Comparative Effects of Turkish Rose Hip (Rosa canina L.) Extracts with Different Polarities on the Healthy and Cancer Cell Lines

Reading the Tea Leaves: Anticarcinogenic Properties of (-)-Epigallocatechin-3-Gallate

Reading the Tea Leaves: Anticarcinogenic Properties of (-)-Epigallocatechin-3-Gallate

Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer, characterized by its lack of the human epidermal growth factor receptor-2 (HER-2), the estrogen receptor (ER), and the progesterone receptor (PR). The high prevalence of triple negative tumors in young women is more commonly observed in young African American (AA) women. Currently, the existing targeted therapy is of minimal benefit in TNBCs. Akt and MAPK have been shown to promote cell proliferation in triple negative breast cancer and MAPK expression may be an underlying mechanism contributing to the generation of chemo-resistance in triple-negative breast cancer. Due to major concerns involving the occurrence of side effects and the emergence of drug-resistant cancer cells, there has been growing interest in the use of naturally occurring molecules with chemo-preventive and chemo- therapeutic properties in cancer treatment. Rosehip extracts have been used as dietary supplements to relieve symptoms associated with diarrhea, gastritis, and rheumatoid arthritis and have been shown to prevent cell proliferation in glioblastomas. This study investigated the efficacy of rosehip extracts in preventing cell proliferation in African American triple negative (HCC70, HCC1806) and luminal (HCC1500) breast cancer cell lines. Each of the breast cancer cell lines were treated with rosehip extracts (1mg/mL -25ng/mL) demonstrated a significant decrease in cell proliferation. The rosehip extract-mediated decrease in cell proliferation was equal to or better than the decrease of cell proliferation observed when known inhibitors of the MAPK (U0126, 10 μM) or AKT (LY294002, 20 μM) signaling pathways were utilized. Additionally, pretreatment of these cell lines with these Rosehip extracts (1mg/mL -25ng/mL) selectively decreased AKT, MAPK, p70S6K, and S6 phosphorylation suggesting these extracts prevent AA TNBC cell proliferation by blocking both the MAPK and AKT signaling mechanisms. Results from cell cycle analysis by flow cytometry, western blot analysis, as well as apoptosis studies demonstrate that rosehip extracts inhibit cell proliferation but do not promote apoptosis. Rosehip extracts also have a synergistic effect with Doxorubicin (20µM), a chemotherapeutic agent used to treat patients with breast cancer, in preventing triple negative breast cancer cell proliferation. Taken together these data suggest that rosehip extracts are capable of decreasing cell proliferation in African American triple negative breast cancer cells without promoting apoptosis and demonstrate a synergistic inhibition of cell proliferation with Doxorubicin, and rosehip extracts may serve as an alternative or compliment to current chemotherapeutic regimens for triple negative breast cancer. Citation Format: Patrice Cagle, Tonisha Coburn, Patrick M. Martin. Rosehip (Rosa canina) extracts prevent MAPK and AKT-mediated cell proliferation in African American triple-negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2014-3213

This paper discusses possible mechanisms for the selective effect of weakly ionized nonequilibrium plasma and currents in electrolytes on healthy and cancerous cells in physiological saline in a Petri dish. The interaction with the plasma source leads to a change in osmotic pressure, which affects the electro-mechanical properties of cell membranes in healthy and cancerous cells in different ways. The currents arising in the electrolyte charge the membranes of healthy and cancerous cells to a different potential difference due to the different values of the membranes’ dielectric constants. We hypothesized: 1. The dielectric permeability of cancer cell membranes is lower than that of healthy cells, as is the capacity of a unit of the membrane surface, and therefore, the additional potential difference acquired by the membrane through charging with currents induced in the intercellular electrolyte is greater in cancer cells. This can lead to electroporation of cancer cell membranes, resulting in their apoptosis, but does not affect healthy cells. 2. It is known from the literature that the equilibrium potential differences on the membrane (resting potential) of cancer and healthy cells are noticeably different. Therefore, a change in the potential difference on the membrane due to currents in the extracellular fluid can affect the permeability and transport properties of the membranes. It can also be a reason for the selective effect of the nonequilibrium plasma interaction with healthy and cancerous cells in physiological saline.

Natural products and their bio-active chemical derivatives have historically been invaluable as a source of novel cancer therapeutic agents. In recent decades, almost half of small-molecule New Chemical Entities (NCEs) were originally from natural products and their derivatives. To use natural products as a rich source of new drug development is a potential trend in the worldwide. In this thesis, we found denbinobin and moscatilin, isolated form traditional Chinese medicine - Herba dendrobii, were effective against the human colorectal cancer cells in vitro and in vivo. Cucurbitacin B, isolated from Begonia nantoensis, can induce apoptosis in human colorectal HT-29 cells. In this thesis, we study if these natural components can be new drugs against human colorectal cancer. Denbinobin, a phenanthraquinone derivative isolated from the stems of Ephemerantha lonchophylla, induces apoptosis in human colon cancer HCT-116 cells. Interestingly, denbinobin induces apoptosis via caspase-independent pathway in HCT-116 cells. Denbinobin triggered the translocation of apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. Denbinobin treatment also caused DNA damage, activation of the p53 tumor suppressor gene, and upregulation of numerous downstream effectors. A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of denbinobin. Taken together, our findings suggest that denbinobin induces apoptosis of human colorectal cancer HCT-116 cells via DNA damage and an AIF-mediated pathway. These results indicate that denbinobin has potential as a novel anti-cancer agent. Moscatilin, purified from the stem of the orchid (Dendrobrium loddigesii), induces apoptosis of human colorectal cancer HCT-116 cells. Moscatilin induced arrest of the cell-cycle at G2/M, with an associated decrease in cells at the G1 and an increase of cells at subG1. In addition, Moscatilin inhibited tubulin polymerization, suggesting that that it might bind to tubulins. Moscatilin also induced the phosphorylation of JNK1/2 and c-Jun, and this may contribute to caspase-dependent apoptosis signaling. Moreover, moscatilin induces DNA damage, activation of p53, and activation of the downstream effector p21WAF1/CIP1. A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of moscatilin. In summary, our results suggest that moscatilin induces apoptosis of colorectal HCT-116 cells via tubulin depolymerization and DNA damage stress and that this leads to the activation of JNK and mitochondria-involved intrinsic apoptosis pathway. These results indicate that moscatilin has potential as a novel anti-cancer agent. Cucurbitacin B, a triterpenoid isolated from Begonia nantoensis, induces apoptosis of human colorectal cancer HT-29 cells at low concentration. Cucurbitacin B reduced mitochondrial membrane potential, induced Bcl-xL phosphorylation, and reduced expression of Mcl-1. Cucurbitacin B-induced apoptosis of HT-29 cells was dependent on activation of the extrinsic and intrinsic apoptosis pathways, as indicated by its activation and processing of procaspase-8, -9, -3, -6 and PARP. Cucurbitacin B blocked HT-29 cells at the G1 phase, elevated the levels of p21WAF1/CIP1 and p27KIP1, and suppressed expression of CDK2, CDK4, and cyclin E. Cucurbitacin B significantly activated MAPKs (ERK, p38, JNK) and Akt, but inhibited STAT3 phosphorylation. Taken together, the results of our studies of HT-29 cells provide insight to the cucurbitacin B-induced arrest of cells at the G1 phase and the apoptosis of these cells.

Event Abstract Back to Event Dendritic pro-drug for local and selective treatment of locally advanced breast cancer Nuria Oliva1, Mariana Atilano1, 2*, João Conde1, 3*, Elazer R. Edelman1, 4* and Natalie Artzi1, 5* 1 MIT, IMES, United States 2 IQS, Chemical Engineering, Spain 3 Queen Mary University of London, School of Engineering and Materials Science, United Kingdom 4 BWH, Harvard Medical School, Cardiovascular Division, United States 5 BWH, Harvard Medical School, Department of Medicine, United States Introduction: Systemic neoadjuvant therapy has been established as the preferred therapeutic approach for locally advanced breast cancer, downstaging the disease and preventing mastectomy. However, complications of systemic chemotherapy are devastating. Local therapy would prevent high concentrations of circulating drug and reduce off-target tissue retention. Yet, the means to attain ideal release kinetics and selective uptake remain elusive. We have developed a novel class of biocompatible and biodegradable adhesive materials based on dendrimers and dextrans[1] that can coat the tumor and locally release drugs in a controlled manner. In this work, I have developed and optimized a dendritic pro-drug capable of discerning between healthy and cancer cells. It selectively enters EGFR-overexpressing breast cancer cells through receptor-mediated endocytosis (RME) and releases doxorubicin inside the cells. They will be added to our adhesive hydrogel for local and sustained delivery. Materials and Methods: PAMAM dendrimer generation 5 (Dendritech) was conjugated to EGF-mimicking peptides[2] (Biopolymer Lab, MIT) and also to doxorubicin (Cayman) through a pH-sensitive linker[3]. Cancer cells (MDA-MB-468, ATCC) and healthy mammary epithelial cells (HMEpC, ATCC) were cultured in their recommended media. Cells were treated with 10 uM tagged-dendrimer solutions and uptake was assessed by FACS. Doxorubicin release from the dendrimer was monitored through UV-VIS spectroscopy in PBS (pH 7.4) and acetate buffer (pH 5.5). Cancer and healthy cells were incubated with 10 uM dendritic pro-drug for 48 hours to study cytotoxicity. Results and Discussion: Fluorescence microscopy showed indiscriminate uptake of naked dendrimer independent of cell type (Fig. 1a-b), while dendrimer-peptide uptake was higher in EGFR+ cancer cells than in EGFR- healthy cells (Fig. 1d-e). Blocking of the receptor using an antibody caused abrogation of dendrimer-peptide uptake, but not naked dendrimer (Fig 1c and f). These results were corroborated by FACS (not shown). Taken together, these data prove that our dendrimer conjugates are being uptaken by RME through EGFR, as opposed to diffusion-driven uptake observed for naked dendrimer. Doxorubicin was conjugated to dendrimer-peptide through a cis-aconityl pH-sensitive linker to form the dendritic pro-drug. Incubation of the dendritic pro-drug in PBS (pH 7.4) showed no statistically significant doxorubicin release over 12 hours, while 45% of the drug was released in acetate buffer (pH 5.5) in the first 3 hours (not shown), thus corroborating pH-triggered release. The dendritic pro-drug showed 86% cytotoxicity after 48 hours in cancer cells, while no toxicity was observed in healthy cells (Fig. 1g-k). Conclusions: We have demonstrated that we can successfully develop a dendritic pro-drug that selectively treats EGFR-overexpressing tumors while minimizing side effects in healthy cells surrounding the tumor. The dendritic pro-drug will be incorporated to our adhesive hydrogel and release kinetics and in vivo efficacy will be assessed. Generalization of this platform with peptides targeting other commonly overexpressed growth factor receptors in cancer (FGF2R, VEGFR or PDGFR) will expand the targeting capabilities of our delivery system. We aim to develop a delivery platform capable of treating tumors in a local and selective manner. Marie Curie International Outgoing Fellowship and Funding (FP7-PEOPLE-2013-IOF, Project 626386); Dr. Dong Soo Yun for cryo-TEM assistance at the Peterson Nanotechnology Materials Core Facility; KI MIT Biopolymers Lab; Dr. Glenn Paradis for FACS assistance with Cancer Center Support (FACS core); KI Genomics Core/ MIT BioMicro Center; Timothy E. Cheng for assistance with computation of methods

Currently, over 80% of children with cancer survive due to effective treatments. However, these treatments can also damage the patient’s healthy cells, leading to side effects. Some of the side effects only develop months to years after the end of treatment. These are known as late effects and include cardiovascular diseases, kidney disease, and second cancers. Reducing the number and severity of late effects of cancer treatment is attempted via two approaches. First, the total dose of the most toxic drugs in a treatment regimen might be reduced. To that end, the most toxic drugs first need to be identified. This thesis describes molecular research that investigates which (chemotherapeutic) drugs cause the most DNA damage in healthy cells, and which lead to the development of second cancers. These drugs might be potential candidates for dose reduction. As toxic effects on normal tissues, but especially blood, can be dose limiting, healthy blood cells, and second cancers of the blood were investigated. We found that a specific group of drugs, called topoisomerase inhibitors, induced most of the genetic alterations that were driving the development of the second cancers. Two other groups, thiopurines and platinum compounds, induced the most DNA damage in healthy cells, which also resulted in mutations that drove second cancer development. In addition, platinum compounds prevented rapid expansion of most second cancers. These could only fully develop after the administration of platinum compounds had ended. Only second cancers with a genetic alteration in the TP53 gene could grow out under platinum compound treatment. Second cancers therefore developed earlier in these patients. This might influence the need and timing for screening of these patients. Surprisingly, not only chemotherapeutic drugs, but also an antiviral agent, ganciclovir, proved to be highly mutagenic and in some patients had likely contributed to cancer development. This drug is administered to transplantation recipients that develop a viral reactivation after transplantation. Ganciclovir belongs to the group of nucleoside analog drugs. The DNA damage caused by other drugs in this group was studied, but none caused DNA damage to the same extend as ganciclovir. The second strategy to minimize late effects involves developing therapies that kill the cancer cells more specifically than conventional chemotherapeutic drugs and that thereby reduce the damage to healthy cells. In this thesis, potential therapy targets in childhood Hodgkin lymphoma were investigated. Molecular processes were identified that were consistently active in the cancer and immune cells of this cancer type but not in healthy reference cells. These could therefore be potential targets for developing targeted therapies. The results of the research in this thesis contribute to our understanding of how late effects develop and which drugs are involved in this process. In addition, it will contribute to more efficient development of future clinical research into the reduction of late effects.

Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer, characterized by its lack of the human epidermal growth factor receptor-2, the estrogen receptor, and the progesterone receptor. AKT has been shown to promote cell proliferation and migration in triple negative breast cancer. Currently, the existing targeted therapy is of minimal benefit in TNBCs. Furthermore, adverse side effects and the emergence of drug-resistant cancer cells are of great concern. Natural products have received growing interest in recent years as an alternative medicine with potential anti-oncogenic properties. Rosehip extracts have been used as dietary supplements to relieve symptoms associated with gastrointestinal disorders and arthritis, and in our laboratory it has been shown to prevent cell proliferation in glioblastomas. This study investigated the efficacy of rosehip extracts in preventing proliferation and migration of a triple negative breast cancer cell line (HCC1395). HCC1395 cells treated with rosehip extracts (1mg/mL -25ng/mL) demonstrated a significant decrease in cell proliferation. The observed decrease in cell proliferation was equal to or better than the decrease of cell proliferation observed when inhibitor of the AKT (LY294002, 20 μM) signaling pathway was utilized. Rosehip extracts also demonstrate anti-migratory potential. Additionally, pretreatment of this cell line with rosehip extracts selectively decreased AKT, p70S6K, and S6K phosphorylation suggesting these extracts prevent TNBC cell proliferation and migration by blocking the AKT signaling mechanism. Western blot analysis and apoptosis studies demonstrate that rosehip extracts inhibit cell proliferation without promoting apoptosis, but induce cell cycle arrest. To investigate the potential clinical application of rosehip extracts we examined whether rosehip extracts could enhance the chemotherapeutic properties of Doxorubicin (20µM), a chemotherapeutic agent used to treat breast cancer. Rosehip extracts demonstrated a greater anti-proliferative effect than Doxorubicin alone and is equally effective in combination with Doxorubicin. These data suggest that rosehip extracts are capable of decreasing cell proliferation and migration in a triple negative breast cancer cell line by blocking the AKT signaling pathway and by regulating the cell cycle. Moreover, rosehip extracts demonstrate a greater inhibition of cell proliferation, when given alone and combination with Doxorubicin. This investigation demonstrates that rosehip extracts may serve as either an alternative or complimentary treatment to current chemotherapeutic regimens for triple negative breast cancer. Citation Format: Patrice Cagle, Patrick Martin. Rosehip (Rosa canina) extracts prevent AKT-mediated cell proliferation and migration in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3477. doi:10.1158/1538-7445.AM2017-3477

FdUMP[N] molecules and conjugates are much more effective at inhibiting the proliferation of human tumor cells than is the widely used anticancer drug 5‐fluorouracil (5FU). We have evaluated the inhibition of thymidylate synthase (TS), the extent of DNA damage, cell cycle arrest, and the induction of apoptosis by FdUMP[10] and 5FU in the human colorectal cancer cell line HT29. The magnitude and duration of TS inhibition following exposure of HT29 cells to FdUMP[10] at 1 × 10− 8 M was greater than that which occurred following exposure of these cells to 5FU at 1 × 10− 6 M. FdUMP[10] exposure also resulted in much more extensive DNA damage to HT29 cells than occurred following exposure to 100‐fold higher concentrations of 5FU. Although exposure of HT29 cells to both drugs resulted in S‐phase arrest, more complete accumulation of cells in S‐phase was achieved following FdUMP[10] exposure at much lower drug concentrations. FdUMP[10] was also much more effective at inducing apoptosis in HT29 cells than was 5FU. The results are consistent with FdUMP[10] being much more efficient that 5FU at inducing DNA damage that results in apoptotic cell death in colon cancer cells. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

Significance High platelet counts and advanced stage of ovarian cancer go hand-in-hand in promoting each other in a feed-forward loop that results in blood coagulation and chemotherapy resistance. This results in a high incidence of death due to thrombosis in ovarian cancer patients, and especially in patients with the ovarian clear cell carcinoma histologic type. Objective and Hypothesis We sought to develop an experimental model of the positive interactions between platelets and cancer cells and test the hypothesis that interference with platelet clotting will inhibit this interaction. Approach The effects of platelets on spheroid formation by cancer or healthy epithelial cells were evaluated using a magnetic 3D cancer spheroids assay. The ES2 and MESOV cell lines, which represent clear cell carcinoma and high grade serous histologies, respectively were used for the ovarian cancer cells. Primary human fallopian tube secretory epithelial cell cultures were used to represent healthy cells. The spheroids were imaged and measured using the Optronix GelCount colony counter and evaluated using metabolic viability (MTT) and protein concentration (SRB) assays. The shear-free platelet aggregation assay was performed in the presence of healthy or cancer cells or their conditioned media. Furthermore, we evaluated possible interruption of this feed-forward loop using antiplatelet agents: aspirin—a cyclooxygenase (COX)-1 and -2 inhibitor, celecoxib—a selective COX-2 inhibitor, clopidogrel—an ADP binding inhibitor, dipyridamole—an ADP uptake inhibitor, eptifibatide—a platelet's GP IIb/IIIa inhibitor, and prostacyclin—a platelet aggregation inhibitor. Results Incubation of platelets with cancer spheroids as they are forming decreased the size and density of the spheres in less than 15 minutes of exposure. The MTT assay indicated that condensed spheres were just as live and viable as the spheres that formed in the absence of platelets. Incubation of cancer cells or cancer cells’ conditioned media with platelets caused clumping of platelets in a cancer cells number-dependent manner. Healthy cells’ conditioned media did not cause platelets aggregation. Pre-treating platelets with up to a 1 mM of aspirin, clopidogrel, dipyridamole, and prostacyclin did not prevent cancer cell-induced aggregation, unlike celecoxib, which prevented aggregation at high concentrations, and eptifibatide, which was able to prevent aggregation at low concentrations as 0.1 µM. Conclusions The positive interaction between platelets and cancer cells can be mimicked in co-culture conditions. This interaction appears to involve platelets GPIIb/IIIa binding.

Introduction: Cancer continues to be the second most common cause of death globally. There is currently any proven treatment medication for cancer. Chemotherapy is one of the modern therapeutic approaches that not only kills cancer cells but also has major negative effects on healthy cells. 80% of people worldwide still get their basic medical care from traditional medications, according to data from the World Health Organization. Scientists have created new approaches to treating cancer by combining radiation, chemotherapy, and surgery with a variety of phytochemicals derived from different plant species. This is because plants and active pharmaceutical ingredients derived from plants have minimal to no side effects and high activity. The Euphorbia macroclada, found in different locations of Turkey, has long been utilized by the public for its traditional medicinal properties in treating a range of illnesses. Research conducted in current scientific databases has established that Euphorbia macroclada exhibits significant antioxidant activity. Methods: This study aims to evaluate the potential antiproliferative effect of the dichloromethane extract of the Euphorbia macroclada on HT-29 and HCT-116 cancer cell lines using the in vitro MTT technique. Additionally, the possible genotoxic activity using the Comet assay on human colon cancer cells were also done. Results: The IC50 concentration of Euphorbia macroclada was determined to be 133,835 µg/mL in HCT-116 and 111,215 µg/mL in HT-29 cells after 24 h. Concurrently with the MTT results, it was established that there was an increase in DNA damage in a dose dependent manner than those of control. Conclusion: Following the collection of these data with the Euphorbia macroclada, it has been determined that dichloromethane extract of the Euphorbia macroclada has the potential to serve as a therapeutic agent for the treatment of colon cancer. On the other hand, in vivo and in vitro studies are needed.

ABSTRACTColorectal cancer therapy with 5-fluorouracil (5-Fu) frequently become ineffective due to resistance to this drug; and thus other effective compounds are essential for therapy. It is well-known marine brown seaweeds contain antioxidant compounds the carotenoid fucoxanthin (Fx) and polyphenolic compound phloroglucinol (Ph) which exerted diverse biological activities including antioxidant and anticancer. The aim of this study was to determine the anticancer activities of Fx or Ph alone as well as combination of each chemical with 5-Fu on two human colorectal cancer cell lines (HCT116 and HT29), with comparison to responses in a normal colon cell line (CCD-18Co). Effects of these compounds on cell viability, induction of DNA damage, and cell death were evaluated using MTT assay, comet assay, nuclear condensation assay, and Western blot. 5-Fu decreased cell viability in a concentration-dependent manner in HCT116 and HT29 cells but was not cytotoxic in CCD-18Co cells. 5-Fu induced DNA damage in HCT116 cells with induction of cell death, while no marked effects on DNA damage and cell death were observed in HT29 cells. Fx or Ph alone also reduced cell viability in both cancer cell lines but no apparent cytotoxic effect in CCD-18Co cells, except for Fx at 50 and 100 µM. Diminished cell viability was accompanied by induction of DNA damage (by Fx) and induction of cell death (by Ph). In combination with 5-Fu, Fx at 10 µM (in HCT116 and HT29 cells), and Ph at 300 µM (in HT29 cells) enhanced the cytotoxic effect of 5-Fu; however, no marked cytotoxicity was noted in CCD-18Co cells. Since Fx and Ph alone reduced cancer cell line viability without an effect on normal cells and when in combination enhanced the cytotoxic effect of 5-Fu only in colon cancer cells, these compounds seem promising as anticancer agents.

Purpose: Conventional cytotoxic therapies are still the standard of care for the treatment of many cancers. However, the associated severe side effects, especially damage to normal proliferating cells like stem and progenitor cells, often lead to drug dose reduction, which limits treatment success. Indeed, chemotherapy-induced myelosuppression is manifested by neutropenia, lymphopenia, anemia, and thrombocytopenia. Although growth factors ameliorate this myelosuppression, their efficacy is still suboptimal and lineage specific. Pre-clinical and clinical studies have shown that AsiDNA, a double-stranded (DS) DNA molecule that mimics DS DNA breaks to interfere with DNA repair by over-activating a false DNA damage signaling through DNA-PK and PARP enzymes (decoy agonist), is extremely well tolerated in standalone in mammals. These observations led us to assess the potential of AsiDNA to protect healthy cells from toxicities of several anti-cancer treatments. Experimental design: In vivo, we analyzed the safety profile of AsiDNA during a recent clinical trial (DRIIV-1b/NCT03579628) in combination with platinum-based chemotherapy. In vitro, we used isolated blood cells from healthy donors, and epithelial and fibroblast cells as models to study if AsiDNA could protect healthy cells to chemo- and radiotherapy-induced toxicity. We monitored cell survival, DNA damage (comet assays) and repair (53BP1 and Rad51 foci), DNA-PK (HSP90 and H2AX phosphorylation) and PARP (PARylation) activation, cell cycle modulation and p53 dependency to identify mechanisms underlying the effects of AsiDNA on healthy cells. Results: In vivo, long-term treatment of several patients with carboplatine+/-paclitaxel + AsiDNA showed no increase of chemotherapies toxicities allowing longer periods of disease control and suggesting a protective effect of AsiDNA. In the in vitro models, we showed that AsiDNA enters non dividing and dividing healthy cells as revealed by intracellular PARylation but induces its nuclear target engagement (H2AX and HSP90 phosphorylation) only in dividing cells. Association of AsiDNA to antitumor treatments increased survival of healthy proliferative cells. Interestingly, AsiDNA displayed two distinct mechanisms of protection depending on the origin of the cells: p53-dependent G1/S cell cycle arrest in fibroblasts and epithelial cells, and DNA repair “doping” in hematological cells revealed by higher recruitment of Rad51 and 53BP1 at damage sites in those cells. Enzymatic inhibition and gene editing revealed that hyperactivation of DNA-PK/p53 pathway by AsiDNA might be required for healthy cells preservation. Conclusion: These findings suggest that the combination of AsiDNA with anticancer treatments should provide a means to attenuate therapy-induced toxicity, while showing the well-documented synergy in tumor cells, thus providing an opportunity to increase the therapeutic window. Citation Format: Wael Jdey, Anouk Sesink, Agathe Cohendet, Juliette Rieu, Chloe Doizelet, Vincent Hayes, Pierre-Marie Girard, Marie Dutreix, Judith Greciet. AsiDNA® treatment protects healthy cells from anticancer treatment toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2600.

It was recently shown that it is possible to exploit the nanoparticle shape to selectively target endocytosis pathways found in cancer and not healthy cells. It is important to understand and compare the endocytosis pathways of nanoparticles in both cancer and healthy cells to restrict the healthy cells from taking up anticancer drugs to help reduce the side effects for patients. Here, the clathrin-mediated endocytosis inhibitor, hydroxychloroquine, and the anticancer drug, doxorubicin, are loaded into the same mesoporous silica nanorods. The use of nanorods was found to restrict the uptake by healthy cells but allowed cancer cells to take up the nanorods via the macropinocytosis pathway. Furthermore, it is shown that the nanorods can selectively deliver doxorubicin to the nucleus of breast cancer cells and to the cytoplasm of pancreatic cancer cells. The dual-drug-loaded nanorods were able to selectively kill the breast cancer cells in the presence of healthy breast cells. This study opens exciting possibilities of targeting cancer cells based on the material shape rather than targeting antibodies.

Despite the early promises of magnetic hyperthermia (MH) as a method for treating cancer, it has been stagnating in the past decade. Some of the reasons for the low effectiveness of superparamagnetic nanoparticles (SPIONs) in MH treatments include (a) low uptake in cancer cells; (b) generation of reactive oxygen species that cause harm to the healthy cells; (c) undeveloped targeting potential; and (d) lack of temperature sensitivity between cancer cells and healthy cells. Here we show that healthy cells, including human mesenchymal stem cells (MSCs) and primary mouse kidney and lung fibroblasts, display an unfavorably increased uptake of SPIONs compared to human brain cancer cells (E297 and U87) and mouse osteosarcomas cells (K7M2). Hydroxyapatite (HAP), the mineral component of our bones, may offer a solution to this unfavorably selective SPION delivery. HAP nanoparticles are commended not only for their exceptional biocompatibility but also for the convenience of their use as an intracellular delivery agent. Here we demonstrate that dispersing SPIONs in HAP using a wet synthesis method could increase the uptake in cancer cells and minimize the risk to healthy cells. Specifically, HAP/SPION nanocomposites retain the superparamagnetic nature of SPIONs, increase the uptake ratio between U87 human brain cancer cells and human MSCs versus their SPION counterparts, reduce migration in a primary brain cancer spheroid model compared to the control, reduce brain cancer cell viability compared to the treatment with SPIONs alone, and retain the viability of healthy human MSCs. A functional synergy between the two components of the nanocomposites was established; as a result, the cancer versus healthy cell (U87/MSC) selectivity in terms of both the uptake and the toxicity was higher for the composite than for SPIONs or HAP alone, allowing it to be damaging to cancer cells and harmless to the healthy ones. The analysis of actin cytoskeleton order at the microscale revealed that healthy MSCs and primary cancer cells after the uptake of SPIONs display reduced and increased anisotropy in their cytoskeletal arrangement, respectively. In contrast, the uptake of SPION/HAP nanocomposites increased the cytoskeletal anisotropy of both the healthy MSCs and the primary cancer cells. In spite of the moderate specific magnetization of HAP/SPION nanohybrids, reaching 15 emu/g for the 28.6 wt % SPION-containing composite, the cancer cell treatment in an alternating magnetic field resulted in an intense hyperthermia effect that increased the temperature by ca. 1 °C per minute of exposure and reduced the cell population treated for 30 min by more than 50%, while leaving the control populations unharmed. These findings on nanocomposites of HAP and SPIONs may open a new avenue for cancer therapies that utilize MH.