Different Concentrations Of As2O3 Research Articles

PB1970: AS2O3 INDUCE IMMUNOGENIC CELL DEATH IN MULTIPLE MYELOMA VIA NLRP3/CASPASE SIGNALING PATHWAY

Background: Multiple myeloma (MM) is a common plasma cell malignant disease, the clonal evolution of myeloma cells and the immune escape caused by it are an important mechanism of refractory drug resistance. Aims: To explored the relationship between NLRP3 / Caspase signaling pathway and immunogenic cell death (ICD) in the intervention of As2O3 on MM cell (a human myeloma-derived KM3 cell line), and explained the inhibitory effect of As2O3 on the proliferation of myeloma cells. Methods: The inhibitory and apoptotic effect of As2O3 on human MM cells was monitored by a CCK-8 method. Meanwhile, the effects of different concentrations of As2O3 on MM cell apoptosis were detected was further determined by flow cytometry. Moreover, the expression of BCL-2, BAX, PARP, HSP70/90, MHC-I, and NLRP3 at mRNA was determined by using quantitative PCR (Q-PCR). The effects of different concentrations of As2O3 on the expression of apoptosis related proteins Bcl-2, Bax, Caspas-3 and PARP were detected by ELISA. Moreover, the expression of ICD related HSP70/90 and MHC-I proteins, as well as the expression of NLRP3 and caspase-1 proteins were explored. Results: The results indicated that As2O3 could significantly inhibit human MM cell proliferation and cell death in a time- and dose-dependent manner in vitro. Meanwhile, the results also showed that As2O3 could effectively increase the expression of Bax, PARP, HSP70/90, MHC-I, and NLRP3 mRNA levels, and decrease the Bcl-2 mRNA level in human MM cells. Moreover, As2O3 could also significantly elevate the expression of NLRP3, caspase-1 proteins and ICD related proteins, leading to increase the expression of apoptosis related proteins Bax, Caspas-3, PARP. Summary/Conclusion: As2O3 can significantly induce human MM cell death in a time- and dose-dependent manner in vitro, and activate NLRP3 / caspase signaling pathway in human MM cells. As2O3 can also increase the expression level of ICD related molecules and apoptosis related molecules Bax, Caspas-3, PARP, and initiate MM cell apoptosis, indicating that As2O3 induce immunogenic cell death in multiple myeloma via NLRP3/Caspase signaling pathway. Overall, As2O3 may be a potential agent in treating human multiple myeloma in clinical practice.

The Hormetic Effect of Arsenic Trioxide on Rat Pulpal Cells: An In Vitro Preliminary Study.

Objectives Despite the agreement that there is no longer any indication for arsenic use in modern endodontics, some concerns are surfacing about the minute amount of arsenic trioxide (As 2 O 3 ) released from Portland cement-based materials. The present study investigated the effect of different concentrations of As 2 O 3 on rat pulpal cells and the efficacy of N -acetylcysteine (NAC) in preventing As 2 O 3 -mediated toxicity. Materials and Methods Cytotoxicities of 50, 10, or 5 µm As 2 O 3 and the effect of cells co-treatment with 50 µm As 2 O 3 and 5,000 µm NAC or 500 µm NAC were tested at 24 hours or 3 days. Cell viability was assessed by means of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and cellular morphological changes were observed under phase contrast microscope. Statistical Analysis Two-way analysis of variance with Tukey’s post-hoc test was used to evaluate differences between the groups (α = 0.05). Results At both exposure times, 50 µm As 2 O 3 resulted in lower optical density (OD) values when compared with 10 or 5 µm As 2 O 3 . At 24 hours, 10 µm As 2 O 3 resulted in a higher OD value compared with the control; however, at 3 days the difference was statistically insignificant. At each exposure time, the OD value of 5 µm As 2 O 3 group was comparable to the control and 10 µm As 2 O 3 group. There were no significant differences between 50 µm As 2 O 3 group and 500 μm NAC+50 μm As 2 O 3 group; however, these two groups had lower OD values when compared with 5,000 μm NAC+50 μm As 2 O 3 group at 24 hours and 3 days. The latter group showed significantly lower OD value in comparison with the control at 24 hours and 3 days. Control cells were polygonal-shaped while 50 µm As 2 O 3 -treated cells exhibited contracted and spherical morphology with increased intercellular spaces. At 24 hours, 10 μm and 5 µm As 2 O 3 -treated cells were slightly hypertrophic. Cells co-treated with NAC and As 2 O 3 showed increased intercellular spaces and lower cellular density compared with the control. Conclusions As 2 O 3 displayed a hormetic effect on pulpal cells; however, the proliferative effect induced by low As 2 O 3 concentrations should be interpreted with caution. NAC did not prevent As 2 O 3 -mediated toxicity; however, it demonstrated potential for ameliorating this toxicity.

Study of As2O3 regulating proliferation and apoptosis of Tca8113 cells by inhibiting the expression of Id-1

Objective: Our study aims to investigate the effect of arsenic trioxide (As2O3) on proliferation and apoptosis of Tca8113 tongue squamous carcinoma cells.Methods: Cell proliferation and the expression of Id-1 mRNA in Tca8113 cells after treatment with different concentrations of As2O3 were detected by MTT and qRT-PCR, respectively. The expression of Id-1, cell proliferation and apoptosis in Id-1 silencing Tca8113 cells were detected by qRT-PCR, Western blot, MTT and flow cytometry, respectively. The pcDNA 3.1-Id-1 overexpression vector was transfected into Tca8113 cells combination with 3 μmol/L As2O3. The detection of cell proliferation, apoptosis and Caspase-3, Bax and Bcl-2 protein expression in transfected Tca8113 cells were performed by MTT, flow cytometry and Western blot assay, respectively.Results: As2O3 of different concentration could inhibit the proliferation of Tca8113 cells and IC50 value was 3.004 ± 0.2379 μmol/L. The expression of Id-1 mRNA was down-regulated in Tca8113 cells treated with 3 μmol/L As2O3 for 48 h. The results of qRT-PCR, Western blot, MTT and flow cytometry indicated that the expression level of Id-1 and cell proliferation ability were decreased while the apoptosis rate was increased in Tca8113 cells after transfection of Id-1 siRNA. Overexpression of Id-1 could attenuate the inhibition or promotion of As2O3 on proliferation, apoptosis and Caspase-3, Bax and Bcl-2 protein expression in Tca8113 cells.Conclusion: As2O3 could regulate the proliferation and apoptosis of Tca8113 cells by inhibiting the expression of Id-1.

Arsenic Trioxide Induces Apoptosis and Regulatory Functions of Plasmacytoid Dendritic Cells

IntroductionArsenic trioxide (As2O3), a well-established drug for acute promyelocytic leukemia, has been recently shown to have therapeutic potential in a number of mouse models of autoimmune diseases such as, systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and chronic graft-versus-host disease. However, the therapeutic mechanism remains largely unknown.Plasmacytoid dendritic cells (pDCs) are a unique subset of dendritic cells specialized in secreting high level of type I interferons and promote anti-viral responses. Clinically, pDCs have also been reported to be implicated in the pathogenesis of a series of autoimmune diseases characterized by a type I IFN signature, such as SSc.Given the pathogenetic and therapeutic role of As2O3 and pDCs in the same spectrum of diseases, we examined the effects of As2O3 on pDCs viability and functions to better understand the therapeutic mechanism of As2O3 on pDC-related autoimmune diseases.Materials and MethodspDCs cells were isolated from fresh peripheral blood mononuclear cells (PBMCs) by magnetic selection. pDCs were cultured in presence of different concentrations of As2O3 to check for viability and functional changes. pDCs viability and apoptosis were checked by flow cytometry with Fixable Viability Dye-Annexin V staining. Phenotype evaluation was done with surface staining. Bcl-2 and Bax protein expressions, reactive oxygen species (ROS) level, and cytokine production of pDCs were measured by intracellular staining. IRF7 and phospho-IRF7 protein levels were measured with cell signaling staining. Cytokine concentrations in supernatants were quantified by ELISA, and IRF7 expression in pDCs was analyzed by real-time RT-PCR.Naïve CD4+ T and CD19+ B cells were isolated from PBMCs. A pDC/T cell co-culture system was constructed to test As2O3 effect on pDCs in inducing T cell proliferation and polarization. A pDC/B cell co-culture system was used to test As2O3 effect on pDCs in inducing B cell differentiation.ResultsWe first investigated the effect of As2O3 on the viability of human pDCs. We observed that As2O3 decreased dose-dependently the percentages of viable pDCs by inducing cell apoptosis. We then checked the protein expression levels of the apoptosis related Bcl-2 family proteins. As2O3 induced a dramatic upregulation of Bax, while slightly down-regulated the expression of Bcl-2, leading to a significantly decreased Bcl-2 to Bax ratio, which determined cell apoptosis.As2O3 is a well-known inducer of oxidative stress, which is one of the major upstream mediators of cell apoptosis. We first demonstrated that As2O3 induced reactive oxygen species (ROS) within pDCs. However, the anti-oxidant NAC pre-treatment only slightly decreased the percentages of apoptotic cells, indicating that As2O3 could induce pDCs apoptosis independently of ROS generation.Then, we investigated the functional effects of As2O3 on pDCs, and observed that culture for 6h with 5μM of As2O3 almost totally inhibited production of IFN-α from gated pDCs within PBMCs (IFN-α positive cells: 28.56±9,99% without As2O3 vs 1.43±1.65% with 5μM As2O3, p<0.05, n=4). ELISA results proved these results and revealed that As2O3 also restrained the secretion of TNF-α, IL-6 and CXCL10 from pDCs. We also demonstrated that As2O3 inhibited IFN-α secretion from pDCs of 12 untreated SSc patients (IFN-α positive cells: 22.48±12.71% without As2O3 vs 1.65±1.24% with 5μM As2O3, p=0.0001, n=12).Further studies revealed that As2O3 inhibited the secretion of IFN-α from activated pDCs by potently down-regulating the total protein and mRNA expression, and also phosphorylation level of inteferon regulatory factor 7 (IRF7), a key adaptor of the type I inteferon pathway.Finally, As2O3 inhibited pDCs maturation by decreasing the expressions of CD80, CD86 and HLA-DR, and increasing PD-L1 expression. As2O3 also down-regulated pDCs capacity to induce CD4+ T cell proliferation, Th1/Th22 polarization, and B cell differentiation towards plasmablasts.ConclusionOur study demonstrated that As2O3, at different concentrations, negatively affects the viability and functions of human pDCs, suggesting that pDCs could be a target for As2O3 treatment efficacy in autoimmune diseases with type-I interferon signature. We are currently running a prospective multicenter clinical trial testing As2O3 in the setting of chronic GVHD (ClinicalTrials.gov Identifier: NCT02966301). DisclosuresMohty:MaaT Pharma: Consultancy, Honoraria.

Effect of As2O3 on colorectal CSCs stained with ALDH1 in primary cell culture in vitro.

Colorectal carcinoma (CRC) is one of the most common types of malignant tumor in humans, and its morbidity is on the increase in economically transitioning countries. Due to its high toxicity, the use of the Chinese Traditional Medicine arsenic (As2O3) is limited. However, certain studies have reported that As2O3 induces differentiation of tumor cells, promotes tumor cell apoptosis and inhibits tumor cell proliferation, although the number of studies on the effects of As2O3 on cancer stem cells (CSCs) of CRC is limited. In order to research the effects of different concentrations of As2O3 on CRC cells and colorectal CSCs in vitro, aldehyde dehydrogenase 1 (ALDH1) was sued to stain the cytoplasm of colorectal CSCs and DAPI was used to stain the nuclei of all tumor cells. Through observing the effect of different concentrations of As2O3 on CRC cells and colorectal CSCs, it was demonstrated that a sufficient concentration of As2O3 clearly inhibited the conversion from colorectal CSCs to CRC cells and increased the density of CSCs.

Metabolite Profiles of Arsenic Tolerant Plants Regenerated from Stem Calli of Andrographis paniculata (Burm.f.) Nees using HPLC and 1D 1H NMR

In vitro culture provide a suitable condition for regeneration of arsenic tolerant plants from stem calli of Andrographis paniculata (Burm.f.) Nees. The regenerated plants could be valuable material applicable to soil remediation. In vitro culture of stem explants on MS basal salts, 3.0% sucrose, 0.8% agar medium fortified with various concentrations of As2O3 (0.0-9.0 µM) along with NAA (3.5 mg L-1) and KIN (1.0 mg L-1) influenced resistant callus formation. Growth of callus was slightly inhibited with increased resistance up to 22% (53±0.8 mg fwt and 27±1.5 mg dwt) on 7.0 µM As2O3 selective medium. The resistant callus line inoculated on MS medium supplemented with different concentrations of As2O3 along with BA (2.5 mg L-1) and NAA (3.0 mg L-1) induced the development of shoots. Shoot organogenesis was slightly inhibited by arsenic metal stress. However the growth tolerance has increased up to 14.5% on medium with 7.0 µM As2O3. The rate of adventitious rooting of plantlets was gradually decreased with more tolerance (11.1±1.4 rootlets per plantlets) in 7.0 µM As2O3 selected media. After acclimatization, about 40% of plants were survived as arsenic tolerance than control plants in pots containing soil treated with 7.0 µM As2O3 solutions. The level of arsenic detectability was 0.96 ppb and 4.67 ppb in control and 7.0 µM As2O3 treated plants, respectively by AAS analysis. Moreover, the production of Andrographolide was found quite high (4.41 mg/g) in tolerant plants grown at 7.0 µM As2O3 treatment than control by HPLC analysis. 1D 1H NMR profile revealed the metabolic changes significantly in control and 7.0 µM As2O3 treated plant samples. This is the first report confirming the suitability of in vitro selection for obtaining of vigorous and proliferative clones of A. paniculata plants tolerant to elevated arsenic concentration.

Arsenic trioxide increases expression of secreted frizzled-related protein 1 gene and inhibits the WNT/β-catenin signaling pathway in Jurkat cells.

The aim of the present study was to investigate the demethylation effect of arsenic trioxide (As2O3) on the secreted frizzled-related protein 1 (SFRP1) gene and its ability to inhibit the Wingless-type MMTV integration site family (WNT) pathway in Jurkat cells. Methylation-specific polymerase chain reaction was used to examine the CpG island methylation status of the SFRP1 gene in leukemia cell lines. In addition, the effects on Jurkat cells of treatment with different concentrations of As2O3 for 48 h were investigated. Reverse transcription-quantitative polymerase chain reaction was employed to measure the expression of mRNAs, while western blot analysis was used to examine protein expression in cells. The SFRP1 gene was methylated in Jurkat cells. However, both methylated and unmethylated SFRP1 genes were detected in HL60 and K562 cells. In normal bone marrow mononuclear cells, the SFRP1 gene was unmethylated. Following treatment with As2O3 for 48 h, the SFRP1 gene was demethylated, and the mRNA and protein expression levels of the SFRP1 gene were increased. By contrast, the mRNA and protein expression levels of β-catenin and cyclin Dl were downregulated. The protein expression of c-myc was also downregulated, but As2O3 exhibited no significant effect on the mRNA expression of c-myc. Abnormal methylation of the SFRP1 gene was detected in Jurkat cells. These results suggest that As2O3 activates SFRP1 gene expression at the mRNA and protein levels in Jurkat cells by demethylation of the SFRP1 gene. Furthermore, they indicate that As2O3 regulates WNT target genes and controls the growth of Jurkat cells through the WNT/β-catenin signaling pathway.

Effect of arsenic trioxide on cortical neuronal migration of rats and its potential mechanism

Objective To explore the effect of arsenic trioxide (As2O3) on the migration of neurons and the potential mechanism through the establishment of primary neuron culture from the brains of neonatal rats. Methods Brain tissues were selected from SD neonatal rats for primary neuron calture.The cells were divided into 4 groups based on the addition of As2O3: normal control group, 1 μmol/L As2O3 group, 10 μmol/L As2O3 group and 20 μmol/L As2O3 group.The primary neurons were treated with different concentrations of As2O3 and cultured for 24 hours.Boyden chamber assay was used to detect the effect of As2O3 on neuronal migration.Immunofluorescence laser confocal microscope was used to observe the structure of actin. Results In the control group, the cultured neurons showed a regular pattern of distribution.In the 3 groups treated with As2O3, the distribution of neurons was loose and disordered, which was most obvious in the 20 μmol/L As2O3 group.The results showed that the higher concentration of As2O3, more difficult it was for the neurons to survive.The number of neuronal migration was 64.6±4.3 for normal control group, 63.0±7.0 for 1 μmol/L As2O3 group, 54.8±3.6 for 10 μmol/L As2O3 group, and 21.6±3.9 for 20 μmol/L As2O3 group.The results showed that As2O3 might inhibit the migration of primary neurons in a dose-dependent manner (F=49.31, P<0.001). The normal actin skeleton was destroyed under the laser confocal microscope in 10 μmol/L As2O3 group and 20 μmol/L As2O3 group, while they remained unaffected in normal control group and 1 μmol/L As2O2 group. Conclusion As2O3 exposure can reduce the neuron migration in a dose-independent manner probably through disrupting the organization of acting cytoskeleton. Key words: Arsenic trioxide; Neuron migration; Actin cytoskeleton

The Effect of Decitabine Combined with Arsenic Trioxide on DAPK Gene and HL-60 Cell Proliferation and Apoptosis

Purpose: Our study was to detect the effect of Decitabine (DAC) combined with arsenic trioxide (AS2O3) on DAPK gene and HL-60 cell proliferation and apoptosis. Methods: DAC and AS2O3 monotherapy, combination treatment and DAC pretreatment were used in this study after incubating with HL-60 cell for 24 h, 48 h, 72 h. CCK8 was used to detect the cell proliferation of HL-60 cell. Flow cytometry was used to detect the cell apoptosis. Then, we used RT-PCR to obtain the gene expression level of DAPK. Results: HL-60 cells were treated with different concentrations of DAC (20 μmol/L, 40 μmol/L, 80 μmol/L), AS2O3 (1 μmol/L, 2.5 μmol/L, 5 μmol/L) monotherapy for 24 h, 48 h, 72 h; along with the extension of the drug concentration and time, proliferation inhibition rate had gradually increased. Monotherapy of DAC, AS2O3 could inhibit the proliferation and induce apoptosis of HL-60 cells, and was time- and dose-dependent. DAC (80 μmol/L) was firstly used for pretreatment, and then, different concentrations of AS2O3 (1 μmol/L, 2.5 μmol/L, 5 μmol/L) were used for 24 h, 48 h, 72 h. It was found that cell proliferation inhibition rate and apoptosis rate had increased significantly. When the two drugs were used together, the increasing proliferation inhibition rate, apoptosis rate and DAPK had become more obvious. Conclusion: DAC and AS2O3 had a synergetic effect for the HL-60 cell proliferation inhibition, apoptosis and expression of DAPK.

Anti-apoptotic and apoptotic pathway analysis of arsenic trioxide‑induced apoptosis in human gastric cancer SGC-7901 cells.

The present study aimed to investigate the effect of arsenic trioxide (As2O3) on human gastric cancer SGC-7901 cells. SGC-7901 cells were treated with different concentrations of As2O3 in the cell growth media for 24, 48 and 72h, and the growth rates were determined by WST-1 cell proliferation assays. Analyses of nuclear morphological changes were performed with DAPI fluorescence staining. Cell apoptosis rates as assessed by flow cytometry were determined after cells were grown in media for 48h containing different As2O3 concentrations. The protein expression patterns of the apoptosis factors, Bax, Fas and caspase-8, and anti-apoptosis factors, Akt, p-Akt, mTOR and p-mTOR, were evaluated by western blot analysis following treatment of the cells with different As2O3 concentrations in the cell growth media for 48h. As a result, As2O3 inhibited the growth of human gastric cancer SGC-7901 cells in concentrations >5µmol/l for longer than 24h. Flow cytometric analysis revealed that the apoptosis of SGC-7901 cells occurred in an As2O3 concentration-dependent manner after 48h (P<0.001). Expression levels of Bax, Fas and caspase-8 were increased, whereas expression levels of Akt, p-Akt, mTOR and p-mTOR were decreased in the SGC-7901 cells after a 48-h incubation with different As2O3 concentrations. In conclusion, As2O3 induced human gastric cancer SGC-7901 cell apoptosis in a time- and concentration dependent manner by inhibiting the activity of anti-apoptosis-related factors.

The inhibitory effect of As₂O₃ combined with phorbol ester on the proliferation of Kasumi-1 cells and its mechanism

To investigate the inhibitory effect of arsenic trioxide (As₂O₃) combined with tetradecanoylphorbol acetate (TPA) on the proliferation of Kasumi-1 cell line and its mechanism. Kasumi-1 cells were treated with 200 nmol/L TPA, different concentrations of As₂O₃ alone and combined with 200 nmol/L TPA. The proliferative inhibition rates were determined with CCK-8. Annexin V was adopted to detect apoptosis. Colony formation assay was used to determine the cloning efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38. The proliferation inhibition rates of Kasumi-1 cells by TPA combined with different concentrations of As₂O₃ (0.2, 2.0 and 20.0 mmol/L)for 48 h were (25.56 ± 7.29)%, (60.63 ± 6.64)%, and (73.37 ± 2.15)%, the apoptosis rates were (61.65 ± 2.62)%, (75.39 ± 1.04)%, and (89.95 ± 1.46)%, and the colony formation rates were (76.17 ± 2.06)%, (38.50 ± 1.87)%, and (18.53 ± 2.20)%, respectively, compared with the different concentrations of As₂O₃ alone groups, the difference was statistically significant (P<0.05). Cells treated with both TPA and As₂O₃ expressed more CD11b antigens compared with the cells exposed to As₂O₃ alone. TPA treated Kasumi-1 cells were arrested at G1 phase compared with the control group, while As₂O₃ increased the percentage of Kasumi-1 cells in the G2 phase. Combination treatment increased the expression of p-P38 of Kasumi-1 cells compared with the cells exposed to As₂O₃ alone. TPA can enhance the effect of As₂O₃ on inducing apoptosis and regulating cell cycle, thereby enhancing its anti-leukemia effect.

Effects of arsenic trioxide on the methylation of TMS1 gene in K562 cells

To detect the methylation status of TMS1 gene and its demethylation by arsenic trioxide (As₂O₃) in K562 cells. K562 cells were treated with different concentrations of As₂O₃ for 48 hours. Methylation-specific PCR (MSP) was used to determine the methylation status of TMS1. RT-PCR and Western blot were used to detect the levels of TMS1 mRNA and protein. TMS1 associated apoptosis proteins Bcl-2/Bax were also analyzed by Western blot. Apoptosis were evaluated by flow cytometry using Annexin V/propium iodide (PI) double staining. TMS1 gene was completely methylated in K562 cells and the levels of TMS1 mRNA and protein were low (0.01±0.01, 0.09±0.02), which could be reversed (mRNA: 0.72±0.04; protein: 1.30±0.06; P<0.01) by 2 μmol/L As2O3 via overt demethylation of TMS1 gene. Apoptosis in experiment group (12.24±1.06) was significantly higher than that in control group (2.05±0.16, P<0.05). In experiment group, the down-expression of antiapoptotic protein Bcl-2 and up-expression of pro-apoptotic protein Bax led to an obvious decline ratio of Bcl-2/Bax (0.56±0.12), as compared to the control group (1.94±0.14, P<0.01). As₂O₃ could up-regulate TMS1 gene expression by reversing its hypermethylation and induced apoptosis by down-regulation of Bcl-2/Bax ratio in K562 cells.

Inhibitory mechanism on growth of MA-891 cells by arsenic trioxide

To investigate the apoptosis in MA-891 cells induced by different concentrations of As2O3 and to study its influence on the activity of telomerase and telomerase mTERT-mRNA, which provide theoretical and experimental basis for breast carcinoma. The MA-891 breast carcinoma cell lines were used as the object. Different concentration of As2O3 was cultivated with MA-891 cells, and the growth inhibition of MA-891 cells was analyzed by MTT. The rate of apoptosis in MA-891 cells was detected by flow cytometry. The telomerase activity was determined by TRAP-PAGE-SILVER staining and was analyzed by specific soft ware. The expression mTERT-mRNA was examined by RT-PCR assay in untreated and As2O3-treated cells. As2O3 could inhibit the growth of MA-891 cells remarkably; the IC50 value of As2O3 for MA-891 was 9.68 micromol x L(-1) and 5.50 micromol x L(-1) respectively during 24,48 h. The percentage of the apoptosis in MA-891 cells was 30.21%, 48.26%, 57.43%, as the cells were treated with 5, 10, 20 micromol x L(-1) As2O3 for 24 hours. Treated with As2O3 for 24 hand 48 h, the telomerase activity of MA-891 cells was inhibited remarkably, which showed obvious time and concentration dependence. The mTERT-mRNA expression of MA-891 cells were significantly inhibited when treated with As2O3 for 24 h and 48h. As2O3 could remarkably inhibit the growth of MA-891 cells and could promote the apoptosis of the cells. Treated with As2O3, the activities of telomerase and telomerase mTERT-mRNA were inhibited remarkably and were obvious dosage-effect correlation.

Arsenic trioxide re-sensitizes ERα-negative breast cancer cells to endocrine therapy by restoring ERα expression in vitro and in vivo

Approximately one-third of breast cancers lack estrogen receptor α (ERα) because of the hypermethylation of the CpG island in the receptor's promoter. These tumors are associated with poorer histological differentiation, a higher growth fraction, are rarely responsive to endocrine therapy and have a worse clinical outcome. Thus, re-expression of ERα in ERα-negative breast cancers may restore the sensitivity of antiestrogen therapy. The ERα-negative breast cancer cell line MDA-MB-435s was treated with different concentrations of arsenic trioxide (As2O3). MS-PCR was used to detect the change in the methylation status of ERα. RT-PCR, immunohistochemistry and Western blot analyses were used to detect changes in the mRNA and protein expression of DNA methyl-transferase-1 (DNMT1) and ERα. Cell proliferation was examined using the MTT assay. A xenograft model in nude mice was used to further examine the results we observed in vitro. The ERα gene was demethylated after As2O3 treatment of MDA-MB-435s cells. RT-PCR, immunohistochemistry and Western blot analyses revealed that DNMT1 expression was inhibited and ERα was re-expressed in a concentration-dependent manner after As2O3 treatment. The MTT assay showed that cell proliferation was significantly suppressed after exposure to different concentrations of As2O3. Addition of tamoxifen (TAM) further suppressed levels of cell proliferation. In vivo, the xenograft tumor volumes of As2O3-treated mice were smaller than those observed in untreated and TAM-treated mice. Treatment with a combination of As2O3+TAM resulted in further suppression. As2O3 can act as a demethylation agent to restore ERα expression in ERα-negative breast cancer cells and re-sensitize these cells to endocrine therapy in vitro and in vivo.

Significance of intracellular arsenic trioxide for therapeutic response in acute promyelocytic leukemia

Arsenic trioxide (As2O3) is an effective drug for treatment of acute promyelocytic leukemia (APL) and malignant tumors. However, it is not commonly known to researchers that sensitivity has been associated with As2O3 concentration in target cells. Cell lines and cell strains of leukemia and solid cancer cells were treated with different concentrations of As2O3, and the concentrations were compared to apoptosis detected by FITC-annexin V and propidium iodide (PI) double staining. Results showed that intracellular and intercellular concentrations of arsenic in different cell lines differed. Our study noted that the cell lines had concentrations of arsenic trioxide in decreasing order, as follows: APL primary cell > K562 > CML primary cell > HL-60 > AML-M2 primary cell > HeLa > H-22. Higher intracellular As2O3 concentrations in cell lines APL, NB4, and K562 can be obtained by treating in culture medium with lower As2O3 concentration for longer times than the transient higher concentration. These results indicate that different leukemia and solid carcinoma cell lines have different intracellular arsenic concentrations, which correlate with different sensitivities to As2O3 in clinical treatment. The intracellular As2O3 concentration is higher; in addition, we note apoptosis, a very important observation in our study. As2O3 inhibited the growth of these cell lines significantly. Novel techniques by maintaining continuous low but effective arsenic levels inside the target leukemic cells in APL may improve the complete remission rate and overall survival with minimum cost and drug toxicity.