Drug efflux transporters, in particular P-glycoprotein (Pgp), limit the success of chemotherapy. We previously found that synthetic doxorubicin conjugated with nitric oxide (NO)-releasing group ...overcomes resistance by inducing a NO-mediated inhibition of Pgp. Here we produced the first liposomal formulations of this nitrooxy-doxorubicin decorated with folic acid (FA), termed LNDF, in order to improve their active targeting against Pgp-expressing tumors. Folate was inserted onto liposomes surface using two different methods and the formulations were compared with respect to their technological features and in vitro behavior. By analyzing human and murine breast cancer cells with different expression of FA receptor (FAR) and Pgp, we demonstrated that LNDF are internalized in a FAR-dependent manner and achieve maximal anti-tumor efficacy against FAR-positive/Pgp-positive cells. Upon uptake of LNDF, nitrooxy-doxorubicin was delivered within nucleus, where it induced cell cycle arrest and DNA damages, and mitochondria, where it impaired the mitochondrial energy metabolism and triggered mitochondria-dependent apoptosis. LNDF reduced the growth of FAR-positive/Pgp-positive tumors and prevented tumor formation in mice, whereas doxorubicin and Caelyx® failed. LNDF cardiotoxicity was comparable to Caelyx®. The sensitivity to LNDF was maintained in tumors exposed to repeated cycles of the drug and in cells derived from the exposed tumors, excluding the onset of secondary resistance.
By combining an innovative multitarget cargo drug, conceived to achieve high efficacy against Pgp-expressing cells, and appropriate strategies of liposome formulation and decoration, we produced a therapeutic tool that may represent a significant advancement in the treatment of FAR-positive/Pgp-positive tumors.
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HSP90 is released by cancer cells in the tumor microenvironment where it associates with different co-chaperones generating complexes with specific functions, ranging from folding and activation of ...extracellular clients to the stimulation of cell surface receptors. Emerging data indicate that these functions are essential for tumor growth and progression. The understanding of the exact composition of extracellular HSP90 complexes and the molecular mechanisms at the basis of their functions in the tumor microenvironment may represent the first step to design innovative diagnostic tools and new effective therapies. Here we review the impact of extracellular HSP90 complexes on cancer cell signaling and behavior.
The loss of muscle mass and force characterizes muscle atrophy in several different conditions, which share the expression of atrogenes and the activation of their transcriptional regulators. ...However, attempts to antagonize muscle atrophy development in different experimental contexts by targeting contributors to the atrogene pathway showed partial effects in most cases. Other master regulators might independently contribute to muscle atrophy, as suggested by our recent evidence about the co-requirement of the muscle-specific chaperone protein melusin to inhibit unloading muscle atrophy development. Furthermore, melusin and other muscle mass regulators, such as nNOS, belong to costameres, the macromolecular complexes that connect sarcolemma to myofibrils and to the extracellular matrix, in correspondence with specific sarcomeric sites. Costameres sense a mechanical load and transduce it both as lateral force and biochemical signals. Recent evidence further broadens this classic view, by revealing the crucial participation of costameres in a sarcolemmal "signaling hub" integrating mechanical and humoral stimuli, where mechanical signals are coupled with insulin and/or insulin-like growth factor stimulation to regulate muscle mass. Therefore, this review aims to enucleate available evidence concerning the early involvement of costamere components and additional putative master regulators in the development of major types of muscle atrophy.
It has been shown that growth hormone-releasing hormone (GHRH) reduces cardiomyocyte (CM) apoptosis, prevents ischemia/reperfusion injury, and improves cardiac function in ischemic rat hearts. ...However, it is still not known whether GHRH would be beneficial for life-threatening pathological conditions, like cardiac hypertrophy and heart failure (HF). Thus, we tested the myocardial therapeutic potential of GHRH stimulation in vitro and in vivo, using GHRH or its agonistic analog MR-409. We show that in vitro, GHRH(1-44)NH₂ attenuates phenylephrine-induced hypertrophy in H9c2 cardiac cells, adult rat ventricular myocytes, and human induced pluripotent stem cell-derived CMs, decreasing expression of hypertrophic genes and regulating hypertrophic pathways. Underlying mechanisms included blockade of Gq signaling and its downstream components phospholipase Cβ, protein kinase Cε, calcineurin, and phospholamban. The receptor-dependent effects of GHRH also involved activation of Gαs and cAMP/PKA, and inhibition of increase in exchange protein directly activated by cAMP1 (Epac1). In vivo, MR-409 mitigated cardiac hypertrophy in mice subjected to transverse aortic constriction and improved cardiac function. Moreover, CMs isolated from transverse aortic constriction mice treated with MR-409 showed improved contractility and reversal of sarcolemmal structure. Overall, these results identify GHRH as an antihypertrophic regulator, underlying its therapeutic potential for HF, and suggest possible beneficial use of its analogs for treatment of pathological cardiac hypertrophy.
RATIONALE:Disrupted proteostasis is one major pathological trait that heart failure (HF) shares with other organ proteinopathies, such as Alzheimer and Parkinson diseases. Yet, differently from the ...latter, whether and how cardiac preamyloid oligomers (PAOs) develop in acquired forms of HF is unclear.
OBJECTIVE:We previously reported a rise in monophosphorylated, aggregate-prone desmin in canine and human HF. We now tested whether monophosphorylated desmin acts as the seed nucleating PAOs formation and determined whether positron emission tomography is able to detect myocardial PAOs in nongenetic HF.
METHODS AND RESULTS:Here, we first show that toxic cardiac PAOs accumulate in the myocardium of mice subjected to transverse aortic constriction and that PAOs comigrate with the cytoskeletal protein desmin in this well-established model of acquired HF. We confirm this evidence in cardiac extracts from human ischemic and nonischemic HF. We also demonstrate that Ser31 phosphorylated desmin aggregates extensively in cultured cardiomyocytes. Lastly, we were able to detect the in vivo accumulation of cardiac PAOs using positron emission tomography for the first time in acquired HF.
CONCLUSIONS:Ser31 phosphorylated desmin is a likely candidate seed for the nucleation process leading to cardiac PAOs deposition. Desmin post-translational processing and misfolding constitute a new, attractive avenue for the diagnosis and treatment of the cardiac accumulation of toxic PAOs that can now be measured by positron emission tomography in acquired HF.
Molecular chaperones are stress proteins that allow the correct folding or unfolding as well as the assembly or disassembly of macromolecular cellular components. Changes in expression and ...post-translational modifications of chaperones have been linked to a number of age- and stress-related diseases including cancer, neurodegeneration, and cardiovascular diseases. Redox sensible post-translational modifications, such as S-nitrosylation, glutathionylation and phosphorylation of chaperone proteins have been reported. Redox-dependent regulation of chaperones is likely to be a phenomenon involved in metabolic processes and may represent an adaptive response to several stress conditions, especially within mitochondria, where it impacts cellular bioenergetics. These post-translational modifications might underlie the mechanisms leading to cardioprotection by conditioning maneuvers as well as to ischemia/reperfusion injury. In this review, we discuss this topic and focus on two important aspects of redox-regulated chaperones, namely redox regulation of mitochondrial chaperone function and cardiac protection against ischemia/reperfusion injury.
Treatment of myocardial ischemia/reperfusion (IR) injury is still an unmet clinical need. A large variability of remote ischemic conditioning (RIC) protection has been reported; however, no studies ...have considered the temperature of the ischemic limb. We analyzed the effects of temperature on RIC protection.
Left hind-limbs of anesthetized male mice were immersed in warm (40 °C, warm-RIC) or cold (20 °C, cold-RIC) water and subjected to a RIC protocol (4 × 5 min limb ischemia/reperfusion). In the control groups (warm-CTR or cold-CTR), the limbs underwent thermic conditions only. Isolated hearts underwent 30 min ischemia and 60 min reperfusion. A PI3K-inhibitor, LY294002 (5 µM), was infused in warm-RIC hearts before the IR protocol (warm-RIC LY). Infarct size was evaluated by nitro blue tetrazolium staining and expressed as the percent of risk area.
While cold-RIC did not reduce the infarct size compared to cold-CTR (51 ± 1.62% vs. 54 ± 1.07% of risk area,
=NS), warm-RIC (44 ± 1.13%) significantly reduced the infarct size with respect to either cold-RIC (
<0.001) or warm-CTR (58 ± 1.41%,
<0.0001). LY294002 infusion revealed the PI3K/Akt involvement in the warm-RIC protection. Infarct size reduction was abrogated by LY294002 pretreatment (warm-RIC: 44 ± 1.13% vs. warm-CTR 58 ± 1.41%
<0.0001; vs. warm-RIC LY 54 ± 1.69%
=0.0002).
our study shows a remarkable difference between warm-RIC and cold-RIC in terms of infarct size reduction, supporting a pivotal role for limb temperature in RIC-induced cardioprotection.
Background
Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, ...such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle‐specific integrin‐associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes.
Methods
Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail‐suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno‐associated virus infection. In vivo interference of exogenous melusin with dominant‐negative kinases and other atrophy attenuators (Grp94 cDNA; 7‐nitroindazole) on size of unloaded rat myofibers was also explored.
Results
Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail‐suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of β1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (−31%), compared with controls (−48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin‐expressing adeno‐associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (−33%) by reducing Atrogin‐1 and MurF‐1 transcripts (P ≤ 0.002), despite of a two‐fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co‐transfection with dominant‐negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7‐nitroindazole treatment or with cDNA transfection of the nNOS‐interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy.
Conclusions
Disuse/unloading‐induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy.
Melusin is a chaperone protein selectively expressed in heart and skeletal muscles. Melusin expression levels correlate with cardiac function in pre-clinical models and in human patients with aortic ...stenosis. Indeed, previous studies in several animal models indicated that Melusin plays a broad cardioprotective role in different pathological conditions. Chaperone proteins, besides playing a role in protein folding, are also able to facilitate supramolecular complex formation and conformational changes due to activation/deactivation of signaling molecules. This role sets chaperone proteins as crucial regulators of intracellular signal transduction pathways. In particular Melusin activates AKT and ERK1/2 signaling, protects cardiomyocytes from apoptosis and induces a compensatory hypertrophic response in several pathological conditions. Therefore, selective delivery of the Melusin gene in heart via cardiotropic adenoviral associated virus serotype 9 (AAV9), may represent a new promising gene-therapy approach for different cardiac pathologies.
Chronic or acute insults to the myocardium are responsible for the onset of cardiomyopathy and heart failure. Due to the poor regenerative ability of the human adult heart, the survival of ...cardiomyocytes is a prerequisite to support heart function. Chaperone proteins, by regulating sarcomeric protein folding, function, and turnover in the challenging environment of the beating heart, play a fundamental role in myocardial physiology. Nevertheless, a number of evidences indicate that, under stress conditions or during cell damage, myocardial cells release chaperone proteins that, from the extracellular milieu, play a detrimental function, by perpetuating inflammation and inducing cardiomyocyte apoptosis. Blocking the activity of extracellular chaperones has been proven to have beneficial effects on heart function in preclinical models of myocardial infarction and cardiomyopathy. The application of this approach in combination with tissue engineering strategies may represent a future innovation in cardiac regenerative medicine.
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