Inhibition of the MALT1-LPCAT3 axis protects cartilage degeneration and osteoarthritis.

The proinflammatory cytokines and arachidonic acid (AA)-derived eicosanoids play a key role in cartilage degeneration in osteoarthritis (OA). The lysophosphatidylcholine acyltransferase 3 (LPCAT3) preferentially incorporates AA into the membranes. Our recent studies showed that MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that LPCAT3 expression was up-regulated in both human and mice articular cartilage of OA, and correlated with severity of OA. The IL-1ß-induces cell death via upregulation of LPCAT3, MMP3, ADAMTS5, and eicosanoids via MALT1. Gene silencing or pharmacological inhibition of LPCAT3 or MALT1 in chondrocytes and human cartilage explants notably suppressed the IL-1ß-induced cartilage catabolism through inhibition of expression of MMP3, ADAMTS5, and also secretion of cytokines and eicosanoids. Mechanistically, overexpression of MALT1 in chondrocytes significantly upregulated the expression of LPCAT3 along with MMP3 and ADAMTS5 via c-Myc. Inhibition of c-Myc suppressed the IL-1ß-MALT1-dependent upregulation of LPCAT3, MMP3 and ADAMTS5. Consistent with the in vitro data, pharmacological inhibition of MALT1 or gene silencing of LPCAT3 using siRNA-lipid nanoparticles suppressed the synovial articular cartilage erosion, pro-inflammatory cytokines, and eicosanoids such as PGE2, LTB4, and attenuated osteoarthritis induced by the destabilization of the medial meniscus in mice. Overall, our data reveal a previously unrecognized role of the MALT1-LPCAT3 axis in osteoarthritis. Targeting the MALT1-LPCAT3 pathway with MALT1 inhibitors or siRNA-liposomes of LPCAT3 may become an effective strategy to treat OA by suppressing eicosanoids, matrix-degrading enzymes, and proinflammatory cytokines.

Bis-arylidene oxindoles for colorectal cancer nanotherapy.

Oxindoles are potent anti-cancer agents and are also used against microbial and fungal infections and for treating neurodegenerative diseases. These oxindoles are earlier established as estrogen receptor (ER)-targeted agents for killing ER (+) cancer cells. Our previously developed bis-arylidene oxindole, Oxifen (OXF) exhibits effective targeting towards ER (+) cancer cells which has a structural resemblance with tamoxifen. Herein, we have designed and synthesized few structural analogues of OXF such as BPYOX, ACPOX and ACPOXF to examine its cytotoxicity in different cancer as well as non-cancer cell lines and its potential to form self- aggregates in aqueous solution. Among these series of molecules, ACPOXF showed maximum toxicity in colorectal cancer cell line which are ER (-) but it also kills non-cancer cell line HEK-293, thereby reducing its cancer cell selectivity. Incidentally, ACPOXF exhibits self-aggregation, without the help of a co-lipid with nanometric size in aqueous solution. ACPOXF self-aggregate was co-formulated with glucocorticoid receptor (GR) synthetic ligand, dexamethasone (Dex) (called, ACPOXF-Dex aggregate) which could selectively kill ER (-) colorectal cancer cells and also could increase survivability of colon-tumour bearing mice. ACPOXF-Dex induced ROS up-regulation followed by apoptosis through expression of caspase-3. Further, we observed upregulation of antiproliferative factor, p53 and epithelial-to-mesenchymal (EMT) reversal marker E-cadherin in tumour mass. In conclusion, a typical structural modification in ER-targeting Oxifen moiety resulted in its self-aggregation that enabled it to carry a GR-ligand, thus broadening its selective antitumor property especially as colon cancer therapeutics.

Etoposide-Entrapped Progesterone-Cationic Lipid Nanoaggregates as Selective Therapeutics against Etoposide-Resistant Colorectal Cancer Cells.

Drug resistance has a major impact on the treatment of several cancers. This is mainly due to the overexpression of cellular drug efflux proteins. Hence, drug-delivery systems that can avoid this resistance are needed. We report PR10, a progesterone-cationic lipid conjugate, as a self-assembling nanoaggregate that delivers a drug cargo of etoposide, a topoisomerase inhibitor, selectively to cancer cells. In this study, we observed that etoposide nanoaggregates (P : E) caused selective and enhanced toxicity in etoposide-resistant CT26 cancer cells (IC50 9 µM) compared to when etoposide (IC50 >20 µM) was used alone. Concurrently, no toxicity was observed in etoposide-sensitive HEK293 cells for P : E treatment (IC50 >20 µM). The P : E-treated cancer cells seem to have no effect on ABCB1 expression, but etoposide-treated cells exhibited a twofold increase in ABCB1 expression, a potent efflux protein for several xenobiotic compounds. This observation supports the notion that the enhanced toxicity of P : E nanoaggregates is due to their ability to keep the expression of ABCB1 low, thus allowing longer intracellular residence of etoposide. In a BALB/c orthotopic colorectal cancer model, the nanoaggregates led to enhanced survival (45 days) compared to etoposide-treated mice (39 days). These findings suggest that PR10 could be used as a potential cancer-selective etoposide delivery vehicle to treat several etoposide-resistant cancers with fewer side effects due to the nonspecific toxicity of the drug.

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.

Significance of Oct-4 transcription factor as a pivotal therapeutic target for CD44+ /24- mammary tumor initiating cells: Aiming at the root of the recurrence.

Breast cancer (BC) remains one of the deadliest and frequently diagnosed metastatic cancers worldwide. Cancer stem cells (CSCs) are the cell population within the tumor niche, having an epithelial to mesenchymal (EMT) transition phenotype, high self-renewal, vigorous metastatic capacity, drug resistance, and tumor relapse. Identification of targets for induction of apoptosis is essential to provide novel therapeutic approaches in BC. Our earlier studies showed that Vitamin C induces apoptotic cell death by losing redox balance in TNBC CSCs. In this study, we have attempted to identify previously unrecognized CSC survival factors that can be used as druggable targets for bCSCs apoptosis regulators isolated from the TNBC line, MDA MB 468. After a thorough literature review, Oct-4 was identified as the most promising marker for its unique abundance in cancer and absence in normal cells and the contribution of Oct-4 to the sustenance of cancer cells. We then validated a very high expression of Oct-4 in the MDA MB 468 bCSCs population using flow-cytometry. The loss of Oct-4 was carried out using small interfering RNA (siRNA)-mediated knockdown in the bCSCs, followed by assessing for cellular apoptosis. Our results indicated that Oct-4 knockdown induced cell death, changes in cellular morphology, inhibited mammosphere formation, and positive for Annexin-V expression, thereby indicating the role of Oct-4 in bCSC survival. Moreover, our findings also suggest the direct interaction between Oct-4 and Vitamin C using in silico docking. This data, hence, contributes towards novel information about Oct-4 highlighting this molecule as a novel survival factor in bCSCs.

Cationic lipid-conjugated bis-arylidene oxindole derivatives as broad-spectrum breast cancer-selective therapeutics.

Breast cancer is a heterogeneous malignancy with wide-ranging variations in therapeutic responses, overall survival etc. Major challenges for available chemotherapeutic agents in achieving clinical success are in maintaining systemic bio-distribution and avoiding non-specific adverse effects. Bis-arylidene oxindoles are estrogen receptor (ER)-selective bioactive molecules with moderate potency. In here, we have designed, synthesized and evaluated a series of twin aliphatic chain cationic lipid-conjugated bis-arylidene oxindole molecules with variations in nature of linker, lengths of carbon spacer and hydrophobic twin chains. We observed that among the various structural analogues, C8 twin-chain containing molecules, PGC8, S2C8 and S3C8 showed effective cancer cell-selective cytotoxicity in different cancer cell lines with an IC50 ranging from 4 to 7 µM. These molecules selectively induced apoptosis, ROS production and cell cycle inhibition at G1/S phase in ER + breast cancer cells but not in non-cancer cells. Additionally, these molecules formed homogenous self-assemblies exhibiting effective hydrodynamic diameter with positive surface charge. The self-assemblies also showed prominent cancer cell-selective uptake and DNA-binding abilities. Hence, we have shown successful incorporation of dexamethasone to the self-assemblies, and its enhanced cytotoxicity even in ER-negative breast cancer cells. All these results indicate that PGC8, S2C8 and S3C8 molecules, albeit their potent and selective ER-positive anti-breast cancer activity, can be repurposed as targeted delivery systems and hold promise as unique, broader spectrum breast cancer cell-selective therapeutic payloads.

Polyketide Quinones Are Alternate Intermediate Electron Carriers during Mycobacterial Respiration in Oxygen-Deficient Niches.

Mycobacterium tuberculosis (Mtb) adaptation to hypoxia is considered crucial to its prolonged latent persistence in humans. Mtb lesions are known to contain physiologically heterogeneous microenvironments that bring about differential responses from bacteria. Here we exploit metabolic variability within biofilm cells to identify alternate respiratory polyketide quinones (PkQs) from both Mycobacterium smegmatis (Msmeg) and Mtb. PkQs are specifically expressed in biofilms and other oxygen-deficient niches to maintain cellular bioenergetics. Under such conditions, these metabolites function as mobile electron carriers in the respiratory electron transport chain. In the absence of PkQs, mycobacteria escape from the hypoxic core of biofilms and prefer oxygen-rich conditions. Unlike the ubiquitous isoprenoid pathway for the biosynthesis of respiratory quinones, PkQs are produced by type III polyketide synthases using fatty acyl-CoA precursors. The biosynthetic pathway is conserved in several other bacterial genomes, and our study reveals a redox-balancing chemicocellular process in microbial physiology.

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.

Mitoapocynin, a mitochondria targeted derivative of apocynin induces mitochondrial ROS generation and apoptosis in multiple cell types including cardiac myoblasts: a potential constraint to its therapeutic use.

Mitoapocynin is a triphenylphosphonium conjugated derivative of apocynin that specifically locates to the mitochondria. It has been developed as a mitochondrially targeted therapeutic antioxidant. We attempted to attenuate the mitochondrial ROS induced in H9c2 cardiac myoblast cells treated with norepinephrine. Mitoapocynin was a poor quencher of total ROS as detected by the fluoroprobe DCFH-DA. Using mitochondrial superoxide specific probe MitoSoxRed, we found that 5-10 µM mitoapocynin itself induces superoxide over and above that is generated by the norepinephrine treatment. A supposedly control molecule to mitoapocynin, the synthetic compound PhC11TPP, having the triphenylphosphonium group and a benzene moiety with C11 aliphatic chain spacer was also found to be a robust inducer of mitochondrial ROS. Subsequent assays with several cell lines viz., NIH3T3, HEK293, Neuro2A, MCF-7 and H9c2, showed that prolonged exposure to mitoapocynin induces cell death by apoptosis that can be partially prevented by the general antioxidant N-acetyl cysteine. Analyses of mitochondrial electron transport complexes by Blue Native Polyacrylamide gel electrophoresis showed that both mitoapocynin and PhC11TPP disrupt the mitochondrial Complex I and V, and in addition, PhC11TPP also damages the Complex IV. Our data thus highlights the limitations of the therapeutic use of mitoapocynin as an antioxidant.

The prospects of nanotherapeutic approaches for targeting tumor-associated macrophages in oral cancer.

OSCC (oral squamous cell carcinoma) is currently one of the most formidable cancers plagued by challenges like low overall survivability, lymph node associated metastasis, drug resistance, and poor diagnostics. The tumor microenvironment (TME) and its constituent stromal elements are crucial modulators of tumor growth and treatment response, more specifically so with regards to resident tumor associated macrophages (TAMs) and their liaison with the different stromal elements in the tumor niche (Figure 1). Interestingly, there isn't much information on TAM-targeted nanotherapy in OSCC where the first line of therapeutics for oral cancer is surgery with other therapeutics such as chemo- and radiotherapy acting only as adjuvant therapy for oral cancer. In the face of this real time situation, there have been some successful attempts at targeted therapy for OSCC cells and we believe they might elicit favorable responses against TAMs as well. Demanding our immediate attention, this review intends to provide a glimpse of the prevailing anti-TAM treatment strategies, which present great prospect for an uncharted territory like OSCC.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells.

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

N-end rule pathway inhibitor sensitizes cancer cells to antineoplastic agents by regulating XIAP and RAD21 protein expression.

Anticancer drugs exert their effects on cancer cells by deregulating many pathways linked to cell cycle, apoptosis, etc. but cancer cells gradually become resistive against anticancer drugs, thereby necessitating the development of newer generation anticancer molecules. N-end rule pathway has been shown to be involved in the degradation of many cell cycle and apoptosis-related proteins. However, the involvements of this pathway in cancer are not well established. Recently, we developed a non-peptide-based N-end rule pathway inhibitor, RF-C11 for type 1 and 2 recognition domains of E3 ubiquitin ligases. The inhibitor significantly increased the half-life of potential N-degrons leading to significant physiological changes in vivo. We hypothesized RF-C11 may be used to decipher the N-end rule pathway's role in cancer towards the development of anticancer therapeutics. In this study, we showed that RF-C11, barring noncancer cells, significantly sensitizes cancer cells towards different anticancer agents tested. We further find that the profound cellular sensitization to anticancer drugs was affected by (a) downregulation of X-linked inhibitor of apoptosis protein, an antiapoptotic protein and (b) by stabilization of RAD21, and thereby inhibiting metaphase to anaphase promotion. The study shows that RF-C11 or its analogs may be used as a novel additive in combination therapy against cancer.

Combating Glioblastoma by Codelivering the Small-Molecule Inhibitor of STAT3 and STAT3siRNA with α5ß1 Integrin Receptor-Selective Liposomes.

Glioblastoma multiforme (GBM) is one of the most aggressive tumors with a median survival of only 15 months. Effective therapeutics need to overcome the formidable challenge of crossing the blood-brain barrier (BBB). Receptors and transporters overexpressed on BCECs are being used for designing liposomes, polymers, polymeric micelles, peptides, and dendrimer-based drug carriers for combating brain tumors. Herein, using the orthotopic mouse glioblastoma model, we show that codelivering a small-molecule inhibitor of the JAK/STAT pathway (WP1066) and STAT3siRNA with nanometric (100-150 nm) α5ß1 integrin receptor-selective liposomes of RGDK-lipopeptide holds therapeutic promise in combating glioblastoma. Rh-PE (red)-labeled liposomes of RGDK-lipopeptide were found to be internalized in GL261 cells via integrin α5ß1 receptors. Intravenously administered near-infrared (NIR)-dye-labeled α5ß1 integrin receptor-selective liposomes of RGDK-lipopeptide were found to be accumulated preferentially in the mouse brain tumor tissue. Importantly, we show that iv injection of WP1066 (a commercially sold small-molecule inhibitor of the JAK/STAT pathway) and STAT3siRNA cosolubilized within the liposomes of RGDK-lipopeptide leads to significant inhibition (>350% compared to the untreated mice group) of orthotopically growing mouse glioblastoma. The present strategy may find future use in combating GBM.

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.

α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection.

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.

Evaluation of the in vivo genotoxicity of liposomal formulation for delivering anticancer estrogenic derivative (ESC8) in a mouse model.

The genotoxic potential of glucocorticoid receptor (GR)-targeted liposomal formulations of the anticancer drug molecule ESC8 was studied in vivo. A methodical literature review discovered no previous studies on the genotoxicity of ESC8. Genotoxicity was assessed in both male and female mice by various assay systems, such as comet assay, chromosomal aberrations and micronuclei assay, which detect different abnormalities. Eleven groups of male mice and eleven groups of female mice, containing six animals per group, were used in the present study: group I served as vehicle control; group II received the positive control (cyclophosphamide 40 mg/kg; CYP); and animals in group III to XI received free drug (ESC8), DX liposome and drug-associated DX liposomal formulation (DXE), respectively, dissolved in 5% solution of glucose at a drug-dose of 1.83, 3.67 and 7.34 mg/kg, respectively. Same drug treatments were followed for the female mice groups. The obtained data revealed the safety of DXE, which did not show substantial genotoxic effects at different dose levels. In contrast, the positive control, CYP, exhibited highly substantial irregular cytogenetic variations in comparison with the control group in different assays.

α-Tocopherol-based cationic amphiphiles with a novel pH sensitive hybrid linker for gene delivery.

Endosomal escape is one of the barriers for the efficient liposomal gene delivery. To address this and based on earlier encouraging results using tocopherol cationic lipids, we elaborated chemical modifications on tocopherol cationic lipids by introducing a novel hybrid pH sensitive linker "ether-ß-hydroxy-triazole" between tocopherol, the anchoring moiety and the basic tris(2-hydroxy ethyl)quaternary ammonium head group (Lp2). As control lipids we designed two lipids (Lp1 and Lp3), one is with only the ether-ß-hydroxy linker in between α-tocopherol and quaternary tris(2-hydroxyethyl)ammonium (Lp1) and the other is with the same novel hybrid linker i.e. "ether-ß-hydroxy-triazole" between the α-tocopherol linked and quaternary tris-ethyl ammonium head group (Lp3). Liposomes were formulated with a combination of a well-known co-lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and biophysical characteristics such as DNA binding, hydrodynamic diameters and global surface charges for liposomes and lipoplexes of respective lipids were evaluated. Cell viability assay and in vitro transfection studies were carried out in NIH3 T3, B16F10, HEK-293, and HepG2 cell lines. In vitro transfection data for the liposomes of lipids (Lp1, Lp2 and Lp3) revealed that the Lp2 lipid with a novel hybrid pH sensitive linker showed superior transfection efficiency when compared with the remaining two analogues. More importantly, Lp2 has shown a similar pattern of transfection efficiency in HepG2 and HEK-293 cell lines when compared with commercially available Lipofectamine 3000.

Mineralocorticoid receptor mediated liposomal delivery system for targeted induction of apoptosis in cancer cells.

Mineralocorticoid receptors (MRs) are nuclear hormone receptors that are ubiquitously present in all cell types and are known to mediate distinct physiological functions like regulating Na(+) and K(+) balance and water excretion. MRs are linked to cell proliferation and can be exploited for the targeted control of cell mass in cancer. The present study is aimed towards extending the concept of using MR ligand spironolactone for selective delivery of genes in cancer cells. The lipoplex (SP) has shown MR mediated targeted transfections as indicated by receptor down-regulation studies using MR antagonists and siRNA. SP-targeted delivery of genes resulted in apoptosis in cell-specific manner while free drug was found to be cytotoxic irrespective of the cancerous or non-cancerous nature. In conclusion, this study presents MR as a target for efficiently delivering anticancer genes and thereby treating cancer through MR-mediated pathway.

Combination of cationic dexamethasone derivative and STAT3 inhibitor (WP1066) for aggressive melanoma: a strategy for repurposing a phase I clinical trial drug.

Glucocorticoid, such as dexamethasone (Dex) is often used along with chemotherapy to antagonize side effects of chemotherapy. However, sustained use of Dex frequently develops drug resistance in patients. As a strategy to re-induce drug sensitivity, we planned to modify Dex by chemically conjugating it with twin ten carbon aliphatic chain containing cationic lipid. The resultant molecule, DX10, inhibited STAT3 activation through lowering the production of IL-6. To enhance the STAT3 inhibitory effect of DX10, we used WP (a commercially available STAT3 inhibitor) along with DX10. Combination treatment of both significantly inhibited STAT3 activation when compared to either of the individual treatment. The effect of DX10, either in combination or alone, was mediated through glucocorticoid receptor (GR), thereby repurposing the role of GR in the context of p-STAT3 inhibition-mediated cancer treatment. Cellular viability study proved the synergistic effect of WP and DX10. Further, combination treatment led to induction of early stage of apoptosis and cell cycle arrest. In vivo melanoma tumor regression study confirmed the enhanced anti-tumor activity of co-treatment over individual treatment of DX10 or WP. Thus, together our result demonstrates that DX10 may be used in combination therapy with STAT3 inhibitor like WP for combating cancer with constitutively active STAT3.

Development of Liposomal Formulation for Delivering Anticancer Drug to Breast Cancer Stem-Cell-Like Cells and its Pharmacokinetics in an Animal Model.

The objective of the present study is to develop a liposomal formulation for delivering anticancer drug to breast cancer stem-cell-like cells, ANV-1, and evaluate its pharmacokinetics in an animal model. The anticancer drug ESC8 was used in dexamethasone (Dex)-associated liposome (DX) to form ESC8-entrapped liposome named DXE. ANV-1 cells showed high-level expression of NRP-1. To enhance tumor regression, we additionally adapted to codeliver the NRP-1 shRNA-encoded plasmid using the established DXE liposome. In vivo efficacy of DXE-NRP-1 was carried out in mice bearing ANV-1 cells as xenograft tumors and the extent of tumor growth inhibition was evaluated by tumor-size measurement. A significant difference in tumor volume started to reveal between DXE-NRP-1 group and DXE-Control group. DXE-NRP-1 group showed ∼4 folds and ∼2.5 folds smaller tumor volume than exhibited by untreated and DXE-Control-treated groups, respectively. DXE disposition was evaluated in Sprague-Dawley rats following an intraperitoneal dose (3.67 mg/kg of ESC8 in DXE). The plasma concentrations of ESC8 in the DXE formulation were measured by liquid chromatography mass spectrometry and pharmacokinetic parameters were determined using a noncompartmental analysis. ESC8 had a half-life of 11.01 ± 0.29 h, clearance of 2.10 ± 3.63 L/kg/h, and volume of distribution of 33.42 ± 0.83 L/kg. This suggests that the DXE liposome formulation could be administered once or twice daily for therapeutic efficacy. In overall, we developed a potent liposomal formulation with favorable pharmacokinetic and tumor regressing profile that could sensitize and kill highly aggressive and drug-resistive cancer stem-cell-like cells.