Lysosomes Of Cancer Cells Research Articles

Profiling Bis(monoacylglycero)phosphate Lipids in Cancer Cell Lysosomes as Therapeutic Targets

BackgroundTumor resistance to apoptosis underlies the clinical failure of conventional chemotherapeutics and commonly employed molecularly targeted drugs. Cancer cells frequently activate survival pathways to evade destruction by apoptosis‐inducing therapies and persist following treatment to seed tumor recurrence and metastatic outgrowth. Lysosomal cell death is an alternative death pathway that triggers non‐apoptotic mechanisms. The lysosome, a major degradative component of eukaryotic cells, is altered by oncogenic transformation. Key to the function and stability of lysosome‐associated enzymes are bis(monoacylglycero)‐phosphate lipids (BMP). BMPs are negatively charged and highly enriched in the internal lysosome membrane. Untargeted analytical methods for the measurement of BMPs and in silico libraries to support the discovery of new BMP species are currently lacking.MethodWe present here an expanded BMP library to include over 1800 BMP species in both positive and negative ESI modes. We have chromatographic separation of BMP from isobaric species, and have used this method to profile cancerous and non‐cancerous immortalized cell lines. Lipidomic analysis was performed using Waters Acquity UPLC CSH C18 (100 mm length × 2.1 mm id; 1.7 μm) column coupled to a Thermo Scientific Q‐Exactive HF mass spectrometer. Data processing was done using MS‐DIAL software and in‐silico structure predictions were done using MS‐FINDER.ResultsIn breast cancer cells (Met‐1), we found that all BMP species measured were significantly less abundant relative to non‐transformed mammary epithelial cells (nMUMG), (n = 10). As BMPs are key to lysosome stability, we hypothesize the dramatic loss of BMPs observed in transformed cells renders cancer‐associated lysosomes unstable. Therefore, targeting BMPs might represent an effective therapeutic strategy for selective cancer cell destruction.Support or Funding InformationThis work was funded by NIH U24 DK097154.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synergistic Lysosomal Activatable Polymeric Nanoprobe Encapsulating pH Sensitive Imidazole Derivative for Tumor Diagnosis.

Developing optical tumor imaging probes with minimal background noise is very important for its early detection of small lesions and accurate diagnosis of cancer. To overcome the bottleneck of low signal to noise ratio and sensitivity, it needs further improvement in fluorescent probe design and understanding of tumor development process. Recent reports reveal that lysosome's acidity in cancer cells can be below 4.5 with high Na+ /H+ exchange activity, which makes it an ideal target intracellular organelle for cancer diagnosis based on the variation of pH. Herein, a boron 2-(2'-pyridyl) imidazole complex derivative (BOPIM-N) is developed, with the ability to show a pH-activatable "OFF-ON" fluorescent switch by inhibiting twisted intramolecular charge transfer upon protonation at pH 3.8-4.5, which is studied for its selective viable cancer cell imaging ability in both in vitro and in vivo experiments. Interestingly, BOPIM-N can specifically emit green fluorescence in lysosomes of cancer cells, indicating its promising cancer cell specific imaging ability. More importantly, nanoformulated BOPIM-N probes can be specifically light-ON in tumor bearing site of nude mice with resolution up to cellular level, indicating its potential application in tumor diagnosis and precision medicine.

Naphthalimide-rhodamine based fluorescent probe for ratiometric sensing of cellular pH

The pH values of lysosomes in cancer cells is slightly lower than that in normal cells, which can be used to distinguish cancer cells from normal cells. According to this, a naphthalimide-rhodamine based fluorescent probe (hereafter referred to as RBN) with a pKa of 4.20 was designed and synthesized for ratiometric sensing of cellular pH via fluorescence resonance energy transfer (FRET), which can respond to different pH precisely through ratiometric fluorescence intensity (I577/I540). RBN can be employed to distinguish cancer cells from normal cells on the basis of different fluorescent response, in particular, RBN showed excellent water solubility and low cell toxicity, all these are quite significant for potential application in cancer diagnose and therapy.

On the synthesis of quinone-based BODIPY hybrids: New insights on antitumor activity and mechanism of action in cancer cells

Fluorescent quinone-based BODIPY hybrids were synthesised and characterised by NMR analysis and mass spectrometry. We measured their cytotoxic activity against cancer and normal cell lines, performed mechanistic studies by lipid peroxidation and determination of reduced (GSH) and oxidized (GSSG) glutathione, and imaged their subcellular localisation by confocal microscopy. Cell imaging experiments indicated that nor-β-lapachone-based BODIPY derivatives might preferentially localise in the lysosomes of cancer cells. These results assert the potential of hybrid quinone-BODIPY derivatives as promising prototypes in the search of new potent lapachone antitumor drugs.

A lysosome-targeted BODIPY as potential NIR photosensitizer for photodynamic therapy

As an indispensable organelle, lysosomes play critical role in cell digestion and are closely relate to cell death and apoptosis. Photosensitizers that target to lysosomes are extremely important to photodynamic therapy. Herein, we report an excellent BODIPY based photosensitizer MBDP for lysosome targeting NIR photosensitizer. MBDP exhibits excellent PDT efficiency with low IC50 of 0.2 μM compared to the classical photosensitizer Ce6 (IC50 = 1.39 μM). Furthermore, MBDP can localize in lysosomes of cancer cells and produce significant singlet oxygen with photoexcitation of 660 nm red light. Additionally, lysosome destruction test and photoinduce cell death assay demonstrate that MBDP-mediated PDT is able to damage the lysosome integrity severely and result in cell apoptosis and death in lysosomal related pathway.

Photothermal therapy of single cancer cells mediated by naturally created gold nanorod clusters

Gold nanorods (GNRs) are generally considered to be nontoxic to normal and cancer cells. They are usually accumulated at lysosomes after entering into cells, forming GNR clusters in which strong plasmonic coupling between GNRs is expected. We investigated the photothermal therapy of single cancer cells by exploiting the significantly enhanced two-photon-induced absorption of GNR clusters naturally created in the lysosomes of cancer cells. It was revealed numerically that the plasmonic coupling between GNRs in GNR clusters can effectively enhance the photothermal conversion efficiency. As a result, the thermal damage of single cancer cells can be induced by using pulse energy as low as ~70 pJ. In experiments, the locations of GNR clusters can be accurately determined through the detection of the two-photon-induced luminescence, which is also significantly enhanced, by using a confocal laser scanning microscope. The photothermal therapy was conducted by focusing femtosecond laser light on the targeted GNR clusters, generating bubbles and deforming cell membranes. The photothermal therapy proposed in this work can lead to the rapid and acute injury of single cancer cells. The dependence of the apoptosis time on the pulse energy of femtosecond laser light was also examined. Our findings suggest a novel strategy for the photothermal therapy of single cancer cells with ultralow energy.

Imaging lysosomal highly reactive oxygen species and lighting up cancer cells and tumors enabled by a Si-rhodamine-based near-infrared fluorescent probe.

Lysosomes have recently been regarded as the attractive pharmacological targets for selectively killing of cancer cells via lysosomal cell death (LCD) pathway that is closely associated with reactive oxygen species (ROS). However, the details on the ROS-induced LCD of cancer cells are still poorly understood, partially due to the absence of a lysosome-targetable, robust, and biocompatible imaging tool for ROS. In this work, we brought forward a Si-rhodamine-based fluorescent probe, named PSiR, which could selectively and sensitively image the pathologically more relavent highly reactive oxygen species (hROS: HClO, HO, and ONOO-) in lysosomes of cancer cells. Compared with many of the existing hROS fluorescent probes, its superiorities are mainly embodied in the high stability against autoxidation and photoxidation, near-infrared exitation and emission, fast fluorescence off-on response, and specific lysosomal localization. Its practicality has been demonstrated by the real-time imaging of hROS generation in lysosomes of human non-small-cell lung cancer cells stimulated by anticancer drug β-lapachone. Moreover, the probe was sensitive enough for basal hROS in cancer cells, allowing its further imaging applications to discriminate not only cancer cells from normal cells, but also tumors from healthy tissues. Overall, our results strongly indicated that PSiR is a very promising imaging tool for the studies of ROS-related LCD of cancer cells, screening of new anticancer drugs, and early diagnosis of cancers.

GE11 peptide conjugated selenium nanoparticles for EGFR targeted oridonin delivery to achieve enhanced anticancer efficacy by inhibiting EGFR-mediated PI3K/AKT and Ras/Raf/MEK/ERK pathways

Selenium nanoparticles (Se NPs) have attracted increasing interest in recent decades because of their anticancer, immunoregulation, and drug carrier functions. In this study, GE11 peptide-conjugated Se NPs (GE11-Se NPs), a nanosystem targeting EGFR over-expressed cancer cells, were synthesized for oridonin delivery to achieve enhanced anticancer efficacy. Oridonin loaded and GE11 peptide conjugated Se NPs (GE11-Ori-Se NPs) were found to show enhanced cellular uptake in cancer cells, which resulted in enhanced cancer inhibition against cancer cells and reduced toxicity against normal cells. After accumulation into the lysosomes of cancer cells and increase of oridonin release under acid condition, GE11-Ori-Se NPs were further transported into cytoplasm after the damage of lysosomal membrane integrity. GE11-Ori-Se NPs were found to induce cancer cell apoptosis by inducting reactive oxygen species (ROS) production, activating mitochondria-dependent pathway, inhibiting EGFR-mediated PI3K/AKT and inhibiting Ras/Raf/MEK/ERK pathways. GE11-Se NPs were also found to show active targeting effects against the tumor tissue in esophageal cancer bearing mice. And in nude mice xenograft model, GE11-Ori-Se NPs significantly inhibited the tumor growth via inhibition of tumor angiogenesis by reducing the angiogenesis-marker CD31 and activation of the immune system by enhancing IL-2 and TNF-α production. The selenium contents in mice were found to accumulate into liver, tumor, and kidney, but showed no significant toxicity against liver and kidney. This cancer-targeted design of Se NPs provides a new strategy for synergistic treating of cancer with higher efficacy and reduced side effects, introducing GE11-Ori-Se NPs as a candidate for further evaluation as a chemotherapeutic agent for EGFR over-expressed esophageal cancers.

Lysosome-Targeting Fluorogenic Probe for Cathepsin B Imaging in Living Cells.

Cathepsin B (CTB) is a lysosomal protease which has been recognized as a promising biomarker for many malignant tumors, and accurate detection of its activity is important in early diagnosis of cancers and predicting metastasis. Herein, we reported a lysosome-targeting fluorogenic small-molecule probe for fluorescence imaging of lysosomal CTB in living cancer cells by incorporating a CTB-recognitive peptide substrate Cbz-Lys-Lys-p-aminobenzyl alcohol (Cbz-Lys-Lys-PABA) and a lysosome locating group morpholine. We demonstrated that the probe could be efficiently activated by CTB to generate ∼73-fold enhancement in fluorescence under acidic lysosomal environment (pH 4.5-6.0), allowing for high sensitivity and specificity to detect CTB. Fluorescence imaging results showed selective accumulation and fluorescence turn-on in the lysosomes of cancer cells, which were capable of reporting on lysosomal CTB activity in cancer cells and normal tissue cells. This study highlights the potential of using a lysosome-targeting group to design a sensitive and specific fluorogenic probe for fluorescence imaging of lysosomal CTB in living cells.

Riccardin D-N induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo

Lysosomes are important targets for anticancer drug discovery. Our previous study showed that Riccardin D-N (RD-N), a natural macrocylic bisbibenzyl derivative produced by Mannich reaction, induced cell death by accumulating in lysosomes. Experiments were performed on human lung squamous cell carcinoma tissue from left inferior lobar bronchus of patient xenografts and H460 cells. RD-N was administrated for 25days. The specimens of xenografts in Balb/c athymic (nu+/nu+) male mice were removed for immunohistochemistry, subcellular fractionation, enzyme activities and Western blotting analysis. mRFP-GFP-LC3 reporter was used to examine autophagy in H460 cells. Sphingomyelin assay was evaluated by thin-layer chromatography and assay kit. Lysosomal membrane permeabilization (LMP) caused by acid sphingomyelinase (ASM) inhibition and subsequent changes of sphingomyelin (SM) metabolism selectively destabilized the cancer cell lysosomes in RD-N-treated H460 cells in vitro and tumor xenograft model in vivo. The destabilized lysosomes induced the release of cathepsins from the lysosomes into the cytosol and further triggered cell death. These results explain the underlying mechanism of RD-N induced LMP. It can be concluded that a more lysosomotropic derivative was synthesized by introduction of an amine group, which could have more potential applications in cancer therapy.

Abstract 3511: Chloroquine-induced lysosomal membrane permeabilization restores sensitivity to cisplatin in refractory lung cancer cells

Abstract Introduction: Lung cancer is the leading cause of cancer-related death. Resistance to treatment contributes to poor outcome. A form of resistance to antineoplastic agents involves the sequestration of chemotherapy inside lysosomes followed by its delivery outside the cell through exocytosis. Lysosomal membrane permeabilization (LMP) not only releases the trapped drug, but also allows the translocation of lysosomal proteases into the cytosol, triggering cell demise. Since lysosomes in cancer cells are more susceptible to LMP than normal cells, we hypothesize that targeting LMP in cisplatin-resistant cells may provide a successful strategy to restore sensitivity to chemotherapy. Materials and Methods: Cisplatin-resistant (cisR) A549 lung cancer cells, obtained by treating parental A549 cells with incremental concentrations of cisplatin, were kindly provided by Dr. Martin Barr. To detect LMP, lysosomes were loaded with FITC-dextran 40KDa and immunofluorescence images were obtained by confocal microscopy. To confirm LMP, cells were homogenized, cytosolic and lysosomal fractions were separated by ultracentrifugation, and cell lysates were immunoblotted with specific antibodies against cathepsin D, α-tubulin and LAMP-1. Cell viability was evaluated using CellTiter blue assay. To measure apoptosis, cells were stained for Annexin-V/Propidium iodide, or FITC-conjugated caspase 3 substrate, and mounted on the Cellometer vision CBA platform, an automated image-based fluorescence microscope equipped with bio-analysis system. Results: cisR A549 cells loaded with FITC-dextran, and incubated with different concentrations of chloroquine (25-100μM) for 24 hours demonstrated increased green haziness in the cytosol and loss of the punctate pattern of the dextran-loaded lysosomes, indicative of dextran leakage following LMP. Release of cathepsins into the cytosol due to LMP was confirmed by the increase in cathepsin D signal in the cytosolic extract on western blot. Next, cisR A549 cells were treated with chloroquine, cisplatin or the drug mix. Cell viability assay revealed a synergistic effect of the combination, with a combination index of 0.70 for cisplatin 25μM and chloroquine 100μM at 48 hours. Notably, there is significant increase in Annexin V stained apoptotic cells, accompanied by a corresponding increase in caspase 3 activity, in the combination. Interestingly, inhibition of lysosomal proteases using E64 was cytoprotective for cells treated with cisplatin and chloroquine, suggesting that chloroquine-induced cell death is LMP mediated. Conclusion: Chloroquine re-sensitizes cisR A549 cells to cisplatin in an LMP-mediated manner. Our preliminary results support the concept that targeting LMP may provide a viable therapeutic strategy to restore sensitivity to chemotherapy in refractory lung cancer. Screening for other repurposed LMP inducer drugs is ongoing. Citation Format: Magdalena Circu, James Cardelli, Glenn Mills, Martin Barr, Hazem E. El-Osta. Chloroquine-induced lysosomal membrane permeabilization restores sensitivity to cisplatin in refractory lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3511.

Copper and conquer: copper complexes of di-2-pyridylketone thiosemicarbazones as novel anti-cancer therapeutics.

Copper is an essential trace metal required by organisms to perform a number of important biological processes. Copper readily cycles between its reduced Cu(i) and oxidised Cu(ii) states, which makes it redox active in biological systems. This redox-cycling propensity is vital for copper to act as a catalytic co-factor in enzymes. While copper is essential for normal physiology, enhanced copper levels in tumours leads to cancer progression. In particular, the stimulatory effect of copper on angiogenesis has been established in the last several decades. Additionally, it has been demonstrated that copper affects tumour growth and promotes metastasis. Based on the effects of copper on cancer progression, chelators that bind copper have been developed as anti-cancer agents. In fact, a novel class of thiosemicarbazone compounds, namely the di-2-pyridylketone thiosemicarbazones that bind copper, have shown great promise in terms of their anti-cancer activity. These agents have a unique mechanism of action, in which they form redox-active complexes with copper in the lysosomes of cancer cells. Furthermore, these agents are able to overcome P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) and act as potent anti-oncogenic agents through their ability to up-regulate the metastasis suppressor protein, N-myc downstream regulated gene-1 (NDRG1). This review provides an overview of the metabolism and regulation of copper in normal physiology, followed by a discussion of the dysregulation of copper homeostasis in cancer and the effects of copper on cancer progression. Finally, recent advances in our understanding of the mechanisms of action of anti-cancer agents targeting copper are discussed.

A linear polyethylenimine (LPEI) drug conjugate with reversible charge to overcome multidrug resistance in cancer cells

Multidrug resistance (MDR) is an important hindrance to efficient cancer chemotherapy. Cancer cell lysosomes play an important role in intrinsic MDR by accumulating chemotherapy drugs and deactivating their therapeutic action. The cationic polymer polyethyleneimine (PEI) can disrupt the endosomal/lysosomal membrane via the proton-sponge effect (PSE). However, its positive charge makes it toxic and so it cannot be used in vivo. Here, linear PEI (LPEI) is used to demonstrate that a pH-triggered charge-reversal carrier can solve this problem. The imines are amidized by masking a lysosomal pH-active agent. LPEI regenerates its positive charge in the acidic endosomal/lysosomal cell compartments and disrupts the endosomal/lysosomal membrane, resulting in delivery of the drugs into the cytoplasm and nuclei where they exert their pharmacologic activity. Folic acid targeting groups are introduced into the polymer to increase its cancer-cell targeting capability. An anticancer drug camptothecin (CPT) conjugated to the carrier by intracellular cleavable disulfide bonds shows improved cytotoxicity over free CPT.

Folate Receptor-Targeted and Cathepsin B-Activatable Nanoprobe for In Situ Therapeutic Monitoring of Photosensitive Cell Death

The integration of diagnostic and therapeutic functions in a single system holds great promise to enhance the theranostic efficacy and prevent the under- or overtreatment. Herein, a folate receptor-targeted and cathepsin B-activatable nanoprobe is designed for background-free cancer imaging and selective therapy. The nanoprobe is prepared by noncovalently assembling phospholipid-poly(ethylene oxide) modified folate and photosensitizer-labeled peptide on the surface of graphene oxide. After selective uptake of the nanoprobe into lysosome of cancer cells via folate receptor-mediated endocytosis, the peptide can be cleaved to release the photosensitizer in the presence of cancer-associated cathepsin B, which leads to 18-fold fluorescence enhancement for cancer discrimination and specific detection of intracellular cathepsin B. Under irradiation, the released photosensitizer induces the formation of cytotoxic singlet oxygen for triggering photosensitive lysosomal cell death. After lysosomal destruction, the lighted photosensitizer diffuses from lysosome into cytoplasm, which provides a visible method for in situ monitoring of therapeutic efficacy. The nanoprobe exhibits negligible dark toxicity and high phototoxicity with the cell mortality rate of 0.06% and 72.1%, respectively, and the latter is specific to folate receptor-positive cancer cells. Therefore, this work provides a simple but powerful protocol with great potential in precise cancer imaging, therapy, and therapeutic monitoring.

Transformation-Associated Changes in Sphingolipid Metabolism Sensitize Cells to Lysosomal Cell Death Induced by Inhibitors of Acid Sphingomyelinase

Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert multidrug resistance. Their cancer selectivity is associated with transformation-associated reduction in ASM expression and subsequent failure to maintain sphingomyelin hydrolysis during drug exposure. Taken together, these data identify ASM as an attractive target for cancer therapy.

Intracellular dissociation of a polymer coating from nanoparticles

Polymer-coated nanoparticles are widely used for drug delivery in cancer therapy. However, it is not clear whether the polymer coating is disrupted in the lipid bilayer or intracellular space. Our current work suggests that the polymer coating of inorganic nanoparticles is disrupted after internalization by cancer cells. Single dispersed red quantum dots (QDs) labeled with 5-carboxyfluorescein (5-FAM) (green) (diameter = 28 nm) were incubated with cancer cells (PC-3) and imaged via fluorescence microscopy. Initially the 5-FAM-labeled polymer coating was attached to the QD and its green fluorescence was quenched when the nanoparticles were internalized after 4 h incubation, but the 5-FAM-labeled polymer became separated from the QD once inside the lysosomes of cells and its fluorescence becomes visible after 8 h. The fluorescence ratio (5-FAM/QDs) was increased 29-fold after 8 h incubation compared to 2 h. The fluorescence quenching effect of PEG-5-FAM after conjugation in solution (quenched by 44%) was compared to free poly(ethylene glycol)-5-FAM (PEG-5-FAM) mixing with QDs, which only exhibited slight (6.9%) quenching of 5-FAM. In addition, the intracellular dissociation of polymer coating from QD loaded micelles (diameter = 300 nm) was also observed. Furthermore, amphiphilic polymer labeled with the hydrophobic dye 6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoic acid (BODIPY® TMR) (red) was applied to encapsulate hydrophobic iron oxide nanoparticles (IONPs). The BODIPY dye was quenched by both the encapsulated IONPs and the hydrophobic region inside the micelles, while an 8-fold fluorescence enhancement was observed after polymeric micelle dissociation. Our in vitro results also reveal the polymeric dissociation after internalization by cancer cells as the dye signal becomes detectable after 24 h incubation. These results suggest that the polymer coating is stable in the lipid bilayer and becomes dissociated from nanoparticles in the lysosome of cancer cells. These data will provide guidance for intracellular drug delivery using polymer coated nanoparticles.

Abstract 2898: Intracellular dissociation of polymer coating from nanoparticles

Abstract Polymer-coated nanoparticles are widely used for drug delivery in cancer therapy. However, it is not known whether the polymer coating will be disrupted in the lipid bilayer or intracellular space. Our current work suggests that the polymer coating of inorganic nanoparticles is disrupted after internalization by cancer cells. Single dispersed red quantum dots (QDs) labeled with 5-FAM (green) (D = 28 nm) were incubated with cancer cells (PC-3) and imaged via fluorescence microscopy. Initially the 5-FAM-labeled polymer coating was attached to the QDs and its green fluorescence was quenched when the nanoparticles were internalized from 2 to 4 hrs, but 5-FAM-labeled polymer became separated from the QDs once they are inside lysosome of cells and its fluorescence suddenly visible from 8 hrs. The fluorescence ratio (5-FAM/QDs) were increased by 29-fold after 8 h incubation compared to 2 h. The fluorescence quenching effect was compared to free PEG-5-FAM mixed with QDs, which only exhibited a 44% decrease in 5-FAM quenching. In addition, the intracellular dissociation of polymer coating from QD loaded micelles (D = 300 nm) was also observed. Furthermore, when labeled amphiphilic polymeric micelles encapsulate hydrophobic iron oxide nanoparticles (IONPs) with the hydrophobic dye BODIPY-TMR (red), double quenching was observed by encapsulated nanoparticles and the hydrophobic region inside the micelles by 8-fold fluorescence reduction. The BODIPY-TMR-labeled nanoparticles also showed polymer dissociation inside of cancer cells after 24 h incubation. These results suggest that polymer coating is stable in the lipid bilayer and it is dissociated from nanoparticles in the lysosome of cancer cells. These data will provide guidance for intracellular drug delivery using polymer coated nanoparticles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2898. doi:1538-7445.AM2012-2898

Translational Research of Photodynamic Therapy with Acridine Orange which Targets Cancer Acidity

During the past 20 years, we have found that acridine orange (AO) selectively accumulates in musculoskeletal sarcomas in vivo or exerts selective cytocidal effects against sarcoma cells in vitro after illumination of the tumor cells with visible light or irradiation of the cells with low-dose X-rays. Based on the data obtained from basic research, we have employed reduction surgery followed by photo- or radiodynamic therapy using AO (AO-PDT & RDT) in 71 patients with musculoskeletal sarcomas, in an attempt to maintain excellent limb function in the patients. We have obtained good local control rates and remarkably better limb functions with this approach as compared to the results obtained with the conventional wide resection surgery. Our basic research demonstrated that AO accumulates densely in intracellular acidic vesicles, especially lysosomes, in an acidity-dependent manner. In cancer cells that proliferate under hypoxic conditions or with Warburg's effect, active glycolysis produces an enormous number of protons, which are released by the cells via proton pumps into the extracellular fluid or lysosomes to maintain a neutral pH of the cytosolic fluid. Cancer cells contain many strongly acidic lysosomes of large sizes; therefore, AO shows marked and prolonged accumulation in the acidic lysosomes of cancer cells. Photon energy excites the AO resulting in the production of activated oxygen species, which oxidize the fatty acids of the lysosomal membrane, resulting in the leakage of lysosomal enzymes and protons, followed by apoptosis of the cancer cells. Based on these observations, we conclude that AO-PDT & RDT target acidic vesicles, especially the lysosomes, in cancer cells, to exhibit selective anti-cancer cell activity. Therefore, it is suggested that AO excited by photon energy has excellent potential as an anticancer "Magic Bullet".

A cyanine based fluorophore emitting both single photon near-infrared fluorescence and two-photon deep red fluorescence in aqueous solution

Optical imaging provides an indispensable way to locate tumors in their early stages with high sensitivity and signal to background ratio. A heptamethine cyanine based fluorophore that emits both single photon near-infrared fluorescence and two-photon deep red fluorescence under physiological conditions was developed. Linear and nonlinear photophysical properties of this fluorophore were investigated and it demonstrated the capability to label lysosomes in cancer cells. The advantages of this fluorophore, including tolerable cytotoxicity, high fluorescence quantum yield, and the ability to emit both near-infrared single photon fluorescence and deep red two photon fluorescence in aqueous solution, give it potential to be used in intra-operatively optical image-guided tumor excision followed by two-photon fluorescence microscopy biopsy analysis after a single administration.

Daunorubicin -TiO2 Nanocomposites As ‘smart’ pH-Responsive Drug Delivery System,

Abstract 3479Daunorubicin (DNR) with a broad spectrum of anti-tumor activity is limited due to the serious side-effects in the clinical application. The aim of this study was to explore the novel pH-responsive drug delivery system (DDS) based on titanium dioxide (TiO2) nanoparticles (Nps) for its potential roles to enable more intelligently controlled release, enhance chemotherapeutic efficiency, and reduce the side-effects of DNR. DNR was loaded onto the TiO2 Nps by forming (six-membered chelate) complexes with transition metal Ti to contract DNR-TiO2 nanocomposites as DDS. The encapsulation efficiency and loading efficiency of DNR loaded TiO2 Nps were assessed and calculated as 65.46±6.82% and 20.63±3.55%, respectively.The DNR was released from the DDS much more rapidly at pH 5.0 and 6.0 than at pH 7.4. The release behavior is a desirable characteristic for tumor-targeted drug delivery. Most DNR will remain in the carrier for a considerable time period at normal physiological conditions (pH 7.4), indicating the potential for the prolonged DNR retention time in the blood circulation and thereby greatly reducing the side effects to the normal tissues. On the other hand, once the DNR loaded TiO2 Nps are taken up by tumor cells via endocytotic process, a faster release may occur at lower local pH, i.e, inside the endosome and lysosome of cancer cells ((pH 4.5∼6.5), leading to the significant improvement in cancer treatment efficacy. The DNR- TiO2 nanocomposites as DDS induced the remarkable improvement in the anti-tumor activity, which were demonstrated by the flow cytometry, MTT assay and nuclear DAPI staining. Furthermore, the possible signaling pathway was explored by Western blot. For instance, in human leukemia cells (K562 cells), our observations demonstrated that the DDS could obviously increase the intracellular concentration of DNR and enhance its potential anti-tumor efficiency through inducing apoptosis in a caspase-dependent manner, indicating that DNR-TiO2 nanocomposites could act as an efficient DDS importing DNR into target cancer cells. These findings revealed that such ‘smart’ DNR delivery strategy represent a promising approach in hematologic malignancy therapy. Disclosures:No relevant conflicts of interest to declare.