microRNA-34a sensitizes lung cancer cell lines to DDP treatment independent of p53 status.

miR-34a was identified as one of the downregulated microRNAs (miRNAs) in human lung cancer. However, the precise biological role of miR-34a in p53 deficient lung cancer cell lines remains largely elusive. In the present study, we aimed to identify the role of miR-34a in the regulation of lung cancer cell proliferation. Using quantitative RT-PCR analysis, we found that miR-34a was highly upregulated in the p53 wild-type A549 human lung cancer cell line when treated with the DNA damaging agent adriamycin (ADR), but not in the SBC-5 cells harboring mutated p53. Transient introduction of miR-34a into A549 and SBC-5 cell lines caused complete suppression of cell proliferation and induced the cell cycle arrested at the G(1) phase. When we knockdown the miR-34a downstream target--Sitr1--using the small-interfering RNA, there was also a cell growth inhibition in both cell lines though not as much as miR-34a did. Moreover, we demonstrated that pretransfection of miR-34a could increase the sensitivity of both lung cancer cell lines to cisplatin (DDP), and this could be reverted by the miR-34a inhibitor. Moreover, when cells pretreated with siR-Sirt1, they are more sensitive to DDP than the control pretreated cells as well. We thus hypothesize the miR-34a/Sirt1 cascade involved with p53-independent functions. Overall, in this study, we found the proliferation inhibition function of miR-34a in vitro in lung cancer cell lines is p53 independent, and also demonstrated the combination therapeutic potential of miR-34a and DDP in lung cancer cell lines.

Serum anti-AEG-1 auto-antibody is a potential novel biomarker for malignant tumors.

Malignant tumors are the leading cause of mortality worldwide. The search for new biomarkers for the early diagnosis of the onset of cancer to reduce high mortality is crucial. The potential of minimal invasive testing using serum from patients renders auto-antibodies promising biomarkers for cancer diagnosis. In this study, a 181 amino acid peptide of extracellular astrocyte elevated gene-1 (AEG-1) was expressed and purified, and the peptide was used in an ELISA assay to detect anti-AEG-1 auto-antibodies (AEG-1-Abs) in 483 serum samples from different cancer patients and 230 serum samples from normal blood donors. The results showed that AEG-1-Abs at titers ≥1:50 were detected in 238 of 483 (49%) cancer patients, and the positive antibody responses in different cancer patients were as follows: 44 of 98 (45%) in breast cancer patients, 48 of 96 (50%) in hepatic carcinoma patients, 43 of 88 (49%) in rectal cancer patients, 51 of 113 (45%) in lung cancer patients, and 52 of 88 (59%) in gastric cancer patients. These results were compared with 0 of 230 (0%) in normal individuals. Moreover, AEG-1-Abs at titers ≥1:50 were also detected in 24 of 94 (26%) cancer patients in TNM stages I and II, and the positive rates of AEG-1-Abs decreased with age. These results suggest that the AEG-1-Ab response acts as a diagnostic biomarker for cancer patients with AEG-1-positive expression, and may also prove to be a possible inducer, with substantial immunity against AEG-1 by immunization boosting with AEG-1 vaccines.

Silencing of Notch3 Using shRNA driven by survivin promoter inhibits growth and promotes apoptosis of human T-cell acute lymphoblastic leukemia cells.

BACKGROUND: As a highly conserved system, the activation of the Notch pathway has been implicated in the tumorigenesis of various hematologic diseases, including leukemias, lymphomas, and multiple myeloma. The Notch3 receptor is frequently expressed in T-cell acute lymphoblastic leukemia (T-ALL). METHODS: To explore its possibility as a therapeutic target for T-ALL, we investigated the effect of Notch3 silencing on Jurkat and SupT1 cells using a novel tumor-specific short hairpin RNA (shRNA) driven by survivin promoters. RESULTS: We found that downregulated expression of Notch3 correlated with significant apoptosis and inhibition of proliferation. CONCLUSION: These facts suggest that downregulating expression of Notch3 could attenuate the Notch signaling activity in T-ALL. All these results indicate that inhibition of Notch3 expression can result in potent antitumor activity in T-ALL.

Effects of sargentgloryvine stem extracts on HepG-2 cells in vitro and in vivo.

AIM: To observe the effects of sargentgloryvine stem extracts (SSE) on the hepatoma cell line HepG-2 in vitro and in vivo and determine its mechanisms of action. METHODS: Cultured HepG-2 cells treated with SSE were analysed by 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-Diphenyltetrazolium bromide and clone formation assay. The cell cycle and apoptosis analysis were conducted by flow cytometric, TdT-Mediated dUTP Nick End Labeling and acridine orange/ethidium bromide staining methods, and protein expression was examined by both reverse transcriptase-polymerase chain reaction and Western blotting. The pathological changes of the tumor cells were observed by haematoxylin and eosin staining. Tumor growth inhibition and side effects were determined in a xenograft mouse model. RESULTS: SSE treatment could not only inhibit HepG-2 cell proliferation in a dose- and time-dependent manner but also induce apoptosis and cell cycle arrest at the S phase. The number of colonies formed by SSE-treated tumor cells was fewer than that of the controls (P < 0.05). SSE induced caspase-dependent apoptosis accompanied by a significant decrease in Bcl-xl and Mcl-1 and elevation of Bak expression (P < 0.05). Tumor necrosis factor α in the xenograft tumor tissue and the liver functions of SSE-treated mice showed no significant changes at week 8 compared with the control group (P > 0.05). Systemic administration of SSE could inhibit the HepG-2 xenograft tumor growth with no obvious toxic side effects on normal tissues. CONCLUSION: SSE can induce apoptosis of HepG-2 cells in vitro and in vivo through decreasing expression of Bcl-xl and Mcl-1 and increasing expression of Bax.

Essential role of cell cycle regulatory genes p21 and p27 expression in inhibition of breast cancer cells by arsenic trioxide.

Arsenic trioxide (As2O3), a component of traditional Chinese medicine, has been used successfully for the treatment of acute promyelocytic leukemia (APL), and As2O3 is of potential therapeutic value for the treatment of other promyelocytic malignancies and some solid tumors including breast cancer. However, the precise molecular mechanisms through which As2O3 induces cell cycle arrest and apoptosis in solid tumors have not been clearly understood. The goal of our study is to gain insight into the general biological processes and molecular functions that are altered by As2O3 treatment in MCF-7 breast cancer cells and to identify the key signaling processes that are involved in the regulation of these physiological effects. In the present study, MCF-7 cells were treated with 5 µM As2O3, and the differential gene expression was then analyzed by DNA microarray. The results showed that As2O3 treatment changed the expression level of several genes that involved in cell cycle regulation, signal transduction, and apoptosis. Notably, As2O3 treatment increased the mRNA and protein levels of the cell cycle inhibitory proteins, p21 and p27. Interestingly, knocking down p21 or p27 individually did not alter As2O3-induced apoptosis and cell cycle arrest; however, the simultaneous down-regulation of both p21 and p27 resulted in attenuating of G1, G2/M arrest and reduction in apoptosis, thus indicating that p21 and p27 as the primary molecular targets of As2O3 against breast cancer. Overall, our results provide new insights into As2O3-related signaling activities, which may facilitate the development of As2O3-based anticancer strategies and/or combination therapies against solid tumors.

Synergistic tumor growth-inhibitory effect of the prostate-specific antigen-activated fusion peptide BSD352 for prostate cancer therapy.

Prostate-specific antigen (PSA), a serine protease, is a promising target for the development of prodrugs in prostate cancer treatment. In this study, we designed a novel fusion peptide, BSD352, containing three functional domains: a protein transduction domain from HIV transactivating regulatory protein (TAT) followed by the BH3 domain of the p53 upregulated modulator of apoptosis (TAT-BH3), an anti-vascular endothelial growth factor peptide (SP5.2), and an anti-basic fibroblast growth factor peptide (DG2). These different domains in BSD352 were linked together by a linker sequence corresponding to a PSA hydrolytic substrate peptide. The BSD352 fusion peptide could be selectively cleaved by PSA in PSA-producing LNCaP prostate cancer cells. Furthermore, the BSD352 fusion peptide was efficiently transduced into tumor cells both in vitro and in vivo, and the BH3 domain was found to induce tumor cell apoptosis by elevating the expression of Bax, cytochrome C release, and caspase-9 cleavage. Moreover, the SP5.2 and DG2 domains in the BSD352 fusion peptide also exhibited in-vitro endothelial cell growth inhibition and in-vivo antiangiogenic activities. Direct injection of BSD352 into an established LNCaP xenograft tumor in mice inhibited tumor growth, whereas a synergistic effect was observed with the combined use of wild-type BH3, SP5.2, and DG2 functional domains. These results suggest that BSD352 could be beneficial for the treatment of accessible prostate tumors and may provide a complementary strategy for prostate cancer therapy.

p21WAF1/CIP1 gene transcriptional activation exerts cell growth inhibition and enhances chemosensitivity to cisplatin in lung carcinoma cell.

BACKGROUND: Non-small-cell lung carcinomas (NSCLCs) exhibit poor prognosis and are usually resistant to conventional chemotherapy. Absence of p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor, has been linked to drug resistance in many in vitro cellular models. RNA activation (RNAa) is a transcriptional activation phenomena guided by double-strand RNA (dsRNA) targeting promoter region of target gene. METHODS: In this study, we explored the effect of up-regulation of p21 gene expression on drug-resistance in A549 non-small-cell lung carcinoma cells by transfecting the dsRNA targeting the promoter region of p21 into A549 cells. RESULTS: Enhanced p21 expression was observed in A549 cells after transfection of dsRNA, which was correlated with a significant growth inhibition and enhancement of chemosensitivity to cisplatin in A549 cells in vitro. Moreover, in vivo experiment showed that saRNA targeting the promoter region of p21 could significantly inhibit A549 xenograft tumor growth. CONCLUSIONS: These results indicate that p21 plays a role in lung cancer drug-resistance process. In addition, this study also provides evidence for the usage of saRNA as a therapeutic option for up-regulating lower-expression genes in lung cancer.

Stathmin is involved in arsenic trioxide-induced apoptosis in human cervical cancer cell lines via PI3K linked signal pathway.

Although the mechanisms of arsenic trioxide (As2O3)-induced apoptosis have been elucidated extensively in hematologic cancers, those in solid tumors have yet to be clearly defined. In the present study, we show that As2O3 triggers apoptosis through the intrinsic pathway and significantly downregulates stathmin expression. Decreased stathmin expression is necessary for the dissipation of mitochondrial membrane potential (Δ ψm), the translocation of cytochrome C from the mitochondria to the cytosol, and subsequent cell death. Overexpression of wild type stathmin effectively delays As2O3-mediated mitochondrial events. Conversely, expression of a small interfering RNA (siRNA) targeting stathmin enhances As2O3-triggered apoptosis in cell culture and in mouse models. Furthermore, we demonstrate that As2O3-induced stathmin downregulation is mediated through the phosphatidylinositol-3-kinase (PI3K) signaling pathway, and that a PI3K inhibitor effectively attenuated stathmin downregulation and cell apoptosis upon As2O3-treatment. These data support a stathmin-dependent pathway of As2O3-mediated cell death in solid tumor cells, and indicate that stathmin is a target of the PI3K/Akt pathway in cervical cancer cells. All these results may provide a rationale for improving the efficacy of As2O3 as a therapeutic agent through combination treatment with stathmin inhibition or PI3K/Akt inhibitors.

Apoptosis induction effects of EGCG in laryngeal squamous cell carcinoma cells through telomerase repression.

Epigallocatechin-3-gallate (EGCG), the major component of green tea polyphenol, has potent efficiency to prevent the growth of a variety of cancer cells. As a novel anticancer agent for treatment of cancers, EGCG is promising and the mechanism has not been fully understood. Laryngeal squamous cell carcinoma (LSCC) is one common tumor in head and neck cancers. In the present study, we assess the effects of EGCG on LSCC cell line Hep-2, and their possible involvement in EGCG-induced apoptosis. The result showed that treatment of Hep-2 cells with EGCG decreased the cell viability, inhibited the growth and proliferation, induced apoptosis and increased the activity of caspase-3 in a dose-dependent manner. Furthermore, we found that EGCG-treatment repressed telomerase activity effectively in a concentration-dependent manner. The combined results show that EGCG induced apoptosis in Hep-2 cells via inhibiting the telomerase activity.

The BH3-only protein PUMA is involved in green tea polyphenol-induced apoptosis in colorectal cancer cell lines.

The green tea polyphenol (GTP) has been shown to possess cancer therapeutic effect through induction of apoptosis, while the underlying molecular mechanism of its anticancer effect is not well understood. PUMA (p53-upregulated modulator of apoptosis) plays an important role in the process of apoptosis induction in a variety of human tumor cells in both p53-dependent and -independent manners. However, whether or not PUMA is involved in the process of GTP-induced apoptosis in cancer cells has not been well reported. In the present study, we treated HT-29 (mutant p53) and LoVo (wild type p53) human colorectal cancer cells with different concentrations of GTP, which led to repression of cell proliferation and induction of apoptosis in both cell lines. Meanwhile, we also observed increased PUMA expression and decreased ERK (extracellular signal-regulated kinase) activity in both of GTP-treated tumor cell lines carrying different genotypes of p53. To determine the role of PUMA in GTP-induced apoptosis, we used stable RNA interference (RNAi) to suppress PUMA expression. As a result, apoptosis was abrogated in response to GTP-treatment. We also found that suppression of ERK activity by either RNAi or its specific inhibitor significantly enhanced GTP-induced PUMA expression. All these results indicate that PUMA plays a critical role in GTP-induced apoptosis pathway in human colorectal cancer cells and can be regulated partly by ERK inactivation. Demonstration of the molecular mechanism involved in the anti-cancer effect of GTP may be useful in the therapeutic target selection for p53 deficient colorectal cancer.

Silencing stathmin gene expression by survivin promoter-driven siRNA vector to reverse malignant phenotype of tumor cells.

Stathmin gene overexpression has been shown to play an important role in maintenance of malignant phenotype in tumor cells, and the blocking efficacy and tumor specificity of this target has been concerned in clinical trails. In this report, we designed survivin promoter-driven siRNA eukaryotic expression vector that expressed the small interfering RNA targeting stathmin gene to selectively knock down the stathmin gene expression in two different kinds of tumor cell lines while sparing normal cell lines. The therapeutic potential of this recombinant vector was tested in human cervical cancer Hela cells and osteosarcoma SSOP-9607 cells, and in human umbilical vein endothelial cell line ECV304 cells as control. The siRNA vector- transfected Hela cells and SSOP-9607 cells revealed marked inhibition of stathmin expression and a dramatic growth inhibition comparing with ECV304 cells, parental-vector transfected cells and untransfected cells. Cell cycle analysis of siRNA vector transfected tumor cells by Flow Cytometry showed G(2)/M phase block, while morphologic analysis by TURNEL staining method showed marked increase of apoptosis. Our study indicates that survivin gene promoter-driven stathmin siRNA expression vector may have potential use in tumor gene therapy with targeted tumor gene silencing effect.

HotKnots: heuristic prediction of RNA secondary structures including pseudoknots.

We present HotKnots, a new heuristic algorithm for the prediction of RNA secondary structures including pseudoknots. Based on the simple idea of iteratively forming stable stems, our algorithm explores many alternative secondary structures, using a free energy minimization algorithm for pseudoknot free secondary structures to identify promising candidate stems. In an empirical evaluation of the algorithm with 43 sequences taken from the Pseudobase database and from the literature on pseudoknotted structures, we found that overall, in terms of the sensitivity and specificity of predictions, HotKnots outperforms the well-known Pseudoknots algorithm of Rivas and Eddy and the NUPACK algorithm of Dirks and Pierce, both based on dynamic programming approaches for limited classes of pseudoknotted structures. It also outperforms the heuristic Iterated Loop Matching algorithm of Ruan and colleagues, and in many cases gives better results than the genetic algorithm from the STAR package of van Batenburg and colleagues and the recent pknotsRG-mfe algorithm of Reeder and Giegerich. The HotKnots algorithm has been implemented in C/C++ and is available from http://www.cs.ubc.ca/labs/beta/Software/HotKnots.

Decoding of synaptic voltage waveforms by specific classes of recombinant high-threshold Ca(2+) channels.

Studies suggest that the preferential role of L-type voltage-sensitive Ca(2+) channels (VSCCs) in coupling strong synaptic stimulation to transcription is due to their selective activation of local chemical events. However, it is possible that selective activation of the L-type channel by specific voltage waveforms also makes a contribution. To address this issue we have examined the response of specific Ca(2+) channel types to simulated complex voltage waveforms resembling those encountered during synaptic plasticity (gamma and theta firing frequency). L-, P/Q- and N-type VSCCs (alpha1C, alpha1A, alpha1B/beta1B/alpha2delta, respectively) were all similarly activated by brief action potential (AP) waveforms or sustained step depolarization. When complex waveforms containing large excitatory postsynaptic potentials (EPSPs), APs and spike accommodation were applied under voltage clamp we found that the integrated L-type VSCC current was approximately three times larger than that produced by the P/Q- or N-type Ca(2+) channels (gamma frequency 1 s stimulation). For P/Q- or N-type channels the complex waveforms led to a smaller current than that expected from the response to a simple 1 s step depolarization to 0 or +20 mV. EPSPs present in the waveforms favoured the inactivation of P/Q- and N-type channels. In contrast, activation of the L-type channel was dependent on both EPSP- and AP-mediated depolarization. Expression of P/Q-type channels with reduced voltage-dependent inactivation (alpha1A/beta2A/alpha2delta) or the use of hyperpolarized intervals between AP stimuli greatly increased their response to complex voltage stimuli. We propose that in response to complex synaptic voltage waveforms P/Q- and N-type channels can undergo selective voltage-dependent inactivation leading to a Ca(2+) current mediated predominantly by L-type channels.