The mode of cytotoxic action of protocatechualdehyde on V79 cells was examined. The cytotoxicity of protocatechualdehyde on V79 cells was time- and cell cycle-dependent. The lethal action is selective for the cells at S phase only. The cells lethally damaged at S phase seemed to complete their DNA synthesis, although the rate of DNA synthesis became lower and the duration of S phase was prolonged. Some cells arriving at G2 phase directly entered the next S phase without cell division and died of karyorrhexis or karyolysis. Cells treated at stages of the cell cycle other than S showed normal progression of their cell cycle to the G1/S boundary. The block point of the cell cycle seemed to lie a little behind that with hydroxyurea. When the cells at phases other than S were blocked with protocatechualdehyde for 14 hr, elongation of the next S phase occurred after removal of protocatechualdehyde. On alkaline sucrose gradient analysis, little scission of parental DNA was observed in the cells treated with protocatechualdehyde.
Induction of apoptosis in CTLL-2 cells by protocatechualdehyde.
Wang Y, Hasuma T, Yano Y, Morishima Y, Matsui-Yuasa I, Otani S.
Department of Biochemistry, Osaka City University Medical School, Asahimachi, Abeno-ku, Osaka, 545-8585, Japan.
The purpose of this study was to investigate the possible molecular mechanisms of the antiproliferative effect induced by protocatechualdehyde (PA, a dihydroxybenzene derivative). The viability of cytotoxic T cells (CfLL-2) stimulated by IL2 was significantly inhibited at 0.12 mM PA. This inhibitory effect was associated with the induction of apoptosis detected by DNA fragmentation assay. DNA ladder appeared at 0.12 mM PA and the intensity of DNA ladder was visible at 0.3 mM PA. PA inhibited the Ib2-dependent tyrosine phosphorylation of 91, 80 and 55 KDa proteins, but did not affect IL2-dependent serine/threonine phosphorylation of proteins. The levels of bcl-2 protein and mRNA were suppressed by PA. An alteration in bax protein expression on the apoptosis process in CTLL-2 cells was not observed. However, caspase-3 activity was increased by PA. Our results demonstrate that PA inhibited cell proliferation and induced apoptosis in CTLL-2 cells. It is concluded that PA is a potent anti-proliferative agent and is expected to be a promising candidate for novel therapeutics.
Modulation of ornithine decarboxylase activity and mitogen-activated protein kinases in protocatechualdehyde-induced apoptosis of CTLL-2 cells.
Wang Y, Zhu Z, Hasuma T, Yano Y, Morishima Y, Matsui-Yuasa I, Otani S.
Department of Biochemistry, Osaka City University Medical School, Asahimachi, Abeno-ku, Osaka 545-8585, Japan.
Protocatechualdehyde (PA, a dihydroxybenzene derivative) has previously been shown to induce apoptotic cell death in cytotoxic T cells (CTLL-2). However, the molecular mechanisms by which PA regulates apoptosis are still unclear. In this study, the possible roles of ornithine decarboxylase activity (ODC) and mitogen-activated protein kinases (MAPKs) in the PA-induced apoptosis process were further investigated. We demonstrated that PA inhibited ODC activity induced by IL2 in a time- and dose-dependent manner. Furthermore, the expression of ODC mRNA stimulated by IL2 was also effectively suppressed. 0.12 mM PA inhibited the activation of ERK1/2 induced by IL2 and enhanced the activation of JNK, which was abrogated by IL2. No alteration in the effect of p38 MAPK on the apoptosis process was observed in the CTLL-2 cells. PD98059 (a specific ERK1/2 inhibitor) inhibited cell growth, led to cell apoptotic death and effectively decreased ODC activity and suppressed ERK1/2 activation induced by IL2. These data indicate that PA induced apoptosis in CTLL-2 cells by two mechanisms; either via inhibiting ODC induction or interfering with MAPK signaling pathways.
[Study of growth inhibitory effect and apoptosis induced by different matches of Tanshinone IIA and Salvianolic Acid B on Acute Promyelocytic Leukemia cells (HL-60)]
[Article in Chinese]
Guo L, Lei CK, Shan M.
College of Life Sciences, Shanxi Normal University, Xi'an 710061, China. glalguo@snnu.edu.cn
OBJECTIVE: To investigate the growth inhibiting and apoptosis inducing mechanisms of different matches of Tanshinone IIA and Salvianolic Acid B on Acute Promyelocytic Leukemia Cells (HL-60).
METHODS: The HL-60s' growth inhibition and apoptosis-induced rates were detected by MTT reduction assay and flow cytometry with various matches of TanIIA and SalB.
RESULTS: The HL-60s' growth inhibition and apoptosis-induced rates were found higher in the group of TanIIA plus SalB than other single groups, and in the group TanIIA-SalB (10-5 microg/ml) they were the highest (P<0.05).
CONCLUSION: TanIIA and SalB both have obviously growth inhibiting and apoptosis inducing effect, and union groups show stronger effect than any single group, while different matching proportion results in different growth inhibiting and apoptosis inducing action.
Salvianolic acid B inhibits growth of head and neck squamous cell carcinoma in vitro and in vivo via cyclooxygenase-2 and apoptotic pathways.
Hao Y, Xie T, Korotcov A, Zhou Y, Pang X, Shan L, Ji H, Sridhar R, Wang P, Califano J, Gu X.
Department of Oral Diagnostic Service, Howard University, Washington, DC 20059, USA.
Overexpression of cyclooxygenase-2 (COX-2) in oral mucosa has been associated with increased risk of head and neck squamous cell carcinoma (HNSCC). Celecoxib is a nonsteroidal anti-inflammatory drug, which inhibits COX-2 but not COX-1. This selective COX-2 inhibitor holds promise as a cancer preventive agent. Concerns about cardiotoxicity of celecoxib, limits its use in long-term chemoprevention and therapy. Salvianolic acid B (Sal-B) is a leading bioactive component of Salvia miltiorrhiza Bge, which is used for treating neoplastic and chronic inflammatory diseases in China. The purpose of this study was to investigate the mechanisms by which Sal-B inhibits HNSCC growth. Sal-B was isolated from S. miltiorrhiza Bge by solvent extraction followed by 2 chromatographic steps. Pharmacological activity of Sal-B was assessed in HNSCC and other cell lines by estimating COX-2 expression, cell viability and caspase-dependent apoptosis. Sal-B inhibited growth of HNSCC JHU-022 and JHU-013 cells with IC(50) of 18 and 50 microM, respectively. Nude mice with HNSCC solid tumor xenografts were treated with Sal-B (80 mg/kg/day) or celecoxib (5 mg/kg/day) for 25 days to investigate in vivo effects of the COX-2 inhibitors. Tumor volumes in Sal-B treated group were significantly lower than those in celecoxib treated or untreated control groups (p < 0.05). Sal-B inhibited COX-2 expression in cultured HNSCC cells and in HNSCC cells isolated from tumor xenografts. Sal-B also caused dose-dependent inhibition of prostaglandin E(2) synthesis, either with or without lipopolysaccharide stimulation. Taken together, Sal-B shows promise as a COX-2 targeted anticancer agent for HNSCC prevention and treatment. (c) 2008 Wiley-Liss, Inc.
Inhibitory effects of salvianolic acid B on the high glucose-induced mesangial proliferation via NF-kappaB-dependent pathway.
Luo P, Tan Z, Zhang Z, Li H, Mo Z.
West China School of Pharmacy, Sichuan University, Sichuan, PR China.
Salvianolic acid B (Sal B) is one of the major water-soluble compounds isolated from Radix Salviae Miltiorrhizae (Danshen in Chinese) that has been reported to be beneficial to treatment of diabetic complications. However, the mechanisms involved in these effects are not discussed in relation to mesangial proliferation via modulation of NF-kappaB. To explain this, human mesangial cells were pretreated with or without Sal B (0.1, 1, 10 microM) for 24 h and stimulated with high glucose (30 mM). Then the effects of Sal B on mesangial cells proliferation, extracellular matrix production and the possible mechanisms were evaluated by methylthiazoletetrazolium assay, flow cytometry assay, enzyme-linked immunosorbent assay, gelatin zymography assay and western blot assay. These results indicated that Sal B could inhibit high glucose-induced mesangial cells proliferation and extracellular matrix production in a dose-dependent manner, partially through modulating the cell-cycle progress and MMP-2 and MMP-9 activities via suppressing NF-kappaB activation, suggesting that Sal B may be a promising agent for treating diabetic nephropathy.
Salvianolic acid B, an antioxidant from Salvia miltiorrhiza, prevents 6-hydroxydopamine induced apoptosis in SH-SY5Y cells.
Tian LL, Wang XJ, Sun YN, Li CR, Xing YL, Zhao HB, Duan M, Zhou Z, Wang SQ.
Laboratory of Biotechnology, Beijing Institute of Radiation Medicine, 27# Taiping Road, Haidian District, Beijing 100850, PR China.
Oxidative stress caused by dopamine may play an important role in the pathogenesis of Parkinson's disease. Salvianolic acid B is an antioxidant derived from the Chinese herb, Salvia miltiorrhiza. In this study, we investigated the neuroprotective effect of salvianolic acid B against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells. Pretreatment of SH-SY5Y cells with salvianolic acid B significantly reduced 6-hydroxydopamine-induced generation of reactive oxygen species, and prevented 6-hydroxydopamine-induced increases in intracellular calcium. Our data demonstrated that 6-hydroxydopamine-induced apoptosis was reversed by salvianolic acid B treatment. Salvianolic acid B reduced the 6-hydroxydopamine-induced increase of caspase-3 activity, and reduced cytochrome C translocation into the cytosol from mitochondria. The 6-hydroxydopamine-induced decrease in the Bcl-x/Bax ratio was prevented by salvianolic acid B. Additionally, salvianolic acid B decreased the activation of extracellular signal-regulated kinase and induced the activation of 6-hydroxydopamine-suppressed protein kinase C. These results indicate that the protective function of salvianolic acid B is dependent upon its antioxidative potential. Our results strongly suggest that salvianolic acid B may be effective in treating neurodegenerative diseases associated with oxidative stress.
Antiproliferative effect of salvianolic acid A on rat hepatic stellate cells.
Lin YL, Lee TF, Huang YJ, Huang YT.
National Research Institute of Chinese Medicine, Taipei 112, Taiwan.
Suppression of activation or proliferation, or induction of apoptosis in hepatic stellate cells (HSCs) have been proposed as therapeutic strategies against liver fibrosis. Salvia miltiorrhiza has been reported to exert antifibrotic effects in rats with hepatic fibrosis, but its mechanisms of action remain to be clarified. We have investigated the effects of salvianolic acid A (Sal A), an active principle from S. miltiorrhiza, on the proliferation-related biomarkers in a cell line of rat HSCs (HSC-T6) stimulated with platelet-derived growth factor-BB homodimer (PDGF-BB). DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of Sal A. The results showed that Sal A (1-10 microM) concentration-dependently attenuated PDGF-BB-stimulated proliferation (BrdU incorporation) in HSC-T6 cells. Sal A at 10 microM induced cell apoptosis in PDGF-BB-incubated HSCs, together with a reduction of Bcl-2 protein expression, induction of cell cycle inhibitory proteins p21 and p27, and down-regulation of cyclins D1 and E, suppression of Akt phosphorylation, reduction in PDGF receptor phosphorylation, and an increase in caspase-3 activity. Sal A exerted no direct cytotoxicity on primary hepatocytes and HSC-T6 cells under experimental concentrations. Our results suggested that Sal A inhibited PDGF-BB-activated HSC proliferation, partially through apoptosis induction.
In vitro protective effects of salvianolic acid B on primary hepatocytes and hepatic stellate cells.
Lin YL, Wu CH, Luo MH, Huang YJ, Wang CN, Shiao MS, Huang YT.
National Research Institute of Chinese Medicine, Taipei 112, Taiwan.
The production of reactive oxygen species (ROS) is believed to be involved in liver injury and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is a key feature of liver fibrosis. Salvia miltiorrhiza is a traditional Chinese herb used in the treatment of cardiovascular and liver diseases to resolve stasis. The effects of salvianolic acid B (Sal B), a major component of Salvia miltiorrhiza, on oxidative damage include free radical DPPH scavenging, malondialdehyde (MDA) formation and ROS generation in primary rat hepatocytes and HSCs, and on alpha-SMA, and collagen expression in transforming growth factor-beta1 (TGF-beta1)-stimulated HSCs were examined. Results indicated that Sal B scavenged DPPH potently with an IC50 2.2+/-0.2 microg/ml (3.06+/-0.3 microM), inhibited lipid peroxidation and eliminated ROS accumulation in a concentration-dependent manner on primary rat hepatocytes and HSCs. Sal B also reduced alpha-SMA and collagen synthesis and deposition in HSCs, and had no direct cytotoxicity on both hepatocytes and HSCs. Our results suggest that Sal B ameliorated oxidative damage and eliminated ROS accumulation in hepatocytes, and attenuated HSC activation, potentially conferring hepatoprotective and anti-fibrogenic effects.
Induction of apoptosis in CTLL-2 cells by protocatechualdehyde.
Wang Y, Hasuma T, Yano Y, Morishima Y, Matsui-Yuasa I, Otani S.
Department of Biochemistry, Osaka City University Medical School, Asahimachi, Abeno-ku, Osaka, 545-8585, Japan.
The purpose of this study was to investigate the possible molecular mechanisms of the antiproliferative effect induced by protocatechualdehyde (PA, a dihydroxybenzene derivative). The viability of cytotoxic T cells (CfLL-2) stimulated by IL2 was significantly inhibited at 0.12 mM PA. This inhibitory effect was associated with the induction of apoptosis detected by DNA fragmentation assay. DNA ladder appeared at 0.12 mM PA and the intensity of DNA ladder was visible at 0.3 mM PA. PA inhibited the Ib2-dependent tyrosine phosphorylation of 91, 80 and 55 KDa proteins, but did not affect IL2-dependent serine/threonine phosphorylation of proteins. The levels of bcl-2 protein and mRNA were suppressed by PA. An alteration in bax protein expression on the apoptosis process in CTLL-2 cells was not observed. However, caspase-3 activity was increased by PA. Our results demonstrate that PA inhibited cell proliferation and induced apoptosis in CTLL-2 cells. It is concluded that PA is a potent anti-proliferative agent and is expected to be a promising candidate for novel therapeutics.
Antiproliferative activity of protocatechualdehyde on Chinese hamster cells grown in culture.
Nishimura M, Umeda M, Kusanagi A, Urakabe R, Yukimatsu K, Otawara S.
The mode of cytotoxic action of protocatechualdehyde on V79 cells was examined. The cytotoxicity of protocatechualdehyde on V79 cells was time- and cell cycle-dependent. The lethal action is selective for the cells at S phase only. The cells lethally damaged at S phase seemed to complete their DNA synthesis, although the rate of DNA synthesis became lower and the duration of S phase was prolonged. Some cells arriving at G2 phase directly entered the next S phase without cell division and died of karyorrhexis or karyolysis. Cells treated at stages of the cell cycle other than S showed normal progression of their cell cycle to the G1/S boundary. The block point of the cell cycle seemed to lie a little behind that with hydroxyurea. When the cells at phases other than S were blocked with protocatechualdehyde for 14 hr, elongation of the next S phase occurred after removal of protocatechualdehyde. On alkaline sucrose gradient analysis, little scission of parental DNA was observed in the cells treated with protocatechualdehyde.