Zong Chao Liu

Senescence and telomere shortening induced by novel potent G-quadruplex interactive agents, quindoline derivatives, in human cancer cell lines.

Agents stabilizing G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalysed by telomerase or telomerase-independent mechanism and could therefore act as antitumor agents. In this study, we found that quindoline derivatives interacted preferentially with intramolecular G-quadruplex structures and were novel potent telomerase inhibitors. Treatment with quindoline derivatives reproducibly inhibited telomerase activity in human leukemia K562 cells and colon cancer SW620 cells. N′-(10H-Indolo [3,2-b] quinolin-11-yl)-N, N-dimethyl-propane-1,3-diamine (SYUIQ-5), (one of quindoline derivatives), when added to K562 and SW620 cell culture at nonacute cytotoxic concentrations, increased time of population doublings of K562 and SW620 cells, induced a marked cessation in cell growth and cellular senescence phenotype after 35 and 18 days, respectively. Growth cessation was accompanied by a shortening of telomere length, and induction of p16, p21 and p27 protein expression. However, another compound SYUIQ-7 with greater IC50 for telomerase had no obvious cellular effect in nonacute cytotoxic concentrations. These results indicate that quindoline derivatives as novel potent G-quadruplex interactive agents induce senescence and telomere shortening in cancer cells and therefore are promising agents for cancer treatment.

Acetylcholinesterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis

It has been shown that acetylcholinesterase (AChE) expression was induced during apoptosis and the anti-sense oligonucleotides and siRNA of AChE may prevent apoptosis in various cell types. However, the mechanisms underlying AChE upregulation remain elusive. We demonstrated here that c-Jun NH2-terminal kinase (JNK) could mediate AChE expression. In this study, both etoposide and excisanin A, two anticancer agents, induced apoptosis in colon cancer cell line SW620 as determined by Annexin V staining, the cleavage of caspase-3 and the proteolytic degradation of poly (ADP-ribose) polymerase (PARP). The results showed that both the agents upregulated AChE in SW620 cells. In the meantime, JNK was also activated and the expression and phosphorylation of c-Jun increased in SW620 cells exposed to the two agents. The induced AChE mRNA and protein expression could be blocked by SP600125, a specific inhibitor of SAPK/JNK, and small interfering RNA directed against JNK1/2. Transfection with adenovirus-mediated dominant negative c-Jun also blocked the upregulation of AChE expression. Together, these results suggest that AChE expression may be mediated by the activation of JNK pathway during apoptosis through a c-Jun-dependent mechanism.

Involvement of caspase-3 activation in squamocin-induced apoptosis in leukemia cell line HL-60

Annonaceous acetogenins have potent antitumor effect in vitro and in vivo. Squamocin is one of the annonaceous acetogenins and has been reported to have antiproliferative effect on cancer cells. Our results from this study showed that squamocin inhibited proliferation of HL-60 cells with IC50 value of 0.17 microg/ml and induced apoptosis of HL-60 cells. Investigation of the mechanism of squamocin-induced apoptosis revealed that treatment of HL-60 cells with squamocin resulted in extensive nuclear condensation. DNA fragmentation, cleavage of the death substrate poly (ADP-ribose) polymerase (PARP) and induction of caspase-3 activity. Pretreatment of HL-60 cells with caspase-3 specific inhibitor DEVD-CHO prevented squamocin-induced DNA fragmentation, PARP cleavage and cell death. The expression levels of protein bcl-2, bax have no change in response to squamocin treatment in HL-60 cells, whereas stress-activated protein kinase (SAPK/JNK) was activated after treatment with squamocin in HL-60 cells. These results suggest that apoptosis of HL-60 cells induced by squamocin requires caspase-3 activation and is related to SAPK activation.

SYUNZ‐16, a newly synthesized alkannin derivative, induces tumor cells apoptosis and suppresses tumor growth through inhibition of PKB/AKT kinase activity and blockade of AKT/FOXO signal pathway

Alkannin is the major bioactive compound of Arnebia euchroma roots, which is used in many therapeutic remedies in Chinese traditional medicine. SYUNZ‐16 is a new derivative of alkannin. In this study, anticancer effects of SYUNZ‐16 on human lung adenocarcinoma cell line GLC‐82 and human hepatocarcinoma cell line Hep3B were tested in vitro. The results showed SYUNZ‐16 could obviously inhibit the proliferation of these cancer cell lines via induction of apoptosis, with the evidence of increasing AnnexinV‐positive cells and cleaved caspase‐3 and PARP fragments. More importantly, we found that SYUNZ‐16 could inhibit AKT activity in cell‐free system. Treatment of cancer cells with SYUNZ‐16 decreased the phosphorylation of AKT. Additionally, SYUNZ‐16 partially attenuated the phosphorylation levels of FKHR and FKHRL1 in a dose‐dependent and time‐dependent fashion, and led to an increase in the nuclear accumulation of exogenous FKHR, and upregulated the mRNA expression of Bim and TRADD in cancer cells. Further study showed that constitutively activated AKT1 transfection could reduce apoptosis induction mediated by SYUNZ‐16. The in vivo experiments showed that SYUNZ‐16 had inhibitory effects on S‐180 sarcoma implanted to mice. And in GLC‐82 xenograft models, SYUNZ‐16 at 20 mg/kg/qod remarkably inhibited the tumor growth with the T/C value of 45.3%. Taken together, SYUNZ‐16 might be a potent inhibitor of AKT signaling pathway in tumor cells. These data provide evidence for the development of SYUNZ‐16 as a potential antitumor drug candidate for further research and development.

Ceramide induces cell cycle arrest and upregulates p27kip in nasopharyngeal carcinoma cells

Ceramide mediates differentiation, growth arrest, apoptosis, proliferation, cytokine biosynthesis and secretion, and a variety of other cellular functions. However, little is known regarding ceramide signaling linked to the cell cycle. In the present study, the effect of ceramide on cell cycle in nasopharyngeal carcinoma cell line CNE2 was investigated. The results showed that ceramide inhibited cell proliferation and induced cell cycle arrest in G1 phase in CNE2 cells. Exposure of CNE2 cells to ceramide resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27 and a decrease of phospho-Akt without reduced expression of total AKT protein. The activation of phosphatidylinositol-3-kinase (PI3K) and the protein expression of PTEN were unaffected following ceramide treatment. We concluded that ceramide induced cell cycle arrest in G1 phase in CNE2 cells and p27 up-regulation was involved in this process. In addition, up-regulation of p27 resulting from ceramide treatment may be due to the interruption of Akt, but decrease of phospho-Akt is independent of PI3K function or PTEN protein expression.

Synergistic cytotoxicity of Bcl-xL inhibitor gossypol and chemotherapeutic agents in non-Hodgkin's lymphoma cells

Anti-apoptotic proteins Bcl-2 and Bcl-xL are overexpressed in 80% of non-Hodgkin’s lymphoma cells and are thought to play an important role in the resistance of lymphoma cells to current chemotherapeutic agents. Gossypol, an orally-active polyphenolic aldehyde derived from the cotton plant, has been known to have potential anti-neoplastic activity. Recently, gossypol was found to bind to the BH3 binding groove of Bcl-xL and with lesser affinity to Bcl-2. The present study was conducted to determine whether gossypol increases the sensitivity of non-Hodgkin’s lymphoma cells to the actions of chemotherapeutic agents by potentiating treatment-induced apoptosis. The interactions observed between gossypol and chemotherapeutic drugs were analyzed using the median effect principle (CalcuSyn analysis). Our data showed that treatment of Ramos cells with gossypol not only induced cell arrest on the G0/G1 phase, but also augmented apoptosis and growth inhibition induced by etoposide (VP-16), doxorubicin hydrochloride (ADM), vincristine (VCR), and paclitaxel (taxol). However, when gossypol was combined with cisplatin (DDP) an antagonistic effect was observed. Gossypol-induced cell cycle arrest was accompanied by decreased expression of cyclin D1 in Ramos cells. In addition, the peroxisome proliferator-activated receptor (PARP) pathway is, at least in part, involved in the gossypol-induced apoptosis when combined with VP-16. These data indicate that single-agent gossypol is effective in inhibiting growth of non-Hodgkin’s lymphoma cells in vitro and combination studies with certain secondary chemotherapeutic agents further demonstrate it’s synergistic cytotoxicity. These findings support future preclinical and clinical studies of gossypol in the treatment of non-Hodgkin’s lymphoma.

The tumor suppressor p33ING1b enhances taxol-induced apoptosis by p53-dependent pathway in human osteosarcoma U2OS cells.

p33ING1b can stimulate cell cycle arrest, DNA repair, apoptosis and chemosensitivity. The actions of p33ING1b involve p53-dependent and p53-independent mechanisms. To investigate if the p33ING1b isoform is involved in the chemosensitivity of osteosarcoma cells, p33ING1b was overexpressed in p53+/+ U2OS cells or p53-mutant MG63 cells, and then cell growth arrest and apoptosis were assessed after treatment with taxol. The results showed that p33ING1b markedly increased taxol-induced growth inhibition and apoptosis in p53+/+ U2OS cells, but not in p53-mutant MG63 cells. Moreover, ectopic expression of p33ING1b could obviously upregulate p53, p21WAF1 and bax protein levels and activate caspase-3 in taxol-treated U2OS cells. Taken together, our data demonstrate that p33ING1b enhances taxol-induced apoptosis through p53-dependent pathway in human osteosarcoma cells. p33ING1b may be an important marker and/or therapeutic target in the prevention and treatment of osteosarcoma.

Houttuyninum, an active constituent of Chinese herbal medicine, inhibits phosphorylation of HER2/neu receptor tyrosine kinase and the tumor growth of HER2/neu-overexpressing cancer cells.

AIMS The overexpression of HER2/neu receptor plays a key role in tumorigenesis and tumor progression. Small molecules targeting HER2/neu have therapeutic value in cancers that overexpress HER2. In this present study, the effect of houttuyninum, a component in the Chinese herbal medicine Houttuynia cordata Thunb, on HER2/neu tyrosine phosphorylation and its in vivo antitumour activity was investigated. MAIN METHODS The phosphorylation and expression of proteins were determined by Western blot analysis. The MTT assay was employed to examine the inhibition of cell proliferation in vitro. Xenografts were established in nude mice for evaluating the antitumour activity of houttuyninum in vivo. KEY FINDINGS Houttuyninum inhibited phosphorylation of HER2 in a dose-dependent manner with an IC50 of 5.52 μg/ml without reducing HER2/neu protein expression in MDA-MB-453 cells. Houttuyninum also inhibited the activation of ERK1/2 and AKT, downstream molecules in the HER2/neu-mediated signal transduction pathway. In contrast, tyrosine phosphorylation of EGFR was unaffected when the concentration of houttuyninum was increased to 40 μg/ml in both A431 cells and MDA-MB-468 cells. Additionally, houttuyninum preferentially inhibited the growth of MDA-MB-453 cells that overexpressed HER2/neu; the MDA-MB-468 cells that overexpress EGFR remained unaffected. Administration of houttuyninum in vivo resulted in a significant reduction of phosphorylated HER2 levels and in tumor volumes of the BT474 and N87 xenografts, which both overexpress HER2/neu. SIGNIFICANCE Our findings showed that houttuyninum can inhibit the HER2/neu signalling pathway and the tumor growth of cancer cells that overexpress HER2/neu. This drug may provide therapeutic value in the treatment of cancers that involve overexpression of HER2/neu.

Rhabdastrellic acid-A inhibited PI3K/Akt pathway and induced apoptosis in human leukemia HL-60 cells

Increasing evidence suggests that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer treatment. Our investigation indicates that Rhabdastrellic acid‐A, an isomalabaricane triterpenoid isolated from the sponge, Rhabdastrella globostellata, inhibits proliferation of HL‐60 cells with an IC50 value of 0.68 μg/ml, and induces apoptosis. Rhabdastrellic acid‐A also induces cleavage of the death substrate poly (ADP‐ribose) polymerase (PARP) and caspase‐3. Pretreatment of HL‐60 cells with the caspase‐3 specific inhibitor, DEVD‐CHO, prevents Rhabdastrellic acid‐A‐induced DNA fragmentation and PARP cleavage. Activated PI3K and Akt significantly decreases after treatment with Rhabdastrellic acid‐A in HL‐60 cells. Expression levels of protein bcl‐2, bax remain unchanged in response to Rhabdastrellic acid‐A treatment in HL‐60 cells. These results suggest that Rhabdastrellic acid‐A inhibits PI3K/Akt pathway and induces caspase‐3 dependent‐apoptosis in HL‐60 human leukemia cells.

Variable Sensitivity of Endothelial Cells to Epirubicin in Xenografts of Human Nasopharyngeal Carcinoma CNE-2 Cells

Conventional anticancer drugs show non-specific vascular toxicity, and using anticancer drugs as angiogenesis inhibitors was suggested. However, our previous study suggested that vascular endothelial growth factor (VEGF) protected endothelial cells against chemotherapy drugs in vitro. To further test whether the vascular toxicity of anticancer drugs is active in vivo, epirubicin was i.p. injected into nude mice with s.c. xenografts of human nasopharyngeal carcinoma CNE-2 once (one-day schedule) or once a day from day 1 to day 7 (seven-day schedule). At 48 hours after the single injection or the 7th injection tumors were removed for detection of apoptosis of vascular endothelial cells vessels and the content of VEGF in tumor tissues. The results showed that epirubicin damaged tumor microvessels when the drug was given as a single dose, whereas epirubicin lost its vascular toxicity when the drug was given continuously for seven days, accompanied by higher levels of VEGF in tumor tissues. These results suggest the sensitivity of endothelial cells lining tumor vessels is variable during chemotherapy, and the protective effect of VEGF on endothelial cells might be related to the schedule of administration.