Apoptotic and autophagic death union by Thuja occidentalis homeopathic drug in cervical cancer cells with thujone as the bioactive principle.

OBJECTIVE: "Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. METHODS: Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. RESULTS: Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC50) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC50 of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. CONCLUSION: Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.

[Inhibitory effects of Gecko alcohol extract on human esophageal squamous carcinoma cell line EC-109 proliferation and associated mechanism].

OBJECTIVE: To explore the proliferation inhibition effects of Gecko alcohol extract (GAE) on human esophageal squamous carcinoma cell line EC-109 and its mechanism. METHODS: The inhibitory effects of GAE on proliferation of EC-109 cells were measured by MTT. Nucleolus change of apoptotic cells was observed by Hoechest33342 fluorescence staining. Apoptosis rate of EC-109 cells was detected by flow cytometry. The expressions of apoptosis protein Caspase-3 and FAS in EC-109 cells were investigated by immunohistochemistry. RESULTS: GAE had the inhibition effects on the proliferation of esophageal carcinoma cell EC-109. The apoptosis rate of EC-109 cell treated with GAE(3.0 mg/mL, 4.0 mg/mL) for 48h was 20.63% and 39.73%, respectively. Compared with control group,the expression of Fas and Caspase-3 was significantly up-regulated in GAE treated group. CONCLUSION: GAE can inhibit the proliferation of esophageal carcinoma EC-109 cells and induce them apoptosis which may be correlated with increasing expression of protein Fas and Caspase-3.

[Active ingredients of Plastrum Testudinis inhibit epidermal stem cell apoptosis in serum-deprived culture].

OBJECTIVE: To investigate the effects of active ingredients of Plastrum Testudinis (PT) on serum deprivation-induced apoptosis of epidermal stem cells (ESCs). METHODS: ESCs were isolated from the back skin of fetal Sprague-Dawley rats with 2 weeks of gestational age and were divided into normal group (10% fetal bovine serum), control group (serum-deprived culture) and groups treated with serum deprivation plus active ingredients of PT, including ethyl acetate extract (2B), stearic acid ethyl ester (S6), tetradecanoic acid sterol ester (S8) and (+)-4-cholesten-3-one (S9). The vitality of ESCs after 24, 48 and 72 h of culture was measured with MTT method; apoptotic ESCs double-stained with Annexin V-FITC and propidium iodine were detected by flow cytometry (FCM); Bcl-2 and caspase-3 expressions were measured by Western blotting. RESULTS: MTT results indicated that the vitality of ESCs in the active ingredients of PT groups at 48 h was increased compared with the control group and 2B had better effects than the others. FCM results indicated that 2B had the most significant anti-apoptotic effect compared with the control as well as S6, S8 and S9. Western blot results indicated that 2B, S6, S8 and S9 up-regulated the expression of Bcl-2 protein and down-regulated the expression of caspase-3 protein compared with the control. CONCLUSION: Ethyl acetate extract of Plastrum Testudinis inhibits epidermal stem cell apoptosis in serum-deprived culture by regulating the expressions of Bcl-2 and caspase-3 proteins and has a stronger anti-apoptotic effect than its constituents S6, S8 and S9.

[Effect of freeze-dried sea cucumber powder of eastern sea on tumor and immune index of S180-bearing mouse].

OBJECTIVE: To study the effect of freeze-dried sea cucumber powder from eastern sea on tumor and immune of S180-bearing mouse. METHODS: S180 tumor cells were subcutaneouly inoculated to fifty health kunming mice and were randomly divided into five groups (model control group, positive control group, and low, medium, high dose groups). After the groups were orally treated with solution of freeze-dried sea cucumber powder for ten days,the weights of tumors and the inhibition rates of tumor were counted, and the pathological changes of tumors were observed and the expressions of caspase-3 proteins were detected in the tumor tissues, and the thymus index and spleen index were observed , the contents of interleukin-2(IL-2) in the serum were detected. RESULTS: (1) In comparison with the control groups,the weights of tumor were declined and the inhibition rates of tumor were remarkably decreased in the treated groups. (2) The pathological changes of model control group were very obvious. The expressions of caspase-3 proteins in the treated group were more higher than those of model control groups, while were more lower than those of positive control groups. (3) The thymus index, spleen index in the treated group indicated an increased tendency. (4) The contents of IL-2 in the treated groups were more higher than those of other groups. CONCLUSION: Freeze-dried sea cucumber powder could be effective antitumor and could protect and recovery the immune system of S180-bearing mice.

Unexpected effect of the vehicle (grain ethanol) of homeopathic FSH on the in vitro survival and development of isolated ovine preantral follicles.

The aims of this study were to investigate the effects of medium replacement system (experiment I) and of FSH presentations (homeopathic - FSH 6cH and allopathic FSH - rFSH; experiment II) on the in vitro development, hormone production and gene expression of isolated ovine preantral follicles cultured for 6 days. In experiment I, secondary follicles were cultured in the α-MEM+ supplemented with FSH 6cH (0.05 fg/ml) or recombinant bovine FSH (100 ng/ml) without/with daily medium addition. The homeopathic FSH treatments with/without medium addition improved (p < .05) follicular development compared to rFSH100 treatment without addition. FSH 6cH with addition showed the highest (p < .05) estradiol production. To verify whether the effects of homeopathic FSH were not due to its vehicle, experiment II was performed. The α-MEM+ was supplemented or not with alcohol (0.2% grain ethanol, v/v), FSH 6cH or rFSH100 with daily medium addition. Surprisingly, we found that all treatments improved follicular development compared to the α-MEM+ (p < .05). Moreover, homeopathic FSH was similar to the other treatments including its vehicle. In conclusion, its vehicle (ethanol) causes the effect of homeopathic FSH on in vitro development of isolated ovine preantral follicles.

Psorinum 6 × triggers apoptosis signals in human lung cancer cells.

OBJECTIVE: To provide in vitro evidence of Psorinum treatment against cancer cells in a controlled study. METHODS: Effects of homeopathic Psorinum 6× on cell viability were initially determined in several cancer cell lines, including A549, HepG2 and MCF-7, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and an ethanol 6× control. The cell line that exhibited highest inhibition was selected and used in the following experiments. A range of Psorinum 6× doses was used to explore treatment effects on cell cycle arrest, cell death (apoptosis), generation of reactive oxygen species (ROS) and change in mitochondrial membrane potential (MMP) using flow cytometry and fluorescence microscopy, respectively. Expression of several signal proteins related to apoptosis and cell survival were quantified with Western blotting and confocal microscopy. Further, circular dichroism (CD) spectroscopy was used to determine possible drug-DNA interactions, as well as the induction of conformational changes. RESULTS: Treatment of cancer cell lines with Psorinum showed greater anticancer effects in A549 cells than in others. In A549 cells Psorinum treatment inhibited cell proliferation at 24 h after treatment, and arrested cell cycle at sub-G1 stage. It also induced ROS generation, MMP depolarization, morphological changes and DNA damage, as well as externalization of phosphatidyl serine. Further, increases in p53 expression, Bax expression, cytochrome c release, along with reduction of Bcl-2 level and caspase-3 activation were observed after Psorinum 6× treatment, which eventually drove A549 cells towards the mitochondria-mediated caspase-3-dependent pathway. CD spectroscopy revealed direct interaction of Psorinum with DNA, using calf thymus-DNA as target. CONCLUSION: Psorinum 6× triggered apoptosis in A549 cells via both up- and down-regulations of relevant signal proteins, including p53, caspase-3, Bax and Bcl-2.

[Effects of cinobufagin on apoptosis in U-2OS osteosarcomas cells].

OBJECTIVE: To investigate the effects of cinobufagin on the apoptosis in U-2OS osteosarcomas cells (U-2OS cells) and explore its potential mechanism. METHODS: The cytostatic effects of cinobufagin (10, 20, 50, 100, 200, and 400 nmol/L) on U-2OS cells were evaluated by MTT assay at 24, 48, and 72 hours after culture; simple U-2OS cells served as control group. The impact of cinobufagin (100 nmol/L) on the apoptosis in U-2OS cells was determined by flow cytometry at 48 hours after culture, which were treated with cinobufagin (experimental group) or with cinobufagin plus Z-VAD-FMK (control group), and simple U-2OS cells served as blank control group. The Caspase-3 activity was measured by Caspase-3 activity assay kit at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. The expression of apoptosis signal pathway related proteins in U-2OS cells treated with cinobufagin were detected by Western blot at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. RESULTS: The results of MTT assay showed that cinobufagin inhibited the proliferation of U-2OS cells in a dose- and time-dependent manners. At each time point, the growth rate of U-2OS cells was significantly reduced with the increasing cinobufagin concentration, and as time prolonged, the growth rate of U-2OS cells behaved the same way in the same group. There were significant differences among different time points and groups (P < 0.05). The apoptotic rate of experimental group (46.87% +/- 11.23%) was significantly higher than that of the control group (2.34% +/- 0.98%) and blank control group (1.04% +/- 0.25%) (P < 0.05). The Caspase-3 activity in 20, 50, and 100 nmol/L groups were 1.14 +/- 0.32, 1.31 +/- 0.41, and 1.92 +/- 0.54, respectively, which were significantly higher than that in control group (P < 0.05). Compared with 20and 50 nmol/L groups, 100 nmol/L group significantly increased the Caspase-3 activity in U-2OS cells (P < 0.05). Compared with the control group, the expressions of cleaved Caspase-3, cleaved Caspase-9, and Bax were obviously up-regulated; the Bcl-2 expression was down-regulated; and the ratio of Bax/Bcl-2 was increased in different cinobufagin-treated groups (P < 0.05). The same tendency was seen in different cinobufagin-treated goups, showing significant differences among groups (P < 0.05). CONCLUSION: Cinobufagin can inhibite the proliferation of U-2OS cells, and induce cell apoptosis. The potential mechanism of cinobufagin-induced apoptosis may be related to the mitochondria-mediated pathway.

The potentized homeopathic drug, Lycopodium clavatum (5C and 15C) has anti-cancer effect on hela cells in vitro.

Cancer is a disease that needs a multi-faceted approach from different systems of medicine. The purpose of this study was to evaluate whether homeopathically-potentized ultra-high dilutions of Lycopodium Clavatum (LC-5C and LC-15C, respectively) have any anti-cancer effects on HeLa cells. Cells were exposed to either LC-5C (diluted below Avogadro's limit, i.e., 10(-10)) or LC-15C (diluted beyond Avogadro's limit, i.e., 10(-30)) (drug-treated) or to 30% succussed ethanol ("vehicle" of the drug). The drug-induced modulation in the percent cell viability, the onset of apoptosis, and changes in the expressions of Bax, Bcl2, caspase 3, and Apaf proteins in inter-nucleosomal DNA, in mitochondrial membrane potentials and in the release of cytochrome-c were analyzed by utilizing different experimental protocols. Results revealed that administration of LC-5C and LC-15C had little or no cytotoxic effect in normal peripheral blood mononuclear cells, but caused considerable cell death through apoptosis in cancer (HeLa) cells, which was evident from the induction of DNA fragmentation, the increases in the expressions of protein and mRNA of caspase 3 and Bax, and the decreases in the expressions of Bcl2 and Apaf and in the release of cytochrome-c. Thus, the highly-diluted, dynamized homeopathic remedies LC-5C and LC-15C demonstrated their capabilities to induce apoptosis in cancer cells, signifying their possible use as supportive medicines in cancer therapy.

Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating caspase-mediated signaling via reactive oxygen species elevation.

OBJECTIVE: Preventive measures against skin melanoma like chemotherapy are useful but suffer from chronic side effects and drug resistance. Ethanolic extract of Phytolacca decandra (PD), used in homeopathy for the treatment of various ailments like chronic rheumatism, regular conjunctivitis, psoriasis, and in some skin diseases was tested for its possible anticancer potential. METHODS: Cytotoxicity of the drug was tested by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on both normal (peripheral blood mononuclear cells) and A375 cells. Fluorescence microscopic study of 4',6-diamidino-2-phenylindole dihydrochloride-stained cells was conducted for DNA fragmentation assay, and changes in cellular morphology, if any, were also recorded. Lactate dehydrogenase activity assay was done to evaluate the percentages of apoptosis and necrosis. Reactive oxygen species (ROS) accumulation, if any, and expression study of apoptotic genes also were evaluated to pin-point the actual events of apoptosis. RESULTS: Results showed that PD administration caused a remarkable reduction in proliferation of A375 cells, without showing much cytotoxicity on peripheral blood mononuclear cells. Generation of ROS and DNA damage, which made the cancer cells prone to apoptosis, were found to be enhanced in PD-treated cells. These results were duly supported by the analytical data on expression of different cellular and nuclear proteins, as for example, by down-regulation of Akt and Bcl-2, up-regulation of p53, Bax and caspase 3, and an increase in number of cell deaths by apoptosis in A375 cells. CONCLUSION: Overall results demonstrate anticancer potentials of PD on A375 cells through activation of caspase-mediated signaling and ROS generation.

Antihyperglycemic drug Gymnema sylvestre also shows anticancer potentials in human melanoma A375 cells via reactive oxygen species generation and mitochondria-dependent caspase pathway.

OBJECTIVE: Ethanolic extract of Gymnema sylvestre (GS) leaves is used as a potent antidiabetic drug in various systems of alternative medicine, including homeopathy. The present study was aimed at examining if GS also had anticancer potentials, and if it had, to elucidate its possible mechanism of action. METHODS: We initially tested possible anticancer potential of GS on A375 cells (human skin melanoma) through MTT assay and determined cytotoxicity levels in A375 and normal liver cells; we then thoroughly studied its apoptotic effects on A375 cells through protocols such as Hoechst 33258, H2DCFDA, and rhodamine 123 staining and conducted ELISA for cytochrome c, caspase 3, and PARP activity levels; we determined the mRNA level expression of cytochrome c, caspase 3, Bcl2, Bax, PARP, ICAD, and EGFR signaling genes through semiquantitative reverse transcriptase polymerase chain reaction and conducted Western blot analysis of caspase 3 and PARP. We also analyzed cell cycle events, determined reactive oxygen species accumulation, measured annexin V-FITC/PI and rhodamine 123 intensity by flow cytometry. RESULTS: Compared with both normal liver cells and drug-untreated A375, the mortality of GS-treated A375 cells increased in a dose-dependent manner. Additionally, GS induced nuclear DNA fragmentation and showed an increased level of mRNA expression of apoptotic signal related genes cytochrome c, caspase 3, PARP, Bax, and reduced expression level of ICAD, EGFR, and the anti-apoptotic gene Bcl2. CONCLUSION: Overall results indicate GS to have significant anticancer effect on A375 cells apart from its reported antidiabetic effect, indicating possibility of its palliative use in patients with symptoms of both the diseases.

Apigenin-induced apoptosis in A375 and A549 cells through selective action and dysfunction of mitochondria.

We isolated apigenin (5,7,4'-trihydroxy flavone) from ethanolic extract of Lycopodium clavatum (LC) used as a homeopathic mother tincture for treatment of various diseases. We assessed the anticancer potentials of the compound using human malignant melanoma cell line A375 and a lung carcinoma cell line A549 and focussed on its putative molecular mechanism of action on apoptosis induction. We examined the cytotoxicity of apigenin in both cancer cells and normal peripheral blood mononuclear cells (PBMC). A375 cells were more prone to apigenin-induced apoptosis, as compared with A549 cells after 24 h of treatment, while PBMC showed little or no cytotoxicity to apigenin. We also evaluated the effects of apigenin on interaction with DNA by comparative analysis of circular dichroism spectral data and melting temperature profiles (Tm) of calf thymus DNA (CT-DNA) treated with or without apigenin. Reactive oxygen species (ROS) accumulation in mitochondria, super-oxide dismutase and total thiol group (GSH) activities were also analyzed. The apoptotic process involved mitochondrial pathway associated with apigenin-DNA interaction, DNA fragmentation, ROS accumulation, cytochrome c (cyt c) release and mitochondrial transmembrane potential depolarization, Bax, caspase 3, 9, PARP, up-regulation, Bcl-2 down-regulation and down-regulation of cyt c in the mitochondrial fraction. Results of mitochondrial inner membrane swelling measurements, intracellular ADP/ATP ratio and ATPase activity showed that in A549 cells, apigenin did not appear to directly target the mitochondrial oxidative phosphorylation system but rather acted at an upstream step to activate the mitochondrial apoptotic pathway. However, apigenin could directly target and impair mitochondrial function in A375 cells by breaking down their oxidative phosphorylation system. Collectively, these results suggest that apigenin exhibits anticancer potential in A375 and A549 cells that may be mediated through DNA interaction, damage and mitochondrial dysfunction either by direct or indirect action on mitochondrial oxidative phosphorylation system.

Polypeptide from Chlamys farreri attenuates murine thymocytes damage induced by ultraviolet B.

AIM: Polypeptide from Chlamys farreri (PCF, molecular mass is 879) is a new marine polypeptide compound isolated from Chlamys farreri. This study investigates the possible protective roles and the mechanism of PCF against ultraviolet B (UVB)-induced apoptosis in murine thymocytes. METHODS: The rate of apoptosis and caspase-3 activation was measured by flow cytometry. The expression of stress-response genes c-fos and c-jun was observed by RT-PCR. Western blot analysis was performed to determine the release of cytochrome c. RESULTS: It was found that UVB induced murine thymocyte death. The cells treated with UVB showed an increase in cytochrome c release, caspase-3 activity, as well as in the expression of c-fos and c-jun. In addition, all were involved in UVB-induced cell apoptosis. CONCLUSION: Our present observations pointed to the ability of PCF to avert UVB-induced apoptosis in thymocytes by modulating c-fos and c-jun expression, cytochrome c release, and the consequent activation of caspase-3, which were essential components of the UV-induced cell apoptotic pathway. The results suggested that PCF is a promising protective substance against UV radiation.