Chitosan gold nanoparticles induce cell death in HeLa and MCF-7 cells through reactive oxygen species production Ana Carolina Martínez-Torres, Diana G Zarate-Triviño, Helen Yarimet Lorenzo-Anota, Andrea Ávila-Ávila, Carolina Rodríguez-Abrego, Cristina Rodríguez-Padilla Laboratory of Immunology and Virology, Faculty of Biological Sciences, Autonomous University of Nuevo Leon, Monterrey, Mexico Background: Nanotechnology has gained important interest, especially in the development of new therapies; the application of gold nanoparticles (AuNPs) in the treatment and detection of diseases is a growing trend in this field. As cancer represents a serious health problem around the world, AuNPs are studied as potential drugs or drug carriers for anticancer agents. Recent studies show that AuNPs stabilized with chitosan (CH) possess interesting biological activities, including potential antitumor effects that could be selective to cancer cells.Materials and methods: In this study, we synthesized sodium citrate-AuNPs and CH-capped AuNPs of 3–10 nm, and analyzed their cytotoxicity in cervical (HeLa) and breast (MCF-7) cancer cells, and in peripheral blood mononuclear cells (PBMCs). Then, we evaluated the clonogenic potential, cell cycle, nuclear alterations, caspase dependence, and reactive oxygen species (ROS) production in HeLa and MCF-7 cells after chitosan gold nanoparticles (CH-AuNPs) exposure.Results: Our data showed that CH-AuNPs are cytotoxic in a dose-dependent manner in the cancer cell lines tested, while they induce low cytotoxicity in PBMCs. Sodium citrate gold nanoparticles did not show cytotoxic effects. In both HeLa and MCF-7 cell lines, CH-AuNPs inhibit clonogenic potential without inducing cell cycle arrest or nuclear alterations. The cell death mechanism is specific for the type of cancer cell line tested, as it depends on caspase activation in HeLa cells, whereas it is caspase independent in MCF-7 cells. In all cases, ROS production is mandatory for cell death induction by CH-AuNPs, as ROS inhibition with N-acetyl cysteine inhibits cell death.Conclusion: Our results show that CH-AuNPs are selective for HeLa and MCF-7 cancer cells, rather than normal PBMCs, and that ROS production seems to be a conserved feature of the cell death mechanism induced by CH-AuNPs. These results improve the knowledge of CH-AuNPs and open the way to the design of new pharmacological strategies using these agents against cancer. Keywords: AuNPs, cancer, PBMC, nuclear alterations, cell cycle, ROS

Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5⁺ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides. In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2⁺-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease. Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL. Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia (cancer of the white blood cells). It accounts for about a third of all adult leukemias, and about one in 200 people will develop CLL during their lifetime, usually in old age. White blood cells (including T and B lymphocytes) are made by the bone marrow (the soft center of certain bones in the body) and help the body fight infections. Leukemia begins when a bone marrow cell acquires genetic changes (mutations and chromosomal abnormalities) that make it grow and develop abnormally. Over time, the abnormal cells accumulate in the bone marrow, blood, and lymphoid organs (lymph nodes and spleen) (malignant B lymphocytes accumulate in the case of CLL) and stop the marrow producing healthy blood cells. CLL does not usually cause any symptoms during its early stages and is often diagnosed as a result of a routine blood test. Some patients, however, develop painless swellings in the lymph nodes in the neck, armpit, or groin. Other symptoms of CLL include tiredness, frequent infections, and weight loss. Treatments for CLL, which are not given unless symptoms develop, include chemotherapy, immunotherapy, and targeted therapies, which attack cancer cells without harming normal cells. CLL develops very slowly, and people with CLL usually live for many years after their diagnosis. However, there is no cure for CLL, and treatment fails in up to a quarter of patients because resistance to chemotherapy develops. The outlook (prognosis) is particularly bad for patients whose cancer cells contain a dysfunctional TP53 gene (TP53 encodes a protein that normally regulates cell division and cell death). Although some new therapies have recently been approved in the US and Europe, additional approaches to the treatment of CLL are still needed. One potential target for new treatments is the cell surface receptor CD47. In CLL cells, activation of CD47 by a small fragment (peptide) of thrombospondin-1 (a protein that binds to CD47) called 4N1K rapidly induces caspase-independent programmed cell death (PCD), a type of cell death in which the cell uses specialized cellular machinery to kill itself. (PCD normally controls cell numbers and eliminates cells that threaten an animal’s survival, but cancer cells often resist PCD.) Unfortunately, peptides are usually degraded quickly in the human body, which limits their usefulness as therapeutic agents. Here, therefore, the researchers investigate whether PKHB1, a variant of 4N1K designed to be stable in the body (a “serum-stable” agonist peptide), might provide a new approach to the treatment of CLL. The researchers tested the ability of PKHB1 to kill malignant CLL B lymphocytes grown from blood samples collected from 80 patients with CLL. PKHB1 efficiently killed these cells, including those from individuals with a dysfunctional TP53 gene but, importantly, did not kill either normal T and B lymphocytes collected from healthy donors or the residual normal T and B lymphocytes present in the patient samples. Genetic and molecular analyses undertaken by the researchers indicated that the differential response of normal and leukemic B lymphocytes to PKHB1 was the result of sustained activation of a protein called phospholipase C gamma-1 (PLCγ1) in the malignant CLL B lymphocytes. Other experiments showed that PLCγ1 activation drove the induction of a new PCD pathway (a calcium-mediated, caspase-independent pathway) that was not down-regulated by survival stimuli provided by the lymphocyte microenvironment. Finally, the researchers report that injection of PKHB1 reduced the tumor burden in a mouse model of CLL. Taken together, these findings suggest that serum-stable CD47 agonist peptides represent a potential new treatment for CLL that could circumvent the problem of chemotherapy resistance and eliminate malignant CLL B lymphocytes while sparing residual normal B and T lymphocytes. These findings also demonstrate that PKHB1, the serum-stable CD47 agonist peptide tested in this study, directly induces a novel PCD pathway, thereby overcoming the innate avoidance of apoptosis by CLL cells. Before peptide-based strategies for the treatment of CLL can enter clinical trials, however, further studies are needed to develop peptides with increased stability and increased ability to bind to and activate CD47. Moreover, because the mouse CLL model used here incompletely recreates the development of CLL in people, the effectiveness and safety of this pioneer approach needs to be tested much more rigorously in animals before any clinical trials are initiated. Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001796.

T‐cell acute lymphoblastic leukemia (T‐ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed TSP1‐derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first‐described serum‐stable CD47‐agonist peptide) on CEM and MOLT‐4 human cell lines (T‐ALL) and on one T‐murine tumor lymphoblast cell‐line (L5178Y‐R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non‐tumor cells after CD47 activation. In vivo, we determined that PKHB1‐treatment in mice bearing L5178Y‐R cell line, increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze if CD47 activation induced immunogenic cell death (ICD), we evaluated damage‐associated molecular patterns (DAMPs) exposure (Calreticulin, CRT) and release (ATP, HSP70, HSP90, HMGB1, CRT). Furthermore, we administered a prophylactic antitumor vaccination, determining immunologic memory. Our data indicate that PKHB1 induces caspase‐independent and calcium‐dependent cell death in leukemic cells while sparing non‐tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMPs release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Altogether our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.

Apoptosis is nowadays what comes first to your scientist mind when someone mentions cellular suicide. However this is not the sole form of programmed cell death and many other alternative or atypical pathways have now been described. These pathways are indeed rather preferred to apoptosis in some instances based on tissue origin, cell type or development stage of the target cell. In this review, we describe many different programmed cell death subtypes according to their characteristics. Discrete, brutal, final or singular cell death pathways all participate in the elimination of unwanted, damaged or dangerous cells in organisms hence contributing to our knowledge of this particular feature of living beings: dying! Through description of anoikis, necroptosis, entosis, netosis, pyroptosis or ferroptosis, we have no choice but to realize that programmed cell death comes in many flavors.

Background: Nanotechnology proposes the use of gold nanoparticles (AuNPs) for drug delivery, diagnosis, and treatment of cancer. Leukemia is a type of hematopoietic cancer that results from the malignant transformation of white blood cells. Chitosan-coated AuNPs (CH-AuNPs) are cell death inductors in HeLa and MCF-7 cancer cells without affecting peripheral blood mononuclear cells (PBMC). Considering the selectivity and versatile cytotoxicity of CH-AuNPs, we evaluated whether their selectivity is due to the cell lineage or the characteristics of the cancer cells, by assessing its cytotoxicity in leukemic cells. Moreover, we further examined the cell death mechanism and assessed the implication of nuclear damage, autophagosome formation, and the cell death mechanism induced in leukemic cells. Materials and methods: We synthesized CH-AuNPs by chemical methods and analyzed their cell death capacity in a T-acute lymphocytic leukemia cell line (CEM), in a chronic myeloid leukemia cell line (K562), and in healthy cells from the same lineage (PBMC and bone marrow, BM, cells). Then, we assessed ROS generation and mitochondrial and nuclear damage. Finally, we evaluated whether cell death occurred by autophagy, apoptosis, or necroptosis, and the role of ROS in this mechanism. Results: We found that CH-AuNPs did not affect PBMC and BM cells, whereas they are cytotoxic in a dose-dependent manner in leukemic cells. ROS production leads to mitochondrial and nuclear damage, and cell death. We found that CH-AuNPs induce apoptosis in CEM and necroptosis in K562, both undergoing autophagy as a pro-survival mechanism. Conclusion: CH-AuNPs are selective cell death inductors in hematologic cancer cells, without affecting their healthy counterparts. Cell death induced by CH-AuNPs is independent of the cancer cell type; however, its mechanism is different depending on the type of leukemic cells.