BCR-ABL1-induced leukemogenesis and autophagic targeting by arsenic trioxide

We have recently shown that arsenic trioxide (As₂O₃) is a potent inducer of autophagic degradation of the BCR-ABL1 oncoprotein, which is the cause of chronic myeloid leukemia (CML) and Ph+ acute lymphoid leukemia (Ph+ ALL). Our recently published work has shown that pharmacological inhibition of autophagy or molecularly targeting of elements of the autophagic machinery partially reverses the suppressive effects of As₂O₃ on primitive leukemic precursors from CML patients. Altogether, our studies have provided direct evidence that arsenic-induced, autophagy-mediated, degradation of BCR-ABL1 is an important mechanism for the generation of the effects of As₂O₃ on BCR-ABL1 transformed leukemic progenitors. These studies raise the potential of future clinical-translational efforts employing combinations of arsenic trioxide with autophagy-modulating agents to promote elimination of early leukemic progenitors and, possibly, leukemia-initiating stem cells.     

Arsenic trioxide inhibits the growth of Adriamycin resistant osteosarcoma cells through inducing apoptosis

Given that arsenic trioxide (As₂O₃) has been successfully used as a chemotherapeutic agent for refractory malignant tumors, this study is aimed at investigating the effect of As₂O₃ on human Adriamycin resistant osteosarcoma cell line Saos-2. The mechanism underlying multi drug resistance (MDR) in osteosarcoma cells and the anti-tumor effect of As₂O₃ on Adriamycin resistant osteosarcoma cells were analyzed. In our experiment, we first selected Adriamycin resistant osteosarcoma cell line by growing the classic osteosarcoma cell line Saos-2 in the medium with increasing drug concentrations. Then, we compared the IC50s of the osteosarcoma cells treated with different anticancer drugs by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Subsequently, we assessed the expression of classic MDR related molecules, Pgp, multidrug resistance-associated protein (MRP) and glutathione (GSH) activity in the wild type and Adriamycin resistant Saos-2 cells. Furthermore, the apoptosis was assessed by concerning DNA fragment and flow cytometry with Annexin-V staining. To elucidate the underlying mechanism of the apoptosis, related proteins Bcl-2, Bcl-xL, Bax, Bak, cleaved Caspase-3 and cleaved Caspase-9 were analyzed by western blotting. The data showed that the resistance to Adriamycin affected the sensitivity of osteosarcoma cell to other chemotherapeutic agents. The IC50s of Saos-2/ADM cells for methotrexate (1.74-fold), Cisplatin (1.43-fold) and As₂O₃ (1.21-fold) were increased compared with Saos-2 control cells. The expression of Pgp was upregulated comparing with the control cells. No significant difference was detected about the MRP and the glutathione-S-transferase activity and intracellular GSH concentration among different treated osteosarcoma cells. Apoptosis was observed and proved. The western blotting showed that the expression of Bcl-2 and Bcl-xL was downregulated. Meanwhile, the level of Bax, Bak, cleaved Caspase-3 and cleaved Caspase-9 was upregulated after treated with As₂O₃. The study suggests that Adriamycin resistant osteosarcoma cells have good response to As₂O₃-based chemotherapy in vitro, probably via the pathway of inducing apoptosis. And As₂O₃ might serve as an excellent alternative candidate for adjuvant chemotherapeutic agent on this incurable pediatric sarcoma.     

The inherent cellular level of reactive oxygen species: One of the mechanisms determining apoptotic susceptibility of leukemic cells to arsenic trioxide

Though reactive oxygen species (ROS) has been noticed to be involved in arsenic trioxide (As₂O₃)-induced apoptosis of tumor cells, its role in apoptosis signaling remained to be elucidated. The objective of this work was to explore the association of the inherent cellular ROS level with the susceptibility of the tumor cells to apoptosis induction by As₂O₃. Low concentration of As₂O₃ was administered to cultured leukemic cell lines NB4, U937, HL60 and K562. The difference in apoptotic sensitivity was displayed among four cell types. ROS probes were incubated with the cells in the absence of As₂O₃, and ROS was thus quantified relatively by flow cytometry. We manifested, in four cell types, the inherently existed difference in whole ROS quantity, and a positive correlation between the inherent ROS level and their apoptotic sensitivity to As₂O₃. Furthermore, by interference using a ROS producer, we demonstrated that an elevation of ROS level would sensitize the cells to As₂O₃-induced apoptosis. The results of the present work suggested that the inherent ROS level might be determinative in tumor cells for their apoptotic susceptibility to As₂O₃.     

Inhibitory role of TGIF in the As<Subscript>2</Subscript>O<Subscript>3</Subscript>-regulated p21<Superscript><Emphasis Type="Italic">WAF1/CIP1</Emphasis></Superscript> expression

Although arsenic is an infamous carcinogen, it has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we had demonstrated that opposing effects of ERK1/2 and JNK on p21 expression in response to arsenic trioxide (As₂O₃) are mediated through the Sp1 responsive elements of the p21 promoter in A431 cells. Presently, we demonstrate that Sp1, and c-Jun functionally cooperate to activate p21 promoter expression through Sp1 binding sites (−84/−64) by using DNA affinity binding, chromatin immunoprecipitation, and promoter assays. Surprisingly, As₂O₃-induced c-Jun(Ser63/73) phosphorylation can recruit TGIF/HDAC1 to the Sp1 binding sites and then suppress p21 promoter activation. We suggest that, after As₂O₃ treatment, the N-terminal domain of c-Jun phosphorylation by JNK recruits TGIF/HDAC1 to the Sp1 sites and then represses p21 expression. That is, TGIF is involved in As₂O₃-inhibited p21 expression, and then blocks the cell cycle arrest.     

Downregulation of hTERT: An Important As2O3 Induced Mechanism of Apoptosis in Myelodysplastic Syndrome

Two myelodysplastic syndrome (MDS) celllines, MUTZ-1 and SKM-1 cells, were used to study the effect of arsenic trioxide (As₂O₃) on hematological malignant cells. As₂O₃ induced this two cell lines apoptosis via activation of caspase-3/8 and cleavage of poly (ADP-ribose) polymerase (PARP), a DNA repair enzyme. As₂O₃ reduced NF-κB activity, which was important for inducing MUTZ-1 and SKM-1 cells apoptosis. As₂O₃ also inhibited the activities of hTERT in MUTZ-1 and SKM-1 cells. Moreover, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), had no effect on caspase-8 activation, although PDTC did inhibit MUTZ-1 and SKM-1 cells proliferation. Incubation of MUTZ-1 cells with a caspase-8 inhibitor failed to block As₂O₃-induced inhibition of NF-κB activity. Our findings suggest that As₂O₃ may induce apoptosis in MUTZ-1 and SKM-1 cells by two independent pathways: first, by activation of caspase-3/8 and PARP; and second, by inhibition of NF-κB activity, which results in downregulation of hTERT expression. We conclude that hTERT and NF-κB are important molecular targets in As₂O₃-induced apoptosis.     

Targeting Catalase but Not Peroxiredoxins Enhances Arsenic Trioxide-Induced Apoptosis in K562 Cells

Despite considerable efficacy of arsenic trioxide (As₂O₃) in acute promyelocytic leukemia (APL) treatment, other non-APL leukemias, such as chronic myeloid leukemia (CML), are less sensitive to As₂O₃ treatment. However, the underlying mechanism is not well understood. Here we show that relative As₂O₃-resistant K562 cells have significantly lower ROS levels than As₂O₃-sensitive NB4 cells. We compared the expression of several antioxidant enzymes in these two cell lines and found that peroxiredoxin 1/2/6 and catalase are expressed at high levels in K562 cells. We further investigated the possible role of peroxirdoxin 1/2/6 and catalase in determining the cellular sensitivity to As₂O₃. Interestingly, knockdown of peroxiredoxin 1/2/6 did not increase the susceptibility of K562 cells to As₂O₃. On the contrary, knockdown of catalase markedly enhanced As₂O₃-induced apoptosis. In addition, we provide evidence that overexpression of BCR/ABL cannot increase the expression of PRDX 1/2/6 and catalase. The current study reveals that the functional role of antioxidant enzymes is cellular context and treatment agents dependent; targeting catalase may represent a novel strategy to improve the efficacy of As₂O₃ in CML treatment.     

Mechanisms of arsenic trioxide induced apoptosis of human cervical cancer HeLa cells and protection by Bcl-2

It was recently reported that arsenic trioxide (As²O³) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of (As²O³) on human cervical cancer HeLa cells and HeLa cells overexpressing Bcl-2 is studied. By MTT and colony forming ability assays, morphology alteration, flow cytometric analysis, DNA gel electrophoresis andin situ cell death detection (TUNEL), it was found that As²O³ inhibited the growth of HeLa cells and induced G2/M arrest and apoptosis of the cells. RT-PCR, Northern blot, Western blot analysis revealed that As²O³ induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As²O³ induced apoptosis, which might be relative to preventing the cells from As²O³ caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted. However, it was found that As²O³ at a high concentration could also induce apoptosis of HeLa cells overexpressing Bcl-2 possibly mainly via downregulating Bcl-2 expression and slightly inhibiting viral gene expression.     

Opposite effect of ERK1/2 and JNK on p53-independent p21 WAF1/CIP1 activation involved in the arsenic trioxide-induced human epidermoid carcinoma A431 cellular cytotoxicity

Summary While arsenic trioxide (As₂O₃) is an infamous carcinogen, it is also an effective chemotherapeutic agent for acute promyelocytic leukemia and some solid tumors. In human epidermoid carcinoma A431 cells, we found that As₂O₃ induced cell death in time- and dose-dependent manners. Similarly, dependent regulation of the p21 ^WAF1/CIP1 (p21) promoter, mRNA synthesis, and resultant protein expression was also observed. Additionally, transfection of a small interfering RNA of p21 could block the As₂O₃-induced cell growth arrest. The As₂O₃-induced p21 activation was attenuated by inhibitors of EGFR and MEK in a dose-dependent manner. Using a reporter assay, we demonstrated the involvement of the EGFR-Ras-Raf-ERK1/2 pathway in the promoter activation. In contrast, JNK inhibitor enhanced the As₂O₃-induced p21 activation, also in a dose-dependent fashion. Over-expression of a dominant negative JNK plasmid likewise also enhanced this activation. Furthermore, MEK inhibitor attenuated the anti-tumor effect of As₂O₃. In contrast, in combination with JNK inhibitor and As₂O₃ enhanced cellular cytotoxicity. Therefore, we conclude that in A431 cells the ERK1/2 and JNK pathways might differentially contribute to As₂O₃-induced p21 expression and then due to cellular cytotoxicity.     

Isocostunolide inhibited glioma stem cell by suppression proliferation and inducing caspase dependent apoptosis

Glioblastoma multiform (GBM) is a highly aggressive brain tumor with poor life expectancy, and glioma stem cells (GSCs) are a small population of tumor cells existed in GBM, in which GSCs response to drive GBM recurrence, invasion and contribute to the anti-cancer resistance. GSCs have been identified and developed as a therapeutic target for GBM and can be used in drugs screening. Isocostunolide is a natural sesquiterpenoid and contained abundant resource in medicinal plants, but the anti-cancer efficacies of it against GSCs are still unexplored. In this investigation, the anti-tumor activity of isocostunolide against GSCs was investigated and the result demonstrated that it inhibited the growth of GSCs (GSC-3#, GSC-12#, GSC-18#) significantly with an IC₅₀ value of 2.80 μg/ml, 2.61 μg/ml, 1.07 μg/ml, respectively. In further mechanism study, isocostunolide inhibited GSCs cell proliferation, induced GSCs apoptosis significantly, as well as increased the proportion of the cleavage of caspase-3. The result suggested that isocostunolide induced GSCs apoptosis via the caspase dependent apoptotic pathway. Moreover, isocostunolide damaged GSCs colony formation capacity significantly and exhibited the anti-cancer efficacy against GSCs in vitro.     

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line

This study aimed to investigate the effects of arsenic trioxide (As₂O₃) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 μmol/L As₂O₃ in vitro, and the primary APL cells were treated with 2.0 μmol/L As₂O₃ in vitro and 0.16 mg kg⁻¹ d⁻¹ As₂O₃ in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As₂O₃ use, but the mutation spots were remarkably increased after As₂O₃ treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r _NB4-As2O3=0.973818, and r _APL-As2O3=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As₂O₃ aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As₂O₃ in APL treatment.     

Arsenic Trioxide and Resveratrol Show Synergistic Anti-Leukemia Activity and Neutralized Cardiotoxicity

Cardiotoxicity is an aggravating side effect of many clinical antineoplastic agents such as arsenic trioxide (As₂O₃), which is the first-line treatment for acute promyelocytic leukemia (APL). Clinically, drug combination strategies are widely applied for complex disease management. Here, an optimized, cardiac-friendly therapeutic strategy for APL was investigated using a combination of As₂O₃ and genistein or resveratrol. Potential combinations were explored with respect to their effects on mitochondrial membrane potential, reactive oxygen species, superoxide dismutase activity, autophagy, and apoptosis in both NB4 cells and neonatal rat left ventricular myocytes. All experiments consistently suggested that 5 µM resveratrol remarkably alleviates As₂O₃-induced cardiotoxicity. To achieve an equivalent effect, a 10-fold dosage of genistein was required, thus highlighting the dose advantage of resveratrol, as poor bioavailability is a common concern for its clinical application. Co-administration of resveratrol substantially amplified the anticancer effect of As₂O₃ in NB4 cells. Furthermore, resveratrol exacerbated oxidative stress, mitochondrial damage, and apoptosis, thereby reflecting its full range of synergism with As₂O₃. Addition of 5 µM resveratrol to the single drug formula of As₂O₃ also further increased the expression of LC3, a marker of cellular autophagy activity, indicating an involvement of autophagy-mediated tumor cell death in the synergistic action. Our results suggest a possible application of an As₂O₃ and resveratrol combination to treat APL in order to achieve superior therapeutics effects and prevent cardiotoxicity.     

SnoN/SkiL,a TGFβ signaling mediator

Effective treatment of cancer cells with chemotherapeutic drugs relies on their ability to induce cell death, making the discovery of their mechanisms of action crucial. Arsenic trioxide (As₂O₃), used in the treatment of promyelocytic leukemia (PML), triggers cell death in several solid tumor cell lines including ovarian carcinomas. While As₂O₃ is remarkably cytotoxic in human ovarian cancer cells, its mechanism of action is poorly understood. We recently investigated the effects of As₂O₃ on several transforming growth factor-β (TGFβ) signaling mediators to better understand its cell death mechanism. Indeed, dysregulated (TGFβ) signaling is typical of ovarian cancers. Based on our findings, we propose that As₂O₃ induces a Beclin 1-independent autophagic pathway in ovarian carcinoma cells by modulating SnoN/SkiL expression, implicating SnoN as a novel therapeutic target for ovarian cancers.     

Effect of conditioned medium from neural stem cells on glioma progression and its protein expression profile analysis

BACKGROUNDEmerging evidence suggests that the spread of glioma to the subventricular zone (SVZ) is closely related to glioma recurrence and patient survival. Neural stem cells (NSCs) are the main cell type in the SVZ region and exhibit tumor-homing ability.AIMTo evaluate the effects of conditioned medium (CM) derived from SVZ NSCs on the cancer-related behaviors of glioma cells.METHODSThe characteristics of SVZ hNSCs were identified by immunofluorescence. The normoxic-hNSC-CM and hypoxic-hNSC-CM (3% O₂, oxygen-glucose deprived [OGD] culturing) were collected from 80%-90% confluent SVZ NSCs in sterile conditions. The CCK8 and Transwell assays were used to compare and evaluate the effects of normoxic-CM and hypoxic-CM on glioma proliferation and invasion. Then proteins secreted from SVZ NSCs into the CM were investigated by mass spectrometry, and the potential effects of candidate protein NCAN in the regulation of glioma progression were examined by CCK8 and Transwell assays.RESULTSThe CM from SVZ NSCs significantly increased the proliferation and invasion of glioma cells, particularly the CM from OGD NSCs induced under hypoxic conditions. Furthermore, the secreted protein neurocan (NCAN) in CM from OGD NSCs was identified by proteomic analysis. NCAN was expressed in glioma cells and played regulatory roles in mediating the progression of glioma cells mainly via the Rho/Rho-associated protein kinase pathway.CONCLUSIONOur study identified a potential interactive mechanism between SVZ NSCs and glioma cells, in which SVZ NSCs promote glioma progression via the secreted protein NCAN. These findings suggested that exploring the CM derived from cells could be a novel strategy for optimizing treatments and that NCAN derived from SVZ NSCs may be a potential new target in glioma progression.     

Low-Concentration Arsenic Trioxide Inhibits Skeletal Myoblast Cell Proliferation via a Reactive Oxygen Species-Independent Pathway

Myoblast proliferation and differentiation are essential for skeletal muscle regeneration. Myoblast proliferation is a critical step in the growth and maintenance of skeletal muscle. The precise action of inorganic arsenic on myoblast growth has not been investigated. Here, we investigated the in vitro effect of inorganic arsenic trioxide (As₂O₃) on the growth of C2C12 myoblasts. As₂O₃ decreased myoblast growth at submicromolar concentrations (0.25–1 μM) after 72 h of treatment. Submicromolar concentrations of As₂O₃ did not induce the myoblast apoptosis. Low-concentration As₂O₃ (0.5 and 1 μM) significantly suppressed the myoblast cell proliferative activity, which was accompanied by a small proportion of bromodeoxyuridine (BrdU) incorporation and decreased proliferating cell nuclear antigen (PCNA) protein expression. As₂O₃ (0.5 and 1 μM) increased the intracellular arsenic content but did not affect the reactive oxygen species (ROS) levels in the myoblasts. Cell cycle analysis indicated that low-concentrations of As₂O₃ inhibited cell proliferation via cell cycle arrest in the G1 and G2/M phases. As₂O₃ also decreased the protein expressions of cyclin D1, cyclin E, cyclin B1, cyclin-dependent kinase (CDK) 2, and CDK4, but did not affect the protein expressions of p21 and p27. Furthermore, As₂O₃ inhibited the phosphorylation of Akt. Insulin-like growth factor-1 significantly reversed the inhibitory effect of As₂O₃ on Akt phosphorylation and cell proliferation in the myoblasts. These results suggest that submicromolar concentrations of As₂O₃ alter cell cycle progression and reduce myoblast proliferation, at least in part, through a ROS-independent Akt inhibition pathway.     

The effect of intracellular ascorbate on the susceptibility of HL60 and Jurkat cells to chemotherapy agents

Chemotherapy agents initiate tumour cell apoptosis and this is thought to involve oxidative stress. In this study we have investigated the effect of the important antioxidant Vitamin C (ascorbate) on the response of HL60 and Jurkat cells to three chemotherapy drugs, namely etoposide, melphalan and arsenic trioxide (As₂O₃). Cells grown in routine culture media are deficient in ascorbate and to determine its effect on chemotherapy drug-induced apoptosis we supplemented the cells prior to drug exposure. We found that ascorbate had a varied effect on apoptosis and cell cycle progression. Etoposide-induced apoptosis in HL60 cells was significantly increased in ascorbate-loaded cells as measured by caspase-3 activation and DNA degradation, and this appeared to reflect a decrease in the number of necrotic cells rather than increased cytotoxicity. In contrast, ascorbate had no effect on etoposide-induced apoptosis in Jurkat cells. In both cell types melphalan-induced apoptosis was unaffected by intracellular ascorbate, whereas both apoptosis and growth arrest with low concentrations of As₂O₃ were diminished. These results indicate that intracellular ascorbate can affect cell responses to chemotherapy drugs in a complex and somewhat unpredictable manner and that it may play an important role in the responsiveness of tumour cells to chemotherapy regimes. This study was supported by the Health Research Council of New Zealand.     

Ellagic acid mitigates arsenic‐trioxide‐induced mitochondrial dysfunction and cytotoxicity in SH‐SY5Y cells

In the current study, neuroprotective significance of ellagic acid (EA, a polyohenol) was explored by primarily studying its antioxidant and antiapoptotic potential against arsenic trioxide (As₂O₃)‐induced toxicity in SH‐SY5Y human neuroblastoma cell lines. The mitigatory effects of EA with particular reference to cell viability and cytotoxicity, the generation of reactive oxygen species, DNA damage, and mitochondrial dynamics were studied. Pretreatment of SH‐SY5Y cells with EA (10 and 20 μM) for 60 min followed by exposure to 2 μM As₂O₃ protected the SH‐SY5Y cells against the harmful effects of the second. Also, EA pre‐treated groups expressed improved viability, repaired DNA, reduced free radical generation, and maintained altered mitochondrial membrane potential than those exposed to As₂O₃ alone. EA supplementation also inhibited As₂O₃‐induced cytochrome c expression that is an important hallmark for determining mitochondrial dynamics. Thus, the current investigations are more convinced for EA as a promising candidate in modulating As₂O₃‐induced mitochondria‐mediated neuronal toxicity under in vitro system.