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

It was recently reported that arsenic trioxide (As_2O_3) can induce complete remission in patients with acute promyelocytic leukemia (APL). In this present article, the biological effect of As_2O_3 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 electrephoresis and in situ cell death detection (TUNEL), it was found that As_2O_3 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 As_2O_3 induced HeLa cell apoptosis possibly via decreasing the expression of c-myc and viral genes. HeLa cells overexpressing Bcl-2 partly resist As_2O_3 induced apoptosis, which might be relative to preventing the cells from As_2O_3 caused G2/M block, downregulation of c-myc gene expression and inhibition of viral gene expression was also noted, However, it was found that As_2O_3 at a high concentratio     

The cell-rounding activity of the vesicular stomatitis virus matrix protein is due to the induction of cell death

The matrix (M) protein of vesicular stomatitis virus (VSV) expressed in the absence of other viral components causes many of the cytopathic effects of VSV, including an inhibition of host gene expression and the induction of cell rounding. It was recently shown that M protein also induces apoptosis in the absence of other viral components. This raises the possibility that the activation of apoptotic pathways causes the inhibition of host gene expression and cell rounding by M protein. To test this hypothesis, host gene expression and cell rounding were analyzed after the transfection of M mRNA into HeLa cells stably overexpressing Bcl-2 (HeLa-Bcl-2 cells). We have shown previously that Bcl-2 inhibits M-protein-induced apoptosis. Here, we show that activation of the apoptotic pathways downstream of Bcl-2 is not required for the inhibition of host gene expression by M protein. In contrast, overexpression of Bcl-2 inhibited cell rounding induced by M protein, indicating that apoptotic pathways downstream of Bcl-2 are required for the cell-rounding activities of M protein.     

Tetraarsenictetrasulfide and Arsenic Trioxide Exert Synergistic Effects on Induction of Apoptosis and Differentiation in Acute Promyelocytic Leukemia Cells

Since arsenic trioxide (As³⁺) has been successfully used in the treatment of acute promyelocytic leukemia (APL), its adverse effects on patients have been problematic and required a solution. Considering the good therapeutic potency and low toxicity of tetraarsenictetrasulfide (As₄S₄) in the treatment of APL, we investigated the effects of combining As₄S₄ and As³⁺ on the apoptosis and differentiation of NB4 and primary APL cells. As₄S₄, acting similarly to As³⁺, arrested the G₁/S transition, induced the accumulation of cellular reactive oxygen species, and promoted apoptosis. Additionally, low concentrations of As₄S₄ (0.1–0.4 μM) induced differentiation of NB4 and primary APL cells. Compared with the As₄S₄- or As³⁺-treated groups, the combination of As₄S₄ and As³⁺ obviously promoted apoptosis and differentiation of NB4 and primary APL cells. Mechanistic studies suggested that As₄S₄ acted synergistically with As³⁺ to down-regulate Bcl-2 and nuclear factor-κB expression, up-regulate Bax and p53 expression, and induce activation of caspase-12 and caspase-3. Moreover, the combination of low concentrations of As₄S₄ and As³⁺ enhanced degradation of the promyelocytic leukemia-retinoic acid receptor α oncoprotein. In summary, As₄S₄ and As³⁺ synergistically induce the apoptosis and differentiation of NB4 and primary APL cells.     

Rosmarinic acid failed to suppress hydrogen peroxide-mediated apoptosis but induced apoptosis of Jurkat cells which was suppressed by Bcl-2

Rosmarinic acid (RosA), frequently found as a secondary metabolite in herbs and medicinal plants, has exhibited antioxidative and anti-inflammatory activities. RosA was shown to inhibit the proliferation and induce apoptosis of Jurkat T cells but the mechanism of action of RosA in apoptosis remains elusive. RosA inhibited the proliferation of Jurkat cells in a dose-dependent manner by suppressing the expression of cyclin D₃ and p21^Cip1/Waf1 and up-regulating p27^Kip1. RosA induced apoptosis of Jurkat cells in a dose-dependent manner and failed to protect them from hydrogen peroxide (H₂O₂)-mediated apoptosis. Induction of apoptosis by RosA correlated with suppression of Bcl-2 but not of Bak or PUMA. Overexpression of Bcl-2 protected Jurkat cells from both H₂O₂- and RosA-induced apoptosis by altering the ratio of anti- to pro-apoptotic members of the Bcl-2 family. In conclusion, RosA inhibited Jurkat cell proliferation by altering the expression of cyclins and cyclin-dependent kinase inhibitors and induced apoptosis most likely acting through the mitochondrial pathway and possessed no anti-oxidant properties.     

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.     

SS-A/Ro52 promotes apoptosis by regulating Bcl-2 production

SS-A/Ro52 (Ro52), an autoantigen in systemic autoimmune diseases such as systemic lupus erythematosus and Sjögren’s syndrome, has E3 ligase activity to ubiquitinate proteins that protect against viral infection. To investigate Ro52’s role during stress, we transiently knocked it down in HeLa cells by siRo52 transfection. We found that Ro52^low HeLa cells were significantly more resistant to apoptosis than wild-type HeLa cells when stimulated by H₂O₂- or diamide-induced oxidative stress, IFN-α, IFN-γ and anti-Fas antibody, etoposide, or γ-irradiation. Furthermore, Ro52-mediated apoptosis was not influenced by p53 protein level in HeLa cells. Depleting Ro52 in HeLa cells caused Bcl-2, but not other Bcl-2 family molecules, to be upregulated. Taken together, our data showed that Ro52 is a universal proapoptotic molecule, and that its proapoptotic effect does not depend on p53, but is exerted through negative regulation of the anti-apoptotic protein Bcl-2. These findings shed light on a new physiological role for Ro52 that is important to intracellular immunity.     

Inhibitory effect of phycocyanin from Spirulina platensis on the growth of human leukemia K562 cells

The effect of phycocyanin from Spirulinaplatensis on the growth of human chronic myelogenousleukemia-blast crisis K562 cells was studied bysemi-solid agar assay and cell viability measurement. Phycocyanin significantly inhibited the growth of K562cells in a dose-dependent manner. The IC50 value ofthe phycocyanin was 72.5 mg L^-1. After the K562cells were cultured with phycocyanin for 6 days, flowcytometric assays showed that more K562 cells wereblocked to progress through S-phase and arrested at G1phase. DNA fragmentation assay indicated that therewas no ladder of DNA fragments of approximately 200-bpmultiples, indicating that apoptosis had not occurred. Western blot analysis showed that Bcl-2 protein wasexpressed, but its level remained unchanged, whereasthe expression level of c-myc increased. Thesefindings suggest that phycocyanin may be able toinhibit the growth of K562 cells by pathways otherthan apoptosis, and that changed a expression patternof the c-myc protein may be involved in such inhibition.     

Physcion from marine-derived fungus Microsporum sp. induces apoptosis in human cervical carcinoma HeLa cells

Recently, the relationship between apoptosis and cancer has been emphasized and the induction of apoptosis is recognized as one of the key mechanisms of anti-cancer agents. Marine-derived fungi are valuable sources of structurally diverse bioactive anticancer agents. In the present study, a marine-derived fungus, Microsporum sp. was cultured and an anthraquinone derivative, physcion (11.8 mg) was isolated from the culture broth extract (1710 mg). Physcion has shown cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction in HeLa cells was investigated by the expressions of p53, p21, Bax, Bcl-2, caspase-9, and caspase-3 proteins. The Western blot analysis has revealed that physcion could significantly induce cell apoptosis through down-regulating of Bcl-2 expression, up-regulating of Bax expression, and activating the caspase-3 pathway. Furthermore, physcion induced the formation of reactive oxygen species (ROS) in HeLa cells. Collectively, these results suggest that physcion could be a potential candidate in the field of anticancer drug discovery against human cervical cancer.     

Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

Arsenic trioxide (As2O3) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB),which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As2O3 ’s access,the safe and effective therapeutic concentration of As2O3 in the CNS ought to be known. The changes of apoptosis biomarkers,[Ca2 ]i and PKC activity of both leukaemia cells and human cortical neurons,were monitored before and after being treated with As2O3 in vitro with laser confocal microscopy and Western blot. NSE concentration,the neuron invasive biomarker,was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As2O3 compared to NB4. 1.0 μmol / L As2O3 showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.     

Various tolerances to arsenic trioxide between human cortical neurons and leukemic cells

Arsenic trioxide (As₂O₃) is very effective for treatment of acute promyelocytic leukaemia (APL) but little can pass through the blood-brain-barrier (BBB), which limits its use in the prevention and treatment of central nervous system leukaemia (CNSL). Before creating a non-invasive method to help As₂O₃’s access, the safe and effective therapeutic concentration of As₂O₃ in the CNS ought to be known. The changes of apoptosis biomarkers, [Ca²⁺]_i and PKC activity of both leukaemia cells and human cortical neurons, were monitored before and after being treated with As₂O₃ in vitro with laser confocal microscopy and Western blot. NSE concentration, the neuron invasive biomarker, was monitored by enzyme immunoassay (NSE-EIA). This study revealed that cortical neuron was more tolerable to As₂O₃ compared to NB4. 1.0 μmol / L As₂O₃ showed little influence on cortical neuron but effectively promoted apoptosis and induced differentiation of NB4.     

JWA enhances As<Subscript>2</Subscript>O<Subscript>3</Subscript>-induced tubulin polymerization and apoptosis via p38 in HeLa and MCF-7 cells

Arsenic trioxide (As₂O₃) has potential anti-cancer activity against a wide range of carcinomas via apoptosis induction or oncoprotein degradation. The mechanisms involved are not fully elucidated. Here, we demonstrated that As₂O₃ induced-apoptosis in HeLa and MCF-7 cancer cells was in part triggered by tubulin polymerization. High expression of JWA promoted tubulin polymerization and increased the sensitivity of the cancer cells to As₂O₃. The activation of the p38 MAPK (mitogen-activated protein kinases) signaling pathway was found to contribute to JWA-promoted tubulin polymerization. Our results suggest that JWA may serve as an effective enhancer of microtubule-targeted As₂O₃ anti-cancer therapy.     

Induction of apoptosis in human cervical carcinoma HeLa cells by neoechinulin A from marine-derived fungus Microsporum sp.

Recently, the relationship between apoptosis and cancer has been emphasized and the induction of apoptosis is recognized as one of the key mechanisms of anti-cancer agents. Marine-derived fungi are valuable sources of structurally diverse bioactive compounds with anticancer activity. In the present study, a marine-derived fungus, Microsporum sp. was cultured and a prenylated indole alkaloid, neoechinulin A was isolated from the culture broth extract. Neoechinulin A has shown cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction in HeLa cells was investigated by the expressions of p53, p21, Bax, Bcl-2, Caspase 9, and Caspase 3 proteins. Western blot analysis has revealed that neoechinulin A could induce cell apoptosis through down-regulating of Bcl-2 expression, up-regulating of Bax expression, and activating the caspase-3 pathway. Collectively, these results suggest that neoechinulin A could be a potential candidate in the field of anticancer drug discovery against human cervical cancer.     

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.     

Synthesis of novel dibenzoxanthene derivatives and observation of apoptosis in human hepatocellular cancer cells

We have synthesized dibenzoxanthene derivatives 2a-2i via nucleophilic substitution of methoxyl group and evaluated underlying antitumor molecular mechanism of target compounds. Compounds showed high cytotoxic activities against BEL-7402, A549, HeLa and MG-63 cancer cells in the µM range. These compounds inhibited the cell growth of BEL-7402 cells at S or G2/M phase. The compounds 2a-2i also induced the apoptosis of BEL-7402 cells. In addition, compounds enhanced the level of intramolecular ROS and decreased the mitochondrial membrane potential. Western blot analysis showed caspase-3 were activated and the expression of Bcl-2 and Bcl-xl was down-regulated. According to given results, these dibenzoxanthenes exhibited a broad spectrum of antiproliferative effects on various tumors and therapeutic efficacy. Molecular mechanism indicated that induction of apoptosis was associated with DNA fragmentation, ROS generation, mitochondria dysfunction. Compounds induced apoptosis in BEL-7402 cells through the intrinsic ROS-mediated mitochondrial pathway.     

A proteomic investigation into the human cervical cancer cell line HeLa treated with dicitratoytterbium (III) complex

Lanthanides have been reported to induce apoptosis in cancer cell lines. Human cervical cancer cell line HeLa was found to be more sensitive to dicitratolanthanum (III) complex ([LaCit₂]³⁻) than other cancer cell lines. However, the effect and mechanism of dicitratoytterbium (III) complex ([YbCit₂]³⁻) on HeLa cells is unknown. Using biochemical and comparative proteomic analyses, [YbCit₂]³⁻ was found to inhibit HeLa cell growth and induce apoptosis. Similar to the effects of [LaCit₂]³⁻, proteomics results from [YbCit₂]³⁻-treated cells revealed profound changes in proteins relating to mitochondria and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [YbCit₂]³⁻-induced apoptosis. This was confirmed by the decreased mitochondrial transmembrane potential and the increased generation of reactive oxygen species in [YbCit₂]³⁻-treated cells. Western blot analysis showed that [YbCit₂]³⁻-induced apoptosis was accompanied by the activation of caspase-9 and specific proteolytic cleavage of PARP, leading to an increase in the pro-apoptotic protein Bax and a decrease in the anti-apoptotic protein Bcl-2. These results suggest a mitochondrial pathway of cell apoptosis in [YbCit₂]³⁻-treated cells, which will help us understand the molecular mechanisms of lanthanide-induced apoptosis in tumor cells.     

Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death

To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H₂O₂-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H₂O₂, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak-1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H₂O₂-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 μM H₂O₂ was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 μM H₂O₂. Meanwhile, H₂O₂ induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulation of Bcl-2 (P < 0.05). When lymphocytes were pretreated with Se before treated with H₂O₂, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P < 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulated Bcl-2 (P < 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress.     

(−)‐Epicatechin rescues the As2O3‐induced HERG K+ channel deficiency possibly through upregulating transcription factor SP1 expression

(?)‐Epicatechin (EPI) has beneficial effects on the cardiovascular disease. The human ether‐a‐go‐go‐related gene (HERG) potassium channel is crucial for repolarization of cardiac action potential. Dysfunction of the HERG channel can cause long QT syndrome type 2 (LQT2). Arsenic trioxide (As₂O₃) has shown efficacy in the treatment of acute promyelocytic leukemia. However, As₂O₃ can induce the deficiency of HERG channel and cause LQT2. In this study, we examined whether EPI could rescue the As₂O₃‐induced HERG channel deficiency. We found that 3 μM EPI obviously increased protein expression and current of HERG channel. EPI was able to recover the protein expression and current of HERG channel disrupted by As₂O₃. EPI was able to increase the expression of SP1 protein and recover the expression of SP1 protein disrupted by As₂O₃. In addition, EPI significantly shortened action potential duration prolonged by As₂O₃. Our data suggest that EPI rescues As₂O₃‐induced HERG channel deficiency through upregulating SP1 expression.     

Enhancement of esculetin on arsenic trioxide-provoked apoptosis in human leukemia U937 cells

In order to overcome chemotherapy resistance, many laboratories are searching for agents that increase the sensitivity of cancer cells to anticancer drugs. Arsenic trioxide (As₂O₃) is widely used in treating human acute polymyelocytic leukemia (APL). However, solid tumors and other leukemia cells such as U937 promonocytic leukemia cells are insensitive to As₂O₃. Esculetin, a coumarin derivative, has previously induced cell cycle arrest and apoptosis of HL-60 cells as well as enhanced taxol-induced apoptosis in HepG2 cells, thereby displaying anticancer potential. In this study, esculetin inhibited proliferation and mitogen activated protein kinases (MAPKs) activation in human leukemia U937 cells. Since inhibitors of MAPKs have modulated the GSH-redox state and enhanced the sensitivity of leukemia cells to As₂O₃-provoked apoptosis, we monitored the effect of combining esculetin and As₂O₃ (2.5 μM) on the GSH level. Our study showed that esculetin, PD98059 (MEK/ERK inhibitor), and SP600125 (JNK inhibitor) similarly enhanced the As₂O₃-induced GSH depletion. We found that the As₂O₃ (2.5 μM) treatment slightly induced apoptosis and the pretreatment of esculetin enhanced the As₂O₃-provoked apoptosis significantly. In addition, esculetin enhanced the effect of As₂O₃ on caspase activation in U937 cells. We compared the combined esculetin and As₂O₃ treatment to the As₂O₃ treated alone. The combined esculetin and As₂O₃ treatment increased Bid cleavage, Bax conformation change and cytochrome C release. The study also indicated that esculetin enhanced the As₂O₃-induced lysosomal leakage and apoptosis. Furthermore, pretreatment with N-acetylcysteine (NAC) reduced these enhanced effects. Based on these studies, esculetin enhances the As₂O₃-provoked apoptosis by modulating the MEK/ERK and JNK pathways and reducing intracellular GSH levels. GSH depletion led to higher oxidative stress which activated lysosomal-mitochondrial pathway of apoptosis.