α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in human, and transgenic (KrasG12D, and KrasG12D/tp53R270H) mice

Activation of sonic hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biological activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway.

Pancreatic cancer is the fourth leading cause of cancer-related deaths in US, with a 5-year survival rate of approximately 6% 1 . Most of the pancreatic cancer is already metastasized to distant organs by the time it is detected; therefore disease is untreatable at such a late stage. Cancer stem cells (CSCs)/tumor initiating cells (TICs) have been found to be the cause of cancer initiation, progression, metastasis, and drug resistance 2 . Several studies using genetically engineered mouse models have demonstrated the aberrant activation of signaling pathways in pancreatic cancer 3 . One such pathway is sonic hedgehog (Shh) pathway which is highly activated in pancreatic cancer and its expression is correlated with poor prognosis 4,5 . Therefore, there is an urgent need to target CSCs by developing strategies which inhibit the population of CSCs and also those pathways which are highly activated.
The development of natural product-based compounds can be an attractive strategy for the treatment and prevention of pancreatic cancer because they are non-toxic, and inhibit pluripotency and self-renewal capacity of CSCs by targeting multiple pathways 2 . The α -mangostin is derived from the plant mangosteen (Garcinia mangostana) which have originated in the Sunda Islands and the Moluccas of Indonesia 6 . Mangosteen contains xanthonoids, such as α -mangostin, and other phytochemicals 7 . It grows mainly in Southeast Asia, and also in South America 8 . It possesses antioxidant, antimicrobial, anticancer, and anti-inflammatory properties [9][10][11][12] . α -Mangostin suppresses EMT and lipopolysaccharide-induced invasion by inhibiting matrix metalloproteinase-2/9 and increasing E-cadherin expression through extracellular signal-regulated kinase signaling in pancreatic cancer cells 13 . It inhibits mammalian DNA polymerase and topoisomerase activities in colon cancer 14 . The beneficial effects of α -mangostin suggest that it can be further developed for the treatment and prevention of cancer.
In spite of these studies, the molecular mechanisms by which it inhibits pancreatic cancer growth by targeting CSCs have never been examined. α -Mangostin is safe and well-tolerated. Furthermore, we have recently demonstrated that α -mangostin can inhibit pancreatic CSCs characteristics by inhibiting Shh pathway i.e. disrupt Gli-DNA binding activity 15 . However, the in vivo application of α -mangostin is limited due to its hydrophobic nature, poor aqueous solubility and stability, and thus low bioavailability and accumulation in the target organs. To overcome this limitation, we have encapsulated α -mangostin into the core of poly (D, L-lactic-co-glycolic acid) (PLGA) nanoparticles (Mang-NPs).
Shh is a member of the Hedgehog (Hh) family of secreted signaling proteins which play diverse functions during vertebrate development and in tissue homeostasis 16,17 . The binding of Shh to Patched (Ptch) results in loss of Ptch activity and consequent phosphorylation and posttranscriptional stabilization of Smoothened (Smo), leading to activation of Gli transcription factor 18,19 . Gli activation via Smo can occur either by Hh protein stimulation or through loss of Ptch activity. We and others have demonstrated that Shh pathway is constitutively active in pancreatic cancer and inhibition of Smoothened or Gli transcription can suppress the ability of pancreatic CSCs to proliferate and self-renew [20][21][22][23][24][25] .
The main objective of this paper is to examine the molecular mechanisms by which Mang-NPs inhibit human and KC (Pdx Cre ;LSL-Kras G12D ) mice pancreatic cancer stem cell (Pan CSC) characteristics in vitro and tumor growth, development and metastasis in KPC (Pdx Cre ;LSLKras G12D ;LSL-Trp53 R172H ) mice. Mang-NPs suppresses pancreatic CSC characteristics by modulating genes involved in cell proliferation, self-renewal, pluripotency, cell cycle, apoptosis and epithelial mesenchymal transition (EMT), and also inhibit pancreatic cancer growth, development and metastasis in KPC mice by targeting CSCs. In conclusion, Mang-NPs can be used as a potential chemotherapeutic agent for the treatment and prevention of pancreatic cancer. 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 26 . The particle charge was quantified as zeta potential by laser Doppler anemometry using the Zetasizer.
Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Kansas City VA Medical Center, and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health. Pdx1-Cre mice (generated by Dr. Lowy, University of Cincinnati) were purchased from the Jackson laboratory (Bar Harbor, Maine). LSL-Trp53R172H and LSL-KrasG12D/+ mice were obtained from MMHCC, NCI/NIH. The KC (Pdx Cre ;LSL-Kras G12D ) and KPC (Pdx Cre ;LSLKras G12D ;LSL-Trp53 R172H ) mice were generated as we and others have described earlier [27][28][29] . All of these genetically engineered mice were bred and genotyped for the presence of Kras, p53, and Cre 27 . Six-week-old breeding pairs of genetically engineered mice, including transgenic Pdx1-Cre, LSLTrp53 R172H, and LSL-KrasG12D mice were used for breeding. To produce compounded transgenic KPC mice, the double transgenic LSLKras G12D/+ -LSL-Trp53 R172/+ mice were first generated, and then further mated with heterozygous Pdx1-Cre transgenic mice 30 . Pancreatic CSCs were isolated from the KC mice as we described elsewhere 28 . Mang-NPs were administrated intraperitoneally into KPC mice (about 4 weeks old males and females) once per day, five days per week for about 10 weeks. Lentiviral particle production and Nanog shRNA transduction. Nanog shRNA plasmids targeting 4 sites were obtained from Open Biosystems, Huntsville, AL) and cloned into TRIPZ vector. Lentiviral production and transduction were performed as we described elsewhere 26 . Cell viability and apoptosis assays. Cells (1.5 × 10 4 ) were incubated with or without Mang-NPs (0-10 μ M) in culture medium for 48 or 72 h. Cell proliferation was determined by trypan blue assay as described previously 26 . The apoptosis was determined by TUNEL assay.
Tumor spheroid assay. For spheroid forming assay, cells were plated in six-well ultralow attachment plates (Corning Inc., Corning, NY) at a density of 1,000 cells/ml in stem cell growth medium as described above. Human pancreatic CSCs were treated with Mang-NPs (0-10 μ M) 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.
Motility assay. Scratch migration assay was performed as we described elsewhere 26 . In brief, a confluent monolayer of cells was established and then a scratch is made through the monolayer, using a standard 200 μ l plastic pipette tip, which gives rise to an in vitro wound, washed twice with PBS, and replaced in media with or without Mang-NPs. CSC migrate into the scratch area as single cell from the confluent sides. The width of the scratch gap is viewed under the microscope in four separate areas each day until the gap is completely filled in the untreated control wells. Three replicate wells from a 6-well plate were used for each experimental condition. Transwell migration and invasion assays. Transwell migration and invasion assays were performed as we described elsewhere 26 . For transwell migration assay, 1 × 10 5 CSCs were plated in the top chamber onto the non-coated membrane (24-well insert; pore size, 8 mm; Corning Inc.) and allowed to migrate in the lower chamber towards serum-containing medium. After 24 h of incubation, cells were fixed with methanol, stained with crystal violet and counted.
For invasion assay, 1 × 10 5 cells were plated on top of a layer of Matrigel in Transwell chambers (BD Biosciences). Pancreatic CSCs were plated in medium without serum or growth factors, and medium supplemented with serum was used as a chemoattractant in the lower chamber. After 48 h of incubation, cells that invaded to the bottom chamber were fixed with methanol, stained with crystal violet and counted.
Western blot analysis. Western blot analysis was performed as we described elsewhere 29 .
Quantitative real-time PCR. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA) and q-RT-PCR was performed as described elsewhere 29 . Briefly, cDNA was synthesized using a high capacity cDNA reverse transcription kit (Applied Biosystems). Primers specific for each of the signaling 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.
Gli reporter assay. Gli reporter activity was measured as we described elsewhere 25,31 . In brief, we have used a construct containing cop-GFP and luciferase genes downstream of Gli-response element (pGreen 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) Mouse pancreatic CSCs were treated with Mang-NPs (0-10 μ M) 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. Statistical analysis. The mean and SD were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA, followed by Bonferoni's multiple comparison tests using PRISM statistical analysis software (GrafPad Software, Inc., San Diego, CA). Significant differences among groups were calculated at P < 0.05.

Formulation and uptake of α-mangostin encapsulated PLGA nanoparticles (Mang-NPs).
PLGA (50:50 PLGA, 14000-16000 MW, Sigma-Aldrich) nanoparticles (NPs) encapsulating α -mangostin and coumarin-6 (fluorescent dye) were prepared using a double emulsion-solvent evaporation method. Mang-NPs possess the following physico-chemical characteristics: % PVA = 0.50 (w/v); Particle Size = 186.3 ± 6.42 nm; Zeta Potential = 0.03 ± 0.005 mV; Drug encapsulation = 51.16 ± 2.61%. We have recently isolated CSCs from the pancreata of human and Kras G12D mice 28,31,32 . We next examined the uptake of Mang-NPs by pancreatic CSCs and cancer cell lines (AsPC-1 and PANC-1). As shown in Fig. 1, pancreatic CSCs and cell lines were able to absorb Mang-NPs in 30 min as demonstrated by the green color. We have also observed the uptake of Mang-NPs as early as 10 min with a maximum fluorescence reaching at 30 min in vitro. These data suggest that Mang-NPs are functional and can easily enter into the cells.   Fig. 2A-C). Mang-NPs were more effective than Mangostin (5 and 10 μ M) in inhibiting cell proliferation of Pan CSCs and pancreatic cancer cell lines. By comparison, PLGA-NPs (NPs) had no effect on cell proliferation of CSCs and cancer cells. Interestingly, HPNE cells were not significantly affected by NPs, Mangostin and Mang-NPs (Fig. 2D). These data suggest that Mang-NPs can be used for the treatment of pancreatic cancer.

Mang-NPs inhibit colony formation and induce apoptosis in pancreatic CSCs and cell lines.
Colony formation and apoptosis induction are commonly used to assess the effectiveness of anticancer drugs. We next examined the effects of Mang-NPs on colony formation and apoptosis. Mang-NPs inhibited colony formation in Pan CSCs and cancer cell lines (PANC-1, AsPC-1 and MIA PaCa-2) in a dose-dependent manner (Fig. 3A). Similarly, Mang-NPs induced apoptosis in Pan CSCs and pancreatic cancer PANC-1, AsPC-1 and MIA PaCa-2 cell lines (Fig. 3B). These data suggest that Mang-NPs are effective in inhibiting both pancreatic CSCs and cell lines.

Mang-NPs inhibit cell viability in spheroids formed by pancreatic CSCs isolated from humans
and Kras G12D mice. Since CSCs play a major role in cancer initiation, progression, metastasis and drug resistance, they can be used to assess the response of anticancer drugs. Spheroid formation in suspension is one of the characteristics of CSCs. Kras G12D mice mimic pancreatic cancer development in humans. Pancreatic CSCs isolated from Kras G12D mice are phenotypically similar to pancreatic CSCs isolated from humans. We next examined the effects of Mang-NPs on growth of pancreatic CSCs isolated from humans and Kras G12D mice by measuring cell viability in spheroids. Mang-NPs inhibited cell viability of primary, secondary and tertiary spheroids formed by pancreatic CSCs isolated from human and Kras G12D mice (Fig. 4A,B). These data suggest that Mang-NPs can be used for the management of pancreatic cancer by targeting pancreatic CSCs.

Mang-NPs inhibit cell motility, migration and invasion and markers of epithelial-mesenchymal transition. 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 and proliferate 33 . Since CSCs have been shown to be the cause of metastasis, we next measured the effects of Mang-NPs on cell motility, migration, and invasion, and on the expression of EMT markers. Mang-NPs inhibited cell motility, migration, and invasion of Pan CSCs (Fig. 5A-C). Similarly, Mang-NPs inhibited cell motility, migration and invasion in pancreatic cancer AsPC-1 and PANC-1 cell lines (data not shown).
Since Mang-NPs inhibited cell motility, migration, and invasion, we next examined the molecular mechanisms of EMT regulation by measuring the expression of proteins which modulate EMT, i.e. E-cadherin, N-cadherin and transcription factor Slug. As shown in Fig. 5D, Mang-NPs induced the expression of E-cadherin, and inhibited the expression of Slug. These data suggest that Mang-NPs has the potential to inhibit EMT by modulating the expression of E-cadherin and Slug.

Mang-NPs inhibit pluripotency maintaining factors in pancreatic CSCs.
Nanog, and c-Myc are essential for maintaining pluripotency in pancreatic CSCs. We therefore examined the effects of Mang-NPs on the expression of Nanog and c-Myc. Pancreatic CSCs were treated with Mang-NPs and the expression of Nanog and c-Myc was measured by the Western blot analysis. As shown in Fig. 6A, Mang-NPs inhibited the expression of Nanog in a dose dependent manner. In addition, while 1 μ M of Mang-NPs was ineffective in inhibiting c-Myc expression, higher doses of Mang-NPs (5 and 10 μ M) significantly inhibited c-Myc expression in Pan CSCs. We next confirmed the expression of these pluripotency maintaining factors by q-RT-PCR. Mang-NPs inhibited the expression of c-Myc, Oct4 and Nanog (Fig. 6B). These data suggest that Mang-NPs can inhibit CSC characteristics by inhibiting the expression of pluripotency maintain factors.
Nanog is required for maintaining pluripotency and self-renewal of CSCs. We therefore examined whether inhibition of Nanog by shRNA can enhance the anticancer activity of Mang-NPs. Pan CSCs were transduced with lentiviral particles expressing either scrambled or Nanog shRNA. Transduced CSCs were treated with Mang-NPs to examine their effects on colony formation. Pan CSCs/Nanog shRNA group had significantly lower number of colonies than Pan CSCs/Scrambled group (Fig. 6C). Furthermore, Mang-NPs inhibited colony formation in Pan CSCs/Scrambled group in a dose-dependent manner. Interestingly, the inhibitory effects of Mang-NPs were significantly higher in Pan CSCs/Nanog shRNA group than Pan CSCs/Scrambled group at all the doses used. These data demonstrate that inhibition of Nanog expression may be a novel strategy to enhance the effectiveness of Mang-NPs.

Mang-NPs inhibit Shh signaling pathway and Gli transcriptional target proteins. Aberrant acti-
vation of Shh signaling pathway has been demonstrated in pancreatic cancer 5,34 . We next examined the effects of Mang-NPs on components of SHH pathway by measuring the expression of Gli1, Gli2, Patched 1, and Patched  (Fig. 6D). Inhibition of Gli regulates its own expression and also the expression of Patched because they are the direct target of transcription factor Gli. These data suggest that Mang-NPs can inhibit self-renewal capacity of pancreatic CSCs by targeting Shh pathway.
Bcl-2 plays a major role in cell survival and also inhibits apoptosis. Further, it is a direct transcriptional target of Gli. We therefore sought to examine whether Mang-NPs can regulate the expression of Bcl-2 in Pan CSCs (Fig. 6E). Low doses of Mang-NPs (up to 5 μ M) had no effect on Bcl-2 expression, probably because the expression of Gli2 was also not affected at the similar doses of Mang-NPs. However, 10 μ M dose of Mang-NPs inhibited the expression of Bcl-2. These data suggest that Bcl-2 can modulate the anti-proliferative activity of Mang-NPs. We next measured the effects of Mang-NPs on Gli transcriptional activity in Pan CSCs, and cancer cell lines (PANC-1, AsPC-1 and MIA PaCa-2) by luciferase reporter assay. Mang-NPs inhibited Gli reporter activity in Pan CSCs and pancreatic cancer cell lines in a dose-dependent manner (Fig. 7A-D). These data suggest that Mang-NPs can inhibit cell proliferation of Pan CSCs and cancer cell lines by inhibiting Shh-Gli pathway.  KPC mice to examine whether Mang-NPs regulate pancreatic cancer growth, development and metastasis. The treatment of Mang-NPs was started when mice were about 4 weeks old. Mang-NPs were administrated intraperitoneally once per day, five days per week for about 10 weeks. We first measured the weight of pancreas from KPC/ control, KPC/Mang-NPs and C57B/L6 mice (untreated). The data demonstrate that KPC/Mang-NPs group had significantly lower pancreas weight than KPC/control group, and C57B/L6 normal group (Fig. 8A). The pancreas weight from KPC/Mang-NPs group was slightly but significantly higher than C57B/L6 mice (untreated). These data suggest that Mang-NPs can inhibit the growth of pancreatic cancer.
Liver metastasis is very common in late stage of pancreatic cancer 2 . We therefore measured liver metastasis by counting the number of nodules in the liver in control and Mang-NPs groups (Fig. 8C). We have observed liver metastasis in KPC/control group. By comparison, KPC/Mang-NPs group showed no liver metastasis. Overall, these data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis in KPC mice.

Inhibition of pancreatic carcinogenesis by Mang-NPs was correlated with suppression of Shh-Gli pathway and its downstream targets, and modulation of protein expression of stem cells markers, pluripotency maintaining factor, and EMT markers. Since Mang-NPs inhibited
growth, development and metastasis of pancreatic cancer in KPC mice, we next examined the molecular mechanisms of these pathological events. We have previously demonstrated the presence of CSCs in the pancreata of Kras G12D and KPC mice 26,29 . Tumor tissues from control and Mang-NPs treated groups were isolated, and tissue lysates were subjected to the Western blot analysis. Since KPC mice have been shown to harbor a small population of stem cells, we first examined the effects of Mang-NPs on the expression of stem cell markers (CD24 and CD133) and pluripotency maintaining factors (c-Myc, Nanog and Oct4). Treatment of KPC mice with Mang-NPs resulted in downregulation of CD24, CD133, c-Myc, Nanog and Oct4 (Fig. 8D). These data suggest that Mang-NPs can inhibit stem cell population within the tumor.
Since Mang-NPs inhibited pancreatic CSC characteristics in vitro by suppressing Shh pathway, we next examined the effects of Mang-NPs on the components of Shh pathway and its downstream targets in tumor tissues derived from KPC mice. Mang-NPs downregulated the expression of Gli1, Gli2, Patched-1, Patched-2 and smoothened in KPC mice (Fig. 8E). Furthermore, Mang-NPs also inhibited the expression of Gli targets Bcl-2, XIAP and cyclin D1 (Fig. 8F). These data suggest that Mang-NPs can regulate pancreatic carcinogenesis by inhibiting Shh pathway and also its targets which control apoptosis (Bcl-2), cell survival (XIAP) and cell cycle (cyclin D1).
Since Mang-NPs inhibited EMT in vitro and liver metastasis in vivo, we sought to examine the effects of Mang-NPs on markers of EMT in tumor tissues. As shown in Fig. 8G, Mang-NPs induced the expression of E-cadherin and inhibited the expression of N-cadherin, Slug, Snail, and Zeb1. These data suggest that Mang-NPs can reverse EMT by causing cadherin switch and inhibiting the EMT transcription factor.

Discussion
In this paper, we have demonstrated for, the first time, that Mang-NPs can inhibit pancreatic CSC characteristics, tumor growth and metastasis. Specifically, (i) Mang-NPs can be easily uptaken by the Panc CSCs and cancer cell lines; (ii) Mang-NPs inhibit cell viability of pancreatic CSCs and cell lines without inducing any toxicity to human normal pancreatic ductal epithelial cells; (iii) Mang-NPs inhibit colony formation and spheroid formation, and induce apoptosis in pan CSCs; (iv) Mang-NPs inhibit EMT by inducing cadherin switch and suppressing the expression of EMT transcription factors; (v) Mang-NPs inhibit self-renewal capacity of Pan CSC by suppressing pluripotency maintaining factors, and components of Shh pathway and its downstream targets; and (vi) Mang-NPs inhibit pancreatic cancer growth, development and metastasis in KPC mice by modulating the expression of stem cell and EMT markers, pluripotency maintain factors, and cell survival, apoptosis and cell cycle proteins. The ability of Mang-NPs to inhibit pancreatic carcinogenesis and CSC characteristics suggests that it not only inhibits bulk tumor mass but also CSCs which have been shown to be the cause of cancer initiation, progression and metastasis.
Kras G12D mutations are very common in pancreatic cancer, and more than 90% pancreatic cancer patients harbor mutations in this gene 35 . There are no effective drugs which have been designed to treat pancreatic cancer by targeting Kras G12D . In our study, Mang-NPs inhibited cell viability of spheroids formed by CSCs isolated from humans and Kras G12D mice, and also inhibit cell proliferation and induce apoptosis in pancreatic cancer cell lines harboring Kras G12D mutations. Furthermore, Mang-NPs also inhibited PanIN formation, PDAC development and metastasis in KPC mice, suggesting Mang-NPs can be a useful drug for the treatment and/or prevention of pancreatic cancer which harbor CSCs and progenitor cells.
Aberrant activation of Shh signaling pathway has been implicated in several cancers such as pancreatic cancer, prostate, glioblastoma, squamous cell carcinoma, basal cell carcinoma, medulloblastoma, prostate cancer, colon cancer, rhabdomyosarcoma and breast cancer 31 Shh pathway and its targets such as Bcl2, XIAP and Cyclin D1. These data suggest that Mang-NPs can inhibit pancreatic carcinogenesis by inhibiting cell survival, proliferation and cell cycle through Shh pathway. Similarly, in another study we have demonstrated that anthothecol-encapsulated PLGA nanoparticles can inhibit pancreatic CSC characteristics by inhibiting Shh pathway 26 .
EMT is an early pathological event which is characterized by the activation of embryonic programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype 42 . During early events, the expression of E-cadherin (CDH1 gene) goes down and the expression of N-cadherin goes up due to activation of transcription factor Snail, Slug and Zeb1 43 . CSCs also follow the similar patterns of cellular movement for sustained metastatic growth where the dissemination of CSCs from the primary tumor site is followed by their re-establishment in a secondary site. Thus, EMT can be considered as an early event during metastasis. In our in vitro studies, 1 μ M dose (a low dose) of Mang-NPs inhibited cell motility, invasion and migration, but had no effect on c-Myc expression. It could be due to the fact that Mang-NPs inhibited Nanog which can inhibit cell motility, invasion and migration. Our previous and present studies establish a key and essential role of the Shh-Gli pathway in promoting pancreatic CSC tumor growth, stem cell self-renewal and metastatic behavior 20,23,25,29,31,44,45 . In the present study, Mang-NPs not only inhibited tumor growth and development (PanINs to PDAC) but also metastasis to liver.
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 pancreatic cancer cell growth and CSC characteristics in vitro, and tumor growth, development and metastasis in KPC mice. Similarly, the clinical significance of α -Mangostin or Garcinia mangostana extract containing nanoparticles using different carriers have been reported in cancer and other diseases. α -Mangostin encapsulated Gold/polyethyleneimine/cyclodextrin (AuNPs/PEI/CD) nanoparticles inhibited prostate cancer PC-3 and DU-145 cell viability 46 . Furthermore, α -Mangostin encapsulated poly(ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles ameliorates Alzheimer's disease neuropathology by elevating low density lipoprotein receptor (LDLR) expression in microglia and liver cells and accelerating amyloid-beta clearance 47 . A clinical trial with the 4-week-randomized, double-blind, placebo-controlled, split-face study in 10 acne patients demonstrated a significant improvement in acne vulgaris condition with cellulose-based α -mangostin nanoparticles 48 . In another study, oral administration of mucoadhesive nanoparticles loaded with Garcinia mangostana extract eradicated Helicobacter pylori infection in mice 49 .
In conclusion, our study has demonstrated for the first time that Mang-NPs can inhibit proliferation of pancreatic CSCs and cancer cell lines, and inhibit the self-renewal capacity of CSCs isolated from pancreata of human and Kras G12D mice. Mang-NPs also inhibited pancreatic cancer progression from PanINs to PDAC and metastasis in KPC mice by suppressing Shh pathway. Since Mang-NPs inhibited the expression of Pan CSC markers and pluripotency maintaining factors in pancreatic tumors, it suggests that Mang-NPs can inhibit carcinogenesis by targeting CSC population. Overall these data suggest that Mang-NPs offer new hope for the treatment and/or prevention of pancreatic cancer by targeting CSCs. Based on our novel findings, clinical trials are needed to validate the role of Mang-NPs for the treatment and/or prevention of pancreatic cancer.