Arsenicum album Induces Cell Cycle Arrest and Apoptosis, and Inhibits Epithelial-Mesenchymal Transition in Hormone-Dependent MCF7 Breast Cancer Cells.

BACKGROUND: Arsenic trioxide (As2O3) has been in therapeutic use since the 18th century for various types of cancers including skin and breast; however, it gained popularity following FDA approval for its use against acute promyelocytic leukemia. This present work was designed to evaluate the anti-cancer potential of a homeopathic potency of arsenic trioxide (Arsenicum album 6C) in hormone-dependent breast cancer. METHODS: Breast cancer cells (MCF7) were treated with Arsenicum album (Ars 6C) to evaluate its anti-proliferative and apoptotic potential. We examined the effect of Ars 6C on the cell cycle, wound healing, reactive oxygen species (ROS) generation, and modulation of expression of key genes which are aberrant in cancer. RESULTS: Treating breast cancer cells with Ars 6C halted the cell cycle at the sub-G0 and G2/M phases, which could be attributed to DNA damage induced by the generation of ROS. Apoptotic induction was associated with upregulation of Bax expression, with concurrent downregulation of the Bcl-2 gene. Ars 6C was also seen to reverse epithelial to mesenchymal transition and reduce the migration of breast cancer cells. CONCLUSION: The findings suggest that Ars has significant anti-proliferative and apoptotic potential against breast cancer cells. Further studies are required to elucidate the mechanism by which Ars exerts its effect in the in vivo setting.

Number of succussion strokes affects effectiveness of ultra-high-diluted arsenic on in vitro wheat germination and polycrystalline structures obtained by droplet evaporation method.

OBJECTIVES: The aim of this study is to investigate whether the number of succussion strokes applied after each dilution step when preparing the homeopathic treatments influences the effectiveness of ultra-high-diluted (UHD) arsenic trioxide at the 45th decimal dilution/dynamization (As2O3 45x). DESIGN: Wheat seeds, previously stressed with ponderal As2O3, were treated with: As2O3 45x, H2O 45x (dynamized control), or pure water (negative control). The succussion was done manually, and various succussion durations (numbers of strokes) were tested for each treatment. Treatment effectiveness was tested blind using the in vitro germination test and the droplet evaporation method (DEM). Data were processed by the Poisson test (germination test) and by two-way analysis of variance (DEM). MAIN OUTCOME MEASURES: We evaluated both the in vitro germination rate, by counting the non-germinated seeds, and the complexity of polycrystalline structures (PCS) (local connected fractal dimension (LCFD)) obtained by evaporating leakage droplets from stressed seeds that had been watered with the different treatments. RESULTS: We observed a highly significant increase in germination rate when the number of strokes (NS) was ≥32 for both As2O3 45x and H2O 45x, and a significant increase in the LCFD of PCS for As2O3 45x when the NS was ≥32 and for H2O 45x when it was 70. CONCLUSIONS: Both experimental approaches showed increased effectiveness for treatments prepared with a higher number of succussion strokes. These results indicate that succussion may have an important influence on treatment effectiveness, and so highlight the need for further research.

Therapeutic effect of Arsenicum album on leukocytes.

The therapeutic effects of homoeopathic Arsenicum album potencies were investigated in-vitro, using a continuous cell line (MT4), pre-intoxicated with arsenic trioxide (As(2)O(3)), and then treated with succussed and unsuccussed homoeopathic potencies, 6CH, 30CH and 200CH. This study aimed to verify the homoeopathic law of similars and to determine whether potencies diluted beyond Avogadro's constant had physiological effects on cells; whether various potencies would cause different effects as suggested by the concept of hormesis; whether succussed and unsuccussed homoeopathic potencies had different effects on the cells; and to establish whether a biotechnological method could be used to evaluate the above. As(2)O(3) was used to pre-intoxicate and the MTT assay was used to measure the percentage cytotoxicity and half maximal inhibitory concentration (IC(50)) of the cells. The homoeopathic potencies of Arsenicum album (6CH, 30CH and 200CH) were prepared by either succussing or allowing to diffuse for 30 s. After pre-intoxication of the MT4 cells with the IC(50) As(2)O(3) and treatment with succussed and unsuccussed Arsenicum album (6CH-200CH), the cell viability increased with increasing potency from 81% to 194% (over 72 h). The treatments and the times of exposure were found to be statistically significant determinants of cell viability, whereas succussion did not cause any significant variation in the results. The study provided evidence that a biotechnological method (namely cell viability) may be used to scientifically evaluate the physiological effects of homoeopathic potencies on human cells; it confirmed that the homoeopathic potencies did have therapeutic effects; and that succussion was not required in the potentization method in order to produce a curative remedy.

Potentized homeopathic drug Arsenicum Album 30C positively modulates protein biomarkers and gene expressions in Saccharomyces cerevisae exposed to arsenate.

OBJECTIVE: This study examines if homeopathic drug Arsenicum Album 30C (Ars Alb 30C) can elicit ameliorative responses in yeast (Saccharomyces cerevisiae) exposed to arsenate. METHODS: The yeast S. cerevisiae 699 was cultured in a standard yeast extract peptone dextrose broth medium. It was exposed to the final concentration of 0.15 mmol/L arsenate for two intervals, 1 h and 2 h, respectively. The cell viability was determined along with the assessment of several toxicity biomarkers such as catalase (CAT), superoxide dismutase (SOD), total thiol (GSH) and glucose-6-phosphate dehydrogenase (G6PDH), lipid peroxidation, protein carbonylation and DNA damage. Reactive oxygen species (ROS) accumulation, expressions of relevant stress transcription activators like Yap-1 and Msn 2, and mRNA expression of yeast caspase-1 (Yca-1) were also measured. RESULTS: Treatment of arsenate increased lipid peroxidation, protein carbonylation, DNA damage, ROS accumulation and expressions of Yap-1, Msn 2 and Yca-1 and decreased GSH, G6PDH, CAT and SOD. Ars Alb 30C administration decreased lipid peroxidation, protein carbonylation, DNA damage, ROS formation and Msn 2 and Yca-1 expressions and increased cell viability, GSH, G6PDH, CAT and SOD significantly (P<0.05), except for a slight increase in Yap-1 expression. CONCLUSION: Ars Alb 30C triggers ameliorative responses in S. cerevisiae exposed to arsenate.

Effects of homeopathic arsenicum album, nosode, and gibberellic acid preparations on the growth rate of arsenic-impaired duckweed (Lemna gibba L.).

This study evaluated the effects of homeopathically potentized Arsenicum album, nosode, and gibberellic acid in a bioassay with arsenic-stressed duckweed (Lemna gibba L.). The test substances were applied in nine potency levels (17x, 18x, 21x-24x, 28x, 30x, 33x) and compared with controls (unsuccussed and succussed water) regarding their influence on the plant's growth rate. Duckweed was stressed with arsenic(V) for 48 h. Afterwards, plants grew in either potentized substances or water controls for 6 days. Growth rates of frond (leaf) area and frond number were determined with a computerized image analysis system for different time intervals (days 0-2, 2-6, 0-6). Five independent experiments were evaluated for each test substance. Additionally, five water control experiments were analyzed to investigate the stability of the experimental setup (systematic negative control experiments). All experiments were randomized and blinded. The test system exhibited a low coefficient of variation (approximately equal to 1%). Unsuccussed and succussed water did not result in any significant differences in duckweed growth rate. Data from the control and treatment groups were pooled to increase statistical power. Growth rates for days 0-2 were not influenced by any homeopathic preparation. Growth rates for days 2-6 increased after application of potentized Arsenicum album regarding both frond area (p < 0.001) and frond number (p < 0.001), and by application of potentized nosode (frond area growth rate only, p < 0.01). Potencies of gibberellic acid did not influence duckweed growth rate. The systematic negative control experiments did not yield any significant effects. Thus, false-positive results can be excluded with high certainty. To conclude, the test system with L. gibba impaired by arsenic(V) was stable and reliable. It yielded evidence for specific effects of homeopathic Arsenicum album preparations and it will provide a valuable tool for future experiments that aim at revealing the mode of action of homeopathic preparations. It may also be useful to investigate the influence of external factors (e.g., heat, electromagnetic radiation) on the effects of homeopathic preparations.

A review of three simple plant models and corresponding statistical tools for basic research in homeopathy.

In this paper, we review three simple plant models (wheat seed germination, wheat seedling growth, and infected tobacco plants) that we set up during a series of experiments carried out from 1991 to 2009 in order to study the effects of homeopathic treatments. We will also describe the set of statistical tools applied in the different models. The homeopathic treatment used in our experiments was arsenic trioxide (As2O3) diluted in a decimal scale and dynamized. Since the most significant results were achieved with the 45th decimal potency, both for As2O3 (As 45x) and water (W 45x), we here report a brief summary of these results. The statistical analysis was performed by using parametric and nonparametric tests, and Poisson distribution had an essential role when dealing with germination experiments. Finally, we will describe some results related to the changes in variability, which seems to be one of the targets of homeopathic treatment effect.

Isopathic treatment effects of Arsenicum album 45x on wheat seedling growth--further reproduction trials.

BACKGROUND: Two experimental studies on wheat preintoxicated with Arsenic trioxide yielded a significant shoot growth increase after an isopathic application of Ars-alb 45x. One independent reproduction trial however, yielded an effect inversion: wheat shoot growth was significantly decreased after application of Ars-alb 45x. AIMS: In this study we investigated the role of three potential confounding factors on the experimental outcome: geographical location of the experiments, influence of the main experimenter, and seed sensitivity to Arsenic poisoning. Laboratory-internal reproducibility was assessed by meta-analysis. MATERIAL AND METHODS: Wheat poisoned with Arsenic trioxide was cultivated in vitro in either Ars-alb 45x, water 45x, or unpotentised water. Treatments were blinded and randomised. Shoot length was measured after 7 days. The stability of the experimental set-up was assessed by systematic negative control (SNC) experiments. RESULTS: The SNC experiments did not yield significant differences between the three groups treated with unpotentised water. Thus the experimental set-up seemed to be stable. We did not observe any shoot growth increase after a treatment with Ars-alb 45x in any of the newly performed experiments. In contrast, the meta-analysis of all 17 experiments performed (including earlier experiments already published) yielded a statistically significant shoot growth decrease (-3.2%, p=0.017) with isopathic Ars-alb 45x treatment. This effect was quantitatively similar across all five series of experiments. CONCLUSIONS: Ultramolecular Ars-alb 45x led to statistically significant specific effects in arsenic poisoned wheat when investigated by two independent working groups. Effect size and effect direction differ, however. The investigated factors (geographical location, experimenter, seed sensitivity to Arsenic poisoning) did not seem to be responsible for the effect inversion. Laboratory external reproducibility of basic research into homeopathic potentisation remains a difficult issue.

[Differentiation of HL-60 cells induced by realgar nano-particles].

OBJECTIVE: To investigate the proliferation inhibition and the differentiation effects of realgar (As4S4) nano-particles on human acute myeloid leukemia cell line HL-60. METHOD: Cell viability was determined by MTT and PI-stained cell cycle assays. The realgar induced morphological changes on cells were examined after Wright-Giemsa staining. The cell differentiation was evaluated with NBT and specific cell surface antigen (CD11b and CD14) expression assays. RESULT: HL-60 cells exhibited obvious morphological features of differentiation after the realgar treatment. A 24 h incubation of the cells with 0.25-1.0 micromol x L(-1) realgar caused a great increase in NBT reduction ability. The expressions of CD11b and CD14 were augmented in cells treated with 0.50 micromol x L(-1) realgar for 48 h, and cell cycles were arrested in G1 phase. CONCLUSION: Low dose realgar induces differentiation in human acute myeloid leukemia cell line HL-60.

A biostatistical insight into the As(2)O(3) high dilution effects on the rate and variability of wheat seedling growth.

BACKGROUND: Most criticism of homeopathy concerns the lack of scientific bases and theoretical models. Fundamental research could make important contributions to our understanding of the mechanisms of action of homeopathic treatments. Plant-based bioassays are suitable for basic research -- lacking the placebo effect and ensuring large data samples for structured statistical analyses. OBJECTIVE: The aim of this study was to reproduce a previous experiment on the effects of arsenic trioxide (As(2)O(3)) high dilutions on wheat seedling growth in order to verify whether the same significant results could be obtained working in a different place and with a different experimental team. A further goal was to investigate high dilution effects on variability. MATERIAL AND METHODS: A structured experiment was performed blind over 9 weeks, using wheat seeds previously stressed with a sublethal dose of As(2)O(3). The seeds were then treated with either potentized As(2)O(3) (5x, 15x, 25x, 35x, 45x), potentized water (equivalent potencies) or diluted As(2)O(3) (10(-5), 10(-15), 10(-25), 10(-35), 10(-45)). The working variable was the stem length, measured after 4, 5, 6 and 7 days. RESULTS: Some potencies (As(2)O(3) 45x and H(2)O 45x) induced a relevant increase in seedling growth and/or a variability decrease. Diluted As(2)O(3) did not induce any significant results. CONCLUSIONS: Confirmation of a significant stimulating effect on seedling growth and a significant decrease of variability was obtained with ultra-high dilutions at the 45x potency. The model of wheat germination and growth has been confirmed to be a good tool for basic research in homeopathy.

The effects of a 45x potency of arsenicum album on wheat seedling growth -- a reproduction trial.

BACKGROUND: Independent replications of preclinical investigations of homeopathic potencies are rare. However, they are a necessary tool to determine the relevant factors modulating the effects of homeopathic potencies in preclinical systems. OBJECTIVE: The goal of the present study was to reproduce a trial published in 1997. An Italian group of researchers investigated the effect of Arsenicum album 45x on the growth of wheat which had been previously poisoned with a material dose of Arsenicum album. The homeopathic treatment was associated with increased wheat shoot growth significantly different from the control group (+24%, p < 0.001). MATERIALS AND METHODS: Wheat poisoned with a sublethal dose of Arsenicum album was cultivated in either Arsenicum album 45x, water 45x, or unpotentized water. After 7 days, shoot length was measured. Reproducibility was assessed in eight independent experiments. RESULTS: Arsenicum album 45x significantly inhibited wheat shoot growth (-3%) compared to treatment with unpotentized water and water 45x (p = 0.011 and p = 0.037). Within the experimental series performed in this reproduction trial, the effects of Arsenicum album 45x proofed to be reproducible. The wheat seed species used did not seem to have a significant impact on the experimental outcome. CONCLUSION: The result of this replication trial is a reversal of the original study, since Arsenicum album 45x inhibited wheat shoot growth instead of enhancing it. Nevertheless, high homeopathic potencies may induce statistically significant effects in biological systems. However, the magnitude and direction of these effects seem to depend on yet unknown parameters.

[Role of arsenolite on 8-isoprostane of asthmatic mice plasm].

OBJECTIVE: Through the establishment of mouse' ovalbumin- sensitized asthmatic model and the observation of the 8-Isoprostane of plasm, to evaluate the therapeutic effects of arsenolite on asthmatic mice. METHOD: Forty-two healthy Kunming male mice were randomly divided into control group and experience groups, the latter were treated with dexamethasone, arsenolite. Lung function were tested, 8-isoprostane of plasm and WBC of BALF were measured. RESULT: Lung function improved after treating with dexamethasone or arsenolite. The WBC of asthmatic mice were significantly higher than those in control group, and decreased after treating with dexamethasone or arsenolite; 8-Isoprostane of plasm in asthmatic mice was higher than that of control group, and decreased after treating with dexamethasone or arsenolite. CONCLUSION: There is oxidant stress status in asthmatic mice. Arsenolite could lighten airway obstruction, reduce airway high response and redress oxidant stress status in asthmatic mice.

[Realgar nano-particles induce apoptosis and necrosis in leukemia cell lines K562 and HL-60].

OBJECTIVE: To examine the growth-inhibitory, apoptosis- and necrosis-inducing effects of realgar nano-particles (RNP) in human chronic myelogenous leukemia cell line K562 and acute myeloid leukemia cell line HL-60, and to find out the chemical species with efficacy. METHOD: A "solvent-relay" strategy was used for the preparation of RNP suspension. Cell viability was determined by MTT assay. Cell apoptosis and necrosis were characterized with Annexin V-PI double staining in association with flow cytometry and with morphological examination with Hoechst 33258 staining. Parallel experiments with arsenous acid (H3AsO3), the dominant form of arsenic trioxide in the solution, were conducted for comparison. RESULT: The mean diameter of RNP was 159.0 nm. RNP showed growth-inhibitory effect on both cell lines. The double staining test indicated that RNP induced both apoptosis and necrosis, and this was further confirmed by morphological examination. CONCLUSION: RNP induced both apoptosis and necrosis in leukemia cell lines K562 and HL-60. Thioarsenite species with both As-O and As-S bonds may be the active intermediates in the RNP.

[Comparative studies on pharmacological effects of angong niuhuang pill with its simplified prescicription].

OBJECTIVE: Based on the therapeutic claims of Angong Niuhuang pill, a series of pharmacodynamic experiments were designed, where pharmacological effects were investigated comparatively with its simplified prescription(realgar and cinnabar are removed from the original pill) as a parallel control in order to explore possible contribution of cinnabar and realgar to pharmacodynamic activities of the pill as a whole. METHOD: Anti-pyretic, sedative, anti-convulsive, and mice-protected effects of the pill and its simplified prescription as a control were observed, respectively, in rabbits with fever induced by typhoid bacillus, in pentobarbital sodium-induced sleeping mice, in mice with convulsion induced by strychnine, or pentylenetetrazole, and in mice with anoxia induced by NaNO2. RESULT: Both the pill and its simplified prescription were found to have Anti-pyretic action and protective effect against the mouse death induced by anoxia, and synergistic interaction with pentobarbital sodium in sedative activity, although neither of them was found to have any effects on the convulsion of mice. CONCLUSION: No significant difference between Angong Niuhuang pill and its simplified prescription was found in the above pharmacodynamic experiments.

[Effects of realgar on tissue factor expression of NB4 and MR2 cells].

OBJECTIVE: To investigate the effects of Realgar on procoagulant activity (PCA), tissue factor expression and tissue factor mRNA transcription in acute promyelocytic leukemia (APL) cell lines NB4 and MR2 cells. METHOD: NB4 and MR2 cells were treated with 300 micrograms.L-1 Realgar PCA of the treated cells was detected using one-stage clotting assay. TF antigen was detected by ELISA and TFmRNA by semi-quantitive RT-PCR. RESULT: The PCA and TF antigen level in NB4 and MR2 cells were significantly higher than that in HL-60 and K562 cells. Realgar could down-regulate the membrane PCA, TF antigen and TF mRNA transcription of NB4 and MR2 cells in a time-dependent manner. CONCLUSION: Down-regulating TF expression and PCA of NB4 and MR2 cells by Realgar may be one of the mechanism of its improvement effect on DIC-related hemorrhage of APL patients.

[Investigation on NB4 cell responses to realgar by cDNA microarray].

OBJECTIVE: To elucidate the molecular mechanism of realgar-induced apoptosis and differentiation of acute promyelocytic leukemia(APL) cell line NB4. METHOD: The response of NB4 cells to realgar was explored with a cDNA microarray representing 1003 different human genes. RESULT: The analysis of gene expression profiles indicated that 9 genes were up-regulated and 37 genes were down-regulated. Among the 9 up-regulated genes, 2 genes were involved in proteasome degradation pathway. CONCLUSION: PSMC2, PSMD1 and ITGB1 genes may play a role in the apoptosis and differentiation of NB4 cells.

[In vitro study on realgar induced apoptosis of all-trans acid resistant acute promeylocytic leukemia cell line (MR2)].

OBJECTIVE: To acquire a deep understanding of the possible mechanisms of realgar in the treatment of acute promyelocytic leukemia (APL). METHOD: All-trans retinoic acid (ATRA) resistant APL cell line MR2 was used as in vitro model. The effect of realgar on MR2 cell was observed by watching cell viability, cell growth, and by using Methy thiazolyl tetrazolium (MTT) assay, cell morphology, DNA gel electrophoresis and flow cytometry assay. RESULT: The viability and growth of MR2 cell were inhibited after the treatment, to some extent, in a dose and time dependent manner. After being treated with realgar, MR2 cell presented morphologically some features of apoptotic cells such as intact cell membrane, chromatin condensation and nuclear fragmentation, and apoptotic body could be found by electron microscopy as well. Sub-G1 cells were observed by flow cytometry, as well as Annexin V FITC+/PI-cells. DNA ladder could be found by DNA gel electrophoresis. CONCLUSION: Realgar can induce apoptosis of ATRA resistant APL cell line MR2, Which shows the therapeutic effect of realgar on APL may be different from that of ATRA.

Effects of homeopathic arsenic on tobacco plant resistance to tobacco mosaic virus. Theoretical suggestions about system variability, based on a large experimental data set.

CONTEXT: This research aimed at verifying the efficacy of homeopathic treatments by plant-based bioassays, which may be suitable for basic research, because they lack placebo effects and provide large datasets for statistical analyses. OBJECTIVE: To evaluate the effects of homeopathic treatments of arsenic trioxide (As2O3) on tobacco plants subjected to tobacco mosaic virus (TMV) inoculation as biotic stress. DESIGN: Blind, randomized experiment using tobacco leaf disks. MATERIALS AND METHODS: Tobacco plants (Nicotiana tabacum L. cultivar Samsun) carrying the TMV resistance gene N. TMV inoculated leaf disks were floated for 3 days in the following: Distilled water (control). H2O 5 and 45 decimal and centesimal potencies. As2O3 5 and 45 decimal and centesimal potencies. The main outcome measures is the number of hypersensitive lesions observed in a leaf disk. RESULTS: Homeopathic treatments of arsenic induce two effects on the plant: (i) increased resistance to TMV; (ii) decrease variability between experiments (system variability). CONCLUSIONS: In this experimental model two actions of homeopathic treatment were detected: decrease in system variability and enhancement of the natural tendency of the system towards an 'equilibrium point'.

[Studies on red orpiment induction of NB4 and HL-60 cell apoptosis].

OBJECTIVE: To study the possible mechanism of red orpiment, which is main component of composite indigo naturalis tablets, in the treatment of acute promyelocytic leukemia(APL). METHODS: The effect of red orpiment on induction of APL cell line NB4 and HL-60 apoptosis were studied by cell morphology, DNA gel electrophoresis and flow cytometry assay. RESULTS: Red orpiment induced NB4 and HL-60 cell apoptosis. When treated with different concentration of red orpiment(25-200 micrograms/ml) for 16 hours, both NB4 and HL-60 cells showed typical apoptosis features. If decreased the concentration of red orpiment to 12.5 micrograms/ml, the NB4 cell still showed apoptosis features while the HL-60 cell did not when cultured for 72 hours. Arsenic disulfide(As2S2) had the same effect as red orpiment did under the same experiment condition. CONCLUSION: It is the main component, As2S2 of the red orpiment that can induces NB4 and HL-60 cell apoptosis. and the red orpiment is responsible for the high CR rate of APL induced by the composite indigo naturalis tablets.