Increased erythrophagocytosis induces ferroptosis in red pulp macrophages in a mouse model of transfusion.

Macrophages play important roles in recycling iron derived from the clearance of red blood cells (RBCs). They are also a critically important component of host defense, protecting against invading pathogens. However, the effects on macrophage biology of acutely ingesting large numbers of RBCs are not completely understood. To investigate this issue, we used a mouse model of RBC transfusion and clearance, which mimics the clinical setting. In this model, transfusions of refrigerator storage-damaged (ie, "old") RBCs led to increased erythrophagocytosis by splenic red pulp macrophages (RPMs). This robust erythrophagocytosis induced ferroptosis, an iron-dependent form of cell death, in RPMs. This was accompanied by increases in reactive oxygen species and lipid peroxidation in vivo, which were reduced by treatment in vitro with ferrostatin-1, a ferroptosis inhibitor. Old RBC transfusions also induced RPM-dependent chemokine expression by splenic Ly6Chi monocytes, which signaled Ly6Chi monocyte migration from bone marrow to spleen, where these cells subsequently differentiated into RPMs. The combination of cell division among remaining splenic RPMs, along with the influx of bone marrow-derived Ly6Chi monocytes, suggests that, following RPM depletion induced by robust erythrophagocytosis, there is a coordinated effort to restore homeostasis of the RPM population by local self-maintenance and contributions from circulating monocytes. In conclusion, these findings may be clinically relevant to pathological conditions that can arise as a result of increased erythrophagocytosis, such as transfusion-related immunomodulation and impaired host immunity.

A systematic review of lifespan studies in rodents using stem cell transplantations.

Organismal aging involves the progressive decline in organ function and increased susceptibility to age-associated diseases. Regardless of its origin, cellular aging is consequently reflected at the level of organ and associated systems dysfunction. Aging of stem cell populations within the body and their decreased ability to self-renew, differentiate, and regenerate damaged tissues, is a key contributor to organismal decline. Based on this, supplementing young stem cells may delay tissue aging, improve frailty and extend health and lifespan. This review investigates studies in rodents using stem cell transplantation from either mice or human donors. The aim is to consolidate available information on the efficacy of stem cell therapies in rodent models and provide insights to guide further research efforts. Out of the 21 studies included in this review, the methodology varied significantly including the lifespan measurement. To enable comparison the median lifespan was calculated using WebPlotDigitizer 4.6 if not provided by the literature. A total of 18 out of 21 studies evidenced significant lifespan extension post stem cell transplant, with 7 studies demonstrating benefits in reduced frailty and other aging complications.

Selective ablation of primary and paracrine senescent cells by targeting iron dyshomeostasis.

Senescent cells can spread the senescent phenotype to other cells by secreting senescence-associated secretory phenotype factors. The resulting paracrine senescent cells make a significant contribution to the burden of senescent cell accumulation with age. Previous efforts made to characterize paracrine senescence are unreliable due to analyses being based on mixed populations of senescent and non-senescent cells. Here, we use dipeptidyl peptidase-4 (DPP4) as a surface maker to isolate senescent cells from mixed populations. Using this technique, we enrich the percentage of paracrine senescence from 40% to 85%. We then use this enriched culture to characterize DPP4+ primary and paracrine senescent cells. We observe ferroptosis dysregulation and ferrous iron accumulation as a common phenomenon in both primary and paracrine senescent cells. Finally, we identify ferroptosis induction and ferrous iron-activatable prodrug as a broad-spectrum senolytic approach to ablate multiple types of primary and paracrine senescent cells.

Fluorescence-Based Detection of Ferrous Iron in Senescent Cells.

A major limitation in aging research is the lack of reliable biomarkers to assess phenotypic changes with age or monitor response to antiaging interventions. This study investigates the role of intracellular ferrous iron (Fe2+) as a potential biomarker of senescence. Iron is known to accumulate in various tissues with age and recent studies have demonstrated that its level increases dramatically in senescent cells. The current techniques used to measure the accumulation of iron are cumbersome and only measure total iron not specific isotopes such as the redox reactive Fe2+. It is still to be determined whether the damaging form of iron (Fe2+) is specifically elevated in senescent cells. In this study, we assessed the potential use of a newly discovered Fe2+ reactive probe (SiRhoNox-1) for selective labeling of senescent cells in vitro. For this we have generated various senescent cell models and subjected them to SiRhoNox-1 labeling. Our results indicate that SiRhoNox-1 selectivity labels live senescent cells and was more specific and faster than current staining such as SA-ßGal or a derived fluorescent probe C12FDG. Together these findings suggest that SiRhoNox-1 may serve as a convenient tool to detect senescent cells based on their ferrous iron level.

Role of the proteolytic hierarchy between cathepsin L, cathepsin D and caspase-3 in regulation of cellular susceptibility to apoptosis and autophagy.

The present investigation was undertaken to measure the relative abilities of pro-death versus pro-survival proteases in degrading each other and to determine how this might influence cellular susceptibility to death. For this, we first carried out in vitro experiments in which recombinant pro-death proteases (caspase-3 or cathepsin D) were incubated with the pro-survival protease (cathepsin L) in their respective optimal conditions and determined the effects of these reactions on enzyme integrity and activity. The results indicated that cathepsin L was able to degrade cathepsin D, which in turn cleaves caspase-3, however the later enzyme was unable to degrade any of the cathepsins. The consequences of this proteolytic sequence on cellular ability to undergo apoptosis or other types of cell death were studied in cells subjected to treatment with a specific inhibitor of cathepsin L or the corresponding siRNA. Both treatments resulted in suppression of cellular proliferation and the induction of a cell death with no detectable caspase-3 activation or DNA fragmentation, however, it was associated with increased accumulation of cathepsin D, cellular vaculolization, expression of the mannose-6-phosphate receptor, and the autophagy marker LC3-II, all of which are believed to be associated with autophagy. Genetic manipulations leading either to the gain or loss of cathepsin D expression implicated this enzyme as a key player in the switch from apoptosis to autophagy. Overall, these findings suggest that a hierarchy between pro-survival and pro-death proteases may have important consequences on cell fate.

Transfusional iron overload and intravenous iron infusions modify the mouse gut microbiota similarly to dietary iron.

Iron is essential for both microorganisms and their hosts. Although effects of dietary iron on gut microbiota have been described, the effect of systemic iron administration has yet to be explored. Here, we show that dietary iron, intravenous iron administration, and chronic transfusion in mice increase the availability of iron in the gut. These iron interventions have consistent and reproducible effects on the murine gut microbiota; specifically, relative abundance of the Parabacteroides and Lactobacillus genera negatively correlate with increased iron stores, whereas members of the Clostridia class positively correlate with iron stores regardless of the route of iron administration. Iron levels also affected microbial metabolites, in general, and indoles, in particular, circulating in host plasma and in stool pellets. Taken together, these results suggest that by shifting the balance of the microbiota, clinical interventions that affect iron status have the potential to alter biologically relevant microbial metabolites in the host.

Novel function of the thyroid hormone analog tetraiodothyroacetic acid: a cancer chemosensitizing and anti-cancer agent.

Previous studies from our laboratory have demonstrated that thyroid hormones play a key role in cancer progression. In addition, a deaminated form, tetraiodothyroacetic acid (tetrac), that antagonizes the proliferative action of these hormones was found to possess anti-cancer functions through its ability to inhibit cellular proliferation and angiogenesis. The present study was undertaken to investigate whether tetrac could also suppress the development of drug resistance, known as a causative factor of disease relapse. Tetrac was shown to enhance cellular response in vitro to doxorubicin, etoposide, cisplatin, and trichostatin A in resistant tumor cell lines derived from neuroblastoma, osteosarcoma, and breast cancer. The mechanism of action of tetrac did not involve expression of classical drug resistance genes. However, radiolabeled doxorubicin uptake in cells was enhanced by tetrac, suggesting that one or more export mechanisms for chemotherapeutic agents are inhibited. Tetrac was also found to enhance cellular susceptibility to senescence and apoptosis, suggesting that the agent may target multiple drug resistance mechanisms. Tetrac has previously been shown to inhibit tumor cell proliferation in vitro. In vivo studies reported here revealed that tetrac in a pulsed-dose regimen was effective in suppressing the growth of a doxorubicin-resistant human breast tumor in the nude mouse. In this paradigm, doxorubicin-sensitivity was not restored, indicating that (1) the in vitro restoration of drug sensitivity by tetrac may not correlate with in vivo resistance phenomena and (2) tetrac is an effective chemotherapeutic agent in doxorubicin-resistant cells.

Control of multidrug resistance gene mdr1 and cancer resistance to chemotherapy by the longevity gene sirt1.

Irreversible growth arrest (also called senescence) has emerged recently as a tumor suppressor mechanism and a key determinant of cancer chemotherapy outcome. Previous work from our laboratory suggested that the cellular ability to undergo or to escape senescence dictates its fate to become drug-sensitive or drug-resistant, respectively. In the present study, we made the hypothesis that longevity genes, by virtue of their ability to inhibit senescence, may contribute to the onset of drug resistance. We report that expression of the longevity gene sirt1 increased both at the RNA and protein levels in all the five drug-resistant cell lines tested when compared with their drug-sensitive counterparts. In addition, biopsies from cancer patients treated with chemotherapeutic agents also expressed high levels of this molecule. These changes were specific for sirt1 because the expression of other members of its family was not affected. More importantly, small interfering RNA-mediated down-regulation of sirt1 significantly reversed the resistance phenotype and reduced expression of the multidrug resistance molecule P-glycoprotein. This was further confirmed by ectopic overexpression of sirt1, which induced expression of P-glycoprotein and rendered cells resistant to doxorubicin. Collectively, these findings uncovered a novel function for the longevity gene sirt1 as a potential target for diagnosis and/or treatment of cancer resistance to chemotherapy. They also describe a proof of principle that signaling pathways implicated in longevity may share similarities with those leading to development of drug resistance in cancer.

Prolonged red cell storage before transfusion increases extravascular hemolysis.

BACKGROUND: Some countries have limited the maximum allowable storage duration for red cells to 5 weeks before transfusion. In the US, red blood cells can be stored for up to 6 weeks, but randomized trials have not assessed the effects of this final week of storage on clinical outcomes. METHODS: Sixty healthy adult volunteers were randomized to a single standard, autologous, leukoreduced, packed red cell transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage (n = 10 per group). 51-Chromium posttransfusion red cell recovery studies were performed and laboratory parameters measured before and at defined times after transfusion. RESULTS: Extravascular hemolysis after transfusion progressively increased with increasing storage time (P < 0.001 for linear trend in the AUC of serum indirect bilirubin and iron levels). Longer storage duration was associated with decreasing posttransfusion red cell recovery (P = 0.002), decreasing elevations in hematocrit (P = 0.02), and increasing serum ferritin (P < 0.0001). After 6 weeks of refrigerated storage, transfusion was followed by increases in AUC for serum iron (P < 0.01), transferrin saturation (P < 0.001), and nontransferrin-bound iron (P < 0.001) as compared with transfusion after 1 to 5 weeks of storage. CONCLUSIONS: After 6 weeks of refrigerated storage, transfusion of autologous red cells to healthy human volunteers increased extravascular hemolysis, saturated serum transferrin, and produced circulating nontransferrin-bound iron. These outcomes, associated with increased risks of harm, provide evidence that the maximal allowable red cell storage duration should be reduced to the minimum sustainable by the blood supply, with 35 days as an attainable goal.REGISTRATION. ClinicalTrials.gov NCT02087514. FUNDING: NIH grant HL115557 and UL1 TR000040.

Identification of midkine as a mediator for intercellular transfer of drug resistance.

Resistance to cytotoxic agents is a major limitation for their clinical use to treat human cancers. Tumors become resistant to chemotherapy when a subset of cells undergoes molecular changes leading to overexpression of drug transport proteins, alterations in drug-target interactions or reduced ability to commit apoptosis. However, such changes may not be sufficient to explain why both resistant and nonresistant cells survive drug's action in tumors that ultimately become drug resistant. We hypothesized that, in such tumors, a cytoprotective relationship may exist between drug-resistant and neighboring drug-sensitive cells. The present study addresses the possibility that drug-resistant cells secrete in their culture medium factors able to protect sensitive cells from drug toxicity. A survival molecule, midkine, was identified by cDNA array to be expressed only in drug-resistant cells. Midkine-enriched fractions obtained by affinity chromatography exert a significant cytoprotective effect against doxorubicin in the wild-type drug-sensitive cells. Moreover, transfection of these cells with the midkine gene caused a decreased response to doxorubicin. The underlying mechanism of this cytoprotection appeared to imply activation of the Akt pathway and inhibition of drug-induced proliferation arrest as well as apoptotic cell death. These findings provide evidence for the existence of intercellular cytoprotective signals such as the one mediated by midkine, originating from cells with acquired drug resistance to protect neighboring drug-sensitive cells and thus contribute to development of resistance to chemotherapy.

Senescence-initiated reversal of drug resistance: specific role of cathepsin L.

The present study was undertaken to verify whether induction of senescence could be sufficient to reverse drug resistance and, if so, to determine the underlying mechanism(s). Our findings indicated that cotreatment of drug-resistant neuroblastoma cells with doxorubicin, at sublethal concentrations, in combination with the pan-caspase inhibitor, Q-VD-OPH, elicited a strong reduction of cell viability that occurred in a caspase-independent manner. This was accompanied by the appearance of a senescence phenotype, as evidenced by increased p21/WAF1 expression and senescence-associated beta-galactosidase activity. Experiments using specific inhibitors of major cellular proteases other than caspases have shown that inhibition of cathepsin L, but not proteasome or cathepsin B, was responsible for the senescence-initiated reversal of drug resistance. This phenomenon appeared to be general because it was valid for other drugs and drug-resistant cell lines. A nonchemical approach, through cell transfection with cathepsin L small interfering RNA, also strongly reversed drug resistance. Further investigation of the underlying mechanism revealed that cathepsin L inhibition resulted in the alteration of intracellular drug distribution. In addition, in vitro experiments have demonstrated that p21/WAF1 is a substrate for cathepsin L, suggesting that inhibition of this enzyme may result in p21/WAF1 stabilization and its increased accumulation. All together, these findings suggest that cathepsin L inhibition in drug-resistant cells facilitates induction of senescence and reversal of drug resistance. This may represent the basis for a novel function of cathepsin L as a cell survival molecule responsible for initiation of resistance to chemotherapy.

Caspase inhibition switches doxorubicin-induced apoptosis to senescence.

The inhibition of apoptosis is generally believed to be a major determinant of resistance to chemotherapy. However, recent findings have shown that caspase inhibitors do not protect cancer cells from death by cytotoxic agents, but may switch drug-induced apoptosis to an alternative 'default death'. The primary goals of this study were to determine the major characteristics of the 'default death' and the mechanism by which this switch is activated. For this purpose, we first investigated putative cell death modes induced by doxorubicin. Molecular markers associated with these death modes were utilized to identify the default death resulting from the inhibition of apoptosis. Our findings demonstrated that doxorubicin induced at least three distinct types of cell death, senescence, apoptosis and a type of necrosis, which were concentration dependent. Specific molecular markers such as p21/WAF1, activated caspase-3 and activated Akt were associated with these death modes. The pan-caspase inhibitor (Q-VD-OPH) greatly reduced doxorubicin-induced caspase-3 activation but did not protect cells against drug toxicity. The combination of doxorubicin and Q-VD-OPH caused an increased expression of p21/WAF1 and senescence -associated -beta-galactosidase activity, but did not alter Akt activation. Collectively, these findings suggest that the inhibition of apoptosis may lead to an increased expression of cell cycle inhibitors and cellular senescence.

Targeting senescence pathways to reverse drug resistance in cancer.

Irreversible proliferation arrest (also called senescence) has emerged recently as a drug-responsive program able to influence the outcome of cancer chemotherapy. Since the drug amounts required for induction of proliferation arrest are much lower than those necessitated for induction of cell death, forcing cancer cells to undergo senescence may represent a less aggressive approach to control tumor progression. However, to achieve a long-standing control of proliferation, the ability of cancer cells to escape senescence and become drug resistant must be inhibited. Therefore, a clear understanding of the mechanisms that govern drug-induced senescence is critical and can lead to discovery of novel approaches to suppress drug resistance. The present review discusses the relevance of senescence in response to chemotherapy and the onset of drug resistance development. Particular emphasis is directed toward the utilization of findings from the field of research on aging, that can be applied to induction of senescence in cancer cells and reversal of their drug resistance phenotype. Proof of principle for this relationship is represented by the identification of inhibitors of aging associated proteases such as the proteasome and cathepsin L as novel and potent cancer drug resistance reversing agents.

Doxorubicin resistant neuroblastoma cells secrete factors that activate AKT and attenuate cytotoxicity in drug-sensitive cells.

The present study aims to verify the hypothesis that tumor cells with acquired resistance to chemotherapy may secrete survival factors and thus, participate in the protection of drug-sensitive cells against drug toxicity. A human neuroblastoma cell line, SK-N-SH, and its doxorubicin resistant derivative, RDOX6, have been used. Conditioned medium from RDOX6 cells attenuated the cytotoxic response of SK-N-SH cells to doxorubicin. This protective effect was associated with activation of the Akt survival pathway and inhibition of caspase-3. These data indicate that secretion, by drug-resistant cells, of factors that activate anti-apoptotic pathways in drug-sensitive cells, may constitute a novel mechanism of drug resistance.

Modulation of Growth Factor response in brain tumors by complex carbohydrates.

It is well established that growth factors and their receptors are overexpressed in brain tumors and play a key role in tumor cell proliferation. Glycoconjugate molecules expressed at the plasma membrane of mammalian cells have been also reported to be associated with tumor progression. Growth factor receptors and glycoconjugate molecules are able to interact with each other and this interaction usually results in modulation of growth factor receptor mediated signaling and the biological function of the cell. This review addresses the expression of both growth factor receptors and glycoconjugates molecules in the brain and brain tumors. The mechanism by which these two entities interact with each other and the consequences of their interaction on the biological function of tumor cell are also discussed. Glycoconjugate molecules seems to act more specifically on growth factor receptor signaling pathways than most of tyrosine kinase inhibitors. The use of glycoconjugates or their derivatives may represent a new approach to modulate the proliferative behavior of tumors that overexpress growth factor receptors such as brain tumors.

Identification, mechanism of action, and antitumor activity of a small molecule inhibitor of hippo, TGF-ß, and Wnt signaling pathways.

Embryonic signaling pathways, in particular those mediated by Wnt and TGF-ß, are known to play key roles in tumor progression through the induction of epithelial-mesenchymal transition (EMT). Their simultaneous targeting could therefore represent a desirable anticancer strategy. On the basis of recent findings that both Wnt and TGF-ß-associated pathways are regulated by Hippo signaling in mammalian cells, we reasoned that targeting the latter would be more effective in inhibiting EMT. In a search for such inhibitors, we identified a small molecule (C19) with remarkable inhibitory activity not only against Hippo, but also against Wnt and TGF-ß pathways. C19 inhibited cancer cell migration, proliferation, and resistance to doxorubicin in vitro, and exerted strong antitumor activity in a mouse tumor model. Mechanistically, C19 induced GSK3-ß-mediated degradation of the Hippo transducer TAZ, through activation of the Hippo kinases Mst/Lats and the tumor suppressor kinase AMPK upstream of the degradation complex. Overall, this study identified C19 as a multi-EMT pathway inhibitor with a unique mechanism of action. The findings that both AMPK and Mst/Lats mediate the antitumor activity of C19 shed light on a potential cross-talk between metabolic and organ size control pathways in regulating cancer progression. By simultaneously targeting these two pathways, C19 may represent a new type of agents to suppress cancer progression and/or its recurrence.

Histone acetylation-mediated regulation of the Hippo pathway.

The Hippo pathway is a signaling cascade recently found to play a key role in tumorigenesis therefore understanding the mechanisms that regulate it should open new opportunities for cancer treatment. Available data indicate that this pathway is controlled by signals from cell-cell junctions however the potential role of nuclear regulation has not yet been described. Here we set out to verify this possibility and define putative mechanism(s) by which it might occur. By using a luciferase reporter of the Hippo pathway, we measured the effects of different nuclear targeting drugs and found that chromatin-modifying agents, and to a lesser extent certain DNA damaging drugs, strongly induced activity of the reporter. This effect was not mediated by upstream core components (i.e. Mst, Lats) of the Hippo pathway, but through enhanced levels of the Hippo transducer TAZ. Investigation of the underlying mechanism led to the finding that cancer cell exposure to histone deacetylase inhibitors induced secretion of growth factors and cytokines, which in turn activate Akt and inhibit the GSK3 beta associated protein degradation complex in drug-affected as well as in their neighboring cells. Consequently, expression of EMT genes, cell migration and resistance to therapy were induced. These processes were suppressed by using pyrvinium, a recently described small molecule activator of the GSK 3 beta associated degradation complex. Overall, these findings shed light on a previously unrecognized phenomenon by which certain anti-cancer agents may paradoxically promote tumor progression by facilitating stabilization of the Hippo transducer TAZ and inducing cancer cell migration and resistance to therapy. Pharmacological targeting of the GSK3 beta associated degradation complex may thus represent a unique approach to treat cancer.

OT-404, multi-targeted anti-cancer agent affecting tumor proliferation, chemo-resistance, and angiogenesis.

There is a need for a comprehensive anti-cancer strategy that simultaneously targets abnormal proliferation, angiogenesis rates, and development of chemotherapy resistance. We have identified a small molecule, OT-404, that effectively inhibited proliferation and angiogenesis of either chemo-sensitive or -resistant human cancer cells and enhanced cancer cell sensitivity to different chemotherapy. In vivo studies of human tumor xenografts in nude mice showed that OT-404, used alone or encapsulated into nanoparticles, inhibited the growth of doxorubicin-resistant breast cancer MCF-7 by more than 80%, and by 95% when combined with doxorubicin. These findings provide evidence for the potential of OT-404 in cancer management.

Role of the beta catenin destruction complex in mediating chemotherapy-induced senescence-associated secretory phenotype.

Cellular senescence is considered as a tumor suppressive mechanism. Recent evidence indicates however that senescent cells secrete various growth factors and cytokines, some of which may paradoxically promote cancer progression. This phenomenon termed senescence-associated secretory phenotype (SASP) must be inhibited in order for anti-proliferative agents to be effective. The present study was designed to determine whether the ß-catenin destruction complex (BCDC), known to integrate the action of various growth factors and cytokines, would represent a suitable target to inhibit the activity of SASP components. For this, we carried out experiments to determine the effect of drug-induced senescence on secretion of SASP, ß-catenin transactivation, and the relationship between these processes. Moreover, genetic and pharmacological approaches were used to define the implication of BCDC in mediating the effects of SASP components on cell migration and resistance to drugs. The findings indicate that drug-induced senescence was associated with expression of various Wnt ligands in addition to previously known SASP components. Beta catenin transactivation and expression of genes implicated in epithelial-mesenchymal transition (EMT) also increased in response to drug-induced SASP. These effects were prevented by Pyrvinium, a recently described activator of BCDC. Pyrvinium also suppressed the effects of SASP on cell migration and resistance to doxorubicin. Together, these findings provide insights on the potential role of BCDC in mediating the effects of drug-induced SASP on cancer cell invasion and resistance to therapy, and suggest that targeting this pathway may represent an effective approach to enhance the activity of current and prospective anti-cancer therapeutics.

Stress resistant human embryonic stem cells as a potential source for the identification of novel cancer stem cell markers.

Cancer stem cells are known for their inherent resistance to therapy. Here we investigated whether normal stem cells with acquired resistance to stress can be used to identify novel markers of cancer stem cells. For this, we generated a human embryonic stem cell line resistant to Trichostatin A and analyzed changes in its gene expression. The resistant cells over-expressed various genes associated with tumor aggressiveness, many of which are also expressed in the CD133+ glioma cancer stem cells. These findings suggest that stress-resistant stem cells generated in vitro may be useful for the discovery of novel markers of cancer stem cells.