Incubation of androgen-impartial PC3 and DU145 and androgen-dependent LNCaP prostate most cancers cells with .000100 M KML001 for seventy two h diminished cell viability in a dose-dependent way. LNCaP cells were being much more delicate than PC3 or DU145 Cy3 NHS Estercells (Fig one Table 1).To figure out whether or not treatment with KML001 (10 M) induces apoptosis, the ultrastructure of KML001-addressed PC3 cells was analyzed by electron microscopy. PC3 cells addressed with KML001 showed standard apoptotic people, which include a smaller nucleus, much more concentrated cytoplasm, crumpled nuclear membrane, and chromosomes condensed into a semilunar shape with attachments to the nuclear and cellular membranes. In addition, apoptotic bodies were being noticed when the chromatin condensed and ruptured to the nuclear margin. Equivalent results were noticed in DU145 and LNCaP cells treated with IC50 concentrations of KML001 (5 M and 1 M, respectively) (Fig two).To investigate the mechanism of KML001-induced cell dying, we carried out annexin V stream cytometry assays. Treatment method of these cells with KML001 yielded cells good for annexin V staining only and cells beneficial for annexin V and PI staining. These outcomes suggest that KML001 induced cell demise via each apoptosis and necrosis (Fig 3A). We also found that exposure of prostate cancer cells to KML001 resulted in a time- and dose-dependent raise in the cleavage of PARP, as nicely as the activation of procaspases-three, which mediates intrinsic apoptosis (Fig 3B). To affirm the part of apoptosis in the KML001-induced demise of prostate cancer cells, we handled the cells with the caspase inhibitor Z-VAD-FMK. We located that addition of twenty M Z-VAD-FMK diminished the activation of procaspases-3 in all prostate cancer mobile strains (information not demonstrated), supporting the speculation that KML001 induced apoptosis in prostate most cancers cells.To establish whether or not remedy with KML001 (ten M) induces autophagy, the ultrastructure of PC3 taken care of cells was examined by electron microscopy. We noticed autophagic vacuoles, including autophagosomes or autolysosomes, in KML001-treated PC3 cells, as nicely as in DU145 and LNCaP cells addressed with their respective IC50 concentrations of KML001 (Fig 2). Prostate cancer cells exposed to KML001 have been examined by Western blotting, making use of an anti-LC3 antibody that recognizes both equally forms of LC3. We found that expression of LC3 and/or conversion to LC3-II enhanced in a time- and dose-dependent method, relative to untreated cells (Fig 4A). To validate the purpose of autophagy in the KML001-induced loss of life of prostate cancer cells, we dealt with the cells with the autophagy inhibitor three-MA. We discovered that addition of two mM 3-MA decreased the expression of LC3-II in all prostate most cancers mobile lines (Fig 4B). In addition, addition of one mM three-MA also attenuated KML-induced mobile dying in all prostate cancer mobile traces (Fig 4C). Taken collectively, these results assist hypothesis that KML001 induced autophagy-mediated mobile demise in prostate most cancers cells.Induction of apoptosis by KML001 in prostate most cancers cells. (A) FACS assessment of annexin V/PI staining. Results display early apoptosis, described as annexin V-beneficial and PI-detrimental cells, and late apoptosis, defined as annexin V-optimistic and PI-good cells. Effects were being expressed as signifies SD of a few independent experiments. (B) Western blot evaluation of the time- and dose-dependent cleavage of PARP and activation of procaspase-3.KML001 induced dose-dependent ROS accumulation in all three prostate most cancers mobile traces (Fig 5A). We also examined the outcomes of the antioxidant NAC on KML001-induced apoptosis and autophagy. We discovered that remedy of all three prostate most cancers cell traces with one mM NAC lowered the expression of LC3-II and the proteolysis of PARP (Fig 5B). In addition, addition induction of autophagy by KML001 in prostate most cancers cells. (A) Western blot examination of the time- and dose-dependent conversion of LC3-I to-II. (B) Inhibition by three-MA of KML001-induced conversion of LC3 in prostate cancer cells. (C) Cells were exposed to ten M (PC3), 5 M (DU145), or two M (LNCaP) KML001 in the existence or absence of one mM three-MA for seventy two h. Benefits ended up expressed as indicates SD of 3 unbiased experiments. p < 0.05 by one-way ANOVA of 1 mM NAC also attenuated KML-induced cell death in all prostate cancer cell lines (Fig 5C). These findings provide further evidence that exposure of prostate cancer cells to KML001 activated both apoptosis and autophagy via oxidative stress.We subcutaneously injected nude mice with 5 106 DU145 cells to test the effects of KML001 on androgen-independent prostate cancer cells in vivo. After the tumor size reached 100 mm3, we administered KML001 (2.5 or 10 mg/kg/d) to them for 4 weeks. In the DU145 xenograft model, tumor growth was inhibited with KML001 as compared to vehicle (Fig 6A). But KML001 treatment had no effect on body weight of mice (Fig 6B) regulation of autophagy and apoptosis by ROS. All 3 prostate cancer cells were treated with the indicated concentration of KML001 in the absence or presence of 5 mM NAC for 24 h. (A) KML001 induces dose-dependent ROS (blue) accumulation. Cells were stained with DCFH-DA and washed with PBS. More than three fields in each cell were observed by fluorescence microscope (200, and representative images are shown. (B) NAC inhibition of KML001-induced conversion of LC and caspase activation in prostate cancer cells. (C) Cells were exposed to 10 M (PC3), 5 M (DU145), or 2 M (LNCaP) KML001 in the presence or absence of 1 mM NAC for 72 h. Results were expressed as means SD of three independent experiments. p < 0.05 by oneway ANOVA.KML001 treatment has (A) a growth inhibitory effect on DU145 prostate cancer cells in mice, and (B) no effect on body weight of mice. Vehicle and KML001 group mice orally received saline and KML001 (2.5 or 10 mg/kg/d) for 4 weeks, respectively. p < 0.05 vs. vehicle.In the next experiment, we determined the effect of KML001 on proliferation, apoptosis, and autophagy in tumor tissues harvested from animals of different treatment groups. In immunohistochemical staining of the proliferation marker Ki-67, significant decreases in Ki67 immunostaining were observed in KML001-treated tumors than in vehicle-treated tumors (Fig 7A). In TUNEL assay, significant greater apoptotic cells were observed in KML001-treated tumors than in vehicle-treated tumors (Fig 7B). In Western blot analysis, we found that expression of LC3 and/or conversion to LC3-II increased in a dose-dependent manner, relative to vehicle-treated tumors (Fig 7C).Prostate cancer is one of the leading causes of death among men in the United States [19]. Although aggressive efforts toward early detection and treatment decrease mortality rate for prostate cancer, prostate cancer is the second most common cause of cancer death among men yet. Although early-stage prostate cancer requires androgen for growth and thus responds to androgen deprivation therapy, the disease may progress to androgen-independence and may be unresponsive to androgen ablation [20]. Docetaxel-based chemotherapies have shown palliative and survival benefits for patients with castration-resistant prostate cancer, but treatment results are generally unsatisfactory with a median survival time of only 16 to 18 months [21,22]. So it is not only important to develop effective ways of preventing or slowing the formation of castration-resistant prostate cancer, but also to develop new chemotherapeutic agents for the treatment of castration-resistant prostate cancer. Since ATO was shown to have dramatic effects in patients with acute promyelocytic leukemia [1,2], this agent has also been tested in patients with solid tumors, including prostate cancer [23]. Both ATO and KML001 are trivalent arsenicals and identical substances in solution, with similar cytotoxicity against androgen-independent prostate cancer cells [13]. However, the oral bioavailability and water solubility of KML001 suggest its clinical applicability, compared with ATO. Moreover, KML001 was shown to have higher LD50 (41.6 mg/kg) in rats, compared with ATO (14.6 mg/kg) [13]. So we tested the ability of KML001 to inhibit the growth and induce cell death of human prostate cancer cell. We found that KML001 treatment KML001 treatment has (A) anti-proliferative effect, (B) apoptotic effect, and (C) autophagic effect on DU145 prostate cancer cells in mice. Three fields in each mice were observed by fluorescence (200 and bright field microscopes (200, respectively. Representative images of each treatment group were shown. Vehicle and KML001 group mice orally received saline and KML001 (2.5 or 10 mg/kg/d) for 4 weeks, respectively. p < 0.05 vs. vehicle reduced the proliferation of all 3 prostate cancer cell lines, being more toxic to androgendependent LNCaP cells than to androgen-independent cells. More importantly, KML001 had an antiproliferative effect on androgen-independent DU145 cells in vitro and in vivo. ATO acts on malignant cells through a variety of mechanisms, targeting multiple signal transduction pathways and resulting in induction of apoptosis, antiproliferative activity, and antiangiogenesis [3]. Several recent studies have reported that ATO and KML001 may target a telomere/telomerase complex [80,13]. KML001 binds to telomeric sequences and erodes telomere, resulting in telomere-associated DNA damage induction and telomere attrition. In addition, ATO was found to induce type II programmed cell death, autophagy, in malignant glioma cells [11,24]. We have shown here that KML001 treatment resulted in the formation of autophagic vacuoles, as documented by electronic microscopy. Moreover, KML001 induced a time- and dose-dependent increase in LC3-II. Using the autophagy inhibitor 3-MA, we corroborated that the mechanism of KML001-induced cell death involves autophagy. Autophagy is not only responsible for cell killing by itself, but also participates in a lethal signaling event inducing apoptosis or necrosis [25,26]. Additionally, we found that the activation of autophagy and apoptosis by KML001 was mediated by oxidative stress. ROS play a pivotal role in mediating the cytotoxicity induced by KML001. Several studies have found that ROS play important roles in regulating both normal cellular processes and disease progression [279]. In addition, accumulated ROS have been known as the key intermediate for the cytotoxicity induced by chemotherapeutic agents, including ATO [3]. One concern regarding the use of arsenic for clinical applications is its toxicity in humans. Clinical studies have shown that ATO at concentrations less than 2 M does not induce severe side effects [30,31]. As mentioned above, KML001 was less toxic to rats at the same 3688248concentration of ATO [13]. In our study, KML001 had no adverse effect on body weight of DU145 xenograft mice. Our results therefore suggest that KML001 may be clinically useful in patients with castration-resistant prostate cancer. Whereas most chemotherapeutic agents are aimed at inhibiting the growth of castrationresistant prostate cancer, androgen-dependent and-independent prostate cancer cells have been reported to be equally susceptible to ATO-induced apoptosis [23]. Moreover, inhibition by ATO was more pronounced in prostate cancer cells expressing androgen receptor than in prostate cancer cells depleted of androgen receptor, and inhibition of androgen receptor activity by ATO and by the androgen receptor antagonist, bicalutamide, was additive [24]. These results may warrant the future assessment of the effects of KML001, alone or in combination with androgen deprivation therapy, on the progression of androgen-dependent LNCaP to androgen independence in a nude mice xenograft model.We have shown here that exposure of androgen-dependent and-independent prostate cancer cells to KML001 activated both apoptosis and autophagic cell death through oxidative stress. In addition, KML001 had an antiproliferative effect on androgen-independent DU145 cells in vivo.Myasthenia gravis (Greek mu “muscle”, ash neia “weakness” Latin: gravis “serious”) (MG) has been traditionally viewed as solely a peripheral neuromuscular disease characterized by fluctuating fatigue and muscle weakness [1,2]. Its primary symptoms arise from damage produced by autoantibodies directed against acetylcholine receptors (AChRs) on the postsynaptic neuromuscular junction [3]. Anti-AChR antibodies can be detected in serum in about 85% of MG patients, whereas the remaining cases are seronegative. However, about 40% of the latter have detectable antibodies against muscle-specific kinase (MuSK), a receptor kinase required for the formation of cholinergic receptors at the neuromuscular junction [3]. The general notion that MG is strictly a peripheral nervous system disease stems historically from findings that this disorder is not accompanied by gross or otherwise obvious brain pathology [6]. Following the discovery that MG is an autoimmune disorder associated with damage to muscle AChRs [3], this view continued following reports that (a) muscle AChR antibodies do not meaningfully cross the blood brain barrier (BBB) [7], (b) MG patients are seronegative for ganglionic neuronal AChR autoantibodies [8], and (c) muscle AChR antibodies do not bind to major cholinergic neuronal receptor subtypes within the human brain [9]. When behavioral and physiological evidence has been presented in support of MG’s involvement in the central nervous system (CNS), lack of replication has been noted in some cases and positive findings have been frequently discounted [10]. For example, while some studies have found MG-related deficits in verbal memory relative to controls, others have not [11]. The higher prevalence of depression and anxiety seen in MG patients relative to controls has been interpreted as psychological responses to a debilitating and incapacitating disease, rather than to diseasespecific CNS changes [10]. Sleep disturbances, which have been found in some, but not all, MG studies, have been considered to originate “ in the periphery rather than in the CNS, the result of hypoxia caused by oropharyngeal, intercostal and diaphragmatic muscle weakness which may worsen during sleep, especially during REM sleep” [10]. Despite this perspective, there is support for the concept that MG may influence CNS cholinergic processes. Thus, electroencephalographic studies show abnormalities in MG patients [12], as well as in animals with experimental autoimmune MG [13]. Prolonged latencies and decreased amplitudes in visual and auditory evoked potentials have been consistently reported [14,15]. Importantly, low levels of MG-related antibodies have been detected in the cerebrospinal fluid (CSF) of MG patients which, in most cases, are proportional to serum antibody levels, suggesting they may cross the BBB from the periphery [16]. Brain nicotinic AChRs, most notably a7 and a3-containing subtypes, have been found to bind antibodies from sera of MG patients [17], and immunization against the ganglionic a3 subunit has been found to produce both muscle and neuronal AChR antibodies [18].