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Sun Apr 24 04:34:54 EDT 2011
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Hyperactivation of NF-kappaB via the MEK signaling is indispensable for the inhibitory effect of cAMP on DNA damage-induced cell death - With cAMP signaling having a profound inhibitory effect on DNA damage-induced apoptosis in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, understanding how this signaling pathway affects the survival capacity of the cell has important implications for cancer therapy. We have recently shown that p53 is critical for the inhibitory effect of cAMP on genotoxic agents-mediated apoptosis in BCP-ALLs. Here, we show that elevation of cAMP levels in cells exposed to DNA damage enhances the nuclear translocation and DNA binding of NF-kappaB by accelerating the phosphorylation of IKKbeta and thereby phosphorylation and degradation of IkappaBalpha. Furthermore, we show that the ability of cAMP to potentiate the ionizing radiation-induced activation of NF-kappaB requires the activity of MEK. Importantly, pharmacological or genetic ablation of NF-kappaB reversed the inhibitory effect of cAMP on DNA damage-induced apoptosis, demonstrating that, in addition to p53, cAMP relies on the activity of NF-kappaB to provide cells with a survival advantage in the face of DNA damage. Collectively, our results uncover a novel and important interaction between the cAMP and NF-kappaB pathways that may have implications for the targeted treatment of lymphoid malignancies, such as BCP-ALL, in which aberrant NF-kappaB activity functions as a driving force for treatment resistance.

Role of bile salt in regulating Mcl-1 phosphorylation and chemoresistance in hepatocellular carcinoma cells - Background: Glycochenodeoxycholate (GCDA) is one of the major human bile salts. Bile salts stimulate cell survival and proliferation through the mitogen-activated protein kinase, but the downstream signaling mechanism(s) remains enigmatic. Mcl-1 is an antiapoptotic molecule of the Bcl2 family that is extensively overexpressed in tumor tissues of patients with hepatocellular carcinoma (HCC). Results: Here we found that exposure of HepG2 cells to GCDA results in activation of ERK1 and ERK2 and phosphorylation of Mcl-1 in a PD98059 (MEK inhibitor)-sensitive manner. GCDA stimulates Mcl-1 phosphorylation in cells expressing WT but not T163A Mcl-1 mutant, indicating that GCDA-induced Mcl-1 phosphorylation occurs exclusively at the T163 site in its PEST region. GCDA-induced Mcl-1 phosphorylation at T163 enhances the half-life of Mcl-1. Treatment of HepG2 cells with GCDA facilitates Mcl-1 dissociation from Mule (a physiological Mcl-1 ubiquitin E3 ligase). Specific depletion of Mcl-1 from HepG2 cells by RNA interference increases sensitivity of HepG2 cells to chemotherapeutic drugs (i.e. cisplatin and irinotecan). In addition to activation of the ERK/Mcl-1 survival pathway, GCDA can also induce dose-dependent apurinic/apyrimidinic (AP) sites of DNA lesions, which may partially neutralize its survival activity. Conclusion: Our findings suggest that bile salt may function as a survival agonist and/or potential carcinogen in the development of HCC. Molecular approaches that inactivate Mcl-1 by blocking its T163 phosphorylation may represent new strategies for treatment of HCC.

HBx Sensitizes Cells to Oxidative Stress-induced Apoptosis by Accelerating the Loss of Mcl-1 Protein via Caspase-3 Cascade - Background: Oxidative stress has been implicated in the pathogenesis of a wide spectrum of human diseases, including Hepatitis B virus (HBV)-related liver disease. Hepatitis B virus X protein (HBx) is a key regulator of HBV that exerts pleiotropic activity on cellular functions. Recent studies showed that HBx alters mitochondrial membrane potential, thereby sensitizing cells to pro-apoptotic signals. However, it remains largely unknown whether susceptibility of hepatocytes could be disturbed by HBx under oxidative stress conditions. The purpose of this study is to determine the apoptotic susceptibility of HBx-expressing hepatocytes upon exposure to pro-oxidant stimuli in vitro and in vivo and explore its underlying mechanism. Results: Although expression of HBx itself did not activate apoptotic signaling, it significantly enhanced oxidative stress-induced cell death both in vitro and in vivo. Interestingly, this phenomenon was associated with a pronounced reduction of protein levels of Mcl-1, but not other anti-apoptotic Bcl-2 members. Importantly, enforced expression of Mcl-1 prevented HBx-triggered cell apoptosis; conversely, specific knockdown of Mcl-1 exacerbated HBx-induced apoptosis upon exposure to oxidative stress. Furthermore, inhibition of caspase-3 not only abrogated HBx-triggered apoptotic killing but also blocked HBx-induced Mcl-1 loss. Additionally, expression of HBx and Mcl-1 was found to be inversely correlated in HBV-related hepatocellular carcinogenesis (HCC) tissues. Conclusions: Our findings indicate that HBx exerts pro-apoptotic effect upon exposure to oxidative stress probably through accelerating the loss of Mcl-1 protein via caspase-3 cascade, which may shed a new light on the molecular mechanism of HBV-related hepatocarcinogenesis.

Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia - Background: The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results: The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5. Conclusions: Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.

Expression patterns of microRNAs associated with CML phases and their disease related targets - Background: MicroRNAs are important regulators of transcription in hematopoiesis. Their expression deregulations were described in association with pathogenesis of some hematological malignancies. This study provides integrated microRNA expression profiling at different phases of chronic myeloid leukemia (CML) with the aim to identify microRNAs associated with CML pathogenesis. The functions of in silico filtered targets are in this report annotated and discussed in relation to CML pathogenesis. Results: Using microarrays we identified differential expression profiles of 49 miRNAs in CML patients at diagnosis, in hematological relapse, therapy failure, blast crisis and major molecular response. The expression deregulation of miR-150, miR-20a, miR-17, miR-19a, miR-103, miR-144, miR-155, miR-181a, miR-221 and miR-222 in CML was confirmed by real-time quantitative PCR. In silico analyses identified targeted genes of these miRNAs encoding proteins that are involved in cell cycle and growth regulation as well as several key signalling pathways such as of mitogen activated kinase-like protein (MAPK), epidermal growth factor receptor (EGFR, ERBB), transforming growth factor beta (TGFB1) and tumor protein p53 that are all related to CML. Decreased levels of miR-150 were detected in patients at diagnosis, in blast crisis and 67% of hematological relapses and showed significant negative correlation with miR-150 proved target MYB and with BCR-ABL transcript level. Conclusions: This study uncovers microRNAs that are potentially involved in CML and the annotated functions of in silico filtered targets of selected miRNAs outline mechanisms whereby microRNAs may be involved in CML pathogenesis.

Pharmacologic disruption of Polycomb Repressive Complex 2 inhibits tumorigenicity and tumor progression in prostate cancer. - Background: Polycomb repressive complex 2 (PRC2) mediates gene silencing through histone H3K27 methylation. PRC2 components are over-expressed in metastatic prostate cancer (PC), and are required for cancer stem cell (CSC) self-renewal. 3-Dezaneplanocin-A (DZNeP) is an inhibitor of PRC2 with broad anticancer activity. Method: we investigated the effects of DZNeP on cell proliferation, tumorigenicity and invasive potential of PC cell lines (LNCaP and DU145). Results: Exploring GEO and Oncomine databases, we found that specific PRC2 genes (EED, EZH2, SUZ12) predict poor prognosis in PC. Non-toxic DZNeP concentrations completely eradicated LNCaP and DU145 prostatosphere formation, and significantly reduced the expression of CSC markers. At comparable doses, other epigenetic drugs were not able to eradicate CSCs. DZNeP was also able to reduce PC cell invasion. Cells pre-treated with DZNeP were significantly less tumorigenic (LNCaP) and formed smaller tumors (DU145) in immunocompromised mice. Conclusion: DZNeP is effective both in vitro and in vivo against PC cells. DZNeP antitumor activity is in part mediated by inhibition of CSC tumorigenic potential.

Berberine modulates AP-1 activity to suppress HPV transcription and downstream signaling to induce growth arrest and apoptosis in cervical cancer cells - Background: Specific types of high risk Human papillomaviruses (HR-HPVs) particularly, HPV types 16 and 18 cause cervical cancer and while the two recently developed vaccines against these HPV types are prophylactic in nature, therapeutic options for treatment and management of already existing HPV infection are not available as yet. Because transcription factor, Activator Protein-1 (AP-1) plays a central role in HPV-mediated cervical carcinogenesis, we explored the possibility of its therapeutic targeting by berberine, a natural alkaloid derived from a medicinal plant species, Berberis which has been shown to possess anti-inflammatory and anti-cancer properties with no known toxicity; however, the effect of berberine against HPV has not been elucidated. Results: We studied the effect of berberine on HPV16-positive cervical cancer cell line, SiHa and HPV18-positive cervical cancer cell line, HeLa using electrophoretic mobility gel shift assays, western and northern blotting which showed that berberine could selectively inhibit constitutively activated AP-1 in a dose- and time-dependent manner and downregulates HPV oncogenes expression. Inhibition of AP-1 was also accompanied by changes in the composition of their DNA-binding complex. Berberine specifically downregulated expression of oncogenic c-Fos which was also absent in the AP-1 binding complex. Treatment with berberine resulted in repression of E6 and E7 levels and concomitant increase in p53 and Rb expression in both cell types. Berberine also suppressed expression of telomerase protein, hTERT, which translated into growth inhibition of cervical cancer cells. Interestingly, a higher concentration of berberine was found to reduce the cell viability through mitochondria-mediated pathway and induce apoptosis by activating caspase-3. Conclusion: These results indicate that berberine can effectively target both the host and viral factors responsible for development of cervical cancer through inhibition of AP-1 and blocking viral oncoproteins E6 and E7 expression. Inhibition of AP-1 activity by berberine may be one of the mechanisms responsible for the anti-HPV effect of berberine. We propose that berberine is a potentially promising compound for the treatment of cervical cancer infected with HPV.

The functional role of long non-coding RNA in human carcinomas - Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer paradigm demonstrating potential roles in both oncogenic and tumor suppressive pathways. These novel genes are frequently aberrantly expressed in a variety of human cancers, however the biological functions of the vast majority remain unknown. Recently, evidence has begun to accumulate describing the molecular mechanisms by which these RNA species function, providing insight into the functional roles they may play in tumorigenesis. In this review, we highlight the emerging functional role of lncRNAs in human cancer.

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy - Background Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer.Results Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n=66 and 57, respectively) and matching normal benign epithelium (n=21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P<0.001).Conclusions Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.

Soluble Vascular Endothelial Growth Factor Receptor-3 Suppresses Lymphangiogenesis and Lymphatic Metastasis in Bladder Cancer - Background: Most bladder cancer patients experience lymphatic metastasis in the course of disease progression, yet the relationship between lymphangiogenesis and lymphatic metastasis is not well known. The aim of this study is to elucidate underlying mechanisms of how expanded lymphatic vessels and tumor microenvironment interacts each other and to find effective therapeutic options to inhibit lymphatic metastasis. Results: The orthotopic urinary bladder cancer (OUBC) model was generated by intravesical injection of MBT-2 cell lines. We investigated the angiogenesis, lymphangiogenesis, and CD11b+/CD68+ tumor-associated macrophages (TAM) by using immunofluorescence staining. OUBC displayed a profound lymphangiogenesis and massive infiltration of TAM in primary tumor and lymphatic metastasis in lymph nodes. TAM flocked near lymphatic vessels and express higher levels of VEGF-C/D than CD11b- cells. Because VEGFR-3 was highly expressed in lymphatic vascular endothelial cells, TAM could assist lymphangiogenesis by paracrine manner in bladder tumor. VEGFR-3 expressing adenovirus was administered to block VEGF-C/D signaling pathway and clodronate liposome was used to deplete TAM. The blockade of VEGF-C/D with soluble VEGF receptor-3 markedly inhibited lymphangiogenesis and lymphatic metastasis in OUBC. In addition, the depletion of TAM with clodronate liposome exerted similar effects on OUBC. Conclusion: VEGF-C/D are the main factors of lymphangiogenesis and lymphatic metastasis in bladder cancer. Moreover, TAM plays an important role in these processes by producing VEGF-C/D. The inhibition of lymphangiogenesis could provide another therapeutic target to inhibit lymphatic metastasis and recurrence in patients with invasive bladder cancer.