Methyl Sartortuoate Inhibits Colon Cancer Cell Growth by Inducing Apoptosis and G2/M-Phase Arrest

The potential anti-neoplastic activity of terpenoids is of continued interest. In this study, we investigate whether methyl sartortuoate, a terpenoid isolated from soft coral, induced cell cycle arrest and apoptosis in a human colon cancer cell line. Culture studies found that methyl sartortuoate inhibited colon cancer cell (LoVo and RKO) growth and caused apoptotic death in a concentration- and time-dependent manner, by activation of caspase-8, caspase-9, caspase-3, p53 and Bax, and inactivation of B-cell lymphoma 2 (Bcl-2) apoptosis regulating proteins. Methyl sartortuoate treatment led to reduced expression of cdc2 and up-regulated p21 and p53, suggesting that Methyl sartortuoate induced G2-M arrest through modulation of p53/p21/cdc2 pathways. Methyl sartortuoate also up-regulated phospho-JNK and phospho-p38 expression levels. This resulted in cell cycle arrest at the G2-M phase and apoptosis in LoVo and RKO cells. Treatment with the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 prevented methyl sartortuoate-induced apoptosis in LoVo cells. Moreover, methyl sartortuoate also prevented neoplasm growth in NOD-SCID nude mice inoculated with LoVo cells. Taken together, these findings suggest that methyl sartortuoate is capable of leading to activation of caspase-8, -9, -3, increasing p53 and Bax/Bcl-2 ratio apoptosis through MAPK-dependent apoptosis and results in G2-M phase arrest in LoVo and RKO cells. Thus, methyl sartortuoate may be a promising anticancer candidate.     

DADS Suppresses Human Esophageal Xenograft Tumors through RAF/MEK/ERK and Mitochondria-Dependent Pathways

Diallyl disulfide (DADS) is a natural organosulfur compound isolated from garlic. DADS has various biological properties, including anticancer, antiangiogenic, and antioxidant effects. However, the anticancer mechanisms of DADS in human esophageal carcinoma have not been elucidated, especially in vivo. In this study, MTT assay showed that DADS significantly reduced cell viability in human esophageal carcinoma ECA109 cells, but was relatively less toxic in normal liver cells. The pro–apoptotic effect of DADS on ECA109 cells was detected by Annexin V-FITC/propidium iodide (PI) staining. Flow cytometry analysis showed that DADS promoted apoptosis in a dose-dependent manner and the apoptosis rate could be decreased by caspase-3 inhibitor Ac-DEVD-CHO. Xenograft study in nude mice showed that DADS treatment inhibited the growth of ECA109 tumor in both 20 and 40 mg/kg DADS groups without obvious side effects. DADS inhibited ECA109 tumor proliferation by down-regulating proliferation cell nuclear antigen (PCNA). DADS induced apoptosis by activating a mitochondria-dependent pathway with the executor of caspase-3, increasing p53 level and Bax/Bcl-2 ratio, and downregulating the RAF/MEK/ERK pathway in ECA109 xenograft tumosr. Based on studies in cell culture and animal models, the findings here indicate that DADS is an effective and safe anti-cancer agent for esophageal carcinoma.     

Bisdemethoxycurcumin Induces Cell Apoptosis and Inhibits Human Brain Glioblastoma GBM 8401/Luc2 Cell Xenograft Tumor in Subcutaneous Nude Mice In Vivo

Bisdemethoxycurcumin (BDMC) has biological activities, including anticancer effects in vitro; however, its anticancer effects in human glioblastoma (GBM) cells have not been examined yet. This study aimed to evaluate the tumor inhibitory effect and molecular mechanism of BDMC on human GBM 8401/luc2 cells in vitro and in vivo. In vitro studies have shown that BDMC significantly reduced cell viability and induced cell apoptosis in GBM 8401/luc2 cells. Furthermore, BDMC induced apoptosis via inhibited Bcl-2 (anti-apoptotic protein) and increased Bax (pro-apoptotic proteins) and cytochrome c release in GBM 8401/luc2 cells in vitro. Then, twelve BALB/c-nude mice were xenografted with human glioblastoma GBM 8401/luc2 cancer cells subcutaneously, and the xenograft nude mice were treated without and with BDMC (30 and 60 mg/kg of BDMC treatment) every 3 days. GBM 8401/luc2 cell xenografts experiment showed that the growth of the tumors was significantly suppressed by BDMC administration at both doses based on the reduction of tumor size and weights. BDMC did not change the body weight and the H&E histopathology analysis of liver samples, indicating that BDMC did not induce systemic toxicity. Meanwhile, treatment with BDMC up-regulated the expressions of BAX and cleaved caspase-3, while it down-regulated the protein expressions of Bcl-2 and XIAP in the tumor tissues compared with the control group. This study has demonstrated that BDMC presents potent anticancer activity on the human glioblastoma GBM 8401/luc2 cell xenograft model by inducing apoptosis and inhibiting tumor cell proliferation and shows the potential for further development to the anti-GBM cancer drug.     

Blockade of c-Met-Mediated Signaling Pathways by E7050 Suppresses Growth and Promotes Apoptosis in Multidrug-Resistant Human Uterine Sarcoma Cells

E7050 is a potent inhibitor of c-Met receptor tyrosine kinase and has potential for cancer therapy. However, the underlying molecular mechanism involved in the anti-cancer property of E7050 has not been fully elucidated. The main objective of this study was to investigate the anti-tumor activity of E7050 in multidrug-resistant human uterine sarcoma MES-SA/Dx5 cells in vitro and in vivo, and to define its mechanisms. Our results revealed that E7050 reduced cell viability of MES-SA/Dx5 cells, which was associated with the induction of apoptosis and S phase cell cycle arrest. Additionally, E7050 treatment significantly upregulated the expression of Bax, cleaved PARP, cleaved caspase-3, p21, p53 and cyclin D1, while it downregulated the expression of survivin and cyclin A. On the other hand, the mechanistic study demonstrated that E7050 inhibited the phosphorylation of c-Met, Src, Akt and p38 in HGF-stimulated MES-SA/Dx5 cells. Further in vivo experiments showed that treatment of athymic nude mice carrying MES-SA/Dx5 xenograft tumors with E7050 remarkably suppressed tumor growth. E7050 treatment also decreased the expression of Ki-67 and p-Met, and increased the expression of cleaved caspase-3 in MES-SA/Dx5 tumor sections. Therefore, E7050 is a promising drug that can be developed for the treatment of multidrug-resistant uterine sarcoma.     

Involvement of AMPKα and MAPK-ERK/-JNK Signals in Docetaxel-Induced Human Tongue Squamous Cell Carcinoma Cell Apoptosis

Cancers of the oral cavity can develop in the anatomic area extending from the lip, gum, tongue, mouth, and to the palate. Histologically, about 85–90% of oral cavity cancers are of the type squamous cells carcinomas (SCCs). The incidence of oral tongue SCC is higher in the tongue than any other anatomic area of the oral cavity. Here, we investigated the therapeutic effects and molecular mechanisms of docetaxel, which is a paclitaxel antitumor agent, on the cell growth of a human tongue SCC-derived SAS cell line. The results showed that docetaxel (10–300 nM) induced cytotoxicity and caspase-3 activity in SAS cells. Moreover, docetaxel (100 nM) promoted the expression of apoptosis-related signaling molecules, including the cleavages of caspase-3, caspase-7, and poly (ADP-ribose) polymerase (PARP). In mitochondria, docetaxel (100 nM) decreased the mitochondrial membrane potential (MMP) and Bcl-2 mRNA and protein expression and increased cytosolic cytochrome c protein expression and Bax mRNA and protein expression. In terms of mitogen-activated protein kinase (MAPK) and adenosine monophosphate-activated protein kinase (AMPK) signaling, docetaxel increased the expression of phosphorylated (p)-extracellular signal-regulated kinase (ERK), p-c-Jun N-terminal kinase (JNK), and p-AMPKα protein expression but not p-p38 protein expression. Moreover, the increase in caspase-3/-7 activity and Bax protein expression and decreased Bcl-2 protein expression and MMP depolarization observed in docetaxel-treated SAS cells could be reversed by treatment with either SP600125 (a JNK inhibitor), PD98059 (an MEK1/2 (mitogen-activated protein kinase kinase 1/2) inhibitor), or compound c (an AMPK inhibitor). The docetaxel-induced increases in p-JNK, p-ERK, and p-AMPKα protein expression could also be reversed by treatment with either SP600125, PD98059, or compound c. These results indicate that docetaxel induces human tongue SCC cell apoptosis via interdependent MAPK-JNK, MAPK-ERK1/2, and AMPKα signaling pathways. Our results show that docetaxel could possibly exert a potent pharmacological effect on human oral tongue SCC cell growth.     

Protective Effect of Quercetin on Sodium Iodate-Induced Retinal Apoptosis through the Reactive Oxygen Species-Mediated Mitochondrion-Dependent Pathway

Age-related macular degeneration (AMD) leads to gradual central vision loss and is the third leading cause of irreversible blindness worldwide. The underlying mechanisms for this progressive neurodegenerative disease remain unclear and there is currently no preventive treatment for dry AMD. Sodium iodate (NaIO₃) has been reported to induce AMD-like retinal pathology in mice. We established a mouse model for AMD to evaluate the effects of quercetin on NaIO₃-induced retinal apoptosis, and to investigate the pertinent underlying mechanisms. Our in vitro results indicated that quercetin protected human retinal pigment epithelium (ARPE-19) cells from NaIO₃-induced apoptosis by inhibiting reactive oxygen species production and loss of mitochondrial membrane potential as detected by Annexin V-FITC/PI flow cytometry. We also evaluated the relative expression of proteins in the apoptosis pathway. Quercetin downregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP and upregulated the expression of Bcl-2 through reduced PI3K and pAKT expressions. Furthermore, our in vivo results indicated that quercetin improved retinal deformation and increased the thickness of both the outer nuclear layer and inner nuclear layer, whereas the expression of caspase-3 was inhibited. Taken together, these results demonstrate that quercetin could protect retinal pigment epithelium and the retina from NaIO₃-induced cell apoptosis via reactive oxygen species-mediated mitochondrial dysfunction, involving the PI3K/AKT signaling pathway. This suggests that quercetin has the potential to prevent and delay AMD and other retinal diseases involving NaIO₃-mediated apoptosis.     

Ziyuglycoside II Inhibits the Growth of Human Breast Carcinoma MDA-MB-435 Cells via Cell Cycle Arrest and Induction of Apoptosis through the Mitochondria Dependent Pathway

Ziyuglycoside II is one of the major active compounds of Sanguisorba officinalis L., which has a wide range of clinical applications including hemostasis, antibiosis, anti-inflammation and anti-oxidation. This study investigated the effect of ziyuglycoside II on the growth of human breast carcinoma MDA-MB-435 cells for the first time. The results showed that ziyuglycoside II could significantly inhibit the growth of MDA-MB-435 cells through blocking cell cycle progression at G0/G1 and S phase as well as via inducing cell apoptosis. Accumulation of reactive oxygen species (ROS) was observed in the progression of cell cycle arrest, which was associated with the increased expression of cell cycle regulating factors, p53 and p21. Subsequent apoptosis induced by ziyuglycoside II was accompanied with the activation of mitochondrial pathway, in particular a decreased mitochondrial membrane potential (MMP) as well as increased Bax/Bcl-2 ratio, cytochrome c release and the activity of caspase-3 and caspase-9. In conclusion, our study was the first to report that ziyuglycoside II has inhibitory effect on the growth of MDA-MB-435 cells, which might become a potential therapeutic approach of breast cancer in the future.     

Myricanol Induces Apoptotic Cell Death and Anti-Tumor Activity in Non-Small Cell Lung Carcinoma in Vivo

This study explored the inhibiting effect and mechanism of myricanol on lung adenocarcinoma A549 xenografts in nude mice. Forty nude mice with subcutaneous A549 xenografts were randomly divided into five groups: high-dose myricanol (40 mg/kg body weight) group; middle-dose myricanol (20 mg/kg body weight) group; low-dose myricanol (10 mg/kg body weight) group; polyethylene glycol 400 vehicle group (1 mL/kg); and tumor model group. Nude mice were sacrificed after 14 days of treatment and the tumor inhibition rate (TIR, %) was then calculated. The relative mRNA expression levels of Bax, Bcl-2, VEGF, HIF-1α, and survivin in the tumor tissues were determined by real-time PCR. TUNEL assay was applied to determine cellular apoptosis, while IHC test was performed to detect the protein expression levels of Bax, Bcl-2, VEGF, HIF-1α, and survivin. The TIR of the three myricanol-treated groups ranged from 14.9% to 38.5%. The IHC results showed that the protein expression of Bcl-2, VEGF, HIF-1α, and survivin were consistently downregulated, whereas that of Bax was upregulated after myricanol treatment. Myricanol also significantly upregulated the mRNA expression of Bax and downregulated that of Bcl-2, VEGF, HIF-1α, and survivin in a dose-dependent manner (p < 0.05 to 0.001). These results are consistent with those of IHC. The TUNEL assay results indicated that apoptotic-positive cells significantly increased in the myricanol-treated tumor tissues compared with the cells of the vehicle control group (p < 0.01 to 0.001). These data suggest that myricanol could significantly decelerate tumor growth in vivo by inducing apoptosis.     

Se-Methylselenocysteine Inhibits Apoptosis Induced by Clusterin Knockdown in Neuroblastoma N2a and SH-SY5Y Cell Lines

Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 μM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.     

Anti-Tumor Effect of Heat-Killed Bifidobacterium bifidum on Human Gastric Cancer through Akt-p53-Dependent Mitochondrial Apoptosis in Xenograft Models

Paraprobiotics, inactivated microbial cells, regulate immune system and exhibit antioxidant and anti-inflammatory activities in patients with weakened immunity or the elderly. This study evaluated the anti-tumor effects of heat-killed Bifidobacterium and Lactobacillus on human gastric cancer MKN1 cells in vitro and in vivo in xenograft animal models. First, cytotoxicity and apoptosis in MKN1 cells of 11 different heat-killed Bifidobacterium or Lactobacillus strains were examined using the MTT assay or flow cytometry, respectively. Then, BALB/c nude mice xenograft animal models were implanted with human gastric cancer MKN1 cells and orally administered a selected single or a mixture of heat-killed bacterial strains to investigate their inhibitory effect on tumor growth. In addition, the expression of p-Akt, p53, Bax, Bak, cleaved caspase-9, -3, and PARP in the tumor tissues was analyzed using Western blotting assay or immunohistochemistry staining. The results show that heat-killed B. bifidum MG731 (MG731), L. reuteri MG5346 (MG5346), and L. rhamnosus MG5200 (MG5200) induced relatively greater apoptosis than other strains in MKN1 cells. Oral administration of a single dose or a mixture of MG731, MG5346, or MG5200 significantly delayed tumor growth, and MG731 had the most effective anti-tumor effect in the xenograft model. Protein expression of p-Akt, p53, Bax, cleaved caspase-3 and -9, and PARP in tumors derived from the xenograft model correlated with the results of the immunohistochemistry staining.     

Inhibition of Carbonic Anhydrase IX Promotes Apoptosis through Intracellular pH Level Alterations in Cervical Cancer Cells

Carbonic anhydrase IX (CAIX) is a hypoxia-related protein that plays a role in proliferation in solid tumours. However, how CAIX increases proliferation and metastasis in solid tumours is unclear. The objective of this study was to investigate how a synthetic CAIX inhibitor triggers apoptosis in the HeLa cell line. The intracellular effects of CAIX inhibition were determined with AO/EB, AnnexinV-PI, and γ-H2AX staining; measurements of intracellular pH (pHi), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP); and analyses of cell cycle, apoptotic, and autophagic modulator gene expression (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin, and LC3), caspase protein level (pro-caspase 3 and cleaved caspase-3, -8, -9), cleaved PARP activation, and CAIX protein level. Sulphonamide CAIX inhibitor E showed the lowest IC₅₀ and the highest selectivity index in CAIX-positive HeLa cells. CAIX inhibition changed the morphology of HeLa cells and increased the ratio of apoptotic cells, dramatically disturbing the homeostasis of intracellular pHi, MMP and ROS levels. All these phenomena consequent to CA IX inhibition triggered apoptosis and autophagy in HeLa cells. Taken together, these results further endorse the previous findings that CAIX inhibitors represent an important therapeutic strategy, which is worth pursuing in different cancer types, considering that presently only one sulphonamide inhibitor, SLC-0111, has arrived in Phase Ib/II clinical trials as an antitumour/antimetastatic drug.     

Chemical Characterization and Antitumor Activities of Polysaccharide Extracted from Ganoderma lucidum

Ganoderma lucidum polysaccharide (GLP) is a biologically active substance reported to possess anti-tumor ability. Nonetheless, the mechanisms of GLP-stimulated apoptosis are still unclear. This study aims to determine the inhibitory and apoptosis-inducing effects of GLP on HCT-116 cells. We found that GLP reduced cell viability on HCT-116 cells in a time- and dose-dependent manner, which in turn, induced cell apoptosis. The observed apoptosis was characterized by morphological changes, DNA fragmentation, mitochondrial membrane potential decrease, S phase population increase, and caspase-3 and -9 activation. Furthermore, inhibition of c-Jun N-terminal kinase (JNK) by SP600125 led to a dramatic decrease of the GLP-induced apoptosis. Western blot analysis unveiled that GLP up-regulated the expression of Bax/Bcl-2, caspase-3 and poly (ADP-ribose) polymerase (PARP). These results demonstrate that apoptosis stimulated by GLP in human colorectal cancer cells is associated with activation of mitochondrial and mitogen-activated protein kinase (MAPK) pathways.     

ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes

It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.     

A Natural Triterpene Derivative from Euphorbia kansui Inhibits Cell Proliferation and Induces Apoptosis against Rat Intestinal Epithelioid Cell Line in Vitro

Kansenone is a triterpene from the root of the traditional Chinese medicine, Euphorbia kansui. However, kansenone exerts serious toxicity, but the exact mechanism was not clear. In this work, the effects of kansenone on cell proliferation, cell cycle, cell damage, and cell apoptosis were investigated. The suppression of cell proliferation was assessed via the colorimetric MTT assay, and cell morphology was visualized via inverted microscopy after IEC-6 cells were incubated with different concentrations of kansenone. Reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) content were detected for evaluating cell damage. RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis. Annexin V-fluorescein isothiocyanate (FITC)/PI and Hoechst 33342/Annexin V-FITC/PI staining assay for cell apoptosis detection were performed using confocal laser scanning microscopy and high content screening. Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis. The results demonstrated that kansenone exerted high cytotoxicity, induced cell arrest at G0/G1 phase, and caused mitochondria damage. In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways.     

FLLL32 Triggers Caspase-Mediated Apoptotic Cell Death in Human Oral Cancer Cells by Regulating the p38 Pathway

Oral cancer is the most common oral malignant tumor in Taiwan. Although there exist several methods for treatment, oral cancer still has a poor prognosis and high recurrence. FLLL32, a synthetic analog of curcumin with antitumor activity, is currently known to induce melanoma apoptosis and inhibit tumor growth in various cancers. However, few studies have examined the mechanisms of FLLL32 in oral cancer. In this study, we explore whether FLLL32 induces apoptosis in oral cancer. We determined that FLLL32 can inhibit the cell viability of oral cancer. Next, we analyzed the effect of FLLL32 on the cell cycle of oral cancer cells and observed that the proportion of cells in the G2/M phase was increased. Additionally, annexin-V/PI double staining revealed that FLLL32 induced apoptosis in oral cancer cells. Data from the Human Apoptosis Array revealed that FLLL32 increases the expression of cleaved caspase-3 and heme oxygenase-1 (HO-1). FLLL32 activates proteins such as caspase-8, caspase-9, caspase-3, PARP, and mitogen-activated protein kinases (MAPKs) in apoptosis-related molecular mechanisms. Moreover, by using MAPK inhibitors, we suggest that FLLL32 induces the apoptosis of oral cancer cells through the p38 MAPK signaling pathway. In conclusion, our findings suggest that FLLL32 is a potential therapeutic agent for oral cancer by inducing caspase-dependent apoptosis and HO-1 activation through the p38 pathway. We believe that the activation of HO-1 and the p38 pathway by FLLL32 represent potential targets for further research in oral cancer.     

Inhibition of Glycolysis Suppresses Cell Proliferation and Tumor Progression In Vivo: Perspectives for Chronotherapy

A high rate of glycolysis is considered a hallmark of tumor progression and is caused by overexpression of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Therefore, we analyzed the possibility of inhibiting tumor and endothelial cell metabolism through the inhibition of PFKFB3 by a small molecule, (E)-1-(pyridin-4-yl)-3-(quinolin-2-yl)prop-2-en-1-one (PFK15), as a promising therapy. The effects of PFK15 on cell proliferation and apoptosis were analyzed on human umbilical vein endothelial cells (HUVEC) and the human colorectal adenocarcinoma cell line DLD1 through cytotoxicity and proliferation assays, flow cytometry, and western blotting. The results showed that PFK15 inhibited the proliferation of both cell types and induced apoptosis with decreasing the Bcl-2/Bax ratio. On the basis of the results obtained from in vitro experiments, we performed a study on immunodeficient mice implanted with DLD1 cells. We found a reduced tumor mass after morning PFK15 treatment but not after evening treatment, suggesting circadian control of underlying processes. The reduction in tumor size was related to decreased expression of Ki-67, a marker of cell proliferation. We conclude that inhibition of glycolysis can represent a promising therapeutic strategy for cancer treatment and its efficiency is circadian dependent.     

Ponicidin Inhibits Monocytic Leukemia Cell Growth by Induction of Apoptosis

In this study two monocytic leukemia cell lines, U937 and THP-1 cells, were used to investigate the anti-proliferation effects caused by ponicidin. Cell viability was measured by an MTT assay. Cell apoptosis was assessed by flow cytometry as well as DNA fragmentation analysis. Cell morphology was observed using an inverted microscope and Hoechst 33258 staining. RT-PCR and Western blot analysis were used to detect survivin as well as Bax and Bcl-2 expressions after the cells were treated with different concentrations of ponicidin. The results revealed that ponicidin could inhibit the growth of U937 and THP-1 cells significantly by induction of apoptosis. The suppression was in both time- and dose-dependent manner. Marked morphological changes of cell apoptosis were observed clearly after the cells were treated with ponicidin for 48∼72 h. RT-PCR and Western blot analysis demonstrated that both survivin and Bcl-2 expressions were down-regulated remarkably while Bax expression remained constant before and after apoptosis occurred. We therefore conclude that ponicidin has significant anti-proliferation effects by inducing apoptosis on leukemia cells in vitro, downregulation of survivin as well as Bcl-2 expressions may be the important apoptosis inducing mechanisms. The results suggest that ponicidin may serve as potential therapeutic agent for leukemia.     

BL-038, a Benzofuran Derivative,Induces Cell Apoptosis in Human Chondrosarcoma Cells through Reactive Oxygen Species/Mitochondrial Dysfunction and the Caspases Dependent Pathway

Chondrosarcoma is a highly malignant cartilage-forming bone tumor that has the capacity to invade locally and cause distant metastasis. Moreover, chondrosarcoma is intrinsically resistant to conventional chemotherapy or radiotherapy. The novel benzofuran derivative, BL-038 (2-amino-3-(2,6-dichlorophenyl)-6-(4-methoxyphenyl)benzofuran-4-yl acetate), has been evaluated for its anticancer effects in human chondrosarcoma cells. BL-038 caused cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353, but not in primary chondrocytes. Treatment of chondrosarcoma with BL-038 also induced reactive oxygen species (ROS) production. Furthermore, BL-038 decreased mitochondrial membrane potential (MMP) and changed mitochondrial-related apoptosis, by downregulating the anti-apoptotic activity members (Bcl-2, Bcl-x_L) and upregulating pro-apoptotic members (Bax, Bak) of the B-cell lymphoma 2 (Bcl-2) family of proteins, key regulators of the apoptotic machinery in cells. These results demonstrate that in human chondrosarcoma cells, the apoptotic and cytotoxic effects of BL-038 are mediated by the intrinsic mitochondria-mediated apoptotic pathway, which in turn causes the release of cytochrome c, the activation of caspase-9 and caspase-3, and the cleavage of poly (ADP-ribose) polymerase (PARP), to elicit apoptosis response. Our results show that the benzofuran derivative BL-038 induces apoptosis in chondrosarcoma cells.     

Neuroprotective Effects of Ischemic Preconditioning and Postconditioning on Global Brain Ischemia in Rats through the Same Effect on Inhibition of Apoptosis

Transient forebrain or global ischemia induces neuronal death in vulnerable CA1 pyramidal cells with many features. A brief period of ischemia, i.e., ischemic preconditioning, or a modified reperfusion such as ischemic postconditioning, can afford robust protection of CA1 neurons against ischemic challenge. Therefore, we investigated the effect of ischemic preconditioning and postconditioning on neural cell apoptosis in rats. The result showed that both ischemic preconditioning and postconditioning may attenuate the neural cell death and DNA fragment in the hippocampal CA1 region. Further western blot study suggested that ischemic preconditioning and postconditioning down-regulates the protein of cleaved caspase-3, caspase-6, caspase-9 and Bax, but up-regulates the protein Bcl-2. These findings suggest that ischemic preconditioning and postconditioning have a neuroprotective role on global brain ischemia in rats through the same effect on inhibition of apoptosis.