As2O3 In Combination Research Articles

Effects of As2O3 and Resveratrol on the Proliferation and Apoptosis of Colon Cancer Cells and the hERG-mediated Potential Mechanisms.

: Background:As2O3 and resveratrol have been widely considered to be effective in anti-cancer therapies and the underlying mechanisms have been reported extensively. However, the combined treatment effect and potential target of As2O3 and resveratrol in the treatment of tumors remains elusive. The purpose of this study was to investigate the benefits and efficacy of As2O3 in combination with resveratrol in the treatment of colon cancer, as well as looking for new targets that could provide alternative explanation of the efficacy of drugs.Methods:The proliferation of cancer cells was measured by the MTT and EdU staining assay, while the apoptosis of cancer cells was determined by the flow cytometry. Western blot and immunoprecipitation were performed to measure the expression levels of proteins and the interaction between hERG and integrin β1, respectively.Results:In this study, we found that both As2O3 and resveratrol can effectively inhibit cell proliferation and promote cell apoptosis in colon cancer, and the combined effect of the two drugs on colon cancer cells is more preeminent. The combination of As2O3 with resveratrol, on the one hand reduced the expression of hERG channels on the membrane, and on the other hand weaken the binding between hERG and integrin β 1, which may be the main cause of downstream signaling pathways alterations, including the activation of the apoptotic pathway.Conclusion:Taken together, hERG, as a subunit of potassium ion channel on the cell membrane, is highly likely to be involved in the As2O3 and resveratrol induced intracellular signaling cascade disorder, and this novel signaling pathway that sustains the progression of colon cancer may be a promising therapeutic target for human colon cancer treatment in the future.

A Long-Term Retrospective Analysis of Arsenic-Containing Triple-Agents Treatment for Acute Promyelocytic Leukemia (APL) in a Cohort from a Single Hospital

Objective: The long-term efficacy (June 2001 - May 2018) of newly diagnosed APL patients (pts) treated with either As4S4 or As2O3 in combination of all-trans retinoid acid (ATRA), and anthracycline-based chemotherapy in Lu Dao-Pei Hospital is analyzed.Methods: Firstly, 8 years from June 2001 to December 2009, a total of 88 newly diagnosed with APL patients were divided into two groups, who were treated with a protocol of either oral(po) As4S4 (n=42) or intravenous (IV) arsenic trioxide (ATO) (n=46) combined with ATRA and anthracycline-based chemotherapy. Secondly, 17 years from June 2001 till June 2018, 63 cases of newly diagnosed with APL patients were all treated with the protocol of oral As4S4 based triple-agents treatment.The oral As4S4 and IV ATO treatment protocols were as follows. In the first month of induction, either As4S4 60mg/kg/day po or ATO 0.15mg / kg IV × 30 days in combination with ATRA 45mg / m2/day po × 30 days and NVT1.5-3mg / kg IV × 5-7 days were given. This was followed by consolidation chemotherapy 3 to 5 cycles after CR, consistence with : ① IDA12mg/m2 IV×3d; ② Aar-C 75mg/m2 IV×7d + ACLA 14mg/m2×7d; ③ NVT 6mg/kg×3d; ④ Ara-C 75mg/m2×7d + HHT 1.4mg/m2×7d; ⑤MD-Ara-C (the fifth was only given to pts with WBC>10×109/L before treatment). After which, maintenance treatment was given either with As4S4 60mg/kg po or ATO 0.15mg / kg IV ×15d every 30 days. Both groups were also given ATRA 45mg/m2 po×15d every 30 days. The interval was increased by more than 15 days every half year. The total treatment duration lasted 3-4 years.Results:(1) All of the two groups who were treated with a protocol of either oral As4S4 (n=42) or IV arsenic trioxide (ATO) (n=46) from 2001 to 2009 reached 100% of molecular complete remission (MCR). The 8-year estimated disease-free survival (DFS) rate was 100% in oral As4S4 group with a median follow-up of 66 (15-109) months(mos). In ATO group, the DFS rate was 87.1% with a median follow-up of 42 (14-102) mos. The difference of the DFS rate between both groups was statistically significant (p=0.028, Log-Rank test).(2) In view of the outstanding DFS result in oral As4S4 group, after 2009, another 21 APL patients were treated with the protocol of oral As4S4. So in total 63 patients treated with the protocol of oral As4S4, there were 9 children and 54 adults, 24 cases with high WBC>10×109/L before treatment, 11 cases of additional chromosomal abnormalities other than t(15;17), 9 cases of central nervous system leukemia(CNSL), and 2 cases of FLT3-ITD positive in the 22 cases with FLT3 results. All the patients attained hematological complete remission (HCR) after induction therapy without treatment-related death and achieved MCR. The median duration of MCR time was 2 (1-7) mos. The long-term overall survival (OS) rate was 98.4%(Fig1) (one died from endometrial carcinoma), and DFS rate was 100%(Fig2) (With 2 cases relapsed, respectively because of hematological and CNS, of which the current DFS time were 40mos and 30mos after CR again). The median follow-up time was 124 (21-198) mos.Conclusions: APL patients treated with either oral As4S4 or IV As2O3 combined with ATRA and anthracycline-based chemotherapy can obtain 100% MCR and high long-term DFS. Compared with IV ATO, patients in oral As4S4 group achieved significantly better efficacy, with a 17-year DFS of 100%. Moreover, oral As4S4 is easy to give. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Additive antitumor effect of arsenic trioxide combined with intravesical bacillus Calmette–Guerin immunotherapy against bladder cancer through blockade of the IER3/Nrf2 pathway

BackgroundAs an inorganic compound used to treat various cancers and other diseases, arsenic trioxide (As2O3) has been reported to induce cellular apoptosis in certain kinds of cancers including bladder cancer. The aim of the present study was to elucidate the crucial cooperative role of As2O3 and intravesical bacillus Calmette-Guerin (BCG) immunotherapy and its ability to protect against bladder cancer by targeting the IER3/Nrf2 pathway. MethodInitially, an orthotopic bladder cancer model was established in mice by means of intravesical instillation of the human bladder cancer cell line 5637. The expression of IL-6/IL-8 in dendritic cells (DCs) and the proportion of CD4+ cells and ratio of CD4+/CD8+ T cells were subsequently determined. RT-qPCR and Western blot assay methods were employed to determine the expressions of IER3, Nrf2, NQO1, IL-6 and IL-8. Finally, tumor cell apoptosis and the volume and weight of the in vivo tumors were evaluated in an attempt to determine the contributory role of As2O3 in combination with BCG immunotherapy in treating bladder cancer. ResultsThe additive effect of As2O3 and BCG was demonstrated to promote the expressions of IL-6/IL-8 among DCs. Additionally, the proportion of CD4+ cells, ratio of CD4+/CD8+ T cells and rate of tumor cell apoptosis were all elevated, while decreased in vivo tumor volume and weight were detected. Of importance, we determined the role that ad-shNrf2 (adenoviral vectors expressing shRNA against Nrf2) played in inhibiting the effects of As2O3 on bladder cancer. ConclusionTaken together, the key findings of the present study provide evidence defining the effect of As2O3 on inducing the inhibitory effect of BCG on the development of bladder cancer via the IER3/Nrf2 pathway, highlighting the potential of As2O3 as a treatment option for bladder cancer through its enhancement of intravesical BCG.

Stimulatory Effect of Indolic Hormone on As2O3 Cytotoxicity in Breast Cancer Cells: NF-κB-dependent Mechanism of Action of Melatonin.

The advent of combination therapy unprecedentedly shifted the paradigm of cancer treatment by reconstructing the conventional protocols. By identifying the anti-tumoral activity for different natural products, recent interest has focused on inventing the combined- modality strategies to increase the cure rates of cancer, while reducing the toxic side effects of current intensive regimens. To evaluate whether melatonin, indolic hormone produced mainly by the pineal gland, could enhance the pro-apoptotic effect of arsenic trioxide (As2O3) in breast cancer, MCF-7 cells were treated with As2O3-plus- melatonin and then the survival, proliferative rate, caspase-3 activity, and mRNA expression level of anti- apoptosis target genes of NF-κB were investigated. Our results delineated that exposure of MCF-7 cells to As2O3 not only reduced the survival of the cells, but also induced a caspsase-3-dependent apoptotic cell death. Noteworthy, an enhanced induction of apoptosis was found using As2O3 in combination with melatonin. Moreover, RQ-PCR analysis revealed that the enhanced cytotoxic effect of As2O3 in the presence of melatonin is mediated, at least partly, through suppressing the expression of NF-κB anti-apoptotic target genes such as MCL-1, BCL-2, survivin, XIAP, and c-IAP1 in breast cancer cells. The resulting data showed that As2O3, either alone or in combination with melatonin, exerted significant cytotoxic effect against MCF-7 cells. However, further investigations are needed to provide valuable clues for expediting this combination as a therapeutic strategy for breast cancer.

HDAC Inhibitors Lead to Significant Survival Benefit in a Novel Fusion Protein TBLR1-Rarα Induced Murine Promyelocytic Leukemia Model

Introduction: TBLR1-RARα is the tenth fusion gene of acute promyelocytic leukemia (APL) first identified in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation in our previous study. The characteristics of its basic structure and functions had been clarified in our previous study. In this study, we successfully established a novel TBLR1-RARα leukemia mouse model (TR mouse) which fully recapitulated the most relevant features of human APLs. The therapeutic effects of retinoic acid (ATRA), arsenic trioxide (As2O3), cytarabine (Ara-C) and histone deacetylase inhibitors (HDACi) on TR mice were examined. The differentially expressed genes (DEGs) between TR mice and normal mice were compared to explore the possible mechanisms and better therapeutic targets for this kind of APL.Methods: pMSCV-TBLR1-RARα-Flag-IRES-GFP (MSCV-TR) and pMSCV-IRES-GFP (vehicle) retroviral plasmids were constructed and transfected 293T packaging cells to produce retroviruses. Lin- cells from C57BL/6 mice bone marrow were purified and infected with MSCV-TR and vehicle retroviral supernatant. For in vitro assay, the GFP+ lin- cells sorted and incubated with or without different concentrations of ATRA were analyzed for the differentiation and proliferation capacity by cell morphology, myeloid markers (CD11b and GR-1) and colony formation assay. For the in vivo experiment, GFP+ lin- cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Cell surface markers were analyzed by flow cytometry. In treatment assays, GFP+ spleen cells from TR leukemia mice were injected intravenously into recipient mice. The mice were randomly separated into groups and received different treatment with ATRA, As2O3, As2O3 in combination with ATRA, Ara-C, Ara-C in combination with ATRA, chidamide and NL101, respectively. The percentage of GFP+ cells in peripheral blood and body weight were measured dynamically. The survival time of every group was recorded and compared. RNA-seq assay was used to identify DEGs between TR mice and normal mice.Results: In vitro assays indicated that TBLR1-RARα could either block the differentiation of HSCs at a relatively early stage or enhanced the clonogenic potential of cells. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARα expressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. The phenotype analysis revealed that the progenitor markers Sca-1, CD34 and C-kit were positive, the myeloid lineage markers Gr-1 and CD11b were also positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3,CD4 and CD8 were all negative. TR mice treated with 1.5-2.5 mg/kg ATRA alone or together with 2.0 mg/kg As2O3 didn’t survive longer than that of control group, although in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. Leukemic mice receiving Ara-C treatment had a much longer survive time. Surprisingly, HDAC inhibitors (12.5 and 25 mg/kg chidamide and 30 mg/kg NL-101) could significantly prolong the survival time of TR mice. Thousands of DEGs had been identified between TR mice and wild type mice, which were widely involved in multiple pathways and participated in various biological functions.Conclusion: The TBLR1-RARα leukemia mouse model was successfully established for the first time, and its main characteristics were clarified. Although the leukemia cells were sensitive to ATRA in vitro, TR mice didn’t benefit from ATRA or As2O3 treatment in vivo. Besides Ara-C, HDAC inhibitors, such as chidamide and NL-101 exhibited potency therapeutic values for TR mice, which provided a new strategy for this kind of refractory APL. What’ more, lots of genes that might be related with the process of leukemogenesis and new therapeutic targets for TR leukemia were identified. This model would serve as a versatile tool to study the mechanisms of leukemogenesis and help to design better strategies for APLs in further studies. DisclosuresNo relevant conflicts of interest to declare.

Differential mechanistic investigation of protective effects from imperatorin and sec-O-glucosylhamaudol against arsenic trioxide-induced cytotoxicity in vitro

Background and purposeThe clinical use of arsenic trioxide (As2O3) for treating acute promyelocytic leukemia (APL) is limited due to its severe cardiotoxicity. The possible mechanisms of As2O3-induced cardiotoxicity include DNA fragmentation, reactive oxygen species (ROS) generation, cardiac ion channel changes and apoptosis. The present study is designed to investigate the protective effects of imperatorin and sec-O-glucosylhamaudol and to explore their mechanistic involvement in As2O3-induced cytotoxicity. Experimental methodsCell viability assay, Lactate dehydrogenase (LDH) release, Acridine orange/ethidium bromide (AO/EB) double staining, Caspase-3 activity assay, ROS generation, cellular calcium levels, mRNA expression levels by qRT-PCR and protein expression levels by Western blotting were measured in H9c2 cells in combination with As2O3 and imperatorin or sec-O-glucosylhamaudol. Key resultsWe observed that H9c2 cells treated with imperatorin or sec-O-glucosylhamaudol were more resistant to As2O3-induced cell death. Both imperatorin and sec-O-glucosylhamaudol reduced H9c2 cell apoptosis, but both imperatorin and sec-O-glucosylhamaudol had no effects on Caspase-3 activity and intracellular calcium accumulation. Furthermore, imperatorin was capable of suppressing ROS generation, while sec-O-glucosylhamaudol did not show this effect. Moreover, imperatorin and sec-O-glucosylhamaudol triggered Nrf2 activation, which resulted in upregulation of downstream phase II metabolic enzymes and antioxidant protein/enzyme, probably offering cellular protection to As2O3-induced cardiotoxicity via the Nrf2 signal pathway. Conclusions and implicationsImperatorin and sec-O-glucosylhamaudol can ameliorate As2O3-induced cytotoxicity and apoptosis in H9c2 cells, the mechanisms probably related to antioxidation. As2O3 in combination with imperatorin or sec-O-glucosylhamaudol could be considered as a novel strategy to expand the clinical application of As2O3.

Arsenic trioxide reduces chemo-resistance to 5-fluorouracil and cisplatin in HBx-HepG2 cells via complex mechanisms.

BackgroundMultidrug resistance is one of the major reasons chemotherapy-based treatments failed in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). Hypoxia is generally associated with tumor chemo-resistance. The aim of the study was to investigate the effect of Arsenic trioxide (As2O3) on the hypoxia-induced chemo-resistance to 5-FU or cisplatin and explored its underlying mechanism in the HBx-HepG2 cells.MethodsMTT assay was used to examine the cell viability. Mitochondrial membrane potential (MMP) and cell cycle was examined by flow cytometry. qRT-PCR was employed to observe the mRNA expression level; and western blot assay was used to determine the protein expression level.ResultsOur results showed that transfection of HBx plasmid established the HBx-HepG2 cells expressing HBx, and the expression of HBx was confirmed by qRT-PCR and western blot. Exposure of HBx-HepG2 cells to hypoxia (5 % O2, 3 % O2, 1 % O2) for 48 h increased the chemo-resistance to 5-fluorouracil (5-FU) (50–1600 µM) and cisplatin (25–800 µM), reduced MMP, and caused the cell cycle arrest at G0/G1 phase in a concentration-dependent manner. Hypoxia also concentration-dependently (5 % O2, 3 % O2, 1 % O2) reduced mRNA expression level of P-glycoprotein (P-gp), multidrug resistance protein (MRP1), lung resistance protein (LRP), and decreased the protein expression level of hypoxia-inducible factor-1α (HIF-1α), P-gp MRP1, and LRP. Following pretreatment with As2O3 at a non-cytotoxic concentration re-sensitized the hypoxia (1 % O2)-induced chemo-resistance to 5-FU and cisplatin in HBx-HepG2 cells. As2O3 pretreatment also prevented MMP reduction and G0/G1 arrest induced by hypoxia. Meanwhile, As2O3 antagonized increase of HIF-1α protein induced by hypoxia, and it also suppresses the increase in expression levels of P-gp, MRP1, and LRP mRNA and proteins. In addition, As2O3 in combination with 5-FU treatment caused up-regulation of DR5, caspase 3, caspase 8, and caspase 9, and down-regulation of BCL-2, but had no effect of DR4.ConclusionsOur results may suggest that As2O3 re-sensitizes hypoxia-induced chemo-resistance in HBx-HepG2 via complex pathways, and As2O3 may be a potential agent that given in combination with other anti-drugs for the treatment of HBV related HCC, which is resistant to chemotherapy.

Effects of As2O3 in combination with TPA on K562 cells

This study was aimed to investigate the effects of arsenic trioxide (As2O3) combined with TPA on cell cycle, cell differentiation and apoptosis of K562 cell line, and their possible mechanisms. K562 cells were treated with 200 nmol/L TPA, 2 µmol/L As2O3 alone and 200 nmol/L TPA combined with 2 µmol/L As2O3. The proliferative inhibition rates were determined with CCK-8. Annexin V and agarose gel electrophoresis were adopted to detect apoptosis. Colony formation test was used to determine the colony-formation 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 proteins. The results showed that combination treatment had synergistic effects on the proliferative inhibition and apoptosis, which were much higher than those treated alone. As2O3 could decrease the colony formation ability of K562 cells. The cells treated with both TPA and As2O3 expressed far more CD11b antigens compared with cells exposed to As2O3 alone. K562 cells treated with TPA were arrested in G1 phase compared with the control group, As2O3 increased the percentage of K562 cells in the G2 phase. The combination treatment increased the expression of p-P38 of K562 cells compared with the cells exposed As2O3 alone. It is concluded that TPA can enhance the effect of As2O3 on inducing apoptosis and adjusting cell cycle , which will expect to provide a new therapeutic program.

Resveratrol attenuates hepatotoxicity of rats exposed to arsenic trioxide

Arsenic trioxide (As2O3) is an environmental pollutant and potent toxicant to humans. However, it also shows substantial anti-cancer activity in individuals with acute promyelocytic leukemia (APL). Unfortunately, As2O3-treated leukemia patients suffer hepatotoxicity. Resveratrol has been demonstrated to have efficient antioxidant and antineoplastic activities. The study that how As2O3 in combination with resveratrol affects hepatotoxicity and arsenic accumulation in the liver is lacking, and the present study tackles this question. Wistar rats were injected with 3mg/kg As2O3 on alternate days; resveratrol (8mg/kg) was administered 1h before As2O3. Rats were killed on the 8th day to determine histological liver damage, the antioxidant enzymes in serum, the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), and arsenic accumulation in the liver. In the resveratrol+As2O3 group, activities of superoxide dismutase, catalase in serum and GSH/GSSG were significantly increased, histopathological effects were reduced, and arsenic accumulation markedly decreased in the liver, compared with the As2O3-treated group. Thus, resveratrol attenuated As2O3-induced hepatotoxicity by decreasing oxidative stress and arsenic accumulation in the liver. These data suggest that use of resveratrol as post-remission therapy of APL and adjunctive therapy in patients with chronic exposure to arsenic may decrease arsenic hepatotoxicity.

Synergic effect of 3′-azido-3′-deoxythymidine and arsenic trioxide in suppressing hepatoma cells

The aim of this study was to investigate the synergic antitumor effects of arsenic trioxide (As2O3) and 3'-azido-3'-deoxythymidine (AZT) on hepatoma cells and explore the possible molecular basis of these effects. These results showed that AZT enhanced the inhibitory effect of As2O3 on HepG2 and SMMC-7721 cell growth. The IC50 of As2O3 in combination with AZT was lower than that of As2O3 alone. A concentration-dependent synergic effect of As2O3 and AZT (CI < 1) was observed in all the tested combinations of these compounds. These results also showed that the combination of As2O3 and AZT dramatically and significantly increased the number of apoptotic cells in HepG2 and SMMC-7721 cells. Studies in vivo showed that the combination of As2O3 and AZT was statistically superior to either As2O3 or AZT alone in the treatment of tumor-bearing mice. As2O3 (1 mg/kg) containing AZT (50 mg/kg) inhibits proliferation of implanted hepatoma 22 by 56.35%. These results suggest that treating hepatoma with a combination of As2O3 and AZT offers the advantages of reduced toxic side effects and improved therapeutic efficacy. To understand the mechanism through which As2O3 and AZT suppress tumors, we studied the effects of these compounds, both separately, and in combination, on telomerase and caspase-3 activity. The results showed that the growth inhibitory and apoptotic effects of As2O3 and AZT on human hepatoma cells could be related to the inhibition of telomerase and the activation of caspase 3.

Effects of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells

Objective To investigate the effect of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells and provide theoretical basis for clinical application.Methods Cell proliferation was assayed by analyzing the growth and viability of the cells.Apoptosis was assayed by performing cell morphology,Annexin-V/PI staining,DNA-PI staining,and DNA gel electrophoresis.Results After exposure to As2O3 and cinobufacini,the growth of K562 cells was inhibited and the viability of K562 cells was decreased. After treated with 1.0μmol/L As2O3,0.125μg/ml cinobufacini,0.25μg/ml cinobufacini,1.0μmol/L As2O3 + 0.125 μg/ml cinobufacini,1.0μmol/L As2O3 + 0.25μg/ml cinobufacini for 24 and 48 hours,the proliferation inhibition rate were(24 ± 1.3)%,(21 ± 1.5)%,(38 ± 3.1)%,(57 ±2.7)%,(66 ±3.3)% and(49 ±2.9)%,(48 ±2.7)%,(61 ±2.1)%,(77 ±3.8)%,(82 ±4.2)%,the apoptosis rate of K562 cells were(4.8 ± 0.5)%,(5.6 ± 0.7)%,(9.8 ± 0.6)%,(11.9 ± 1.2)%,(15.2±1.5)% and(11.0 ±0.9)%,(12.9 ±1.1)%,(18.4 ±1.5)%,(21.0 ±2.0)%,(28.0 ±1.9)%.The percentage of apoptotic cells was a time- and dose-dependent manner.Typical DNA ladder was shown by DNA gel electrophoresis.Conclusions As2O3 combined with cinobufacini inhibited the proliferation of K562 cells and induced apoptosis of the K562 cells,the combination of the two drugs had better effect. Key words: Arsenicals/AD; CINOBU FAGIN/AD; Leukemia,myeloid,acute/DT; Cell proliferation/DE; Apoptosis/DE

Effects and Mechanisms of FTI-277 Inhibiting Proliferation of Myeloid Leukemia Cells and Enhancing Apoptosis Induced by As2O3.

Objective:To investigate the effects and mechanisms of farnesyltransferase inhibitor FTI-277 inhibiting proliferation of myeloid leukemia cells and enhancing apoptosis induced by arsenic trioxide(As2O3).Methods:HL-60, K562 cells and BMMNCs from 31 AML patients at diagnosis, 21 CML patients at diagnosis were examined. Cell viability and cell proliferation were detected by trypan blue exclusion assay and MTT assay, respectively. Cell apoptosis were determined by light microscope, DNA agrose gel electrophoresis and annexinV-FITC/PI double-staining. Cell cycle was analyzed by flow cytometry. The expression of phospho-ERK1/2 (phospho-P44/42MAPK) and ERK2 (P42MAPK) were detected by flow cytometry and Western blot. The expression of Bcl-2 and active caspapse-3 were measured by flow cytometry.Results: FTI-277 inhibited proliferation of HL-60, K562 cells and BMMNCs from AML, CML patients at diagnosis in a time-dose dependent manner (P<0.05). However, this inhibitor had little effect on proliferation of normal cells. Treatment of myeloid leukemia cells with FTI-277 alone couldn't induce apoptosis (P>0.05). FTI-277 and As2O3 in combination significantly induced apoptosis of myeloid leukemia cells compared with either FTI-277 or As2O3(P<0.05). Furthermore, the percentage of apoptosis increased with increasing concentration of FTI-277 in the combination group (P<0.05). In addition, the apoptosis ladder and the morphological features of apoptosis were readily observed in the combination group. Incubation with FTI-277 led to an increase of myeloid leukemia cells in G2/M (P<0.05), whereas the sub-G1 fraction remained unchanged(P>0.05). Incubation of myeloid leukemia cells with FTI-277 decreased the expression of phospho-ERK1/2(phospho-P44/42MAPK)(P<0.05), whereas the expression of ERK2 (P42MAPK) in myeloid leukemia cells and the expression of phospho-ERK1/2 in normal cells were unaffected by FTI-277(P>0.05). FTI-277 had no effect on Bcl-2 expression in HL-60 and K562 cells(P>0.05). As2O3 decreased Bcl-2 expression in HL-60 cells(P<0.05), but didn't affect Bcl-2 expression in K562 cells(P>0.05). As2O3 alone or FTI-277 and As2O3 in combination significantly increased active caspapse-3 expression in HL-60 and K562 cells(P<0.05), the combination group being more significantly increased compared with As2O3 alone (P<0.05).Conclusion:FTI-277 exerts proliferation inhibition on myeloid leukemia cells by inhibiting constitutive ERK/MAPK activation and resulting in a G2/M block. FTI-277 doesn't inhibit proliferation of normal cells with low steady-state levels of ERK/MAPK activity. Combination of FTI-277 and As2O3 results in the activities of caspases and the synergistic induction of apoptosis in HL-60 cells by simultaneous targeting of the ERK/MAPK and Bcl-2 pathways. As2O3 interferes with the BCR/ABL protein signal transduction, whereas FTI-277 lowers the apoptotic threshold, thereby increasing the activation of caspase-3 and generating the synergistic induction of apoptosis in K562 cells.

Myeloperoxidase Is Required for the Combined Apoptotic Effects of Arsenic Trioxide with Ascorbic Acid or Epigallocatechin-3-Gallate in Myeloid Leukemia Cells.

Arsenic trioxide (As2O3) is a drug used world-wide that selectively causes the death of acute promyelocytic leukemia (APL) cells by novel mechanisms and induces complete clinical remission in 90% of patients without significant toxicity. The outstanding success of As2O3 therapy in relapsed APL patients has not been replicated by an equivalent success in other types of acute myeloid leukemia (AML). We have studied As2O3 mechanism(s) of action and identified agents to use in combination to improve the use of As2O3 as a treatment for other types of AML. As2O3 produces higher levels of H2O2 and apoptosis in APL NB4 cells than in other AML cells at therapeutic concentrations of 1-2 uM. As2O3 does not induce apoptosis in HL-60 cells but is synergistic with ascorbic acid (AA) or epigallocatechin-3-gallate (EGCG). Both AA and EGCG produce H2O2 which is augmented by the addition of As2O3. Apoptosis induction by As2O3 in combination with AA or EGCG is inhibited by catalase and the antioxidant N-acetylcysteine. Myeloperoxidase (MPO), a major neutrophil enzyme, augments H2O2-induced apoptosis by converting it into more potent reactive oxygen species. HL-60, NB4, SKNO-1 and PLB985 cells which express high level of MPO, but not U937 and K562 cells without expression of MPO, are responsive to As2O3 plus AA or EGCG-induced apoptosis. HP-100 cells, a subclone of HL-60 cells without MPO expression, are resistant to both combination treatments. MPO stable transfection sensitizes K562 cells to apoptosis following treatment with As2O3 and AA or EGCG. NADPH oxidase is an enzyme complex which generates O2− and H2O2 in neutrophils. In APL NB4 cells, As2O3 induces expression of NADPH oxidase members and is thought to participate in H2O2 production required for As2O3 response. X-CGD cells, a subclone of PLB-985 with targeted disruption of the gp91phox gene (Zhen et al, PNAS 90:9832, 1993) and loss of NADPH oxidase activity but with high levels of MPO activity, are responsive to treatment with As2O3 and AA or EGCG. These results suggest that AA and EGCG enhance As2O3-induced apoptosis through a synergistic production of H2O2 which is independent NADPH oxidase activation in non-APL AML cells. MPO augments the apoptotic effect of H2O2 produced by As2O3 plus AA or EGCG and it could be used as a marker to predict the sensitivity of AML cells to both combination treatments.

Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by L-buthionine sulfoximine.

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemic patients and apoptosis in various tumor cells in vitro. To develop As2O3-based combination chemotherapy for renal cell carcinoma (RCC), we investigated the cytotoxic effects of As2O3 in combination with chemotherapeutic agents or L-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor. Cytotoxicity and synergy were assessed by the MTT assay and isobolographic analysis, respectively. Apoptosis was monitored by Hoechst 33342 staining, flow cytometrical analysis, and DNA fragmentation assay. Treatment of ACHN cells with As2O3 in combination with adriamycin, vinblastine, or 5-fluorouracil induced an antagonistic effect. However, combination treatment with As2O3 and BSO resulted in a synergistic cytotoxic effect. Synergy was also obtained in Caki-1, Caki-2, NC65 cells and freshly derived RCC cells from 6 patients. Simultaneous treatment of ACHN cells with As2O3 and BSO caused significantly more cytotoxicity than the As2O3 first BSO second or the reverse treatment. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of As2O3 and BSO was realized by inducing apoptosis. This combination markedly decreased intracellular GSH content and GSH-S-transferase (GST) activity. However, neither the intracellular GSH nor GST was decreased by As2O3 with adriamycin, vinblastine, or 5-fluorouracil. Furthermore, the GSH-increasing agents N-acetylcysteine and lipoic acid significantly inhibited the combined cytotoxicity of As2O3 and BSO. These findings indicate that BSO sensitizes RCC cells to As2O3-induced apoptosis through the down-regulation of the intracellular GSH redox system, suggesting the potential application of a combination of As2O3 and BSO for the treatment of RCC.

Targeted removal of PML-RARα protein is required prior to inhibition of histone deacetylase for overcoming all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia

AbstractAll-trans retinoic acid (tRA)–induced differentiation in NB4 cells, a cell line derived from an acute promyelocytic leukemia patient with t(15;17) translocation, is markedly facilitated by sodium butyrate (NaB), a histone deacetylase inhibitor (HDACI), or by hexamethylene bisacetamide (HMBA), a non–HDACI tRA-differentiation inducer, as determined by nitroblue tetrazolium reduction. The tRA-induced expression of RIG-G, Bfl-1/A1, and p21waf1 and, to a lesser extent, of CCAAT/enhancer binding protein–ε (C/EBPε) are also enhanced by such combined treatments. Both responses are associated with a facilitated diminution of the leukemogenic PML-RARα protein and retained ΔPML-RARα, a cleavage product. Treatment with tRA in tRA differentiation–resistant NB4 subclones R4 and MR-2 does not result in PML-RARα diminution and the tested gene expressions. Moreover, the addition of HMBA or NaB with tRA results in only minimal increase of differentiation in the tRA differentiation–resistant subclones. The increases in acetylated histone H3 (AcH3) and AcH4 in NaB-treated NB4, R4, and MR-2 cells are similar and do not correlate with the extent of differentiation induction when NaB and HMBA are given in combination with tRA. Arsenic trioxide (As2O3) treatment results in the total degradation of PML-RARα without increasing AcH3 or AcH4 or inducing differentiation in R4 cells. As2O3 in combination with tRA induces gene (Bfl-1/A1 and C/EBPε) expression and partial differentiation. Both NaB and HMBA addition to As2O3-plus-tRA–treated R4 cells further enhances differentiation. These results suggest that elimination of the dominant negative PML-RARα protein is required prior to inhibition of histone deacetylase to fully overcome tRA-differentiation resistance in APL cells.

Arsenic trioxide: a new immunomodulatory agent in the management of multiple myeloma.

Arsenic has been used as a medicinal for thousands of years. Several reports from China relative to its use mainly in acute promyelocytic leukemia, especially from the Shanghai group, has caused a resurgence in the investigation of the drug in the management of malignancies with focus on malignancies of hematologic origin. Arsenic is eliminated by many routes (urine, feces, sweat, milk, hair, skin, and lungs), although most is ultimately excreted in urine. Multiple myeloma is characterized by the clonal proliferation of malignant plasma cells in the bone marrow associated with bone loss, renal disease, and immunodeficiency. Preclinical evidence suggests an immunologic mechanism behind the therapeutic effects of As2O3 on myeloma cells. This appears to be achieved by a marked increase in lymphokine-activated killers mediated killing and up-modulation of CD38 and Cd54, two molecules involved in cell-cell interactions. Moreover, As2O3 alone or administered with ascorbic acid may provide a novel therapy for lymphoproliferative disorders. Preliminary clinical data in relapsed/refractory multiple myeloma suggest that As2O3 does have a role in the management of multiple myeloma; however, preclinical data show that the addition of ascorbic acid, and using As2O3 in combination with other active chemotherapeutic agents will enhance its role in managing the disease, and this is probably the position the drug will occupy in the armamentarium against myeloma.

Treatment of Acute Promyelocytic Leukemia with Arsenic Trioxide: Clinical and Basic Studies

Arsenic trioxide (As2O3) has recently been identified as an effective drug in the treatment of newly diagnosed and relapsed acute promyelocytic leukemia (APL) without cross-resistance to all-trans retinoic acid and achieved complete remission rates of 80–90% according to most reports. With intravenous infusion at a dose of 0.08–0.16 mg/kg daily, a course of 28–42 days is required to induce remission. As2O3 in combination with chemotherapy as postremission therapy results in longer survival than arsenic alone. In vitro, As2O3 exerts dose-dependent dual effect; triggering apoptosis at relatively high concentration (0.5–2.0 μmol/1), which is associated with the disruption of mitochondrial transmembrane potentials, while inducing partial differentiation at low concentration (0.1–0.5 μmol/1), which might be related to retinoic acid signaling pathway. Importantly, at both concentrations, As2O3 can degrade PML (promyelocytic leukemia)-RARα (retinoic acid receptor), an oncoprotein that has a central role in leukemogenesis.

Role of p38 MAPK and JNK in enhanced cervical cancer cell killing by the combination of arsenic trioxide and ionizing radiation

Enhancement of radiation response is possible to be achieved by combination treatment with therapeutic drugs. Previously we have shown that As2O3 in combination with ionizing radiation enhanced radiation response in cervical cancer cells. In this study, we further investigated molecular mechanism of synergistic enhancement of radiation response in combination with As2O3. The combination treatment of HeLa cells induced translocation of Bax to the mitochondria and a marked phosphorylation of Bcl-2. p38 MAPK and JNK were also found to be activated in response to the combination treatment. Pretreatment of PD169316, a p38 MAPK specific inhibitor, completely attenuated the combination treatment-induced mitochondrial relocalization of Bax, and altered Bcl-2 phosphorylation. Moreover, pretreatment of SP600125, JNK specific inhibitor, clearly attenuated Bcl-2 phosphorylation, but did not affect Bax translocation to the mitochondria. In addition, N-acetyl-L-cysteine (NAC), a thiol-containing anti-oxidant, completely blocked p38 MAPK and JNK activations, Bax relocalization and Bcl-2 phosphorylation. These results indicate that activation of p38 MAPK is specifically required for translocation of Bax to the mitochondria, and both JNK and p38 MAPK are involved in phosphorylation of Bcl-2 in response to combination treatment with gamma-radiation and As2O3, and that ROS is a critical regulator of p38 MAPK and JNK activations. The molecular mechanism elucidated in this study may provide insight into the design of future combination cancer therapies to cells intrinsically less sensitive to radiation treatment.