLGA nanoparticles (Mang‐NPs) and examined the molecular mechanisms by which Mang‐NPs inhibit CRC cell viability, colony formation, epithelial‐mesenchymal transition (EMT) and induce apoptosis. Mang‐NPs inhibited cell viability, colony formation and induced apoptosis. Mang‐NPs also inhibited EMT by up‐regulating E‐cadherin and inhibiting N‐cadherin and transcription factors Snail, Slug and Zeb1. As dysregulated signalling through the Notch receptors promotes oncogenesis, we measured the effects of Mang‐NPs on Notch pathway. Mang‐NPs inhibited Notch signalling by suppressing the expression of Notch receptors (Notch1 and Notch2), their ligands (Jagged 1 and DLL4), γ‐secretase complex protein (Nicastrin) and downstream target (Hes‐1). Notch receptor signalling regulates cell fate determination in stem cell population. Finally, Mang‐NPs inhibited the self‐renewal capacity of CSCs, stem cell markers (CD133, CD44, Musashi and LGR5) and pluripotency maintaining factors (Oct4, Sox‐2, KLF‐4, c‐Myc and Nanog). Overall, our data suggest that Mang‐NPs can inhibit CRC growth, EMT and CSCs’ population by suppressing Notch pathway and its target. Therefore, Mang‐NPs can be used for the treatment and prevention of CRC.


| INTRODUC TI ON
Colorectal cancer (CRC) is the second most common type of malignancy and the fourth leading cause of the cancer-related death worldwide. 1  The development of CRC is a complex multistage process which involves sequential mutational events occurring along with progression of the cancer. 2 Notch and Wnt signalling pathways in CRC development are implicated in the regulation of several biological processes, including cell proliferation, differentiation, angiogenesis, apoptosis and survival. 3,4 The activation of these pathways is correlated with poor prognosis. 2,5 Like normal colorectal epithelium, CRCs are organized hierarchically and include populations of CSCs which play significant role in cancer development. 6,7 Therefore, there is an urgent need to target those pathways which regulate CSCs pool during CRC growth, development and metastasis.
Notch pathway is one of highly conserved cellular pathways responsible for direct cell to cell interaction in multicellular organisms. 8 Proper function of Notch pathway is essential for normal cell development, differentiation, proliferation and apoptosis. [9][10][11] Notch signalling pathway consists of five ligands including Jagged-1, Jagged-2, Delta-like-1 (Dll1), Delta-like-3 (Dll-3) and Delta-Like-4 (Dll-4), and four receptors: Notch1, Notch2, Notch3 and Notch4. 12 Notch signalling pathway also contains several downstream target genes including Hes-1, Hey-1 and p21. While the Notch ligands are the single-pass transmembrane proteins of DSL family, the Notch receptors are transmembrane proteins containing both types of extracellular and intracellular domains. 12 Ligand-binding causes a conformational change leading to ADAMmediated ectodomain shedding (S2 cleavage) and subsequent γ-secretase-mediated proteolysis within the transmembrane domain (S3/S4 cleavage) resulting in the release Notch intracellular domain (NICD). Subsequently, NICD translocates to the nucleus and associates with the DNA-binding protein CSL (RBPjκ in mammals) to form a composite interface where the coactivator Mastermind (MAML), p300 and the histone acetyltransferase (HAT) bind, leading to the activation of the transcriptional complex and induction of genes. 13 Notch signalling mediates the maintenance of intestinal development and homeostasis through the regulation of the differentiation of colonic goblet cells and stem cells/ progenitor cells. 14,15 Notch signalling pathway has a diverse role in tumorigenesis. 16 Depending on the cellular context, activation of the Notch pathway can exert either an oncogenic or tumour suppressor function. [17][18][19][20] Moreover, Notch1 plays an oncogenic role in CRC. 4 The overexpression of Notch1 in CRC cells increased the expression of the downstream targets Hes-1 and Hey-1. A recent study has reported that Notch expression in the primary stage of CRC is relatively higher than the later stage. Notch signalling promotes CRC growth through regulation of cell cycle and apoptosis. 21 Therefore, Notch signalling pathway represents a novel target for cancer therapeutic intervention in CRC.
The development of natural product-based compounds can be an attractive strategy for the treatment and prevention of CRC because they are non-toxic, and regulate stem cell population by inhibiting pluripotency and self-renewal capacity of CSCs via multiple signalling pathways. 22,23 The α-mangostin is derived from the plant mangosteen (Garcinia mangostana) which was originated in the Sunda Islands and the Moluccas of Indonesia. 24 Xanthonoid such as α-mangostin is found in Mangosteen, which grows mainly in Southeast Asia and South America. 25 Mangostin exerts antioxidant, antimicrobial, anticancer and anti-inflammatory activities. [26][27][28][29] Mangostin inhibits mammalian DNA polymerase and topoisomerase activities in cancer cells. 30 Based on these beneficial effects of mangostin, it can be developed for the treatment and prevention of CRC. Although several studies have demonstrated that α-Mangostin is safe and well-tolerated, the in vivo use of α-mangostin is limited due to its hydrophobic nature, poor aqueous solubility, stability, bioavailability and accumulation in the target organs. To overcome these limitations, we have encapsulated α-mangostin into the core of poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles (Mang-NPs). Generation of NPs allowed us to increase the efficacy and therapeutic benefits of α-mangostin for the treatment and prevention of CRC.
The main objective of this paper is to examine the molecular mechanisms by which Mang-NPs inhibit CRC growth and inhibit the stem cell characteristics. Mang-NPs inhibited growth of cancer cells and CSCs by suppressing Notch pathway. Mang-NPs inhibited those genes which play major roles in cell proliferation, self-renewal, pluripotency, cell cycle, apoptosis and EMT. In conclusion, Mang-NPs can be used as a potential chemotherapeutic agent for the treatment and prevention of CRC.

| Particle size and zeta potential analysis
Freeze-dried nanoparticles were suspended in deionized water.
The mean particle diameter and width (polydispersity index) were determined by photon correlation spectroscopy using a Zetasizer 3000. 31,32 The particle charge was quantified as zeta potential by laser Doppler anemometry using the Zetasizer.

| Cell culture
Human colorectal cancer cells (HCT116 and HT29) and normal epithelial cells CRL-1831 were purchased from American Type Culture Collection (ATCC) and maintained in culture conditions as recommended by ATCC (Manassas, VA). We have previously described the isolation and characterization of human CRC CSCs (CD133 + /CD44 + / LGR5 + ). 33 Colorectal CSCs were cultured in stem cell growth medium at 37°C in a humidified atmosphere of 95% air and 5% CO 2 . 34

| Colonosphere (spheroid formation) assay
For colonosphere assay, cells were plated in six-well ultralow attachment plates (Corning Inc, Corning, NY) at a density of 1000 cells/mL in stem cell growth medium as described. 33 Human CRC CSCs were treated with Mang-NPs (0-10 µmol/L) for 7 days to obtain primary spheroids. Spheroids were collected after 7 days and dissociated with Accutase (Innovative Cell Technologies, Inc). The CSCs obtained from dissociation of spheroids were counted. At the end of incubation period, spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days (primary, secondary and tertiary colonosphere, respectively).

| Motility assay
Cell motility assay was performed as we described elsewhere. 31 In brief, cells were grown to a confluent monolayer in a 6-well plate, scratched with a 200-μL tip and washed twice with PBS. After incubation with medium containing 1% FBS following treatment with or without Mang-NPs, the cells were photographed at 0, and 24 hours under an inverted microscope (Olympus, Tokyo, Japan) at 40× magnification. The width of the scratch gap is viewed under the microscope in four separate areas each day until the gap is filled in the untreated control wells.

| Transwell migration assay
Transwell migration assays were performed as we described elsewhere. 31 In brief, 1 × 10 5 cells in 200 μL of medium with 1% FBS were plated in the top chamber onto the non-coated membrane (6.5-mm diameter, 8-μm pores; Corning Costar, Corning, NY) and allowed to migrate in the lower chamber towards 10% FBS (as chemoattractant)-containing medium. Cell was treated with Mang-NPs. After 48 hours of incubation at 37°C in 5% CO 2 , cells were fixed with methanol, stained with crystal violet and counted under an inverted microscope (100 × magnification).

| Transwell invasion assay
Transwell invasion assays were performed as we described elsewhere. 31 In brief, 1 ×

| Western blot analysis
Western blot analysis was performed as we described elsewhere. 36,37 Proteins from the cell lysates were prepared from cells treated with or without Mang-NPs. Equal amounts of protein were denatured and separated by SDS-PAGE, transferred onto PVDF membranes and incubated with primary antibody (1:100-500) followed by secondary antibody (1:5000). The peak intensity of each band was visualized using an Enhanced Chemiluminescence kit (Sigma-Aldrich).

| Quantitative real-time PCR
Total RNA was extracted from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The concentration, purity and integrity of the RNA were measured using a Nano Drop2000 spectrophotometer (Thermo Scientific). q-RT-PCR was performed as described elsewhere. 38 F I G U R E 1 Uptake of α-Mang-NPs by colorectal cancer cell line. Colorectal cancer cells (HCT-116) were treated with coumarin-6 containing Mang-NPs for 2 h. Cells were incubated with Hoechst 33 342 for nuclear staining. After washing cells, they were visualized to examine uptake of Mang-NPs, and photographs were taken by the fluorescence microscope. Green colour = Mang-NPs. Blue colour = Nucleus F I G U R E 2 α-Mang-NPs inhibit cell proliferation and colony formation, and induce apoptosis in colorectal cancer cells. A, CRC cells were treated with Mang-NPs (0-10 µmol/L) for 72 hours, and cell viability was measured as described in Materials and Methods. Data represent mean (n = 4) ± SD. *, #, and % = significantly different from respective control (NPs group), P < 0.05. B, HCT-116 and HT29 cells were treated with NPs or Mang-NPs (0-10 µmol/L) for 21 days. Colonies were photographed. C, Colorectal normal CRL-1831 and cancer cell lines (HT116 and HT29) were treated with Mang-NPs (0-10 µmol/L) for 48 hours. Apoptosis was measured by TUNEL assay. Data represent mean ± SD. *, # and % = significantly different from respective control, P < 0.05 Briefly, cDNA was synthesized using a high capacity cDNA reverse transcription kit (Applied Biosystems). Primers specific for each of the signalling molecules were designed using NCBI/Primer-BLAST and used to generate the PCR products. For the quantification of gene amplification, real-time PCR was performed using an ABI 7300 Sequence Detection System in the presence of SYBR-Green.

| Notch reporter assay
Notch reporter activity was measured as we described elsewhere. 39,40 In brief, a pGreenFire1-Notch plasmid that expressed copGFP reporter and firefly luciferase under the control of four Notch response elements and a minimal CMV promoter (pGreen Fire1-4xNotch-mCMV-EF1-Puro) was purchased from System Biosciences. Lentiviral production and transduction were performed as we described elsewhere. 31

| Immunofluorescence
Colorectal cancer cells were grown in 12-well plates (Beckton Dickinson, Bedford, MA) and treated with or without a mixture

| Statistical analysis
The mean and SD were calculated for each experimental group.
Differences between groups were analysed by ANOVA or t tests using PRISM statistical analysis software (GrafPad Software, Inc, San Diego, CA). Significant differences among groups were calculated at P < 0.05.

| Uptake of α-mangostin-encapsulated PLGA nanoparticles (Mang-NPs) by colorectal cancer cells
In order to improve the efficacy and improve the half-life of a drug, nanotechnology has successfully been used. 31

| Mang-NPs inhibit cell viability and colony formation, and induce apoptosis in colorectal cancer cells, but have no effect on human normal colorectal epithelial cells
To

| Mang-NPs inhibit cell motility, migration and invasion and regulate markers of epithelial-mesenchymal transition in colorectal cancer cells
Epithelial-mesenchymal transition (EMT) is a biological process by which cells undergo through genetic changes that allow them to leave the primary site and migrate to distant location (secondary site) to reestablish, proliferate and survive. 42 We next measured the effects of Mang-NPs on cell motility, migration and invasion.

| Mang-NPs inhibit cell viability in spheroids, stem cell markers and pluripotency maintaining factors in colorectal CSCs
As CSCs play a major role in colorectal carcinogenesis and are responsible for cancer initiation, progression, metastasis and drug resistance, they can be used to evaluate the efficacy of anticancer drugs. Spheroid formation in suspension has been used to measure the stem cell characteristics in vitro. Mang-NPs inhibited cell viability of primary, secondary and tertiary spheroids formed by colorectal CSCs isolated from primary tumours ( Figure 6A). These data suggest that Mang-NPs can be used for the treatment of CRC because they target self-renewal capacity of CSCs.
As Mang-NPs inhibited spheroid formation, we next sought to measure its effects on the expression of commonly used CRC stem cell markers CD133, CD44, Musashi and LGR5 by q-RT-PCR. Transduced cells were treated with Mang-NPs (0-10 µmol/L) for 24 h. CSL reporter activity was measured as we described. 40 Data represent mean ± SD. *, # and @ = significantly different from control, P < 0.05 As shown in Figure 6B

| Mang-NPs inhibit cell motility, migration and invasion and markers of epithelial-mesenchymal transition in colorectal CSCs
During epithelial-mesenchymal transition (EMT), cells undergo through genetic changes that allow them to leave the primary site and migrate to distant location where they reestablish, create a suitable environment and proliferate. 42  F I G U R E 6 α-Mang-NPs inhibit cell viability in spheroids formed by CSCs isolated from human CRC tissues, and expression of stem cell markers and pluripotency maintaining factors. (A), Human colorectal CSCs were treated with Mang-NPs (0-10 µmol/L) for 7 days to obtain primary spheroids. At the end of incubation period, spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain secondary spheroids. Secondary spheroids were collected, reseeded and treated with Mang-NPs for another week to obtain tertiary spheroids. Cell viability in spheroids was measured by trypan blue assay at the end of 7, 14 and 21 days. Data represent mean ± SD. *, # and @ = significantly different from control, P < 0.05. (B), Expression of stem cell markers. Colorectal CSCs were treated with Mang-NPs (0-10 µmol/L) for 36 h. The expression of CD133, CD44, Musashi and LGR5 was measured by q-RT-PCR. Data represent mean ± SD. *, # and @ = significantly different from control, P < 0.05. (C), Expression of pluripotency maintaining factors. Colorectal CSCs were treated with Mang-NPs (0-10 µmol/L) for 36 h. The expression of Oct-4, Sox-2, KLF-4, c-Myc and Nanog was measured by q-RT-PCR. Data represent mean ± SD. *, # and @ = significantly different from control, P < 0.05

| Mang-NPs inhibit Notch signalling pathway and target genes in colorectal CSCs
Notch pathway regulates stem cell characteristics and plays significant role in CRC carcinogenesis. 43 We therefore sought to examine the effects of Mang-NPs on the components of Notch pathway and its downstream targets. Mang-NPs inhibited the expression of Notch1, Notch2, Jagged1, Hes1 and Dll4 in CSCs isolated from primary CRC ( Figure 8A). As activation of Notch pathway regulates CSL transcription, we next measured the effects of Mang-NPs on CSL transcriptional activity ( Figure 8B). These data suggest that Mang-NPs can inhibit self-renewal capacity of colorectal CSCs by targeting Notch pathway and its target.  44,45 In addition, Notch pathway activation was also shown to drive chemoresistance in cancer. The combination of chemotherapy with NOTCH1 inhibitor synergistically attenuated chemotherapy-enriched CSC population. 46,47 In the present study, we have demon- Nanotechnology has been successfully used to deliver anticancer drugs because nanoparticles enhance the bioavailability and distribution, preserve the integrity of compounds and improve the biological activity of drugs. In the present study, Mang-NPs inhibited CRC growth and CSC characteristics by suppressing Notch signalling pathway. In another studies, we have demonstrated that Mang-NPs and Antho-NPs (anthothecol-encapsulated NPs) suppressed proliferation of pancreatic CSCs and cancer cells, and inhibited the self-renewal capacity of CSCs isolated from pancreatic cancer tissues from human and Kras G12D mice. 31,32 Similarly, α-Mangostin-encapsulated F I G U R E 8 Regulation of Notch pathway and CSL transcription by α-Mang-NPs. (A), Colorectal CSCs were treated with Mang-NPs (0-10 µmol/L) for 36 h. The expression of Notch1, Notch2, Jagged1, Hes1 and DLL4 was measured by the q-RT-PCR. Data represent mean ± SD. *, # and @ = significantly different from control, P < 0.05. (B), Regulation of CSL transcription by Mang-NPs. CSCs cells were transduced with Notch-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Notch with EF1, System Biosciences). Transduced cells were treated with Mang-NPs (0-10 µmol/L) for 24 h. CSL reporter activity was measured as we described elsewhere. 40 Data represent mean ± SD. *, # and @ = significantly different from control, P < 0.05 Gold/polyethyleneimine/cyclodextrin (AuNPs/PEI/CD) nanoparticles inhibited prostate cancer growth. 48 Clinical trials are needed to demonstrate the safety and efficacy of Mang-NPs for the treatment and prevention of solid tumours.

| D ISCUSS I ON
In conclusion, our study has demonstrated that α-Mang-NPs can inhibit the growth of CRC and self-renewal capacity of CSCs. As α-Mang-NPs inhibited stem cell markers and pluripotency maintaining factors, it offers a great potential to inhibit CRC growth and metastasis by targeting CSC population. Therefore, α-Mang-NPs offer new hope for the treatment and/or prevention of CRC.

ACK N OWLED G EM ENTS
We thank our laboratory members for critical reading of the manuscript.

CO N FLI C T O F I NTE R E S T
VCB, RKV and SS have declared that no competing interests exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.