Curcumin: A Multi-dimensional Approach to Pancreatic Cancer Targeting Cell Death and Exosomes

Pancreatic cancer is currently one of the most difficult diseases to treat due to difficulty of detection and the aggressive nature of the disease. In addition, pancreatic cancer has the highest mortality rates compared to other cancer types. These mortality rates are attributable in part to increasing resistance to cancer therapy. Cancer therapy resistance is caused by adaptations that favor survival within cancer cells and their environment, termed the tumor microenvironment. Intracellular adaptations include the overexpression of resistance-linked genes, such as the inhibitor of apoptosis (IAP) family of proteins and overall resistance to cell death. Adaptations in the tumor microenvironment include altered intercellular vesicular signaling through exosomes, resulting in tumor growth and progression. However, recent studies have shown that exosomes can also be used as a delivery mechanism for drugs with poor bioavailability, thus providing a therapeutic advantage for these compounds. Currently, researchers are moving toward a multi-dimensional approach to pancreatic cancer therapy that incorporates compounds that target crucial players in chemotherapy resistance and in the tumor microenvironment, such as exosomes. Our studies are centered on the anti-cancer properties of curcumin, a turmeric derivative, on these intracellular and intercellular resistance mechanisms. The long term goal of this research is to determine the mechanisms by which curcumin modulates intracellular pathways related to pancreatic cancer survival and therapy resistance and exosome composition and release to improve the understanding of pancreatic cancer pathology and support the development of novel therapeutic approaches for pancreatic cancer patients. The specific objective of this research was to determine curcumin’s role in modulating intracellular proteins imperative for pancreatic cancer chemotherapy resistance such as the IAP proteins. Moreover, this research addressed the effects of curcumin on exosome release and function, specifically in the context of delivery to recipient pancreatic cancer cells. We have established that curcumin reduces expression of the IAPs in pancreatic cancer cells, inhibiting their survival and growth. Furthermore, curcumin not only attenuates pro-survival signaling through exosomes, but also itself carried within the nanovesicles and delivered to recipient pancreatic cancer cells, resulting in pancreatic cancer cell death

FAK activity sustains intrinsic and acquired ovarian cancer resistance to platinum chemotherapy.

Gene copy number alterations, tumor cell stemness, and the development of platinum chemotherapy resistance contribute to high-grade serous ovarian cancer (HGSOC) recurrence. Stem phenotypes involving Wnt-β-catenin, aldehyde dehydrogenase activities, intrinsic platinum resistance, and tumorsphere formation are here associated with spontaneous gains in Kras, Myc and FAK (KMF) genes in a new aggressive murine model of ovarian cancer. Adhesion-independent FAK signaling sustained KMF and human tumorsphere proliferation as well as resistance to cisplatin cytotoxicity. Platinum-resistant tumorspheres can acquire a dependence on FAK for growth. Accordingly, increased FAK tyrosine phosphorylation was observed within HGSOC patient tumors surviving neo-adjuvant chemotherapy. Combining a FAK inhibitor with platinum overcame chemoresistance and triggered cell apoptosis. FAK transcriptomic analyses across knockout and reconstituted cells identified 135 targets, elevated in HGSOC, that were regulated by FAK activity and β-catenin including Myc, pluripotency and DNA repair genes. These studies reveal an oncogenic FAK signaling role supporting chemoresistance

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Enhancement of Gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant

Background: Current therapeutic options for advanced pancreatic cancer have been largely disappointing with modest results at best, and though adjuvant therapy remains controversial, most remain in agreement that Gemcitabine should stand as part of any combination study. The inhibitor of apoptosis (IAP) protein Survivin is a key factor in maintaining apoptosis resistance, and its dominant-negative mutant (Survivin-T34A) has been shown to block Survivin, inducing caspase activation and apoptosis. Methods: In this study, exosomes, collected from a melanoma cell line built to harbor a tetracycline-regulated Survivin-T34A, were plated on the pancreatic adenocarcinoma (MIA PaCa-2) cell line. Evaluation of the presence of Survivin-T34A in these exosomes followed by their ability to induce Gemcitabine-potentiative cell killing was the objective of this work. Results: Here we show that exosomes collected in the absence of tetracycline (tet-off) from the engineered melanoma cell do contain Survivin-T34A and when used alone or in combination with Gemcitabine, induced a significant increase in apoptotic cell death when compared to Gemcitabine alone on a variety of pancreatic cancer cell lines. Conclusion: This exosomes/Survivin-T34A study shows that a new delivery method for anticancer proteins within the cancer microenvironment may prove useful in targeting cancers of the pancreas

Curcumin Modulates Pancreatic Adenocarcinoma Cell-Derived Exosomal Function.

Pancreatic cancer has the highest mortality rates of all cancer types. One potential explanation for the aggressiveness of this disease is that cancer cells have been found to communicate with one another using membrane-bound vesicles known as exosomes. These exosomes carry pro-survival molecules and increase the proliferation, survival, and metastatic potential of recipient cells, suggesting that tumor-derived exosomes are powerful drivers of tumor progression. Thus, to successfully address and eradicate pancreatic cancer, it is imperative to develop therapeutic strategies that neutralize cancer cells and exosomes simultaneously. Curcumin, a turmeric root derivative, has been shown to have potent anti-cancer and anti-inflammatory effects in vitro and in vivo. Recent studies have suggested that exosomal curcumin exerts anti-inflammatory properties on recipient cells. However, curcumin's effects on exosomal pro-tumor function have yet to be determined. We hypothesize that curcumin will alter the pro-survival role of exosomes from pancreatic cancer cells toward a pro-death role, resulting in reduced cell viability of recipient pancreatic cancer cells. The main objective of this study was to determine the functional alterations of exosomes released by pancreatic cancer cells exposed to curcumin compared to exosomes from untreated pancreatic cancer cells. We demonstrate, using an in vitro cell culture model involving pancreatic adenocarcinoma cell lines PANC-1 and MIA PaCa-2, that curcumin is incorporated into exosomes isolated from curcumin-treated pancreatic cancer cells as observed by spectral studies and fluorescence microscopy. Furthermore, curcumin is delivered to recipient pancreatic cancer cells via exosomes, promoting cytotoxicity as demonstrated by Hoffman modulation contrast microscopy as well as AlamarBlue and Trypan blue exclusion assays. Collectively, these data suggest that the efficacy of curcumin may be enhanced in pancreatic cancer cells through exosomal facilitation

Glucocorticoid Receptor Regulates and Interacts with LEDGF/p75 to Promote Docetaxel Resistance in Prostate Cancer Cells

Patients with advanced prostate cancer (PCa) invariably develop resistance to anti-androgen therapy and taxane-based chemotherapy. Glucocorticoid receptor (GR) has been implicated in PCa therapy resistance; however, the mechanisms underlying GR-mediated chemoresistance remain unclear. Lens epithelium-derived growth factor p75 (LEDGF/p75, also known as PSIP1 and DFS70) is a glucocorticoid-induced transcription co-activator implicated in cancer chemoresistance. We investigated the contribution of the GR–LEDGF/p75 axis to docetaxel (DTX)-resistance in PCa cells. GR silencing in DTX-sensitive and -resistant PCa cells decreased LEDGF/p75 expression, and GR upregulation in enzalutamide-resistant cells correlated with increased LEDGF/p75 expression. ChIP-sequencing revealed GR binding sites in the LEDGF/p75 promoter. STRING protein–protein interaction analysis indicated that GR and LEDGF/p75 belong to the same transcriptional network, and immunochemical studies demonstrated their co-immunoprecipitation and co-localization in DTX-resistant cells. The GR modulators exicorilant and relacorilant increased the sensitivity of chemoresistant PCa cells to DTX-induced cell death, and this effect was more pronounced upon LEDGF/p75 silencing. RNA-sequencing of DTX-resistant cells with GR or LEDGF/p75 knockdown revealed a transcriptomic overlap targeting signaling pathways associated with cell survival and proliferation, cancer, and therapy resistance. These studies implicate the GR–LEDGF/p75 axis in PCa therapy resistance and provide a pre-clinical rationale for developing novel therapeutic strategies for advanced PCa

Proteomic Profiling of Serum-Derived Exosomes from Ethnically Diverse Prostate Cancer Patients

<p>Prostate cancer (PCa) remains the most frequently diagnosed male malignancy in Western countries and the second most common cause of male cancer death in the United States. The relatively elevated PCa incidence and mortality among African American men makes this cancer type a challenging health disparity disease. To increase the chance for successful trea tment, earlier detection and prediction of tumor aggress iveness will be important and need to be resolved. This study demonstrates that small membrane-bound vesicles shed from the tumor called exosomes contain ethnically and tumor-specific biomarkers, and could be exploited for their diagnostic and therapeutic potential.</p

Spectrophotometric detection of curcumin within exosomes from PANC-1 cells.

<p>Exosomes were isolated from untreated PANC-1 cells (curcumin-negative exosomes) or PANC-1 cells treated with 50 μM of curcumin for 24 hours (curcumin-positive exosomes). (A) Whole (non-lysed, blue) exosomes from curcumin-treated PANC-1 cells were subjected to spectral analysis compared to vehicle (1X PBS blank, red), in which optical density (OD) at 420 nm was measured. No peak in absorbance was detected at 420 nm from whole (non-lysed) exosomes. (B) Methanol and sonication were used to lyse exosomes from curcumin-treated PANC-1 cells (lysed curcumin-positive exosomes, green) or exosomes from untreated PANC-1 cells (lysed curcumin-negative exosomes, blue). A methanol-only blank (red) was used as a negative control for this assay. A characteristic peak in OD at 420 nm was detected in lysed curcumin-positive exosomes, but not in lysed curcumin-negative exosomes or the methanol-only blank. Data are representative of three independent experiments.</p

Exosomal curcumin reduces recipient pancreatic adenocarcinoma cell viability.

<p>Naïve recipient PANC-1 or MIA PaCa-2 cells were cultured for the indicated times with exosomes isolated from untreated cells (curcumin-negative exosomes) or exosomes isolated from cells treated with 50 μM of curcumin (curcumin-positive exosomes). In a separate experiment, naïve recipient cells were treated with 10 μg/mL heparin prior to and during incubation with curcumin-positive exosomes (heparin + curcumin-positive exosomes). Viability was determined via (A) AlamarBlue and (B) Trypan blue exclusion assays and exosome treatments were compared to naïve cells not exposed to exosomes or heparin (untreated). Data are represented as mean + SEM of three independent experiments, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p