Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer

Objective

To investigate the effect of parthenolide on apoptosis and autophagy and to study the role of the PI3K/Akt signaling pathway in cervical cancer.

Results

Parthenolide inhibits HeLa cell viability in a dose dependent-manner and was confirmed by MTT assay. Parthenolide (6 µM) induces mitochondrial-mediated apoptosis and autophagy by activation of caspase-3, upregulation of Bax, Beclin-1, ATG5, ATG3 and down-regulation of Bcl-2 and mTOR. Parthenolide also inhibits PI3K and Akt expression through activation of PTEN expression. Moreover, parthenolide induces generation of reactive oxygen species that leads to the loss of mitochondrial membrane potential.

Conclusion

Parthenolide induces apoptosis and autophagy-mediated growth inhibition in HeLa cells by suppressing the PI3K/Akt signaling pathway and mitochondrial membrane depolarization and ROS generation. Parthenolide may be a potential therapeutic agent for the treatment of cervical cancer.

     

ERK-mediated autophagy promotes inactivated Sendai virus (HVJ-E)-induced apoptosis in HeLa cells in an Atg3-dependent manner

Background

Apoptosis and autophagy are known to play important roles in cancer development. It has been reported that HVJ-E induces apoptosis in cancer cells, thereby inhibiting the development of tumors. To define the mechanism by which HVJ-E induces cell death, we examined whether HVJ-E activates autophagic and apoptotic signaling pathways in HeLa cells.

Methods

Cells were treated with chloroquine (CQ) and rapamycin to determine whether autophagy is involved in HVJ-E-induced apoptosis. Treatment with the ERK inhibitor, U0126, was used to determine whether autophagy and apoptosis are mediated by the ERK pathway. Activators of the PI3K/Akt/mTOR/p70S6K pathway, 740 Y-P and SC79, were used to characterize its role in HVJ-E-induced autophagy. siRNA against Atg3 was used to knock down the protein and determine whether it plays a role in HVJ-E-induced apoptosis in HeLa cells.

Results

We found that HVJ-E infection inhibited cell viability and induced apoptosis through the mitochondrial pathway, as evidenced by the expression of caspase proteins. This process was promoted by rapamycin treatment and inhibited by CQ treatment. HVJ-E-induced autophagy was further blocked by 740 Y-P, SC79, and U0126, indicating that both the ERK- and the PI3K/Akt/mTOR/p70S6K-pathways were involved. Finally, autophagy-mediated apoptosis induced by HVJ-E was inhibited by siRNA-mediated Atg3 knockdown.

Conclusion

In HeLa cells, HVJ-E infection triggered autophagy through the PI3K/Akt/mTOR/p70S6K pathway in an ERK1/2-dependent manner, and the induction of autophagy promoted apoptosis in an Atg3-dependent manner.

     

MiR-9 enhances the sensitivity of A549 cells to cisplatin by inhibiting autophagy

Objectives

To demonstrate that miR-9 inhibits autophagy by down-regulating Beclin1 and thus enhances the sensitivity of A549 cells to cisplatin.

Results

MiR-9 inhibited Beclin1 expression by binding to its 3′UTR. The inhibition decreased the cisplatin-induced autophagy in A549 cells, evidenced by the decreased expression of LC3II and GFP-LC3 puncta and the increased expression of P62. Upregulation of miR-9 level enhanced the sensibility of A549 cells to cisplatin and increased the cisplatin-induced apoptosis. Overexpression of Beclin1 reversed above effects of miR-9 mimics, cisplatin-induced autophagy was increased and apoptosis was decreased.

Conclusions

MiR-9 inhibits autophagy via targeting Beclin1 3′UTR and thus enhances cisplatin sensitivity in A549 cells.

     

An LC–MS-based lipidomics approach for studying the impact of dietary eicosapentaenoic acid on modulating methylmercury toxicity in mice

Introduction

Methylmercury (MeHg) exposure has been a public health problem for many decades. There is a growing interest in searching for possible dietary nutrients that may affect MeHg toxicity.

Objectives

The study aims to evaluate the impact of eicosapentaenoic acid (EPA) on modulating MeHg toxicity in mice.

Methods

The study was based on a two-level factorial design, where the factors were presence or absence of EPA and MeHg in the feed. A liquid chromatography-mass spectrometry-based lipidomics approach was used to identify and quantify the main phospholipid species in mouse liver and plasma. The effects of EPA and MeHg on phospholipid species were evaluated by principal component analysis and statistics. Some biochemical and toxicological markers were measured and hepatic histological assay was carried out.

Results

EPA treatment significantly elevated the phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) species that contain n-3 PUFA, and reduced the levels of PtdCho and PtdEtn species that contain arachidonic acid (ARA), while MeHg exhibited opposite effects on these specific PtdCho species in liver. MeHg induced higher prostaglandin E₂ and lower prostaglandin E₃, thus increasing pro-inflammatory factors, while EPA decreased these ARA-derived inflammatory factors. Moreover, MeHg induced chronic inflammatory symptoms in mice, including severe hepatic necrosis, higher aspartate aminotransferase and alanine aminotransferase activities in plasma, higher thiobarbituric acid reactive substances and lower glutathione in liver. These symptoms were all alleviated by EPA treatment.

Conclusion

EPA may have protective effect against MeHg-induced toxicity due to the favorable modification of membrane phospholipid composition and inhibition of inflammatory factors.

     

Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

Background

Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid.

Results

Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclx_s, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclx_s oligomerization, accumulation of Ca²⁺, and increased oxygen consumption.

Conclusion

Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions.

     

RAC3 influences the chemoresistance of colon cancer cells through autophagy and apoptosis inhibition

Background

RAC3 coactivator overexpression has been implicated in tumorigenesis, contributing to inhibition of apoptosis and autophagy. Both mechanisms are involved in resistance to treatment with chemotherapeutic agents. The aim of this study was to investigate its role in chemoresistance of colorectal cancer.

Methods

The sensitivity to 5-fluorouracil and oxaliplatin in colon cancer cells HT-29, HCT 116 and Lovo cell lines, expressing high or low natural levels of RAC3, was investigated using viability assays.

Results

In HCT 116 cells, we found that although 5-fluorouracil was a poor inducer of apoptosis, autophagy was strongly induced, while oxaliplatin has shown a similar ability to induce both of them. However, in HCT 116 cells expressing a short hairpin RNA for RAC3, we found an increased sensitivity to both drugs if it is compared with control cells. 5-Fluorouracil and oxaliplatin treatment lead to an enhanced caspase 3-dependent apoptosis and produce an increase of autophagy. In addition, both process have shown to be trigged faster than in control cells, starting earlier after stimulation.

Conclusions

Our results suggest that RAC3 expression levels influence the sensitivity to chemotherapeutic drugs. Therefore, the knowledge of RAC3 expression levels in tumoral samples could be an important contribution to design new improved therapeutic strategies in the future.

     

Copper oxide nanoparticles induce anticancer activity in A549 lung cancer cells by inhibition of histone deacetylase

Objectives

Copper oxide nanoparticles (CuO NPs) promoting anticancer activity may be due to the regulation of various classes of histone deacetylases (HDACs).

Results

Green-synthesized CuO NPs significantly arrested total HDAC level and also suppressed class I, II and IV HDACs mRNA expression in A549 cells. A549 cells treated with CuO NPs downregulated oncogenes and upregulated tumor suppressor protein expression. CuO NPs positively regulated both mitochondrial and death receptor-mediated apoptosis caspase cascade pathway in A549 cells.

Conclusion

Green-synthesized CuO NPs inhibited HDAC and therefore shown apoptosis mediated anticancer activity in A549 lung cancer cell line.

     

Lipoxin A4 receptor agonist BML-111 induces autophagy in alveolar macrophages and protects from acute lung injury by activating MAPK signaling

Background

Acute lung injury (ALI) is a life-threatening lung disease where alveolar macrophages (AMs) play a central role both in the early phase to initiate inflammatory responses and in the late phase to promote tissue repair. In this study, we examined whether BML-111, a lipoxin A4 receptor agonist, could alter the phenotypes of AM and thus present prophylactic benefits for ALI.

Methods

In vitro, isolated AMs were treated with lipopolysaccharide (LPS) to induce ALI. In response to BML-111 pre-treatment, apoptosis and autophagy of AMs were examined by flow cytometry, and by measuring biomarkers for each process. The potential involvement of MAPK1 and mTOR signaling pathway was analyzed. In vivo, an LPS-induced septic ALI model was established in rats and the preventative significance of BML-111 was assessed. On the cellular and molecular levels, the pro-inflammatory cytokines TNF-α and IL-6 from bronchoalveolar lavage were measured by ELISA, and the autophagy in AMs examined using Western blot.

Results

BML-111 inhibited apoptosis and induced autophagy of AMs in response to ALI inducer, LPS. The enhancement of autophagy was mediated through the suppression of MAPK1 and MAPK8 signaling, but independent of mTOR signaling. In vivo, BML-111 pre-treatment significantly alleviated LPS-induced ALI, which was associated with the reduction of apoptosis, the dampened production of pro-inflammatory cytokines in the lung tissue, as well as the increase of autophagy of AMs.

Conclusions

This study reveals the prophylactic significance of BML-111 in ALI and the underlying mechanism: by targeting the MAPK signaling but not mTOR pathway, BML-111 stimulates autophagy in AMs, attenuates the LPS-induced cell apoptosis, and promotes the resolution of ALI.

     

MiR-320 regulates cardiomyocyte apoptosis induced by ischemia–reperfusion injury by targeting AKIP1

Background

MicroRNAs play important roles in regulation of the cardiovascular system. The purpose of this study was to investigate microRNA-320 (miR-320) expression in myocardial ischemia-reperfusion (I/R) injury and the roles of miR-320 in cardiomyocyte apoptosis by targeting AKIP1 (A kinase interacting protein 1).

Methods

The level of miR-320 was detected using quantitative real-time polymerase chain reaction (qRT-PCR), and cardiomyocyte apoptosis was detected via terminal dUTP nick end-labeling assay. Cardiomyocyte apoptosis and the mitochondrial membrane potential were evaluated via flow cytometry. Bioinformatics tools were used to identify the target gene of miR-320. The expression levels of AKIP1 mRNA and protein were detected via qRT-PCR and Western blot, respectively.

Results

Both the level of miR-320 and the rate of cardiomyocyte apoptosis were substantially higher in the I/R group and H9c2 cells subjected to H/R than in the corresponding controls. Overexpression of miR-320 significantly promoted cardiomyocyte apoptosis and increased the loss of the mitochondrial membrane potential, whereas downregulation of miR-320 had an opposite effect. Luciferase reporter assay showed that miR-320 directly targets AKIP1. Moreover, knock down and overexpression of AKIP1 had similar effects on the H9c2 cells subjected to H/R.

Conclusions

miR-320 plays an important role in regulating cardiomyocyte apoptosis induced by I/R injury by targeting AKIP1 and inducing the mitochondrial apoptotic pathway.

     

The altered human serum metabolome induced by a marathon

Introduction

Endurance races have been associated with a substantial amount of adverse effects which could lead to chronic disease and long-term performance impairment. However, little is known about the holistic metabolic changes occurring within the serum metabolome of athletes after the completion of a marathon.

Objectives

Considering this, the aim of this study was to better characterize the acute metabolic changes induced by a marathon.

Methods

Using an untargeted two dimensional gas chromatography time-of-flight mass spectrometry metabolomics approach, pre- and post-marathon serum samples of 31 athletes were analyzed and compared to identify those metabolites varying the most after the marathon perturbation.

Results

Principle component analysis of the comparative groups indicated natural differentiation due to variation in the total metabolite profiles. Elevated concentrations of carbohydrates, fatty acids, tricarboxylic acid cycle intermediates, ketones and reduced concentrations of amino acids indicated a metabolic shift between various fuel substrate systems. Additionally, elevated odd-chain fatty acids and α-hydroxy acids indicated the utilization of α-oxidation and autophagy as alternative energy-producing mechanisms. Adaptations in gut microbe-associated markers were also observed and correlated with the metabolic flexibility of the athlete.

Conclusion

From these results it is evident that a marathon places immense strain on the energy-producing pathways of the athlete, leading to extensive protein degradation, oxidative stress, mammalian target of rapamycin complex 1 inhibition and autophagy. A better understanding of this metabolic shift could provide new insights for optimizing athletic performance, developing more efficient nutrition regimens and identify strategies to improve recovery.

     

Impaired mitochondrial calcium uptake caused by tacrolimus underlies beta-cell failure

Background

One of the most common side effects of the immunosuppressive drug tacrolimus (FK506) is the increased risk of new-onset diabetes mellitus. However, the molecular mechanisms underlying this association have not been fully clarified.

Methods

We studied the effects of the therapeutic dose of tacrolimus on mitochondrial fitness in beta-cells.

Results

We demonstrate that tacrolimus impairs glucose-stimulated insulin secretion (GSIS) in beta-cells through a previously unidentified mechanism. Indeed, tacrolimus causes a decrease in mitochondrial Ca²⁺ uptake, accompanied by altered mitochondrial respiration and reduced ATP production, eventually leading to impaired GSIS.

Conclusion

Our observations individuate a new fundamental mechanism responsible for the augmented incidence of diabetes following tacrolimus treatment. Indeed, this drug alters Ca²⁺ fluxes in mitochondria, thereby compromising metabolism-secretion coupling in beta-cells.

     

Association of mitochondrial dysfunction and lipid metabolism with type 2 diabetes mellitus: A review of literature

Background

Diabetes mellitus (DM) is one of the most prevalent chronic diseases, and its prevalence continues to increase globally. The impact of mitochondrial dysfunction and lipid metabolism on diabetes mellitus and insulin resistance (IR) has been implicated in several previous reports; however, the results of studies are confusing despite four decades of study.

Methods/Results

This review has evaluated updated understanding of the role of mitochondrial dysfunction and lipid metabolism on type 2 diabetes, and found that mitochondrial dysfunction and lipid metabolism disorder induce the dysregulation of liver and pancreatic beta cells, insulin resistance, and type 2 diabetes.

Conclusion

Mitochondrial dysfunction and lipid metabolism induce metabolic dysregulation and finally increasing the possibility of diabetes.

     

Reduction of apoptosis and preservation of mitochondrial integrity under ischemia/reperfusion injury is mediated by estrogen receptor β

Background

Estrogen improves cardiac recovery after ischemia/reperfusion (I/R) by yet incompletely understood mechanisms. Mitochondria play a crucial role in I/R injury through cytochrome c-dependent apoptosis activation. We tested the hypothesis that 17β-estradiol (E2) as well as a specific ERβ agonist improve cardiac recovery through estrogen receptor (ER)β-mediated mechanisms by reducing mitochondria-induced apoptosis and preserving mitochondrial integrity.

Methods

We randomized ovariectomized C57BL/6N mice 24h before I/R to pre-treatment with E2 or a specific ERβ agonist (ERβA). Isolated hearts were perfused for 20min prior to 30min global ischemia followed by 40min reperfusion.

Results

Compared with controls, ERβA and E2 treated groups showed a significant improvement in cardiac recovery, i.e. an increase in left ventricular developed pressure, dP/dtmax and dP/dtmin. ERβA and E2 pre-treatment led to a significant reduction in apoptosis with decreased cytochrome c release from the mitochondria and increased mitochondrial levels of anti-apoptotic Bcl2 and ACAA2. Protein levels of mitochondrial translocase inner membrane (TIM23) and mitochondrial complex I of respiratory chain were increased by ERβA and E2 pre-treatment. Furthermore, we found a significant increase of myosin light chain 2 (MLC2) phosphorylation together with ERK1/2 activation in E2, but not in ERβA treated groups.

Conclusions

Activation of ERβ is essential for the improvement of cardiac recovery after I/R through the inhibition of apoptosis and preservation of mitochondrial integrity and can be a achieved by a specific ERβ agonist. Furthermore, E2 modulates MLC2 activation after I/R independent of ERβ.

     

Metabolic profiling of isolated mitochondria and cytoplasm reveals compartment-specific metabolic responses

Introduction

Subcellular compartmentalization enables eukaryotic cells to carry out different reactions at the same time, resulting in different metabolite pools in the subcellular compartments. Thus, mutations affecting the mitochondrial energy metabolism could cause different metabolic alterations in mitochondria compared to the cytoplasm. Given that the metabolite pool in the cytosol is larger than that of other subcellular compartments, metabolic profiling of total cells could miss these compartment-specific metabolic alterations.

Objectives

To reveal compartment-specific metabolic differences, mitochondria and the cytoplasmic fraction of baker’s yeast Saccharomyces cerevisiae were isolated and subjected to metabolic profiling.

Methods

Mitochondria were isolated through differential centrifugation and were analyzed together with the remaining cytoplasm by gas chromatography–mass spectrometry (GC–MS) based metabolic profiling.

Results

Seventy-two metabolites were identified, of which eight were found exclusively in mitochondria and sixteen exclusively in the cytoplasm. Based on the metabolic signature of mitochondria and of the cytoplasm, mutants of the succinate dehydrogenase (respiratory chain complex II) and of the F_OF₁-ATP-synthase (complex V) can be discriminated in both compartments by principal component analysis from wild-type and each other. These mitochondrial oxidative phosphorylation machinery mutants altered not only citric acid cycle related metabolites but also amino acids, fatty acids, purine and pyrimidine intermediates and others.

Conclusion

By applying metabolomics to isolated mitochondria and the corresponding cytoplasm, compartment-specific metabolic signatures can be identified. This subcellular metabolomics analysis is a powerful tool to study the molecular mechanism of compartment-specific metabolic homeostasis in response to mutations affecting the mitochondrial metabolism.

     

Mitochondria-cytoskeleton associations in mammalian cytokinesis

Background

The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown.

Methods

We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells.

Results

Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division.

Conclusions

Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.

     

Deferiprone targets aconitase: Implication for Friedreich's ataxia treatment

Background

Friedreich ataxia is a neurological disease originating from an iron-sulfur cluster enzyme deficiency due to impaired iron handling in the mitochondrion, aconitase being particularly affected. As a mean to counteract disease progression, it has been suggested to chelate free mitochondrial iron. Recent years have witnessed a renewed interest in this strategy because of availability of deferiprone, a chelator preferentially targeting mitochondrial iron.

Method

Control and Friedreich's ataxia patient cultured skin fibroblasts, frataxin-depleted neuroblastoma-derived cells (SK-N-AS) were studied for their response to iron chelation, with a particular attention paid to iron-sensitive aconitase activity.

Results

We found that a direct consequence of chelating mitochondrial free iron in various cell systems is a concentration and time dependent loss of aconitase activity. Impairing aconitase activity was shown to precede decreased cell proliferation.

Conclusion

We conclude that, if chelating excessive mitochondrial iron may be beneficial at some stage of the disease, great attention should be paid to not fully deplete mitochondrial iron store in order to avoid undesirable consequences.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

TNFα expressed on the surface of microparticles modulates endothelial cell fate in rheumatoid arthritis

Background

Rheumatoid arthritis (RA) is associated with a high prevalence of atherosclerosis. Recently increased levels of microparticles (MPs) have been reported in patients with RA. MPs could represent a link between autoimmunity and endothelial dysfunction by expressing tumor necrosis factor alpha (TNFα), a key cytokine involved in the pathogenesis of RA, altering endothelial apoptosis and autophagy. The aim of this study was to investigate TNFα expression on MPs and its relationship with endothelial cell fate.

Methods

MPs were purified from peripheral blood from 20 healthy controls (HC) and from 20 patients with RA, before (time (T)0) and after (T4) 4-month treatment with etanercept (ETA). Surface expression of TNFα was performed by flow cytometry analysis. EA.hy926 cells, an immortalized endothelial cell line, were treated with RA-MPs purified at T0 and at T4 and also, with RA-MPs in vitro treated with ETA. Apoptosis and autophagy were then evaluated.

Results

RA-MPs purified at T0 expressed TNFα on their surface and this expression significantly decreased at T4. Moreover, at T0 RA-MPs, significantly increased both apoptosis and autophagy levels on endothelial cells, in a dose-dependent manner. RA-MPs did not significantly change these parameters after 4 months of in vivo treatment with ETA.

Conclusions

Our data demonstrate that MPs isolated from patients with RA exert a pathological effect on endothelial cells by TNFα expressed on their surface. In vivo and in vitro treatment with ETA modulates this effect, suggesting anti-TNF therapy protects against endothelial damage in patients with RA.

     

Autophagy regulated by lncRNA HOTAIR contributes to the cisplatin-induced resistance in endometrial cancer cells

Objectives

To identify whether lncRNAs (long non-coding RNA) participate in the regulation of cisplatin-resistant induced autophagy in endometrial cancer cells.

Results

Autophagy activity was significantly boosted in cisplatin-resistant Ishikawa cells, a human endometrial cancer cell line, compared with that in parental Ishikawa cells. After analyzing the overall long noncoding RNA (lncRNA) profiling, a meaningful lncRNA, HOTAIR, was identified. It was down-regulated simultaneously in cisplatin-resistant Ishikawa cells and parental Ishikawa cells treated with cisplatin. RNA interference of HOTAIR reduced the proliferation of cisplatin-resistant Ishikawa cells and enhanced the autophagy activity of cisplatin-resistant Ishikawa cells with or without cisplatin treatment, in addition, beclin-1, multidrug resistance (MDR), and P-glycoprotein (P-gp) were mediated by lncRNA HOTAIR.

Conclusions

It is clear that lncRNAs, specifically HOTAIR, can regulate the cisplatin-resistance ability of human endometrial cancer cells through the regulation of autophagy by influencing Beclin-1, MDR, and P-gp expression.

     

WRN-targeted therapy using inhibitors NSC 19630 and NSC 617145 induce apoptosis in HTLV-1-transformed adult T-cell leukemia cells

Background

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATLL), a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells.

Methods

Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential.

Results

Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor) induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145.

Conclusions

WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.