Search

Since the current methods of treatment for malignant glioma, radiotherapy and chemotherapy, are unsatisfactory, the development of novel therapeutic compounds is required. In the present study, the inhibitory effect of tetrandrine citrate (TetC) on the proliferation of human glioma U87 cells, as well as its mechanism of action, were investigated. An MTT assay was used to assess cell viability in vitro, and the production of intracellular reactive oxygen species (ROS) was determined by assessing the fluorescence intensity of 2,7‑-dichlorofluorescein (DCF). Flow cytometry was used to determine the level of apoptosis and cell cycle status, and the protein expression levels of apoptosis‑associated proteins were determined using western blotting. Additionally, the antitumor activity of TetC was assessed in vivo using a nude mouse xenograft model. The results revealed that in vitro, the proliferative rate of U87, U251 and human umbilical vein endothelial cells (HUVECs) was significantly reduced in a dose‑dependent manner following treatment with TetC, although TetC had the greatest inhibitory effect on U87 cells. The vacuolization and apoptosis of U87 cells was induced using 10 and 20 µmol/l TetC, respectively. The overall proliferative inhibition was associated with an increase in the levels of ROS and apoptosis. In TetC‑treated cells, the expression levels of apoptosis‑related proteins, including cleaved (CL) caspase‑3, Fas, phosphorylated (p)‑p38 and p‑JNK, were increased, whereas those of caspase‑3 and Bcl‑2 were decreased. In vivo, TetC was highly effective at inhibiting the growth of human glioma U87 xenografts in BALB/c nude mice, with a percentage growth inhibition of ≥68.7%. These findings indicated that the potent antitumor activity of TetC may be mediated through an increase in ROS levels, the downregulation of Bcl‑2, and the upregulation of CL caspase‑3, Fas, p‑p38 and p‑JNK expression levels.

The purpose of the present study was to assess the protective effects of rhein lysinate (RHL) in a KK/HlJ mouse model of diabetic nephropathy (DN) and to explore its mechanism of action. A total of 4 groups were established: C57BL/J control, the KK/HlJ model and 25 and 50 mg/kg/day RHL-treated KK/HlJ groups. The KK/HlJ mouse model of DN was established by streptozotocin injection, followed by maintenance on a specific diet. The albumin-to-creatinine ratio (ACR) was determined at 5 weeks and at 16 weeks, the kidneys were harvested, and morphological examination and immunohistochemical analysis were performed. The levels of malondialdehyde (MDA), as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities in the kidneys were measured using appropriate assay kits. The expression of inflammatory factors and associated proteins was analyzed using western blot analysis. At 5 weeks, the levels of ACR in KK/HlJ mice were increased, which was inhibited by treatment with RHL. Treatment with RHL (50 mg/kg/day) decreased the body weight of KK/HlJ mice. Compared with the C57BL/J control, the KK/HlJ model mice had a significantly lower activity of SOD and GSH-px in the kidneys, but had significantly higher levels of MDA. Treatment of KK/HlJ mice with RHL significantly increased the activities SOD and GSH-px, and reduced the MAD level in the kidneys. Renal tubular epithelial cell edema was observed in KK/HlJ mice but not in C57BL/J mice. RHL decreased the incidence of renal tubular epithelial cell edema and significantly decreased the expression of TNF-α and IL-6 as well as the expression and phosphorylation of NF-κB in the kidneys. Therefore, DN is associated with the expression of inflammatory factors, renal tubular epithelial cell edema and renal dysfunction in KK/HlJ mice. RHL improves renal function by decreasing kidney inflammation.

In previous studies, we demonstrated that rhein lysinate (RHL), the salt of rhein and lysine that is easily dissolved in water, inhibited the growth of tumor cells derived from breast and ovarian cancer, hepatocellular carcinoma, cervical cancer and lung carcinoma. Based on these observations, human glioma U87 cells and a xenograft model in BALB/c nude mice were used to examine the antitumor activity of RHL against human glioma. Notably, RHL statistically significantly suppressed the growth of human glioma U87 xenografts in BALB/c nude mice. In vitro, there was a significant reduction in cell proliferation after treatment with RHL in a dose- and time-dependent manner. The overall growth inhibition was correlated with the increase in reactive oxygen species (ROS) production and cell apoptosis. The apoptosis- and cell cycle-related proteins including BAX and Bim were increased, whereas Bcl-2 and cyclin D were decreased in the RHL-treated cells. The results demonstrated that RHL is highly effective against the growth of human glioma U87 xenografts in BALB/c nude mice. The potent antitumor activity of RHL may be mediated through downregulation of Bcl-2 and cyclin D expression and upregulation of BAX and Bim expression.

The aim of the present study was to investigate the anti-aging effects of rhein lysinate (RHL), and to explore its mechanism of action in a D-galactose-induced aging mouse model. Aging was induced by D-galactose (100 mg/kg/day) that was subcutaneously injected to animals for 8 weeks. RHL was simultaneously administered once a day by intragastric gavage. The appetite, mental condition, body weight and organ index of the mice were monitored. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined, and the levels of malondialdehyde (MDA) in the liver, kidney and serum were measured by appropriate assay kits. Western blot analysis was used to detect proteins associated with age. The results indicated that RHL may improve the appetite, mental state and organ conditions of the model mice, improve the activities of SOD and GSH-Px, reduce MDA levels and modulate the expression of age-associated proteins (Sirtuin 1, p21 and p16) in D-galactose-induced mice. Therefore, RHL may be effective at suppressing the aging process through a combination of enhancing antioxidant activity, scavenging free radicals and modulating aging-associated gene expression.

In a previous study, it was demonstrated that Rhein lysinate (RHL) inhibited HeLa cell proliferation via a specific mechanism. The aim of the present study was to clarify the mechanism of RHL by investigating its effect on mitochondrial damage and cell apoptosis. The results indicated that RHL inhibited cell growth and proliferation in HeLa cells. HeLa cells treated with RHL developed extensive vacuolization in a dose- and time-dependent manner. Ultrastructure analysis using transmission electron microscopy revealed that the vacuoles observed were damaged mitochondria and endoplasmic reticulum. The effects of RHL on mitochondria were further confirmed by a decrease in mitochondrial membrane potential and increased generation of reactive oxygen species. The mitochondrial proteome was analyzed, and the results demonstrated that the expression of the cytoskeletal protein keratin and dermal papilla derived protein 12 (associated with the oxidation- reduction process), which are associated with mitochondrial structure and function, were decreased compared with the untreated control group. Hoechst staining, flow cytometry and western blotting also revealed that apoptosis was induced at 24 h following RHL treatment. These results confirm that RHL toxicity in HeLa cells is a dynamic process. Vacuolar degeneration appeared in HeLa cells treated with 160 µmol/l RHL during the first 6 h and with the extension of RHL treatment, cell apoptosis was presented at ~24 h in HeLa cells.

Rhein lysinate (RHL) is the salt of lysine and rhein and the objective of this study was to investigate the protection of RHL to liver in diabetic mice. The model of type 2 diabetes was established by high-fat diet and streptozotocin treatment. Malondialdehyde, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were measured using a spectrophotometer. Inflammatory factors (TNF-α and IL-6) and related proteins (ERK1/2 and SREBP-1c) were analyzed by Western blot. Tissue profile was determined by hematoxylin and eosin staining and accumulation of fat was examined by Nile red staining. The results indicated that plasma glucose levels of type 2 diabetic mice were over 13.9 mM. Compared with model group, plasma glucose levels were decreased, however insulin levels were increased in RHL (25 and 50 mg/kg)-treated group. Elevated plasma triglyceride and cholesterol were also markedly attenuated after RHL treatment. The activities of SOD and GSH-Px of livers were increased after RHL treatment. Livers of RHL-treated mice had more normal structure and less steatosis than that of diabetic mice. Moreover, RHL decreased the expression of TNF-α and IL-6 and the phosphorylation of SREBP-1c and ERK1/2. In conclusion, RHL has a noticeable hepatic protection in diabetic mice.

To investigate the protective effects of rhein lysinate (RHL), a major bioactive constituent of the rhizome of rhubarb (Rheum palmatum Linn or Rheum tanguticum Maxim), against kidney impairment in senescence-prone inbred strain 10 (SAMP10) mice. SAMP10 mice were orally administered RHL (25 or 50 mg/kg) daily until 50% of the mice died. Senescence-resistant inbred strain 1 (SAMR1) mice administered no drug were taken as control. The kidneys were harvested after animal death, and examined morphologically and with immunochemical assays. The levels of MAD, SOD and GSH-px in the kidneys were measured with a photometric method. The expression of inflammatory factors and related proteins in the kidneys was analyzed using Western blotting. Treatment of SAMP10 mice with RHL had no effect on the body weight or phenotype. However, RHL significantly prolonged the median survival time of SAMP10 mice by approximately 25%, as compared to untreated SAMP10 mice. Compared SAMR1 mice, SAMP10 mice had a significantly lower level of SOD in the kidneys, but had no significant difference in the MDA or GSH-px levels. Treatment of SAMP10 mice with RHL significantly reduced the MAD level, and increased the SOD and GSH-px levels in the kidneys. Glomerulonephritis was observed in SAMP10 mice but not in SAMR1 mice. RHL decreased the incidence of glomerulonephritis, and significantly decreased the levels of TNF-α, IL-6, NF-κB, collagen types I and III in the kidneys. Accelerated senescence is associated with glomerulonephritis in SAMP10 mice, and RHL prolongs their median survival time by reducing the severity of glomerulonephritis.

Reducing the expression of endothelial cell adhesion molecules (ECAMs) is known to decrease inflammation-induced vascular complications. In this study, we explored whether rhein can reduce the inflammation-induced expression of ECAMs in human umbilical vein endothelial cells (HUVECs) with or without lipopolysaccharide (LPS) stimulation. HUVECs were treated with different concentrations of rhein with or without 2.5 μg/ml LPS stimulation. Cell viability was assayed using the MTT method. Real-time PCR and Western blot analysis were used to measure the transcription and expression levels of ECAMs, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-SELECTIN and related signaling proteins. The results indicated that rhein (0–20 μmol/L) and LPS (0–10 μg/ml) had no effect on the viability of HUVECs. LPS could promote the expression of VCAM-1, ICAM-1 and E-SELECTIN. Rhein appeared to target VCAM-1, ICAM-1 and E-SELECTIN, with the transcription and expression of all three factors being reduced by the rhein treatment (10 and 20 μmol/L). The transcription and expression of VCAM-1 were also reduced by treatment with rhein (10 and 20 μmol/L) in the presence of LPS stimulation. In conclusion, rhein treatment reduced the expression of VCAM-1 in HUVECs via a p38-dependent pathway.

The aim of this work was to construct a human recombinant p66Shc adenovirus and to investigate the inhibition of recombinant p66Shc adenovirus on MCF-7 cells. The recombinant adenovirus expression vector was constructed using the Adeno-X Adenoviral System 3. Inhibition of MCF-7 cell proliferation was determined by MTT. Intracellular ROS was measured by DCFH-DA fluorescent probes and 8-OHdG was detected by ELISA. Cell apoptosis and the cell cycle were assayed by flow cytometry. Western blot were used to observe protein expression. p66Shc expression was upregulated in 4 cell lines after infection. The inhibitory effect of p66Shc recombinant adenovirus on MCF-7 cells was accompanied by enhanced ROS and 8-OHdG. However, no significant differences were observed in the cell apoptosis rate. The ratio of the cell cycle G2/M phase showed a significant increase. Follow-up experiments demonstrated that the expressions of p53, p-p53, cyclin B1 and CDK1 were upregulated with the overexpression of p66Shc. The Adeno-X Adenoviral System 3 can be used to efficiently construct recombinant adenovirus containing p66Shc gene and the Adeno-X can inhibit the proliferation of MCF-7 cells by inducing cell cycle arrest at the G2/M phase. These results suggested that p66Shc may be a key target for clinical cancer therapy.

In previous studies, we found that rhein lysinate (RHL; the salt of rhein and lysine, easily dissolved in water) inhibited the growth of tumor cells in breast and ovarian cancer and hepatocellular carcinoma. This study aimed to investigate the effect of RHL on the growth of human cervical carcinoma HeLa cells and any underlying mechanisms. RHL inhibited the growth of HeLa cells in a dose- and time-dependent manner. It was also noted that RHL induced apoptosis in HeLa cells in a dose-dependent manner. Mechanistically, RHL triggered HeLa cell apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in RHL-induced growth inhibition. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed RHL-induced growth inhibition by decreasing the level of cleaved PARP and caspase-3/7. Phosphorylation of the extracellular signal-related kinase (ERK) was increased by RHL; conversely, the MEK inhibitor which inhibits ERK activity, synergistically enhanced RHL-induced growth inhibition in HeLa cells. The results showed that RHL inhibits Hela cell growth through the activation of p38 MAPK and JNK, and is a potential chemotherapeutic agent for cervical cancer.

Lidamycin (LDM, also known as C-1027) as an anti-cancer agent inhibits growth in a variety of cancer cells by inducing apoptosis and cell cycle arrest. In this study we demonstrated that inhibition of mouse embryonic carcinoma (EC) cell growth using LDM at low concentrations can be attributed to a loss of the cell's self-renewal capability but not to apoptosis or cell death, which can be correlated to the down-regulation of embryonic stem (ES) cell-like genes Oct4, Sox2 and c-Myc. MTT assays showed that LDM inhibited the growth of mouse P19 EC cells in a time- and dose-dependent manner. The EC cells exposed to a low dose (0.01 nM) of LDM lost their capability to generate colonies, as evidenced by the colony forming assay. Flow cytometer analyses demonstrated that LDM induced G1 arrest in exposed EC cells without apoptosis. Real-time qPCR, Western blotting and immunocytochemistry revealed that Oct4, Sox2 and c-Myc were down-regulated in LDM-exposed EC cells, but not adriamycin (ADM)-exposed cells. Furthermore, a combination of the low dose of LDM and ADM significantly reduced the proliferation of the cancer cells than single-agent treatment. This suggested that synergy of ADM and LDM improved chemotherapy. Taking together, our results indicate that LDM can reduce the capability for self-renewal that mouse EC cells possess through the repression of ES cell-like genes, thereby inhibiting carcinoma cell growth. This data also suggests that LDM might have potential for application in CSC-based therapy and be a useful tool for studying ES cell pluripotency and differentiation.

To investigate the effects of lidamycin (LDM) on a mouse myeloma cell line (SP2/0) and human multiple myeloma cell lines (U266 and SKO-007), and provide the basis for the use of LDM in cancer therapy.A 3-[4,5-dimethylthiazol-2-yl]5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]2H-tetrazolium inner salt (MTS) assay was used to determine the degree of growth inhibition by the drugs analyzed in this study. Cell cycle distribution and analysis were measured by flow cytometry combined with propidium iodide (PI) staining. The effects on apoptosis were measured by Hoechst 33342 staining and by flow cytometry combined with fluorescein-isothiocyanate-Annexin V/propidium iodide (FITC-Annexin V/PI) staining. Protein expression was determined by Western blot analysis. In vivo antitumor activity was measured using a murine myeloma model in BALB/c mice.There was a significant reduction in cell proliferation after treatment with LDM. The overall growth inhibition correlated with increased apoptotic cell death. LDM-induced cell apoptosis was associated with the activation of c-Jun-N-terminal kinase (JNK), and cleavage of caspase-3/7 and poly (ADP-ribose) polymerase (PARP). LDM markedly suppressed tumor growth in a murine myeloma model.LDM induces apoptosis in murine myeloma SP2/0 cells as well as in human myeloma U266 and SKO-007 cell lines. The sustained activation of JNK might play a critical role in LDM-induced apoptosis in the SP2/0 cell line. LDM demonstrates significant antitumor efficacy against myeloma SP2/0 cells in mice. Taken together, our data provide some clues for further research of the effects of LDM on human multiple myeloma.Acta Pharmacologica Sinica (2009) 30: 1025-1032; doi: 10.1038/aps.2009.75.

In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC(50) values of LDM for the U266 and SKO-007 cells were 0.0575 +/- 0.0015 and 0.1585 +/- 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM.

KNDC1 (kinase noncatalytic C-lobe domain containing 1), a brain-specific Ras guanine nucleotide exchange factor, controls the negative regulation of neuronal dendrite growth. However, the effect of KNDC1 on cellular senescence remains to be elucidated. The present study investigated the impact of KNDC1 knockdown on human endothelial cell senescence and the mechanisms underlying this effect. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as a model of biological aging. Senescence-associated β-galactosidase staining was used to detect cellular senescence and flow cytometry was employed to determine cell cycle progression. Quantitative polymerase chain reaction (qPCR) and western blot analysis were utilized to investigate mRNA transcription and protein expression. In the HUVECs, a senescence-like phenotypes developed with increasing passage number in vitro, which were associated with a progressive increase in the transcription and expression of KNDC1. KNDC1 knockdown promoted cell proliferation and partially reversed cellular senescence and cell cycle arrest in the G0/G1 phase in aging HUVECs. Investigations into the mechanism underlying this effect demonstrated that KNDC1 knockdown promoted HUVEC proliferation via the extracellular signal-regulated kinase signaling pathway and delayed HUVEC senescence by inhibiting the p53-p21-p16 transduction cascade. In addition, the promotion of the capillary tube network formation and the increased expression of endothelial nitric oxide synthase revealed that the activity and function of endothelial cells were enhanced. In conclusion, KNDC1 knockdown delayed endothelial cell senescence and promoted HUVEC activity and function. These results demonstrated that KNDC1 may be a novel therapeutic target for the development of agents to extend human life.

The objective of this study was to investigate the effect of rhein lysinate (RHL) on monocyte adhesion and its mechanism. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth inhibition by drugs. The monocyte chemoattractant protein (MCP)-1 levels were assayed using MCP-1 ELISA. The expression of proteins was detected by Western blotting analysis. The results indicated that RHL inhibited monocyte adhesion in a dose- and time-dependent manner. RHL (

Aim: To observe the effect of Rhein lysinate (RHL) on cellular senescence of human umbilical vascular endothelial cells (HUVECs) and elucidate its action mechanism. Methods: Cell viability was determined using MTT assay. The expression levels of Sirt1 mRNA and protein were measured by RT-PCR and Western blot, respectively. Senescence associated (SA)-β-galactosidase activity was detected to evaluate cell senescence. Apoptosis and cell cycle progression were determined using flow cytometry. Results: Treatment with RHL (10 μmol/L) for 48 h significantly increased the proliferation of HUVECs. In contrast, treatment with H 2 O 2 (25, 50 and 100 μmol/L) for 6 d dose-dependently increased β-galactosidase positive cells. Spontaneous cell senescence appeared as the cell passage increased. Pre-treatment with RHL (10 μmol/L) reversed H 2 O 2 or increased cell passage-induced cell senescence. H 2 O 2 (100 μmol/L) significantly arrested HUVECs at G1 phase (73.8% vs 64.6% in the vehicle group), which was blocked by RHL (10 μmol/L). RHL (5 and 10 μmol/L) enhanced both mRNA transcription and protein expression of Sirt1. H 2 O 2 (100 μmol/L) significantly decreased Sirt1 expression, and induced up-regulation of p53 acetylation and p16 INK4a , which were blocked by pre-treatment with RHL (10 μmol/L). Interference with siRNA for Sirt1 abolished the effect of RHL. H 2 O 2 (100 μmol/L) did not induce HUVEC apoptosis. The expression of apoptosis-associated proteins, such as p53, p21, Bcl-2, and Bax, did not significantly change in the presence of H 2 O 2 (100 μmol/L) or RHL (10 μmol/L). Conclusion: RHL protected HUVECs against cellular senescence induced by H 2 O 2 , via up-regulation of Sirt1 expression and down-regulation of the expression of acetyl-p53 and p16 INK4a .

Rhein lysinate (RHL), easily dissolved in water, is one of the anthraquinones, and has been shown to have anti-tumor activity in different human cancer cell lines. In the present study, we observed that RHL could cause vacuolar degeneration in HeLa cells, which was not observed in human umbilical vein endothelial cells (HUVECs) and other cell lines (SKOV-3 and SK-BR-3). Therefore, the purpose of this study was to investigate the anti-tumor effect of rhein lysinate on human cervix cancer HeLa cells. The results indicated that RHL could induce HeLa cell S-phase arrest and RHL (higher than 80 μM) also induced HeLa cell G2/M-phase arrest in a dose-dependent manner. Compared to the HeLa cells, RHL induced HUVECs G1-phase arrest at all dose levels tested in a dose-dependent manner. Treatment with RHL led to a significant S or G2/M-phase arrest through promoting the expression of p53 and p21 and the phosphorylation of p53. Moreover, 80 μM RHL could increase 5-FU anti-tumor activity. In conclusion, RHL could be a novel chemotherapeutic drug candidate for the treatment of human cervix cancer in the future.