Low Concentrations Of Doxorubicin Research Articles

Occupational Exposure during Intraperitoneal Pressurized Aerosol Chemotherapy Using Doxorubicin in a Pig Model

BackgroundThis study evaluated occupational exposure levels of doxorubicin in healthcare workers performing rotational intraperitoneal pressurized aerosol chemotherapy (PIPAC) procedures. MethodsAll samples were collected during PIPAC procedures applying doxorubicin to an experimental animal model (pigs). All procedures were applied to seven pigs, each for approximately 44 min. Surface samples (n = 51) were obtained from substances contaminating the PIPAC devices, surrounding objects, and protective equipment. Airborne samples were also collected around the operating table (n = 39). All samples were analyzed using ultra-high performance liquid chromatography-mass spectrometry. ResultsAmong the surface samples, doxorubicin was detected in only five samples (9.8%) that were directly exposed to antineoplastic drug aerosols in the abdominal cavity originating from PIPAC devices. The telescopes showed concentrations of 0.48–5.44 ng/cm2 and the trocar showed 0.98 ng/cm2 in the region where the spraying nozzles were inserted. The syringe line connector showed a maximum concentration of 181.07 ng/cm2, following a leakage. Contamination was not detected on the surgeons' gloves or shoes. Objects surrounding the operating table, including tables, operating lights, entrance doors, and trocar holders, were found to be uncontaminated. All air samples collected at locations where healthcare workers performed procedures were found to be uncontaminated. ConclusionsMost air and surface samples were uncontaminated or showed very low doxorubicin concentrations during PIPAC procedures. However, there remains a potential for leakage, in which case dermal exposure may occur. Safety protocols related to leakage accidents, selection of appropriate protective equipment, and the use of disposable devices are necessary to prevent occupational exposure.

DDEL-13. ULTRASOUND-ENHANCED DELIVERY OF LIPOSOMAL DOXORUBICIN ACROSS THE BLOOD BRAIN BARRIER INDUCES AN IFN-GPHENOTYPE IN MICROGLIA, MACROPHAGES, AND T CELLS AND IMPROVES RESPONSE TO PD-1 BLOCKADE IN GLIOMAS

Abstract INTRODUCTION Given the limited drug penetration across the blood-brain barrier (BBB), the therapeutic potential of new and existing therapies has not been fully exploited for the benefit of glioblastoma (GBM) patients. METHODS Here we employed a novel drug delivery technology based on low-intensity pulsed ultrasound combined with intravenous microbubbles (LIPU/MB) that temporarily opens the BBB to deliver liposomal doxorubicin (DOX) and anti-PD-1 therapy (aPD-1) in mouse glioma models and 3 recurrent GBM patients. Immunological variables were evaluated in tumor and immune cells as well as efficacy in glioma-bearing mice treated with DOX delivered by LIPU. These included measurement of HLA ABC and HLA DR protein expression by tumor cells, microglia, and macrophages and IFN-g production by glioma-associated microglia and macrophages in mouse and human tumors. We also assessed efficacy of LIPU/MB enhanced combination therapy in glioma-bearing mice. RESULTS Upregulation of HLA ABC and HLA DR was observed in GBM cell lines at low concentrations of DOX. Tumor cells from GBM patients treated with DOX, aPD-1 and LIPU/MB showed increased expression of HLA ABC and HLA DR compared to paired pretreatment samples. In both mice and humans, LIPU/MB liposomal DOX increased absolute brain drug concentrations and elicited a specific IFN-g phenotype and MHC I expression in glioma-associated microglia and macrophages in mice and humans. Furthermore, LIPU/MB-mediated BBB opening increased brain concentrations of aPD-1 in mice and in peritumoral regions of GBM patients. Combined treatment with liposomal DOX and aPD-1 delivered with LIPU/MB resulted in long-term survival of glioma-bearing mice that relied on the activity of CD8+ T cells for its efficacy. CONCLUSIONS Overall, this translational study demonstrates the utility of LIPU/MB to stimulate intracranial immune responses in the context of treatment with DOX and aPD-1 for gliomas.

Chitosan-Boric Acid Scaffolds for Doxorubicin Delivery in the Osteosarcoma Treatment.

Biologically compatible chitosan-based scaffolds have been considered a promising platform for tissue regeneration, tumor treatment, and targeted drug delivery. Chitosan-based scaffolds can be utilized as pH-sensitive drug carriers with targeted drug delivery resulting in less invasive tumor treatments. Further improvement with bioactive ions, such as borate ions, can result in the dual functionality of chitosan carriers provided by simultaneous antitumor efficacy and tissue regeneration. Here, boric acid-containing crosslinked chitosan scaffolds were prepared as delivery systems of doxorubicin, a chemotherapy drug used in the treatment of osteosarcoma. The encapsulation of boric acid was indicated by FTIR spectroscopy, while the ICP-MS analysis indicated the rapid release of boron in phosphate buffer (pH 6.0) and phosphate-buffered saline solution (pH 7.4). The obtained chitosan-boric acid scaffolds exhibit a highly porous and interconnected structure responsible for high swelling capacity, while enzymatic degradation indicated good scaffolds stability during four weeks of incubation at pH 6.0 and 7.4. Furthermore, the release of doxorubicin investigated in phosphate buffers indicated lower doxorubicin concentrations at pH 7.4 with respect to pH 6.0. Finally, the cytotoxicity of prepared doxorubicin-encapsulated scaffolds was evaluated on human sarcoma cells indicating the scaffolds' potential as cytostatic agents.

The effect of ciprofloxacin on doxorubicin cytotoxic activity in the acquired resistance to doxorubicin in DU145 prostate carcinoma cells.

The present study aimed to assess the influence of ciprofloxacin (CIP) against the doxorubicin (DOX)-resistant androgen-independent prostate cancer DU145 cells. The DOX-resistant DU145 (DU145/DOX20) cells were established by exposing DU145 cells to the increasing concentrations of DOX. The antiproliferative effect of CIP was examined through employing MTT, colony formation, and 3D culture assays. DU145/DOX20 cells exhibited a twofold higher IC50 value for DOX, an increased ABCB1 transporter activity, and some morphological changes accompanied by a decrease in spheroid size, adhesive and migration potential compared to DU145 cells. CIP (5 and 25µgmL-1) resulted in a higher reduction in the viability of DU145/DOX20 cells than in DU145 cells. DU145/DOX20 cells were more resistant to CIP in 3D culture compared to the 2D one. No spheroid formation was observed for DU145/DOX20 cells treated with DOX and CIP combination. CIP and DOX, alone or in combination, significantly reduced the growth of DU145 spheroids. CIP in combination with 20nM DOX prevented the colony formation of DU145 cells. The clonogenicity of DU145/DOX20 cells could not be estimated due to their low adhesive potential. CIP alone caused a significant reduction in the migration of DU145 cells and resulted in a more severe decrease in the wound closure ability of DOX-exposed ones. We identified that CIP enhanced DOX sensitivity in DU145 and DU145/DOX20 cells. This study suggested the co-delivery of low concentrations of CIP and DOX may be a promising strategy in treating the DOX-resistant and -sensitive hormone-refractory prostate cancer.

Multi-target tracheloside and doxorubicin combined treatment of lung adenocarcinoma

Chemotherapy is one of the main methods for malignant lung cancer treatment. However, the side effects of chemotherapy drugs are serious and it is prone to drug resistance. Therefore, multi-drug combination chemotherapy is popular in lung cancer treatment. This study found that tracheloside (TCS) was a novel inhibitor of TMEM16A which was specific high expressed in lung cancer tissues. TCS concentration dependently inhibited TMEM16A with an IC50 of 3.09 ± 0.21 μM. It inhibited lung cancer cells proliferation, migration, and induced cells apoptosis targeting TMEM16A. In addition, molecular docking combined with site-directed mutagenesis confirmed that the binding sites of TCS to TMEM16A were S387, E623, E624. Subsequently, multi-target combined drug administration was conducted based on the different drug targets of TCS and doxorubicin (DOX). Both in vitro and in vivo experiments indicated that the combined administration of low concentration of TCS and DOX achieved satisfactory anticancer effect, and it offset the side effects caused by high concentration of DOX. Therefore, TCS is a safe and efficient anticancer lead compound which can enhance the effect of DOX.

Stepwise responsive carboxymethyl chitosan-based nanoplatform for effective drug-resistant breast cancer suppression

Efficient delivery systems for co-delivery of P-glycoprotein (P-gp) inhibitors and chemotherapeutic drugs are essential for inhibiting multi-drug resistance (MDR) breast cancers. Herein, we present a multi-functional carboxymethyl chitosan (CMC) based core-shell nanoplatform to co-deliver MDR1 gene-silenced small interfering RNA (siMDR1) and doxorubicin (DOX) for optimal combinatorial therapy. DOX is linked to CMC through a disulfide bond to model redox-responsive prodrug (CMC-DOX) as the inner core. siMDR1 is encapsulated in oligoethylenimine (OEI), which is electrostatically adsorbed on CMC-DOX as the pH-responsive sheddable shielding shell. AS1411 aptamer and GALA peptide functionalised hyaluronic acid (AHA/GHA) are provided on the surface for tumour-targeting and endo/lysosomal escape. The nanoplatform could stepwise release payloads with acid/redox triggered fashion. AHA effectively improves nanoplatform intracellular uptake and tumour accumulation. GHA facilitates cargos escape from endo/lysosomes to cytoplasm. The multi-functional nanoplatform provides 86.3 ± 2.2% siMDR1 gene silencing and significantly downregulates P-gp expression. Moreover, it ensures 55.7 ± 1.6% MCF-7/ADR cell apoptosis at a low concentration of DOX (30 μg/mL) in vitro and performs synergistic therapeutic effects suppressing tumour growth in vivo. Overall, the multi-functional CMC-based biopolymers can be efficient siRNA/drug co-delivery carriers for cancer chemotherapy.

Cytotoxic and Radiosensitizing Effects of Folic Acid-Conjugated Gold Nanoparticles and Doxorubicin on Colorectal Cancer Cells.

Purpose: Radiotherapy is one of the most important therapeutic options used to treat cancers. Radiation effects can be improved using nanoparticles and chemotherapeutic drugs as radiosensitizing agents. The aim of the present study was to evaluate the effects of folic acid-conjugated gold nanoparticles (GNP-F) in combination with doxorubicin (DOX) and x-Ray irradiation in colorectal cancer (CRC) cell line (HT-29). Methods: The cell viability assay (WST-1) was performed to study the cytotoxic effects of different concentrations of DOX and GNP-F after 24 and 48 hours treatments. Then, the effects of the GNP-F, X-Ray irradiation, and DOX drug in single and combined treatments were examined after 24 and 48 hours treatment with effective doses. Likewise, the caspase 3 gene expression ratio and the caspase 3 activity were assessed after 48 h treatment. Moreover, the malondialdehyde (MDA) level was determined in treated and untreated cells. Results: When GNP-F (at a concentration of 70 μM) was combined with X-ray irradiation (2 Gy) and DOX drug, induced more cytotoxic effects compared to the control group. The results of cell viability assay showed that GNP-F + X-Ray in combination with a low concentration of DOX (0.25 × IC50) enhanced the cytotoxic effects of cells compared to related single treatments. Caspase 3 gene expression ratio and caspase 3 activity increased in double and triple combination treatments in comparison with the single groups. Moreover, the MDA level increased in triple combination compared to the single treatments. Conclusion: Our findings confirmed the potential anti-cancer effects of the GNP-F and DOX in combination with X-Ray irradiation in CRC cells.

Additive effects of resveratrol and doxorubicin on bladder cancer cells.

The treatment of bladder cancer remains a challenge in clinical practice. Different chemotherapeutic protocols can be used; however, it is common to observe tumor recurrence and secondary effects that result in toxicity. Doxorubicin (DOX), one of the most effective anticancer agents used to treat bladder cancer, can cause chronic cardiotoxicity, limiting its use in clinical practice. Resveratrol (RES), a natural product with potential antitumor activity against bladder cancer, is associated with rapid metabolism and low bioavailability and needs to be combined with chemotherapeutic drugs to improve its use. Our study aimed to assess the therapeutic effect of a low concentration of DOX (2 µM) in combination with RES (150, 200 and 250 µM) on two bladder cancer cell lines. We investigated the mechanism of interaction between the drugs by performing cytotoxicity, clonogenic, oxidative stress, cell migration, cell morphology and nuclear division index (NDI) assays. Cytotoxicity evaluation revealed an additive interaction between RES and DOX for both cell lines. Additionally, the results of cell colony formation, oxidative stress, cell migration, cell morphology and NDI assays showed that a combination of DOX and RES was more effective than RES or DOX alone. In conclusion, a low concentration of DOX combined with RES could potentiate the antitumor effects of the drugs on bladder cancer cells, thus overcoming the secondary effects caused by DOX and the low bioavailability of resveratrol.

Hydrophilic Polyhedral Oligomeric Silsesquioxane, POSS(OH)32, as a Complexing Nanocarrier for Doxorubicin and Daunorubicin.

A novel strategy, recently developed by us, to use polyhedral oligomeric silsesquioxanes (POSS) as an anti-cancer drug carrier is presented. Anthracycline:POSS complexes were prepared by simple co-addition of doxorubicin (DOX) or daunorubicin (DAU) with hydrophilic POSS(OH)32. Co-delivery of POSS and anthracyclines led to higher anti-cancer activity towards HeLa (cervical cancer endothelial) and MCF-7 (human breast adenocarcinoma) cell lines. The obtained supramolecular hybrid complexes were characterised by nuclear magnetic resonance (NMR) spectroscopy (nuclear Overhauser effect spectroscopy [NOESY] and homonuclear correlation spectroscopy [COSY]), Fourier transform infrared spectroscopy (FTIR), and dynamic light scattering (DLS). The two-dimensional (2D) NOESY spectra of the complexes showed the cross-correlation peaks for hydroxyl groups of POSS (~4.3–4.8 ppm) with OH groups of DOX and DAU. FTIR showed that hydroxyl group of POSS can interact with amine and hydroxyl groups of DOX and DAU. The viability of HeLa and MCF-7 was analysed with the MTT assay to evaluate the cytotoxicity of free DOX and DAU and the relevant complexes with POSS at different molar ratios. At a low DOX concentration (2.5 µM), for molar ratios 1:1, 1:4, and 1:8 (POSS:DOX), the complexes showed two and three times higher cytotoxicity towards HeLa and MCF-7 cells, respectively, than DOX itself after both 24- and 48-h incubation. The 1 µM concentration for a 1:4 POSS:DOX molecular ratio and the 2.5 µM concentration for all complexes were more toxic towards MCF-7 cells than free DOX after 48-h incubation. In the case of POSS:DAU complexes, there was higher toxicity than that of free drug after 48-h incubation. It can be concluded that the formation of non-covalent complexes increases toxicity of anthracycline drugs towards Hela and MCF-7 cells. The novel complexes are inexpensive to prepare and more effective than free drugs at low systemic toxicity.

Platelet Activation Is Triggered by Factors Secreted by Senescent Endothelial HMEC-1 Cells In Vitro.

Aging is one of the main risk factors for the development of chronic diseases, with both the vascular endothelium and platelets becoming functionally altered. Cellular senescence is a form of permanent cell cycle arrest initially described in primary cells propagated in vitro, although it can also be induced by anticancer drugs and other stressful stimuli. Attesting for the complexity of the senescent phenotype, senescent cells synthesize and secrete a wide variety of bioactive molecules. This “senescence-associated secretory phenotype” (SASP) endows senescent cells with the ability to modify the tissue microenvironment in ways that may be relevant to the development of various physiological and pathological processes. So far, however, the direct role of factors secreted by senescent endothelial cells on platelet function remains unknown. In the present work, we explore the effects of SASP factors derived from senescent endothelial cells on platelet function. To this end, we took advantage of a model in which immortalized endothelial cells (HMEC-1) were induced to senesce following exposure to doxorubicin, a chemotherapeutic drug widely used in the clinic. Our results indicate that (1) low concentrations of doxorubicin induce senescence in HMEC-1 cells; (2) senescent HMEC-1 cells upregulate the expression of selected components of the SASP and (3) the media conditioned by senescent endothelial cells are capable of inducing platelet activation and aggregation. These results suggest that factors secreted by senescent endothelial cells in vivo could have a relevant role in the platelet activation observed in the elderly or in patients undergoing therapeutic stress.

Non-porous phosphonated ionic silica nanospheres as nanocarriers for efficient intracellular delivery of doxorubicin

A novel nanoscale drug delivery system based on monodispersed non-porous ionic silica nanospheres (NISNs) decorated with phosphonated active sides homogeneously distributed all over their surface, was developed. Doxorubicin (DOX), a well-known antitumor drug, was successfully loaded on the surface of the silica nanoparticles via electrostatic interactions. The final drug vehicle possesses excellent solubility, while enhancing significantly the efficacy of the drug. The administration of DOX-loaded NISNs against two aggressive DOX-resistant human prostate adenocarcinoma cell lines DU145 and PC3 leads to increased medicinal efficacy with extremely low DOX concentrations (0.1 μM) that could significantly reduce DOX side effects. In addition, NISNs was found to be non-toxic. The efficient cellular uptake of NISNs_DOX was confirmed by flow cytometry analysis and visualized by confocal microscopy. The translocation of DOX inside cells drastically changed, when DOX was bound to NISNs nanoparticles. Specifically, DOX loaded to NISNs nanoparticles is preferentially localized in the cytosol and significant efficacy was observed due to slow controlled release of DOX to the nucleus. The results reported in this work strongly support the potential utilization of NISNs derivatives as non-toxic nanocarriers for high loading efficiency and intracellular delivery of therapeutic drugs.

Doxorubicin-verapamil dual loaded PLGA nanoparticles for overcoming P-glycoprotein mediated resistance in cancer: Effect of verapamil concentration

Despite of various technical advances in cancer treatment, chemotherapy is still the promising option. One of the most obstacles in cancer chemotherapy process is drug resistance due to over expression of drug efflux pumps such as P-glycoprotein (P-gp). So to overcome these problems, P-gp inhibitors such as verapamil (VER) have been examined in different studies. Designing drug delivery systems to achieve concurrent delivery of chemotherapeutic agent and efflux pump inhibitors may improve therapeutic efficacy. Nanoparticles are potential carriers to encapsulate two or more drugs in a single system. Doxorubicin (DOX), an anticancer agent is an anthracycline antibiotic that blocks DNA synthesis and transcription. In present study co-encapsulated DOX and VER in PLGA nanoparticles were prepared and optimized using Design expert® software. The selected nanoparticles showed suitable diameter (407 nm), polydispersity index (0.68) and a stable zeta potential (−31mv) with acceptable DOX and VER loading%. Nanoparticles released 39% of DOX and 32% VER within 72 h. Cell cytotoxicity studies on MCF-7 cell line indicated that DOX-VER loaded nanoparticles presented higher cytotoxicity and lower IC50 at lower DOX and VER concentrations after 24 and 72 h compared to free-DOX. Results suggest that DOX-VER loaded PLGA NPs may potentially be useful carriers for co-encapsulation of DOX and VER with sufficient cytotoxic effect at lower DOX concentration which may decrease their side effects. Indeed, in the low and safe VER concentration, side effects of both drugs will be reduced. It means VER higher efficacy was in lower DOX concentration and VER to DOX mole ratios.

Evaluation of the combined effects of doxorubicin and bortezomib on the human acute lymphoblastic leukemia cell line

Abstract Increasing numbers of oncological patients and growing drug resistance ensure that new methods of cancer treatment are intensively sought. Combining drugs for a synergistic effect is one of several possible ways to mitigate this problem. This leads to reducing the effective drug dose and the occurrence of side effects. Doxorubicin (DOX) is an antineoplastic agent that has several mechanisms of action. DOX intercalates between base pairs of DNA helix, inhibits topoisomerase II and also forms reactive oxygen species. Bortezomib (BZT) is an antitumor agent belonging to the group of proteasome inhibitors. It has been observed that BZT triggers an oxidative stress response in vitro and in vivo. Accumulation of oxidatively damaged proteins and the simultaneously blocking of the proteasome can be very damaging to the tumour cell. For this reason, the aim of the study was to assess the potentially synergistic effect of DOX and BZT on human acute lymphoblastic leukemia (ALL). In the work, the cells were treated with both agents and their combinations and the effect was evaluated on the basis of morphological assessment, MTT assay and level of reduced glutathione measurement. The study has shown that on acute lymphoblastic leukemia cells, synergistic effects came about in the combination of 1nM BZT with a wide range of concentrations of DOX. Herein, the visible, coactive effect of DOX and BZT was observed on oxidative stress levels. This phenomenon can be essential in blunting the possibility of rapid manifestation of resistance seen in BZT monotherapy. In addition, the needed very low concentrations of DOX reduce the risk of therapy side effect.

The P-glycoprotein inhibitor diltiazem-like 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one inhibits esterase activity and H3 histone acetylation

With the aim to reduce multidrug resistance several molecules were synthesized and tested for their ability to inhibit ATP-binding cassette (ABC) proteins, which are responsible for drugs transport out from cells. The compound 8-(4-chlorophenyl)-5-methyl-8-[(2Z)-pent-2-en-1-yloxy]-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one namely 2c, is structurally related to the myocardial-calcium-channel-modulator diltiazem and is considered one of the most efficient P-glycoprotein inhibitors, able to induce apoptosis at low concentrations of doxorubicin in multidrug resistant ovarian cells. In this study experiments were carried out to evaluate other biological activities of compound 2c. We verified the ability of 2c to inhibit ABC transporters do not involved in drug resistance and considering the inhibitory effect of diltiazem on recombinant human carboxylesterase, we observed its inhibitory effect on the esterase activity. Our findings demonstrated that 2c exhibits broad-spectrum activity as ABC transporters inhibitor being able to inhibit ABCC6, a protein belonging to the ABC family although poorly involved in drug resistance. 2c also inhibits cell esterase activity, acetylcholine esterase activity in vitro and cell histone H3 acetylation according to its structural homology with some known HAT inhibitors. The results obtained provide new knowledge on the biological activities of 2c and represent useful information when it is used as an inhibitor of drug resistance.

A Triphenylphosphonium-Functionalized Mitochondriotropic Nanocarrier for Efficient Co-Delivery of Doxorubicin and Chloroquine and Enhanced Antineoplastic Activity.

Drug delivery systems that target subcellular organelles and, in particular, mitochondria are considered to have great potential in treating disorders that are associated with mitochondrial dysfunction, including cancer or neurodegenerative diseases. To this end, a novel hyperbranched mitochondriotropic nanocarrier was developed for the efficient co-delivery of two different (both in chemical and pharmacological terms) bioactive compounds. The carrier is based on hyperbranched poly(ethyleneimine) functionalized with triphenylphosphonium groups that forms ~100 nm diameter nanoparticles in aqueous media and can encapsulate doxorubicin (DOX), a well-known anti-cancer drug, and chloroquine (CQ), a known chemosensitizer with arising potential in anticancer medication. The anticancer activity of this system against two aggressive DOX-resistant human prostate adenocarcinoma cell lines and in in vivo animal studies was assessed. The co-administration of encapsulated DOX and CQ leads to improved cell proliferation inhibition at extremely low DOX concentrations (0.25 μΜ). In vivo experiments against DU145 human prostate cancer cells grafted on immunodeficient mice resulted in tumor growth arrest during the three-week administration period and no pervasive side effects. The findings put forward the potential of such targeted low dose combination treatments as a therapeutic scheme with minimal adverse effects.

Multiple emulsions as effective platforms for controlled anti-cancer drug delivery.

Developing pH-responsive multiple emulsion platforms for effective glioblastoma multiforme therapy with reduced toxicity, a drug release study and modeling. Cancer cell line: U87 MG, multiple emulsions with pH-responsive biopolymer and encapsulated doxorubicin (DOX); preparation of multiple emulsions in a Couette-Taylor flow biocontactor, in vitro release study of DOX (fluorescence intensity analysis), in vitro cytotoxicity study (alamarBlue cell viability assay) and numerical simulation of DOX release rates. The multiple emulsions offered a high DOX encapsulation efficiency (97.4 ± 1%) and pH modulated release rates of a drug. Multiple emulsions with a low concentration of DOX (0.02 μM) exhibited broadly advanced cell (U87 MG) cytotoxicity than free DOX solution used at the same concentration. Emulsion platforms could be explored for potential delivery of chemotherapeutics in glioblastoma multiforme therapy.

SL2B aptamer and folic acid dual-targeting DNA nanostructures for synergic biological effect with chemotherapy to combat colorectal cancer.

DNA nanostructures prepared by self-assembly possess good stability, high biocompatibility, and low immunogenicity as drug delivery vehicles. In this work, DNA tetrahedron (TD) was constructed and modified with SL2B aptamer (S) and folic acid (F). TD possessed a small diameter (~6 nm) and entered into the nucleus quickly. SL2B aptamer can inhibit cancer cell growth by disturbing vascular endothelial growth factor/Notch signaling pathways. To explore the effect of SL2B number on colorectal cancer inhibition, SL2B multimers (dimer, trimer, and tetramer) were constructed by functionalization of TD with different numbers of SL2B. One SL2B per TD was the most efficient anticancer strategy and showed significantly better anticancer efficacy than SL2B, probably due to the enhanced stability of SL2B by TD. Doxorubicin (DOX) is a potent anticancer agent that can intercalate into DNA double strands. Results showed that TD could facilitate DOX entrance into the nucleus and the intracellular delivery of DOX was further enhanced by functionalization of SL2B and F. DOX-intercalated TD modified with two F and two S (DOX@TD-2F2S) could cause sufficient HT-29 cell inhibition at a much lower DOX concentration. In sum, DOX@TD-2F2S exhibited a synergic anticancer biological effect with chemotherapy and can be a promising strategy for treating colorectal cancer.

Abstract P3-03-16: Molecular iodine impairs chemoresistance and invasive mechanisms, enhances doxorubicin retention and induces downregulation of CD44+/CD24+ and E-Cad+/Vim+ subpopulations in MCF-7 cells resistant model

Abstract The main problem in the treatment of breast cancer is the ability of the cells to become chemoresistant and metastatic. Both components involved complex mechanisms based on alterations of apoptosis, the cell cycle and drug metabolism, and its correlates with the cancer stem cell phenotype and/or epithelial-mesenchymal transition. In this study, Doxorubicin (DOX), a member of the anthracycline family used as first-line therapy for several cancers, given chronically to establish a low-dose DOX-resistant mammary cancer model to analyze the effect of molecular iodine (I2) on the chemoresistant and invasive mechanisms. I2 exerts an antitumor effect on different types of iodine-capturing neoplasm through its oxidant/antioxidant properties and formation of iodolipids. Wild-type breast carcinoma cells (MCF-7/W) were treated chronically with low DOX concentration (10 nM) to generate a clinically relevant model of DOX-resistance (MCF-7/D). After treatment with I2 and DOX alone or in combination, the proliferation assay (Trypan Blue exclusion), RT-qPCR expression (p21, Bax, Bcl-2, Survivin, PPARγ, E-Cadherin and Vimentin), flow cytometry (CD24, CD44, E-Cadherin, and Vimentin) and DOX retention (DOX fluorescence) were performed. Tumorogenic capacity was analyzed by xenograft induction in Foxn1 nu/nu mice. MCF-7/D was established after 30 days of treatment when the culture showed a proliferation rate similar to that of MCF-7/W. These DOX-resistant cells also showed increases in p21, Bcl-2 and MDR-1 expression. Supplementation with 200 μM I2 exerted similar effects in both cell lines: it decreased the proliferation rate by approximately 40%, and I2 co-administration with DOX significantly increased the inhibitory effect (~ 60%) and also increased apoptosis (Bax/Bcl-2 index), principally by inhibiting Bcl-2 expression. The inhibition by I2+DOX was also accompanied by impaired MDR-1 induction as well as by a significant increase in PPARγ expression. All these changes could be attributed to enhanced DOX retention and differential down selection of CD44+/CD24+ and E-Cad+/Vim+ subpopulations. I2+DOX selected cells (CD44-/CD24- and E-Cad+/Vim-) showed a weak induction of xenografts, indicating that the iodine supplements reversing the tumorogenic capacity of MCF-7/D. In conclusion, molecular iodine reduces the drug resistance and invasive capacity of mammary cancer cells exposed to DOX and represents an anti-chemoresistance agent with clinical potential. This work was partially supported by grant PAPIIT-UNAM IN201516. We thank Juana Cardenas for technical support. Alexander Bontempo is a student of Programa de Doctorado en Ciencias Biomédicas, UNAM and receives a fellowship from CONACYT 262489. Citation Format: Carmen A, Alexander B, Brenda U-V, Evangelina D-G, Ángel Luis R. Molecular iodine impairs chemoresistance and invasive mechanisms, enhances doxorubicin retention and induces downregulation of CD44+/CD24+ and E-Cad+/Vim+ subpopulations in MCF-7 cells resistant model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-16.

Notch signaling is important for epithelial-mesenchymal transition induced by low concentrations of doxorubicin in osteosarcoma cell lines.

Osteosarcoma is an aggressive pediatric tumor affecting growing bones that typically occurs in adolescents and young adults. Although advances in treatment have been made in recent years, a high proportion of patients relapse due to metastases, which are frequently resistant to chemotherapy and pose a significant threat to long-term survival. Previous studies have demonstrated that the epithelial-mesenchymal transition (EMT) is associated with cancer occurrence and metastasis, and our previous study demonstrated the occurrence of EMT in osteosarcoma. Notch is a regulator involved in several cellular processes, and previous studies have identified that the Notch signaling pathway may be activated during chemotherapy. However, whether chemotherapy affects the EMT remains to be elucidated. To address this issue, in the present study osteosarcoma cells were exposed to sublethal doses of doxorubicin, which resulted in upregulation of the expression of genes in the Notch signaling pathway and its target genes. Furthermore, doxorubicin treatment promoted mesenchymal-like properties and enhanced the migration and invasion of cells. In addition, treatment with the selective γ-secretase inhibitor DAPT was able to prevent the EMT and inhibit the in vitro migration of osteosarcoma cells. The results of this study suggested that there is a significant correlation between the Notch signaling pathway and the EMT, and revealed an underlying molecular mechanism by which doxorubicin may induce the EMT via Notch signaling.

Heterologous expression and functional characterization of phytaspase, a caspase-like plant protease

Following the cloning and expression of tobacco (Nicotiana tabacum) phytaspase gene in Escherichia coli BL21, the recombinant protease was purified by affinity chromatography for further characterization. Circular dichroism (CD) spectroscopy and in silico analysis revealed structural similarities of recombinant phytaspase with other plant serine-proteases. Molecular docking studies showed favourable binding of synthetic peptide substrate for caspase 8 (Ac-VETD-AMC) to the reactive pocket of recombinant phytaspase indicating its potential in assessing functional activity of recombinant phytaspase. In silico findings were supported by caspase 8-like activity of purified phytaspase demonstrated in vitro. The Michaelis constant (KM) and specificity constant (kcat/KM) of phytaspase for hydrolyzing Ac-VETD-AMC were found to be 1.587μM and 4.67×103M−1min−1, respectively. Transient expression of phytaspase in lung epithelial adenocarcinoma cells (A549) resulted in reduced IC50 value of doxorubicin. This is the first report of functional expression of mature phytaspase in bacterial system as well as its transfection to sensitize A549 cells at lower doxorubicin concentration.