CREB activation drives acinar to ductal reprogramming and promote pancreatic cancer progression in animal models of alcoholic chronic pancreatitis.

BACKGROUND AND AIMS: In vivo induction of alcoholic chronic pancreatitis (ACP) causes significant acinar damage, increased fibroinflammatory response, and heightened activation of cyclic response element binding protein 1 (CREB) when compared with alcohol (A) or chronic pancreatitis (CP) mediated pancreatic damage. However, the study elucidating the cooperative interaction between CREB and the oncogenic Kras G12D/+ (Kras*) in promoting pancreatic cancer progression with ACP remains unexplored. METHODS: Experimental ACP induction was established in multiple mouse models, followed by euthanization of the animals at various time intervals during the recovery periods. Tumor latency was determined in these mice cohorts. Here, we established CREB deletion (Creb fl/fl ) in Ptf1a CreERTM/+ ;LSL-Kras G12D+/-(KC) genetic mouse models (KCC-/-). Western blot, phosphokinase array, and qPCR were used to analyze the pancreata of Ptf1a CreERTM+/-, KC and KCC -/- mice. The pancreata of ACP-induced KC mice were subjected to single-cell RNA sequencing (scRNAseq). Further studies involved conducting lineage tracing and acinar cell explant cultures. RESULTS: ACP induction in KC mice had detrimental effects on the pancreatic damage repair mechanism. The persistent existence of acinar cell-derived ductal lesions demonstrated a prolonged state of hyperactivated CREB. Persistent CREB activation leads to acinar cell reprogramming and increased pro-fibrotic inflammation in KC mice. Acinar-specific Creb ablation reduced advanced PanINs lesions, hindered tumor progression, and restored acinar cell function in ACP-induced mouse models. CONCLUSIONS: Our findings demonstrate that CREB cooperates with Kras* to perpetuate an irreversible ADM and PanIN formation. Moreover, CREB sustains oncogenic activity to promote the progression of premalignant lesions toward cancer in the presence of ACP.

Progesterone-Cationic Lipid Conjugate-Based Self-Aggregates for Cancer Cell-Selective Uptake through Macropinocytosis and the Antitumour Effect.

Progesterone (PR) is an endogenous steroid hormone that activates the progesterone receptor (PgR) and is known to play a critical role in cancer progression. Herein, we report the development of cationic lipid-conjugated PR derivatives by covalently conjugating progesterone with cationic lipids of varying hydrocarbon chain lengths (n = 6-18) through a succinate linker. Cytotoxicity studies performed on eight different cancer cell lines reveal that PR10, one of the lead derivatives, exerts notable toxicity (IC50 = 4-12 µM) in cancer cells irrespective of their PgR expression status and remains largely nontoxic to noncancerous cells. Mechanistic studies show that PR10 induces G2/M-phase cell cycle arrest in cancer cells, leading to apoptosis and cell death by inhibiting the PI3K/AKT cell survival pathway and p53 upregulation. Further, in vivo study shows that PR10 treatment significantly reduces melanoma tumor growth and prolongs the overall survival of melanoma tumor-bearing C57BL/6J mice. Interestingly, PR10 readily forms stable self-aggregates of ∼190 nm size in an aqueous environment and exhibits selective uptake into cancerous cell lines. In vitro uptake mechanism studies in various cell lines (cancerous cell lines B16F10, MCF7, PC3, and noncancerous cell line HEK293) using endocytosis inhibition proves that PR10 nanoaggregates enter selectively into the cancer cells predominantly using macropinocytosis and/or caveolae-mediated endocytosis. Overall, this study highlights the development of a self-aggregating cationic derivative of progesterone with anticancer activity, and its cancer cell-selective accumulation in nanoaggregate form holds great potential in the field of targeted drug delivery.

Modification of α-Tocopherol Succinate with a Tumor-targeting Peptide Conjugate Enhances the Antitumor Efficacy of a Paclitaxel-loaded Lipid Aggregate.

Paclitaxel (PTX) is a widely used chemotherapeutic agent in the clinic. However, its clinical benefit is limited due to its low water solubility, off-target toxicity, and for being a multidrug-resistant (MDR) substrate. To overcome these limitations in this study, a tumor-targeting peptide (CRGDK peptide, a ligand for NRP-1 receptor) conjugate of α-tocopheryl succinate (α-TOS) was synthesized and modified on PTX-loaded lipid aggregate (TL-PTX) to leverage the benefits of α-TOS, which include a) anti-cancer activity, b) increased PTX loading, and c) inhibition of MDR activity. Use of peptide conjugate of α-TOS (α-TOS-CRGDK) in lipid aggregate increased PTX entrapment efficiency by 20%, helped in NRP-1 specific cellular uptake and significantly enhanced apoptotic and cell killing activity (p <0.01) of PTX compared to control formulation (CL-PTX) by inhibiting MDR-activity in melanoma resulting in ∼70% increment in overall survival of melanoma tumor-bearing mice. In conclusion, CRGDK- α-TOS conjugate in association with PTX-loaded liposome provided a unique NRP-1 targeted, drug-resistant reversing anticancer regimen for treating aggressive melanoma.

Enriched pharmacokinetic behavior and antitumor efficacy of thymoquinone by liposomal delivery.

Background: Thymoquinone (TQ) has potential anti-inflammatory, immunomodulatory and anticancer effects but its clinical use is limited by its low solubility, poor bioavailability and rapid clearance. Aim: To enhance systemic bioavailability and tumor-specific toxicity of TQ. Materials & methods: Cationic liposomal formulation of TQ (D1T) was prepared via ethanol injection method and their physicochemical properties, anticancer effects in orthotopic xenograft pancreatic tumor model and pharmacokinetic behavior of D1T relative to TQ were evaluated. Results: D1T showed prominent inhibition of pancreatic tumor progression, significantly greater in vivo absorption, approximately 1.5-fold higher plasma concentration, higher bioavailability, reduced volume of distribution and improved clearance relative to TQ. Conclusion: Encapsulation of TQ in cationic liposomal formulation enhanced its bioavailability and anticancer efficacy against xenograft pancreatic tumor.

Glucocorticoid receptor-targeted liposomal delivery system for delivering small molecule ESC8 and anti-miR-Hsp90 gene construct to combat colon cancer.

High mortality rate in colon cancer patients is often attributed to late diagnosis. To overcome the conventional chemotherapy associated challenges, chemotherapeutic drugs (single or combination) or genetic drugs are often delivered using ligand-modified delivery systems that selectively target over expressed receptors or particular receptors that act abnormally in cancer cells. In the current investigation, first we assessed anti-colon cancer effect of a cationic estrogenic molecule, ESC8 which was earlier shown to act against estrogen receptor (ER) ± breast cancer cells. We found that against both colon and breast cancer cells the anticancer activity is intervened by AMPK-mTOR pathway and at the same time it acts as anti-angiogenic agent. It also showed enhancement of mesenchymal-to-epithelial (MET) transition as well as reduction of cyclin D in both cells. Earlier we demonstrated the use of glucocorticoid receptor (GR) targeted cationic liposomal delivery system carrying anti-Hsp90 plasmid and ESC8 to act as potent anti-skin cancer therapeutics. As ESC8 demonstrated anti-colon cancer effect in vitro, in here, we used the same GR-targeted liposomal formulation but carrying a more fusogenic cationic lipid D1 and used against colon tumor orthotopic model in mice. We show that GR targeted formulation (D1XE-Hsp90) exhibited efficient cellular uptake, transfection and selective cytotoxicity in colon cancer cells, tumor-targeted bio-distribution and enhanced survivability, reduced tumor size in orthotopic colon tumor-bearing mice. The tumor sections exhibited reduced tumor proliferation as well as neo-vascularization, thus supporting the holistic antitumor effect of the D1XE-Hsp90 formulation. Over all our results establish the GR-targeted D1XE-Hsp90 formulation as potent anti-colon cancer therapeutics.

Self-Assembling Derivative of Hydrocortisone as Glucocorticoid Receptor-Targeted Nanotherapeutics for Synergistic, Combination Therapy against Colorectal Tumor.

Hydrocortisone, a natural glucocorticoid secreted by adrenal and extra-adrenal tissues, locally governs the transcription of genes involved in inflammation, immune response, metabolism, and energy homeostasis via binding to its cognate glucocorticoid receptor (GR). In this study, we show that modified hydrocortisone (HC16), a cancer-selective cytotoxic molecule, showed synergism in combination with drugs like Doxorubicin and docetaxel, self-assembled into vesicles, entrapped docetaxel and complexed with anti-cancer plasmid DNA for enhanced killing of cancer cells. These vesicles exhibited GR-mediated nuclear localization, delivery of the p53 gene, and also inhibited cell viability selectively in RKO, HCT15, and CT26 colon cancer cells but not in normal cells like CHO and HEK293T. Apart from exerting its own anti-cancer activity, the self-assembled HC16 vesicles loaded with docetaxel sensitized the cancer cells to its drug cargo by downregulating the drug metabolizing CYP3A4 gene. This indirectly reduces the risk of nonspecific adverse effects in normal cells, as the viability of sensitized cancer cells could be significantly reduced even in low doses of cytotoxic docetaxel. The near infrared (NIR)-dye-associated self-assemblies accumulated in a colon tumor with higher orders of NIR intensity compared to those in a colon of healthy mice. Thereafter, the treatment of HC16-docetaxel-p53 vesicle/DNA complex led to significant tumor regression, which resulted in a cecum/body weight ratio in tumor-bearing mice similar to that of healthy mice measured at 24 h postcompletion of treatment. There was an up to 2.5-fold enhancement in the overall survivability of colon-tumor-bearing mice treated with HC16-docetaxel-p53 vesicle/DNA complexes when compared against the pristine docetaxel-treated groups. Further, the HC16-docetaxel-p53 vesicle/DNA complex-treated group showed reduced nuclear accumulation of cell proliferation marker Ki67, reduced protein levels of prosurvival and mesenchymal proteins like Bcl-2, PARP, vimentin, and N-cadherin, and increased the levels of pro-apoptotic activated caspases as compared to the pristine docetaxel-treated groups. The therapeutic package described herein is expected to find future use as a rational, multifaceted, GR-targeted approach for inhibiting colon tumor progression.

Methoxy-enriched cationic stilbenes as anticancer therapeutics.

Stilbene-based compounds are largely described for their antioxidant activity. But their use as anticancer chemotherapeutics is hampered by poor pharmacokinetic properties and non-selectivity towards cancer and non-cancer potency. To overcome these drawbacks, twin chain cationic lipid conjugated, methoxy-enriched stilbene derivatives were designed, synthesized and evaluated for their anticancer potency. Our findings reveal that HMSC16, a molecule with the highest number of methoxy groups and with C16-twin chain lipid, is the most potent as well as the most selective anticancer agent when compared to the other synthesized derivatives and commercially available stilbene-based drug, tamoxifen, and resveratrol. To justify these results, we have conducted a series of mechanistic experiments where we found that HMSC16 induced ROS generation, apoptosis, and autophagy by affecting the mitochondrial, lysosomal and nuclear pathways. Further cell cycle analysis data reveals that HMSC16 not only induces cell death but is also involved in the arrest of the cell cycle at the sub-G1 phase. Moreover, HMSC16 showed self-aggregation property owing to a possibly favorable hydrophilic-lipophilic balance. The self-aggregation property of HMSC16 allowed it to entrap hydrophobic drugs, withaferin. With entrapped withaferin, HMSC16 showed additive if not synergistic cell killing effect in HeLa cells. From the above results, we concluded that HMSC16 can be used not just as a drug but also as a drug delivery agent.

Efficient anti-tumor nano-lipoplexes with unsaturated or saturated lipid induce differential genotoxic effects in mice.

Cationic lipids are well-known excipients for nanometric liposomal gene delivery systems. However, because of the suspected, collateral toxicity in normal cells, the use of cationic lipids for the treatment of human tumor is largely limited. Recently, we developed a glucocorticoid receptor (GR)-targeted liposomal, anticancer delivery system (DXE nano-lipoplex), which carried cationic lipid of saturated twin aliphatic chains. It exhibited efficient anti-tumor effect in aggressive and drug-resistant tumor models. Toward exploring lipoplex's human clinical use, we incorporated another nano-lipoplex (D1XE) group that carried cationic lipid with one of its aliphatic chain carrying unsaturation and compared in vivo genotoxicological profiling-based safety assessment and the respective anti-tumor efficacy of the lipoplexes. Thus, both the lipoplexes differ only by the chemical identity of one of their constituent cationic lipid. Unsaturated aliphatic chains in lipid generally impart efficient cell surface fusogenic property in lipid formulations. Herein, we report that nanoplex with unsaturated cationic lipid (D1XE) exhibited better physical appearance with less flocculent behavior than nanoplex with saturated lipid (DXE). Upon multiple injections, D1XE nanoplex imparted better tumor regression but most importantly, exhibited much lower overall toxicity (e.g. genotoxicity, weight loss, etc.) than DXE nanoplex. With a higher antitumor effect but a lower genotoxic effect, D1XE is proved to be a better nanoplex than DXE for the potential clinical trial. Thus, this study clearly delineates the importance of incorporating a constituent lipid that carries a single unsaturated aliphatic chain toward developing efficient anti-tumor nano-lipoplexes with reduced genotoxicity.

Phenazine-1-carboxamide functionalized mesoporous silica nanoparticles as antimicrobial coatings on silicone urethral catheters.

Microbial infections due to biofilms on medical implants can be prevented by antimicrobial coatings on biomaterial surfaces. Mesoporous silica nanoparticles (MSNPs) were synthesized via base-catalyzed sol-gel process at room temperature, functionalized with phenazine-1-carboxamide (PCN) and characterized by UV-visible, FT-IR, DLS, XRD spectroscopic techniques, SEM, TEM, TGA and BET analysis. Native MSNPs, PCN and PCN-MSNPs were evaluated for anti-Candida minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), Candida albicans (C. albicans) biofilms and C. albicans-Staphylococcus aureus (S. aureus) polymicrobial biofilm inhibition. PCN-MSNPs were four-fold effective (MIC 3.9 µg mL-1; 17.47 µM) and MFC (7.8 µg mL-1; 34.94 µM) as compared to pure PCN (MIC 15.6 µg mL-1; 69.88 µM) and MFC (31.2 µg mL-1; 139.76 µM). PCN-MSNPs inhibited in vitro C. albicans MTCC 227-S. aureus MTCC 96 biofilms at very low concentration (10 µg mL-1; 44.79 µM) as compared to pure PCN (40 µg mL-1; 179.18 µM). Mechanistic studies revealed that PCN induced intracellular ROS accumulation in C. albicans MTCC 227, S. aureus MTCC 96 and S. aureus MLS-16 MTCC 2940, reduction in total ergosterol content, membrane permeability, disruption of ionic homeostasis followed by Na+, K+ and Ca2+ leakage leading to cell death in C. albicans MTCC 227 as confirmed by confocal laser scanning micrographs. The silicone urethral catheters coated with PCN-MSNPs (500 µg mL-1; 2.23 mM) exhibited no formation of C. albicans MTCC 227 - S. aureus MTCC 96 and C. albicans MTCC 227 - S. aureus MLS -16 MTCC 2940 biofilms. This is the first report on PCN-MSNPs for use as antimicrobial coatings against microbial adhesion and biofilm formation on silicone urethral catheters.

In vivo targeting of DNA vaccines to dendritic cells using functionalized gold nanoparticles.

The clinical success of dendritic cell (DC)-based genetic immunization remains critically dependent on the availability of effective and safe nano-carriers for targeting antigen-encoded DNA vaccines to DCs, the most potent antigen-presenting cells in the human body in vivo. Recent studies revealed the efficacies of mannose receptor-mediated in vivo DC-targeted genetic immunization by liposomal DNA vaccine carriers containing both mannose-mimicking shikimoyl and transfection enhancing guanidinyl functionalities. However, to date, the efficacies of this approach have not been examined for metal-based nanoparticle DNA vaccine carriers. Herein, we report for the first time, the design, synthesis, physico-chemical characterization and bioactivities of gold nanoparticles covalently functionalized with a thiol ligand containing both shikimoyl and guanidinyl functionalities (Au-SGSH). We show that Au-SGSH nanoparticles can deliver DNA vaccines to mouse DCs under in vivo conditions. Subcutaneous administration of near infrared (NIR) dye-labeled Au-SGSH showed significant accumulation of the NIR dye in the DCs of the nearby lymph nodes compared to that for the non-targeting NIR-labeled Au-GSH nanoconjugate containing only a covalently tethered guanidinyl group, not the shikimoyl-functionality. Under prophylactic settings, in vivo immunization (s.c.) with the Au-SGSH-pCMV-MART1 nanoplex induced a long-lasting (180 days) immune response against murine melanoma. Notably, mannose receptor-mediated in vivo DC-targeted immunization (s.c.) with the Au-SGSH-MART1 nanoplex significantly inhibited established melanoma growth and increased the overall survivability of melanoma-bearing mice under therapeutic settings. The Au-SGSH nanoparticles reported herein have potential use for in vivo DC-targeted genetic immunization against cancer and infectious diseases.

Novel Amphiphilic G-Quadruplex Binding Synthetic Derivative of TMPyP4 and Its Effect on Cancer Cell Proliferation and Apoptosis Induction.

Porphyrins are well-known anticancer agents because of their high binding affinity for G-quadruplex DNA and excellent photophysical properties. Several studies carried out using TMPyP4 established it as an efficient chemotherapeutic and a photodynamic therapeutic (PDT) agent, but its use as a lead molecule has been restricted because of its high level of binding to double-stranded DNA (dsDNA), which may have side effects on normal cells and tissues. To minimize its interaction with dsDNA and to enhance internalization into cells, an analogue of TMPyP4 (5Me) was synthesized. Its selectivity for G-quadruplex DNA over dsDNA was evaluated by spectroscopic methods, and its role in stabilizing G-quadruplex DNA was assessed by fluorescence lifetime and thermal melting experiments. Biophysical studies indicated that 5Me interacts well with G-quadruplex DNA. In vitro cytotoxicity experiments with tumor cell lines (PANC-1, B16F10, and MDA MB 231) have revealed that 5Me can inhibit the growth of cancer cells comparable to TMPyP4. MTT and apoptotic assays demonstrated the ability of 5Me to specifically affect cancer cells over normal cells. Cell cycle analysis showed that 5Me, like TMPyP4, induces G2/M phase cell cycle arrest. In addition, 5Me is more effectively taken up by both cancer and normal cells than TMPyP4. In addition, we have noticed that 5Me is more efficient than TMPyP4 in inhibiting the growth of the cancer cells after irradiation with light (600-720 nm, 20 J/cm2, 50 mW/cm2). By and large, these experimental results indicate that 5Me can be an efficient chemotherapeutic as well as a PDT agent.

Glucocorticoid Receptor-Targeted Liposomal Codelivery of Lipophilic Drug and Anti-Hsp90 Gene: Strategy to Induce Drug-Sensitivity, EMT-Reversal, and Reduced Malignancy in Aggressive Tumors.

Many cancers including the late stage ones become drug-resistant and undergo epithelial-to-mesenchymal transition (EMT). These lead to enhanced invasion, migration, and metastasis toward manifesting its aggressiveness and malignancy. One of the key hallmarks of cancer is its overdependence on glycolysis as its preferred energy metabolism pathway. The strict avoidance of alternate energy pathway gluconeogenesis by cancer cells points to a yet-to-be hoisted role of glucocorticoid receptor (GR) especially in tumor microenvironment, where cells are known to become drug-sensitive through induction of gluconeogenesis. However, since GR is involved in metabolism, anti-inflammatory reactions, immunity besides inducing gluconeogenesis, a greater role of GR in tumor microenvironment is envisaged. We have shown previously that GR, although ubiquitously expressed in all cells; afford to be an effective cytoplasmic target for killing cancer cells selectively. Herein, we report the therapeutic use of a newly developed GR-targeted liposomal concoction (DXE) coformulating a lipophilic drug (ESC8) and an anti-Hsp90 anticancer gene against aggressive tumor models. This induced drug-sensitivity and apoptosis while reversing EMT in tumor cells toward effective retardation of aggressive growth in pancreas and skin tumor models. Additionally, the ESC8-free lipid formulation upon cotreatment with hydrophilic drugs, gemcitabine and doxorubicin, could effectively sensitize and kill pancreatic cancer and melanoma cells, respectively. The formulation-triggered EMT-reversal was GR-dependent. Overall, we found a new strategy for drug sensitization that led to the advent of new GR-targeted anticancer therapeutics.