Mark Kester - Academia.edu (original) (raw)
Papers by Mark Kester
Elsevier's Integrated Pharmacology, 2007
The FASEB Journal
Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human... more Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human health due to its ability to induce colitis and cause colon tumor formation in mice through the production of B. fragilis toxin (BFT). The formation of tumors is dependent on a pro‐inflammatory signaling cascade, which begins with the disruption of epithelial barrier integrity through cleavage of E‐cadherin. Here, we show that BFT increases levels of glucosylceramide, a vital intestinal sphingolipid, both in mice and in colon organoids (colonoids) generated from the distal colons of mice. When colonoids are treated with BFT in the presence of an inhibitor of glucosylceramide synthase (GCS), the enzyme responsible for generating glucosylceramide, colonoids become highly permeable, lose structural integrity, and eventually burst, releasing their contents into the extracellular matrix. By increasing glucosylceramide levels in colonoids via an inhibitor of glucocerebrosidase (GBA, the enzyme that degrades glucosylceramide), colonoid permeability was reduced, and bursting was significantly decreased. In the presence of BFT, pharmacological inhibition of GCS caused levels of tight junction protein 1 (TJP1) to decrease. However, when GBA was inhibited, TJP1 levels remained stable, suggesting that BFT‐induced production of glucosylceramide helps to stabilize tight junctions. Taken together, our data demonstrate a glucosylceramide‐dependent mechanism by which the colon epithelium responds to BFT.
Cancer Letters, 2013
Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In... more Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In human liver and colon cancer cells, treatment with an autophagy inducer, nanoliposomal C6ceramide, in combination with the autophagy maturation inhibitor, vinblastine, synergistically enhanced apoptotic cell death. Combination treatment resulted in a marked increase in autophagic vacuole accumulation and decreased autophagy maturation, without diminution of the autophagy flux protein P62. In a colon cancer xenograft model, a single intravenous injection of the drug combination significantly decreased tumor growth in comparison to the individual treatments. Most importantly, the combination treatment did not result in increased toxicity as assessed by body weight loss. The mechanism of combination treatment-induced cell death both in vitro and in vivo appeared to be apoptosis. Supportive of autophagy flux blockade as the underlying synergy mechanism, treatment with other autophagy maturation inhibitors, but not autophagy initiation inhibitors, were similarly synergistic with vinblastine. Additionally, knockout of the autophagy protein Beclin-1 suppressed combination treatment-induced apoptosis in vitro. In conclusion, in vitro and in vivo data support a synergistic antitumor activity of the nanoliposomal C6-ceramide and vinblastine combination, potentially mediated by an autophagic mechanism.
Cancer Biology & Therapy, 2011
poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-bas... more poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-based nanotherapeutics due to metabolic pathways which neutralize ceramide to less toxic or pro-oncogenic metabolites. We have recently developed a novel 80 nanometer diameter liposomal formulation that incorporates 30 molar percent C 6-ceramide, a bioactive lipid that is pro-apoptotic to many cancer cells, but not to normal cells. In this manuscript, we evaluated the efficacy of combining nanoliposomal C 6-ceramide (Lip-C 6) with either gemcitabine or an inhibitor of glucosylceramide synthase. We first assessed the biological effect of Lip-C 6 in paNC-1 cells, a gemcitabine-resistant human pancreatic cancer cell line, and found that low doses alone did not induce cell toxicity. however, cytotoxicity was achieved by combining Lip-C 6 with either non-toxic sub-therapeutic concentrations of gemcitabine or with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (pDMp). Furthermore, these combinations with Lip-C 6 cooperatively inhibited paNC-1 tumor growth in vivo. Mechanistically, Lip-C 6 inhibited pro-survival akt and erk signaling, whereas the nucleoside analog gemcitabine did not. Furthermore, by including pDMp within the nanoliposomes, which halted ceramide neutralization as evidenced by LC-Ms/Ms/Ms, the cytotoxic effects of Lip-C 6 were enhanced. Collectively, we have demonstrated that nanoliposomal ceramide can be an effective anti-pancreatic cancer therapeutic in combination with gemcitabine or an inhibitor of ceramide neutralization.
European Journal of Pharmacology, 1989
ACS nano, Jan 27, 2018
Detection of monolayer morphology on nanoparticles smaller than 10 nm has proven difficult with t... more Detection of monolayer morphology on nanoparticles smaller than 10 nm has proven difficult with traditional visualization techniques. Here matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is used in conjunction with atomistic simulations to detect the formation of Janus-like monolayers on noble metal nanoparticles. Silver metal nanoparticles were synthesized with a monolayer consisting of dodecanethiol (DDT) and mercaptoethanol (ME) at varying ratios. The nanoparticles were then analyzed using MALDI-MS, which gives information on the local ordering of ligands on the surface. The MALDI-MS analysis showed large deviations from random ordering, suggesting phase separation of the DDT/ME monolayers. Atomistic Monte Carlo (MC) calculations were then used to simulate the nanoscale morphology of the DDT/ME monolayers. In order to quantitatively compare the computational and experimental results, we developed a method for determining an expected MALDI-MS spectrum from...
Molecular Cancer Therapeutics
Ceramides are bioactive lipids that mediate cell death in cancer cells, and ceramide-based therap... more Ceramides are bioactive lipids that mediate cell death in cancer cells, and ceramide-based therapy is now being tested in dose-escalating phase I clinical trials as a cancer treatment. Multiple nanoscale delivery systems for ceramide have been proposed to overcome the inherent toxicities, poor pharmacokinetics, and difficult biophysics associated with ceramide. Using the ceramide nanoliposomes (CNL), we now investigate the therapeutic efficacy and signaling mechanisms of this nanoscale delivery platform in refractory ovarian cancer. Treatment of ovarian cancer cells with CNL decreased the number of living cells through necroptosis but not apoptosis. Mechanistically, dying SKOV3 ovarian cancer cells exhibit activation of pseudokinase mixed lineage kinase domain-like (MLKL) as evidenced by oligomerization and relocalization to the blebbing membranes, showing necroptotic characteristics. Knockdown of MLKL, but not its upstream protein kinases such as receptor-interacting protein kinases, with siRNA significantly abolished CNL-induced cell death. Monomeric MLKL protein expression inversely correlated with the IC 50 values of CNL in distinct ovarian cancer cell lines, suggesting MLKL as a possible determinant for CNL-induced cell death. Finally, systemic CNL administration suppressed metastatic growth in an ovarian cancer cell xenograft model. Taken together, these results suggest that MLKL is a novel pronecroptotic target for ceramide in ovarian cancer models.
PLOS ONE
Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages ... more Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A 2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE 2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14 hi CD163 hi CD206 hi ). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1β, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression.
Advances in cancer research, 2018
Sphingolipids are bioactive lipids that participate in a wide variety of biological mechanisms, i... more Sphingolipids are bioactive lipids that participate in a wide variety of biological mechanisms, including cell death and proliferation. The myriad of pro-death and pro-survival cellular pathways involving sphingolipids provide a plethora of opportunities for dysregulation in cancers. In recent years, modulation of these sphingolipid metabolic pathways has been in the forefront of drug discovery for cancer therapeutics. About two decades ago, researchers first showed that standard of care treatments, e.g., chemotherapeutics and radiation, modulate sphingolipid metabolism to increase endogenous ceramides, which kill cancer cells. Strikingly, resistance to these treatments has also been linked to altered sphingolipid metabolism, favoring lipid species that ultimately lead to cell survival. To this end, many inhibitors of sphingolipid metabolism have been developed to further define not only our understanding of these pathways but also to potentially serve as therapeutic interventions. ...
Signal transduction and targeted therapy, 2017
The pathogenesis of chronic lymphocytic leukemia (CLL) is poorly understood and it remains incura... more The pathogenesis of chronic lymphocytic leukemia (CLL) is poorly understood and it remains incurable with current therapies. We have previously shown that nanoliposomal C6-ceramide (CNL) is an effective therapy in anmurine model of CLL. However, the key signaling pathways mediating CNL-induced cell death in CLL remains unknown. We hypothesized that CNL targets STAT3, a critical regulator of hematopoietic biology. We observed that CNL treatment reduced phosphorylated STAT3 at both Y705 and S727 residues in CLL cell lines and patient cells. This, in turn, reduced STAT3 transcriptional activity and expression of critical STAT3-dependent survival factors like Mcl-1 and survivin. The effect of CNL on STAT3 was further confirmedas shown by reduced STAT3 phosphorylation in xenograft tumors obtained from mice treated with CNL. CNL suppressed STAT3 phosphorylation at Y705 and S727 through reduction in BTK activity and MEK1/2 kinase/PKC activities, respectively. Moreover, a synergistic reduct...
Journal of Leukemia
Novel approaches to enhancing the efficacy of ceramide-based therapeutics are of interest for the... more Novel approaches to enhancing the efficacy of ceramide-based therapeutics are of interest for the treatment of acute myeloid leukemia (AML). Ceramide is a bioactive sphingolipid that has long been established as an inducer of apoptosis. We have pioneered the effort to develop nanoliposomal C6-ceramide (Lip-C6) as an anticancer therapeutic and recently have engaged in efforts to enhance its therapeutic efficacy. Ceramide catabolism by acid ceramidase and subsequently sphingosine kinase 1 yields the metabolite oncogenic sphingosine-1-phosphate (S1P). Therefore, in the present study we hypothesized that targeting of this metabolic pathway with the sphingosine kinase 1 inhibitor safingol would augment the anti-AML efficacy of Lip-C6. We generated and evaluated nanoliposomes encapsulating safingol (Lip-Saf), in combination with Lip-C6 using AML cell lines and primary AML patient samples. This combination exerted synergistic therapeutic efficacy using HL-60 and KG-1 cells, and additive efficacy using HL-60/VCR cells. In contrast, the combination of Lip-C6 and Lip-Saf yielded an antagonistic effect using the murine AML cell line C1498 and this effect was correlated with an increase in autophagy as a potent leukemia survival mechanism. Intriguingly, by using an inhibitor of ceramide glycosylation, nanoliposomal tamoxifen, we observed synergistic anti-AML efficacy using the C1498 cell line demonstrating that unique manipulations of ceramide metabolism may be prevalent in different AMLs. Lastly, we evaluated both favorable prognosis and poor prognosis primary patient AML samples and observed combinatorial efficacy of Lip-C6 and Lip-Saf. This was reflected by a decrease in autophagy and concomitant increase in apoptosis, as well as the ability to block colony forming capacity. Altogether, these results demonstrated that the efficacy of Lip-C6 as an experimental anti-AML therapy can be dramatically enhanced through combination with inhibitors of ceramide metabolism.
Nanomedicine
Breast cancer is a major ongoing public health issue among women in both developing and developed... more Breast cancer is a major ongoing public health issue among women in both developing and developed countries. Significant progress has been made to improve the breast cancer treatment in the past decades. However, the current clinical approaches are invasive, of low specificity and can generate severe side effects. As a rapidly developing field, nanotechnology brings promising opportunities to human cancer diagnosis and treatment. The use of nanoparticulate-based platforms overcomes biological barriers and allows prolonged blood circulation time, simultaneous tumor targeting and enhanced accumulation of drugs in tumors. Currently available and clinically applicable innovative nanoparticulate-based systems for breast cancer nanotherapies are discussed in this review.
Cancer Translational Medicine
To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the nove... more To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the novel small molecule inhibitor, SKI-178. Using the state-of-the-art Cellular Thermal Shift Assay (CETSA) technique to detect "direct target engagement" of proteins intact cells, in vitro and in vivo assays, pharmacological assays and multiple mouse models of acute myeloid leukemia (AML). Herein, we demonstrate that SKI-178 directly target engages both Sphingosine Kinase 1 and 2. We also present evidence that, in addition to its actions as a Sphingosine Kinase Inhibitor, SKI-178 functions as a microtubule network disrupting agent both in vitro and in intact cells. Interestingly, we separately demonstrate that simultaneous SphK inhibition and microtubule disruption synergistically induces apoptosis in AML cell lines. Furthermore, we demonstrate that SKI-178 is well tolerated in normal healthy mice. Most importantly, we demonstrate that SKI-178 has therapeutic efficacy in several mouse models of AML. SKI-178 is a multi-targeted agent that functions both as an inhibitor of the SphKs as well as a disruptor of the microtubule network. SKI-178 induced apoptosis arises from a synergistic interaction of these two activities. SKI-178 is safe and effective in mouse models of AML, supporting its further development as a multi-targeted anti-cancer therapeutic agent.
Nanomedicine : nanotechnology, biology, and medicine, 2017
Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and n... more Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive, phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. These findings demonstrate a nov...
Biochemical pharmacology, Apr 15, 2017
The anticancer properties of ceramide, a sphingolipid with potent tumor-suppressor properties, ca... more The anticancer properties of ceramide, a sphingolipid with potent tumor-suppressor properties, can be dampened via glycosylation, notably in multidrug resistance wherein ceramide glycosylation is characteristically elevated. Earlier works using the ceramide analog, C6-ceramide, demonstrated that the antiestrogen tamoxifen, a first generation P-glycoprotein (P-gp) inhibitor, blocked C6-ceramide glycosylation and magnified apoptotic responses. The present investigation was undertaken with the goal of discovering non-anti-estrogenic alternatives to tamoxifen that could be employed as adjuvants for improving the efficacy of ceramide-centric therapeutics in treatment of cancer. Herein we demonstrate that the tamoxifen metabolites, desmethyltamoxifen and didesmethyltamoxifen, and specific, high-affinity P-gp inhibitors, tariquidar and zosuquidar, synergistically enhanced C6-ceramide cytotoxicity in multidrug resistant HL-60/VCR acute myelogenous leukemia (AML) cells, whereas the selective...
Journal of leukemia (Los Angeles, Calif.), 2014
Acute Myeloid Leukemia (AML) is a highly heterogeneous and poor prognosis disease with few availa... more Acute Myeloid Leukemia (AML) is a highly heterogeneous and poor prognosis disease with few available therapeutic options. Novel advances are urgently needed, however effective models to test experimental therapeutics have been lacking. Recently, NOD/SCID/IL2rγnull (NSG) mice were shown to engraft primary human AML in a manner that recapitulated the natural disease and its progression. Additionally, integrated genomic profiling was used to refine risk stratification of AML. In this study, we demonstrated the engraftment of molecularly defined primary AML in NSG mice. We showed that AML that express DNMT3A mutations, which predict for adverse outcome, engrafted with exceptional efficacy. Lastly, we demonstrated that human AML-engrafted NSG mice can be effectively used to study novel ceramide-based therapeutics. Ceramide is a bioactive sphingolipid that has been implicated as an inducer of apoptosis. Elevation in cancer cell ceramide levels either via exogenous delivery or by provoking...
Cell reports, Nov 1, 2016
Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; howeve... more Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, Sphk1 also appears to facilitate endosomal fusion independent of its catalytic activity, given that catalytically inactive Sphk1(G82D) is recruited to endocytic membranes by sphingosine or sphingosine-based Sphk1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceram...
Oncotarget, Jan 4, 2016
There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard th... more There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased ov...
Nucleic acid therapeutics, Jan 18, 2016
Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecysto... more Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against "exposed" CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bi...
Elsevier's Integrated Pharmacology, 2007
The FASEB Journal
Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human... more Enterotoxigenic Bacteroides fragilis (ETBF) is a commensal bacterium of great importance to human health due to its ability to induce colitis and cause colon tumor formation in mice through the production of B. fragilis toxin (BFT). The formation of tumors is dependent on a pro‐inflammatory signaling cascade, which begins with the disruption of epithelial barrier integrity through cleavage of E‐cadherin. Here, we show that BFT increases levels of glucosylceramide, a vital intestinal sphingolipid, both in mice and in colon organoids (colonoids) generated from the distal colons of mice. When colonoids are treated with BFT in the presence of an inhibitor of glucosylceramide synthase (GCS), the enzyme responsible for generating glucosylceramide, colonoids become highly permeable, lose structural integrity, and eventually burst, releasing their contents into the extracellular matrix. By increasing glucosylceramide levels in colonoids via an inhibitor of glucocerebrosidase (GBA, the enzyme that degrades glucosylceramide), colonoid permeability was reduced, and bursting was significantly decreased. In the presence of BFT, pharmacological inhibition of GCS caused levels of tight junction protein 1 (TJP1) to decrease. However, when GBA was inhibited, TJP1 levels remained stable, suggesting that BFT‐induced production of glucosylceramide helps to stabilize tight junctions. Taken together, our data demonstrate a glucosylceramide‐dependent mechanism by which the colon epithelium responds to BFT.
Cancer Letters, 2013
Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In... more Autophagy, a catabolic survival pathway, is gaining attention as a potential target in cancer. In human liver and colon cancer cells, treatment with an autophagy inducer, nanoliposomal C6ceramide, in combination with the autophagy maturation inhibitor, vinblastine, synergistically enhanced apoptotic cell death. Combination treatment resulted in a marked increase in autophagic vacuole accumulation and decreased autophagy maturation, without diminution of the autophagy flux protein P62. In a colon cancer xenograft model, a single intravenous injection of the drug combination significantly decreased tumor growth in comparison to the individual treatments. Most importantly, the combination treatment did not result in increased toxicity as assessed by body weight loss. The mechanism of combination treatment-induced cell death both in vitro and in vivo appeared to be apoptosis. Supportive of autophagy flux blockade as the underlying synergy mechanism, treatment with other autophagy maturation inhibitors, but not autophagy initiation inhibitors, were similarly synergistic with vinblastine. Additionally, knockout of the autophagy protein Beclin-1 suppressed combination treatment-induced apoptosis in vitro. In conclusion, in vitro and in vivo data support a synergistic antitumor activity of the nanoliposomal C6-ceramide and vinblastine combination, potentially mediated by an autophagic mechanism.
Cancer Biology & Therapy, 2011
poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-bas... more poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-based nanotherapeutics due to metabolic pathways which neutralize ceramide to less toxic or pro-oncogenic metabolites. We have recently developed a novel 80 nanometer diameter liposomal formulation that incorporates 30 molar percent C 6-ceramide, a bioactive lipid that is pro-apoptotic to many cancer cells, but not to normal cells. In this manuscript, we evaluated the efficacy of combining nanoliposomal C 6-ceramide (Lip-C 6) with either gemcitabine or an inhibitor of glucosylceramide synthase. We first assessed the biological effect of Lip-C 6 in paNC-1 cells, a gemcitabine-resistant human pancreatic cancer cell line, and found that low doses alone did not induce cell toxicity. however, cytotoxicity was achieved by combining Lip-C 6 with either non-toxic sub-therapeutic concentrations of gemcitabine or with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (pDMp). Furthermore, these combinations with Lip-C 6 cooperatively inhibited paNC-1 tumor growth in vivo. Mechanistically, Lip-C 6 inhibited pro-survival akt and erk signaling, whereas the nucleoside analog gemcitabine did not. Furthermore, by including pDMp within the nanoliposomes, which halted ceramide neutralization as evidenced by LC-Ms/Ms/Ms, the cytotoxic effects of Lip-C 6 were enhanced. Collectively, we have demonstrated that nanoliposomal ceramide can be an effective anti-pancreatic cancer therapeutic in combination with gemcitabine or an inhibitor of ceramide neutralization.
European Journal of Pharmacology, 1989
ACS nano, Jan 27, 2018
Detection of monolayer morphology on nanoparticles smaller than 10 nm has proven difficult with t... more Detection of monolayer morphology on nanoparticles smaller than 10 nm has proven difficult with traditional visualization techniques. Here matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is used in conjunction with atomistic simulations to detect the formation of Janus-like monolayers on noble metal nanoparticles. Silver metal nanoparticles were synthesized with a monolayer consisting of dodecanethiol (DDT) and mercaptoethanol (ME) at varying ratios. The nanoparticles were then analyzed using MALDI-MS, which gives information on the local ordering of ligands on the surface. The MALDI-MS analysis showed large deviations from random ordering, suggesting phase separation of the DDT/ME monolayers. Atomistic Monte Carlo (MC) calculations were then used to simulate the nanoscale morphology of the DDT/ME monolayers. In order to quantitatively compare the computational and experimental results, we developed a method for determining an expected MALDI-MS spectrum from...
Molecular Cancer Therapeutics
Ceramides are bioactive lipids that mediate cell death in cancer cells, and ceramide-based therap... more Ceramides are bioactive lipids that mediate cell death in cancer cells, and ceramide-based therapy is now being tested in dose-escalating phase I clinical trials as a cancer treatment. Multiple nanoscale delivery systems for ceramide have been proposed to overcome the inherent toxicities, poor pharmacokinetics, and difficult biophysics associated with ceramide. Using the ceramide nanoliposomes (CNL), we now investigate the therapeutic efficacy and signaling mechanisms of this nanoscale delivery platform in refractory ovarian cancer. Treatment of ovarian cancer cells with CNL decreased the number of living cells through necroptosis but not apoptosis. Mechanistically, dying SKOV3 ovarian cancer cells exhibit activation of pseudokinase mixed lineage kinase domain-like (MLKL) as evidenced by oligomerization and relocalization to the blebbing membranes, showing necroptotic characteristics. Knockdown of MLKL, but not its upstream protein kinases such as receptor-interacting protein kinases, with siRNA significantly abolished CNL-induced cell death. Monomeric MLKL protein expression inversely correlated with the IC 50 values of CNL in distinct ovarian cancer cell lines, suggesting MLKL as a possible determinant for CNL-induced cell death. Finally, systemic CNL administration suppressed metastatic growth in an ovarian cancer cell xenograft model. Taken together, these results suggest that MLKL is a novel pronecroptotic target for ceramide in ovarian cancer models.
PLOS ONE
Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages ... more Pancreatic ductal adenocarcinoma (PDAC) tumor growth is enhanced by tumor-associated macrophages (TAMs), yet the mechanisms by which tumor cells and TAMs communicate are not fully understood. Here we show that exosomes secreted by PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages in vitro. Exosomes from AsPC-1, an ascites-derived human PDAC cell line, were enriched in ICAM-1, which mediated their docking to macrophages through interactions with surface-exposed CD11c on macrophages. AsPC-1 exosomes also contained much higher levels of arachidonic acid (AA), and they fused at a higher rate with THP-1-derived macrophages than did exosomes from other PDAC cell lines or from an immortalized normal pancreatic ductal epithelial cell line (HPDE) H6c7. Phospholipase A 2 enzymatic cleavage of arachidonic acid from AsPC-1 exosomes reduced fusion efficiency. PGE 2 secretion was elevated in macrophages treated with AsPC-1 exosomes but not in macrophages treated with exosomes from other cell lines, suggesting a functional role for the AsPC-1 exosome-delivered arachidonic acid in macrophages. Non-polarized (M0) macrophages treated with AsPC-1 exosomes had increased levels of surface markers indicative of polarization to an immunosuppressive M2-like phenotype (CD14 hi CD163 hi CD206 hi ). Furthermore, macrophages treated with AsPC-1 exosomes had significantly increased secretion of pro-tumoral, bioactive molecules including VEGF, MCP-1, IL-6, IL-1β, MMP-9, and TNFα. Together, these results demonstrate that compared to exosomes from other primary tumor-derived PDAC cell lines, AsPC-1 exosomes alter THP-1-derived macrophage phenotype and function. AsPC-1 exosomes mediate communication between tumor cells and TAMs that contributes to tumor progression.
Advances in cancer research, 2018
Sphingolipids are bioactive lipids that participate in a wide variety of biological mechanisms, i... more Sphingolipids are bioactive lipids that participate in a wide variety of biological mechanisms, including cell death and proliferation. The myriad of pro-death and pro-survival cellular pathways involving sphingolipids provide a plethora of opportunities for dysregulation in cancers. In recent years, modulation of these sphingolipid metabolic pathways has been in the forefront of drug discovery for cancer therapeutics. About two decades ago, researchers first showed that standard of care treatments, e.g., chemotherapeutics and radiation, modulate sphingolipid metabolism to increase endogenous ceramides, which kill cancer cells. Strikingly, resistance to these treatments has also been linked to altered sphingolipid metabolism, favoring lipid species that ultimately lead to cell survival. To this end, many inhibitors of sphingolipid metabolism have been developed to further define not only our understanding of these pathways but also to potentially serve as therapeutic interventions. ...
Signal transduction and targeted therapy, 2017
The pathogenesis of chronic lymphocytic leukemia (CLL) is poorly understood and it remains incura... more The pathogenesis of chronic lymphocytic leukemia (CLL) is poorly understood and it remains incurable with current therapies. We have previously shown that nanoliposomal C6-ceramide (CNL) is an effective therapy in anmurine model of CLL. However, the key signaling pathways mediating CNL-induced cell death in CLL remains unknown. We hypothesized that CNL targets STAT3, a critical regulator of hematopoietic biology. We observed that CNL treatment reduced phosphorylated STAT3 at both Y705 and S727 residues in CLL cell lines and patient cells. This, in turn, reduced STAT3 transcriptional activity and expression of critical STAT3-dependent survival factors like Mcl-1 and survivin. The effect of CNL on STAT3 was further confirmedas shown by reduced STAT3 phosphorylation in xenograft tumors obtained from mice treated with CNL. CNL suppressed STAT3 phosphorylation at Y705 and S727 through reduction in BTK activity and MEK1/2 kinase/PKC activities, respectively. Moreover, a synergistic reduct...
Journal of Leukemia
Novel approaches to enhancing the efficacy of ceramide-based therapeutics are of interest for the... more Novel approaches to enhancing the efficacy of ceramide-based therapeutics are of interest for the treatment of acute myeloid leukemia (AML). Ceramide is a bioactive sphingolipid that has long been established as an inducer of apoptosis. We have pioneered the effort to develop nanoliposomal C6-ceramide (Lip-C6) as an anticancer therapeutic and recently have engaged in efforts to enhance its therapeutic efficacy. Ceramide catabolism by acid ceramidase and subsequently sphingosine kinase 1 yields the metabolite oncogenic sphingosine-1-phosphate (S1P). Therefore, in the present study we hypothesized that targeting of this metabolic pathway with the sphingosine kinase 1 inhibitor safingol would augment the anti-AML efficacy of Lip-C6. We generated and evaluated nanoliposomes encapsulating safingol (Lip-Saf), in combination with Lip-C6 using AML cell lines and primary AML patient samples. This combination exerted synergistic therapeutic efficacy using HL-60 and KG-1 cells, and additive efficacy using HL-60/VCR cells. In contrast, the combination of Lip-C6 and Lip-Saf yielded an antagonistic effect using the murine AML cell line C1498 and this effect was correlated with an increase in autophagy as a potent leukemia survival mechanism. Intriguingly, by using an inhibitor of ceramide glycosylation, nanoliposomal tamoxifen, we observed synergistic anti-AML efficacy using the C1498 cell line demonstrating that unique manipulations of ceramide metabolism may be prevalent in different AMLs. Lastly, we evaluated both favorable prognosis and poor prognosis primary patient AML samples and observed combinatorial efficacy of Lip-C6 and Lip-Saf. This was reflected by a decrease in autophagy and concomitant increase in apoptosis, as well as the ability to block colony forming capacity. Altogether, these results demonstrated that the efficacy of Lip-C6 as an experimental anti-AML therapy can be dramatically enhanced through combination with inhibitors of ceramide metabolism.
Nanomedicine
Breast cancer is a major ongoing public health issue among women in both developing and developed... more Breast cancer is a major ongoing public health issue among women in both developing and developed countries. Significant progress has been made to improve the breast cancer treatment in the past decades. However, the current clinical approaches are invasive, of low specificity and can generate severe side effects. As a rapidly developing field, nanotechnology brings promising opportunities to human cancer diagnosis and treatment. The use of nanoparticulate-based platforms overcomes biological barriers and allows prolonged blood circulation time, simultaneous tumor targeting and enhanced accumulation of drugs in tumors. Currently available and clinically applicable innovative nanoparticulate-based systems for breast cancer nanotherapies are discussed in this review.
Cancer Translational Medicine
To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the nove... more To further characterize the selectivity, mechanism-of-action and therapeutic efficacy of the novel small molecule inhibitor, SKI-178. Using the state-of-the-art Cellular Thermal Shift Assay (CETSA) technique to detect "direct target engagement" of proteins intact cells, in vitro and in vivo assays, pharmacological assays and multiple mouse models of acute myeloid leukemia (AML). Herein, we demonstrate that SKI-178 directly target engages both Sphingosine Kinase 1 and 2. We also present evidence that, in addition to its actions as a Sphingosine Kinase Inhibitor, SKI-178 functions as a microtubule network disrupting agent both in vitro and in intact cells. Interestingly, we separately demonstrate that simultaneous SphK inhibition and microtubule disruption synergistically induces apoptosis in AML cell lines. Furthermore, we demonstrate that SKI-178 is well tolerated in normal healthy mice. Most importantly, we demonstrate that SKI-178 has therapeutic efficacy in several mouse models of AML. SKI-178 is a multi-targeted agent that functions both as an inhibitor of the SphKs as well as a disruptor of the microtubule network. SKI-178 induced apoptosis arises from a synergistic interaction of these two activities. SKI-178 is safe and effective in mouse models of AML, supporting its further development as a multi-targeted anti-cancer therapeutic agent.
Nanomedicine : nanotechnology, biology, and medicine, 2017
Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and n... more Drug resistant cancers like pancreatic ductal adenocarcinoma (PDAC) are difficult to treat, and nanoparticle drug delivery systems can overcome some of the limitations of conventional systemic chemotherapy. In this study, we demonstrate that FdUMP and dFdCMP, the bioactive, phosphorylated metabolites of the chemotherapy drugs 5-FU and gemcitabine, can be encapsulated into calcium phosphosilicate nanoparticles (CPSNPs). The non-phosphorylated drug analogs were not well encapsulated by CPSNPs, suggesting the phosphate modification is essential for effective encapsulation. In vitro proliferation assays, cell cycle analyses and/or thymidylate synthase inhibition assays verified that CPSNP-encapsulated phospho-drugs retained biological activity. Analysis of orthotopic tumors from mice treated systemically with tumor-targeted FdUMP-CPSNPs confirmed the in vivo up take of these particles by PDAC tumor cells and release of active drug cargos intracellularly. These findings demonstrate a nov...
Biochemical pharmacology, Apr 15, 2017
The anticancer properties of ceramide, a sphingolipid with potent tumor-suppressor properties, ca... more The anticancer properties of ceramide, a sphingolipid with potent tumor-suppressor properties, can be dampened via glycosylation, notably in multidrug resistance wherein ceramide glycosylation is characteristically elevated. Earlier works using the ceramide analog, C6-ceramide, demonstrated that the antiestrogen tamoxifen, a first generation P-glycoprotein (P-gp) inhibitor, blocked C6-ceramide glycosylation and magnified apoptotic responses. The present investigation was undertaken with the goal of discovering non-anti-estrogenic alternatives to tamoxifen that could be employed as adjuvants for improving the efficacy of ceramide-centric therapeutics in treatment of cancer. Herein we demonstrate that the tamoxifen metabolites, desmethyltamoxifen and didesmethyltamoxifen, and specific, high-affinity P-gp inhibitors, tariquidar and zosuquidar, synergistically enhanced C6-ceramide cytotoxicity in multidrug resistant HL-60/VCR acute myelogenous leukemia (AML) cells, whereas the selective...
Journal of leukemia (Los Angeles, Calif.), 2014
Acute Myeloid Leukemia (AML) is a highly heterogeneous and poor prognosis disease with few availa... more Acute Myeloid Leukemia (AML) is a highly heterogeneous and poor prognosis disease with few available therapeutic options. Novel advances are urgently needed, however effective models to test experimental therapeutics have been lacking. Recently, NOD/SCID/IL2rγnull (NSG) mice were shown to engraft primary human AML in a manner that recapitulated the natural disease and its progression. Additionally, integrated genomic profiling was used to refine risk stratification of AML. In this study, we demonstrated the engraftment of molecularly defined primary AML in NSG mice. We showed that AML that express DNMT3A mutations, which predict for adverse outcome, engrafted with exceptional efficacy. Lastly, we demonstrated that human AML-engrafted NSG mice can be effectively used to study novel ceramide-based therapeutics. Ceramide is a bioactive sphingolipid that has been implicated as an inducer of apoptosis. Elevation in cancer cell ceramide levels either via exogenous delivery or by provoking...
Cell reports, Nov 1, 2016
Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; howeve... more Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, Sphk1 also appears to facilitate endosomal fusion independent of its catalytic activity, given that catalytically inactive Sphk1(G82D) is recruited to endocytic membranes by sphingosine or sphingosine-based Sphk1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceram...
Oncotarget, Jan 4, 2016
There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard th... more There is an urgent unmet need for new therapeutics in acute myeloid leukemia (AML) as standard therapy has not changed in the past three decades and outcome remains poor for most patients. Sphingolipid dysregulation through decreased ceramide levels and elevated sphingosine 1-phosphate (S1P) promotes cancer cell growth and survival. Acid ceramidase (AC) catalyzes ceramide breakdown to sphingosine, the precursor for S1P. We report for the first time that AC is required for AML blast survival. Transcriptome analysis and enzymatic assay show that primary AML cells have high levels of AC expression and activity. Treatment of patient samples and cell lines with AC inhibitor LCL204 reduced viability and induced apoptosis. AC overexpression increased the expression of anti-apoptotic Mcl-1, significantly increased S1P and decreased ceramide. Conversely, LCL204 induced ceramide accumulation and decreased Mcl-1 through post-translational mechanisms. LCL204 treatment significantly increased ov...
Nucleic acid therapeutics, Jan 18, 2016
Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecysto... more Pancreatic ductal adenocarcinomas (PDACs) constitutively express the G-protein-coupled cholecystokinin B receptor (CCKBR). In this study, we identified DNA aptamers (APs) that bind to the CCKBR and describe their characterization and targeting efficacy. Using dual SELEX selection against "exposed" CCKBR peptides and CCKBR-expressing PDAC cells, a pool of DNA APs was identified. Further downselection was based on predicted structures and properties, and we selected eight APs for initial characterizations. The APs bound specifically to the CCKBR, and we showed not only that they did not stimulate proliferation of PDAC cell lines but rather inhibited their proliferation. We chose one AP, termed AP1153, for further binding and localization studies. We found that AP1153 did not activate CCKBR signaling pathways, and three-dimensional Confocal microscopy showed that AP1153 was internalized by PDAC cells in a receptor-mediated manner. AP1153 showed a binding affinity of 15 pM. Bi...