Intercellular nanotubes mediate mitochondrial trafficking between cancer and immune cells.

Cancer progresses by evading the immune system. Elucidating diverse immune evasion strategies is a critical step in the search for next-generation immunotherapies for cancer. Here we report that cancer cells can hijack the mitochondria from immune cells via physical nanotubes. Mitochondria are essential for metabolism and activation of immune cells. By using field-emission scanning electron microscopy, fluorophore-tagged mitochondrial transfer tracing and metabolic quantification, we demonstrate that the nanotube-mediated transfer of mitochondria from immune cells to cancer cells metabolically empowers the cancer cells and depletes the immune cells. Inhibiting the nanotube assembly machinery significantly reduced mitochondrial transfer and prevented the depletion of immune cells. Combining a farnesyltransferase and geranylgeranyltransferase 1 inhibitor, namely, L-778123, which partially inhibited nanotube formation and mitochondrial transfer, with a programmed cell death protein 1 immune checkpoint inhibitor improved the antitumour outcomes in an aggressive immunocompetent breast cancer model. Nanotube-mediated mitochondrial hijacking can emerge as a novel target for developing next-generation immunotherapy agents for cancer.

Surface-Enhanced Biocompatibility and Adsorption Capacity of a Zirconium Phosphate-Coated Polyaniline Composite.

The present study deals with the synthesis, characterization, and testing of a novel composite, zirconium(IV) phosphate-coated polyaniline (ZrPO4@PANI), toward the adsorption- and surface-controlled toxicity applications. Following the synthesis of the ZrPO4@PANI composite using the sol-gel route, various characterization techniques such as Fourier transform infrared spectroscopy, scanning electron microscopy, and powder X-ray diffraction were employed to confirm its surface functionality, morphology and agglomeration, and crystallinity and crystal nature, respectively. The composite was found to be effective toward the adsorptive removal of the methylene blue dye (an organic pollutant) as against the changes in the dye concentration, dose, pH, and so forth. Also, to understand the MB adsorption kinetics, the experimental data were evaluated using the Langmuir and Freundlich models and the results were described in accordance with the Langmuir isotherm model (an adsorption capacity of 120.48 mg/g at ambient temperature). In addition, the tests conducted using pseudo-first- and pseudo-second-order kinetic models confirmed the existence of pseudo-second-order rates. Furthermore, the calculation of thermodynamic parameters for the MB adsorption, namely, changes in enthalpy, entropy, and Gibbs' free energy, exhibited a spontaneous, feasible, and exothermic nature. Finally, the comparative studies of in vitro toxicity and flow cytometry confirmed that the copresence of ZrPO4 along with PANI significantly improved the biocompatibility. The outcome of the experimental results implies that the composite is capable enough of serving as the safe and low-cost adsorbent, in addition to supporting the effective capping of the surface toxicity of PANI.

Investigation of the Tribological Characteristics of Aluminum 6061-Reinforced Titanium Carbide Metal Matrix Composites.

The current trend in the materials engineering sector is to develop newer materials that can replace the existing materials in various engineering sectors in order to be more and more efficient. Therefore, the present research work is aimed at fabricating and determining the physical, mechanical, and dry sliding wear properties of titanium carbide (TiC)-reinforced aluminum alloy (Al6061) metal matrix composites (MMCs). For the study, the Al6061-TiC microparticle-reinforced composites were fabricated via the liquid metallurgy route through the stir casting method, where the reinforcement of the TiC particles into the Al6061 alloy matrix was added in the range of 0 to 8.0 wt.%, i.e., in the steps of 2.0 wt.%. The synthesis procedure followed the investigation of the various mechanical properties of Al6061-TiC MMCs, such as the density and structure, as well as mechanical and dry wear experimentation. The tests performed on the casted Al6061, as well as its TiC composites, were in harmony with ASTM standards. As per the experimental outcome, it can be confirmed that the increase in the weight percentage of TiC into the Al6061 alloy substantially increases the density, hardness, and tensile strength, at the expense of the percentage of elongation. In addition, the dry wear experiments, performed on a pin-on-disc tribometer, showed that the Al6061-TiC MMCs have superior wear-resistance properties, as compared to those of pure Al6061 alloy. Furthermore, optical micrograph (OM), powdered X-ray diffraction (XRD), energy dispersive spectroscopy (EDS), and scanning electron microscopy (SEM) analyses were employed for the developed Al6061-TiC MMCs before and after the fracture and wear test studies. From the overall analysis of the results, it can be observed that the Al6061-TiC composite material with higher TiC reinforcement displays superior mechanical characteristics.

Adsorption, Equilibrium Isotherm, and Thermodynamic Studies towards the Removal of Reactive Orange 16 Dye Using Cu(I)-Polyaninile Composite.

To overcome some of the limitations of activated carbon like efficiency, cost-effectiveness, and reusability, the present work deals with Cu(I)-based polyaniline (PANI) composite for the removal of reactive orange 16 (RO16) dye. Following the synthesis and characterization of formed Cu(I)-PANI composite, the batch experiments performed for the removal of RO16 dye indicated that the composite has the capacity to reduce the coloring from RO16. The experiments were conducted for the study of effects against changes in pH, time, and dose at room temperature, where we observed for a pH impact on the dye adsorption capacity in the range of 2-12. Among all, the optimal RO16 removal was found to be 94.77% at a pH of 4 and in addition, the adsorption kinetics confirmed to be pseudo-second-order with more suitability towards the Langmuir isotherm, where it is presumed to be the formation of a monolayer of dye molecule at the homogeneous absorbent surface. The calculated maximum capacity, qm, determined from the Langmuir model was 392.156 mg/g. Further application of isotherms to attain thermodynamic parameters, a slight positive value of ΔS° for RO16 adsorption was observed, meaning that there is an increased randomness in the irregular pattern at the specific Cu(I)-PANI interface for an adsorption process. This mechanism plays an essential role in maintaining the effects of water pollution; and, based on the analysis therefore, it is prominent that the Cu(I)-PANI composite can be employed as a promising and economical adsorbent for the treatment of RO16 and other dye molecules from the sewage in wastewater.

Drug Release Profiles of Mitomycin C Encapsulated Quantum Dots-Chitosan Nanocarrier System for the Possible Treatment of Non-Muscle Invasive Bladder Cancer.

Nanotechnology-based drug delivery systems are an emerging technology for the targeted delivery of chemotherapeutic agents in cancer therapy with low/no toxicity to the non-cancer cells. With that view, the present work reports the synthesis, characterization, and testing of Mn:ZnS quantum dots (QDs) conjugated chitosan (CS)-based nanocarrier system encapsulated with Mitomycin C (MMC) drug. This fabricated nanocarrier, MMC@CS-Mn:ZnS, has been tested thoroughly for the drug loading capacity, drug encapsulation efficiency, and release properties at a fixed wavelength (358 nm) using a UV-Vis spectrophotometer. Followed by the physicochemical characterization, the cumulative drug release profiling data of MMC@CS-Mn:ZnS nanocarrier (at pH of 6.5, 6.8, 7.2, and 7.5) were investigated to have the highest release of 56.48% at pH 6.8, followed by 50.22%, 30.88%, and 10.75% at pH 7.2, 6.5, and 7.5, respectively. Additionally, the drug release studies were fitted to five different pharmacokinetic models including pesudo-first-order, pseudo-second-order, Higuchi, Hixson-Crowell, and Korsmeyers-Peppas models. From the analysis, the cumulative MMC release suits the Higuchi model well, revealing the diffusion-controlled mechanism involving the correlation of cumulative drug release proportional to the function square root of time at equilibrium, with the correlation coefficient values (R2) of 0.9849, 0.9604, 0.9783, and 0.7989 for drug release at pH 6.5, 6.8, 7.2, and 7.5, respectively. Based on the overall results analysis, the formulated nanocarrier system of MMC synergistically envisages the efficient delivery of chemotherapeutic agents to the target cancerous sites, able to sustain it for a longer time, etc. Consequently, the developed nanocarrier system has the capacity to improve the drug loading efficacy in combating the reoccurrence and progression of cancer in non-muscle invasive bladder diseases.

Nanotherapeutic approaches to overcome distinct drug resistance barriers in models of breast cancer.

Targeted delivery of drugs to tumor cells, which circumvent resistance mechanisms and induce cell killing, is a lingering challenge that requires innovative solutions. Here, we provide two bioengineered strategies in which nanotechnology is blended with cancer medicine to preferentially target distinct mechanisms of drug resistance. In the first 'case study', we demonstrate the use of lipid-drug conjugates that target molecular signaling pathways, which result from taxane-induced drug tolerance via cell surface lipid raft accumulations. Through a small molecule drug screen, we identify a kinase inhibitor that optimally destroys drug tolerant cancer cells and conjugate it to a rationally-chosen lipid scaffold, which enhances anticancer efficacy in vitro and in vivo. In the second 'case study', we address resistance mechanisms that can occur through exocytosis of nanomedicines. Using adenocarcinoma HeLa and MCF-7 cells, we describe the use of gold nanorod and nanoporous vehicles integrated with an optical antenna for on-demand, photoactivation at ~650 nm enabling release of payloads into cells including cytotoxic anthracyclines. Together, these provide two approaches, which exploit engineering strategies capable of circumventing distinct resistance barriers and induce killing by multimodal, including nanophotonic mechanisms.

Aptasensor for the Detection of Mycobacterium tuberculosis in Sputum Utilising CFP10-ESAT6 Protein as a Selective Biomarker.

A portable electrochemical aptamer-antibody based sandwich biosensor has been designed and successfully developed using an aptamer bioreceptor immobilized onto a screen-printed electrode surface for Mycobacterium tuberculosis (M. tuberculosis) detection in clinical sputum samples. In the sensing strategy, a CFP10-ESAT6 binding aptamer was immobilized onto a graphene/polyaniline (GP/PANI)-modified gold working electrode by covalent binding via glutaraldehyde linkage. Upon interaction with the CFP10-ESAT6 antigen target, the aptamer will capture the target where the nano-labelled Fe3O4/Au MNPs conjugated antibody is used to complete the sandwich format and enhance the signal produced from the aptamer-antigen interaction. Using this strategy, the detection of CFP10-ESAT6 antigen was conducted in the concentration range of 5 to 500 ng/mL. From the analysis, the detection limit was found to be 1.5 ng/mL, thereby demonstrating the efficiency of the aptamer as a bioreceptor. The specificity study was carried out using bovine serum albumin (BSA), MPT64, and human serum, and the result demonstrated good specificity that is 7% higher than the antibody-antigen interaction reported in a previous study. The fabricated aptasensor for M. tuberculosis analysis shows good reproducibility with an relative standard deviation (RSD) of 2.5%. Further analysis of M. tuberculosis in sputum samples have shown good correlation with the culture method with 100% specificity and sensitivity, thus making the aptasensor a promising candidate for M. tuberculosis detection considering its high specificity and sensitivity with clinical samples.

Nanoengineered Disruption of Heat Shock Protein 90 Targets Drug-Induced Resistance and Relieves Natural Killer Cell Suppression in Breast Cancer.

Drug-induced resistance, or tolerance, is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor, the consequence on tumor growth, and therapeutic strategies to address these challenges remain undescribed. Here, we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatiotemporally controlled nanomedicines, which boost therapeutic efficacy and invigorate "disabled" NK cells. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis, NK group 2 member D, and MHC class I polypeptide-related sequence A, B. Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. On the basis of this evidence, we engineered a "chimeric" nanotherapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor, radicicol, which targets the tumor, reduces tolerance, and optimally reprimes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically approved agents. These findings highlight a convergence between drug-induced resistance, the tumor immune contexture, and engineered approaches that consider the tumor and microenvironment to improve the success of combinatorial therapy. SIGNIFICANCE: This study uncovers a molecular mechanism linking drug-induced resistance and tumor immunity and provides novel engineered solutions that target these mechanisms in the tumor and improve immunity, thus mitigating off-target effects.

Rubusoside-assisted solubilization of poorly soluble C6-Ceramide for a pilot pharmacokinetic study.

Although C6-Ceramide has attracted much attention as a possible tumor suppressor, the delivery of C6-Ceramide is still challenging due to its inherent hydrophobicity and insolubility. In this study we explored the use of a natural compound rubusoside (RUB) as a solubilizer to enhance the solubility of a fluorescence-labeled C6-Ceramide (NBD C6-Ceramide) and to characterize its pharmacokinetics and tissue distribution in an animal model. RUB significantly enhanced the solubility of NBD C6-Ceramide by forming nanomicelles, and efficiently delivered NBD C6-Ceramide in rats by oral and intravenous administration. RUB loaded 1.96 % of NBD C6-Ceramide in the nanomicelles and solubilized it to a concentration of 3.6 mg/mL in water. NBD C6-Ceramide in nanomicelles remained stable in aqueous solutions, allowing intravenous administration without the use of any organic solvents or surfactants. After oral administration, NBD C6-Ceramide rapidly rose to peak plasma concentrations within the first 90 min, distributed to tissues, and remained in vivo for more than 24 h. Tissular levels of NBD C6-Ceramide from high to low were associated with heart, lung, cerebellum, testicle, spleen, liver, kidney, and brain. Altogether, our study demonstrated that RUB-assisted nanomicelles can serve as an efficient and convenient delivery system for short-chain C6-Ceramide and enable in vivo evaluation of potential new cancer treatments.

Ceramide-Rubusoside Nanomicelles, a Potential Therapeutic Approach to Target Cancers Carrying p53 Missense Mutations.

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer in vivo bioavailability and restore p53-dependent tumor suppression in cancer cells carrying a p53 missense mutation. Natural RUB encapsulated short-chain C6-Cer so as to form Cer-RUB nanomicelles (∼32 nm in diameter) that substantially enhanced Cer solubility and its levels in tissues and tumors of mice dosed intraperitoneally. Intriguingly, Cer-RUB nanomicelle treatments restored p53-dependent tumor suppression and sensitivity to cisplatin in OVCAR-3 ovarian cancer cells and xenograft tumors carrying p53 R248Q mutation. Moreover, Cer-RUB nanomicelles showed no signs of significant nonspecific toxicity to noncancerous cells or normal tissues, including bone marrow. Furthermore, Cer-RUB nanomicelles restored p53 phosphorylated protein and downstream function to wild-type levels in p53 R172H/+ transgenic mice. Altogether, this study, for the first time, indicates that natural Cer-RUB nanomicelles offer a feasible approach for efficaciously and safely targeting cancers carrying p53 missense mutations.

Targeting tumor phenotypic plasticity and metabolic remodeling in adaptive cross-drug tolerance.

Metastable phenotypic state transitions in cancer cells can lead to the development of transient adaptive resistance or tolerance to chemotherapy. Here, we report that the acquisition of a phenotype marked by increased abundance of CD44 (CD44Hi) by breast cancer cells as a tolerance response to routinely used cytotoxic drugs, such as taxanes, activated a metabolic switch that conferred tolerance against unrelated standard-of-care chemotherapeutic agents, such as anthracyclines. We characterized the sequence of molecular events that connected the induced CD44Hi phenotype to increased activity of both the glycolytic and oxidative pathways and glucose flux through the pentose phosphate pathway (PPP). When given in a specific order, a combination of taxanes, anthracyclines, and inhibitors of glucose-6-phosphate dehydrogenase (G6PD), an enzyme involved in glucose metabolism, improved survival in mouse models of breast cancer. The same sequence of the three-drug combination reduced the viability of patient breast tumor samples in an explant system. Our findings highlight a convergence between phenotypic and metabolic state transitions that confers a survival advantage to cancer cells against clinically used drug combinations. Pharmacologically targeting this convergence could overcome cross-drug tolerance and could emerge as a new paradigm in the treatment of cancer.

An N6-methyladenosine at the transited codon 273 of p53 pre-mRNA promotes the expression of R273H mutant protein and drug resistance of cancer cells.

Mutant p53 proteins that promote cancer cell invasive growth, metastasis and drug resistance emerge as therapeutic targets. Previously, we reported that suppression of ceramide glycosylation restored wild-type p53 protein and tumor suppressing function in cancer cells heterozygously carrying p53 R273H, a hot-spot missense mutation; however, the mechanisms underlying the control of mutant protein expression remain elusive. Herein, we report that an N6-methyladenosine (m6A) at the point-mutated codon 273 (G > A) of p53 pre-mRNA determines the mutant protein expression. Methylation of the transited adenosine was catalyzed by methyltransferase like 3 (METTL3), and this m6A-RNA promoted a preferential pre-mRNA splicing; consequently, the produced p53 R273H mutant protein resulted in acquired multidrug resistance in colon cancer cells. Furthermore, glycosphingolipids (particularly globotriaosylceramide) generated from serial ceramide glycosylation were seen to activate cSrc and ß-catenin signaling so as to upregulate METTL3 expression, in turn promoting expression of p53 R273H mutant protein, with consequent drug resistance. Conversely, either silencing METTL3 expression by using small interfering RNA (siRNA) or inhibiting RNA methylation with neplanocin A suppressed m6A formation in p53 pre-mRNA, and substantially increased the level of phosphorylated p53 protein (Ser15) and its function in cells heterozygously carrying the R273H mutation, thereby re-sensitizing these cells to anticancer drugs. Concordantly, suppression of ceramide glycosylation repressed METTL3 expression and m6A formation in p53 pre-mRNA, thus sensitizing cells carrying R273H to anticancer drugs. This study uncovers a novel function of pre-mRNA m6A as a determinant of mutant protein expression in cancer cells heterozygously carrying the TP53 R273H mutation. Suppressing both RNA methylation and ceramide glycosylation might constitute an efficacious and specific approach for targeting TP53 missense mutations coding for a G > A transition, thereby improving cancer treatments.

Fluorescence HPLC Analysis of the in-vivo Activity of Glucosylceramide Synthase.

Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the in-vivo activities that directly represent the cellular effects of enzymes in live organs are critical importance to appreciate the roles enzymes play in modulating physiological or pathological processes, although assessments of such in-vivo enzyme activity are more difficult than typical test-tube assays. Recently, we, for the first time, developed a direct and easy-handling method for HPLC analyzing the in-vivo activity of glucosylceramide synthase (GCS). GCS that converts ceramide into glucosylceramide is a limiting-enzyme in the syntheses of glycosphingolipids and is one cause of cancer drug resistance. In our method developed, rubusoside nanomicelles delivers fluorescence N-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl]-d-erythro-sphingosine (NBD C6-ceramide) into mice, tissues uptake the cell-permeable substrate, and GCS converts it into NBD C6-glucosylceramide in all organs simultaneously. Further, HPLC analyzes the extracted NBD C6-glucosylceramide to assess alterations of the in-vivo GCS activities in tissues. This method can be broadly used to assess the in-vivo GCS activities in any kind of animal models to appreciate either the role GCS plays in diseases or the therapeutic efficacies of GCS inhibitors.

Protein-polysaccharide nanohybrids: Hybridization techniques and drug delivery applications.

Complex nanosystems fabricated by hybridization of different types of materials such as lipids, proteins, or polysaccharides are usually superior to simple ones in terms of features and applications. Proteins and polysaccharides hold great potential for development of nanocarriers for drug delivery purposes based on their unique biocompatibility, biodegradability, ease of functionalization, improved biodistribution and minimal toxicity profiles. Protein-polysaccharide nanohybrids have gained a lot of attention in the past few years particularly for drug delivery applications. In this review, different hybridization techniques utilized in the fabrication of such nanohybrids including electrostatic complexation, Maillard conjugation, chemical coupling and electrospinning were thoroughly reviewed. Moreover, various formulation factors affecting the characteristics of the formed nanohybrids were discussed. We also reviewed in depth the outcomes of such hybridization ranging from stability enhancement, to toxicity reduction, improved biocompatibility, and drug release modulation. We also gave an insight on their limitations and what hinders their clinical translation and market introduction.

Inhalable lactoferrin-chondroitin nanocomposites for combined delivery of doxorubicin and ellagic acid to lung carcinoma.

AIM: The use of inhalable nanomedicines can overcome the Enhanced permeation and retention effect (EPR)-associated drawbacks in lung cancer therapy via systemic nanomedicines. METHODS: We developed a lactoferrin-chondroitin sulfate nanocomplex for the co-delivery of doxorubicin and ellagic acid nanocrystals to lung cancer cells. Then, the nanocomplex was converted into inhalable nanocomposites via spray drying. RESULTS: The resulting 192.3 nm nanocomplex exhibited a sequential faster release of ellagic acid, followed by doxorubicin. Furthermore, the nanocomplex demonstrated superior cytotoxicity and internalization into A549 lung cancer cells mediated via Tf and CD44 receptors. The inhalable nanocomposites exhibited deep lung deposition (89.58% fine particle fraction [FPF]) with powerful antitumor efficacy in lung cancer bearing mice. CONCLUSION: Overall, inhalable lactoferrin-chondroitin sulfate nanocomposites would be a promising carrier for targeted drug delivery to lung cancer.

Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo.

Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate with stem cell pluripotency, cancer drug resistance, GSL storage disorders and other diseases. Enzyme activities measured conventionally with currently available ex-vivo methods do not enable reliable assessment of the roles played by GCS in vivo. We report herein a substrate-incorporation method enabling rapid and efficient assessment of GCS in-vivo activity. Upon nanoparticle-based delivery, fluorescent NBD C6-ceramide was efficiently converted to NBD C6-glucosylceramide in live cells or in mouse tissues, whereupon an HPLC assay enabled detection and quantification of NBD C6-glucosylceramide in the low-femtomolar range. The enzyme kinetics of GCS in live cells and mouse liver were well-described by the Michaelis-Menten model. GCS activities were significantly higher in drug-resistant cancer cells and in tumors overexpressing GCS, but reduced after silencing GCS expression or inhibiting this enzyme. Our studies indicate that this rapid and efficient method provides a valuable means for accurately assessing the roles played by GCS in normal vs. pathological states, including ones involving cancer drug resistance.

Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells.

Missense mutation of tumor suppressor p53, which exhibits oncogenic gain-of-function (GOF), not only promotes tumor progression, but also diminishes therapeutic efficacies of cancer treatments. However, it remains unclear how a p53 missense mutant contributes to induced pluripotency of cancer stem cells (CSCs) in tumors exposed to chemotherapeutic agents. More importantly, it may be possible to abrogate the GOF by restoring wild-type p53 activity, thereby overcoming the deleterious effects resulting from heterotetramer formation, which often compromises the efficacies of current approaches being used to reactivate p53 function. Herewith, we report that p53 R273H missense mutant urges cancer cells to spawn CSCs. SW48/TP53 cells, which heterozygously carry the p53 R273H hot-spot mutant (R273H/+, introduced by a CRISPR/Casp9 system), were subchronically exposed to doxorubicin in cell culture and in tumor-bearing mice. We found that p53-R273H (TP53-Dox) cells were drug-resistant and exhibited epithelial-mesenchymal transition (EMT) and increased numbers of CSCs (CD44v6+/CD133+), which resulted in enhanced wound healing and tumor formation. Inhibition of glucosylceramide synthase with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) sensitized p53-R273H cancer cells and tumor xenografts to doxorubicin treatments. Intriguingly, PDMP treatments restored wild-type p53 expression in heterozygous R273H mutant cells and in tumors, decreasing CSCs and sensitizing cells and tumors to treatments. This study demonstrated that p53-R273H promotes EMT and induced pluripotency of CSCs in cancer cells exposed to doxorubicin, mainly through Zeb1 and ß-catenin transcription factors. Our results further indicate that restoration of p53 through inhibition of ceramide glycosylation might be an effective treatment approach for targeting cancers heterozygously harboring TP53 missense mutations.

Ceramide modulates pre-mRNA splicing to restore the expression of wild-type tumor suppressor p53 in deletion-mutant cancer cells.

Mutants of tumor suppressor p53 not only lose the activity in genome stabilizing and in tumor suppression, but also exhibit oncogenic function in cancer cells. Most efforts in restoring p53 biological activity focus on either altering mutant-protein conformation or introducing an exogenous p53 gene into cells to eliminate p53-mutant cancer cells. Being different from these, we report that ceramide can restore the expression of wild-type p53 and induce p53-dependent apoptosis in deletion-mutant cancer cells. We show that endogenous long-carbon chain ceramide species (C16- to C24-ceramides) and exogenous C6-ceramide, rather than other sphingolipids, restore wild-type mRNA (intact exon-5), phosphorylated protein (Ser15 in exon-5) of p53, and p53-responsive proteins, including p21 and Bax, in ovarian cancer cells, which predominantly express a deleted exon-5 of p53 mutant before treatments. Consequently, the restored p53 sensitizes these p53-mutant cancer cells to DNA damage-induced growth arrest and apoptosis. Furthermore, we elucidate that ceramide activates protein phosphatase-1, and then the dephosphorylated serine/arginine-rich splicing-factor 1 (SRSF1) is translocated to the nucleus, thus promoting pre-mRNA splicing preferentially to wild-type p53 expression. These findings disclose an unrecognized mechanism that pre-mRNA splicing dysfunction can result in p53 deletion-mutants. Ceramide through SRSF1 restores wild-type p53 expression versus deletion-mutant and leads cancer cells to apoptosis. This suggests that heterozygous deletion-mutants of p53 can be restored in posttranscriptional level by using epigenetic approaches.