Pancreatic cancer is the fourth largest cause of cancer deaths in the Unites States and the prognosis is grim with <5% survival chances upon diagnosis. The objective of this study was to assess the combined chemopreventive effect of solid lipid nanoparticle (SLN) encapsulated drugs aspirin (ASP), curcumin (CUR) and free sulforaphane (SFN) for the chemoprevention of pancreatic cancer. Experiments were carried out (1) to evaluate the feasibility of encapsulation of these chemopreventive agents within solid lipid systems and (2) to measure the synergistic effects of a combination of ASP with CUR in SLNs mixed with free SFN against cell proliferation and apoptosis in pancreatic cancer cells, MIA PaCa-2 and Panc-1. The SLNs were prepared using a modified solvent evaporation technique and were characterized for particle sizing, encapsulation efficiency and drug release. ASP and CUR SLNs were formulated within the particle size range of 150–250 nm and were found to have an encapsulation efficiency of 85 and 69%, respectively. Sustained release of drugs over a 96 h period from SLNs was observed. The SLNs were stable over a 3-month storage period at room temperature. Cell viability studies demonstrated that combinations of low doses of ASP SLN (25 μM), CUR SLN (2.5 μM) and free SFN (5 μM) significantly reduced cell viability by 43.6 and 48.49% in MIAPaca-2 and Panc-1 cell lines, respectively. Furthermore, increased apoptosis of 61.3 and 60.37% was found in MIA Paca-2 and Panc-1 cell lines, respectively, in comparison to the individual doses administered. Synergistic effects were demonstrated using MTS and apoptosis assays. Thus, this study successfully demonstrated the feasibility of using a solid lipid nanoparticulate system for the first time to deliver this novel combination chemoprevention regimen, providing valuable evidence for the usability of nanotechnology-based drug regimens towards pancreatic cancer chemoprevention.

Pancreatic cancer ranks as the fourth most deadly form of cancer in the United States with ~37,000 deaths each year. The present study evaluated the chemopreventive potential of a combination of aspirin (ASP), curcumin (CUR) and sulforaphane (SFN) in low doses to human pancreatic cancer cells, MIA PaCa-2 and Panc-1. Results demonstrated that low doses of ASP (1 mM), CUR (10 μM) and SFN (5 μM) (ACS) combination reduced cell viability by ~70% (P<0.001), and also induced cell apoptosis by ~51% (P<0.001) accompanied by activation of caspase-3 and Poly(ADP-ribose) polymerase (PARP) proteins. The NF-κB DNA binding activity was inhibited by ~45% (P<0.01) and ~75% (P<0.001) in MIA PaCa-2 and Panc-1 cells, respectively. Mechanistic studies revealed that ACS promoted increase expression of phospho extracellular signal-regulated kinase 1/2 (P-ERK1/2), c-Jun, p38 MAPK and p53 proteins. Furthermore, the cells pretreated with U0126 (ERK1/2 inhibitor) partially abolished the effect of ACS on cell viability. Data from this study demonstrate that a low-dose ACS combination inhibits cell growth by inducing cell apoptosis, and proposes sustained activation of the ERK1/2 signaling pathway as one of the possible mechanisms.

Pancreatic cancer is a deadly disease killing 37,000 Americans each year. Despite two decades of research on treatment options, the chances of survival are still <5% upon diagnosis. Recently, chemopreventive strategies have gained considerable attention as an alternative to treatment. We have previously shown significant in vitro chemopreventive effects with low dose combinations of aspirin (ASP), curcumin (CUR) and sulforaphane (SFN) (ACS) on pancreatic cancer cell lines. Here, we report the results of 24-week chemopreventive study with the oral administration of ACS combinations on the N-nitrosobis (2-oxopropyl) amine (BOP)-treated Syrian golden hamster model to suppress the progression of pancreatic intraepithelial neoplasms (PanINs) using (1) unmodified (free drug) combinations of ACS, and (2) nanoencapsulated (solid-lipid nanoparticles; SLN) combinations of ASP, CUR and free SFN. The use of three different doses (low, medium and high) of unmodified ACS combinations exhibited reduction in tumor incidence by 18%, 50% and 68.7% respectively; whereas the modified nano-encapsulated ACS regimens reduced tumor incidence by 33%, 67% and 75%, respectively, at 10X lower dose compared to the free drug combinations. Similarly, while the unmodified free ACS demonstrated a notable reduction in cell proliferation, the SLN encapsulated ACS regimens, showed significant reduction in cell proliferation at 6.3%, 58.6 % and 72.8 % as evidenced by PCNA expression. Cell apoptotic indices were also up regulated by 1.5X, 2.8X and 3.2X respectively, compared to BOP control. These studies provide a proof-of-concept for the use of an oral, low dose, nanotechnology-based combinatorial regimen for the long term chemoprevention of pancreatic cancer.

Pancreatic cancer ranks as the fourth most deadly form of cancer in the United States with approximately 37,000 deaths each year. The present study aims to evaluate the combinatorial chemopreventive effects of low doses of chloroquine (CQ) and aspirin (ASP) alone and in combination CQA (CQ+ASP) on three pancreatic cancer cell lines, MIA PaCa-2, Panc-1 and BxPC-3. Results demonstrated that low doses of CQ (6.25μM) and ASP (1mM) alone did not reduce cell viability, whereas combination CQA reduced cell viability by ∼80% (P&lt;0.001). CQA combination blocked cell cycle progression at G0/G1 checkpoint. The CQA treatment for 24 h inhibited the NF-κB p50 DNA binding activity by ∼45% (P&lt;0.01) in MIA PaCa-2 and BxPC-3 cells, but no NF-κB inhibition observed in Panc-1 cells. Mechanistic studies revealed that treatment with CQA combination increased the expression of LC3-II protein, indicates inhibition of autophagy. In order to verify the involvement of PI3K and mTOR pathway in CQA-induced apoptosis, we used PI3K inhibitor (3-MA) and mTOR inhibitor (Rapamycin) and analyzed by cell viability assay. The inhibition of PI3K by 3-MA pretreatment enhanced the cell growth inhibition of CQA treatment, whereas the inhibition of m-TOR by rapamycin pretreatment attenuated CQA induced reduction in cell viability. Overall, our results for the first time, illustrate that low dose CQA combinations impart synergistic suppressive activity than chemopreventive agents alone, thus confirming the importance of novel combination chemoprevention strategies against pancreatic cancer. Citation Format: Arvind Thakkar, B. Karthik Grandhi, Jeffrey Wang, Sunil Prabhu. Inhibition of PI3K-mTOR pathway using combination of chloroquine and aspirin regimen leads to apoptosis in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2596. doi:10.1158/1538-7445.AM2013-2596

Pancreatic cancer caused 40,000 deaths in 2010 and is currently ranked as the fourth leading cause of deaths in the United States. Due to poor prognosis of this disease, it is imperative that chemopreventive strategies be developed to control the formation of the initial neoplastic lesions. We have previously reported the effectiveness of a low dose combination regimen of aspirin, curcumin and sulforaphane (ACS) on MIA-PaCa-2 and Panc-1 pancreatic cancer cell lines. The objective of the current study was to use the ACS combination regimens in vivo to demonstrate the molecular targets responsible for prevention of pancreatic cancer in N-nitrosobis(2-oxopropyl) amine (BOP) carcinogen treated Syrian golden hamsters. BOP treated Syrian golden hamsters (n=6/group) were sacrificed after 24 weeks of treatment with a low (20+45+1.6), medium (67+150+5) and high (200+450+16) doses (in mg/kg) of ACS, respectively. Saline and BOP controls were included in the study. The pancreatic tissues were processed for isolation of nuclear and cytoplasmic proteins, and 20 mg of tissue was used for isolation of total RNA. Results from the Western blot show that low dose of ACS combined regimen down-regulated active NF-κB by inhibiting phosphorylation of IαBβ and p65. Low dose ACS also inhibited phosphorylation of Akt, needed for IKK activation. Consequently at the transcription level, low dose ACS significant down-regulated NF-κB regulated gene products TNFα, IL1α and IL6 compared to BOP controls (p&lt;0.05). Thus, low dose ACS inhibited the translocation of NF-κB into the nucleus by inhibiting the phosphorylation of IαBβ and p65. In addition, RT qPCR analysis of proliferation markers such as proliferating cell nuclear antigen (PCNA) and cyclin D1 show &gt;2 fold down-regulation in low dose regimen compared to BOP controls. With the high and medium doses, a trend similar to the low dose ACS was evident, whereby the effects were more significant at higher doses (p&lt;0.001), thus demonstrating a dose-dependent response. The significance of this work is in the demonstration of the effectiveness in gene regulation of ACS even at the low-dose level which shows its potential as a powerful combinatorial regimen for pancreatic cancer prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2589. doi:1538-7445.AM2012-2589

Gastrointestinal cancers, in particular colon and pancreatic cancer, account for almost 100,000 deaths each year in the United States, ranking third and fourth respectively, in the most common cause of cancer related deaths. Our lab has been focused on developing strategies for chemoprevention rather than treatment after diagnosis. For pancreatic cancer, the prognosis is usually poor with high mortality rates after diagnosis. Thus, the objective of this work was to study the effects of chemopreventive combination regimens of a non-steroidal anti-inflammatory drug (NSAID) naproxen (N), in combination with resveratrol (R, an antioxidant) and sulforaphane (S, a broccoli extract) on human colon (HT-29 and HCT-116) and pancreatic (MIA PaCa-2 and Panc-1) cancer cell lines. MTS cell proliferation assay was conducted on all cell lines. For the MTS assay, upon 75 % confluence, 100µL of 2.5X103 cells each of HT-29, HCT116 and MIA PaCa-2 and 4X10 3 cells Panc-1 cells were transferred into each well of 96-well plates. The NRS chemopreventive agents alone, or in combination, were added to the cells and incubated for 72 h. The plates were then incubated for 1-4 h at 37°C in a humidified, 5% CO2 atmosphere. Absorbance was recorded at 490 nm using an ELISA plate reader. For HT-29 and HCT 116 colon cancer cell lines, the combinations of naproxen (500µM/L) with resveratrol (50µM/L) and sulforaphane (5µM/L) showed reduction in cell viability of 56.74% and 52.9%, respectively. The IC50 values for NRS were 1.46 mM/L,104.54µM/L and 17.81µM/L respectively on HT-29 cell line. Similarly, for MIA PaCa-2 and Panc-1 pancreatic cancer cell lines, combinations of naproxen (500µM/L) with resveratrol (6µM/L) and sulforaphane (5µM/L) showed reduction in cell viability of 66.67% and 58.8% respectively. The IC50 values for NRS were 778µM/L, 74.8µM/L and 11.82µM/L respectively, on MIA Paca-2 cell line. When used individually, none of the chemopreventive agents at the above concentrations demonstrated any decrease in cell viability, however when combined together, a synergistic effect was observed. Our results demonstrate that NRS drug combinations at low concentrations exhibit significant decrease in cell proliferation in both human colon and pancreatic cancer cell lines. This data provides compelling evidence of the potential of these combination regimens for the chemoprevention of colon and pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1632. doi:1538-7445.AM2012-1632

According to the American Cancer Society (ACS), an estimated 37,660 deaths will occur in 2011 due to complications from pancreatic cancer. The onset of pancreatic cancer is often devoid of symptoms and is mostly diagnosed at a stage where survival rate is less than 5%. Thus, chemoprevention could play an important role in the fight against this silent yet deadly disease. The objective of our study was to use combinations of chemopreventive agents namely piroxicam (a non-steroidal anti-inflammatory drug), curcumin (a natural spice with significant cancer chemopreventive properties) and sulforaphane (a potent broccoli extract) in very low doses to inhibit cell proliferation and promote apoptosis on two human pancreatic cancer cell lines, MIA PaCA-2 and Panc-1. More importantly, we compared the effects of the chemopreventive combinations in both, unmodified (free drug) and modified forms, encapsulated within solid-lipid nanoparticles (SLNs). SLNs were prepared using a process of hot-melt oil in water (o/w) emulsion technique using stearic acid as the lipid backbone to which the chemopreventive agents were added and then recovered as nanoparticles (∼200 nm size) after a process of freeze-drying. MTS cell proliferation, apoptosis and cell colony forming assays were conducted on cultured cells. For the MTS assay, upon 75 % confluence, 2.5 × 103 cells for MIA PaCa-2 and 4 × 103 cells for Panc-1 were transferred into each well of 96-well plates. The PCS chemopreventive agents alone or in combination were added to the cells and incubated for 72 h. Absorbance was recorded at 490 nm using an ELISA plate reader. For the apoptosis assay, 3 × 105 cells were cultured in 6-well plates for both MIA PaCa-2 and Panc-1 cell-lines. PCS, individual or combinations, were added and the plates were incubated for 72 h. Cells were analyzed using flow cytometry, measuring the fluorescence emission at 530 nm (FL1) and &gt;575 nm (FL3). For the cell colony formation assays, MIA PaCa-2 and Panc-1 cells were seeded at a density of 1 ml containing 3 × 104 cells, incubated for a period of 24 h. After treatment with PCS alone and in combinations, cells were trypsinized then incubated for a period of 10 days. The number of colonies with &gt;50 cells were counted under a dissecting microscope. IC50 values obtained for the unmodified and modified forms of piroxicam were (663.1μM; 52.85μM), curcumin (16.05μM; 4.93μM) respectively and free sulforaphane at 11.82 μM/L for MIA PaCa-2 cells. For Panc-1 cells, the IC50 values for unmodified and modified piroxicam were (389.3μM; 66.96μM), curcumin (24.01μM; 7.5μM) respectively and free sulforaphane at 17.81μM/L. For MIA PaCa-2 cell line, unmodified combinations of piroxicam (250μM) with curcumin (10μM) and sulforaphane (5μM) showed significant reduction in cell viability of &gt; 60% using MTS assay. In comparison, lower concentrations of piroxicam SLN (25μM) and curcumin SLN (2.5μM) with free sulforaphane (5μM) mixtures showed decrease in cell viability by &gt;47.4%. Flow cytometry analysis demonstrated apoptosis of 71% (unmodified) and 38.4% (modified) for the combinations, at the same concentration ranges as above. Panc-1 cell line treated with similar concentrations of unmodified and modified SLN combinations showed a &gt;82% and &gt;45.79% decrease in cell viability and 91% and 54.6% apoptotic cells, respectively. Colony formation assays in both cell lines showed a significant decrease (p value &lt;0.0001) in cell counts. From this study, we believe the action of nanotechnology-based chemopreventive regimens on human pancreatic cell lines provides strong evidence of its potential as a viable tool to prevent the onset of pancreatic cancer. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B64.