Differentiation of promyelocytic leukemia cells HL-60 induced by daidzein in vitro and in vivo.

We have screened more than one thousand synthetic and natural chemicals to explore differentiation inducers and found that daidzein has potent differentiation-inducing activity for human leukemia HL-60 cells, both in vitro and in vivo. In vitro study showed that daidzein at concentrations exceeding 10 micrograms/ml caused inhibition of HL-60 cells; and it induced differentiation of the cells into granulocytic lineage as judged by NBT reduction activity, phagocytic ability and morphological characteristics. Flow cytometry study indicated that daidzein arrested HL-60 cells in the G1 phase. The growth of HL-60 cells in the subrenal capsules of mice and in diffusion chambers implanted into the peritoneal cavities of mice was inhibited by 50 mg/kg daidzein. HL-60 cells treated with daidzein in vivo also exhibited characteristic morphological changes of matured cells. Moreover, the colony forming efficiency of HL-60 cells in diffusion chambers in mice was markedly inhibited by the administration of daidzein.

Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro.

Extracts of Helleborus species are used as phytopreparations with immunostimulatory properties in Romanian traditional medicine. In Germany, Helleborus niger is used in homeopathy and as an adjuvant therapy in the treatment of tumor patients in anthroposophical medicine. In vitro application of an aqueous extract from Helleborus niger resulted in a slight induction of sister chromatid exchanges (SCE) in cultured peripheral blood mononuclear cells (PBMC) from healthy individuals, an effect associated with a slight increase of the [3H]thymidine uptake in the DNA of isolated lymphocytes. Since the cytokines interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha were reported to increase the number of SCE, we measured the concentrations of these cytokines in the supernatants of cultured PBMC treated with the plant extract. Here, no significant changes were observed as compared with the controls, but a trend to higher supernatant concentrations of TNF-alpha in six out of ten individuals was noted. Compared with lymphocytes treated with the alkylating substance, cyclophosphamide, the increase of the SCE levels induced by the plant extract is weak. The relevance of this DNA destabilizing property remains to be clarified.

The effect of homeopathic plant extract solutions on the cell proliferation of human cutaneous fibroblasts in vitro.

BACKGROUND: According to homotoxicology illness is defined as an overload of the connective tissue matrix with toxic substances, the homotoxins. In order to support elimination of these homotoxins, complex homeopathic medicines were developed. Fibroblasts are the local cells of this matrix and produce and modulate the composition of the extracellular matrix (ECM) in every organ. OBJECTIVE: In this study, the effect of 17 potentiated plant extracts, which are components of some antihomotoxic remedies, on the cell proliferation of human cutaneous fibroblasts (F54) was analyzed as an indication of their influence on the ECM. MATERIAL AND METHODS: Cell proliferation of the F54 cells was measured by a colorimetric XTT-based assay kit. RESULTS: Six of the tested agents had no effect on the cell proliferation of fibroblasts; 11 plant extracts had a dose-dependent inhibitory effect on cell proliferation. CONCLUSIONS: As the modulation of the ECM is dependent on the activity of the fibroblasts, these results indicate that the plant extracts may modulate the composition of the ECM via the inhibitory effect on fibroblasts cell growth.

[Growth capability of epithelial cell line of human poorly differentiated nasopharyngeal carcinoma and its response to Chinese medicinal herbs and marine drugs].

By 3H-TdR incorporation, dye exclusion and cell colony-forming tests, the capability of short-term in vitro growth of the epithelial cell line of human poorly differentiated nasopharyngeal carcinoma (CNE-2Z) was assayed. At the same time, its response to 54 kinds of Chinese medicinal herbs and marine drugs was studied. The results showed that the 3H-TdR incorporation rate of cells was 1.8 +/- 0.02%, reproduction rate was 60.9 +/- 13.0% and colony-forming rate, 40.8 +/- 3.5%. As to the ratios of the three cell growth indexes and response to medicines, the Chinese medicinal herbs and marine drugs causing the reduction of colony-forming and cell survival ratios were predominant (64.8% and 40.7%). The results indicate that the majority of drugs possess the cytotoxic and inhibitory effect on cell reproduction to different degrees. The composite cell response to every kind of drug could be divided into 6 types: descending, ascending, peaked, valley-like, depressed and stable. The depressing type drugs might inhibit or arrest the cell growth of nasopharyngeal carcinoma and are worthy of further study.

[Experimental study of hirudo and Salvia miltiorrhiza on smooth muscle cell hyperplasia after balloon angioplasty].

OBJECTIVE: To find the preventive method of restenosis after transluminal angioplasty (TA). METHODS: Sixty rabbit iliac arteries of 30 rabbits were divided randomly into three groups, group A, B and C, 20 in each. After experimental atherosclerotic stenosis and TA performing, ordinary forage plus 2 g of hirudo powder was fed to the group A every day, to the group B, 2 g Salvia miltiorrhiza powder was added and to the group C, ordinary forage only. Angiography and pathomorphological examination were carried out 30 days later. RESULTS: (1) Hirudo significantly reduced the thickness of iliac intima as compared with the other two groups (P < 0.05), the thickness in the group A, B and C was 40.1 +/- 9.8 microns, 48.2 +/- 8.2 microns and 69.3 +/- 9.2 microns respectively. (2) The incidence of restenosis after TA in both group A (31.4%) and group B (48.4%) were significantly lower than that in the group C (62.8%, P < 0.05). And significant difference was also found in comparison of group A and group B (P < 0.05). (3) Ultrastructure change examined by transmission electron microscopy showed that less rough endoplasmic reticulum and more myofilaments in well differentiated smooth muscle cell (SMC) of the arterial intima in the group A and B, while in the group C, more rough endoplasmic reticulum, more synthetic cells were seen in SMC. CONCLUSIONS: Hirudo and red sage root could effectively inhibit the hyperplasia of SMC in arterial intima and reduce the intima thickness and the incidence of restenosis after TA significantly, thus might play a role in the prevention of restenosis after TA. The effect of hirudo is more potent than that of red sage root.

[Pharmacological study of Limax extract on inhibiting H14 cell].

OBJECTIVE: To study the inhibitive effect of Limax extract on H14 and inducement effect of apoptosis and differentiation of H14 cells in vitro. METHODS: MTT method, DNA gel electrophoresis, cell stain and microscope observation. RESULTS: After H14 cell line was induced by Limax extract, the growth of H14 cell was inhibited in vitro and the morpha of the cells changed. CONCLUSION: Limax extract had inhibitive effect on H14 cell, partially by apoptosis, and partially by differentiation.

[Study on anticancer activity of Syngnathus in vitro].

OBJECTIVE: To study the anticancer activity of Syngnathus in vitro. METHOD: Observing the influence of different extracts from Syngnathus on growth of different cancer cell strains by MTT method. RESULT: It has been found out that the fat-soluble nonsaponified extract from Temminck et Schlegel and the alcoholic extract from Syngnathus acus have cytotoxic activities. The nonsaponified extract from Temminck et Schlegel can inhibit the growth of cancer cell strains KB, Hela, PAA, K562, and Bcap37, and the alcoholic extract from Syngnathus acus can inhibit the growth of cancer cell strin KB, But Bloch shows no apparent anticancer activity. CONCLUSION: Syngnathus has promising prospects as an anticancer Chinese medicine.

[A comparison of chemical composition and bioactivity of polypeptides from velvet antlers of Cervus nippon Temminck and Cervus elaphus Linnaeus].

OBJECTIVE: To compare the chemical composition and bioactivity of polypeptides(PPs) isolated from velvet antlers of sika deer (Cervus nippon Temminck) and red deer (Cervus elaphus Linnaeus). METHOD: The two kind of polypeptides were isolated from the above mentioned velvet antlers with same technology. The chemical composition was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF mass spectrometry. Stimulant activity of cells proliferation was measured by [3H] TdR incorporation into DNA. RESULT: The graphs of SDS-PAGE and MALDI-TOF MS of velvet antler polypeptides (VAPPs) from Chinese and New zealand red deer were very similar, but there were obvious difference in respect of graph between sika deer and red deer. VAPPs 25-50 mg.L-1 showed marked proliferation-promoting activity for rabbit costed chondrocytes, either sika deer or red deer. However, the activity of sika deer VAPPs 12.5 mg.L-1 for epidermal cells was weaker than that of red deer (12.5 mg.L-1). CONCLUSION: The chemical property and bioactivity of VAPPs from sika deer and red deer are significantly different.

[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.

[Morphological changes and inhibiting effect on human gastric cancer cell SGC-7901 caused by aining].

OBJECTIVE: To investigate the inhibiting effect of Aining on the human gastric cancer cells. METHODS: Morphological and MTT methods were adopted to explore the inhibiting effect of Aining and cisplatin (DDP) on the proliferation of SGC-7901 cancer cell. RESULTS: Apoptotic morphological changes were seen after the cells cultured with Aining and DDP; inhibiting rate of 1 g/L Aining group was 51%, which had no significant differences with the inhibiting rate 53% of the 25 mg/L DDP group. But both the Aining and the DDP groups were significantly different from the blank group (P < 0.01). CONCLUSION: Not only DDP but also Aining could inhibit the proliferation activity of the human gastric cancer cells, and the traditional Chinese medicine compound prescription Aining is very likely to become a new medicine which is utilized to inhibit cancer.

Biosynthesized silver nanoparticles by ethanolic extracts of Phytolacca decandra, Gelsemium sempervirens, Hydrastis canadensis and Thuja occidentalis induce differential cytotoxicity through G2/M arrest in A375 cells.

The capability of crude ethanolic extracts of certain medicinal plants like Phytolacca decandra, Gelsemium sempervirens, Hydrastis canadensis and Thuja occidentalis used as homeopathic mother tinctures in precipitating silver nanoparticles from aqueous solution of silver nitrate has been explored. Nanoparticles thus precipitated were characterized by spectroscopic, dynamic light scattering, X-ray diffraction, atomic force and transmission electron microscopic analyses. The drug-DNA interactions of silver nanoparticles were analyzed from data of circular dichroism spectroscopy and melting temperature profiles using calf thymus DNA (CT-DNA) as target. Biological activities of silver nanoparticles of different origin were then tested to evaluate their effective anti-proliferative and anti-bacterial properties, if any, by exposing them to A375 skin melanoma cells and to Escherichia coli C, respectively. Silver nanoparticles showed differences in their level of anti-cancer and anti-bacterial potentials. The nanoparticles of different origin interacted differently with CT-DNA, showing differences in their binding capacities. Particle size differences of the nanoparticles could be attributed for causing differences in their cellular entry and biological action. The ethanolic extracts of these plants had not been tested earlier for their possible efficacies in synthesizing nanoparticles from silver nitrate solution that had beneficial biological action, opening up a possibility of having therapeutic values in the management of diseases including cancer.

FMS*Calciumfluor specifically increases mRNA levels and induces signaling via MAPK 42,44 and not FAK in differentiating rat osteoblasts.

The homeopathic compound of resonance FMS*Calciumfluor (FMS*) reportedly promotes osteogenic differentiation of rat pre-osteoblasts in vitro. Here, we show that the continuous exposure of differentiating rat osteogenic cells (ROB) to FMS* modulates the level of expression of mRNAs for 7 of the 8 osteogenic markers tested. Alkaline phosphatase (AP), osteocalcin (OC), metalloproteinases (MMP-2 and -14), procollagenase C (BMP-1), biglycan (BG) and integrin 1 are expressed at higher levels in FMS*-treated osteoblasts than in control cultures. MMP-2 and -14 mRNA are not down-modulated at mineralization. Also, the pattern of expression induced by FMS* for some of these genes (BMP-1, BG and integrin 1) is changed, but collagen type I (Coll I) mRNA levels are not affected by treatment with FMS*. This suggests that FMS* modulates mRNA levels and that this is not generalized, but gene(s) specific. We also report that exposure to FMS* rapidly and transiently induces activation of mitogen-activated protein kinases (MAPKs) 42,44 in populations of early osteoblasts, but not in pre-osteoblasts, with a cell differentiation stage-dependent and pertussis toxin (PTX)-sensitive response. Subsequent to FMS* MAPK signaling activation, an increase in AP and MMP-14 mRNA is detected, which is also inhibited by PTX, suggesting that FMS* activation of MAPK signaling could be an early event required for the induction of these genes. Exposure to FMS* does not cause changes in the activity of p125 (FAK)-mediated signaling.

Effects of bufalin and cinobufagin on the proliferation of androgen dependent and independent prostate cancer cells.

BACKGROUND: Cardiac glycosides may induce oncolytic effects in cancers. This study was to evaluate bufalin and cinobufagin effects on the proliferation of prostate cancer cell lines named LNCaP, DU145, and PC3. METHODS: Cell proliferation was measured by MTT assay. The cytotoxic effects were determined by lactate dehydrogenase measurements. The intracellular calcium concentration ([Ca(2+)](i)) was measured by a dual-wavelength spectrometer system. TUNEL assay and flow cytometry were performed to measure percentage of apoptotic cells. A colorimetric assay was to measure caspases activities. RESULTS: Bufalin and cinobufagin inhibited proliferation of cancer cells at doses of 0.1, 1, or 10 microM after 2-4 days of culture. Cytotoxicity of bufalin and cinobufagin on the DU145 and LNCaP cells was dose-dependent. Bufalin or cinobufagin increased [Ca(2+)](i) and apoptosis in cancer cells after a 24-hr culture as well as caspase 3 activities in DU145 and PC3 cells and caspase 9 activities in LNCaP cells. CONCLUSIONS: Bufalin and cinobufagin may inhibit the proliferation of prostate cancer cell lines associated with sustained elevation of the [Ca(2+)](i) and that of apoptosis.

Osteogenesis in vitro in rat tibia-derived osteoblasts is promoted by the homeopathic preparation, FMS*Calciumfluor.

We studied the effects of in vitro treatment of differentiating osteogenic cells with FMS*Calciumfluor, to determine whether it caused changes in proliferative or differentiation potential of osteoblasts. FMS*Calciumfluor was developed for the therapy of post-menopausal and age-related osteoporosis on the basis of the principles of resonance homeopathy and VTR Vega test. Its daily prescribed therapeutical usage is about 30,000-fold less in fluoride concentration than that recommended for NaF associated with calcium monophosphate. Rat tibial osteoblast (ROB) primary cultures represent populations of early osteoblasts and their derivative cultures of more than 60 cumulative population doubling (CPD) represent more mature osteogenic cells. Both these populations were shown to undergo in vitro differentiation, as monitored by the sequential expression of markers that define the stages of the osteogenic progression. Here we report that continual treatment of ROB during osteogenesis with FMS*Calciumfluor modulated the expression of critical osteogenic markers: alkaline phosphatase (AP), an indicator of osteoblast maturation, and(45)Ca incorporation into the matrix and nodule formation, events of the last phase of osteogenesis and a measure of osteoid mineralization. Treatment did not affect proliferation, or expression and activation of metalloproteinases (MMP). AP activity and levels of AP mRNA were increased by treatment with FMS*Calciumfluor; the incorporation of radiolabelled Ca into the matrix was also increased and the formation of nodules occurred in a shorter time and with higher frequency than in untreated control cultures. The effects of FMS*Calciumfluor were concentration dependent and specific for its modalities of preparation, and were observed at a concentration about three orders of magnitude lower than similar effects reported in the literature by treatment of osteoblast cultures in vitro with NaF.

The role of black seed in the proliferation and biochemical marker levels of Hep-2 cells.

For centuries, people in the Middle East and Southeast Asia have used Nigella sativa, also known as black seed (BS), for its homeopathic effects. The objective of this study was to investigate the role BS might have on the metabolic biomarkers of the Hep-2 cell line. The experimental design entailed six groups of five wells each (50,000 cells). Groups II through VI were treated with BS, lipopolysaccharide (LPS), cortisol, LPS + cortisol, and BS + LPS + cortisol, respectively. Group I was the untreated control group. At the end of 24, 48, and 72 hours, the total cell count, protein and MDA levels were measured by following standard lab protocols. Data collected from this study revealed that Hep-2 cells exposed to LPS and cortisol (group V) resulted in a decrease in cell proliferation compared to the control. BS treatment induced a higher proliferation rate than group V. Similar trends were observed in the metabolic behavior of Hep-2 cells as evidenced by the total protein and MDA levels. The exposure of BS showed a shift in the metabolic pathways. In conclusion, this study showed that exposure to LPS resulted in an alteration in the metabolic function and this phenomenon was further escalated under stressful conditions (increased cortisol exposure). In addition, the use of BS reversed the traumatic condition.

Velvet antler polypeptides promoted proliferation of chondrocytes and osteoblast precursors and fracture healing.

AIM: To study the effects of velvet antler (VA) total polypeptides (VATP) and VA polypeptides, VAP-A, VAP-B, and VAP-C on proliferation of chondrocytes and osteoblast precusors. METHODS: Chondrocytes (rabbit and human fetus) and osteoblast precusors (chick embryo) were incubated in the culture medium containing VATP or VAP-A, VAP-B, and VAP-C. [3H]TdR incorporation into DNA was measured. Fracture healing-promoting action of VATP was determined in rats. RESULTS: VATP 50-200 mg.L-1 and VAP-B 12.5, 25, and 50 mg.L-1 showed most marked proliferation-promoting activity for rabbit costed chondrocytes and increased incorporation of [3H]TdR from (73 +/- 9) Bq (control group) to (272 +/- 55), (327 +/- 38), and (415 +/- 32) Bq, respectively (P < 0.01). The activity of VAP-A was weaker than that of VAP-B, and VAP-C had no activity. VATP 10 and 20 mg.kg-1 by local injection into the cross-section fracture area accelerated healing of radial fracture. The healing rate of VATP-treated group was higher (75%) than that of control group (25%) (P < 0.05). CONCLUSION: VATP accelerated fracture healing by stimulating proliferation of chondrocytes and osteoblast precursors.

The in vitro action of a succussed substance on the proliferative response of human lymphocytes stimulated with phytohemagglutinin.

Human peripheral blood lymphocytes from healthy controls, immunodepressed patients presenting chronic bacterial infections or neoplasias and from allergic patients were stimulated in vitro with phytohemagglutinin (PHA) in culture medium supplemented or not with 1 x 10(-7), 1 x 10(-15) or 1 x 10(-30) succussed dilutions or bee venom or phosphorus in tridistilled water. The most significant inhibition due to DNA incorporation was noted in lymphocytes from allergic patients cultivated in media supplemented with 1 x 10(-30) succussed substance dilution in the presence of PHA. The cells from immunodepressed patients did not show a significant inhibition at 1 x 10(-30) dilution. Hypothetically, we try to explain these findings as the expression of the changes induced by the succussed solution on the water molecule which in turn, influences the chemical structure of the cellular membrane and implicitly, its functions.

Selective inhibitory effect of bufalin on growth of human tumor cells in vitro: association with the induction of apoptosis in leukemia HL-60 cells.

We found that bufalin, an active principle of the Chinese medicine chan'su, has selective inhibitory effects on the growth of various human cancer cells. In order to examine whether the growth-inhibitory effect of bufalin on human cancer cells is associated with apoptosis, human leukemia cells were treated with bufalin. HL-60, ML1, and U937 leukemia cells treated with bufalin at 10(-8) M and above had condensed and fragmented nuclei. Flow cytometric analysis of these cells treated with bufalin showed fragmented DNA smaller than that of the G1 phase. DNA of HL-60 cells treated with bufalin showed a ladder pattern characteristic of apoptosis, as analyzed by agarose gel electrophoretic analysis. DNA synthesis and topoisomerase II activity of HL-60 cells were markedly inhibited as the concentration of bufalin was increased. The concentration needed for inducing apoptosis of HL-60 cells was 10(-8) M, which is comparable to that of camptothecin, but lower than those of other antitumor drugs such as cisplatin, VP16 and all-trans retinoic acid. Apoptosis was not observed when human mononuclear and polymorphonuclear cells were treated with 10(-6) M bufalin for 24 h. These results indicate the association of the growth-inhibitory effect of bufalin with the induction of apoptosis, at least in HL-60 cells, and suggest the usefulness of bufalin for differentiation-apoptosis-inducing therapy for cancer.